opioid peptides and blood pressure control

Opioid Peptides and Blood Pressure Control
Springer-Verlag Berlin Heidelberg New York London Paris Tokyo
Prof. Dr. med. K. O. Stumpe Dr. med. Karin Kraft Prof. med. A. I. Faden Med. Univ.-Poliklinik WilhelmstraBe 35-37 5300 Bonn 1
11th Scientific Meeting of the International Society of Hypertension Satellite Symposium· Bonn· September 6-7, 1986
ISBN-13:978-3-540-18935-0 e-ISBN-13:978-3-642-73429-8 DOl: 10.1007/978-3-642-73429-8
Ubrary of Congress Cataloging·in-Publication Data Opioid peptides and blood pressure controll K. O. Stumpe (ed.).
p.em. Papers presented at a meeting held in Bonn, Sept. 6-7, 1986 as a satellite symposium to the 11th
Scientific Meeting of the International Society of Hypertension. ISBN-13:978-3-540-l8935-0 (U.S.) 1. Blood pressure--Regulation--Congresses. 2. Opioids--PhysiologicaI effect--Congresses. 3. Hyper
tension--Pathophysiology--Congresses. I. Stumpe, K. O. (Klaus Otto), 1938 -. II. International Society of Hypertension. Scientific Meeting (11th: 1985 : Heidelberg, Germany)
[DNLM: 1. Blood Pressure--drug effects--congresses. 2. Endorphins--pharmacology--congresses. 3. Endorphins--physiology--congresses. 4. Hypertension- physiopathology--congresses. WG 106 0611986] OP 109.065 1988 616.1'32061--dc19 DNLMIDLC for Ubrary of Congress 88-15945
CIP
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Contents
A. I. FADEN, K. KRAFf,andK.O. STUMPE. . . . . . . . . . . . . . . . . . . . . . . . 1
Anatomy
Distribution of Opioid Peptides Functionally Related to the Cardiovascular System
W. KUMMER, M. REINECKE, C. HEYM, and W. G. FORSSMANN . . . . . . . . . . . . 5
Studies on Enkephalinergic Mechanisms in Cardiovascular Centers of the Medulla Oblongata of the Rat and their Interactions with Centrally Administered Neuropeptide Y
A. HARFSTRAND, K. FuxE, L. F. AGNATI, A. CINTRA, M. KALlA, and L. TERENIUS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Multiplicity of Opioidergic Pathways Related to Cardiovascular Innervation: Differential Contribution of All Three Opioid Precursors
E. WEIHE, D. NOHR, W. HARTSCHUH, B. GAUWEILER, and T. FINK. . . . . . . .. 27
Physiology
A. I. FADEN. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 53
Adrenergic Opioid Interaction in the Brain Stem: Role in Cardiovascular Regulation
G. KUNos, andR. MOSQUEDA-GARCIA. . . . . . . . . . . . . . . . . . . . . . . . .. 62
Influence of the Opioid System on Sympathetic Activity and the Renin-Aldosterone System in Healthy Males
M. BRAMNERT, and B. HOKFELT .......... . . . . . . . . . . . . . . . . . . .. 71
VI Contents
Role of Leu-morphin, an Opioid Peptide, in the Central Regulation of Fluid Balance and Blood Pressure in Rats
H. IMuRA, K. NAKAO, T. YAMADA, H. bOH, S. SHIONO,~. SAKAMOTO, N. MORII, A. SUGAWARA, Y. SAITO, andM. MUKOYAMA ............... 83
Endogenous Opioids in the Dorsal Vagal Complex and Resting Cardiovascular Function in the Anesthetized Rat
A. H. HASSEN, and E. P. BROUDY . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 90
Influence of Opiate Peptides on Blood Pressure Regulation and on Hypothalamic Blood Flow
W.DEJoNG,J.COXVANPuT,andP.SANDOR ...................... 98
Opioid Peptides in Human Adrenal Medulla: Their Role in the Modulation of Catecholamine Secretion
E. BALDI, M. MAGGI, M. L. DE FEO, C. PUPILLI, C. SELLI, R. ZIMLICHMAN, E. FORSBERG, V. CARLA, andM. MANNELLI . . . . . . . . . . . . . . . . . . . . . .. 103
Cardiovascular Effects of Neuropeptide Yin the Caudal Ventrolateral Medulla
I.M. MAcRAE,andJ.L. REID . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 112
G.R. VANLoON,K. PIERZCHALA,L. V. BROWN, andD.R. BROWN. . . . . . . .. 117
Pharmacology
Opioid Receptors in the Sympathetic Supply to Blood Vessels and the Heart
B. SZABO, D. RAMME, andK. STARKE. . . . . . . . . . . . . . . . . . . . . . . . . .. 129
Interactions of Opioid Peptides and Adrenergic Agents in the Regulation of Blood Pressure
H. M. RHEE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 141
Effect of Opiate Receptor Blockade on the Cardiovascular and Plasma Noradrenaline Response to Intravenous Tyramine in Man
P. M. G. BouLOux, A. GROSSMAN, and G. M. BESSER. . . . . . . . . . . . . . . .. 150
Effects of Mu- and Delta-Opiate Receptor Agonists on Systemic and Regional Hemodynamics in Conscious Rats
O. S. MEDVEDEV, E. R. MARTYNOVA, and A. HOQuE. . . . . . . . . . . . . . . . .. 159
Retardment of Development of Hypertension in the Spontaneously Hypertensive Rat by Long-Term Kappa-Opioid Receptor Antagonism
J. DIEHL, K. KRAFT, andK. O. STUMPE. . . . . . . . . . . . . . . . . . . . . . . . .. 168
Naltrexone Inhibits Alpha-Methydopa-Induced Hypotension in a Dose-Dependent Manner
Contents VII
P.L.M.VANGIERSBERGEN,G.A.HEAD,andW.DEJoNG .............. 174
W. R. DIXON, and A. CHANDRA .............................. 183
Opioid Receptor Types at Noradrenergic Neurons and their Roles in Blood Pressure Regulation
P. ILLES, andB. BUCHER .................................. , 190
Effect of Opioids on Plasma Levels of Immunoreactive Atrial Natriuretic Factor
J. GUTKOWSKA, B. BARANOWSKA, K. RAcz, R. GARCIA, G. THIBAULT, M. CANTIN, andJ. GENEST ................................. 206
Production by Systemic Enkephalin of Hemodynamic Effects by Afferent Modulation of Autonomic Nervous System Tone
T.D.GILEs,andG.E.SANDER ............................... 212
Pathophysiology and Clinical Aspects
Endogenous Opioids in the Pathophysiology of Shock: Sites of Action, Autonomic Involvement, and Receptor Interactions
J.W. HOLADAY,D.S. MALCOLM,andJ.B. LONG . . . . . . . . . . . . . . . . . . .. 221
Endogenous Opioids and Blood Pressure in Man
P. C. RUBIN. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 233
Effects of Hemorrhagic Shock on Plasma Met-enkephalin, Vasopressin, Catecholamines, and Cardiovascular Functions in Intact and Adrenalectomized Dogs
T. KIMURA, M. INOUE, K. MATSUI, K. OTA, M. SHon, and K. YOSHINAGA. . . .. 236
Effect of Hypertension on the Response of Plasma Beta-Endorphin to the Cold Pressor Test
R. FUKUNAGA,N. HANDA, S. YONEDA,K. KIMURA, andT. KAMADA . . . . . . .. 247
Normalization by Clonidine of Reduced Plasma Beta-endorphin and Leu-enkephalin Concentrations and Elevated Blood Pressure in Young Patients with Mild Essential Hypertension
K. KRAFT, R. THEOBALD, R. KOLLOCH, and K. O. STUMPE . . . . . . . . . . . . .. 253
VIII Contents
C. FARSANG .......................................... 260
Effect of Low Dosage of Naloxone on Clonidine-Induced Changes in Blood Pressure, Catecholamines, Renin, and Aldosterone in Essential Hypertension
M. BRAMNERT, andB. HOKFELT . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 275
Effect of Lisinopril on Circulating Neuropeptides in Essential Hypertensive Patients
S. BRANDMAN, W. T. WISEMAN, J.D. STEPHENS, C. LONG, D.R. GLOVER, andM.J. VANDENBURG .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 282
Endogenous Opioids and Reversal of Renovascular Hypertension
M. E. EDMUNDS, G. 1. RUSSELL, R. F. BING, H. THURSTON, andJ. D. SWALES.. 287
Comparison of Pain Threshold as Assessed by Tooth Pulp Stimulation in Normotensives with Different Hypertensive Hereditary Backgrounds and in Borderline and Established Hypertensives
S. GHIONE, C. RosA, L. MEZZASALMA, andE. PANATTONI . . . . . . . . . . . . .. 294
Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 299
A.!, FADEN, K. KRAFr, and K. O. STUMPE
Following the discovery of the pentapeptide enkephalins in 1975, a number of endogenous opioid peptides and opiate receptors have been identified. Endogenous opioids and opiate-receptor mechanisms have been implicated in a variety of regulat ory and dysregulatory functions including analgesia, cardiovascular regulation, shock, hypertension, traumatic spinal cord and brain injury, stroke, immune func tion, feeding behavior, diuresis, gastrointestinal motility, and respiratory control, among others.
