The receptor for the orexigenic peptide melanin-concentrating hormone is a G-protein-coupled receptor.

Gene-knockout studies of melanin-concentrating hormone (MCH) and its effect on feeding and energy balance have firmly established MCH as an orexigenic (appetite-stimulating) peptide hormone. Here we identify MCH as the ligand for the orphan receptor SLC-1. The rat SLC-1 is activated by nanomolar concentrations of MCH and is coupled to the G protein G alpha i/o. The pattern of SLC-1 messenger RNA expression coincides with the distribution of MCH-containing nerve terminals and is consistent with the known central effects of MCH. Our identification of an MCH receptor could have implications for the development of new anti-obesity therapies.

Vasopressin receptors in human adrenal medulla and pheochromocytoma.

The nature of vasopressin (VP) receptors present in normal and tumoral human adrenal was investigated using various experimental approaches. Specific VP-binding sites were detected by autoradiography using [3H]arginine VP as a radioligand in adrenal cortex and medulla. The V1a receptor subtype was expressed in the two parts of the gland, as shown by pharmacological studies and RT-PCR experiments. By contrast, the V1b receptor subtype was only expressed in medullary chromaffin cells. This was confirmed by the characterization of V1b transcripts detected in adrenal medulla tissues. In pheochromocytoma, we also detected functional V1b receptors. These receptors triggered intracellular calcium mobilization from intracellular pools and were involved in catecholamine secretion. Binding experiments performed on pheochromocytoma plasma membrane preparations also revealed V1a vasopressin-binding sites, whose roles and cellular localization have not yet been determined. RT-PCR experiments confirmed these data; 100% and 80% of the five tumors tested exhibited V1a and V1b transcripts, respectively. Perifusion experiments also demonstrated that some pheochromocytomas may secrete large amounts of VP. Our findings imply that VP locally secreted by human adrenal medulla may regulate adrenal function by acting on V1a or V1b receptors. More interestingly, we demonstrate that one pheochromocytoma oversecretes VP. In this particular case, this may contribute to the increase in blood pressure observed.

Signaling mechanisms induced by endothelin agonists in human adrenal glomerulosa cells.

Endothelin-1 (ET-1) exhibits secretagogue and trophic actions on the adrenal zona glomerulosa (ZG). Little information is available on the intracellular signaling events that follow stimulation of ET receptors on ZG cells. This study examined the expression of ET receptor subtypes and their involvement in transduction mechanisms induced by ET agonists on human ZG cells in primary culture. RT-PCR allowed the detection of both ETA and ETB receptor mRNAs in these cells. ET-1 induced a concentration-dependent increase in inositol phosphate (IP) accumulation in the presence of LiCl, whereas ETB agonists were inactive. The ET-1-induced increase in IP accumulation was prevented by BQ-123. ET-1 evoked an increase in [Ca2+]i, which was partially prevented by BQ-788. IRL 1620 also delayed the rise in [Ca2+]i. These results show that in human adrenal ZG cells, ET-1 induces an increase in IP accumulation through ETA receptor activation and evokes a rise in [Ca2+]i via stimulation of both ETA and ETB receptors.

Inhibition of oxytocin receptor function by direct binding of progesterone.

The steroid hormone progesterone (P4) is essential for establishing and maintaining pregnancy in mammals. One of its functions includes maintenance of uterine quiescence by decreasing uterine sensitivity to the uterotonic peptide hormone oxytocin. Although it is generally held that steroid hormones such as P4 act at a genomic level by binding to nuclear receptors and modulating the expression of specific target genes, we show here that the effect of P4 on uterine sensitivity to oxytocin involves direct, non-genomic action of P4 on the uterine oxytocin receptor (OTR), a member of the G-protein-coupled receptor family. P4 inhibits oxytocin binding to OTR-containing membranes in vitro, binds with high affinity to recombinant rat OTR expressed in CHO cells, and suppresses oxytocin-induced inositol phosphate production and calcium mobilization. These effects are highly steroid- and receptor-specific, because binding and signalling functions of the closely related human OTR are not affected by P4 itself but by the P4 metabolite 5beta-dihydroprogesterone. Our findings provide the first evidence for a direct interaction between a steroid hormone and a G-protein-coupled receptor and define a new level of crosstalk between the peptide- and steroid-hormone signalling pathways.

