Naloxone potentiates ACTH and angiotension II but not potassium stimulated aldosterone secretion, in vitro

The effects of naloxone on basal and ACTH, Angiotensin II (AII) and [K+] o stimulated aldosterone secretion from superfused rat adrenocortical tissue were investigated. A high dose (10(-6) M) of naloxone inhibited while a smaller dose (10(-10) M) potentiated and doses of 10(-8) or 10(-12) M naloxone were without an effect on ACTH stimulated aldosterone secretion. A potentiation of AII stimulated aldosterone secretion was observed beginning 2 hrs after 10(-6) or 10(-10) M naloxone was administered while no effect was observed with 10(-4) M naloxone. No effects of 10(-6), 10(-8), 10(-12) M naloxone were detected on aldosterone secretion stimulated by transiently elevating extracellular potassium. Naloxone from 10(-4) to 10(-12) M did not appear to significantly influence basal steroidogenic activity under these conditions. These findings demonstrate that the "opioid antagonist" naloxone has prominent actions on adrenocortical tissue. Both the specificity and lack of specificity of the action of this agent to influence the activity of the 3 secretagogues suggest that naloxone and possibly a naturally occurring endogenous ligand interacts with one or more membrane receptor distinct from the ACTH receptor. A naturally occurring ligand for this receptor could play a prominent role in the physiological regulation of adrenal steroid secretion.     

Direct effects of heparin on basal and stimulated aldosterone production in rat adrenal glomerulosa cells

Direct effects of heparin (0.1-10 IU/ml) on basal and stimulated aldosterone production have been studied using intact rat adrenal glomerulosa cells. Heparin at any dose did not affect basal aldosterone production when added to the incubation medium. Heparin at a 0.01 IU/ml dose had no effect on aldosterone production maximally stimulated by angiotensin II (AII, 4.8 X 10(-8) M), ACTH (4.3 X 10(-9) M) or potassium (8.0 mM). However, heparin at 0.1 and 0.3 IU/ml doses selectively blocked aldosterone production maximally stimulated by AII but not by ACTH or potassium, while the compound at 1 and 10 IU/ml doses inhibited aldosterone production maximally stimulated by these three stimuli. In addition, the inhibitory effect of 0.3 IU/ml heparin occurred as early as 30 min after incubation with heparin. These data suggest that heparin at 0.1 and 0.3 IU/ml doses acts directly on adrenal zona glomerulosa to selectively block the stimulatory action of AII, while the compound at 1 and 10 IU/ml doses inhibits all the stimulatory actions of AII, ACTH and potassium.     

Compared effects of ACTH, angiotensin II and POMC peptides on isolated human adrenal cells

Human adrenocortical tissue obtained, on eight occasions, at the time of nephrectomy for renal carcinoma (outside the adrenal pole) was treated by collagenase to dissociate the cells. These were hen submitted to a short, 2-h, incubation with the N-terminal fragment (16 K) of POMC, its derivative, gamma 3-MSH, beta-lipotropin and beta-endorphin, in parallel with ACTH 1-24 (Synacthen Ciba) and angiotensin II (AII, Hypertensin Ciba). Under the influence of ACTH (10(-10) M), and AII (10(-10) M), basal glucocorticoid output, including more than 80% cortisol, was increased by factors of 3 +/- 0.51 (SEM) and 1.35 +/- 0.12 (SEM), respectively. The corresponding aldosterone responses were 1.60 +/- 0.13 for ACTH and 1.38 +/- 0.09 for AII. With the exception of gamma 3-MSH, the POMC peptides under study had no steroidogenic effect. gamma 3-MSH (10(-9) M) and AII (10(-10) M) stimulated aldosterone production to approximately similar levels of, respectively, 1.23 +/- 0.05 and 1.38 +/- 0.09 times the basal production. In contrast to AII however, gamma 3-MSH showed no apparent effect on glucocorticoid output. Steroidogenic response to ACTH was potentiated by gamma 3-MSH at a concentration of 10(-10) M which, when used alone, proved ineffective. This potentiating effect was pronounced for the aldosterone response, whereas the glucocorticoid production was hardly affected. This action ceased to be visible when the cells reached maximal stimulation by ACTH. These findings suggest that gamma 3-MSH--a portion of the 16 K fragment--may have a possible role in aldosterone secretion.     