Over the past 10 years, many studies have demonstrated a relationship between endogenous opioids and the cardiovascular system under both homeostatic and pathophysiological conditions. Opioids and opiate receptors have been found in various cardioregulatory sites within the brain and spinal cord, as well as in peripheral tissues such as sympathetic ganglia, adrenal gland, and heart. Both endogenous opioids and exogenous opiates have been shown to produce potent cardiovascular effects following central nervous system or systemic administration. Opiate-receptor antagonists have been demonstrated to reverse hypotension from sepsis, hypo volemia, and anaphylaxis; such studies have been used to infer activity of endogenous opioid systems in shock. Changes in tissue concentrations of endogenous opioids and! or opiate receptors have been found after shock and hypertension, further implying a role for opioid systems in the etiology of these conditions. In addition, modification of opiate receptor regulation, receptor binding, or opioid metabolism has also been used to establish a potential role for endogenous opioid systems in cardiovascular control and dyscontrol.
Although the relationship between opioids and cardiovascular regulation has received increasing attention, there has not previously been an international meeting devoted to this topic. In September 1986, such a meeting was held in Bonn as a Satellite Symposium of the International Society of Hypertension. Its purpose was to permit anatomists, pharmacologists, physiologists, and clinicians to interact in a critical analysis of the role of the opioids on cardiovascular control in physiological and pathological conditions. It was hoped that such a multidisciplinary symposium would both serve as a state-of-the-art review and promote further experimental and clinical research efforts in this area. Clearly, we need to know far more about the interactions of opioids and the cardiovascular system before establishing a clear role for the opioid system in the physiology and pathophysiology of cardiovascular con trol.
2 Introduction
We were most gratified by the participation of many outstanding investigators in the scientific program. The present volume contains the proceedings of this sym posium. It is our hope that the book will serve both as reference for use within this field and as a stimulus for further research.
Anatomy
w. KUMMER, M. REINECKE, C. HEYM, and W. G. FORSSMANN
Department of Anatomy, University of Heidelberg, 1m Neuenheimer Feld 307, D-6900 Heidelberg, FRO
Introduction
Endogenous opioid peptides exert multiple modulatory effects in the regulation of cardiovascular function at both central [11] and peripheral sites [49]. A crucial basis for the understanding of the complex mechanisms involved in this regulatory system is the detailed knowledge of the morphological distribution of opioid peptides. The morphological methods appropriate for this purpose require antisera raised against the different opioid peptides and the use of immunohistochemistry. However, dif ficulties arise from the structural similarities of opioid peptides. To our present knowledge, opioid peptides are cleavage products of three large precursor molecules: a) proopiomeianocortin (POMC) processing results in the production of endorphins, b) prodynorphin is the precursor of neoendorphins and dynorphins, and c) proenkephalin contains one copy of leu-enkephalin (LE) and several opioids
sharing the met-enkephalin (ME) sequence at their N-terminus.
However, the ME sequence is also part of endorphins, and LE represents the N terminus ofneoendorphins and dynorphins [5, 21, 31, 32]. The multiple occurrence of the ME and LE fragment and cross-reactions of most enkephalin (ENK) antisera with both ME and LE complicate the interpretation of immunohistochemical studies. Specific antisera raised against peptides characteristic for a distinct precursor [e. g., beta-endorphin (END), alpha-neo-endorphin (NEO) , met-enkephalin-arg-phe (MEAP)], therefore, have to be used additionally. Thus, our immunohistochemical study on the distribution of opioid peptides in cardiovascular regulatory centers and in the sympathoadrenal system is focussed mainly on precursor-specific peptides. The specificity of the antisera used is presented elsewhere [25, 26]. The results are compared with previous findings on ME and LE distributions and the recent literature is included to present a survey on the distribution of opioid peptides in the spinal cord and peripheral nervous system with respect to cardiovascular function. The morphol ogy of brain opioid systems has been extensively reviewed recently [22, 33].
Paravertebral Ganglia
After colchicine treatment, principal neurons of the superior cervical ganglion (SCG) and the stellate ganglion of the rat exhibit moderate immunoreactive (IR)-MEAP.
6 W. Kummer et al.
b
t ·
0'
", , . j
, o'
f.
Fig. 1 a, b. Guinea pig stellate ganglion. a Dyn-IR principal neurones and few immunorective fibres. b regional accumulation of MEAP-IR pericellular fibres. Bar = 50 I!m
This is not the case in the same ganglia of the guinea pig. The immunohistochemical findings are in accordance with data obtained from high-performance liquid chromatography (HPLC) studies combined with radioimmunoassay (RIA), showing a high MEAP content of rat SCG when compared with guinea pig SCG [40]. Another cleavage product of proenkephalin, ME, has been immunohistochemically localized in rat SCG neurons [1, 2, 9, 37] . Alpha-NEO-IR neurons are visible in the stellate ganglion of colchicine-treated rats, and both dynorphin (DYN) A 1-17-IR and alpha NEO-IR neurons are visible in the guinea pig stellate ganglion (Fig. 1 a) . These results correlate with the demonstration of DYN A 1-17-IR and alpha-NEO-IR within preganglionically denervated neurons of the guinea pig SCG and within swollen nerve fibers at the proximal end of transected postganglionic branches [27] . Prodynorphin processing in guinea pig paravertebral sympathetic neurons was concluded from the marked depletion of alpha-NEO-IR and DYN A 1-8-IR within heart extracts follow ing treatment with the neurotoxin 6-hydroxydopamine (6-0HDA) demonstrated by RIA and HPLC analysis [28, 49] . At present, the alpha-NEO-IR material in rat sympathetic ganglia has not been characterized by analytical biochemical methods.
Small intensely fluorescent (SIF) cells of paravertebral ganglia display ENK-IR in most species, but not in rats [16, 37]. Both ME and LE antisera were used, but due to cross-reacting properties of the antisera with both enkephalins, with DYN, and with alpha-NEO it is still unclear whether these pentapeptides in fact are present in SIF cells [27,42] . In the guinea pig, peptides derived from prodynorphin, i. e., DYN A 1- 17 and alpha-NEO, were immunohistochemically demonstrated to occur in more SIF cells than LE or ME [4, 42] . So far we have not detected proenkephalin-related peptides in SIF cells of guinea pig paravertebral ganglia using MEAP-specific anti bodies. ME-IR and LE-IR varicosities terminating on principal neurons are a com mon feature of mammalian paravertebral ganglia [14, 17, 27, 37, 42]. In addition,
Distribution of Opioid Peptides Functionally Related to the Cardiovascular System 7
MEAGL-IR-beaded fibers have been reported in the rat [15], and MEAP-IR fiber baskets occur in human paravertebral ganglia [14]. In the guinea pig stellate ganglion regional accumulations of ENK-IR [42] and MEAP-IR fibers (Fig. 1 b) are striking. Costorage of ME-IR, LE-IR, and MEAP-IR substances in pericellular varicosities suggests proenkephalin processing in those perikarya which give rise to these ENK-IR fibers. LE-IR [27] and MEAP-IR fiber baskets are absent in the denervated guinea pig SCG, suggesting a preganglionic origin ofthese fibers. ENK -IR nerve terminals of yet unknown origin approach SIF cell clusters in rat paravertebral ganglia [16]. In contrast to rat, DYN A 1-17-IR [27, 42] and alpha-NEO-IR varicosities [27] can be found in guinea pig paravertebral ganglia. They probably represent processes of SIF cells as deduced from studies on denervated, axotomized, 6-0HDA- or reserpine treated animals [27]. Alpha- and beta-END-IR elements have not been detected in paravertebral ganglia [37] .
Prevertebral Ganglia
Less than 1 % of the neurons in the guinea pig inferior mesenteric ganglion contain alpha-NEO-IR (Fig. 2a). Recently Jule et al. [20] have detected numerous ENK-IR perikarya in the celiac ganglion of colchicine-treated cats, but cross-reactivity of the applied antibodies to DYN and alpha-NEO was not excluded. Most SIF cells of guinea pig prevertebral ganglia exhibit alpha-NEO-IR ([4]; Fig. 2a), whereas DYN A-IR cell bodies are less frequent [4, 42]. MEAP-IR SIF cells occur rarely [4]. The question of coexistence of pro en kephalin- and prodynorphin-derived opioid peptides in these SIF cells has so far not been clarified. Only proenkephalin-related peptides [ME, MEAP, met-enkephalin-arg-gly-Ieu, (MEAGL)] can be identified immunohis tochemically in beaded nerve fibers within large SIF cell clusters (Fig. 2b; [4, 15, 16,
~ ' a
" .. ,
Fig.2a,b. Consecutive sections of guinea pig inferior mesenteric ganglion, a MEAP-IR fibers surrounding principal neurons and penetrating a large SIF cell cluster; b Numerous NEO-IR nerve fibers and SIF cells. Two principal neurons display NEO-IR (arrows) . Bar = 50 !!m
8 W. Kummer et al.
37, 42]). In contrast, extremely dense networks of DYN-IR and alpha-NEO-IR varicosities surround the principal neurons (Fig. 2a; [7,42]). Degeneration experi ments carried out on guinea pig inferior mesenteric ganglia reveal a peripheral (intestinal) origin of these fibers, whereas the numerous ENK-IR [7,8] and MEAP IR (Fig. 2b; [46]) fiber baskets stem from central (preganglionic) neurons. Schultz berg et al. [37] observed a very dense immunoreactive nerve terminal network in the guinea pig inferior mesenteric ganglion after incubation with one beta-END anti serum whereas negative results were obtained with two other beta-END antisera. No additional evidence for the occurrence of POMC-derived opioids in prevertebral ganglia has been presented as yet.
