Adrenomedullin and proadrenomedullin N-terminal 20 peptide (PAMP) in adrenal chromaffin cells.

Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are peptides having multiple physiological functions and are most abundantly expressed in the adrenal medulla. In addition to PAMP, PAMP12, a 12 amino acid peptide with sequence identity to PAMP between amino acids 9-20, has also been shown to be expressed in the adrenal medulla. AM, PAMP and PAMP12 are released along with catecholamines by regulated exocytosis upon stimulation of adrenal chromaffin cells. PAMP and PAMP12 regulate catecholamine release and synthesis by interfering with nicotinic cholinergic receptors in these chromaffin cells. AM may also cause gradual release of catecholamine from these cells. AM, PAMP and PAMP12 are endogenous peptides that modulate chromaffin cell function via different mechanisms.     

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.     

Binding of prostaglandin E2 to cultured bovine adrenal chromaffin cells and its effect on catecholamine secretion

We have previously shown that plasma membranes from adrenal medulla possess specific high-affinity binding sites for prostaglandins (PGs) E1 and E2. We have now investigated the binding of PGE2 to intact bovine adrenal chromaffin cells and the effects of prostaglandins on the release of catecholamines from these cells. Adrenal chromaffin cells specifically bound PGE2 with a dissociation constant of 2 nM and a concentration of about 40,000 binding sites per cell. Low concentrations of PGE2 inhibited the nicotine-stimulated release of catecholamines from these cells. The effect of PGE2 was biphasic, the maximal inhibitory effect being observed at a concentration of between 1 and 10 nM. Higher concentrations (1 microM) of PGE2 had minimal inhibitory effects on nicotine-evoked noradrenaline release, but instead had a direct stimulatory effect in the absence of cholinergic agonists. Although the stimulatory effects of high concentrations of PGE2 were reproducibly observed in all cell preparations, only about one-half of the cultures tested responded to the inhibitory effects of this prostaglandin. It is possible that PGE2 plays a modulatory role in the regulation of catecholamine secretion from the adrenal medulla.     

Chromogranin A Synthesis and Secretion in Chromaffin Cells

A sensitive and selective radioimmunoassay for chromogranin A (Chrg A) has been developed to quantitate content, release, and biosynthesis of this secretory protein in neuroendocrine tissues. An antiserum raised against Chrg A from bovine adrenal medulla was found to detect predominantly only the Mr 70-75 kilodalton Chrg A in its native form, allowing the use of this antiserum as a quantitatively specific probe for Chrg A in cell-free extracts of the adrenal medulla and chromaffin cells. Chrg A comprises about 10% of the total protein of the chromaffin cell. It is released in parallel with Met-enkephalin and catecholamines from the bovine chromaffin cell in primary culture in response to nicotine and nicotinic cholinergic agonists. From 14 to 22% of total Chrg A is released from the cell during a 15-min exposure to a maximally stimulatory dose of nicotine (10-100 microM). Chrg A release on nicotinic stimulation is blocked by D-600 and hexamethonium to the same extent as Met-enkephalin and catecholamine release. The parallel time course and percent release of Chrg A and Met-enkephalin indicate that these secretory polypeptides are contained in, and released from, functionally identical cellular compartments. Chrg A and Met-enkephalin pentapeptide sequences are present in the chromaffin cell at a ratio of about 2:1, although Chrg A is far more abundant on a mass basis. Chrg A and Met-enkephalin biosynthesis appear to be differentially regulated within the chromaffin cell, since chronic treatment of cells with nicotine and forskolin causes an elevation of Met-enkephalin pentapeptide without a concomitant elevation of intracellular levels of Chrg A.     

Angiotensin II Increases Catecholamine Release from Bovine Adrenal Medulla but Does Not Enhance That Evoked by K+ Depolarization or by Carbachol

The effect of angiotensin II on catecholamine release from bovine adrenal medulla has been investigated. In retrogradely perfused, isolated bovine adrenal glands, angiotensin II increased basal efflux of catecholamines, but the presence of angiotensin II did not increase the release of catecholamines evoked either by bolus injections of the secretagogue carbachol or by depolarization with a perfusing solution containing a raised concentration of K+. In chromaffin cells maintained in primary tissue culture, angiotensin II increased 3H release from cells preloaded with [3H]-noradrenaline but did not enhance the release evoked by carbachol or by depolarization with K+. The increase in 3H release evoked by angiotensin II from chromaffin cells in tissue culture was inhibited by its analogue antagonist Sar1,Ala8-angiotensin II (saralasin) and was entirely dependent on the presence of Ca2+ in the experimental medium. These findings suggest that, in the chromaffin cells of the bovine adrenal medulla, angiotensin II acts on specific receptors to cause a calcium-dependent catecholamine release but triggers no additional response that acts synergistically with depolarizing or nicotinic stimuli to augment catecholamine release.     

