Phospholamban ablation rescues the enhanced propensity to arrhythmias of mice with CaMKII-constitutive phosphorylation of RyR2 at site S2814.

KEY POINTS: Mice with Ca(2+) -calmodulin-dependent protein kinase (CaMKII) constitutive pseudo-phosphorylation of the ryanodine receptor RyR2 at Ser2814 (S2814D(+/+) mice) exhibit a higher open probability of RyR2, higher sarcoplasmic reticulum (SR) Ca(2+) leak in diastole and increased propensity to arrhythmias under stress conditions. We generated phospholamban (PLN)-deficient S2814D(+/+) knock-in mice by crossing two colonies, S2814D(+/+) and PLNKO mice, to test the hypothesis that PLN ablation can prevent the propensity to arrhythmias of S2814D(+/+) mice. PLN ablation partially rescues the altered intracellular Ca(2+) dynamics of S2814D(+/+) hearts and myocytes, but enhances SR Ca(2+) sparks and leak on confocal microscopy. PLN ablation diminishes ventricular arrhythmias promoted by CaMKII phosphorylation of S2814 on RyR2. PLN ablation aborts the arrhythmogenic SR Ca(2+) waves of S2814D(+/+) and transforms them into non-propagating events. A mathematical human myocyte model replicates these results and predicts the increase in SR Ca(2+) uptake required to prevent the arrhythmias induced by a CaMKII-dependent leaky RyR2. ABSTRACT: Mice with constitutive pseudo-phosphorylation at Ser2814-RyR2 (S2814D(+/+) ) have increased propensity to arrhythmias under ß-adrenergic stress conditions. Although abnormal Ca(2+) release from the sarcoplasmic reticulum (SR) has been linked to arrhythmogenesis, the role played by SR Ca(2+) uptake remains controversial. We tested the hypothesis that an increase in SR Ca(2+) uptake is able to rescue the increased arrhythmia propensity of S2814D(+/+) mice. We generated phospholamban (PLN)-deficient/S2814D(+/+) knock-in mice by crossing two colonies, S2814D(+/+) and PLNKO mice (SD(+/+) /KO). SD(+/+) /KO myocytes exhibited both increased SR Ca(2+) uptake seen in PLN knock-out (PLNKO) myocytes and diminished SR Ca(2+) load (relative to PLNKO), a characteristic of S2814D(+/+) myocytes. Ventricular arrhythmias evoked by catecholaminergic challenge (caffeine/adrenaline) in S2814D(+/+) mice in vivo or programmed electric stimulation and high extracellular Ca(2+) in S2814D(+) /(-) hearts ex vivo were significantly diminished by PLN ablation. At the myocyte level, PLN ablation converted the arrhythmogenic Ca(2+) waves evoked by high extracellular Ca(2+) provocation in S2814D(+/+) mice into non-propagated Ca(2+) mini-waves on confocal microscopy. Myocyte Ca(2+) waves, typical of S2814D(+/+) mice, could be evoked in SD(+/+) /KO cells by partially inhibiting SERCA2a. A mathematical human myocyte model replicated these results and allowed for predicting the increase in SR Ca(2+) uptake required to prevent the arrhythmias induced by a Ca(2+) -calmodulin-dependent protein kinase (CaMKII)-dependent leaky RyR2. Our results demonstrate that increasing SR Ca(2+) uptake by PLN ablation can prevent the arrhythmic events triggered by SR Ca(2+) leak due to CaMKII-dependent phosphorylation of the RyR2-S2814 site and underscore the benefits of increasing SERCA2a activity on SR Ca(2+) -triggered arrhythmias.

G protein receptors 7 and 8 are expressed in human adrenocortical cells, and their endogenous ligands neuropeptides B and w enhance cortisol secretion by activating adenylate cyclase- and phospholipase C-dependent signaling cascades.