Vasopressin : a potent autocrine/paracrine regulator of mammal adrenal functions.

The control of adrenal functions by locally secreted neuropeptides or neurotransmitters is of great physiological importance. Vasopressin (VP) is one of these autocrine/paracrine regulators. We demonstrated by RT-PCR and perifusion experiments that rat and human adrenal medulla expressed and released vasopressin under basal conditions and under stimulation by acetylcholine. Intra-adrenal concentrations of VP may be sufficient to activate adrenal VP receptors. In the cortex, only the V1a receptor subtype has been detected. It triggered both steroid secretion and cortical growth. In the medulla, both V1a and V1b receptor subtypes were expressed. V1b receptors were mainly present on chromaffin cells and stimulated catecholamine secretion. The role of the V1a receptor remains unclear. Pathophysiological studies also revealed that human pheochromocytoma did not overexpress vasopressin receptors but might oversecrete vasopressin causing high plasma VP concentrations and elevated blood pressure.

Genomic and non-genomic mechanisms of oxytocin receptor regulation.

Our recent studies have shown that regulation of uterine oxytocin (OT) binding involves at least two different mechanism: Estradiol (E2)-induced upregulation is accompanied by an increase in OT receptor (OTR) mRNA accumulation, implying that the E2 effect is mediated via increased OTR gene transcription and/or OTR mRNA stabilization. In contrast, P (P)-induced OTR down-regulation occurs via a novel non-genomic mechanism, involving a direct interaction of P with the OTR at the level of the cell membrane. We found that P specifically binds to the OTR and inhibits its ligand binding and signalling functions. Physiological levels of P repress in vitro the ligand binding capacity (Bmax) of the OTR by > 50%. When expressed in CHO cells, the OTR provides a high affinity (Kd: 20nM) membrane binding site for P. OT-induced inositol phosphate production and intracellular calcium mobilization is inhibited 85% and 90%, respectively, by P. These effects are specific as signalling and binding functions of the closely related V1a vasopressin receptor remain unaffected by P, and as other, related steroids are devoid of any effect on OTR binding or signalling functions. The present observation of a specific interaction of a steroid with a G-protein-linked receptor defines a new mechanism of non-genomic steroid action and uncovers a novel level of crosstalk between steroid and peptide hormone action.

Vasopressin regulates adrenal functions by acting through different vasopressin receptor subtypes.

In mammals, vasopressin is known to be synthesized in the hypothalamus and released in the blood stream at the pituitary level. This neuropeptide is also synthesized and secreted by the adrenal medulla in many species including human. Moreover, agents like acetylcholine and corticotropin releasing factor stimulates its basal secretion. V1a vasopressin receptors are present in the adrenal cortex and are involved in steroids secretion (aldosterone in the zona glomerulosa and glucocorticoids in the zona fasciculata of some species). These receptors are coupled to phospholipase C beta and to dihydropyridine-sensitive calcium channels via heterotrimeric G proteins differing by their sensitivities to pertussis toxin. The adrenal medulla, from many species, exhibits V1a vasopressin receptors. In rat adrenal medulla, functional V1b vasopressin receptors could also be characterized. These receptors stimulate catecholamines secretion via activation of phospholipase C beta and subsequent mobilization of intracellular calcium. The adrenal medulla secretes AVP and exhibits functional vasopressin receptors. The adrenal cortex also possesses functional vasopressin receptors and is in contact with adrenal medulla via "medullary rays". We may thus reasonably conclude that AVP physiologically regulates adrenal gland functions via autocrine/paracrine mechanisms.

Somatostatin blocks the potentiation of TRH-induced TSH secretion from perifused pituitary fragments and the change in intracellular calcium concentrations from dispersed pituitary cells elicited by prepro-TRH (PS4) or by tri-iodothyronine.

