Evidence that the Peripheral-Type Benzodiazepine Receptor Ligand Ro 5–4864 Inhibits β-Endorphin Release from AtT-20 Cells by Blockade of Voltage-Dependent Calcium Channels

The demonstrations that Ro 5-4864, a ligand selective for the peripheral-type benzodiazepine (BZD) binding site, inhibited cellular differentiation and proliferation and that occupancy of the peripheral-type BZD binding site likely mediated the observed BZD effects on diverse endocrine tissues suggested that Ro 5-4864 disrupted a common cellular regulatory event. Using a well-characterized anterior pituitary-derived tumor cell line (AtT-20 cells), which synthesizes and secretes adrenocorticotropic hormone (ACTH), beta-lipotropin hormone (beta-LPH), and beta-endorphin (BE), we have investigated the molecular mechanism of action of Ro 5-4864's capacity to alter BE secretion. Ro 5-4864 inhibits basal and induced BE release from AtT-20 cells, through a cyclic AMP-independent mechanism. Ro 5-4864 completely blocked the corticotropin-releasing hormone and forskolin-induced release of BE without altering the concomitant production of cyclic AMP. The addition to AtT-20 cells of CGP 28392, a dihydropyridine that has been demonstrated in other systems to specifically activate voltage-dependent Ca2+ channels, resulted in a cyclic AMP-independent, dose-related increase in BE secretion. This CGP-induced BE release was blocked by increasing concentrations of Ro 5-4864. In contrast to the capacity of Ro 5-4864 to block CGP-induced BE release, Ro 5-4864 lacked the capacity to block enhanced BE secretion due to the calcium ionophore A23187, which increases intracellular Ca2+ levels independent of the voltage-dependent Ca2+ channels. Our findings suggest that Ro 5-4864 inhibits BE secretion from AtT-20 cells through a blockade of the voltage-dependent membrane Ca2+ channels and this mechanism of action may be responsible for Ro 5-4864's diverse effects observed on other cell types.     

Evidence That Receptor-Linked G Protein Inhibits Exocytosis by a Post-Second-Messenger Mechanism in AtT-20 Cells

In AtT-20 cells somatostatin inhibits the secretion of adrenocorticotropic hormone (ACTH) through the activation of GTP binding proteins (G proteins) linked to second messengers such as calcium and cyclic AMP (cAMP). Recently, it has been proposed that there may be G proteins that regulate directly the exocytotic machinery. We have investigated whether somatostatin could inhibit secretion at a step distal to second messengers through a GTP binding protein. For these studies two experimental paradigms were used: (1) intact cells stimulated by calcium ionophores and (2) digitonin-permeabilized cells exposed to buffers of increasing Ca2+ concentrations. Somatostatin inhibited by 70% the ACTH release caused by the calcium ionophore ionomycin without modifying the ionophore-induced elevation in cytosolic [Ca2+]. This effect was cAMP independent because (1) it was observed in the presence of high concentrations of membrane-permeant cAMP analogues, and (2) it was not accompanied by a change in cAMP levels. The effect was also independent of the levels of activators of protein kinase C because it could be produced in the presence of high concentrations of phorbol esters. The action of somatostatin was prevented by pertussis toxin. In digitonin-permeabilized AtT-20 cells somatostatin inhibited release induced by calcium buffers in a GTP-dependent manner. These two observations indicate the involvement of a G protein. It is proposed that a G protein coupled to somatostatin receptors inhibits the intracellular machinery of secretion at a step distal to second messengers, perhaps at the exocytotic site.     

Interactions Among Lithium, Calcium, Diacylglycerides, and Phorbol Esters in the Regulation of Adrenocorticotropin Hormone Release from AtT-20 Cells