Role of calcium in stimulus-secretion coupling on isolated frog interrenal gland

The influence of extracellular calcium concentration on the steroidogenic response to ACTH and to the angiotensin II analogue [Sar1-Val5]AII has been studied in the frog, using a perfusion system technique. The release of corticosterone and aldosterone in the effluent medium was measured by specific radioimmunoassays. In calcium-free medium the stimulatory effect of ACTH (10(-9) M) was completely abolished whereas the response to dbcAMP (5 mM) was unchanged indicating that the role of calcium takes place before the formation of cAMP. Conversely, in the absence of calcium, angiotensin II (10(-7) M) was still able to stimulate corticosterone and aldosterone production. Addition of Co2+ (4 mM), a calcium antagonist, to the perfusion medium, inhibited partially the response of adrenal tissue to ACTH, dbcAMP and angiotensin. The voltage-dependent calcium channel blocker verapamil (10(-6) induced a dose-related inhibition of the corticotropic effect of ACTH. At the higher dose (10(-4) M), verapamil totally inhibited the stimulation of corticosterone and aldosterone production induced by ACTH. By contrast, at the same dose it did not alter the stimulatory effect of forskolin (2.4 X 10(-7)M) on corticosterone output, but significantly diminished forskolin-induced aldosterone response. Similarly, angiotensin-stimulated corticosterone production was slightly inhibited by 10(-4) M verapamil, whereas aldosterone response to angiotensin was totally abolished, indicating that verapamil may act intracellularly to block the conversion of corticosterone to aldosterone. Taken together, these results indicate that, in amphibians extracellular calcium is essential for the action of ACTH, either for the binding of the hormone to its receptor and/or for the transduction of the information from hormone-receptor complex to the adenylate cyclase moiety and that the mechanism of action of angiotensin does not involve calcium uptake by adrenocortical cells.     

The effects of extracellular K+ and angiotensin II on cytosolic Ca++ and steroidogenesis in adrenal glomerulosa cells

We evaluated changes in cytosolic calcium concentration (Ca++) and steroidogenesis in rat adrenal glomerulosa cells (GC) stimulated with potassium (K+) or angiotensin II (AII). Cytosolic Ca++ concentration was determined using the Ca++-sensitive, fluorescent dye QUIN 2. Raising extracellular K+ increased cytosolic Ca++ from 267 +/- 23 nM at 3.7 mM K+ to a maximum of 377 +/- 40 nM at 8.7 mM K+ (p less than 0.01, N = 23). AII also increased cytosolic Ca++ from 238 +/- 20 nM to a maximum of 427 +/- 42 nM at 10(-7) M (p less than 0.01, N = 16). In parallel studies, K+ and AII stimulated aldosterone secretion from QUIN 2-loaded GC at concentrations similar to those which raised cytosolic Ca++. QUIN 2-loaded cells were as responsive steroidogenically as unloaded cells and showed trypan blue exclusion of 98% suggesting that QUIN 2 did not compromise cellular viability. These results provide direct support for a role of cytosolic Ca++ as a second messenger during stimulation of aldosterone secretion by both K+ and AII.     

Aldosterone and corticosterone stimulation by ACTH in isolated rat adrenal glomerulosa cells: interaction with vasopressin

An interaction between ACTH and vasopressin on steroidogenesis was observed in isolated rat adrenal zona glomerulosa cell preparations. 1. The presence of 10(-11) M vasopressin further increased by 52% the output of aldosterone produced by 10(-12) M ACTH on those cells. 2. At a pharmacological concentration of ACTH (10(-7) M), the aldosterone output was increased 5 fold while the addition of 10(-12) M or 10(-8) M vasopressin decreased it by 17% and 48% respectively. 3. Vasopressin also produced a dose-dependent inhibition of the stimulatory effect of ACTH on the output of corticosterone. 4. We have thus shown for the first time, that vasopressin acts directly on adrenal zona glomerulosa cell preparations to modify the aldosterone output by modulating the action of ACTH. It is postulated that, in addition to other known aldosterone regulating factors, ACTH and vasopressin might synergistically act to regulate the secretion of aldosterone in vivo.     

Local generation of angiotensin II as a mechanism of aldosterone secretion in rat adrenal capsules.

