Receptor activity modifying proteins regulate the activity of a calcitonin gene-related peptide receptor in rabbit aortic endothelial cells

In Xenopus oocytes with an endogenous calcitonin gene-related peptide (CGRP) receptor, a receptor activity modifying protein (RAMP1) enhancing CGRP stimulated chloride currents of the cystic fibrosis transmembrane regulator was recently cloned [McLatchie, L.M. et al. (1998) Nature 393, 333-339]. Here, transient expression of RAMP1 in rabbit aortic endothelial cells (RAEC) brought about stimulation of cAMP accumulation by human (h) alphaCGRP with an EC50 of 0.41 nM. This was antagonized by a CGRP receptor antagonist alphaCGRP(8-37). Co-expression of RAMP3 together with RAMP1 reduced the maximal cAMP response to h alphaCGRP by 47% (P < 0.05). The cells also express RAMP2 encoding mRNA and an adrenomedullin (ADM) receptor coupled to stimulation of cAMP formation by hADM (EC50 0.18 nM). The latter was antagonized by an ADM receptor antagonist hADM(22-52). In conclusion, expression of a CGRP receptor in RAEC requires RAMP1. The same receptor presumably recognizes ADM making use of endogenous RAMP2. The results reveal competition between the different RAMPs in the regulation of CGRP/ADM receptor activity.     

Pharmacological and functional characterization of muscarinic receptors in the frog pars intermedia

The secretion of alphaMSH from the intermediate lobe of the frog pituitary is regulated by multiple factors, including classical neurotransmitters and neuropeptides. In particular, acetylcholine (ACh), acting via muscarinic receptors, stimulates alphaMSH release from frog neurointermediate lobes (NILs) in vitro. The aim of the present study was to characterize the type of receptor and the transduction pathways involved in the mechanism of action of ACh on frog melanotrope cells. The nonselective muscarinic receptor agonists muscarine and carbachol both stimulated alphaMSH release from perifused frog NILs, whereas the M1-selective muscarinic agonist McN-A-343 was virtually devoid of effect. Both the M1>M3 antagonist pirenzepine and the M3>M1 antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide inhibited muscarine-induced alphaMSH release. Administration of a brief pulse of muscarine in the vicinity of cultured melanotrope cells provoked a 4-fold increase in the cytosolic calcium concentration ([Ca2+]i). Suppression of Ca2+ in the culture medium or addition of 3 mM Ni2+ abrogated the stimulatory effect of muscarine on [Ca2+]i and alphaMSH release. In contrast, omega-conotoxin GVIA and nifedipine did not significantly reduce the stimulatory effect of muscarine on [Ca2+]i and alphaMSH secretion. Exposure of NILs to muscarine provoked an increase in inositol phosphate formation, and this effect was dependent on extracellular Ca2+. The inhibitor of polyphosphoinositide turnover neomycin significantly attenuated the muscarine-evoked alphaMSH release. Similarly, pretreatment of frog NILs with phorbol ester markedly reduced the secretory response to muscarine. In contrast, the stimulatory effect of muscarine on alphaMSH release was not affected by the phospholipase A2 inhibitor dimethyl eicosadienoic acid or by the tyrosine kinase inhibitors lavendustin A, genistein, and tyrphostin 25. Muscarine at a high concentration (10(-4) M) only produced a 40% increase in cAMP formation. Preincubation of frog NILs with pertussis toxin did not significantly affect the muscarine-induced stimulation of alphaMSH release. These results indicate that frog melanotrope cells express a muscarinic receptor subtype pharmacologically related to the mammalian M3 receptor. Activation of this receptor causes calcium influx through Ni2+-sensitive Ca2+ channels and subsequent activation of the phopholipase C/protein kinase C transduction pathway.     

