Ontogeny of adrenal VIP content and release from adrenocortical cells in the ovine fetus

The present study was designed to investigate the presence of VIP in fetal adrenals, to determine the changes in adrenal VIP content associated with maturation, and to explore the factors which regulate fetal adrenal VIP release. Adrenal glands from ovine fetuses at 70 to 140 days gestation were used. Adrenal VIP content, as measured by radioimmunoassay, were low at 70 and 80 days of gestation. This was followed by a rapid increase in VIP content from 80 to 110 days reaching a plateau between 110 and 130 days at levels comparable to that in the adult. A significant fall in adrenal VIP content occurred at 140 days, immediately prior to term. Release of VIP from fetal adrenocortical cells in vitro was significantly elevated by angiotensin II at 10(-5) M, while ACTH had no effect. Acetylcholine at 50 microM and high potassium stimulated fetal adrenal VIP release while norepinephrine did not. These results suggest that the VIP neuronal system in the ovine fetal adrenal matures between 80 and 110 days of gestation. Furthermore, the release of VIP from the fetal adrenocortical cells may be regulated by angiotensin II and cholinergic neurotransmitters.     

Regulation of catecholamine release in human adrenal chromaffin cells by β-adrenoceptors

The adrenal gland plays a fundamental role in the response to a variety of stress situations. After a stress condition, adrenal medullary chromaffin cells release, by exocytosis, high quantities of catecholamine (epinephrine, EP; norepinephrine, NE), especially EP. Once in the blood stream, catecholamines reach different target organs, and induce their biological actions through the activation of different adrenoceptors. Adrenal gland cells may also be activated by catecholamines, through hormonal, paracrine and/or autocrine system. The presence of functional adrenoceptors on human adrenal medulla and their involvement on catecholamines secretion was not previously evaluated. In the present study we investigated the role of β(1)-, β(2)- and β(3)-adrenoceptors on catecholamine release from human adrenal chromaffin cells in culture. We observed that the β-adrenoceptor agonist (isoproterenol) and β(2)-adrenoceptor agonist (salbutamol) stimulated catecholamine (NE and EP) release from human adrenal chromaffin cells. Furthermore, the β(2)-adrenoceptor antagonist (ICI 118,551; 100 nM) and β(3)-adrenoceptor antagonist (SR 59230A; 100 nM) inhibited the catecholamine release stimulated by isoproterenol and nicotine in chromaffin cells. The β(1)-adrenoceptor antagonist (atenolol; 100 nM) did not change the isoproterenol- neither the nicotine-evoked catecholamine release from human adrenal chromaffin cells. Moreover, our results show that the protein kinase A (PKA), protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and phospholipase C (PLC) are intracellular mechanisms involved in the catecholamine release evoked by salbutamol. In conclusion, our data suggest that the activation of β(2)- and β(3)-adrenoceptors modulate the basal and evoked catecholamine release, NE and EP, via an autocrine positive feedback loop in human adrenal chromaffin cells.     

Effects of orexin on cultured porcine adrenal medullary and cortex cells

New orexigenic peptides called orexins have recently been described in the neurons of the lateral hypothalamus and perifornical area. No orexins have been found in the adipose tissues or visceral organs, including the adrenal gland. However, expression of the orexin receptor (OXR) in the rat adrenal gland has been reported. With regard to the effects of orexins on peripheral organs, we previously reported that orexins suppress catecholamine synthesis and secretion in the rat pheochromocytoma cell line PC12. To further clarify the pharmacological effects of orexins on peripheral organs, we examined the effects of orexin-A on catecholamine, cortisol, and aldosterone secretion, using cultured porcine adrenal glands. We initially confirmed the expression of the orexin receptor (OXR-1) in cultured porcine adrenal medulla and cortex. Orexin-A (1000 nM) significantly increased the release of both epinephrine (E) and norepinephrine (NE) from porcine adrenal medullary cells. Similarly, orexin-A (> or = 100 nM) significantly increased the release of both cortisol and aldosterone from porcine adrenal cortex cells. Orexin-A (100 nM) significantly inhibited basal and the PACAP-induced increase in cAMP levels in adrenal medullary cells. Conversely, orexin-A (>o = 100 nM) significantly increased the cAMP level in adrenal cortex cells. These results indicate that orexin-A induces the release of catecholamine from porcine adrenal medullary cells, and aldosterone and cortisol from the cortex cells and has opposite effects on cAMP levels in adrenal medulla and cortex.     