Angiotensin-converting enzyme (ACE) is found in the adrenal gland, but the role of adrenal ACE in the formation of angiotensin II (AII) and subsequent stimulation of aldosterone is unclear. We examined the effect of adrenal ACE activity on aldosterone secretion by superfusing rat adrenal capsules with angiotensin I (AI) in the presence and absence of the ACE inhibitor, lisinopril. Angiotensin I (10 microM) stimulated aldosterone secretion from 914 +/- 41 to 1465 +/- 118 pg/min/capsule (P less than 0.05). Simultaneous superfusion of AI plus lisinopril (100 microM) inhibited the stimulation of aldosterone by 73% (P less than 0.05). Perfusion of the capsules with angiotensin II (1 microM) stimulated aldosterone from 893 +/- 180 to 1466 +/- 181 pg/min/capsule (P less than 0.01). In contrast, simultaneous superfusion of AII plus lisinopril (100 microM) did not inhibit the AII stimulation of aldosterone. The failure of lisinopril to inhibit AII stimulation of aldosterone argues against a toxic or nonspecific action of lisinopril. The inhibition of AI stimulation of aldosterone release by lisinopril is mostly due to lisinopril inhibition of ACE and resulting decreased conversion of AI to AII. These results demonstrate that adrenal ACE may generate AII from AI in the adrenal gland, and this locally produce AII stimulates aldosterone.     

Mineralocorticoid-induced increase in beta-adrenergic receptors of cultured rat arterial smooth muscle cells

We have investigated the effect of mineralocorticoids on beta-adrenergic receptors in cultured arterial smooth muscle cells. Mineralocorticoid (aldosterone) treatment resulted in a significant increase in beta-adrenergic receptors measured by [3H]dihydroalprenolol (DHA) binding. This effect required at least 20 hours of incubation with aldosterone and was completely blocked by cycloheximide (10 micrograms/ml), indicating protein synthesis was required for this response. Aldosterone at the concentration range of 10(-8)-10(-6) M increased [3H]DHA binding, but was ineffective at 10(-9) M. Scatchard analysis of [3H]DHA binding revealed that the observed significant increase in binding was due to an increased number of binding sites (P less than 0.05), and that the affinity was unchanged. The aldosterone (1 x 10(-8) M) effect was completely blocked by the combination of RU 38486 (10(-6) M) and spironolactone (10(-7) M), but not by the glucocorticoid antagonist RU 38486 alone. While basal c-AMP levels were not changed by aldosterone (10(-6) M) treatment, the isoproterenol (10(-6) M) stimulated level of c-AMP was significantly higher in cells treated with aldosterone (P less than 0.05). We conclude that aldosterone, acting through the mineralocorticoid receptor, has a direct effect on arterial smooth muscle cells mediated through modulation of beta-adrenergic receptors of these cells.     

Calcium-dependence of serotonin-mediated aldosterone secretion and differential effects of calcium antagonists

The requirement for calcium in the serotonin-mediated aldosterone secretion was investigated using rat adrenal capsular cells. In the calcium-free medium both basal as well as serotonin-stimulated aldosterone secretion (at concentrations of 10(-7) M and 10(-8) M of serotonin) were significantly impaired. The effects of calcium-channel blockers were then examined. Verapamil (10(-5) M and 10(-6) M markedly inhibited basal and serotonin-evoked aldosterone secretion. In equimolar concentrations nifedipine had much less effect and diltiazem produced no apparent attenuation of either basal or serotonin-stimulated aldosterone secretion. These results indicate the calcium-dependence of serotonin-induced aldosterone secretion. The variable effects of the calcium-channel blockers suggest different or multiple mechanisms of action of these agents.     