Interactions among lithium, calcium, and phorbol esters in the regulation of adrenocorticotropin hormone (ACTH) release were examined in a tumor cell line (AtT-20) of the anterior pituitary. Lithium, which blocks the phosphatase that converts inositol phosphates (IPs) to inositol, increases the levels of IPs in these cells and stimulates ACTH release. This ion potentiates the ability of calcium, an activator of phospholipase C, to raise levels of IPs in these cells and to stimulate ACTH secretion. Pretreatment of AtT-20 cells with calcium specifically abolishes the ACTH release response to lithium or calcium, a result suggesting that these secretagogues may act through a common mechanism to induce hormone secretion. Prior exposure of AtT-20 cells to either lithium or calcium also attenuates the ACTH release induced by phorbol ester, an activator of protein kinase C. To examine the link among lithium, calcium, phosphatidylinositol (PI) turnover, and phorbol ester-evoked ACTH secretion, AtT-20 cells were treated with 1-oleoyl-2-acetoyl-sn-3-glycerol (OAG), an analogue of the diacylgylcerols that are formed by phospholipase C during PI metabolism and that also activate protein kinase C. OAG itself does not alter ACTH release or the levels of IPs in AtT-20 cells. Pretreatment of AtT-20 cells with OAG, however, selectively blocks the ACTH release response to lithium, calcium, or phorbol ester. Furthermore, such pretreatment reduced the ability of lithium to increase levels of IPs. The results suggest that one mechanism of action of lithium is to potentiate selectively an action of calcium, possibly the stimulation of phospholipase C activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multireceptor-induced release of adrenocorticotropin from anterior pituitary tumor cells

Corticotropin releasing factor (CRF), (?) isoproterenol and vasoactive intestinal peptide (VIP) induced cyclic AMP synthesis and the release of immunoreactive adrenocorticotropin hormone (ACTH) from clonal mouse AtT-20 pituitary tumor cells. CRF and (?) isoproterenol together produced an additive increase in cyclic AMP formation but a less than additive effect on ACTH secretion. VIP with either CRF or (?) isoproterenol produced additive increases in both cyclic AMP and ACTH secretion. Forskolin, an activator of adenylate cyclase stimulated the release of ACTH suggesting that cyclic AMP mediates some of the effects of hormone-receptor activation on ACTH secretion. The action of all three receptor agonists and forskolin on ACTH release was blocked by dexamethasone treatment. The release process, but not the changes in cyclic AMP synthesis was calcium dependent with all these hormones. The calcium ionophore, A-23187, increased ACTH secretion without altering intracellular cyclic AMP content. Its effect on secretion was not additive with either CRF, (?) isoproterenol or VIP. These observations indicate that hormone-induced regulation of ACTH secretion converges at varying intracellular locations.     

Correlation between electrical activity and ACTH/beta-endorphin secretion in mouse pituitary tumor cells

The electrical and secretory activities of mouse pituitary tumor cells (AtT-20/D-16v), which contain and release the ACTH/beta-endorphin family of peptides, were studied by means of intracellular recordings and radioimmunoassays. Injection of depolarizing current pulses evoked action potentials in all cells and the majority (82%) displayed spontaneous action potential activity. Action potentials were found to be calcium-dependent. Barium increased membrane resistance, action potential amplitude and duration, and release of ACTH and beta- endorphin immunoactivity. Isoproterenol increased both action potential frequency and hormone secretion. Raising the external calcium concentration increased the frequency and amplitude of the action potentials and stimulated secretion of ACTH and beta-endorphin immunoactivity. Thus, stimulation of secretory activity in AtT-20 cells was closely correlated with increased electrical activity. However, a complete blockade of action potential activity had no effect on basal hormone secretion in these cells. These results suggest that the mechanisms underlying stimulated hormone secretion are different from those responsible for basal secretory activity. It is proposed that the increased influx of calcium due to the increased action potential frequency initiates the stimulated release of hormone from these cells.     

Control of PTH secretion is mediated through calcium channels and is blocked by pertussis toxin treatment of parathyroid cells

Parathyroid hormone secretion is negatively regulated by calcium. We utilized calcium channel agents: +202-791, a calcium channel agonist and -202-791, a calcium channel antagonist, to evaluate the role of calcium channels in PTH secretion. +202-791 inhibited PTH release from bovine parathyroid cells and the antagonist stimulated release. Incubation with pertussis toxin which ADP-ribosylates and inactivates a guanine nucleotide regulatory protein (G-protein) releases the inhibition by the calcium channel agonist. These findings indicate that a G-protein is interposed between the calcium channel and a putative intracellular site controlling PTH secretion.     