Glucose-dependent insulinotropic polypeptide stimulation of lipolysis in differentiated 3T3-L1 cells: wortmannin-sensitive inhibition by insulin

GIP is an important insulinotropic hormone (incretin) that has also been implicated in fat metabolism. There is controversy regarding the actions of GIP on adipocytes. In the current study, the existence of GIP receptors and effects of GIP on lipolysis were studied in differentiated 3T3-L1 cells. GIP receptor messenger RNA was detected by RT-PCR and RNase protection assay. Receptors were detected in binding studies (IC50 26.7 +/- 0.7 nM). GIP stimulated glycerol release with an EC50 of 3.28 +/- 0.63 nM. GIP (10(-9)-10(-7) M) +/- IBMX increased cAMP production by 1180-2246%. The adenylyl cyclase inhibitor MDL 12330A (10(-4) M) inhibited GIP-induced glycerol production by >90%, and reduced cAMP responses to basal. Preincubation of 3T3-L1 cells with insulin inhibited glycerol responses to GIP, and the inhibitory effect of insulin was blocked by the phosphatidylinositol 3'-kinase inhibitor, wortmannin. It is concluded that GIP stimulates glycerol release in 3T3-L1 cells primarily via stimulation of cAMP production, and that insulin antagonizes GIP-induced lipolysis in a wortmannin-sensitive fashion. It is suggested that effects of GIP on fat metabolism in vivo may depend upon the circulating insulin level, and that meal-released GIP may elevate circulating fatty acids, thus optimizing pancreatic beta-cell responsiveness to stimulation by glucose and GIP.     

Intracranial facial nerve neurinoma: surgical strategy of tumor removal and functional reconstruction

The effects of a new forskolin derivative, (13R)-spiroforskolin, on the ventricular cAMP-activated chloride current (I(Cl(cAMP))) and the atrial L-type calcium current (I(Ca,L)) were measured by means of whole-cell recording from isolated guinea-pig cardiac myocytes at 30 degrees C and 20-22 degrees C, respectively. In contrast to forskolin, the derivative contains a tetrahydrofuran rather than a tetrahydropyran moiety. (13R)-spiroforskolin activated I(Cl(CAMP)) in 58% of the ventricular myocytes studied. The concentration required for the half maximal effect (EC50 value) amounted to 9.6x10(-11) M and was lower than the EC50 value for forskolin (2.4x10(-8) M). (13R)-spiroforskolin evoked a smaller maximal I(Cl(cAMP)) amplitude than forskolin. The rundown of the (13R)-spiroforskolin-activated I(Cl(cAMP)) was faster than that of the forskolin-induced current. Neither forskolin nor (13R)-spiroforskolin in maximally effective concentrations increased I(Cl(cAMP)) in cells containing high concentrations of cAMP. Furthermore, as an activator of atrial I(Ca,L) (13R)-spiroforskolin displayed a smaller activation and a lower EC50 value (5.8x10(-10) M) than forskolin (EC50 value: 3.7x10(-7) M). The effect of (13R)-spiroforskolin was observed in only 30% of the atrial cells studied. None of the drugs exerted a stimulatory effect in atrial cells containing a high [cAMP]. The washout of the drug effect was significantly faster in (13R)-spiroforskolin- than in forskolin-treated atrial myocytes. We conclude that (13R)-spiroforskolin as a forskolin derivative displays unique characteristics. It is a more potent but less efficacious activator of cardiac ionic conductances than the parent compound. The results suggest that (13R)-spiroforskolin, like forskolin, most probably exerts its effects via stimulation of the adenylyl cyclase.     

B2-kininergic action of linear and cyclic tryptophan6- and tyrosine6-kallidin

This study was undertaken to determine the importance of the central aromatic moiety in the kallidin and cyclokallidin molecules, using the relaxation of the isolated duodenum of the rat. Replacement in kallidin of the central phenylalanine by tryptophan increased the potency from an EC50 of 3 x 10(-10)M to 2 x 10(-12)M. Replacement by tyrosine decreased the potency to an EC50 of 8 x 10(-8)M. In cyclo-kallidin (EC50: 10(-8)M) the potencies were decreased: cyclo-Trp6-kallidin showed an EC50 of 10(-6)M and cyclo-Tyr6-kallidin of 3 x 10(-7)M. The relaxation of the rat duodenum by linear and cyclic kinins was potentiated by the bradykinin potentiating peptide BPP5a and antagonized by the B2 antagonist HOE-140. At a concentration of 10(-9)M, HOE-140 significantly decreased the potencies of bradykinin and cyclo-kallidin, but not of the B1 agonist desArg9-bradykinin.     