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.     

Alpha-adrenergic stimulants, prostaglandins and catecholamine release from the adrenal gland in vitro.

In rat adrenal glands incubated in Locke's solution in vitro norepinephrine and phenylephrine inhibited the release of epinephrine. PGE2 and PGE1 also inhibited the release of catecholamines but PGFalpha1 had no effect on the adrenal. Thus, catecholamine release from adrenal cells may be regulated by the same mechanisms as in adrenergic nerve endings.     

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.     

Agouti-related protein has an inhibitory paracrine role in the rat adrenal gland

alpha-Melanocyte-stimulating-hormone (alpha-MSH) is an agonist at the melanocortin 3 receptor (MC3-R) and melanocortin 4 receptor (MC4-R). alpha-MSH stimulates corticosterone release from rat adrenal glomerulosa cells in vitro. Agouti-related protein (AgRP) an endogenous antagonist at the MC3-R and MC4-R, is expressed in the adrenal gland. We investigated the expression of the MC3-R and MC4-R and the role of AgRP in the adrenal gland. MC3-R and MC4-R expression was detected in rat adrenal gland using RT-PCR. The effect of AgRP on alpha-MSH-induced corticosterone release was investigated using dispersed rat adrenal glomerulosa cells. AgRP administered alone did not affect corticosterone release, but co-administration of AgRP and alpha-MSH attenuated alpha-MSH-induced corticosterone release. To investigate glucocorticoid feedback, adrenal AgRP expression was compared in rats treated with dexamethasone to controls. AgRP mRNA was increased in rats treated with dexamethasone treatment compared to controls. Our findings demonstrate that adrenal AgRP mRNA is regulated by glucocorticoids. AgRP acting via the MC3-R or MC4-R may have an inhibitory paracrine role, blocking alpha-MSH-induced corticosterone secretion.     

Parallel but Separate Release of Catecholamines and Acetylcholinesterase from Stimulated Adrenal Chromaffin Cells in Culture

Abstract: A pharmacological study was made of the effects of veratridine and lasalocid on the release of catecholamines, acetylcholinesterase (AChE) and dopamine-β-hydroxylase (DBH) from cultures of isolated bovine adrenal chromaffin cells. Exposure of the cultures to veratridine resulted in concomitant release of catecholamines and AChE into the external medium in a dose-dependent and Ca^{2 +}-dependent manner. A Ca²⁺ iono-phore, lasalocid, also produced a dose-dependent and parallel release of both catecholamines and AChE. The release of the two components was accompanied by release of DBH. The present results provide pharmacological evidence for a parallel release of catecholamines, AChE, and DBH from cultured adrenal chromaffin cells, and the stoichiometry of the release evoked by different secretagogues suggests that AChE and catecholamines are released from different cellular compartments.     

α-Adrenergic stimulants, prostaglandins and catecholamine release from the adrenal gland in vitro

In rat adrenal glands incubated in Locke's solution in vitro norepinephrine and phenylephrine inhibited the release of epinephrine. PGE₂ and PGE₁ also inhibited the release of catecholamines but PGF_α1 had no effect on the adrenal. Thus, catecholamine release from adrenal cells may be regulated by the same mechanisms as in adrenergic nerve endings.     