The release of catecholamines from the adrenal medulla and its modulation by alpha 2-adrenoceptors in the anaesthetized dog

The adrenal nerve of anaesthetized and vagotomized dogs was electrically stimulated (10 V pulses of 2 ms duration for 10 min) at frequencies of 1, 3, 10, and 25 Hz. There was a correlation between the frequency of stimulation and the plasma concentrations of epinephrine, norepinephrine, and dopamine in the adrenal vein, mainly after the 1st min of stimulation and the maximal concentration was reached sooner with higher frequencies of stimulation. Moreover, the relative percentage of catecholamines released in response to the electrical stimulation was not changed by the frequency of stimulation. To test the hypothesis that a local negative feedback mechanism mediated by alpha 2-adrenoceptors exists in the adrenal medulla, the effects of the systemic administration of clonidine (alpha 2-antagonist) on the concentrations of catecholamines in the adrenal vein were evaluated during the electrical stimulation of the adrenal nerve (5 V pulses of 2 ms duration for 3 min) at 3 Hz. Moreover, the effects of the systemic injections of more specific alpha 2-agonist and antagonist (oxymetazoline and idazoxan) were tested on the release of catecholamines in the adrenal vein in response to electrical stimulation of the splanchnic nerve at 1 and 3 Hz frequencies. The injection of 0.5 mg/kg of yohimbine caused a significant increase in the concentrations of epinephrine and norepinephrine in the adrenal vein induced by the electrical stimulation of the adrenal nerve and the injection of 15 micrograms/kg of clonidine had no effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

PACAP regulates immediate catecholamine release from adrenal chromaffin cells in an activity-dependent manner through a protein kinase C-dependent pathway

Adrenal medullary chromaffin cells are a major peripheral output of the sympathetic nervous system. Catecholamine release from these cells is driven by synaptic excitation from the innervating splanchnic nerve. Acetylcholine has long been shown to be the primary transmitter at the splanchnic-chromaffin synapse, acting through ionotropic nicotinic acetylcholine receptors to elicit action potential-dependent secretion from the chromaffin cells. This cholinergic stimulation has been shown to desensitize under sustained stimulation, yet catecholamine release persists under this same condition. Recent evidence supports synaptic chromaffin cell stimulation through alternate transmitters. One candidate is pituitary adenylate cyclase activating peptide (PACAP), a peptide transmitter present in the adrenal medulla shown to have an excitatory effect on chromaffin cell secretion. In this study we utilize native neuronal stimulation of adrenal chromaffin cells in situ and amperometric catecholamine detection to demonstrate that PACAP specifically elicits catecholamine release under elevated splanchnic firing. Further data reveal that the immediate PACAP-evoked stimulation involves a phospholipase C and protein kinase C-dependant pathway to facilitate calcium influx through a Ni²⁺ and mibefradil-sensitive calcium conductance that results in catecholamine release. These data demonstrate that PACAP acts as a primary secretagogue at the sympatho-adrenal synapse under the stress response.     

Pituitary adenylate cyclase-activating peptide enhances electrical coupling in the mouse adrenal medulla

Neuroendocrine adrenal medullary chromaffin cells receive synaptic excitation through the sympathetic splanchnic nerve to elicit catecholamine release into the circulation. Under basal sympathetic tone, splanchnic-released acetylcholine evokes chromaffin cells to fire action potentials, leading to synchronous phasic catecholamine release. Under elevated splanchnic firing, experienced under the sympathoadrenal stress response, chromaffin cells undergo desensitization to cholinergic excitation. Yet, stress evokes a persistent and elevated adrenal catecholamine release. This sustained stress-evoked release has been shown to depend on splanchnic release of a peptide transmitter, pituitary adenylate cyclase-activating peptide (PACAP). PACAP stimulates catecholamine release through a PKC-dependent pathway that is mechanistically independent of cholinergic excitation. Moreover, it has also been reported that shorter term phospho-regulation of existing gap junction channels acts to increase junctional conductance. In this study, we test if PACAP-mediated excitation upregulates cell-cell electrical coupling to enhance chromaffin cell excitability. We utilize electrophysiological recordings conducted in adrenal tissue slices to measure the effects of PACAP stimulation on cell coupling. We report that PACAP excitation increases electrical coupling and the spread of electrical excitation between adrenal chromaffin cells. Thus PACAP acts not only as a secretagogue but also evokes an electrical remodeling of the medulla, presumably to adapt to the organism's needs during acute sympathetic stress.     