Melatonin action in neonatal gonadotrophs

Neonatal pituitary cells express MT1 and MT2 subtype of melatonin receptors that are coupled to pertussis toxin-sensitive G proteins. Their activation by melatonin leads to a decrease in cAMP production and activity of protein kinase A, and attenuation of gonadotropin-releasing hormone (GnRH)-induced gonadotropin secretion. Single cell calcium and electrophysiological recordings have revealed that a reduction in gonadotropin release results from melatonin-induced inhibition of GnRH-stimulated calcium signaling. Melatonin inhibits both calcium influx through voltage-dependent calcium channels and calcium mobilization from intracellular stores. Inhibition of calcium influx, probably in a cAMP/protein kinase C-dependent manner, and the accompanying calcium-induced calcium release from ryanodine-sensitive intracellular pools by melatonin results in a delay of GnRH-induced calcium signaling. Melatonin-induced attenuation of GnRH-induced and inositol (1,4,5)-trisphosphate-mediated calcium release from intracellular pools attenuates the amplitude of calcium signal. The potent inhibition of GnRH-induced calcium signaling and gonadotropin secretion by melatonin provides an effective mechanism to protect premature initiation of pubertal changes that are dependent on plasma gonadotropin levels. During the development, such tonic inhibitory effects of melatonin on GnRH action gradually decline due to a decrease in expression of functional melatonin receptors. In adult animals, melatonin does not have obvious direct effects on pituitary functions, whereas the connections between melatonin release and hypothalamic functions, including GnRH release, are preserved, and are critically important in synchronizing the external photoperiods and reproductive functions through still not well characterized mechanisms.     

Characterization of the cyclic AMP-independent actions of somatostatin in GH cells. II. An increase in potassium conductance initiates somatostatin-induced inhibition of prolactin secretion

The neuropeptide somatostatin inhibits prolactin release from GH4C1 pituitary cells via two mechanisms, inhibition of stimulated adenylate cyclase activity and an undefined cAMP-independent process. Somatostatin also hyperpolarizes GH4C1 cells and reduces their intracellular free Ca2+ concentration ([Ca2+]i) in a cAMP-independent manner. To determine whether these ionic changes were involved in the cAMP-independent mechanism by which somatostatin inhibited secretion, changes in cAMP levels were prevented from having any biological consequences by performing experiments in the presence of a maximal concentration of a cAMP analog. Under these conditions, inhibition of prolactin release by somatostatin required a transmembrane concentration gradient for K+ but not one for either Na+ or Cl-. However, elimination of the outward K+ gradient did not prevent somatostatin inhibition of vasoactive intestinal peptide-stimulated hormone release. Therefore, somatostatin's cAMP-mediated mechanism does not require a K+ gradient, whereas its cAMP-independent inhibition of secretion appears to result from a change in K+ conductance. Consistent with this conclusion, membrane hyperpolarization with gramicidin (1 microgram/ml) mimicked somatostatin inhibition of prolactin release. In addition, the K+ channel blocker tetrabutylammonium prevented the effects of somatostatin on the membrane potential, the [Ca2+]i and hormone secretion. Nonetheless, a K+ gradient was not sufficient for somatostatin action. Even in the presence of a normal K+ gradient, somatostatin was only able to inhibit prolactin release when the extracellular Ca2+ concentration was at least twice the [Ca2+]i. Furthermore, the calcium channel blocker, nifedipine (10 microM), which prevents the action of somatostatin to reduce the [Ca2+]i, specifically blocked inhibition of prolactin release via somatostatin's cAMP-independent mechanisms. Therefore, a decrease in Ca2+ influx through voltage-dependent Ca2+ channels produces both the fall in [Ca2+]i and inhibition of hormone secretion in response to somatostatin.     