Effect of angiotensin II and losartan on the phagocytic activity of peritoneal macrophages from Balb/C mice

Angiotensin II (AII), a product of rennin-angiotensin system, exerts an important role on the function of immune system cells. In this study, the effect of AII on the phagocytic activity of mouse peritoneal macrophages was assessed. Mice peritoneal macrophages were cultured for 48 h and the influence of different concentrations of AII (10(-14) to 10(-7) M) and/or losartan, 10(-16) to 10(-6) M), an AT1 angiotensin receptor antagonist, on phagocytic activity and superoxide anion production was determined. Dimethylthiazoldiphenyltetrazolium bromide reduction and the nucleic acid content were used to assess the cvtotoxicity of losartan. A stimulatory effect on phagocytic activity (P < 0.05) was observed with 10(-13) M and 10(-12 M) AII concentrations. The addition of losartan (up to10(-14) M) to the cell cultures blocked (P < 0.001) the phagocytosis indicating the involvement of AT1 receptors. In contrast, superoxide anion production was not affected by AII or losartan. The existence of AT1 and AT2 receptors in peritoneal macrophages was demonstrated by immunofluorescence microscopy. These results support the hypothesis that AII receptors can modulate murine macrophage activity and phagocytosis, and suggest that AII may have a therapeutic role as an immunomodulatory agent in modifying the host resistance to infection.     

Evidence for a tonic inhibitory role of nifedipine-sensitive calcium channels in aldosterone biosynthesis.

This study investigated the effects of the calcium channel blockers nifedipine (a dihydropyridine) and verapamil (a papaverine derivative), on aldosterone production utilizing isolation of the early and late phases of aldosterone biosynthesis. Pregnenolone production (the early phase of aldosterone biosynthesis) was assessed in trilostane-treated bovine glomerulosa cells, used to inhibit the conversion of pregnenolone onwards to aldosterone. Conversion of exogenous corticosterone to aldosterone, an index of late phase activity, was assessed using aminoglutethimide to inhibit endogenous aldosterone production. Low concentrations of nifedipine, 10(-11)-10(-9) M, stimulated basal total aldosterone biosynthesis by enhancing the late phase although the early phase was inhibited. In the presence of 12 mM potassium (K+), which is less effective in stimulating aldosterone production than lower K+ concentrations, aldosterone production was enhanced by nifedipine, 10(-8) M, by an effect on the late phase. At K+ 6 and 8 mM, nifedipine, 10(-4) M, inhibited the early phase. Nifedipine 10(-5) inhibited angiotensin II (AII)-stimulated total aldosterone biosynthesis by independent effects on the early and late phases. Verapamil, 10(-4) M, inhibited total and early phase aldosterone production at K+, 4 mM and inhibited both phases at K+, 8 mM, stimulation was not observed using verapamil. Verapamil, 10(-4) M, also inhibited AII-stimulated aldosterone production. Basal and AII-stimulated pregnenolone production were inhibited by verapamil, 10(-4) M (basal) and 10(-6) M (AII-stimulated). These studies using nifedipine have revealed subtle calcium-dependent mechanisms involved in the tonic inhibition of activity in the late phase of aldosterone biosynthesis and the reversal of the inhibitory effect of high K+ concentrations also on the late phase. In addition, the data reported indicate that both AII and K+ independently enhance activity in the early and late phases of aldosterone production by calcium-dependent mechanisms.     

Greater importance of Ca(2+)-calmodulin in maintenance of ang II- and K(+)-mediated aldosterone secretion: lesser role of protein kinase C.

In this study we have investigated various components of the stimulus-secretion coupling process leading to aldosterone secretion from the calf adrenal glomerulosa cells as evoked by angiotensin II (AII) and potassium (K+). The roles of Ca2+, calmodulin and protein kinase C in the sustained phase rather than initiation of aldosterone secretion were of special interest. Our investigations revealed that the reduction of extracellular Ca2+ by EGTA or interruption of Ca2+ influx by nitrendipine at various time points after stimulation with either AII or K+ markedly compromised aldosterone secretion. Calmodulin inhibitors, calmidazolium and W-7 reduced aldosterone secretion profoundly. Agonists of protein kinase C, phorbol ester or diacylglycerol analogues failed to stimulate aldosterone secretion while the protein kinase C inhibitor, H-7, only partially inhibited aldosterone secretion at a concentration which completely inhibited protein kinase C activity. Calmodulin inhibitors produced significantly greater inhibition of aldosterone secretion than inhibitors of protein kinase C.     