Neuropeptides B and W (NPB and NPW) are regulatory peptides that act via two subtypes of G protein-coupled receptors, named GPR7 and GPR8. RT-PCR demonstrated the expression of these receptors in both zona glomerulosa and zona fasciculata-reticularis (ZF/R) cells of the human adrenal cortex. NPB and NPW did not affect aldosterone secretion from dispersed zona glomerulosa cells but enhanced cortisol production from ZF/R cells, NPB being more effective than NPW. NPB evoked sizable cAMP and inositol triphosphate responses from ZF/R cells, which were abrogated by the adenylate cyclase inhibitor SQ-22536 and the phospholipase C inhibitor U-73122, respectively. Cortisol response to NPB was lowered by either SQ-22536 and the protein kinase (PK) A inhibitor H-89 or U-73122 and the PKC inhibitor calphostin-C and abolished by the simultaneous exposure to H-89 and calphostin-C. NPW elicited only a rise in cAMP production from dispersed ZF/R cells, and its cortisol response was suppressed by both SQ-22536 and H-89. PreproNPB and preproNPW mRNAs were detected in human adrenal cortexes. We conclude that: 1) NPB and NPW exert a secretagogue action on human ZF/R cells, probably acting in an autocrine-paracrine manner; and 2) the effect of NPB is mediated by both the adenylate cyclase/PKA and the phospholipase C/PKC cascades, whereas that of NPW involves only the activation of the former signaling pathway.

Immune-endocrine interactions in the mammalian adrenal gland: facts and hypotheses.

Several cytokines, which are the major mediators of the inflammatory responses, are well-known to stimulate the hypothalamopituitary corticotropin-releasing hormone (CRH)/adrenocorticotropic hormone (ACTH) system, thereby evoking secretory responses by the adrenal cortex. Many of these cytokines, including interleukin-1 (IL-1), IL-2, IL-6, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (INF-gamma) are synthesized in the adrenal gland by both parenchymal cells and resident macrophages, and the release of some of them (e.g., IL-6 and TNF-alpha) is regulated by the main agonists of steroid hormone secretion (e.g., ACTH and angiotensin-II) and bacterial endotoxins. Adrenocortical and adrenomedullary cells are provided with specific receptors for IL-1, IL-2, and IL-6. IL-1 and TNF-alpha directly inhibit aldosterone secretion of zona glomerulosa cells, whereas IL-6 enhances it. IL-2, IL-3, IL-6, and INF-alpha are able to directly stimulate glucocorticoid production by zona fasciculata and zona reticularis cells, whereas IL-1 exerts an analogous effect through an indirect mechanism involving the stimulation of catecholamine release by chromaffin cells and/or the activation of the intramedullary CRH/ACTH system; again, TNF-alpha depresses glucocorticoid synthesis. IL-6 raises androgen secretion by inner adrenocortical layers. IL-1 enhances the proliferation of adrenocortical cells, and findings suggest that cytokines may control the apoptotic deletion of senescent zona reticularis cells. The relevance of the intraadrenal cytokine system in the fine-tuning of the secretion and growth of the adrenal cortex under normal conditions remains to be explored. However, indirect proof is available that local immune-endocrine interactions may play an important role in modulating adrenal responses to inflammatory and immune challenges and stresses.

Zona glomerulosa morphology and function in streptozotocin-induced diabetic rats.

Streptozotocin-induced diabetic rats showed a significant lowering in both PRA (-31%) and basal plasma aldosterone concentration (-59%), coupled with a notable atrophy of the zona glomerulosa (-30%) and its parenchymal cells (-36%). Kalaemia and the blood level of ACTH were not affected. Insulin infusion reversed all the streptozotocin-evoked effects. Analogous, though less conspicuous, changes were induced by experimental diabetes also in rats whose hypothalamo-hypophyseal-adrenal axis and renin-angiotensin system had been pharmacologically interrupted by the simultaneous administration of dexamethasone-captopril and maintenance doses of ACTH-angiotensin II: the drops in the basal plasma aldosterone concentration and in the volume of zona glomerulosa and its cells ranged from -20% to -22%. In these animals, experimental diabetes significantly depressed the aldosterone response to the acute stimulation with angiotensin II (-55%), potassium (-50%), and ACTH (-43%). These findings indicate that the well known impairment of renin release may only partially account for the antiadrenoglomerulotrophic effect of experimental diabetes in rats. The hypothesis is advanced that the chronic lack of insulin may directly depress both the growth of the zona glomerulosa and the newly synthesis of some enzymes of aldosterone synthesis.

Effects of prolonged cyclosporine-A treatment on the morphology and function of rat adrenal cortex.