Dual regulation of ACTH secretion by guanine nucleotides in permeabilized AtT-20 cells

1. We have examined the effects of guanine nucleotides on ACTH secretion from digitonin-permeabilized AtT-20 cells, with the aim of analyzing the involvement of GTP-binding proteins (G proteins) in the secretory process. 2. AtT-20 cells permeabilized with 20 microM digitonin displayed calcium-dependent secretion. The EC50 of calcium was approximately 2 microM and the maximal stimulation was 350% of basal release. 3. Nonhydrolyzable guanine nucleotides also stimulated ACTH release, in a virtually Ca2+-free medium. The EC50 of guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) was approximately 15 microM and the maximal stimulation was approximately 230% of basal release. The effects of calcium and guanine nucleotides were not additive. 4. In the presence of the inhibitory hormone, somatostatin guanine nucleotides inhibited the calcium-stimulated secretion. 5. Both the stimulatory and the inhibitory effects on secretion of guanine nucleotides were independent of changes in cyclic AMP (cAMP) and calcium. It is suggested that G proteins influence an unknown step in the secretion process, which would be near or at the exocytotic site. 6. The results can be explained by assuming the existence of two types of G proteins, one with stimulatory effects on exocytotic release (GeS) and another with inhibitory effects (GeI).     

Red pigment-concentrating hormone induces a calcium-mediated retraction of distal retinal pigments in the crayfish

The octapeptide red pigment-concentrating hormone is capable of eliciting the aggregation of intracellular pigment granules in distal retinal pigment cells of isolated retinas of the crayfish Procambarus clarkii (Girard). The final level and the time course of pigment aggregation are dose dependent within a range of 10(-10) mol l(-1) to 10(-4) mol l(-1). The effect of red pigment-concentrating hormone is prevented by previous incubation with an anti- red pigment-concentrating hormone antibody; however, application of the antibody after the onset of the red pigment-concentrating hormone effect, does not prevent its full development. A similar effect to that elicited by red pigment-concentrating hormone is induced by the calcium ionophores ionomycin and A-23187. Red pigment-concentrating hormone evokes entry of 45Ca2+ to retinal cells. However, the red pigment-concentrating hormone-induced pigment aggregation persists in the presence of the calcium channel blocker verapamil and in Ca2+-free solutions. Caffeine and thapsigargin, known to release calcium from intracellular stores, elicit distal pigment aggregation, while ryanodine and dantrolene, blockers of intracellular calcium release, as well as the intracellular calcium chelator bapta-AM suppress the effect of red pigment-concentrating hormone. These results suggest that red pigment-concentrating hormone elicits distal retinal pigment aggregation by increasing intracellular calcium concentration, acting via a dual mechanism: (1) promoting calcium entry, and (2) releasing intracellular calcium.     

Prenylated Rab acceptor domain family member 1 is involved in stimulated ACTH secretion and inhibition

Dexamethasone (Dex) inhibits stimulated adrenocorticotrophic hormone (ACTH) secretion in AtT-20 cells, a mouse corticotroph tumor cell line. Dexras1 protein expression is induced in corticotrophs by Dex. The function of Dexras1 is unknown; however, it may be involved in corticotrophic negative feedback. Here we report the identification of a Dexras1 interactor, prenylated Rab acceptor domain family member 1 (PRAF1), a protein that localizes to the Golgi complex, post-Golgi vesicles, and endosomes. We determined that amino acids 54–175 of PRAF1 are essential for interaction with Dexras1 and that specific point mutations located within this region enhance PRAF1–Dexras1 interactions. AtT-20 cells stably transfected with truncated or mutated PRAF1 constructs had altered responses to corticotrophin-releasing hormone and Dex, upregulated expression of the ACTH prohormone pro-opiomelanocortin (POMC), altered POMC processing, and altered Golgi complex morphology with decreased intra-Golgi and intracellular co-localization of PRAF1 and ACTH proteins. Our findings indicate that PRAF1 plays a novel role in ACTH stimulated secretion. We propose a model whereby Dexras1 interaction with PRAF1 may lock the sites necessary for PRAF1–Rab3A–VAMP2 interaction resulting in Dex-mediated inhibition of ACTH secretion.     