Stable expression of the recombinant human VIP1 receptor in clonal Chinese hamster ovary cells: pharmacological, functional and molecular properties

We stably transfected Chinese hamster ovary (CHO) cells with the recombinant human vasoactive intestinal peptide (VIP)1 receptor. A clone referred to as Clone 15 was isolated and studied for receptor properties. The following data were obtained: (1) one class of binding site was identified by Scatchard analysis of [125I]VIP binding to cell membranes with a Kd of 0.41 nM and a Bmax of 1.62 pmol/mg protein; (2) the constant Ki for the inhibition of [125I]VIP binding by VIP and related peptides was: VIP (0.9 nM) = pituitary adenylate cyclase-activating peptide (PACAP)-27 (1.3 nM) < PACAP-38 (6.8 nM) < helodermin (46.0 nM) < human growth hormone-releasing factor (GRF) (0.6 microM) < peptide histidine methionineamide (2.0 microM) < secretin (> 10 microM); (3) cross-linking experiments using [125I]VIP identified a single M(r) 67000 recombinant receptor; (4) VIP stimulated cAMP production in Clone 15 cells with an EC50 of 0.20 nM; (5) some previously described VIP receptor antagonists including [4-Cl-D-Phe6, Leu17]VIP, [Ac-Tyr1,D-Phe2]GRF-(1-29) amide and VIP-(10-28) inhibited [125I]VIP binding with a Ki of 0.7, 1.6 and 2.5 microM, respectively. They failed to stimulate cAMP production in Clone 15 cells and inhibited, at least partially, the VIP (0.3 nM)-evoked cAMP production; (6) exposure of Clone 15 cells to 10 nM VIP for 24 h resulted in a sharp decrease in Bmax in Clone 15 cells (0.43 vs. 1.62 pmol/mg protein in control cells) and in the potency and efficacy of VIP in stimulating cAMP. Moreover, immunofluorescence studies using confocal microscopy indicated that the receptor was internalized and sequestered in vesicular structures within the cells. It is concluded that Clone 15 cells provide a valuable tool to further characterize various functional and pharmacological aspects of the human VIP1 receptor.     

Inhibitory activity of loratadine and descarboxyethoxyloratadine on histamine-induced activation of endothelial cells

BACKGROUND: The allergic inflammatory reaction is characterized by leucocyte adherence and infiltration processes which are controlled by the expression of adhesion molecules on the surface of vascular endothelium. One of the main mediators implicated in allergic reactions is represented by histamine. Histamine is a potent activator of endothelial cells (EC): it induces the expression of P-selectin on the surface of endothelium and the secretion of IL-6 and IL-8. OBJECTIVES: Loratadine (L), a histamine H1-antagonist, and one of its active metabolites, descarboxyethoxyloratadine (DCL), were studied at different concentrations for their ability to reduce the histamine-induced activation of human umbilical vein EC (HUVEC). METHODS: HUVEC were stimulated in the presence of histamine at 10(-6) M, 10(-5) M and 10(-4) M. We assessed by ELISA the expression of P-selectin on EC surface, as well as cytokine production in EC supernatants of 24 h culture. RESULTS: Our results showed that for a 10(-4) M-histamine stimulation, L and DCL have a similar inhibitory effect on P-selectin expression (IC50 = 13 x 10[-9] M and 23 x 10[-9] M, respectively). L and DCL inhibited significantly IL-6 and IL-8 secretion induced by histamine with a more powerful efficiency of the active metabolite. For the dose of 10(-4) M histamine, a 50% inhibition of IL-6 secretion was obtained for a dose of DCL equal to 2.6 x 10(-12) M whereas the same magnitude of effects were only reached for a higher concentration of L (0.3 x 10[-6] M). Similar results were obtained for IL-8 (IC50 = 0.2 x 10[-6] M for L and 10[-9] M for DCL). Analysis of IL-8 mRNA expression by RT-PCR was in accordance with these data. CONCLUSION: These results demonstrate that both L and DCL are active to reduce the histamine-induced activation of EC. Interestingly, DCL seems to be effective at lesser concentrations especially to inhibit cytokine secretion.     