The effects of prolonged (30 day) treatment with daily therapeutical doses of cyclosporine A (CSA) (20 mg/kg) on the function and morphology of adrenal cortex were studied in adult male rats. CSA-treated animals developed a notable hypertension, along with a striking rise in PRA, which was not coupled with significant changes in the plasma concentrations of aldosterone and corticosterone (hyperreninemic hypoaldosteronism). Morphometry showed that zona glomerulosa (ZG) and zona fasciculata, and their parenchymal cells were atrophic. Isolated capsular (ZG) and inner (zona fasciculata/reticularis) cells displayed reduced basal and stimulated secretory responses. However, while the response of ZG cells to angiotensin II was almost completely suppressed (96%), basal steroid secretion of isolated cells, as well as the aldosterone and corticosterone response of ZG cells to potassium and ACTH, and corticosterone production of inner cells in response to ACTH were decreased by only about 30-40%. The hypothesis is advanced that CSA exerts a dual effect on rat adrenal cortex: 1) a general inhibitory effect on the growth and steroidogenic capacity of adrenocortical cells, which manifests itself only after very prolonged treatment and may be caused by an impairment of protein synthesis; and 2) an acute effect involving the specific blockade of the angiotensin-II-induced stimulation of the secretory activity of ZG cells.

Direct secretagogue effect of corticotropin-releasing factor on the rat adrenal cortex: the involvement of the zona medullaris.

CRF dose-dependently enhanced corticosterone (B) secretion by rat adrenal slices including both cortex and medulla. Conversely, CRF did not exert any B response by fragments of adrenocortical autotransplants, which are completely deprived of chromaffin tissue. However, autotransplant quarters exhibited a dose-dependent response to ACTH qualitatively similar to that of adrenal slices, although markedly less intense. The maximal B response of adrenal slices to CRF (10(-8) M) was completely annulled by corticotropin-inhibiting peptide (10(-6) M), a competitive inhibitor of ACTH, which totally blocked the secretory response to ACTH (10(-8) M) of both kinds of preparations. ACTH immunoreactivity was present in the adrenal gland of control rats, but was undetectable in autotransplanted adrenocortical nodules. Moreover, adrenal fragments mainly composed of chromaffin tissue released detectable amounts of ACTH in response to high concentrations of CRF (10(-8)/10(-6) M). These findings suggest that chromaffin medullary cells play a pivotal role in the direct adrenocortical secretagogue effect of CRF, probably by releasing ACTH, which, in turn, may evoke, in a paracrine manner, the glucocorticoid response.

Endothelins stimulate deoxyribonucleic acid synthesis and cell proliferation in rat adrenal zona glomerulosa, acting through an endothelin A receptor coupled with protein kinase C- and tyrosine kinase-dependent signaling pathways.

The effects of endothelins (ET) on the proliferative activity of the rat adrenal cortex have been investigated in vivo, using an in situ perfusion technique of the intact left gland. The chemicals were dissolved in the perfusion medium, and the perfusion continued for 120 min. ET-1 concentration dependently increased the mitotic index and [3H]thymidine incorporation into DNA in the zona glomerulosa (ZG; 6- and 3-fold increases, respectively, at a 10(-8) M concentration), but not in the inner adrenocortical layers, where the basal proliferative activity was negligible. The effect of 10(-8) M ET-1 was blocked by the ETA receptor antagonist BQ-123, whereas the ETB receptor antagonist BQ-788 was ineffective. ET-2 and ET-3 (10(-8) M) enhanced DNA synthesis in the ZG, but their effects were less intense than that of 10(-8) M ET-1 and were directly related to their binding potency for the ETA receptor subtype (ET-1 > ET-2 >> ET-3). The selective ETB receptor agonists BQ-3020, IRL-1620, and sarafotoxin-6B were ineffective. The ZG proliferogenic action of 10(-8) M ET-1 was reversed by both the protein kinase C inhibitor Ro31-8220 and the tyrosine kinase inhibitor tyrphostin-23; a complete blockade was obtained at a 10(-6)-M concentration of each inhibitor. In contrast, neither the protein kinase A inhibitor H-89 (10(-5) M) nor the cyclooxygenase and lipoxygenase inhibitors indomethacin and phenidone (10(-5) M) affected ET-1 action. Collectively, our findings indicate that ETs stimulate the proliferation of rat adrenal ZG cells, acting through ETA receptors coupled with protein kinase C- and tyrosine kinase-dependent signaling pathways. The results of the present study are in keeping with the view that in mammals, ZG is the proliferative layer involved in the maintenance of growth of the entire adrenal cortex and with the previous autoradiographic demonstration that ZG is the only adrenocortical layer provided with ETA receptors.