A correlative study of the binding of dexamethasone in hypothalamic blocks in vitro with its ability to inhibit the release of bioactive corticotrophin-releasing factor

Rat hypothalamic blocks incubated in vitro were used to study the characteristics of binding of [3H]dexamethasone and other steroids to cytosolic binding sites. Cytosols prepared following incubation of the tissue with [3H]dexamethasone for 2 h contained specifically bound steroid in amounts that depended upon the concentration of potassium (but not sodium) ions in the extracting buffer. There was an increase in bound [3H]dexamethasone extracted as the potassium ion concentration increased up to 0.1 M, but not beyond. Dexamethasone, when added to hypothalami in vitro caused a biphasic inhibition of bioactive corticotrophin-releasing factor (CRF) release, and the extent of the second phase of inhibition was dose-related. 11-Epicortisol, when added in a 100-fold molar excess over dexamethasone was able to prevent the second phase of inhibition caused by the latter steroid, as in the binding studies it was able to cause a 50% reduction in the binding of [3H]dexamethasone. In the functional studies it was shown that 11-epicortisol was able to "rescue" the tissue from dexamethasone-mediated delayed inhibition of CRF secretion if added to the blocks 30 min (but not later) after the agonistic steroid.     

Modulation of active renin secretion by renin-binding protein (RnBP) in mouse pituitary AtT-20 cells transfected with human renin and RnBP cDNAs.

To investigate the role of renin-binding protein (RnBP) in renin metabolism, RnBP expression plasmid, which was constructed to express human RnBP under the control of mouse mammary tumor virus long terminal repeat, was transfected into mouse pituitary AtT-20 cells together with the expression plasmid encoding human renin. The transfectant secreted prorenin and active renin, whereas RnBP was expressed only in the presence of dexamethasone and without secretion into the medium. The secretion of active renin was stimulated by forskolin, and the stimulation was repressed by dexamethasone. The secretion of prorenin, however, was insensitive to forskolin irrespective of the presence or absence of dexamethasone. Moreover, the forskolin-stimulated release of active renin was hardly repressed by dexamethasone in AtT-20 cells transfected with the renin expression plasmid and a selectable plasmid pMAMneo. Coexistence of RnBP and renin mRNAs in human Wilms' tumor G-401 cells was shown by means of polymerase chain reaction of respective cDNAs from the cells. These results suggest that RnBP modulates the release of active renin in renin-producing cells.     

Mechanism of inhibitory action of TMB-8 [8-(NN-diethylamino)octyl-3,4,5-trimethoxybenzoate] on aldosterone secretion in adrenal glomerulosa cells.

The mechanism of 8-(NN-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) action was evaluated in isolated adrenal glomerulosa cells. TMB-8 inhibits both angiotensin II- and K+-stimulated aldosterone secretion in a dose-dependent manner. The ID50 for angiotensin II- and K+-stimulated aldosterone secretion is 46 and 28 microM, respectively. In spite of the fact that 100 microM-TMB-8 inhibits angiotensin II-stimulated aldosterone secretion almost completely, TMB-8 (100 microM) does not inhibit angiotensin II-induced 45Ca2+ efflux from prelabelled cells nor does it affect inositol 1,4,5-trisphosphate-induced calcium release from non-mitochondrial pool(s) in saponin-permeabilized cells. TMB-8 has no inhibitory effect on A23187-induced aldosterone secretion, but 12-O-tetradecanoylphorbol 13-acetate-induced aldosterone secretion is completely abolished. TMB-8 effectively inhibits both angiotensin II- and K+-induced increases in calcium influx but has no effect on A23187-induced calcium influx. TMB-8 inhibits the activity of protein kinase C dose-dependently. These results indicate that TMB-8 inhibits aldosterone secretion without inhibiting mobilization of calcium from an intracellular pool. The inhibitory effect of TMB-8 is due largely to an inhibition of plasma membrane calcium influx, but this drug also inhibits the activity of protein kinase C directly.     

Cortisol rapidly suppresses intracellular calcium and voltage-gated calcium channel activity in prolactin cells of the tilapia (Oreochromis mossambicus)

