Met5]-enkephalin-like peptides of the adrenal medulla: release by nerve stimulation and functional implications

In adrenal chromaffin cells, various molecular forms of polypeptides cross-reacting with [met5]-enkephalin antisera are stored in granules together with catecholamines and soluble proteins. Splanchnic nerve stimulation increases the release of enkephalin-like peptides from the adrenal gland into the adrenal vein. The release of these peptides appears to be mediated by a cholinergic nicotinic receptor. Fractionation of plasma extracts on Bio-gel P-2 shows the presence of only low molecular weight peptides in the resting condition. The low molecular weight fraction contains mainly [met5]-enkephalin and [leu5]-enkephalin. When the splanchnic nerve is stimulated high and low molecular weight peptides are released and the amount of low molecular weight peptides in plasma is increased. The content of enkephalin-like peptides in adrenal venous plasma was similar in control and reserpinized dogs. Splanchnic nerve stimulation increased the peptide content but not the epinephrine content of plasma in reserpinized dogs. This also caused a fall of arterial blood pressure which could be prevented by pretreatment with naloxone. A decrease in blood pressure was also directly elicited by the injection of [met5]-enkephalin-[arg6-phe7]. The duration of the hypotensive effect of this peptide was prolonged by prior injection of captopril.     

Appearance of enkephalin-immunoreactivity in rat adrenal medulla following treatment with nicotinic antagonists or reserpine

Various neuroendocrine factors known to be important in the regulation of adrenal catecholamine biosynthesis were investigated for possible effects on enkephalin-like immunoreactivity (Enk-IR) in the adrenal medulla of the rat. In normal rats, the adrenal chromaffin cells were not stained for either methionine (met-) or leucine (leu-) Enk-IR. Staining for Enk-IR appeared in many chromaffin cells following denervation of the adrenal or treatment of rats with the nicotinic receptor antagonists chlorisondamine or pempidine. These observations suggest that splanchnic nerve activity normally depresses the levels of enkephalin-like peptides in chromaffin cells through a trans-synaptic mechanism involving acetylcholine release and nicotinic receptor stimulation. Paradoxically, treatment with reserpine also increased Enk-IR in chromaffin cells. However, this increase did not appear to result from the well known effect of reserpine to increase presynaptic nerve firing and tyrosine hydroxylase (TOH) activity, since no increase in Enk-IR was observed following treatment with phenoxybenzamine or 6-hydroxydopamine, drugs which also increase TOH activity through trans-synaptic mechanisms. The reserpine effect also did not appear to be mediated by a stress-induced increase in glucocorticoid hormones since glucocorticoid therapy alone did not increase adrenal Enk-IR. It is suggested that the increase in adrenal Enk-IR following reserpine may result from a direct action of reserpine on chromaffin cells.     

Biochemical characterization of enkephalin-like immunoreactive peptides of adrenal glands

The total enkephalin-like immunoreactive peptide content of adrenal glands from dog, cattle, guinea pig and rat was investigated by radioimmunoassay using a (met⁵)-enkephalin antiserum. Dog adrenals contain the highest amount of peptides, cattle and guinea pig adrenals contain lesser amounts, and the rat adrenals had the least amount (0.05% that of the dog). Comparison of the (met⁵)-enkephalin content of the adrenal cortex and medulla with that of whole bovine adrenal gland indicates that the peptides are concentrated in the medulla. Analysis of the chromaffin granules from bovine adrenal medulla indicates this to be the primary storage site for (met⁵)-enkephalin-like peptides. Gel chromatography reveals a molecular heterogeneity of the immunoreactive peptides in all species tested; high molecular weight peptides account for a larger proportion of the immunoreactivity when compared with the low molecular weight peptides.     

Opioid peptides in adrenal gland

Enkephalin-like immunoreactive peptides have been observed in adrenal glands of all species studied with the highest contents found in dogs and cows, and the lowest in rats. These peptides are located both in gland cells and in afferent nerve terminals. Bovine adrenal glands contain opioid peptides in many molecular forms. The peptides include a group of low molecular weight forms (M.W. <1000) which are capable of binding to the opiate receptor, and a group of high molecular weight forms (M.W. >1000) which contain enkephalin within their peptide sequence, but are devoid of opioid activity unless treated with trypsin. The physiological role(s) of the adrenal enkephalin-like material is not clear at present. However, it has been observed that nicotine-stimulated release of catecholamine from isolated chromaffin cells can be reduced by opiate agonists, suggesting that enkephalin-like peptide in nerve terminals may act on chromaffin cells. Several lines of evidence suggest that enkephalin-like peptides in gland cells can be released into the bloodstream.     