Adrenomedullin enhances cell proliferation and deoxyribonucleic acid synthesis in rat adrenal zona glomerulosa: receptor subtype involved and signaling mechanism.

The effect of adrenomedullin (ADM) on the proliferative activity of the rat adrenal cortex has been investigated in vivo, using an in situ perfusion technique of the intact left gland. ADM and other chemicals were dissolved in the perfusion medium, and the perfusion was continued for 180 min. ADM infusion concentration dependently increased the mitotic index and [3H]thymidine incorporation into DNA in the zona glomerulosa (ZG; the maximal effective concentration was 10(-8) M), but not in inner adrenocortical layers, where basal proliferative activity was negligible. The effect of 10(-8) M ADM was equipotently counteracted by both the calcitonin gene-related peptide (CGRP) type 1 receptor antagonist CGRP-(8-37) and ADM-(22-52). The adenylate cyclase inhibitor SQ-22536 (10(-4) M), the cAMP blocker Rp-cAMP-S (10(-3) M), and the protein kinase A inhibitor H-89 (10(-5) M), although counteracting the ZG proliferogenic action of 10(-9) M ACTH, did not affect the 10(-8) M ADM-elicited increase in ZG DNA synthesis. Similar results were obtained using the phospholipase C inhibitor U-73122 (10(-5) M), the inositol-1,4,5-trisphosphate antagonist D,L-myo-inositol-1,4,5-trisphosphothiate (10(-4) M), and the protein kinase C inhibitor calphostin C (10(-5) M), which, however, significantly inhibited the ZG proliferogenic effect of 10(-9) M angiotensin II. The growth-promoting action of 10(-8) M ADM was not affected by the phospholipase A2 inhibitor AACOCF3 (10(-5) M), the cyclooxygenase (COX) inhibitor indomethacin (10(-5) M), or the mixed COX/lipoxygenase inhibitor phenidone (10(-5) M). In contrast, the ZG proliferogenic effect of 10(-8) M ADM was abolished by either the tyrosine kinase (TK) inhibitor tyrphostin-23 (10(-5) M) or the mitogen-activated protein kinase (MAPK) antagonists PD-98059 and U0216 (10(-4) M). ADM (10(-8) M) stimulated TK and p42/p44 MAPK activity in dispersed ZG, but not ZF, cells, and the effect was reversed by either 10(-6) M CGRP-(8-37) and ADM-(22-52) or preincubation with 10(-5) M tyrphostin-23. Collectively, our findings indicate that 1) ADM stimulates cell proliferation in the rat ZG, through CGRP-(8-37)- and ADM-(22-52)-sensitive receptors, probably of the CGRP1 subtype; and 2) the mitogenic effect of ADM is mediated by activation of the TK-MAPK cascade, without any involvement of the adenylate cyclase/protein kinase A-, phospholipase C/protein kinase C-, and COX- or lipoxygenase-dependent signaling pathways.

Interleukin-1 beta enhances corticosterone secretion by acting directly on the rat adrenal gland.

Interleukin-1 (IL-1), a monokine released by activated monocytes during the acute phase of the inflammatory responses, has been reported to enhance hypophyseal ACTH release mainly by stimulating hypothalamic CRF secretion. We investigated a possible direct effect of IL-1 beta on the adrenal gland of the rat. IL-1 beta was found to dose-dependently (4-8 micrograms/kg) raise corticosterone (B) blood concentration in hypophysectomized rats, without inducing any significant increase in the level of circulating ACTH. IL-1 beta did not affect B production by either isolated rat inner adrenocortical cells or fragments of adrenocortical autotransplants lacking chromaffin cells, but dose-dependently (10(-8)-10(-6) M) enhanced that by adrenal slices including both cortex and medulla. The secretory effect of IL-1 beta (10(-6) M) was completely blocked by both alpha-helical-CRF (10(-6) M) and corticotropin-inhibiting peptide (10(-6) M), two competitive inhibitors which (at these concentrations) were able to annul B response of adrenal slices to CRF (10(-6) M) and ACTH (10(-8) M), respectively. In light of many findings indicating that adrenal medulla contains and releases CRF and numerous POMC-derived peptides (including ACTH), the hypothesis is advanced that the mechanism underlying the direct secretory effect of IL-1 beta on the adrenal gland may involve the activation of an intraadrenal CRF/ACTH system.