TRH and somatostatin (SRIH) are well known to stimulate and to inhibit TSH secretion respectively. However, the mechanisms underlying the effect of SRIH on thyrotrophs are still not understood. We have previously shown in vitro that the TSH response to TRH is potentiated in a Ca(2+)-dependent fashion through the activation of dihydropyridine (DHP)-sensitive Ca2+ channels by the prepro-TRH (160-169) cryptic peptide (PS4) and tri-iodo-L-thyronine (T3), when the hormone was added shortly before a TRH pulse in order to avoid its genomic effect. Using perifused rat pituitary fragments, the present study has shown that SRIH inhibits, in a dose-dependent manner, the TSH response to physiological concentration of TRH (10 nM) and reverses the Ca(2+)-dependent potentiation of that response induced either by PS4 or by T3. We have also demonstrated that the inhibition by SRIH of the T3 potentiation of TRH-induced TSH secretion is pertussis toxin-sensitive. Our data suggest that SRIH inhibits the PS4 and T3 potentiation of TRH-induced TSH secretion through the inactivation of DHP-sensitive Ca2+ channels. Using primary cultures of rat anterior pituitary cells and videomicroscopy, we have already demonstrated that TRH, as well as PS4 and T3, are able to increase intracellular Ca2+ concentration ([Ca2+]i) rapidly, in 15 s. Our study has shown that SRIH is able to abolish the acute rise in [Ca2+]i induced either by PS4 or by T3. Since [Ca2+]i responses to PS4 and T3 are also abolished by the DHP nifedipine, our results suggest that [Ca2+]i changes in PS4- or T3-sensitive pituitary cells depend directly or indirectly on the activation of DHP-sensitive Ca2+ channels and that the inhibitory effect of SRIH may be mediated by inactivation of this type of channel.

Membrane-delimited G protein-mediated coupling between V1a vasopressin receptor and dihydropyridine binding sites in rat glomerulosa cells.

In rat glomerulosa cells, vasopressin stimulates intracellular calcium mobilization via at least two distinct mechanisms: the release of calcium from inositol-1,4,5-P3-sensitive stores and the activation of transmembrane calcium influx. In this study, we focused on the second mechanism through three experimental approaches. By videomicroscopically examining Fura-2-loaded cells, we demonstrate that vasopressin induces a dose-dependent and receptor-mediated calcium influx fully inhibited by either 1 microM nifedipine or a pertussis toxin pretreatment and potentiated by 1 microM BAY K 8644. Patch-clamp experiments also indicate that vasopressin stimulates L-type calcium current by 87% and only weakly inhibits T-type calcium current. To further characterize the coupling between the vasopressin receptor and the dihydropyridine calcium channel, we performed binding studies using tritiated nitrendipine. With this technique, we showed that on intact cells, vasopressin is able to increase the specific binding of tritiated nitrendipine in a dose-dependent manner (Kact = 2 nM). Pharmacological studies using a series of vasopressin analogs revealed that this effect is mediated via a V1a vasopressin receptor subtype. Furthermore, the vasopressin-stimulated nitrendipine binding was sensitive to pertussis toxin pretreatment, which affected only the maximum binding capacity of nitrendipine-binding sites. More interestingly, we demonstrate that vasopressin still increases nitrendipine binding to plasma membrane preparation and that GTP is absolutely necessary for such a hormonal effect. Altogether, these data confirm the existence of a tight and direct coupling between the V1a vasopressin receptor and a dihydropyridine calcium channel via a pertussis toxin-sensitive G protein.

Molecular and functional characterization of V1b vasopressin receptor in rat adrenal medulla.

In rat adrenal medulla, PCR experiments reveal the expression of messenger RNA encoding the gene for the V1b vasopressin receptor. Complementary DNA amplified sequences corresponded to the cloned rat pituitary V1b vasopressin receptor. Video microscopy experiments performed on fura-2-loaded adrenal medullary or adrenal glomerulosa cell primary cultures showed that vasopressin dose dependently mobilized intracellular calcium, suggesting that functional vasopressin receptors are expressed in these tissues. The use of d[D-3-Pal]vasopressin, a specific V1b vasopressin agonist, and SR 49059, a specific V1b vasopressin antagonist, revealed that V1b receptors are exclusively expressed in adrenal medulla. Using an indirect immunological approach (plasma membrane localization of dopamine-beta-hydroxylase), we demonstrated that stimulation of rat adrenal medulla V1b receptor leads to catecholamine secretion. More interestingly, PCR experiments performed on rat adrenal medulla RNA revealed that the arginine vasopressin-encoding gene is also expressed in this tissue. In addition, perifusion experiments indicated that [Arg8] vasopressin is released by the adrenal medulla. Together, these data suggest that vasopressin may regulate the adrenal functions by paracrine/autocrine mechanisms involving distinct vasopressin receptor subtypes: V1a in the adrenal cortex and V1b in the adrenal medulla.