Cortisol was previously shown to rapidly (10-20 min) reduce the release of prolactin (PRL) from pituitary glands of tilapia (Oreochromis mossambicus). This inhibition of PRL release by cortisol is accompanied by rapid reductions in (45)Ca(2+) and cAMP accumulation. Cortisol's early actions occur through a protein synthesis-independent pathway and are mimicked by a membrane-impermeable analog. The signaling pathway that mediates rapid, nongenomic membrane effects of glucocorticoids is poorly understood. Using the advantageous characteristics of the teleost pituitary gland from which a nearly pure population of PRL cells can be isolated and incubated in defined medium, we examined whether cortisol rapidly reduces intracellular free calcium (Ca(i)(2+)) and suppresses L-type voltage-gated ion channel activity in events that lead to reduced PRL release. Microspectrofluorometry, used in combination with the Ca(2+)-sensitive dye fura 2 revealed that cortisol reversibly reduces basal and hyposmotically induced Ca(i)(2+) within seconds (P < 0.001) in dispersed pituitary cells. Somatostatin, a peptide known to inhibit PRL release through a membrane receptor-coupled mechanism, similarly reduces Ca(i)(2+). Under depolarizing [K(+)], the L-type calcium channel agonist BAY K 8644, a factor known to delay the closing of L-type Ca(2+) channels, stimulates PRL release in a concentration-dependent fashion (P < 0.01). Cortisol (and somatostatin) blocks BAY K 8644-induced PRL release (P < 0.01; 30 min), well within the time course over which its actions occur, independent of protein synthesis and at the level of the plasma membrane. Results indicate that cortisol inhibits tilapia PRL release through rapid reductions in Ca(i)(2+) that likely involve an attenuation of Ca(2+) entry through L-type voltage-gated Ca(2+) channels. These results provide further evidence that glucocorticoids rapidly modulate hormone secretion via a membrane-associated mechanism similar to that observed with the fast effects of peptides and neurotransmitters.     

Effects of testosterone on hormonal content and calcium-dependent basal secretion in female rat pituitary cells

In vivo and in vitro effects of elevated androgens on agonist-induced gonadotropin secretion have been addressed previously. Here we investigated the effects of testosterone on hormonal content and basal (in the absence of agonists) hormone release in pituitary lactotrophs, somatotrophs and gonadotrophs from female rats. Furthermore we tested the hypothesis that testosterone action is dependent on the pattern of spontaneous and Bay K 8644 (a L-type calcium channel agonist) -induced calcium signalling. Mixed anterior pituitary cells were cultured in steroid containing or depleted media, and testosterone (1pM to 10nM) was added for 48h. Cells were studied for their spontaneous and Bay K 8644-induced calcium signalling pattern and total hormone levels (release and hormonal content). In lactotrophs, somatotrophs and gonadotrophs testosterone did not affect the pattern of spontaneous calcium signalling. Bay K 8644-induced calcium signalling and hormone release were not affected by testosterone. In both steroid-depleted and -containing medium, testosterone inhibited prolactin (PRL), luteinizing hormone (LH) and growth hormone (GH) cellular content and release in a dose-dependent manner, with IC(50)s in a sub-nanomolar concentration range. These results indicate that testosterone inhibits basal hormone release from lactotrophs, somatotrophs and gonadotrophs without affecting intracellular calcium signalling. This action of testosterone is not dependent on the presence of other steroid hormones.     

5,6-Epoxyeicosatrienoic acid mobilizes Ca2+ in anterior pituitary cells

Luteinizing hormone releasing hormone stimulates the concomitant release of luteinizing hormone and 45Ca2+ from prelabeled anterior pituitary cells. Indomethacin (10 microM) and nordihydroguaiaretic acid (10 microM) had no effect on the luteinizing hormone releasing hormone-stimulated release of either luteinizing hormone or 45Ca2+. Eicosatetraynoic acid (10 microM) blocked both luteinizing hormone releasing hormone-stimulated luteinizing hormone secretion and luteinizing hormone releasing hormone-stimulated 45Ca2+ efflux. 5,6-Epoxyeicosatrienoic acid stimulated both luteinizing hormone secretion and 45Ca2+ efflux from anterior pituitary cells. Additionally, 5,6-epoxyeicosatrienoic acid closely mimics the ability of luteinizing hormone releasing hormone to increase intracellular free calcium. These results are consistent with the hypothesis that 5,6-EET alters calcium homeostasis in a manner similar to that observed during luteinizing hormone releasing hormone stimulation of luteinizing hormone release.     