Investigations on the morphology and function of adrenocortical tissue regenerated from gland capsular fragments autotransplanted in the musculus gracilis of the rat.

This paper describes the function and morphology of regenerated adrenocortical nodules obtained by implanting, in the musculus gracilis of rats, several (n = 6-7) fragments of the capsular tissue of their excised adrenal glands. Four months after the operation, each bilaterally adrenalectomized rat developed six or seven well encapsulated adrenocortical nodules about 2-3 mm in diameter and always lacking chromaffin cells, and displayed almost complete normalization of basal and stimulated blood levels of corticosterone, but not of aldosterone. In vitro study showed that regenerated nodules were well functioning as far as glucocorticoid production was concerned. Accordingly, electron microscopy and stereology indicated that the majority of the parenchymal cells (independently of their location in the outer subcapsular, middle, or inner portions) closely resembled those of the zonae fasciculata/reticularis of the adrenal gland of age-matched sham-operated rats. By contrast, regenerated nodules evidenced a relative impairment in aldosterone secretion, and this was coupled with the presence of only a few zona glomerulosa-like cells. Such cells were grouped in small islets located near the few connective trabeculae detaching from the capsule, and autoradiography showed that they were the only parenchymal elements of the nodule able to bind [125I]angiotensin-II. The possibility is suggested that the paucity of zona glomerulosa-like cells in regenerated nodules could be ascribed to the absence of zona medullaris, which is currently thought to exert a paracrine control on the growth and secretion of zona glomerulosa in the rat adrenal glands.

Proadrenomedullin N-terminal 20 peptide inhibits aldosterone secretion of human adrenocortical and Conn's adenoma cells: comparison with adrenomedullin effect.

Adrenomedullin (ADM) and proadrenomedullin N-terminal 20 peptide (PAMP) are two vasoactive peptides, which are highly expressed in human adrenal gland. Autoradiography showed the presence of abundant [125I]ADM and [125I]PAMP binding sites in both the outer cortex and medulla of human adrenals. ADM, but not PAMP binding was completely displaced by the specific CGRP1 receptor antagonist CGRP(8-37). ADM and PAMP concentration-dependently inhibited angiotensin-II (ANG-II)-stimulated, but not basal aldosterone secretion of dispersed human adrenocortical cells. PAMP was significantly more potent than ADM (IC50, 0.98 x 10(-11) vs. 3.16 x 10(-9) mol/L). CGRP(8-37) abolished the inhibitory action of ADM, without affecting that of PAMP. Qualitatively analogous findings were obtained using aldosteronoma dispersed cells. However, tumor cells were more sensitive than normal adrenocortical cells (IC50 were 1.32 x 10(12) and 1.51 x 10(-9) mol/L for PAMP and ADM, respectively). Moreover, PAMP was found to also depress basal aldosterone secretion (IC50, 4.27 x 10(-11) mol/L). Neither basal nor ANG-II-stimulated cortisol production by both normal and tumorous adrenocortical cells was altered by ADM or PAMP. Collectively, these findings confirm that ADM (CGRP1) and PAMP receptors are present in the human outer adrenal cortex and allow us to draw the following conclusions: 1) because of its potency, PAMP may a better candidate for being considered a physiological regulator of aldosterone secretion than ADM; and 2) under pathological conditions, both peptides may be capable of reversing overproduction of aldosterone.

Orexin A stimulates cortisol secretion from human adrenocortical cells through activation of the adenylate cyclase-dependent signaling cascade.