Blood Vessels
Our studies on the distribution of opioid peptides in the guinea pig were carried out to identify this type of peptidergic innervation in the perivascular plexus of many organs. Alpha-NEO-IR and, less numerous, DYN A l-l7-IR varicosities are present mainly around small arteries and arterioles, and sometimes related to small veins. Skin vessels of guinea pig paw and snout are innervated by LE-IR, DYN A l-8-IR, and alpha-NEO-IR fibers [47]. As for large arteries, alpha-NEO-IR and DYN A l-l7-IR varicosities can be observed along the guinea pig inferior mesenteric artery (Fig. 3 a), but not in perivascular nerves of the common carotid artery. Differential supply with DYN-IR fibers even along a single artery has been described recently by Morris et al. [30], who colocalized DYN A l-l3-IR with both NPY-IR and VIP-IR in non noradrenergic axons along a circumscribed segment of the guinea pig uterine artery. The origin of perivascular DYN-IR and alpha-NEO-IR fibers is still unclear, because both sympathetic [27] and sensory ganglia [25, 47] of the guinea pig contain DYN-IR and alpha-NEO-IR neurons.
a . ,
Fig. 3a, b. NEO-IR varicosities along a first-order branch of the guinea pig inferior mesenteric artery (a), and within the guinea pig atrial myocardium (b). Bar = 10 Itm
Heart
Enkephalin-like material has been detected in heart extracts of various mammals by different methods [19, 28, 46, 48, 49]. Surprisingly, in the guinea pig heart compara tively high ME-IR levels were obtained in combined HPLC and RIA study [28] but almost no ME-like activity was detected in the respective HPLC fraction using the mouse vas deferens assay [48]. Immunohistochemically, ENK-IR nerve fibers supply the coronary arteries of the guinea pig heart [12, 34, 35] with a preference for the arterial system of the atria. While no ENK-IR fibers seem to contact myocardiocytes or nodal cells, they are, however, present in high numbers within the intracardiac ganglia [34]. Prodynorphin-related peptides were extracted from rat [38] and guinea pig heart [48, 49]. Immunohistochemical investigation of the guinea pig heart reveals beaded alpha-NEO-IR nerve fibers mainly associated with arterioles and small arteries. Fibers without a clear relationship to blood vessels are only occasionally observed (Fig. 3 b). In general, alpha-NEO-IR terminals are less numerous than those displaying LE-IR. No immunoreactive fibers as yet have been observed after incuba tion with antisera directed against DYN A 1-17. These results correspond to biochem ical findings, revealing high levels of alpha-NEO- and low concentrations of DYN A 1-17-like material in guinea pig heart [48, 49], which is possibly due to the degradation of DYN A 1-17 into smaller fragments. In the guinea pig, the marked depletion of both extractable ENK-IR and prodynorphin-derived peptide-IR in response to appli cation of the neurotoxin 6-0HDA suggests a sympathetic origin of the respective nerve fibers [28,49].
Adrenal Medulla
Opioid peptides are costored and coreleased with catecholamines from adrenal medullary cells [29, 43]. Since the first immunohistochemical demonstration ofENK IR in the adrenal medulla [36], numerous studies have dealt with the distribution of ENK-IR, MEAP-IR, and MEAGL-IR cell bodies and fibers within the adrenal gland of various species and have revealed remarkable species differences (see review [23]). Recently, the messenger RNA encoding preproenkephalin has been detected in the rat adrenal medulla using the in situ hybridization technique [3]. Endorphins are not common to all species, but have been described in adrenal medullary cells of pig, cow, dog, and man [10, 39]. RIA measurements of fractionated bovine adrenal medullary cells indicated costorage of several DYN A fragments with noradrenaline as well as costorage of LE with adrenaline [29]. Recent work of our group [4] was focussed on the guinea pig adrenal, which almost completely lacks typical noradrenaline-contain ing cells [41]. Previously, DYN A 1-13-IR cells were demonstrated in this organ [44]; however, the immunoreactions obtained could be suppressed by preabsorption with LE. After applying antisera specific for the C-terminus of DYN A 1-17 and alpha NEO, many cells display alpha-NEO-IR and few cells exhibit DYN A-IR. Both immunoreactions cannot be blocked by ME or LE. Investigations using immunos tained, plastic-embedded semithin sections (0.5 !tm) reveal that LE-IR, ME-IR, and MEAP-IR are coexistent with prodynorphin-related peptide-IR in some of the
10 W. Kummer et al.
adrenal medullary cells. This indicates that no separate DYNergic and ENKergic cell systems as proposed for the bovine adrenal medulla [29] exist in the guinea pig.
Thoracolumbar Sympathetic Nuclei of the Spinal Cord
Preganglionic sympathetic neurons as identified by retrograde labeling are richly supplied by ENK-IR fibers [13, 18]. Denervation experiments in the rat revealed a supraspinal origin of these fibers [18]. In agreement with findings on rat and sheep [33], we observed an identical distribution pattern of MEAP-IR axons in the guinea pig spinal cord, suggesting proenkephalin as the source of the previously described ENK-IR material. Similarly, a previous report on ENK-IR within perikarya of preganglionic sympathetic neurons [6] has recently been substantiated by the use of antisera directed against MEAGL [23]. From the ultrastructural appearance of MEAGL-IR preganglionic fibers in the rat adrenal medulla, coexistence ofproenke phalin-related peptides and acetylcholine was suggested [24]. This hypothesis is strongly supported by the simultaneous demonstration of MEAGL-IR and choline acetyltransferase within identical neurons of thoracic sympathetic nuclei [23]. Neither POMC-related nor prodynorphin-related peptides have been described as yet within spinal cord sympathetic nuclei.
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Studies on Enkephalinergic Mechanisms in Cardiovascular Centers of the Medulla Oblongata of the Rat and their Interactions with Centrally Administered N europeptide Y
A.lliRFSTRAND, K. FUXE, L.F. AGNATI, A. aNTRA, M. KALlA and L. TERENIUS
Department of Histology, Karolinska Institutet, Box 60400, S-10401 Stockholm, Sweden
Introduction
In our previous work beta-endorphin, morphine, and d-ala2-met-enkephalinamide in the nanomolar range were found to produce preferential vasodepressor responses and bradycardia upon intracisternal (i. c.) injection into the alpha-chloralose anesthetized rat [1]. Leu- and met-enkephalin and alpha neo-endorphin administered in the same way preferentially produced vasopressor actions, which with the two latter peptides were associated with bradycardia. Both the pressor and depressor responses were counteracted by naloxone pretreatment i. c., but the depressor actions were preferen tially sensitive to the blocking activity of naloxone. These results indicated the existence of two types of opiate receptors in central cardiovascular regulation, both innervated by enkephalin immunoreactive (IR) terminals [2]. Subsequent work has also demonstrated the existence of high densities of the dynorphin IR nerve terminals and cell bodies within the nucleus tractus solitarii (nTS) and in the nucleus ambiguus [3,4]. The dynorphin synapses may inter alia operate via kappa-opiate-receptors, and an injection of preferential kappa-opiate agonists into the nucleus ambiguus and nTS produces cardiovascular actions [5]. Both enkephalin and dynorphin peptides and their associated opiate receptors may therefore be involved in the regulation of the activity of the cardiovascular centers of the medulla oblongata. In a recent study by Kalia et al. [6] it was found that enkephalin IR terminals densely innervate the subnuclei of the nTS receiving baroreceptor and chemoreceptor afferents (dorsal strip, dorsal subnucleus, dorsal parasolitary region) and also subnuclei receiving cardiac afferents (commissural nucleus of the nTS) and gastrointestinal afferents (medial subnucleus of the nTS). Substantial numbers of enkephalin IR nerve cell bodies have also been observed in all the various subnuclei, including the lateral respiratory subnuclei, which also receive sparse to moderate enkephalin innervation [6].
These results indicated that there may be large numbers of enkephalin IR inter neurons participating in the integration of information in the cardiovascular, respira tory, and gastrointestinal subnuclei of the nTS.
14 A. Hlirfstrand et al.
In the present paper, we have analyzed the codistribution of enkephalin IR nerve terminals within the nTS, the dorsal motor nucleus of the vagus, the nucleus ambi guus, and the C1 area in relation to the distribution of [3H]etorphin and [3H]D-ala2-
D-Ieu5-enkephalin-binding sites, which represent markers for mu- and delta-opiate receptors respectively [10], using immunocytochemistry and receptor auto radiography.
Materials and Methods
Specific pathogen-free, 200- to 250-g male Sprague-Dawley rats (ALAB, Stockholm, Sweden) were used.
Immunocytochemical Experiments
The rats underwent transcardiac perfusion with 150 ml 0.1 M sodium phosphate buffer containing 4% (w/v) paraformaldehyde and 0.4% (w/v) picric acid. The brain stem was then kept in the fixative for 4 h and then transferred to a 10% sucrose solution. Twenty-micrometer-thick serial cryotome sections of the medulla oblongata were made at levels 1 mm caudal to 2 mm rostral to the obex. For further details of the immunocytochemical procedures, see Fuxe et al. [14].