Stimulation of retinal pigment epithelial cell growth by neuropeptides in vitro

PURPOSE: The proliferation of many cell types are regulated by cytokines and neuropeptides by autocrine and paracrine mechanisms. Retinal pigment epithelial (RPE) cells are also regulated by cytokines. But RPE cells are very close to the neural retina which has some neuropeptides. The present study was to investigate the effects of neuropeptides on the growth of RPE cells. METHODS: RPE cells were obtained from the eyes of 11 day old chick embryos and cultured in Dulbecco's modified Eagle's culture medium containing 10% fetal calf serum. The growth of RPE cells was evaluated by [3H]-thymidine uptake. RESULTS: Substance P, beta-endorphin and calcitonin gene-related peptide markedly stimulated the growth of RPE cells. The effects of methionine-enkephalin, somatostatin and vasoactive intestinal peptide were intermediate. The strongest effects of substance P, beta-endorphin and calcitonin gene-related peptide were observed at 10(-6) to 10(-7) M. The stimulation of RPE cells with beta-endorphin was inhibited by naloxone, suggesting that the stimulation with beta-endorphin is mediated by an opioid receptor. beta-endorphin and substance P induced RPE cell growth stimulating activity. Leucine-enkephalin and neuropeptide Y did not affect the growth of RPE cells. CONCLUSIONS: These results suggest that neuropeptides play an important role in the regulation of RPE cell growth.     

Effects of ethanol on basal and prostaglandin E1-induced increases in beta-endorphin release and intracellular cAMP levels in hypothalamic cells

Our recent studies determining the effect of cAMP-elevating agents forskolin and dibutyryl cAMP on ethanol-induced immunoreactive beta-endorphin (IR-beta-EP) release from hypothalamic cells in primary cultures suggested the possibility that both stimulatory and adaptive secretory responses of beta-EP neurons after ethanol exposure may involve the cAMP system. To determine further the role of cAMP, the effects of prostaglandin E1 (PGE1) on basal and ethanol-regulated IR-beta-EP secretion and cAMP productions were determined in primary cultures of hypothalamic cells. The results presented in this study show that a 50 mM dose of ethanol, which is within the EC50 dose of ethanol required to elevate IR-beta-EP release from hypothalamic cells, increased cellular levels of cAMP and elevated IR-beta-EP release simultaneously from the cultured neurons for a period of 6 hr. The cAMP and IR-beta-EP secretory responses developed desensitization to ethanol challenge after 24 hr of constant ethanol incubation. The cAMP-elevating agent PGE1 increased the cellular content of cAMP and IR-beta-EP release in a dose-dependent manner. The EC50 dose of PGE1 for elevation of IR-beta-EP and cAMP was approximately 0.5 microM. As with ethanol, chronic treatment with PGE1 desensitized the cAMP and IR-beta-EP responses of hypothalamic neurons to PGE1. Acute exposure to ethanol increased the PGE1-stimulated levels of cAMP and IR-beta-EP, whereas chronic exposure to ethanol resulted in diminished cAMP responses to PGE1. These data provide evidence that the cAMP system may be involved in controlling hypothalamic beta-EP secretion, as well in regulating the stimulatory and adaptive responses of beta-EP neurons to ethanol.     