Adrenal versus nonadrenal sympathetic preganglionic neurones in the lower thoracic intermediolateral nucleus of the cat: physiological properties

Adrenal and nonadrenal sympathetic preganglionic neurones (SPNs) in the intermediolateral nucleus of spinal segments T8-T10 in the cat were compared according to a number of physiological properties. An SPN was classified as "adrenal" (n = 37) if it could be antidromically activated by electrical stimulation of the adrenal medulla. An SPN that could not be activated from the adrenal medulla yet could be antidromically activated by electrical stimulation of the greater splanchnic nerve was classified as "nonadrenal" (n = 123). Approximately 50% of adrenal SPNs (17 out of 37) were activated antidromically by stimulation of both the greater splanchnic nerve and adrenal medulla, suggesting that these neurones projected to the adrenal medulla via the greater splanchnic nerve, with the other adrenal SPNs taking a different route. The mean conduction velocities of adrenal (6.7 +/- 1.8 (SD) m/s) and nonadrenal (6.7 +/- 1.5 m/s) sympathetic preganglionic axons were similar. Over 80% of adrenal (31 out of 37) and nonadrenal (104 out of 116) SPNs were spontaneously active. The two types of neurone were indistinguishable in terms of the rates and patterns of discharge. Adrenal SPNs discharged with a mean rate of 1.4 +/- 1.1 spikes/s, and nonadrenal SPNs discharged with a mean rate of 1.8 +/- 1.4 spikes/s. With both types of SPN, the pattern of spontaneous activity was either irregular or phasic. With the latter pattern, periodic bursts of discharge were at the same frequency as oscillations in arterial pressure, frequency of ventilation, or phrenic nerve discharge. These data suggest that adrenal and nonadrenal sympathetic preganglionic neurones in the intermediolateral nucleus in caudal thoracic segments share a number of common physiological properties.     

Vasoactive intestinal peptide and substance P increase levels of enkephalin-containing peptides in adrenal chromaffin cells

The neuropeptides substance P and vasoactive intestinal peptide (VIP), reported to exist in the splanchnic nerve terminals innervating the adrenal medulla, elevate the levels of enkephalin-containing peptides (ECPs) in cultured bovine adrenal medullary chromaffin cells. Cellular ECP stores were increased over 48 hr by 72 and 46 percent, respectively, following incubation with 5 microM VIP or 10 microM substance P, maximally effective concentrations. The results suggest that VIP and substance P may be trans-synaptic modulators of chromaffin cell ECP stores.     

Localization of enkephalins in adrenaline cells and the nerves innervating adrenaline cells in rat adrenal medulla

The coexistence of met5- and leu5-enkephalin-like immunoreactivities with catecholamines in the rat adrenal medulla was studied with combined fluorescence microscopy and immunocytochemistry. Both met5- and leu5-enkephalin-like immunoreactivities were localized in few heavily stained adrenaline cells and in a population of nerves innervating adrenaline cells and as well as ganglion cells among the adrenaline cells. Only occasionally single noradrenaline cells exhibited light immunostaining for both enkephalins but no positive fibers could be found around the noradrenaline cells. In electron microscope the immunoreaction was seen in the granules of the adrenaline cells and in the large synaptic vesicles of the nerve terminals around the adrenaline cells. The present findings suggest that enkephalin-like immunoreactivity coexists mainly with adrenaline in rat adrenal medulla and that the enkephalin immunoreactive terminals regulate secretion of adrenaline from rat adrenal medulla.     