Role of adrenal renin-angiotensin system in the control of aldosterone secretion in sodium-restricted rats

This study examined the effect of the pharmacological manipulation of adrenal renin-angiotensin system (RAS) on aldosterone secretion from in situ perfused adrenals of rats kept on a normal diet and sodium restricted for 14 days. Neither the angiotensin-converting enzyme inhibitor captopril nor the nonselective angiotensin II receptor antagonist saralasin and the AT(1) receptor-selective antagonist losartan affected basal aldosterone output in normally fed rats. In contrast, they concentration dependently decreased aldosterone secretion in sodium-restricted animals, with maximal effective concentration ranging from 10(-7) to 10(-6) M. Captopril (10(-6) M), saralasin (10(-6) M), and losartan (10(-7) M) counteracted aldosterone response to 10 mM K(+) in sodium-restricted rats but not in normally fed animals. Collectively, these findings provide evidence that adrenal RAS plays a role in the regulation of aldosterone secretion, but only under conditions of prolonged stimulation of zona glomerulosa probably leading to overexpression of adrenal RAS.     

Okadaic acid inhibits angiotensin II, adrenocorticotropin and potassium-dependent aldosterone secretion

The present study was designed to assess the effect of okadaic acid (OA), a protein phosphatase inhibitor, on aldosterone secretion in response to angiotensin II (AII), adrenocorticotropin (ACTH) and rises in external potassium concentration (K+). AII (10nM) caused a 20-fold increase in aldosterone production and OA reduced this response by 45%. ACTH (10nM) caused an 8.6-fold increase in aldosterone secretion and OA reduced this by 83%. Increasing K+ concentration from 3 to 12mM caused a 13-fold increase in aldosterone production, which OA inhibited by 36%. These results suggest that protein phosphatases participate in the control of adrenal steroid production, even though ACTH, AII and K+ act via different intracellular messenger systems.     

Effects of prolactin on aldosterone secretion in rat zona glomerulosa cells

Acute effects and action mechanisms of prolactin (PRL) on aldosterone secretion in zona glomerulosa (ZG) cells were investigated in ovariectomized rats. Administration of ovine PRL (oPRL) increased aldosterone secretion in a dose-dependent manner. Incubation of [3H]-pregnenolone combined with oPRL increased the production of [3H]-aldosterone and [3H]-deoxycorticosterone but decreased the accumulation of [3H]-corticosterone. Administration of oPRL produced a marked increase of adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in ZG cells. The stimulatory effect of oPRL on aldosterone secretion was attenuated by the administration of angiotensin II (Ang II) and high potassium. The Ca2+ chelator, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, 10(-2) M), inhibited the basal release of aldosterone and completely suppressed the stimulatory effects of oPRL on aldosterone secretion. The stimulatory effects of oPRL on aldosterone secretion were attenuated by the administration of nifedipine (L-type Ca2+ channel blocker) and tetrandrine (T-type Ca2+ channel blocker). These data suggest that the increase of aldosterone secretion by oPRL is in part due to (1) the increase of cAMP production, (2) the activation of both L- and T-type Ca2+ channels, and (3) the activation of 21-hydroxylase and aldosterone synthase in rat ZG cells.     

Evidence for a paracrine role of adrenomedullin in the physiological resetting of aldosterone secretion by rat adrenal zona glomerulosa

Adrenomedullin (ADM) has been recently found to directly inhibit agonist-stimulated aldosterone secretion by dispersed zona glomerulosa (ZG) cells and to stimulate basal catecholamine release by adrenomedullary fragments. In light of the fact that catecholamines enhance aldosterone secretion acting in a paracrine manner, we have investigated whether these two effects of ADM may interact when the integrity of the adrenal gland is preserved. ADM increased basal aldosterone output by adrenal slices containing a core of adrenal medulla, and the effect was blocked by the beta-adrenoceptor antagonist l-alprenolol. In contrast, ADM evoked a moderate inhibition of K(+)-stimulated aldosterone production, and the blockade was complete in the presence of l-alprenolol. The in vivo bolus injection of ADM did not affect plasma aldosterone concentration (PAC) in rats under basal conditions. Conversely, when rat ZG secretory function was enhanced (by sodium restriction or infusion with angiotensin-II [ANG-II]) or depressed (by sodium loading or infusion with the angiotensin-converting enzyme inhibitor captopril), ADM evoked a sizeable decrease or increase in PAC, respectively. The prolonged infusion with the ADM receptor antagonist ADM(22-52) caused a further enhancement of PAC in sodium-restricted or ANG-II-treated rats, and a further moderate decrease of it in sodium-loaded or captopril-administered animals. RIA showed that ADM plasma concentration did not exceed a concentration of 10(-11) M in any group of animals. Under basal conditions, ADM adrenal content was 1.2-2.0 pmol/g, which may give rise to local concentrations higher than 10(-8) M (i.e. well above the minimal effective ones in vitro). ADM adrenal concentration was markedly increased (from two-fold to three-fold) by both ZG stimulatory and suppressive treatments. Collectively, our findings suggest that in vivo 1) ADM, in addition to directly inhibit aldosterone secretion, may enhance it indirectly by eliciting catecholamine release, the two actions annulling each other under basal conditions; 2) under conditions leading to enhanced aldosterone secretion, the direct inhibitory effect of ADM prevails over the indirect stimulatory one, and the reverse occurs when aldosterone secretion is decreased; and 3) the modulatory action of ADM on the aldosterone secretion has a physiological relevance, endogenous ADM being locally synthesized in adrenals.     