Differential control of adrenal and sympathetic catecholamine release by alpha 2-adrenoceptor subtypes

In the adrenergic system, release of the neurotransmitter norepinephrine from sympathetic nerves is regulated by presynaptic inhibitory alpha2-adrenoceptors, but it is unknown whether release of epinephrine from the adrenal gland is controlled by a similar short feedback loop. Using gene-targeted mice we demonstrate that two distinct subtypes of alpha2-adrenoceptors control release of catecholamines from sympathetic nerves (alpha 2A) and from the adrenal medulla (alpha 2C). In isolated mouse chromaffin cells, alpha2-receptor activation inhibited the electrically stimulated increase in cell capacitance (a correlate of exocytosis), voltage-activated Ca2+ current, as well as secretion of epinephrine and norepinephrine. The inhibitory effects of alpha2-agonists on cell capacitance, voltage-activated Ca2+ currents, and on catecholamine secretion were completely abolished in chromaffin cells isolated from alpha 2C-receptor-deficient mice. In vivo, deletion of sympathetic or adrenal feedback control led to increased plasma and urine norepinephrine (alpha 2A-knockout) and epinephrine levels (alpha 2C-knockout), respectively. Loss of feedback inhibition was compensated by increased tyrosine hydroxylase activity, as detected by elevated tissue dihydroxyphenylalanine levels. Thus, receptor subtype diversity in the adrenergic system has emerged to selectively control sympathetic and adrenal catecholamine secretion via distinct alpha2-adrenoceptor subtypes. Short-loop feedback inhibition of epinephrine release from the adrenal gland may represent a novel therapeutic target for diseases that arise from enhanced adrenergic stimulation.     

Temperature effects in the stimulus-secretion process from isolated chromaffin cells

Temperature effects on the stimulus-secretion coupling process was studied by inducing release of catecholamines (CA) from isolated chromaffin cells of the bovine adrenal medulla. Use was made of three different secretagogues: acetylcholine (ACH), high potassium concentration, and the calcium ionophore A23187, at various incubation temperatures. The latter two agents induced a monotonic increase in secretion with rise in temperature, suggesting different regions of the dependence of total release on temperature. The ACH-induced secretion was, however, markedly different and exhibited a maximal release at 30 degrees C. Kinetic experiments using ACH stimulus revealed that this maximum is produced by different temperature dependence in the stages of activation and desensitization. A proposed model for the total release process yields temperature-dependent parameters that can be divided into three regions of initial rates of secretory activity corresponding to the above independent findings using high K+ concentration and the calcium ionophore. The transitions between the various regions indicate possible transitions in the physical properties of the plasma and secretory granule membranes. Elucidation of the interaction between the membranes is of primary importance in the determining mechanism of CA secretion from the isolated adrenal medulla cell.     

Catecholamine secretion from the adrenal medulla of the fetus, regulation by hormones

In the ovine fetus, the adrenal medulla activity secretes catecholamines into the circulation under normal and stress conditions. Little is known regarding the endocrine regulation of adrenal medullary catecholamine secretion in the fetus. The present study was undertaken to investigate the direct effects of the hormones prolactin, angiotensin II and cortisol on catecholamine release from fetal adrenal medulla, and to determine whether the effect of the hormones change during development into adulthood. Adrenal medulla from fetal, newborn and adult pregnant sheep was collected, dispersed into single cells and plated. Following preincubation, the cells were treated with ovine prolactin or angiotensin II at 8, 40 and 200 micrograms/ml; or cortisol at 10(-8), 10(-7) and 10(-6)M for 24 h. Catecholamine release into the medium were measured at 3, 6, 12 and 24 h. Ovine prolactin at 8 to 200 micrograms/ml significantly stimulated the release of total catecholamines after 12 h of incubation. The effect of prolactin was dose-dependent such that the magnitude of the response increased and the response time shortened with increasing concentrations of prolactin. In addition, the release of all three catecholamines--dopamine, norepinephrine and epinephrine--was significantly elevated. In newborn cells, only the highest concentration of 200 micrograms/ml ovine prolactin stimulated total catecholamine release at 6 h and 12 h, with significant increases of the three catecholamines at 12 h. In maternal cells, stimulation of catecholamine release was observed also with the highest concentration of prolactin tested (200 micrograms/ml) and after 12 h of incubation, when only the release of epinephrine was significantly enhanced by 324%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional and morphological characterization of isolated bovine adrenal medullary cells