Localization of enkephalins in adrenaline cells and the nerves innervating adrenaline cells in rat adrenal medulla

Summary The coexistence of met5- and leu5-enkephalinlike immunoreactivities with catecholamines in the rat adrenal medulla was studied with combined fluorescence microscopy and immunocytochemistry. Both met5- and leu5-enkephalin-like immunoreactivities were localized in few heavily stained adrenaline cells and in a population of nerves innervating adrenaline cells and as well as ganglion cells among the adrenaline cells. Only occasionally single noradrenaline cells exhibited light immunostaining for both enkephalins but no positive fibers could be found around the noradrenaline cells. In electron microscope the immunoreaction was seen in the granules of the adrenaline cells and in the large synaptic vesicles of the nerve terminals around the adrenaline cells. The present findings suggest that enkephalin-like immunoreactivity coexists mainly with adrenaline in rat adrenal medulla and that the enkephalin immunoreactive terminals regulate secretion of adrenaline from rat adrenal medulla.     

Simultaneous monitoring of acetylcholine and catecholamine release in the in vivo rat adrenal medulla

To simultaneously monitor acetylcholine release from pre-ganglionic adrenal sympathetic nerve endings and catecholamine release from post-ganglionic adrenal chromaffin cells in the in vivo state, we applied microdialysis technique to anesthetized rats. Dialysis probe was implanted in the left adrenal medulla and perfused with Ringer's solution containing neostigmine (a cholinesterase inhibitor). After transection of splanchnic nerves, we electrically stimulated splanchnic nerves or locally administered acetylcholine through dialysis probes for 2 min and investigated dialysate acetylcholine, choline, norepinephrine and epinephrine responses. Acetylcholine was not detected in dialysate before nerve stimulation, but substantial acetylcholine was detected by nerve stimulation. In contrast, choline was detected in dialysate before stimulation, and dialysate choline concentration did not change with repetitive nerve stimulation. The estimated interstitial acetylcholine levels and dialysate catecholamine responses were almost identical between exogenous acetylcholine (10 microM) and nerve stimulation (2 Hz). Dialysate acetylcholine, norepinephrine and epinephrine responses were correlated with the frequencies of electrical nerve stimulation, and dialysate norepinephrine and epinephrine responses were quantitatively correlated with dialysate acetylcholine responses. Neither hexamethonium (a nicotinic receptor antagonist) nor atropine (a muscarinic receptor antagonist) affected the dialysate acetylcholine response to nerve stimulation. Microdialysis technique made it possible to simultaneously assess activities of pre-ganglionic adrenal sympathetic nerves and post-ganglionic adrenal chromaffin cells in the in vivo state and provided quantitative information about input-output relationship in the adrenal medulla.     

Chromatographic Evidence for the Presence of Multiple Tachykinins in the Bovine Adrenal Medulla

Among the mammalian tachykinins, substance P (SP) has been shown to be the most potent at modulating the response due to nicotinic acetylcholine receptor stimulation of bovine adrenal chromaffin cells. SP-like immunoreactivity has been detected in nerve terminals innervating the adrenal medulla; however, little is known of the presence of other tachykinins in this tissue. In this study, reverse-phase HPLC was used to fractionate peptides in bovine adrenal medullary extracts, and the fractions were analyzed by radioimmunoassay using antisera to SP or neurokinin A (NKA). The results show that both NKA- and SP-like immunoreactivities are present in the adrenal medulla. The presence of neurokinin B is also indicated. The presence of multiple tachykinins in this tissue raises questions as to their functions in the adrenal medulla.     

Developmental and Stress-Induced Remodeling of Cell–Cell Communication in the Adrenal Medullary Tissue

The adrenal medullary tissue contributes to maintain body homeostasis in reaction to stressful environmental changes via the release of catecholamines into the blood circulation in response to splanchnic nerve activation. Accordingly, chromaffin cell stimulus-secretion coupling undergoes temporally restricted periods of anatomo-functional remodeling in response to prevailing hormonal requirements of the organism. The postnatal development of the adrenal medulla and response to stress are remarkable physiological situations in which the stimulus-secretion coupling is critically affected. Catecholamine secretion from rat chromaffin cells is under a dual control involving an incoming initial command arising from the sympathetic nervous system that releases acetylcholine at the splanchnic nerve terminal-chromaffin cell synapses and a local gap junction-mediated intercellular communication. Interestingly, these two communication pathways are functionally interconnected within the gland and exhibit coordinated plasticity mechanisms. This article reviews the physiological and molecular evidence that the adrenal medullary tissue displays anatomical and functional adaptative remodeling of cell–cell communications upon physiological (postnatal development) and/or physiopathological (stress) situations associated with specific needs in circulating catecholamine levels.     