Gastric inhibitory polypeptide stimulates glucocorticoid secretion in rats, acting through specific receptors coupled with the adenylate cyclase-dependent signaling pathway.

Gastric inhibitory polypeptide (GIP) is a 42-amino acid peptide, belonging to the VIP-secretin-glucagon superfamily, some members of this group are able to regulate adrenocortical function. GIP-receptor mRNA has been detected in the rat adrenal cortex, but investigations on the effect of GIP on steroid-hormone secretion in this species are lacking. Hence, we have investigated the distribution of GIP binding sites in the rat adrenal gland and the effect of their activation in vivo and in vitro. Autoradiography evidenced abundant [125I]GIP binding sites exclusively in the inner adrenocortical layers, and the computer-assisted densitometric analysis of autoradiograms demonstrated that binding was displaced by cold GIP, but not by either ACTH or the selective ACTH-receptor antagonist corticotropin-inhibiting peptide (CIP). The intraperitoneal (IP) injection of GIP dose-dependently raised corticosterone, but not aldosterone plasma concentration: the maximal effective dose (10 nmol/rat) elicited a twofold increase. GIP did not affect aldosterone and cyclic-AMP release by dispersed zona glomerulosa cells. In contrast, GIP enhanced basal corticosterone secretion and cyclic-AMP release by dispersed inner adrenocortical cells in a concentration-dependent manner, and the maximal effective concentration (10(-7) M) evoked 1.5- and 2.4-fold rises in corticosterone and cyclic-AMP production, respectively. GIP (10(-7) M) did not display any additive or potentiating effect on corticosterone and cyclic-AMP responses to submaximal or maximal effective concentrations of ACTH. The corticosterone secretagogue action of 10(-7) M GIP was abolished by the protein kinase A (PKA) inhibitor H-89 (10(-5)M), and unaffected by CIP (10(-6)M). Collectively, these findings indicate that GIP exerts a moderate but statistically significant stimulatory effect on basal glucocorticoid secretion in rats, acting through specific receptors coupled with the adenylate cyclase/PKA-dependent signaling pathway.     

Angiotensin II increases cytosolic calcium and stimulates catecholamine release in cultured bovine adrenomedullary cells

In bovine adrenomedullary cells in primary culture, angiotensin II (AII) elicited virtually immediate, dose-related increments in cytosolic calcium [( Ca++]i) measured by the Quin 2 technique and stimulated approximately proportional secretion of norepinephrine, epinephrine, and dopamine measured by liquid chromatography with electrochemical detection. Peak responses of [Ca++]i to AII were similar to peak responses to nicotine or KCl. Pre-treatment with verapamil or washing the cells in calcium-free medium attenuated the stimulatory effect of AII on [Ca++]i. Pre-treatment with nicotine, which temporarily inactivates cholinergic receptor-activated calcium channels, did not affect [Ca++]i responses to AII. The results indicate functional effects of AII on cultured chromaffin cells. The mechanism of cellular activation by AII appears to include increases in [Ca++]i due to opening of membrane calcium channels which may be unrelated to cholinergic receptor-operated calcium channels.     

Studies on the alpha-adrenergic activation of hepatic glucose output. I. Studies on the alpha-adrenergic activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase in isolated rat liver parenchymal cells.