The location of enkephalin IR cell bodies, nerve terminals, and preterminal proces ses was examined by the biotin-avidin peroxidase method (Vecta Stain ABC, Vector Laboratories, Burlington, California. The same procedure was also used to study tyrosine hydroxylase (TH) IR in adjacent coronal sections. Immunocytochemical analysis was combined with cytoarchitectural identification of the various nuclear subgroups, performed on adjacent sections using thionine staining [6]. For characteri zation ofthe enkephalin antiserum used, see Schultzberg et al. [11], forcharacteriza tion of the TH antiserum, see Hokfelt et al. [12], and for purification of TH, see Markey et al. [13].
Receptor Autoradiographic Experiments
The rats underwent transcardiac perfusion with ice-cold sodium chloride solution (0.9% w/v) under methohexital sodium anesthesia. The medulla oblongata was dis sected out and frozen and coronal 14-J.tm-thick sections were made in a Leitz cryostat at various rostrocaudal levels of the medulla oblongata, matching those taken for immunocytochemistry. For further details, see Hiirfstrand et al. [8]. In the [3H]etor phin-binding experiments the radioligand concentration was 1.5 nm, and the sections were incubated with the radioligand for 45 min at room temperature. Nonspecific binding was defined as the binding in the presence of naloxone (2 J.tm) [10]. In the [3H]D-ala2-D-leu5 (DADL)-enkephalin experiments the radioligand concentration was 10 nm. The binding procedure was performed for 60 min at room temperature. Nonspecific binding was defined as the binding in the presence of levalorphane (1 J.tm) [10]. A tritium-sensitive sheet film eH Ultrofilm, LKB Stockholm, Sweden),
Studies on Enkephalinergic Mechanisms in Cardiovascular Centers 15
was used. Exposure time was 4-6 weeks. The specific activity was 44 Cilmmol for the [3H]DADL-enkephalin and 36 Cilmmol for the [3H]etorphin. Both ligands were purchased from NEN, United States.
Physiological Experiments
Arterial blood pressure (ABP) and heart rate (HR) were recorded as described [16, 17]. Briefly, alpha-chloralose anesthesia was introduced by an injection into the lingual vein using a dose of 100 mg/kg after an initial anesthesia with halothane (3% in air). Mean arterial blood pressure (MAP) and HR were measured by means of a heparinized catheter positioned in the common carotid artery. The catheter was connected to a Statham PC23DC transducer connected to a Grass polygraph (Model 7).
Measurement of the respiration rate (RR) and indirect measurement of tidal volume were carried out by means of an intraesophageal catheter positioned at the mid-level of the mediastinum. This catheter was also connected via a Statham trans ducer to the Grass polygraph. The basal values were recorded for a 30-min period prior to the i. c. treatment with the morphiceptine and/or neuropeptide (NPY). The i. c. injections were made by means of a stereotaxic device. All substances were dissolved in mock CSF and the injection volume was 10 Ill. The body temperature was maintained at 370-37.5 0C, and each rat was used only once. Morphiceptine was purchased from Peninsula (Belmont Ca., United States) and NPY from Bachem (Bubendorf, Switzerland). In the statistical analysis Dunn's test was used.
Results
Immunocytochemical Studies
In Fig. 1 a - f the distribution of enkephalin IR nerve terminals is demonstrated within both the dorsal and ventral cardiovascular areas of the medulla. In Fig. 1 c the distribution of the enkephalin IR nerve cell bodies is also shown, since this section was taken from an animal which had been treated with 120 mg colchicine i. c. 48 h previously. The dense enkephalin innervation of the part of the nTS medial to the TS is shown together with the dense innervation of the dorsal motor nucleus of the vagus (mnX). A sparse to moderate enkephalin innervation is shown in the lateral subnuclei of the nTS as well as in the part of the reticular formation extending from the nTS toward the Al and CI areas. In Fig. 1 a-c a dense innervation by enkephalin IR terminals is also found within the subtrigeminal part of the lateral reticular nucleus. At these levels a moderate enkephalin innervation is also found in the adjacent part of the caudal subnucleus of the nucleus tractus spinalis nervi trigemini.
In Fig. 1 c enkephalin IR nerve cell bodies are found in large parts of the reticular formation of the medulla oblongata. They are usually scattered but aggregated within the Cl area.
In Fig. 2 (lower half) the distribution of enkephalin IR nerve cell bodies is shown in great detail within the nTS and the area postrema at a level - 0.2 mm caudal to obex.
16 A. Harfstrand et al.
A TS I PT
E ity is demonstrated at various rostrocaudal
dmnX levels of the medulla oblongata of the male rat. In c the section is taken from a colchicine- treated rat (120 Jlg, i. c.,
C3 48 h before killing). The level is indicated in the
- nSpVI lower left part of each figure and indicates the distance in millimeters from the obex. The biotin-avidin immuno- peroxidase procedure
Oi was used. Primaryanti- serum was diluted at
1.6 py 1: 1000. ap, area postrema; TS, tractus sol-
B
-0.05
1.1. itarius; nXII, hypoglos- sal nucleus; cc, central canal; PT, paratrigemi- F nal nucleus; nRtpc, par vocellular reticular nucleus; LRt, lateral reticu lar nucleus; LRtPC, parvocellular part of the lateral reticular nucleus; LRtS5, subtrigeminal part of the lateral reticu larnucleus;py, pyrami dal tract; Oi, inferior olive; nSp VC, caudal part of the nucleus trac tus spinalis nervi trige mini; nSp VI, inter positus part of the nuc leus tractus spinalis nervi trigemini; aA, 2.0 ambiguus nucleus
1mm
I
1mm
18 A. Hiirfstrand et at.
Enkepbalin IR nerve cell bodies are found in almost all subnuclei of the nTS with the exception of the dorsolateral subnucleus. Large numbers are also found in the external zone of the area postrema. Within the dmnX, however, only enkephalin IR terminals are found and the low density within the lateral subnuclei of the nTS relative to the medially located subnuclei is also further illustrated.
TR IV , ./.' I
ENK IV
200~m .
Fig. 2. Tyrosine hydoxylase and enkephalin-like immunoreactivity are shown in adjacent coronal sections of the medulla oblongata at the level of the area postrema. The biotin-avidin immunoperoxid ase procedure was used. The TH antiserum was diluted 1: 1500 and the enkephalin antiserum at 1:1000. The rat had been pretreated with colchicine i.c. 48 h before killing (120 flg). Substantial numbers of enkephalin IR nerve cell bodies are found in all the subnuclei of the nTS and in the outer zone of the area postrema. The TH IR cell bodies are predominantly found within the medial subnucleus ofthe nTS (mnTS) and within the dorsal subnuclei (dorsal strip) (ds), dorsal subnucleus of the tractus solitarius (dnTS), dorsal parasolitary region (dPSR), and area postrema. The enkephalin IR nerve terminals appear as fine dots in the background within all the various subnuclei of the tractus solitarius and within the dmnX. dinPS, dorsolateral subnucleus of the tractus solitarius; ni, interstitial subnucleus; vinTS, ventrolateral subnucleus of the tractus solitarius; vnTS, ventral subnucleus of the tractus solitarius; PVR, periventricular region; ncom, commissural nucleus; cc, central canal; dmnX, dorsal motor nucleus of the vagus; nRtpc, parvocellular reticular nucleus. Asterisks indicate the same vessels
In Fig. 2 (upper half) the distribution of the catecholamine (CA) nerve cell bodies as demonstrated by TH IR is shown in the nTS in an adjacent section. There is an obvious codistribution with enkephalin IR cell bodies within the medial subnucleus, the dorsal strip, the dorsal subnucleus, and the dorsal parasolitary region. However, the enkephalin IR cell bodies are found in much larger numbers within the laterally located subnuclei and within the intestitial nucleus of the tractus solitarius. Also in the area postrema a substantial difference exists, since the TH IR cell bodies are found to be located all over the area postrema and in a high density.
In Fig. 3 the existence of substantial numbers of large enkephalin IR nerve cell bodies is shown within the reticular paragigantocellular nucleus, within the ventral part of the gigantocellular reticular nucleus, and within the nucleus raphe magnus. Also the dense enkephalin innervation of the nucleus ambiguus is demonstrated. At this level the ventrolateral medulla as seen in the adjacent section contains the adrenaline cell group CI, which thus codistributes with large numbers of enkephalin IR nerve cell bodies (see also Fig. 4). However, no evidence for coexistence of enkephalin and TH IR has been found in this region, using the occlusion method of Agnati et al. [18] . In Fig. 4 the existence of a dense plexus of enkephalin IR nerve terminal within the nucleus ambiguus is shown together with a further illustration of the distribution of enkephalin cell bodies in the ventral medulla (see also Fig. 1 e, f).