Role of Ca2+ in the action of adrenocorticotropin in cultured human adrenal glomerulosa cells.

The present report details the role of Ca2+ in the early events of ACTH action in human adrenal glomerulosa cells. Threshold stimulations of both aldosterone and cAMP production were obtained with a concentration of 10 pM ACTH, an ED50 of 0.1 nM, and maximal aldosterone stimulation (5.5-fold increase over control) at 10 nM ACTH. ACTH also induced a sustained increase of intracellular calcium ([Ca2+]i) with maximal stimulation of 1.6 +/- 0.1-fold over control values. This increase does not involve mobilization of calcium from intracellular pools since no response was observed in Ca2+-free medium or in the presence of nifedipine, suggesting the involvement of Ca2+ influx by L-type Ca2+ channels. This was confirmed by patch clamp studies that demonstrated that ACTH stimulates L-type Ca2+ channels. Moreover, the Ca2+ ion is not required for ACTH binding to its receptor, but is essential for sustained cAMP production and aldosterone secretion after ACTH stimulation. These results indicate that, in human adrenal glomerulosa cells, a positive feedback loop between adenylyl cyclase-protein kinase A-Ca2+ channels ensures a slow but sustained [Ca2+]i increase that is responsible for sustained cAMP production and aldosterone secretion.

Absence of calcium channels in neonatal rat aortic myocytes.

We have investigated whole-cell Ba2+ currents through Ca2+ channels (IBa) in single myocytes freshly isolated from the aortic media of neonatal (1-day-old) and adult (12-week-old) rats. In neonatal myocytes, (IBa) was undetectable even in presence of the dihydropyridine (DHP) agonist Bay K 8644. Binding of [3H]Nitrendipine on crude plasma membrane preparation of media confirmed the absence of DHP-receptors. By contrast, a robust DHP-sensitive 'L-type' IBa was recorded in adults which was consistent with the presence of specific [3H]Nitrendipine binding sites. In conclusion, neonatal aortic myocytes do not express any Ca2+ channels. The acquisition of L-type Ca2+ channels may be related to cell differentiation and acquisition of contractility during postnatal development.

Dual effects of fluoroaluminate on activation of calcium influx and inhibition of agonist-induced calcium mobilization in rat glomerulosa cells.

Results presented in this study demonstrate that, in rat glomerulosa cells, fluoroaluminate (AlF4-) alone stimulates both cAMP accumulation (maximal stimulation 10-fold, ED50, 24 mM) and total inositol phosphate accumulation (maximal stimulation 12-fold, ED50 14 mM). Despite a transient accumulation of Ins(1,4,5)P3 after AlF4- stimulation, no rapid and transient intracellular calcium mobilization was observed. In contrast to angiotensin II (Ang II) or vasopressin (AVP), AlF4- induces only a slow and sustained increase in intracellular Ca2+. We demonstrate that this increase results from a Ca2+ influx mediated by cAMP-protein kinase A (PKA) pathway since preincubation with H-89, a potent PKA inhibitor, inhibits this influx. Moreover, a short preincubation (15 min at 37 degrees C) of cells with AlF4- or ACTH prevents the initial release of Ca2+ from intracellular stores induced by Ang II, but does not affect the amount of InsPs accumulated under Ang II stimulation. This rapid inhibition of Ang II action is mediated by ACTH- or AlF4(-)-stimulated cAMP production since pretreatment with H-89 leads to a complete reversal. cAMP most likely acts at the level of Ins(1,4,5)P3 receptors since an increase in intracellular cAMP blunts the calcium response induced by addition of exogenous Ins(1,4,5)P3 to permeabilized cells. These results point out that, in rat glomerulosa cells, activation of the cAMP pathway can induce a rapid desensitization of the phospholipase C pathway by acting downstream of inositol phosphate accumulation.

Dihydropyridine-like effects of amiodarone and desethylamiodarone on thyrotropin secretion and intracellular calcium concentration in rat pituitary.