Orexins A and B are two hypothalamic peptides that increase food intake and body weight and probably play a role in the sleep regulation. They act through two subtypes of G protein-coupled receptors, called OX1-R and OX2-R. OX1-R selectively binds orexin-A, whereas OX2-R is nonselective for both orexins. Orexins did not affect the in vitro secretion of either catecholamine or aldosterone from human adrenals. Conversely, orexin A, but not orexin B, concentration dependently increased basal cortisol secretion from dispersed adrenocortical cells; the maximal effective concentration was 10(-8) mol/L. Orexin A (10(-8) mol/L) enhanced the cortisol response to maximal effective concentrations (10(-9) mol/L) of angiotensin II and endothelin-1, but only to low concentrations of ACTH (10(-12)/10(-11) mol/L). Orexin A (10(-8) mol/L) increased basal cAMP release by dispersed adrenocortical cells, and the effect was blocked by the adenylate cyclase inhibitor SQ-22536. The cortisol response to 10(-8) mol/L orexin A was unaffected by the ACTH receptor antagonist corticotropin-inhibiting peptide, but was abolished by either SQ-22536 or the protein kinase A inhibitor H-89. RT-PCR demonstrated high levels of OX1-R messenger ribonucleic acid and very low levels of OX2-R messenger ribonucleic acid in human adrenal zona fasciculata-reticularis and adrenal medulla. Collectively, our findings suggest that orexins selectively stimulate glucocorticoid secretion from human adrenocortical cells, acting through OX1-R coupled with the adenylate cyclase-dependent signaling pathway.

Expression and function of vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide, and their receptors in the human adrenal gland.

VIP and pituitary adenylate cyclase-activating polypeptide (PACAP) are two regulatory peptides that possess remarkable amino acid sequence homology and act through common receptors, named PAC(1), VPAC(1), and VPAC(2). PAC(1) receptor is selective for PACAP, whereas VPAC(1) and VPAC(2) receptors bind both VIP and PACAP. We have investigated the expression and function of VIP, PACAP, and their receptors in the zona glomerulosa (ZG), zonae fasciculata and reticularis, and adrenal medulla (AM) of the human adrenal cortex. RT-PCR and RIA detected VIP and PACAP expression exclusively in AM cells. RT-PCR demonstrated the presence of PAC(1) mRNA only in AM and of VPAC(1) and VPAC(2) mRNAs in both ZG and AM cells. VIP and PACAP concentration-dependently increased aldosterone and catecholamine secretion from cultured ZG and AM cells. The catecholamine response to both peptides was higher than the aldosterone response, and the secretagogue action of PACAP was more intense than that of VIP. The aldosterone response of cultured ZG cells to VIP or PACAP was unaffected by the PAC(1) receptor antagonist PACAP-(6-38) (PAC(1)-A), but was significantly decreased by the VPAC(1) receptor antagonist [Ac-His(1),D-Phe(2),Lys(15),Arg(16)]VIP-(3-7),GH-releasing factor-(8-27)-NH(2) (VPAC(1)-A). The catecholamine response of cultured AM cells to VIP was lowered by VPAC(1)-A and unaffected by PAC(1)-A; conversely, the catecholamine response to PACAP was reduced by both PAC(1)-A and VPAC(1)-A. Simultaneous exposure to both antagonists did not abolish the catecholamine response to PACAP. Collectively, our findings allow us to conclude that in human adrenals 1) VIP and PACAP biosynthesis exclusively occurs in AM cells; 2) ZG cells are provided with functional VPAC(1) and VPAC(2) receptors, whose activation by VIP or PACAP elicits a moderate aldosterone response; 3) AM cells possess PAC(1), VPAC(1), and VPAC(2) receptors, whose activation evokes a marked catecholamine response; and 4) the catecholamine response to PACAP is more intense than that to VIP, because it is mediated by all subtypes of VIP/PACAP receptors.

Cholecystokinin (CCK) stimulates aldosterone secretion from human adrenocortical cells via CCK2 receptors coupled to the adenylate cyclase/protein kinase A signaling cascade.

Cholecystokinin (CCK) IS a regulatory peptide that acts via two receptor subtypes, CCK1-R and CCK2-R. RT-PCR demonstrated the expression of both CCK1-R and CCK2-R in the zona glomerulosa (ZG), but not zona fasciculata-reticularis cells of the human adrenal cortex. CCK and the CCK2-R agonist pentagastrin enhanced basal aldosterone secretion from ZG cells without affecting cortisol production from zona fasciculata-reticularis cells. The aldosterone response to CCK and pentagastrin was suppressed by a CCK2-R antagonist, but not by a CCK1-R antagonist. Pentagastrin evoked a sizeable cAMP, but not inositol triphosphate, response from ZG cells, whereas CCK plus CCK2-R antagonist was ineffective. The cAMP response to pentagastrin was abrogated by CCK2-R antagonist or the adenylate cyclase inhibitor SQ-22536, and the aldosterone response was abolished by both SQ-22536 and the protein kinase A inhibitor H-89. Both CCK and pentagastrin increased steroidogenic acute regulatory protein mRNA expression in ZG cells; the effect was abrogated by CCK2-R antagonist. We conclude that CCK exerts secretagogue action on human ZG cells, acting through CCK2-Rs coupled to the adenylate cyclase/protein kinase A signaling cascade, which, in turn, stimulates the expression of steroidogenic acute regulatory protein, the rate-limiting step of steroidogenesis.