Epinephrine and the alpha-adrenergic agonist phenylephrine activated phosphorylase, glycogenolysis, and gluconeogenesis from lactate in a dose-dependent manner in isolated rat liver parenchymal cells. The half-maximally active dose of epinephrine was 10-7 M and of phenylephrine was 10(-6) M. These effects were blocked by alpha-adrenergic antagonists including phenoxybenzamine, but were largely unaffected by beta-adrenergic antagonists including propranolol. Epinephrine caused a transient 2-fold elevation of adenosine 3':5'-monophosphate (cAMP) which was abolished by propranolol and other beta blockers, but was unaffected by phenoxybenzamine and other alpha blockers. Phenoxybenzamine and propranolol were shown to be specific for their respective adrenergic receptors and to not affect the actions of glucagon or exogenous cAMP. Neither epinephrine (10-7 M), phenylephrine (10-5 M), nor glucagon (10-7 M) inactivated glycogen synthase in liver cells from fed rats. When the glycogen synthase activity ratio (-glucose 6-phosphate/+ glucose 6-phosphate) was increased from 0.09 to 0.66 by preincubation of such cells with 40 mM glucose, these agents substantially inactivated the enzyme. Incubation of hepatocytes from fed rats resulted in glycogen depletion which was correlated with an increase in the glycogen synthase activity ratio and a decrease in phosphorylase alpha activity. In hepatocytes from fasted animals, the glycogen synthase activity ratio was 0.32 +/- 0.03, and epinephrine, glucagon, and phenylephrine were able to lower this significantly. The effects of epinephrine and phenylephrine on the enzyme were blocked by phenoxybenzamine, but were largely unaffected by propranolol. Maximal phosphorylase activation in hepatocytes from fasted rats incubated with 10(-5) M phenylephrine preceded the maximal inactivation of glycogen synthase. Addition of glucose rapidly reduced, in a dose-dependent manner, both basal and phenylephrine-elevated phosphorylase alpha activity in hepatocytes prepared from fasted rats. Glucose also increased the glycogen synthase activity ratio, but this effect lagged behind the change in phosphorylase. Phenylephrine (10-5 M) and glucagon (5 x 10(-10) M) decreased by one-half the fall in phosphoryalse alpha activity seen with 10 mM glucose and markedly suppressed the elevation of glycogen synthase activity. The following conclusions are drawn from these findings. (a) The effects of epinephrine and phenylephrine on carbohydrate metabolism in rat liver parenchymal cells are mediated predominantly by alpha-adrenergic receptors. (b) Stimulation of these receptors by epinephrine or phenylephrine results in activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase by mechanisms not involving an increase in cellular cAMP. (c) Activation of beta-adrenergic receptors by epinephrine leads to the accumulation of cAMP, but this is associated with minimal activation of phosphorylase or inactivation of glycogen synthase...     

The effects of hormones on cyclic adenosine 3':5'-monophosphate accumulation in transitional epithelium of the urinary bladder.

Transitional epithelium lining rabbit urinary bladders was isolated and studied in vitro. The homogeneity of the isolated epithelium was demonstrated by light and electron microscopical monitoring as well as cell culture studies. Transitional epithelium responded to epinephrine and prostaglandin E1 (PGE1) in the presence of 2mM 1-methyl, 3-isobutylxanthine (MIX) with increases in intracellular levels of cyclic adenosine 3':5'-monophosphate (cyclic AMP). Corticotropin, aldosterone, insulin, parathyroid hormone and vasopressin were slightly but significantly stimulatory under similar conditions. Glucagon and oxytocin were not stimulatory at the concentrations tested. The effects of epinephrine and PGE1 were potentiated by 2mM MIX 20-fold or greater. The cells were slightly more sensitive to PGE1 then to epinephrine. The prostaglandin produced a noticeable response at about 10nM, while effects of epinephrine were discernible at 0.1muM. Maximal responses to both effectors were seen at about 10muM. The action of 10muM epinephrine, but not 10muM PGE1, was completely abolished by 0.1mM propranolol. Responses to combinations of epinephrine and PGE1 were additive. Cyclic AMP accumulated in the incubation medium of transitional epithelial cells exposed to epinephrine, PGE1, MIX, or combinations of the agonists. The appearance of cyclic AMP in the medium was slow compared to the rate of intracellular accumulation, but reached significant levels following prolonged stimulation.