Fetal adrenal VIP: distribution and effect on medullary catecholamine secretion

Vasoactive intestinal peptide (VIP) was found in the adrenal gland of ovine fetuses at 130-135 days gestation and was shown to stimulate catecholamine secretion. VIP was demonstrated by immunocytochemistry using the indirect antibody-enzyme method. VIP-immunoreactive nerve fibers were observed in the capsule, zona glomerulosa and inner layer of the cortex as well as in the medulla; furthermore small clusters of VIP-containing cell bodies were found at the corticomedullary border. To study the direct effect of VIP on catecholamine release, fetal adrenal medulla was dispersed into single cells and incubated in vitro with VIP for 6 hours. Catecholamine release into the medium was measured at 1, 3 and 6 hours. At 6 hours of incubation, VIP stimulated total catecholamine release from fetal adrenomedullary cells in a dose-dependent manner at concentrations ranging from 10(-8) to 10(-4) M. The release of norepinephrine and epinephrine, but not dopamine, was significantly enhanced. The presence of VIP in the fetal adrenal cortex and medulla, and the ability of VIP to stimulate catecholamine release from fetal adrenomedullary cells in vitro suggest that VIP may be an important modulator of medullary catecholamine secretion during fetal life.     

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.     

Effects of imidazole compounds on catecholamine release in adrenal chromaffin cells

1. Effects of imidazole compounds and guanabenz on the stimulus-evoked release of catecholamine (CA) were studied in cultured bovine adrenal chromaffin cells. 2. Clonidine, oxymetazoline, phentolamine, chlorpheniramine, and guanabenz inhibited acetylcholine (ACh)-evoked CA release in a dose-dependent manner, but not high K(+)-evoked release. 3. The inhibition by these compounds was not antagonized by nonimidazole and nonguanidine alpha 2-antagonists (yohimbine and phenoxybenzamine) but was significantly antagonized by tolazoline (imidazole alpha 2-antagonist) and cimetidine (imidazole H2-antagonist). Moreover, tolazoline by itself augmented the ACh-evoked, but not the high K(+)-evoked, CA release. 4. Although chlorpheniramine and cimetidine are antagonists for H1 and H2 histaminergic receptors, the site of action for these compounds in our results seemed to differ from the histamine receptors. 5. These results suggest that the inhibitory action of imidazole compounds and guanabenz on ACh-evoked CA release in adrenal chromaffin cells is mediated through an imidazole receptor. Adrenal chromaffin cells may contain an endogenous clonidine-displacing substance (CDS) which has been found in adrenal gland and brain as an endogenous ligand for imidazole receptors. Thus, CDS may have a regulatory role in the stimulus-secretion coupling in these cells.     

Relationship between arachidonic acid release and Ca2(+)-dependent exocytosis in digitonin-permeabilized bovine adrenal chromaffin cells.

The relationship between Ca2(+)-dependent arachidonic acid release and exocytosis from digitonin-permeabilized bovine adrenal chromaffin cells was investigated. The phospholipase A2 inhibitors mepacrine, nordihydroguaiaretic acid and indomethacin had no effect on either arachidonic acid release or secretion. The phospholipase A2 activator melittin had no effect on secretion. The specific diacylglycerol lipase inhibitor RG80267 had no effect on secretion, but decreased basal arachidonic acid release to such an extent that the level of arachidonic acid in treated cells in response to 10 microM-Ca2+ was equivalent to that of control cells in the absence of Ca2+. Staurosporine, a protein kinase C inhibitor, was found to abolish Ca2(+)-dependent arachidonic acid release completely, but had only a slight inhibitory effect on Ca2(+)-dependent secretion. It is concluded that arachidonic acid is not essential for Ca2(+)-dependent exocytosis in adrenal chromaffin cells.     

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.     

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.     