Meta-analysis and cost comparison of polyethylene glycol lavage versus sodium phosphate for colonoscopy preparation

Alpha-MSH, a proopiomelanocortin (POMC)-derived peptide, is known to be produced in the pituitary, the skin, and melanoma tumors and to possess many biological effects, mainly on melanocyte pigmentation and growth. Moreover, the melanocyte expresses adhesion molecules, including ICAM-1. The latter has been reported to play a role in melanoma spread and associated metastatic process. We conducted a study in order to evaluate the possible effect of MSH on ICAM-1 expression in human cultured malignant and normal melanocytes. Our data show that alpha-MSH inhibits ICAM-1 expression stimulated by TNF in a concentration-dependent manner, both at the protein and gene expression level. Ninety percent inhibition was obtained with 10 nM MSH, while 50% inhibition was achieved with 1 nM. Endogenous cAMP elevation with forskolin as well as an exogenous cAMP stable analogue (Sp-cAMPS) produced the same inhibitory effect. A screening of malignant melanocytes showed that inhibition of ICAM-1 expression could be achieved only in those cells expressing detectable MSH receptors and seemed to correlate with the number of binding sites. In conclusion, our data strongly suggest alpha-MSH as a potent inhibitor of ICAM-1 expression in malignant melanocytes acting through MSH receptor stimulation and subsequent cAMP increase.     

Mechanism of prostaglandin E2-, F2alpha- and latanoprost acid-induced relaxation of submental veins

The mechanism of prostaglandin E2-, prostaglandin F2alpha- and latanoprost acid (13,14-dihydro-17-phenyl-18,19,20-trinor-prostaglandin F2alpha)-induced relaxation of the rabbit submental vein was studied. Prostaglandin E2 caused maximum relaxation of endothelin-1 precontracted vessels (EC50: 1.8 x 10(-8) M). Much of the relaxation could be abolished by denuding the endothelium with the nitric oxide synthase inhibitor, L-NAME (N(G)-Nitro-L-arginine methylester). CGRP-(8-37) (calcitonin gene-related peptide fragment (8-37)), a calcitonin gene-related peptide receptor antagonist, exhibited a partial blocking effect, whereas the tachykinin NK1 receptor blocker, GR 82334 ([D-Pro9[Spiro-gamma-Lactam]Leu10,Trp11]physalaemin (1-11)), markedly attenuated the response. Both prostaglandin F2alpha and the relatively selective FP receptor agonist, latanoprost acid, caused relaxation of the veins to about 50% of the precontracted state in the presence of GR 32191B ([1R-[1alpha(Z),2beta,3beta,5alpha]]-(+)-7-[5-([1,1'-b iphenyl]-4-ylmethoxy)-3-hydroxy-2-(1-piperidinyl)cyclopentyl]-4-he ptenoic acid), a thromboxane receptor antagonist (EC50: for prostaglandin F2alpha 7.9 x 10(-9) M, and for latanoprost acid 4.9 x 10(-9) M). L-NAME, as well as denuding the endothelium, completely abolished the effect. In addition, most or at least a large part of the relaxation was also blocked by CGRP-(8-37) as well as GR 82334. These results indicate that the FP receptor-mediated relaxation of veins is based on release of nitric oxide in addition to involvement of calcitonin gene-related peptide and substance P, or some other tachykinin, probably released from perivascular sensory nerves. The more pronounced relaxation induced by prostaglandin E2 could be due to vasodilator EP receptors in the smooth muscle layer of the veins.     

Activation of phospholipase C and guanylyl cyclase by endothelins in human trabecular meshwork cells