Transsynaptic Regulation of Galanin, Neurotensin, and Substance P in the Adrenal Medulla: Combinatorial Control by Second-Messenger Signaling Pathways

The adrenomedullary content of neurotensin and substance P was examined 1, 6, and 12 days after hypoglycemic shock. The neurotensin content was increased 60-fold within 24 h and remained elevated for up to 12 days, whereas the substance P content was increased approximately sevenfold within 24 h of insulin treatment and returned to control levels by 12 days poststimulation. Because protein kinase A, protein kinase C, and calcium influx in the rat adrenal medulla are all stimulated following splanchnic nerve stimulation, the differential regulation of neurotensin and substance P biosynthesis following stimulation of these three pathways was examined in bovine chromaffin cells in vitro. Neurotensin levels were up-regulated by elevated potassium, forskolin, and phorbol ester in bovine chromaffin cells. Substance P levels were up-regulated by elevated potassium and forskolin but not by phorbol ester treatment. When chromaffin cells were treated with phorbol ester in combination with forskolin, neurotensin levels were increased in a synergistic fashion, whereas phorbol ester antagonized the forskolin-induced elevation of substance P levels. Earlier, it was reported that galanin biosynthesis, like neurotensin biosynthesis, is upregulated by depolarization, phorbol ester stimulation, and forskolin treatment in chromaffin cells in vitro. Here we report that galanin is also, like neurotensin, increased greater than 60-fold after stimulation of the rat adrenal medulla in vivo. Neuropeptide-specific combinatorial effects of stimulating the calcium, protein kinase A, and protein kinase C signaling pathways may underlie the quantitative differences between galanin and neurotensin compared with substance P up-regulation in rat adrenal medulla after splanchnic nerve stimulation in vivo.     

Effects of clentiazem (TA-3090) and nifedipine on basal circulating catecholamine levels and on stimulation-evoked adrenal catecholamine secretion in anesthetized dogs.

The effects of TA-3090 (clentiazem) and nifedipine on basal sympathoadrenal activity and on the adrenal medullary response during splanchnic nerve stimulation were studied in dogs anesthetized with sodium pentobarbital. Plasma concentrations of epinephrine and norepinephrine were measured in aortic and adrenal venous blood before and after acute administration of the drugs, as well as during left splanchnic nerve stimulation before and after administration of drugs. Following intravenous injections, TA-3090 (30, 100, and 300 micrograms/kg) did not affect basal circulating catecholamine levels, whereas nifedipine (10, 30, and 100 micrograms/kg) markedly increased aortic epinephrine and norepinephrine concentrations in a dose-dependent manner in correlation with progressive decreases in mean arterial pressure. The changes in aortic epinephrine and norepinephrine concentrations were inversely related to those in mean arterial pressure (r = 0.603, p < 0.01; r = 0.536, p < 0.01; respectively). In response to direct splanchnic nerve stimulation (2 Hz, 2 ms, 1 min, 12 V), adrenal venous epinephrine and norepinephrine concentrations significantly increased, with a high degree of reproducibility. The catecholamine responses to splanchnic nerve stimulation were not affected by either TA-3090 or nifedipine at any dose tested. The present results suggest that the increases in circulating catecholamine levels following nifedipine administration are due to baroreflex activation secondary to the drug-induced hypotension. The study indicates that both TA-3090 and nifedipine did not significantly affect L-type Ca2+ channels related to catecholamine release in the adrenal medulla under the present experimental conditions.     

Revisiting the Stimulus-Secretion Coupling in the Adrenal Medulla: Role of Gap Junction-Mediated Intercellular Communication

The current view of stimulation-secretion coupling in adrenal neuroendocrine chromaffin cells holds that catecholamines are released upon transsynaptic sympathetic stimulation mediated by acetylcholine released from the splanchnic nerve terminals. However, this traditional vertical scheme would merit to be revisited in the light of recent data. Although electrical discharges invading the splanchnic nerve endings are the major physiological stimulus to trigger catecholamine release in vivo, growing evidence indicates that intercellular chromaffin cell communication mediated by gap junctions represents an additional route by which biological signals (electrical activity, changes in intracellular Ca²⁺ concentration,…) propagate between adjacent cells and trigger subsequent catecholamine exocytosis. Accordingly, it has been proposed that gap junctional communication efficiently helps synapses to lead chromaffin cell function and, in particular, hormone secretion. The experimental clues supporting this hypothesis are presented and discussed with regards to both interaction with the excitatory cholinergic synaptic transmission and physiopathology of the adrenal medulla.     