Effect of Forskolin and Prostaglandin E1 on Stimulus Secretion Coupling in Cultured Bovine Adrenal Chromaffin Cells

Treatment of adrenal chromaffin cells with forskolin (0.1-10 microM) stimulated cyclic AMP levels, reduced the maximal stimulation of release of noradrenaline by nicotine, and increased release in response to elevated external potassium and the calcium ionophore A23187. The presence of the phosphodiesterase inhibitor Ro 20-17-24 with forskolin potentiated both the stimulation of cyclic AMP and the inhibition of nicotine-induced noradrenaline release. Dibutyryl cyclic AMP, and the elevation of cyclic AMP with prostaglandin E1, also attenuated nicotine-stimulated release. However, when the stimulation of intracellular cyclic AMP production by prostaglandin E1 was potentiated by low levels of forskolin, there was not a concomitant potentiation of effect on noradrenaline release. Dideoxyforskolin, an analogue of forskolin which does not stimulate adenylate cyclase, inhibited both potassium- and nicotine-stimulated release, probably by a mechanism unrelated to the action of forskolin in these experiments. Using Fura-2 to estimate free intracellular calcium levels, both forskolin and dideoxyforskolin (at 10 microM) reduced the calcium transient in response to nicotine. These results support a model in which elevation of cyclic AMP inhibits the activation of nicotinic receptors, but augments stimulus secretion coupling downstream of calcium entry. The data, however, do not indicate a simple relationship between total intracellular cyclic AMP levels and the attenuation of nicotinic stimulation of release.     

Protection of Human Pancreatic Islets from Lipotoxicity by Modulation of the Translocon

Type 2 diabetes is characterized by peripheral insulin resistance and pancreatic beta cell dysfunction. Elevated free fatty acids (FFAs) may impair beta cell function and mass (lipotoxicity). Altered calcium homeostasis may be involved in defective insulin release. The endoplasmic reticulum (ER) is the major intracellular calcium store. Lipotoxicity induces ER stress and in parallel an ER calcium depletion through unknown ER calcium leak channels. The main purposes of this study is first to identify one of these channels and secondly, to check the opportunity to restore beta cells function (i.e., insulin secretion) after pharmacological inhibition of ER calcium store depletion. We investigated the functionality of translocon, an ER calcium leak channel and its involvement on FFAs-induced alterations in MIN6B1 cells and in human pancreatic islets. We evidenced that translocon acts as a functional ER calcium leak channel in human beta cells using anisomycin and puromycin (antibiotics), respectively blocker and opener of this channel. Puromycin induced a significant ER calcium release, inhibited by anisomycin pretreatment. Palmitate treatment was used as FFA model to induce a mild lipotoxic effect: ER calcium content was reduced, ER stress but not apoptosis were induced and glucose induced insulin secretion was decreased in our beta cells. Interestingly, translocon inhibition by chronic anisomycin treatment prevented dysfunctions induced by palmitate, avoiding reticular calcium depletion, ER stress and restoring insulin secretion. Our results provide for the first time compelling evidence that translocon actively participates to the palmitate-induced ER calcium leak and insulin secretion decrease in beta cells. Its inhibition reduces these lipotoxic effects. Taken together, our data indicate that TLC may be a new potential target for the treatment of type 2 diabetes.     

The Adenosine Analogue N6-L-Phenylisopropyladenosine Inhibits Catecholamine Secretion from Bovine Adrenal Medulla Cells by Inhibiting Calcium Influx

We reported earlier that adenine nucleotides and adenosine inhibit acetylcholine-induced catecholamine secretion from bovine adrenal medulla chromaffin cells. In this article, we used an adenosine analogue, N6-L-phenylisopropyladenosine (PIA), to study the mechanism underlying inhibition of catecholamine secretion by adenosine. PIA inhibits secretion induced by a nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium, or by elevated external K+. The half-maximal effect on 1,1-dimethyl-4-phenylpiperazinium-induced secretion occurred at approximately 5 x 10(-5) M. The inhibition is immediate and reversible. Fura-2 measurements of cytosolic free Ca2+ indicate that PIA inhibits Ca2+ elevation caused by stimulation; measurements of 45Ca2+ influx show that PIA inhibits uptake of Ca2+. PIA does not inhibit calcium-evoked secretion from digitonin-permeabilized cells, nor does PIA cause any significant change in the dependence of catecholamine secretion on calcium concentration. These data suggest that inhibition by PIA occurs at the level of the voltage-sensitive calcium channel.