Fig. 3. Tyrosine hydroxylase and enkephalin IR are demons trated in the ventrolateral medulla of the colchicine-tre ated rat (for details, see text to previous figure legends). The immunoreactivities are demon strated in adjacent sections at a level + 1.8 mm rostral to obex. Both enkephalin and TH IR cell bodies are demonstrated in the Cl area but without any obvious codistribution within that area. The Cl area consists mainly of the paragigantocellular reticu lar nucleus (PGi) and the reticular gigantocellular nuc leus, ventral part (Giv). For other abbreviations, see text to previous figures. Asterisks indi cate the same vessels
TH 1:2500 .
i l~ *
EN~ 1:1000
) py J ./ Fig. 4. Enkephalin-like IR is
demonstrated in the ventral medulla with the ventral mid line region and the nucleus ambiguus in a coronal section of the medulla oblongata at a ros trocaudallevel + 1.8 mm rostral to obex. The biotin-avidin peroxidase procedure was used. Large numbers of enkephalin IR nerve cell bodies are found within the PGi and Giv as well as in the ventral midline area mainly nucleus raphe magnus. A dense enkephalin innerva tion is demonstrated in the nuc leus ambiguus
Fig.5a-d. The nTS, the dmnX, and adjacent parts of the medulla oblongata are shown in coronal section at the rostrocaudallevel - 0.20 mm caudal to obex following staining for nerve cell bodies (thionin staining) (a) and following incubation with pH]D-ala2 -leus -enkephalin (DADL) (10 nm) and following incubation with [3H]etorphin (ETO) (1 nm). It is shown that thereis dense labeling of all the subnuclei of the nTS medial to the tractus solitarius and moderate labeling in the dmnX following incubation with [3H]DADL en Kephalin (10 nm). As for the incubation with PH]ETO the labeling is mainly confined to the dorsal subnuclei of the nTS and the periventricular region. Note the relative absence of labeling in the dmnX. For abbreviations, see text to previous figures
3H - DADL 10nM
Receptor Autoradiographic Experiments
The results are summarized in Fig. 5 a, band 6. At the area postrema level [3H]D-ala2-
D-leu5-enkephalin seems to label strongly the entire nTS with the exception of the lateral subnuclei and the dmnX, while the [3H]etorphin densely labels only the periventricular regions surrounding the area postrema and the dorsal cardiovascular subnuclei. The dmnX is only weakly labeled, which is true also for the lateral
3H-ETO 1nM
3H-DADL 10nM
+1.7 Fig. 6. Visualization of [3Hletorphin (ETO) (1 nm), and [3H1D-ala2-D-leu5-enkephalin (10 nm) binding in two paralle114-!lm coronal sections of the rostral part (obex + 1.7 mm) of the medulla oblongata of the rat. Note the intense [3H1ETO binding in the lateral part of the nTS. TS, tractus solitarius; nA, nucleus ambiguus
subnuclei. [3H]D-ala2-D-Ieu5 enkephalin also substantially labels the area postrema. In Fig. 6a and b both [3H]etorphin and [3H]D-ala2-D-leu5 enkephalin are shown to label strongly the nucleus ambiguus. However, at this rostral level (1.7 mm rostral to obex) only [3H]etorphin strongly labels the lateral subnuclei of the nTS. Instead the dmnX and the medial subnuclei with adjacent reticular formation and the ventral parasolitary region are moderately labeled by both radioligands. Also both radioligands only weakly label the ventrolateral medulla.
Functional Experiments
The results obtained in the studies on the interaction between NPY and morphiceptin have demonstrated additive cardiovascular responses obtained upon their coad ministration i.c. into the a-chloralose-anesthetized male rat (Fig. 7.). Thus there appears to develop an additive action, when morphiceptine and NPY are given together in low doses, producing weak hypotensive responses by themselves. No such interactions were observed with heart rate and respiratory rate (Fig. 7).
Discussion
Overall the present study demonstrates a codistribution of enkephalin IR nerve terminals and mu- and delta-opiate receptors within the nTS, nucleus ambiguus, and CI area. This appears to be especially true in the cardiovascular nuclei of the nTS, i. e., dorsal strip, dorsal parasolitary region, dorsal subnucleus and adjacent periventricu lar region, and in the nucleus ambiguus that may control the HR. The baroreceptor afferents are known to terminate within the dorsal cardiovascular nuclei of the nTS. Therefore, enkephalins released in these regions, probably reach both the mu- and delta type of opiate receptors, both of which may participate in modulation of baroreceptor reflex activity [24]. Thus, these results strongly support the view of the existence of multiple opiate receptors involved in central cardiovascular regulation [1, 5,20,21].
It seems possible that the two types of opiate receptors which have been shown to overlap in many nuclei in the present study may regulate the sensitivity of one another by a receptor-receptor interaction [22] so that a more selective response can be obtained. The results indicate that these subtypes of opiate receptors are both reached by enkephalins.
It is of substantial interest that the lateral nuclei of the nTS, which are involved with respiratory regulation, in the rostral parts of the nTS are characterized by a high density of mu-opiate receptors, while only a sparse plexus of enkephalin IR terminals and a relatively low density of delta-opiate receptors are present. Thus, in this region there is a mismatch between the pre- and postsynaptic elements of enkephalin neurons. Furthermore the mu-opiate receptors dominate. Such a mismatch has also been noted in previous studies by Agnati et al. [23] in a correlation analysis between the distribution of enkephalin IR and beta-endorphin IR nerve terminals and the distribution of [3H]etorphin and of [3H]d-ala2-d-Ieu5-enkephalin-binding sites in the tel- and diencephalon. The mismatch was predominantly observed within the
24 A. Harfstrand et al.
MAP
~ 10
0
-10
-20
Ba8al valuee: MAP HR RR n:
~
NPY 75pmol. 107t 6 427t16 81t6 6 ..... ~~~~:; Morphlceptln 1nmol
Fig. 7. Cardiovascular and respiratory effects of NPY and morphiceptine are shown after their combined i. c. administration in the alpha-chloralose-anesthetized male rat. The time curves are shown in the left part in the figure. The values are given as means ± SEMs and taken as percentage of respective mean basal value. In the right part of the figure the means ± SEMs are given for the vasodepressor areas (D P A) and vasopressor areas (VP A) (upper part), for the tachycardic (TCA) and the bradycardic (BPA) areas (middle part), and for the tachypneic and bradypneic areas (lower part) in arbitrary units calculated by an IBM XT (cardiovascular software by GUNA Consult Stockholm, Sweden). The statistical analysis was performed by means of Dunn's test for multiple comparisons. • P < 0.05; *. P < 0.01
thalamus and hypothalamus. Based on these observations it was suggested that in these areas enkephalin synapses may mainly operate via volume transmission, i. e., that the opiate receptors are reached by enkephalins, which have diffused from distant enkephalin nerve terminals present in the same or adjacent regions. Such a volume transmission could result in a high plasticity and a long-term action [23]. Thus it should be considered that also in the lateral nTS and other parts of the nTS
enkephalins may be released also in a paracrine fashion to reach distant opiate receptors. It seems likely that the [3H]etorphin-binding sites within the lateral nTS are reached by enkephalins, since they appear to have a similar affinity to those located within the medial nTS and the dorsal subnuclei and in the nucleus ambiguus.
In contrast, the dmnX appears to be innervated by enkephalin synapses which predominantly operate via the delta-opiate receptors relative to mu-opiate receptors. These results further underline the view that each enkephalin IR nerve terminal system may operate with its own unique set of opiate receptors. It will be of substantial interest also to evaluate how the kappa-opiate receptors within the dorsal and ventral cardiovascular centers are distributed in relation to the mu- and delta-opiate recep tors, in order to possibly further understand interactions between these three main types of opiate receptors in the brain and their involvement in cardiovascular control.
In previous work we have demonstrated that a high density of 125I-NPY-binding sites exists within the dorsal subnuclei of the nTS [8]. In the present study a high density of mu-opiate receptors was also demonstrated at this site. It was therefore of substantial interest to evaluate the cardiovascular effects of coadministered NPY and morphiceptine given i. c. In the doses tested, NPY and morphiceptine were both found to produce weak hypotensive actions when given alone, and additivity was observed in the coadministration experiments. Thus, unlike adrenaline and NPY, which when given together centrally show no additivity and even antagonistic interac tions with regard to their hypotensive actions, the mu-opiate receptors and the NPY receptors appear to be able to regulate cardiovascular responses without any obvious interactions; that means neither antagonism nor enhancement of their cardiovascular effects [25]. Thus, central NPY and mu-opiate receptor mechanisms may show additivity in their ability to lower arterial blood pressure.
Acknowledgments. This work was supported by a grant from the Swedish Medical Research Council (04X-715). We are grateful to Sylvia Oliphant for excellent secre tarial assistance.