Substance P-like immunoreactivity in adrenal chromaffin cells and intra-adrenal nerve fibers of rats

The present peroxidase-antiperoxidase immunohistochemical study demonstrated a relatively small number of cells with substance P(SP)-like immunoreactivity in the adrenal medulla of rats. These cells were found alone or in small groups, were polygonal in shape and lacked long cytoplasmic processes. At immunoelectron microscopy, the immunoreactive cells were characterized by abundant granular vesicles, and the immunoreactive material was confined to the round core of the vesicles. Thus, it is suggested that SP co-exists with catecholamines in a population of chromaffin cells of the rat adrenal medulla. In addition a few SP-immunoreactive nerve fibers with varicosities were found in the adrenal medulla of rats. They extended between small clusters of chromaffin cells and had their dot-like terminals around and within the cell clusters. The SP-immunoreactive nerve fibers were characterized by the presence of abundant small clear vesicles mixed with a few large granular vesicles; the immunoreactivity appeared in the latter, but was also perfused throughout the entire axoplasm. The nerve fibers formed synapses on nonimmunoreactive chromaffin cells. Judging from the presence of bundles of SP-immunoreactive nerve fibers penetrating the adrenal capsule and cortex as well as the absence of SP-immunoreactive ganglion cells in the medulla, the intramedullary SP-immunoreactive nerve fibers seem to be extrinsic in origin.     

Effects of substance P on the long-term regulation of tyrosine hydroxylase activity and catecholamine levels in cultured adrenal chromaffin cells

Acetylcholine, released from splanchnic nerve terminals innervating adrenal chromaffin cells, is known to increase synthesis of adrenal tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis. The neuropeptide substance P is also present in the splanchnic nerve innervating the adrenal medulla, and this study examined whether substance P has any long-term effects on tyrosine hydroxylase activity and catecholamine levels in cultures of adult bovine adrenal chromaffin cells. When cultures were incubated for 3 days with substance P and carbachol, a cholinergic agonist, substance P (10(-6) M, and greater) completely inhibited the increase in tyrosine hydroxylase activity normally induced by carbachol. Long-term stimulation with carbachol also depleted endogenous catecholamines from the cells and substance P prevented this carbachol-induced depletion of catecholamine content. Substance P by itself, in the absence of carbachol, had only a slight effect on tyrosine hydroxylase activity. 8-Bromoadenosine 3':5'-cyclic monophosphate, an analogue of adenosine 3':5'-cyclic monophosphate, also increases tyrosine hydroxylase activity in chromaffin cells; however, substance P had no effect on the increase in tyrosine hydroxylase activity induced by this analogue. These results indicate that substance P's effects are relatively specific for the carbachol-induced increased in tyrosine hydroxylase activity and that the primary site of action of substance P is not a site common to the mechanism of tyrosine hydroxylase induction by carbachol and 8-bromoadenosine 3':5'-cyclic monophosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical localization of Met-enkephalin, Met-enkephalin-Arg6-Gly7-Leu8, Met-enkephalin-Arg6-Phe7 and Leu-enkephalin in human adrenal medulla and pheochromocytomas

Immunohistochemical localization of Met-enkephalin, Met-enkephalin-Arg6-Gly7-Leu8, Met-enkephalin-Arg6-Phe7 and Leu-enkephalin was studied in human adrenal medulla and pheochromocytomas at the light and electron microscopic levels. Both adrenal medulla and pheochromocytomas (4 adrenal, 1 extra-adrenal) showed scattered or clustered cells which contained all of the above peptides and suggested the production of proenkephalin A. The presence of these peptides predominantly in the secretory granules suggested that proenkephalin A is processed to final products mainly in the secretory granules. The localization of Met-enkephalin-Arg6-Gly7-Leu8 and Met-enkephalin-Arg6-Phe7 in cisternae of rough endoplasmic reticula indicated their actual production in pheochromocytomas.     