Single bovine adrenal medullary cells have been obtained by retrograde perfusion of adrenal medullae with a solution of 0.05% collagenase in Ca++-free Krebs Henseleit buffer. Chromaffin cells were obtained in high yield (5 X 10(6) cells/g medulla), and more than 95% of these were viable as shown by exclusion of trypan blue. The isolated cells were capable of respiring at a linear rate for a minimum of 120 min. Ultrastructural examination revealed that the cells were morphologically intact, and two distinct types of adrenal medullary cells were identified, on the basis of the morphology of their electron-dense vesicles, as (a) adrenaline-containing and (b) noradrenaline-containing cells. Biochemical analysis showed that the cells contained catecholamines and dopamine-beta-hydroxylase (DBH). The cells released catecholamines and DBH in response to acetylcholine (ACh), and this release was accompanied by changes in the vesicular and surface membranes observed at the ultrastructural level. The time-course of ACh-stimulated catecholamine and DBH release, and the dependence of this release on the concentration of ACh and extracellular Ca++ have been investigated. The isolated cells were pharmacologically sensitive to the action of the cholinergic blocking agents, atropine and hexamethonium.     

Regulated exocytosis in chromaffin cells and cytotoxic T lymphocytes: How similar are they?

Chromaffin cells from the adrenal medulla secrete catecholamines into the blood stream as part of the fight-or-flight response. Cytotoxic T lymphocytes from the immune system release cytotoxic substances to kill antigen-presenting cells. While at first glance these two cell types do not seem to have much in common, evidence from human diseases indicates that the molecular mechanisms of exocytosis of the respective granules share many similar features. In this review we highlight the similarities and differences of individual aspects of granule maturation and release in both cell types. In addition, we discuss established and putative molecules involved in distinct steps and suggest technical approaches which might facilitate future studies in chromaffin cells and cytotoxic T lymphocytes.     

Induction of Adrenal Tyrosine Hydroxylase mRNA by Single Immobilization Stress Occurs Even After Splanchnic Transection and in the Presence of Cholinergic Antagonists

Abstract: Immobilization (IMO) stress elevates plasma catecholamines and increases tyrosine hydroxylase (TH) gene expression in rat adrenals. This study examined the mechanism(s) of IMO-induced changes in adrenal TH mRNA levels. Innervation of the adrenal medulla is predominantly cholinergic and splanchnicotomy as well as nicotinic receptor antagonists prevent the cold-induced rise in TH mRNA levels. In this study, the IMO-induced rise in plasma catecholamines, but not TH mRNA levels, was reduced by the antagonist chlorisondamine. Muscarinic antagonist atropine also did not prevent the IMO stress-elicited rise in TH mRNA. Furthermore, denervation of the adrenals by unilateral splanchnicotomy did not block the IMO-induced rise in TH mRNA but completely prevented the induction of neuropeptide Y mRNA. These results suggest that (1) the large increase in adrenal TH gene expression elicited by a single IMO stress is not regulated via cholinergic receptors or splanchnic innervation, and (2) there is a dissociation between regulatory mechanisms of catecholamine secretion and elevation of TH gene expression in the adrenal medulla of rats during IMO stress.     

Protein kinase c-β-like immunoreactivity in the developing and adult rat adrenal gland

Summary Protein kinase c--like immunoreactivity was studied in the adrenal gland of adult rats and at different pre- and postnatal stages of development (E17-P21) with an antibody specific to both the ₂₁ and - subtypes of the kinase. In the adult rat adrenal gland, the immunoreactivity was seen in numerous nerve fibres in the adrenal medulla both in bundles and individually forming occasionally dense networks around chromaffin cell groups. Several protein kinase c--immunoreactive fibres were also observed transversing the adrenal cortex towards the medulla. No remaining immunoreactive fibres two weeks after transection of the splanchnic nerve could be seen; nor was any immunoreactivity observed in the chromaffin cells of the adrenal medulla or in the cortical cells, but some faintly immunoreactive ganglion cells were detected in the adrenal medulla. The amount and distribution of protein kinase c--like immunoreactivity in the fetal and developing adrenals was very similar to that seen in the adrenal glands of adult rats. On the basis of its localization, the -subtype of protein kinase c does not appear to be directly involved in the release of catecholamines from the adrenal medulla, but it might have a role in the regulation of neurotransmitter release from preganglionic cholinergic neurons.     