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11. Schultzberg M, Lundberg JM, Hokfelt T, Terenius L, Brandt J, Elde RP, Goldstein M (1978) Enkephalin-like immunoreactivity in gland cells and nerve terminals of the adrenal medulla. Neuroscience 3: 1169-1186
12. Hokfelt T, Fuxe K, Goldstein M (1975) Applications of immunohistochemistry to studies on monoamine cell systems with special reference to nervous tissues. Ann NY Acad Sci 254: 407 -432
13. Markey KA, Kondo S, Shenkman I, Goldstein M (1980) Purification and characterization of tyrosine hydroxylase from a clonal phaechromocytoma cell line. Mol Pharmacol17: 79-85
14. Fuxe K, Wikstrom AC, Okret S, Agnati LF, Hiirfstrand A, Yu ZY, Granholm L, Zoli M, Vale W, Gustafsson JA (1983) Mapping of glucocorticoid receptor immunoreactive neurons in the rat tel- and diencephalon using a monoclonal antibody against liver glucocorticoid receptor. Endoc rinology 117 (5): 1803-1812
15. Agnati LF, Fuxe K, Benfenati F, Zini I, Zoli M, Fabbri L, Hiirfstrand A (1984) I. Methodological aspects. Acta Physiol Scand 532: 5-32
16. Fuxe K, Agnati LF, Hiirfstrand A, Zini A, Tatemoto I, Merlo Pich E, Hokfelt T, Mutt V, Terenius L (1983) Central administration of neuropeptide Y induces hypotension bradypnea and EEG synchronization in the rat. Acta Physiol Scand 118: 189-192
17. Hiirfstrand A, Fuxe K, Agnati LF, Ganten D, Eneroth P, Tatemoto K, Mutt V (1984) Studies on neuropeptide-Y catecholamine interactions in central cardiovascular regulation in the a-chloral ose anaesthetized rat. Evidence for a possible new way of activating the a-2 adrenergic transmis sion line. Clin Exp Hypertens [A) 6 (10,11): 1947-1950
18. Agnati LF, Fuxe K, Locatelli V, Benfenati F, Zini I, Panerai AE, EI Etreby MF, Hokfelt T (1982) Neuroanatomical methods for the quantitative evaluation of coexistence of transmitters in nerve cells. Analysis of the ACTH- and beta-endorphin immunoreactive nerve cell bodies of the mediobasal hyphothalamus of the rat. J Neurosci Methods 5: 203-214
19. Kalia M, Welles RV (1980) Brainstem projection ofthe aortic nerve in the cat. A study using the tetramethyl benzidine as the substrate for horseradish peroxidase. Brain Res 188: 23-32
20. Florez J, McCarthy LE, Borison HL (1979) A comparative study in the cat of the respiratory effects of morphine injected intravenously and into the cerebrospinal fluid. J Pharmacol Exp Ther 163: 448-455
21. Laubie M, Schmitt H, Vincent M, Remond G (1977) Central cardiovascular effects of mor phinomimetic peptides in dogs. Eur J Pharmacol46: 67-71
22. Fuxe K, Agnati LF (1985) Receptor-receptor interactions in the central nervous system. A new integrative mechanism in synapses. Med Res Rev 5 (4): 441-482
23. Agnati LF, Fuxe K, Zoli M, Ferraguti F, Zini 1 (1986) Studies on central enkephalin and ~ endorphin immunoreactive neurons of adult and old male rats give evidence for the existence of two main types of communication in the central nervous system: the volume transmission and the wiring transmission. Acta Physiol Scand (in press)
24. Holaday JW (1983) Cardiovascular effects of endogenous opiate systems. Ann Rev Pharmacol Toxicol23: 541-594
25. Fuxe K, Agnati LF, Hiirfstrand A et al. (1986) Morphofunctional studies on the neuropeptide Y/ adrenaline costoring nerve terminal systems in the dorsal cardiovascular region of the medulla oblongata. Focus on receptor-receptor interactions in cotransmission. Prog Brain Res 68: 303-313
Multiplicity of Opioidergic Pathways Related to Cardiovascular Innervation: Differential Contribution of All Three Opioid Precursors
E. WEIHE*, D. NOHR*, w. HARTSCHUH**, B. GAUWEILER*, and T. FINK*
• Department of Anatomy, Johannes Gutenberg-University, Mainz, FRG ** Department of Dermatology, University of Heidelberg, Heidelberg, FRG
Introduction
The endogenous opioid family consists of the three precursors proenkephalin (proenkephalin A), prodynorphin (proenkephalin B), and proopiomelanocortin (POMC) , from which various opioid and nonopioid peptides can be processed, apparently in a tissue-specific manner (cf. Civelli et al. 1984; Goldstein 1984; Herz 1984; Udenfriend and Kilpatrick 1984; Civelli et al. 1985; Khachaturian et al. 1985; Kosterlitz 1985). Their distribution in areas of the CNS which are involved in car diovascular regulation is well documented. The biochemistry and functions of endoc rine (pituitary and adrenal) opioids have also been well characterized (cf. Millan and Herz 1985). The conception that endocrine and CNS opioid peptides and receptors may play an important role in various physiological and pathophysiological car diovascular regulatory mechanisms is widely accepted (cf. Holaday 1983; McQueen 1983; Holaday, this volume).
Peripheral neuronal, paracrine, and perhaps even nonadrenal endocrine opioid systems related to the heart itself as well as to the vasculature were investigated by biochemical and immunohistochemical methods (McQueen 1983; North and Egan 1983; Weihe and Reinecke 1983; Weihe et al. 1983; Vincent et al. 1984; Xiang et al. 1984; Weihe et al. 1985, b, c; Bumstock 1986; Hartschuh et al. 1986; Howells et al. 1986). Their molecular identities, tissue-specific distribution, and precise actions in cardiovascular regulation under resting or pathophysiological conditions are still unclear. Differential presynaptic opioid sympathoinhibitory receptors apparently playa crucial role in the heart and in different vascular beds (cf. Starke 1977; Fukuda et al. 1985; Krumins et al. 1985; Starke et al. 1985, Fuderet al. 1986; Illes et aI.1987).
The present study is based on the consideration that there may be also differential processing and distribution of the various opioid peptides in intrinsic or extrinsic efferent or afferent nerves supplying the peripheral cardiovascular system. To investi gate this hypothesis we envisaged a systematic immunohistochemical study which was aimed to determine the preponderant molecular forms and peripheral cardiovascular histotopography of the potential plethora of peptides derived from the three opioid precursors. Particular attention was paid to characterize peptides of the opioid family in primary sensory afferents which are one source of cardiovascular innervation. Their interrelation with nonopioid peptides, in particular substance P, which are sensory transmitter candidates of antidromic vasodilation and peripheral inflammat ory mechanisms (cf. Salt and Hill 1983; Lundberg and Hokfelt 1986) will be also
28 E. Weihe et al.
determined. Qur study concentrated on the guinea pig, but questions of interspecies variations are also addressed.
Materials and Methods
Tissue Processing for Immunohistochemistry
Various tissues of several adult mammalian species (ten guinea-pigs, five rats, four cats, two dogs, and three rabbits) were fixed by perfusion or immersion with Bouin's solution. In some cases a prefixation with a freshly prepared 4% paraformaldehyde/ 1 % glutaraldehyde solution was employed. Some tissues of pig, in particular skin, were obtained within 15 min from a local slaughter house and fixed by immersion in Bouin's solution. One isolated pig heart (about 20 min postmortem) was perfused with the same fixative. Human tissue (skin) was fixed by immersion in Bouin's solution. Tissues were processed for immunohistochemistry using various enzymatic (horseradish peroxidase) or immunofluorescence methods as described (Weihe et al. 1984; 1985a; 1986).
Antisera
A plethora of commercial and donated antisera against various opioid and nonopioid peptide sequences which are contained in the three opioid precursors was used. 1. Polyclonal rabbit antisera against proenkephalin (PRO-ENK)-opioid sequences:
Met-enkephalin (Immunonuclear); Met-enkephalyl-Arg-Phe, Met-enkephalyl Arg-Gly-Leu, metorphamide, Leu-enkephalin, BAM 12 P (Weber et al. 1983a, b); amidorphin (Seizinger et al. 1985).
2. Polyclonal rabbit antisera against prodynorphin (PRO-DYN)-opioid sequences: Leu-enkephalin, dynorphin A 1-8, alpha-neoendorphin, dynorphin B (cf. Weber et al. 1983a, b); alphalbeta-neoendorphin, dynorphin A 1-17 (cf. Millan et al. 1986).
3. Polyclonal rabbit antisera against POMC-derived opioid peptide ~-endorphin (Immunonuclear, Peninsula) and against the non opioid sequences adrenocor ticotrophic hormone (ACTH) (Weber et al. 1983a) and alpha-melanocyte stimulating hormone (alpha-MSH, Immunonuclear, cf. Khachaturian et al. 1985) werde also used.
In addition, monoclonal mouse antibodies against the opioid message sequence Tyr-Gly-Gly-Phe (Meo et al. 1983) and against Leu-enkephalin (Seralab, Cuello et al. 1984) were employed.
Rabbit polyclonal antisera against calcitonin gene-related peptide (CGRP) were obtained from Peninsula or Amersham and a monoclonal rat antibody against subst ance P (SP) from Serotec. A rabbit polyclonal antiserum against atrial natriuretic factor (ANF) was also used (Arendt et al. 1985).
Working dilutions of polyclonal antisera in immunoenzymatic procedures were in the range from 1 : 6000 to 1 : 80000. The monoclonal SP antibody (ascites) was used in
Multiplicity of Opioidergic Pathways Related to Cardiovascular Innervation 29
a dilution of 1 :50 to 1 : 400. The dilution of the monoclonal antibody (ascites) against Leu-enkephalin varied from 1: 5000 to 1: 40000.
The specificities of all antisera were tested in various tissues under our immunohis tochemical conditions by preabsorption with homologous synthetic antigens and a plethora of heterologous antigens having varying degrees of sequence homology (Weihe et al. 1986).