Identification of pro-opiomelanocortin and the secretion of its peptide fragments in the adrenals of the bull

Immunoreactive alpha-, beta- and gamma-endorphins and beta-lipotropin--C-terminal peptide fragments of pro-opiomelanocortin (POMC)--were discovered and measured by RIA in the bovine adrenal medulla and cortex. These peptides were also discovered in perfusates of the adrenal gland. POMC proper and some intermediate forms of its processing not differing in electrophoretic mobility from the respective molecular forms of hypophyseal POMC were identified in the medulla and cortex of the adrenals by the immunoblotting technique with the use of antiserum to beta-lipoprotein. It is concluded that POMC gene is expressed in the adrenal medulla and cortex and that as a result of POMC processing a noticeable amount of its peptide fragments is formed and secreted in adrenal cells. The authors thus suggest the presence of existence of the pituitary-unrelated mechanisms of adrenal function control with participation of POMC peptides synthesized in the adrenals.     

Substance P-like immunoreactivity in adrenal chromaffin cells and intra-adrenal nerve fibers of rats

Summary The present peroxidase-antiperoxidase immunohistochemical study demonstrated a relatively small number of cells with substance P(SP)-like immunoreactivity in the adrenal medulla of rats. These cells were found alone or in small groups, were polygonal in shape and lacked long cytoplasmic processes. At immunoelectron microscopy, the immunoreactive cells were characterized by abundant granular vesicles, and the immunoreactive material was confined to the round core of the vesicles. Thus, it is suggested that SP co-exists with catecholamines in a population of chromaffin cells of the rat adrenal medulla. In addition a few SP-immunoreactive nerve fibers with varicosities were found in the adrenal medulla of rats. They extended between small clusters of chromaffin cells and had their dotlike terminals around and within the cell clusters. The SP-immunoreactive nerve fibers were characterized by the presence of abundant small clear vesicles mixed with a few large granular vesicles; the immunoreactivity appeared in the latter, but was also perfused throughout the entire axoplasm. The nerve fibers formed synapses on nonimmunoreactive chromaffin cells. Judging from the presence of bundles of SP-immunoreactive nerve fibers penetrating the adrenal capsule and cortex as well as the absence of SP-immunoreactive ganglion cells in the medulla, the intramedullary SP-immunoreactive nerve fibers seem to be extrinsic in origin.     

Identification and characterization of N-glycosylated and phosphorylated variants of proenkephalin A-derived peptides in bovine adrenal medulla, spinal cord and ileum

Recent studies have shown that during its biosynthesis in bovine adrenal medulla, the opioid precursor proenkephalin A, may be both N-glycosylated and phosphorylated. To investigate whether these chemical modifications were common to proenkephalin A processing in other tissues, we have sought to characterize enkephalin-containing peptides from bovine adrenal medulla, spinal cord and ileum. The peptides were identified using antiserum L189, specific for the C-terminus of Met-enkephalin Arg6Gly7Leu8 (MERGL), and L152, specific for the C-terminus of Met-enkephalin Arg6Phe7 (MERF). Glycosylated MERGL-immunoreactive peptides of 23, 20, 16 and 13 kDa were identified in adrenal medulla using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and concanavalin A-Sepharose affinity chromatography. Sephadex G50 gel filtration fractionated the glycosylated peptides into two immunoreactive peaks. Similar peaks of concanavalin A-binding MERGL immunoreactivity were detected in extracts of spinal cord and ileum, although there were differences in relative proportions of the two peaks. Antiserum L152 identified phosphorylated N-terminally extended variants of MERF when boiling water extracts of adrenal medulla, spinal cord and ileum were separated by anion exchange chromatography. In adrenal medulla these peptides were more than 99% phosphorylated, whereas in both ileum and spinal cord there was a relatively higher proportion of the unphosphorylated peptide. The results indicate that N-glycosylation and phosphorylation of proenkephalin A occurs in adrenal medulla, spinal cord and ileum, although there are tissue-specific differences in the relative proportions of the modified and unmodified peptides.