Endothelial cell nitric oxide inhibits aldosterone synthesis in zona glomerulosa cells: modulation by oxygen

The regulation of aldosterone synthesis by endogenous nitric oxide (NO) was examined in cultured cells of the adrenal cortex. Endothelial NO synthase (eNOS) was detected by Western blot in cultured adrenal endothelial cells (ECs) but not in zona glomerulosa (ZG) cells or adrenal fibroblasts. Neither inducible (iNOS) nor neuronal NOS (nNOS) isoforms were detected in the cells. Only ECs had NOS activity and converted [(3)H]L-arginine to [(3)H]L-citrulline. Angiotensin II (ANG II, 100 nM) increased EC production of nitrate/nitrite by 2.4-fold. Coincubation with ECs or treatment with DETA nonoate increased the fluorescence of ZG cells loaded with an NO-sensitive dye, diaminofluorescein 2 diacetate (DAF-2 DA). DETA nonoate inhibited ANG II (1 nM) and potassium (10 mM) -stimulated aldosterone release in a concentration-related manner. This inhibitory effect of NO was enhanced >10-fold by decreasing the oxygen concentration from 21 to 8%. Coincubation of EC and ZG cells in 8% oxygen inhibited ANG II-induced aldosterone release, and inhibition was reversed by blockade of NOS. These findings indicate that adrenal EC-derived NO inhibits aldosterone release by cultured ZG cells and that the sensitivity to NO inhibition is increased at low oxygen concentrations.     

The release of 3H-1-methyl-4-phenylpyridinium from bovine adrenal chromaffin cells is modulated by somatostatin

Besides cholinergic regulation, catecholamine secretion from adrenal chromaffin cells can be elicited and/or modulated by noncholinergic neurotransmitters and hormones. This study was undertaken to investigate the influence of somatostatin and octreotide on [3H]MPP+ secretion evoked by KCl or cholinergic agents, from bovine adrenal chromaffin cells. The release of [3H]MPP+ was markedly increased by excess KCl (50 mM), acetylcholine (50 microM-10 mM) and by the nicotinic agonists, nicotine (5-100 microM) and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP, 10-100 microM), but not by the muscarinic agonist, pilocarpine (10-100 microM). Acetylcholine-evoked release of [3H]MPP+ from these cells was mainly mediated by nicotinic receptors: a) nicotine and DMPP stimulated the release of [3H]MPP+, b) a nicotinic antagonist, hexamethonium, markedly blocked the acetylcholine-evoked response and c) pilocarpine was devoid of effect on [3H]MPP+ secretion. At all concentrations tested, somatostatin and octreotide interfered neither with [3H]MPP+ basal release nor with KCl-induced release of [3H]MPP+. However, somatostatin (0.01-0.3 microM) increased the release of [3H]MPP+ induced by a high concentration of acetylcholine (10 mM). Octreotide (1-10 microM) had no effect. These results, showing that somatostatin potentiates acetylcholine-induced [3H]MPP+ release, support the hypothesis that somatostatin may increase the release of catecholamines from adrenal medullary cells.     

Dopamine Receptors on Adrenal Chromaffin Cells Modulate Calcium Uptake and Catecholamine Release

The presence of dopamine-containing cells in sympathetic ganglia, i.e., small, intensely fluorescent cells, has been known for some time. However, the role of dopamine as a peripheral neurotransmitter and its mechanism of action are not well understood. Previous studies have demonstrated the presence of D2 dopamine receptors on the surface of bovine adrenal chromaffin cells using radioligand binding methods and dopamine receptor inhibition of catecholamine release from perfused adrenal glands. In the present study, we provide evidence confirming a role of dopamine receptors as inhibitory modulators of adrenal catecholamine release from bovine chromaffin cell cultures and further show that the mechanism of modulation involves inhibition of stimulated calcium uptake. Apomorphine gave a dose-dependent inhibition (IC50 = 1 microM) of 45Ca2+ uptake stimulated by either nicotine (10 microM) or membrane depolarization with an elevated K+ level (60 mM). This inhibition was reversed by a series of specific (including stereospecific) dopamine receptor antagonists: haloperidol, spiperone, sulpiride, and (+)-butaclamol, but not (-)-butaclamol. In addition, the calcium channel agonist Bay K 8644 was used to stimulate uptake of 45Ca2+ into chromaffin cells, and this uptake was also inhibited by the dopamine receptor agonist apomorphine. The combined results suggest that dopamine receptors on adrenal chromaffin cells alter Ca2+ channel conductance, which, in turn, modulates catecholamine release.