Stability of Bovine Adrenal Medulla Cells in Culture

The functional stability of primary cultures of adrenal medulla cells was investigated. Isolated cells were prepared by treatment of bovine adrenal glands with collagenase followed by purification on Percoll density gradients and were maintained in Dulbecco's medium containing 10% fetal calf serum. Within 12 h after plating on plastic culture dishes, the cells became firmly attached and exhibited good survival for periods of time up to 3 weeks, as indicated by their morphology using light and electron microscopy, by maintenance of their content of catecholamines, tyrosine hydroxylase, dopamine-beta-hydroxylase, and phenylethanolamine N-methyltransferase, and their ability to respond to secretagogues. During the first 10 days to 2 weeks in culture there was little or no change in any of these parameters. During the 3rd week there were progressive losses of catecholamine and enzyme activities and increased vacuolization of medullary cells. The cells synthesized protein and RNA with no apparent loss in activities over the period studied, but did not incorporate [3H]thymidine into PCA-precipitable material. The cells responded to secretagogues and secretory antagonists similarly to isolated perfused adrenal glands. The studies described here demonstrate that primary cultures of adrenal medulla cells provide an excellent experimental system for obtaining more detailed information on stimulus-secretion coupling and other functional aspects of the adrenal medulla.     

Central angiotensin II increases biosynthesis of tyrosine hydroxylase in the rat adrenal medulla

Angiotensin II acting centrally contributes to the regulation of blood pressure and water intake and stimulates the release of catecholamines from the adrenal medulla. We hypothesized that the central angiotensin II is one mediator of biosynthesis of catecholamines in the adrenal medulla. Rats were administered i.c.v. angiotensin II or saline, and TH mRNA and protein levels in adrenal medulla were measured 1 or 3 h later. Angiotensin II did not change TH mRNA or protein 1 h later. However, by 3 h, angiotensin II increased TH mRNA and protein levels. Centrally administered angiotensin II elevates TH mRNA expression and protein levels in the adrenal medulla. In conclusion, one component of central angiotensin II elevation of blood pressure may be the result of increased catecholamine synthesis in the adrenal gland and elevated TH synthesis represents one underlying mechanism.     

Regulation of the neuronal nicotinic acetylcholine receptor by SRC family tyrosine kinases

Src family kinases (SFKs) are abundant in chromaffin cells that reside in the adrenal medulla and respond to cholinergic stimulation by secreting catecholamines. Our previous work indicated that SFKs regulate acetylcholine- or nicotine-induced secretion, but the site of modulatory action was unclear. Using whole cell recordings, we found that inhibition of SFK tyrosine kinase activity by PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine) treatment or expression of a kinase-defective c-Src reduced the peak amplitude of nicotine-induced currents in chromaffin cells or in human embryonic kidney cells ectopically expressing functional neuronal alpha3beta4alpha5 acetylcholine receptors (AChRs). Conversely, the phosphotyrosine phosphatase inhibitor, sodium vanadate, or expression of mutationally activated c-Src resulted in enhanced current amplitudes. These results suggest that SFKs and putative phosphotyrosine phosphatases regulate the activity of AChRs by opposing actions. This proposed model was supported further by the findings that SFKs physically associate with the receptor and that the AChR is tyrosine-phosphorylated.     

Dynamin I plays dual roles in the activity-dependent shift in exocytic mode in mouse adrenal chromaffin cells

Under low stimulation, adrenal chromaffin cells release freely soluble catecholamines through a restricted granule fusion pore while retaining the large neuropeptide-containing proteinacious granule core. Elevated activity causes dilation of the pore and release of all granule contents. Thus, physiological differential transmitter release is achieved through regulation of fusion pore dilation. We examined the mechanism for pore dilation utilizing a combined approach of peptide transfection, electrophysiology, electrochemistry and quantitative imaging techniques. We report that disruption of dynamin I function alters both fusion modes. Under low stimulation, interference with dynamin I does not affect granule fusion but blocks its re-internalization. In full collapse mode, disruption of dynamin I limits fusion pore dilation, but does not block membrane re-internalization. These data suggest that dynamin I is involved in both modes of exocytosis by regulating contraction or dilation of the fusion pore and thus contributes to activity-dependent differential transmitter release from the adrenal medulla.