PURPOSE: To characterize effects of endothelins on activities of phospholipase C (PLC) and nucleotide cyclases in human trabecular meshwork (TM) cells. METHODS: Cultured simian virus 40-transformed human TM (HTM-3) or non-transformed (HTM-16) cells were used. Changes in the PLC activity were determined by assaying the production of [3H] inositol phosphates. Accumulation of cyclic GMP or cyclic AMP in cell lysate was measured by radioimmunoassay. RESULTS: Endothelin-1 (ET-1; 1 microM) stimulated PLC in HTM-16 cells, but Sarafotoxin S6c (SRTX), an ET(B) receptor subtype-selective agonist (1 microM), did not. Similar results were obtained in HTM-3 cells: ET-1, but not ET-3 or SRTX, activated PLC in a dose-dependent manner, with a calculated EC50 of 646 pM. The peptide also stimulated the accumulation of cGMP in a concentration-dependent manner with an EC50 of 37.2 pM. ET-3 or SRTX was not effective except at much higher concentrations. Both the PLC and guanylyl cyclase stimulation induced by ET-1 (10 nM) were completely inhibited by pretreating the cells with BQ-123 (<10 microM), an ET(A) receptor selective antagonist, but not by BQ-788 (10 microM), an ET(B) receptor subtype-specific antagonist. Neither ET-1 nor ET-3 stimulated adenylyl cyclase activity in HTM-3 cells at concentration as high as 1 microM. CONCLUSION: ET-1 activates PLC and guanylyl cyclase in TM cells. Potency profiles of ET receptor agonists and antagonists suggest that the ET(A) receptor subtype is involved in both actions of ET-1. The effects of the ET peptides in TM cells are interesting and could be part of the mechanism of their IOP-lowering effect.     

Effects of adrenomedullin on rat cerebral arterioles

The effects of adrenomedullin on isolated rat intracerebral arterioles were investigated and compared with those of calcitonin gene-related peptide (CGRP) and amylin. Adrenomedullin produced dose-dependent vasodilation (maximum dilation 27.1 +/- 2.1% at 3 x 10(-7) M, median effective dose (EC50)) 1.6 x 10(-9) M). CGRP produced similar vasodilation (19.8 +/- 4.1%) at 10(-7) M with a lower EC50 of 2.8 x 10(-11) M. Amylin did not cause vasodilation at concentrations up to 10(-6) M. Adrenomedullin-induced vasodilation was significantly suppressed by CGRP-(8-37). These data suggest that adrenomedullin is a potent vasodilator for arterioles in the cerebral microcirculation that acts through CGRP receptors.     

Clarion cochlear implant: short-term effects on voice parameters

The effect of acetylcholine on the neurointermediate lobe beta-endorphin secretion was studied in the neonatal and in the adult rat in vitro. Acetylcholine stimulated beta-endorphin secretion from the 2-day- and 5-day-old neurointermediate lobe, the effect was dose dependent and more pronounced in the presence of the cholinesterase inhibitor eserine. The 10-day-, the 21-day-old and the adult rat neurointermediate lobes did not respond to acetylcholine, even in the presence of eserine. Basal beta-endorphin secretion was elevated by the D2 receptor antagonist sulpiride, but acetylcholine was without effect in the 10-day-old and in the adult neurointermediate lobe even after dopamine receptor blockade. The beta-endorphin stimulatory response to acetylcholine was diminished by the M1 muscarinic receptor antagonist pirenzepine and blocked by the M3 > M1 antagonist 4-diamino-phenyl-piperidine (4-DAMP). The selective M2 antagonist methoctramine and nicotine had no effect. These data indicate that the neurointermediate lobe beta-endorphin secretion is under special muscarinic cholinergic regulation for a relatively short time after birth. The disappearance of this stimulatory cholinergic effect in later life might be due to changes in the intracellular secretory machinery in the IL and/or to the uncoupling of the cholinergic receptors from the intracellular signal transduction system(s) responsible for the stimulated secretion in the rat melanotrope cells.     

Phospholipase D- and protein kinase C isoenzyme-dependent signal transduction pathways activated by the calcitonin receptor