Sections were analyzed and photographed on a Leitz-Orthoplan light microscope if not otherwise mentioned.
Results
Specificity of Antisera
In most cases the specificity of antisera was found to be essentially similar to that determined by other authors or that indicated on commercial data sheets.
The main specificity characteristics were: The antiserum against Met-enkephalin (ME) cross-reacted with Leu-enkephalin (LE) and with other peptide sequences containing either pentapeptide at the N-terminus to a certain extent. The polyclonal antiserum against LE also dit not fully differentiate LE-, ME-, or C-terminal extended forms of either pentapeptides including heptapeptide (ME-RF), octapep tide (ME-RGL), dynorphin A 1-17 (DYN A 1-17), DYN A 1-8, DYN A 1-13, or alphalbeta-neoendorphin (alpha/~-NEO). Thus, the antisera against the two pen tapeptides were to be expected to stain PRO-ENK as well as PRO-DYN-opioid peptides to an extent which could not be neglected.
The antisera against ME-RF or ME-RGL did not cross-react with PRO-DYN sequences or with POMC sequences and therefore could be regarded as being table to stain PRO-ENK sequences rather specifically. The antiserum against metorphamide (METOR) was very specific since no cross-reactions with other opioid peptides could be observed. The amidorphin (AMID OR) antiserum recognized neither PRO-DYN nor POMC sequences, but cross-reacted with peptide F. It therefore could also be regarded as being able to stain the PRO-ENK family specifically.
The antisera against DYN A 1-17, DYN A 1-8, and NEO did not cross-react with any PRO-ENK or POMC-opioid sequences. They did not recognize the penta peptides and therefore appear to be very specific in staining only the PRO-DYN family. In addition they appeared to discriminate different dynorphins and neoendor phins.
The monoclonal antibody against LE did not discriminate between ME and LE. Since it did not recognize larger molecular forms of the pentapeptides it was found to be very specific for the two pentapeptides as described (Cuello et al. 1984). The alpha MSH antiserum cross-reacted with des-acetyl-alpha-MSH but not with ACTH. The beta-endorphin antiserum (Peninsula) showed some cross-reactivity with beta lipotropins and with Met-enkephalin although the commercial data sheet indicated zero cross-reaction with ME in radioimmunoassay (RIA). Immunoreactions obtained with the beta-endorphin antiserum were partly but not completely preabsorbable with up to 10 !-tmol beta-lipotropin or ME. The monoclonal antibody against SP did not recognize opioid peptides.
30 E. Weihe et al.
Histotopography of Immunoreactivities
Paravertebral and Prevertebral Sympathetic Ganglia of Guinea Pig
In stellate ganglia immunoreactive(IR)-ME-RF, IR-ME-RGL, IR-AMIDOR, and IR-peptide F were equally present in fibers and varicosities, surrounding about one third of principal ganglionic cells. The intraganglionic distribution of these codistri buted IR -opioid peptides overlapped with that of the equally frequent IR-ME and IR LE (Fig. la, Table 1). As a rule, the density of IR-METOR or IR-BAM 12 P
Table 1. Distribution of selected IR derivatives of the three opioid precursors in extrinsic and intrinsic neurons and paracrine systems related to peripheral cardiovascular innervation in guinea pig
Sympathetic ganglia (stellate) Principal neurons Paraganglionic cells Fibers Axosomatic contacts
(mesenteric) Principal neurons Paraganglionic cells Fibers
PRO-ENK PRO-DYN PRO-ENKI POMC PRO-DYN
ME-RGL METORDYN NEO LE MSH* ~-END
- /+ +++ +++ ++ ++ +++ ++ ++
++++ +++ ++++ ++ +++
+++ +++ ++ +++ +++ ++ ++ +++ ++
-/+ +++ ++++ +++ ++++ +++ ++
Fig. la, b Adjacent sections of guinea pig stellate ganglion, a Immunoreactive LE in fibers and endings surrounding a major population of non-IR principal ganglionic cells and in a group of para ganglionic cells (arrow), x 150. b Immunoreactive-alpha-MSH is present in large-diameter intraganglionic fibers but absend from cells, x 150
Table 1. (Continued)
ME-RGL METORDYN NEO LE MSH* ~-END
Sensory (spinal and trigeminal) ganglia
Small cells +++ +++ +++ Intermediate cells + + + -/+ Large cells -/+ -/+ ++++ - Fibers ++ ++ ++ ++++ -
Somatik nerve trunks -/+ + + ++ +++
Vagal nerve -/+ -/+ -/+ ++ +++
Phrenic nerve -/+ -/+ ++ +++
Heart (intrinsic ganglia) Ganglionic cells -/+ NT Paraganglionic cells -/+ ++ ++ +++ NT Fibers + -/+ ++ ++ +++ +++ NT
(chemoreceptor glomera) Paraganglionic cells -/+ + + +++ NT Fibers -/+ -/+ +++ -/+ NT
(neuroeffector junctions) Aorta, pulmonary trunk + + ++ NT Coronary arteries + + ++ NT Microvasculature + + ++ NT Myoendocrine atrial cells -/+ -/+ NT Conduction system + + ++ NT
(nonneuroeffector fibers) Interstitial and paravascular + + ++ +++ NT
Neurovascular fibers in Skin + + + ++/+++ ++++ NT Mucocutaneous junctions + + + ++/+++ ++++ NT Skeletal muscle + +++ NT Joint tissues NT NT + ++ NT Digestive system
Tongue + -/+ -/+ +++ ++++ NT Salivary glands ++++ NT ++++ ++ NT Esophagus ++ ++ ++ +++ + NT GEP system +++ ++ +++ ++++ + ?
Urogenital tract +++ +++ +++ +++ ++ -/+ Respiratory tract +++ ++ NT Lymphatic system + + NT Adrenal gland +++ + ++ ++ ++++ +++ NT
Intrinsic neurons GI tract +++ + ++ ++ ++++ + +
Paracrine cells Epithelium of GI tract +++ Respiratory epithelium +++
Subjective rating of relative densities of IR fibers or IR cells: -, absent; -/+, very low; +, low; + +, moderate; +++, high; + +++, very high. (?) presence questionable; NT, not tested; note thatIR-MSH* occupied myelinated fibers (nonvaricose) larger than A delta whereas IR-opioid peptides were restricted to C and also some A delta fibers (varicose). For more details, the constellation of other IR peptides, and comparison with other species see text
32 E. Weihe et al.
surrounding a significantly minor proportion of principal ganglionic cells was lower. In mesenteric cells was lower. In mesenteric ganglia, the vast majority of principal ganglionic cells were innervated by an extremely dense plexus containing IR-ME-RF, IR-ME-RGL, IR-AMIDOR (peptide F), IR-ME, and IR-LE as well as IR-SP and IR-CGRP. IR-METOR was less frequent. In comparison with IR-PRO-ENK-sequ ences, IR-PRO-DYN sequences (alpha/beta-neo, DYN A 1-17, DYN A 1-8) were less frequent in stellate but almost equally frequent in mesenteric ganglia. Innervation of the ganglionic microvasculature was a regular finding. IR-PRO-ENK and IR-PRO DYN sequences were codistributed, but coexistence in individual fibers or endings could not be assessed unequivocally. Relatively infrequent, and apparently merely trespassing, IR-SP and IR-CGRP fibers, but no IR-primary ganglionic cells, were present in stellate and mesenteric ganglia whereas mesenteric ganglia contained abundant IR-SP and IR -CG RP fibers which not only trespassed but also targeted non IR ganglionic cells.
Paraganglionic cells in stellate ganglia contained IR-ME, IR-LE, IR-ME-RGL, IR-DYN A 1-17, IR-DYN A 1-8, and IR-alphalbeta-NEO but apparently no IR METOR (Fig. 1 a). It was not unequivocally clear whether IR-PRO-ENK and IR PRO-DYN sequences coexisted in individual paraganglionic cells. Almost all para ganglionic cells in the abdominal paraganglia in the vicinity of mesenteric sympathetic ganglia contained IR-PRO-ENK sequences (ME, LE, ME-RGL, AMID OR) but apparently no IR-METOR, which was restricted to paraganglionic fibers. The IR PRO-DYN sequences (DYN A 1-17, DYN A 1-8, alpha/beta-NEO) were clearly present in about one-half of the paraganglionic cells. The staining produced by the monoclonal LE-antibody was equivalent to that of the polyclonal antisera against ME or LE. No clear evidence was obtained for the presence of opioid immunoreactivity in principal ganglionic cells. Some of them showed faint staining with the alpha/beta NEO antisera, but this was not drastically different from background staining to allow classification as being unequivocally specific at the current stage of our investigations using non-colchicine-treated animals. In some lumbar paravertebral ganglia we observed some principal ganglionic cells faintly stained with the antiserum against ME-RGL.
Some IR-beta-endorphin fibers were seen in stellate ganglia. However, this immunoreactivity was partly pre absorbable with ME. Large-diameter fibers contain ing IR-alpha-MSH were visualized trespassing stellate and prevertebral sympathetic ganglia. No IR-MSH principal ganglionic or paraganglionic cells were observed (Fig.1b).