Participation of endogenous opiates in regulation of the hypothalamic-pituitary-testicular axis in normal men.

To assess the influence of endogenous opioids on human gonadotropin secretion, integrated concentrations of gonadotropins during 24-h naloxone infusion (2.08 mg/h) were examined in a group of six normal men. Naloxone significantly stimulated LH secretion in all subjects, whereas serum FSH levels were similar during both saline and naloxone infusion. Serum testosterone, dihydrotestosterone, and 17 alpha-hydroxyprogesterone levels increased during the infusion of naloxone, but the delta 4-androstenedione concentration was not modified. The frequency and amplitude of LH secretory episodes were clearly increased when an equal amount of the drug was given and LH concentrations were measured every 15 min for 6 h. The results suggest the endogenous opioids inhibit the hypothalamic-pituitary-gonadal axis in normal men.

Cerebellin enhances in vitro secretory activity of human adrenal gland.

Cerebellin is a 16-amino acid peptide, originally isolated from rat cerebellum, whose presence has been recently demonstrated in the human adrenal glands and especially in medullary chromaffin cells. Cerebellin concentration dependently increased basal catecholamine (norepinephrine and epinephrine) release by human adrenal slices, containing medullary chromaffin tissue, minimal and maximal effective concentrations being 10(-9) and 10(-7) mol/L. Cerebellin (10(-7) mol/L) markedly enhanced cAMP release by adrenal slices, and the protein kinase A inhibitor H-89 (10(-5) mol/L) blocked catecholamine response to cerebellin. Cerebellin did not affect basal steroid secretion of dispersed human adrenocortical cells, but it concentration dependently increased aldosterone and cortisol production by adrenal slices. Again minimal and maximal effective concentrations were 10(-9) and 10(-7) mol/L. Aldosterone and cortisol responses to 10(-7) mol/L cerebellin was suppressed by both the beta-adrenoceptor antagonist l-alprenolol (10(-6) mol/L) and H-89 (10(-5) mol/L). Collectively, the present findings allow us to conclude that 1) cerebellin exerts a sizable secretagogue action on both cortex and medulla of human adrenals; 2) the peptide directly stimulates catecholamine release via the adenylate cyclase/protein kinase A-dependent signaling pathway; and 3) the mechanism underlying the adrenocortical stimulatory effect of cerebellin is indirect and probably involves the release of catecholamines, which in turn, acting in a paracrine manner, enhance steroid-hormone secretion.

Human pheochromocytomas express orexin receptor type 2 gene and display an in vitro secretory response to orexins A and B.

Orexins A and B are hypothalamic peptides, that act through two receptor subtypes, called OX1-R and OX2-R. OX1-R selectively binds orexin A, whereas OX2-R is nonselective for both orexins. High levels of OX1-R mRNA and low levels of OX2-R mRNA have been previously detected in the human adrenal cortex and medulla. Here we demonstrated by RT-PCR the expression of the OX2-R, but not the OX1-R, gene in 10 benign secreting pheochromocytomas. Both orexins A and B stimulated catecholamine secretion from pheochromocytoma slices; the maximal effective concentration was 10(-8) mol/liter. Orexins A and B (10(-8) mol/liter) increased IP3, but not cAMP production, by tumor slices, and the effect was blocked by the PLC inhibitor U-73122. The catecholamine response to 10(-8) mol/liter orexins A and B was abolished by either U-73122 or the PKC antagonist calphostin C and was unaffected by the adenylate cyclase inhibitor SQ-22536 and the PKA inhibitor H-89. Collectively, these findings suggest that orexins stimulate catecholamine secretion from human pheochromocytomas, acting through OX2-R coupled to the PLC-PKC signaling pathway.