Amiodarone (AM) and its major metabolite desethylamiodarone (DEA) are structurally similar to biologically active thyroid hormones. Amiodarone therapy is frequently associated with impairment of thyrotropic function, whose mechanisms are still controversial. Besides its effect on nuclear thyroid hormone binding. AM is able to displace dihydropyridine (DH) binding on membrane preparations from several tissues. By perifusing rat pituitary fragments and measuring thyrotropin (TSH) release we examined: the effect of AM on Ca(2+)-dependent and DHP-sensitive potentiation of the TSH response to thyrotropin-releasing hormone (TRH) induced by either triiodothyronine (T3, perifused for only 30 min before a TRH pulse) or by the prepro-TRH peptide 160-169 (PS4); and the effect of DEA on TRH-induced TSH response in the presence or absence of the DHP nifedipine. We show that AM reverses T3 or PS4 potentiation of the TSH response to TRH; this effect is specific because AM does not modify ionomycin potentiation of that response. In contrast, DEA significantly potentiates the TSH response to TRH and the DHP nifedipine reverses that potentiation. We also tested whether AM would change the acute T3-induced increase in intracellular Ca2+ concentration by measuring intracellular Ca2+ ([Ca2+])i with fura-2 imaging on primary cultures of pituitary cells. We show that AM reverses the effect of T3 on [Ca2+]i as well as the PS4-induced increase in [Ca2+]i. In contrast, DEA increases [Ca2+]i and nifedipine reverses this effect. Our results suggest that AM and DEA display DHP-like effects on TRH-induced TSH release, behaving either as a Ca2+ channel blocker (AM) or as a Ca2+ channel agonist (DEA).

Triiodo-L-thyronine enhances TRH-induced TSH release from perifused rat pituitaries and intracellular Ca2+ levels from dispersed pituitary cells.

There is now increasing evidence that Ca2+ serves as the first messenger for the prompt and non-genomic effects of 3,5,3' triiodo-L-thyronine (T3) in several tissues. We have previously shown that the first phase of thyroid stimulating hormone (TSH) release in response to thyrotropin-releasing hormone (TRH) can be potentiated by messengers of hypothalamic origin, by a Ca(2+)-dependent phenomenon involving the activation of dihydropyridine-sensitive Ca2+ channels. By perifusing rat pituitary fragments, we have investigated whether T3 would modify TSH release when the hormone is applied for a short time (i.e. 30 min) before a 6 min pulse of physiological concentration of TRH, thus excluding the genomic effect of T3. We show that: (1) increasing concentrations of T3 (100 nM-10 microM) in the perifused medium potentiates the TRH-induced TSH release in a dose-dependent manner; (2) the T3 potentiation is not reproduced by diiodothyronine and T3 does not potentiate the increase if TSH release induced by a depolarizing concentration of KCl; (3) the protein synthesis inhibitor cycloheximide, does not significantly modify the effect of T3; (4) addition of Co2+, nifedipine, verapamil, or omega-conotoxin in the medium, at a concentration which does not modify the TSH response to TRH, reverses the T3 potentiation of that response. We also tested whether T3 would change intracellular concentrations of Ca2+, by measuring [Ca2+]i with fura-2 imaging on primary cultures of dispersed pituitary cells, either in basal conditions or after stimulation by TRH or/and T3. Both substances induced a fast increase of [Ca2+]i, with a peak at 15 s, followed by a subsequent progressive decay with TRH and a rapid return with T3. Our data suggest that T3 enhances TRH-induced TSH release by a protein synthesis-independent and Ca(2+)-dependent phenomenon, probably due to an increase in Ca2+ entry through the activation of dihydropyridine- and omega-conotoxin-sensitive Ca2+ channels. They also show that T3 may acutely enhance [Ca2+]i in pituitary cells. These findings support the idea of the occurrence of a prompt and stimulatory role of T3 at the plasma membrane level in normal rat pituitary gland.

Vasopressin stimulates steroid secretion in human adrenal glands: comparison with angiotensin-II effect.