Sensory ganglia
In guinea-pig, in all segmental dorsal root ganglia and in trigeminal ganglia, mainly small cells « 20 !lm), but also some cells of intermediate (20-40 !lm) diameter, contained coexisting IR-PRO-DYN sequences (LE, alpha/beta-NEO, DYN A 1-17), but no IR-PRO-ENK-sequences. The staining pattern of the monoclonal antibody against the opioid message sequence was equal. Interestingly, the antiserum angainst ME produced no staining in the sensory cells in spite of its potential cross reactivity to LE. The frequency of opioid-IR cells varied depending on the segmental level. The highest number of opioid-IR cells was in the lumbosacral ganglia (Fig. 2).
Fig. 2a, b Adjacent sections oflumbosacral dorsal root ganglion L5 of guinea pig. a Immunoreactive DYN A 1-17 in mainly small diameter ganglionic cells and fibers, x 360. b Immunoreactive SP in small and medium-sized ganglionic cells and fibers, x 360. Identical cells labeled with IR-DYN and IR-SP are marked by arrows. Normarsky optics (Zeiss Axiophot)
Also centrally and peripherally directed processes and small-diameter fibers in the C or A delta-range contained IR-PRO-DYN sequences. A part of them supplied the ganglionic microvasculature. The monoclonal antibody against LE visualized almost the same number of cells and fibers as the other antisera against prodynorphin sequences. Almost all large (> 40 !lm) cells and myelinated, but not unmyelinated, fibers contained IR-alpha-MSH whereas other IR-POMC pep tides were absent (Fig. 3).
In a substantial, but not total, number of small and also some medium-sized ganglionic cells IR-PRO-DYN sequences, IR-SP and IR-CGRP were found to coexist (Fig. 2a, b). There were also nonopioid IR cells containing IR-SP or IR-CGRP and vice versa, as revealed by analyzing adjacent sections. Nonopioid-IR cells containing IR-SP or IR-CGRP were mainly of medium or large diameter. The overall number of opioid IR cells was lower than of those containing IR-SP or IR-CGRP. IR-MSH was found to be independent of opioid-IR or SP/CGRP-IR cell and fiber populations.
In rats neither IR-PRO-DYN sequences nor IR-PRO-ENK sequences could be detected unequivocally. Lumbosacral dorsal root ganglia of cat contained equal
34 E. Weihe et aI.
Fig. 3. Immunoreactive alpha-MSH in the majority of large diameter cells and fibers of cervical dorsal root ganglion C4 of guinea pig, x 180
numbers of IR-PRO-DYN sequences in mainly small but also medium-sized cells. In ganglionic cells of cervical dorsal root ganglia of rabbits IR-LE and IR-DYN A 1-17 were present.
Pig trigeminal ganglia contained some IR-LE, but also some IR-ME-RGL cells. Coexistence patterns in non-guinea-pig species have not yet been analyzed. IR-MSH was found in the sensory ganglia of all species investigated, and it was always localized in most of the large cells and larg-diameter myelinated fibers. The immunostaining of sensory ganglionic cells was successfully performed without colchicine treatment.
Peripheral Somatic and Visceral Nerve Trunks
It was very difficult to visualize opioid-IR fibers in peripheral nerves because their diameter is very small, mainly in the nonmyelinated range. In contrast the population of SP-IR and CGRP-IR fibers, which were in the larger-diameter range (A delta), was visualized more easily. Thick myelinated alpha-MSH-IR fibers were distinguished without any difficulty. The most suitable antiserum for visualization of opioid-IR fibers was the polyclonal antiserum against LE, which, however, cross-reacted with several PRO-ENK and PRO-DYN sequences. LE-IR fibers were seen in the cervical and thoracic vagal nerve, in the phrenic nerves, in the trigeminal branches, and in the brachial plexus, and in the sciatic nerves of guinea pig. The vasa nervorum appeared to be innervated with IR-LE fibers. Results with other species were equivocal and will be published elsewhere.
Heart and Macrovasculature
Immunoreactive LE was the predominant opioid immunoreactivity which could be visualized in guinea pig heart. IR-LE nerve fibers were sparse and present in small diameter fibers supplying coronary arteries, some intramural vessels, endocardium, valves, epicardium, and pericardium. IR-LE was present in neuroeffctor and non neuroeffector fibers (Table 1). Some IR-LE was found in guinea pig, dog, rabbit, and pig sinoatrial node. It was also present in nerves supplying coronary vessels of pig and dog. The situation in the cat was equivocal.
Immunoreactive LE predominated in intrinsic cardiac ganglia where numerous IR LE fibers and endings surrounding ganglionic cells were seen in guinea pig, dog, and pig (Fig. 4a, b). Equivocal faint staining of some ganglionic cells was observed. IR ME-RGL in some cardiac nerves was rare. Some intracardiac ganglia contained IR NEO and IR-DYN A fibers which were also seen in a few coronary nerves of guinea pig and cat.
Paraganglionic cells of various cardiac ganglia contained IR-ME, -LE, and -DYN A 1-8 particularly in guinea pig, but also in cat (Fig. 4 b). Guinea pig paraganglionic cells of aortic, pulmonary, and coronary cardiac glomera as well as small cells in the carotid body contained IR-ME, IR-LE, IR-DYN A 1-8, and IR-alpha-NEO. Results with other species and antisera were equivocal.
Immunoreactive LE small-diameter fibers exhibiting some varicosities were regul ary seen in the thoracic aorta, pulmonary trunk, and carotid artery of guinea pig, cat, and dog (Fig. 4c). IR-LE fibers were denser adjacent to cardiac, aortic, or carotid glomera. Some of the IR-LE fibers penetrated the outer media; also contacts to the vasa vasorum were seen. IR-SP or IR-CGRP fibers were much more frequent in these locations. IR-MSH was regularly seen in numerous large-diameter myelinated fibers of paravascular nerves whereas other IR-POMC peptides were not visualized .
b c
. .
Fig. 4a-c Guinea pig intrinsic cardiac ganglia and macrovasculature: a Immunoreactive LE in varicose fibers and endings surrounding juxtasinunodal intrinsic ganglionic cells, x 750. b Immunoreactive DYN A 1-8 in a paraganglionic cell (arrow) of a juxtacoronary intrinsic ganglion and in a few fiber profiles, x 750. c Immunoreactive LE small-diameter fibers in the media-adventitia border of juxtacardiac ascending aorta, x 750
36 E. Weihe et al.
Somatic and Mucocutaneous Tissues
In the skin and in various mucocutaneous tissues (nose, ear, and anogenital regions), in striated muscle, and in joint tissues IR-LE was the predominant opioid IR form in nerves supplying the vasculature which could be clearly visualized (Table 1). In skin, some IR-DYN A 1-8 and IR-NEO fibers were observed to be associated with the subepidermal microvasculature and other cutaneous blood vessels. IR-LE was regu larly seen in close contact with papillary venules in skin and mucocutaneous tissues. It was also present in association with the microvasculature of some striated muscles and of various joint tissues. This pattern was observed in guinea pigs and to a certain extent also in pig and cat. In these locations codistribution with some of the much more frequent IR-SP and IR-CGRP fibers was obvious. There were many large diameter fibers containing IR-MSH in these tissues and in all species investigated including human skin (Fig. 5).
Vasculature of the Digestive System
Several molecular forms of opioid peptides were visualized throughout the digestive tract. The tongue of guinea pig, dog, and in particular cat was extremely rich in IR-LE fibers supplying arteries, veins, and the microvasculature, including that of subepithe lial areas and of seromucous glands (Fig. 6). The vasculature of the submandibular
.. '
6
Fig. 6. Immunoreactive LE in abundant small diameter varicose fibers supplying cat intraling ual artery (tangential-longitudinal section), x 160
v
7
Fig. 7. Immunoreactive LE in small-diameter fibers supplying the microvasculature (venule, v) of guina pig submandibular gland, x 2000
\ .~ ,
8
glands was also richly innervated with IR-LE fibers in guinea pig, cat, and dog (Fig. 7). In guinea pigs numerous IR-ME-RGL varicosities were seen in the salivary glands and their microvasculature whereas IR-PRO DYN sequences were absent in this location.
Opioidergic fibers were seen throughout the gastroenteropancreatic (GEP) sys tem. Numerous fibers containing IR-PRO-ENK sequences (ME, LE, ME-RGL, ME-RF, AMIDORIPEPTIDE F) and IR-PRO-DYN sequences (DYN A 1-17, DYN A 1-8, DYN B, NEO) supplied the vasculature - including the microvascula ture - of the lamina propria, submucosa, and muscle cell layers in esophagus, stomach, duodenum, jejunum, ileum, and colon of guinea pig and rat (Fig. 8, Table 1). The guinea pig gastrointestinal (GI) tract seemed to contain more IR-PRO-DYN sequences than that of the rat . Duodenum of cat and dog contained IR-PRO-ENK and IR-PRO-DYN proportions similar to guinea pig. Intrinsic ganglia of the entire digestive tract contained IR-PRO-ENK and IR-PRO-DYN sequences in many fibers and perikarya. Interestingly, IR-METOR was almost restricted to ganglia of the myenteric plexus where it was present in numerous perikarya and fibers. IR-DYN A 1-8 predominated in perikarya of the submucous plexus whereas the IR-PRO-ENK sequences showed so

opioid peptides and blood pressure control

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