The calcitonin receptor expressed by the porcine LLC-PK1 renal tubule cells is a seven-transmembrane domain, G protein-coupled receptor activating adenylyl-cyclase and phospholipase C. Salmon calcitonin stimulated dose- and time-dependent release of the phospholipase D-dependent phosphatidylcholine product [3H] choline with an EC50 = 2.5 +/-0.3 x 10(-8) M, similar to that determined for phosphoinositide metabolism (EC50 = 4.5 +/-1.0 x 10(-8)M). The hormone failed to induce release of [3H]phosphocholine and [3H]glycerophosphocholine, ruling out activation of phosphatydilcholine-specific phospholipase C and phospholipase A. Calcitonin stimulated phosphatidic acid, a product of phospholipase D-dependent phosphatydilcholine hydrolysis. Activation of phospholipase D was confirmed by release of [3H]phosphatydilethanol, a specific and stable product in the presence of a primary alcohol. Activation of calcitonin receptor induced diacylglycerol formation, with a rapid peak followed by a prolonged increase, due to activation of phospholipase C and of phospholipase D. Consequently, the protein kinase-C alpha, but not the delta isoenzyme, was cytosol-to-membrane translocated by approximately 50% after 20 min exposure to calcitonin, whereas protein kinase-C zeta, which was approximately 40% membrane-linked in unstimulated cells, translocated by approximately 19%. The human calcitonin receptor expressed by BIN-67 ovary tumor cells, although displaying higher affinity for calcitonin, failed to activate phospholipase D and protein kinase-C in response to the hormone. This receptor lacks the G protein binding consensus site due to the presence of a 48-bp cassette encoding for a 16-amino acid insert in the predicted first intracellular loop. This modification is likely to prevent the calcitonin receptor from associating to phospholipase-coupled signaling.     

Effects of endothelin on phospholipases and generation of second messengers in cat iris sphincter and SV-CISM-2 cells

In both immortalized cat iris sphincter smooth muscle cells (SV-CISM-2 cells) and cat iris sphincter, endothelin-1 (ET-1) markedly increased the activities of phospholipase A2 (PLA2), as measured by the release of arachidonic acid (AA), phospholipase C (PLC), as measured by the production of inositol trisphosphate (IP3), and phospholipase D (PLD), as measured by the formation of phosphatidylethanol (PEt). In SV-CISM-2 cells, ET-1 induced AA release, IP3 production and PEt formation in a dose- and time-dependent manner. The dose-response studies showed that the peptide is more potent in activating PLD (EC50 = 1.2 nM) than in activating PLC (EC50 = 1.5 nM) or PLA2 (EC50 = 1.7 nM). The time course studies revealed that ET-1 activated the phospholipases in a temporal sequence in which PLA2 was stimulated first (t1/2 = 12 s), followed by PLC (t1/2 = 48 s) and lastly PLD (t1/2 = 106 s). In SV-CISM-2 cells, in contrast to the intact iris sphincter, sarafotoxin-c, an ETB receptor agonist, had no effect on the phospholipases, and indomethacin, a cyclooxygenase inhibitor, had no effect on the stimulatory effect of ET-1 on the phospholipases. These results suggest that in this smooth muscle cell line, ET-1 interacts with the ETA receptor subtype to activate, via G proteins, phospholipases A2, C and D in a temporal sequence.     

Desensitization of CGRP and adrenomedullin receptors in SK-N-MC cells: implications for the RAMP hypothesis

Recent evidence (1) suggests that the related peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) bind to the same heptahelical transmembrane receptor, with receptor specificity being determined by a receptor associated modifying protein (RAMP). If correct, this hypothesis would predict that each peptide should desensitize the cellular response to subsequent stimulation by itself or the other peptide. We have therefore studied the patterns of desensitization of these receptors in SK-N-MC cells. SK-N-MC cells were stimulated for 20 minutes in either serum free medium alone (control) or SFM containing AM 10(-8) M or CGRP 10(-7) M. Cells were then incubated for a further 20 minutes in SFM containing a second agonist and 1 mM isobutyryl methylxanthine (IBMX), before harvesting and assay for cAMP. Pre-exposure of cells to CGRP or AM decreased cAMP generation in response to subsequent stimulation with CGRP by 58% (+/-14) and 42% (+/-14) (SD) respectively. Pre-incubation of cells with 100 nM H-89 abolished this effect, indicating that desensitization was mediated through PKA. In contrast, there was no attenuation of the cAMP response to stimulation with AM by pre-exposure to AM or CGRP. These results suggest that CGRP and AM receptors exhibit different patterns of desensitization in SK-N-MC cells: a finding with significant implications for the RAMP hypothesis.