Autoradiographic experiments using iodinated vasopressin analog revealed the presence of specific vasopressin-binding sites in the human adrenal cortex (zona glomerulosa and zona fasciculata). These receptors exhibited a good affinity for arginine vasopressin (3.3 nM), with classical V1a pharmacology and densities of 65 and 135 fmol/mg protein-enriched membranes from zona glomerulosa and fasciculata, respectively. Vasopressin receptors present in both glomerulosa and fasciculata cell-enriched primary cultures were coupled to phospholipase C (ED50, 0.9 and 1.8 nM; maximal stimulation, 4.3- and 5.8-fold, respectively). Vasopressin also stimulated an increase in intracellular calcium through at least two distinct mechanisms: the mobilization of intracellular pools via vasopressin-stimulated inositol phosphate accumulation and the activation of calcium influx. In glomerulosa cell-enriched primary cultures, vasopressin increased aldosterone secretion (ED50, 0.4 nM; maximal stimulation, 2.5-fold) and was found to be as potent as angiotensin-II in stimulating aldosterone secretion, phosphoinositide turnover, and calcium mobilization. In fasciculata cells, vasopressin and angiotensin-II were also able to stimulate cortisol secretion and inositol phosphate accumulation. Moreover, perifusion experiments demonstrated that vasopressin was released from the adrenal medulla. Together, these results indicate that vasopressin can be considered a potent paracrine modulator of adrenal steroid secretion in man.

Dual effects of nordidemnin on WRK1 cells: inhibition of phosphoinositide metabolism and cell proliferation.

Nordidemnin (NorD), a cyclodepsipeptide isolated from marine invertebrates, exhibits antiproliferative and antitumoral properties identical to didemnin B on many cell lines. On WRK1 cells, a rat mammary tumor cell line, NorD considerably reduced the vasopressin-stimulated accumulation of inositol phosphates. This effect was more pronounced on dividing cells and of weak amplitude on quiescent ones. It was observed with nanomolar concentrations of NorD and became significative after 3 hr of incubation at 37 degrees C. The maximal effect was observed after a 14-hr incubation period. In contrast, the inactive analog epinordidemnin, as well as the structurally related immunosuppressive cyclosporin A, had no significant effect on phosphoinositide metabolism. More detailed analysis demonstrated that NorD reduced the amounts of all intracellular inositol phosphate isomers, including inositol pentakisphosphate and inositol hexakisphosphate. Vasopressin-stimulated inositol (1,4,5)-trisphosphate accumulation was reduced by 80% and, as a consequence, the intracellular calcium mobilization was strongly affected. Similarly, NorD reduced both the level of inositol (1,4,5)-trisphosphate and the intracellular free calcium concentration of unstimulated cells. NorD blocked phosphoinositide metabolism by reducing the myoinositol transporter and, by a consequence, the pool of inositol lipids. NorD also strongly inhibited WRK1 cell proliferation with the same EC50 as that observed for the effect on phosphoinositide metabolism. Epinordidemnin, which was unable to inhibit inositol phosphate accumulation, had no effect on cell growth. Cyclosporin A, which slightly inhibited WRK1 cell growth, did not significantly affect the calcium-phosphatidylinositol cascade. Taken together, these results suggest that NorD might interfere with WRK1 cell growth by inhibiting phosphoinositide turnover.

Reversible inhibition of acetylcholinesterase by eseroline, an opioid agonist structurally related to physostigmine (eserine) and morphine.

The action of eseroline--(3aS,8aR)-1,2,3,3a,8,8a-hexahydro-1,3a,8-trimethylpyrrolo[2,3-b]indo l-5-ol--salicylate was tested on preparations of ChE from different sources and on the longitudinal muscle of guinea-pig ileum. While eseroline is eseroline is extremely weak-acting on horse serum BuChE (Ki = 208 +/- 42 microM), it is a rather strong competitive inhibitor of AChE's, its Ki being 0.15 +/- 0.08 microM, 0.22 +/- 0.10 microM and 0.61 +/- 0.12 microM in electric eel, human RBC and rat brain, respectively. Eseroline inhibitory action in AChE in independent of the duration of pre-incubation and appears fully developed in less than 15 sec. This action is also rapidly reversible; after pre-incubation followed by dilution, maximum enzymic activity is regained within 15 sec. The electrically-evoked contractions of the longitudinal strip were inhibited by concentrations of eseroline in the range 0.2-15 microM, while they were increased by concentrations over 20 microM. In the same preparation, without electrical stimulation, but in the presence of naloxone, eseroline induced contractions at concentrations higher than 5 microM. This effect was antagonized by atropine. The inhibitory activity of eseroline parallels, as regards selectivity, potency and kinetics, that of the phenolic anticurare agent edrophonium, while it differs markedly from that of physostigmine.