Endothelin-1[1-31], acting as an ETA-receptor selective agonist, stimulates proliferation of cultured rat zona glomerulosa cells.

Endothelin-1 (ET-1)[1-31] is a novel hypertensive peptide that mimics many of the vascular effects of the classic 21 amino acid peptide ET-1[1-21]. However, at variance with ET-1[1-21] that enhances aldosterone secretion from cultured rat zona glomerulosa (ZG) cells by acting via ETB receptors, ET-1[1-31] did not elicit such effect. Both ET-1[1-21] and ET-1[1-31] raised the proliferation rate of cultured ZG cells, the maximal effective concentration being 10(-8) M. This effect was blocked by the ETA-receptor antagonist BQ-123 and unaffected by the ETB-receptor antagonist BQ-788. Quantitative autoradiography showed that ET-1[1-21] displaced both [(125)I]PD-151242 binding to ETA receptors and [(125)I]BQ-3020 binding to ETB receptors in both rat ZG and adrenal medulla, while ET-1[1-31] displaced only [(125)I]BQ-3020 binding. The tyrosine kinase (TK) inhibitor tyrphostin-23 and the p42/p44 mitogen-activated protein kinase (MAPK) inhibitor PD-98059 abolished the proliferogenic effect of ET-1[1-31], while the protein kinase-C (PKC) inhibitor calphostin-C significantly reduced it. ET-1[1-31] (10(-8) M) stimulated TK and MAPK activity of dispersed ZG cells, an effect that was blocked by BQ-123. The stimulatory action of ET-1[1-31] on TK activity was annulled by tyrphostin-23, while that on MAPK activity was reduced by calphostin-C and abolished by either tyrphostin-23 and PD-98059. These data suggest that ET-1[1-31] is a selective agonist of the ETA-receptor subtype, and enhances proliferation of cultured rat ZG cells through the PKC- and TK-dependent activation of p42/p44 MAPK cascade.

Paracrine control of steroid hormone secretion by chromaffin cells in the adrenal gland of lower vertebrates.

The adrenal glands of lower vertebrates display a notable intermingling between steroidogenic and chromaffin tissues, which increases from Pisces to Aves. As in mammals, adrenal chromaffin cells contain and release, in addition to catecholamines, serotonin and several peptides, which may affect the secretory activity of steroidogenic cells in a paracrine manner. Stimulatory molecules include serotonin, arginine-vasotocin, tachykinins, vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide and calcitonin gene-related peptide; inhibitory molecules are dopamine, somatotropic hormone-release inhibiting hormone and galanin. Epinephrine and norepinephrine appear to stimulate steroid secretion in Aves and to inhibit it in Pisces, while their action in Amphibia is controversial. Likewise, atrial natriuretic peptide exerts an anti-secretagogue action in Amphibia and a marked secretagogue effect in Pisces and Aves. The effects of opioids (enkephalins and endorphins) have scarcely been investigated and the findings obtained are highly questionable. Compared with the amazing mass of investigations carried out in mammals, studies in lower vertebrates are few, and in large part performed in Amphibia and Aves. It appears that much further work has to be done by comparative endocrinologists to fully clarify the physiological relevance of the functional interactions between chromaffin and steroidogenic cells in the adrenal glands of lower vertebrates.

Effects of 4-aminopyrazolo (3,4-d) pyrimidine on rat hepatocytes: an ultrastructural morphometric study.

The prolonged administration of the hypolipidemic drug 4-aminopyrazolo (3,4-d) pyrimidine (4APP) induced conspicuous morphological changes in rat hepatocytes, which are clearly demonstrated by stereology. We observed a significant decrease in the rough endoplasmic reticulum, and an accumulation of lipid droplets, which was coupled with a comparable rise in the hepatic concentrations of total cholesterol and triglycerides. These changes were interpreted as the expression of the 4APP-provoked impairment of the synthesis of the polypeptide chains of lipoproteins and of the consequent suppression of the assembly of lipid molecules in exportable lipoproteins. We also noted a neto lowering in smooth endoplasmic reticulum and peroxisomes, a decrease which was hypothesized to be the morphological counterpart of a reduced de novo cholesterol synthesis, due to the 4APP-induced rise in the intracellular cholesterol concentration.