Stimulation and homologous desensitization of calcitonin gene-related peptide receptors in cultured beating rat heart cells

Addition of calcitonin gene-related peptide (CGRP), 1 X 10(-7) M, to cultured neonatal rat heart cells resulted in rapid increases in beating rate and cellular concentrations of cAMP. Calcitonin (1 X 10(-7) M), in contrast, had no significant effect on heart cell beating rate or cAMP content. CGRP-stimulated increases in heart cell cAMP content were rapid, transient, dose dependent, and potentiated by isobutyl-methylxanthine (1 X 10(-4) M). Half-maximal increases in heart cell cAMP content occurred at 1 X 10(-8) M CGRP. Heart cell adenylate cyclase responses to CGRP were desensitized in a rapid (i.e. within 5 min) and dose-dependent manner by prior exposure to CGRP. Complete and half-maximal desensitization of heart cells to CGRP occurred at 1 X 10(-8) and 3 X 10(-10) M CGRP, respectively. Desensitization of heart cells to CGRP did not modify the cAMP response of heart cells to beta-adrenergic agonist stimulation, and beta-adrenergic agonist desensitization of heart cells did not modify responses to CGRP. Heart cell cAMP responses to CGRP were additive to those of the beta-adrenergic agonist isoproterenol and occurred in the presence of beta-adrenergic blockade. These observations demonstrate that CGRP exerts specific and potent agonist actions in cardiac myocytes and that regulation of myocardial responses to CGRP may occur by mechanisms involving increases in cAMP and receptor desensitization.     

The lack of gonadotrophin-releasing hormone (GnRH) receptor desensitisation in alphaT3-1 cells is not due to GnRH receptor reserve or phosphatidylinositol 4,5-bis-phosphate pool size

The phospholipase C (PLC)-activating gonadotrophin-releasing hormone (GnRH) receptor is thought not to rapidly desensitise in alphaT3-1 cells. This extremely unusual characteristic raises the concern that it might be a feature of the cell type, rather than the receptor per se. Here we have used video imaging to establish whether the effects of endogenous PLC-activating G-protein coupled receptors (GPCRs) on Ca2+ ion concentration [Ca2+]i desensitise in these cells. Oxytocin, endothelin-1, methacholine, and UTP all caused [Ca2+]i increases which underwent rapid homologous desensitisation in that they were transient and responses to repeat stimuli were attenuated whereas subsequent responses to GnRH were not. To test whether receptor reserve obscures functional desensitisation of GnRH receptors, a photoaffinity antagonist (Pant-1), was used to effect a partial and irreversible receptor blockade. UV crosslinking in medium with 1000 nM Pant-1 reduced GnRH receptor number to 20 +/- 5% and reduced maximal buserelin-stimulated [3H]IP(X) accumulation to 57 +/- 5%, demonstrating removal of receptor reserve. In control alphaT3-1 cells the initial rate of GnRH-stimulated [3H]IP(X) accumulation was maintained for at least 5 min and GnRH caused a sustained increase in Ins(1,4,5)P3 mass (confirming the resistance of GnRH receptors to desensitisation) and Pant-1 pre-treatment reduced the magnitude of these responses without altering their temporal profiles. In alphaT3-1 cells stably transfected with recombinant human muscarinic receptors (alphaT3-1/M3), responses to methacholine were characteristic of desensitising GPCRs (transient Ins(1,4,5)P3 and curvilinear [3H]IP(X) responses) and were unaltered by Pant-1. To test the relevance of phospholipid pool size, alphaT3-1/M3 cells were pre-treated with GnRH or methacholine in medium with LiCl (to deplete PtdIns(4,5)P2 pools). These pre-treatments reduced subsequent responses to methacholine and GnRH comparably, indicating access to a shared PtdIns(4,5)P2 pool. Partial depletion of this pool (GnRH pre-treatment in medium with LiCl) reduced the magnitude of the [3H]IP(X) and Ins(1,4,5)P3 responses to methacholine and GnRH, without altering their temporal profiles. Thus the GnRH receptor does not undergo rapid homologous desensitisation in alphaT3-1 cells in spite of the fact that they can desensitise other endogenous (and recombinant) PLC-activating GPCRs, and the lack of desensitisation cannot be attributed to the existence of GnRH receptor reserve or access to an atypically large or rapidly re-cycled PtdIns(4,5)P2 pool. This unique functional characteristic (mammalian GnRH receptors are the only PLC-activating GPCRs known not to rapidly desensitise) almost certainly therefore reflects the atypical structure of these receptors (mammalian GnRH receptors are the only PLC-activating GPCRs known to lack C-terminal tails).     

Rapid desensitisation of the GH secretagogue (ghrelin) receptor to hexarelin in vitro

Ghrelin, the recently identified hormone with GH-secreting and appetite-inducing effects, acts on the GH secretagogue receptor (GHS-R). GHS-R belongs to the G protein-coupled 7 transmembrane domain receptors and activates the phospholipase C pathway; it then leads to the release of GH from somatotroph cells via an elevation of intracellular calcium concentration. Both in vivo and in vitro studies demonstrated that the effect of GH secretagogues (GHS) could be desensitised similar to most receptor stimulation systems. We have studied whether acute desensitisation of the GHS-R occurs in response to the GHS hexarelin in vitro in terms of intracellular calcium concentration. Chinese hamster ovary cells were transiently transfected with cDNA encoding the human type 1a GHS-R. The presence of messenger RNA was confirmed with RT-PCR, while no GHS-R was observed in mock-transfected cells. Calcium responses to the peptide GHS analogue hexarelin were measured using the fluorescent indicator fura-2. Cells were stimulated with the peptide GHS, hexarelin, at concentrations between 10(-10) and 10(-7) M. Cells transfected with the GHS-R cDNA demonstrated a significant and specific calcium response to hexarelin that was not observed in mock-transfected cells. Marked desensitisation of the calcium response to hexarelin was observed 2-5 min after the first dose of hexarelin (10(-7) M) was administered. These data show directly for the first time the desensitisation of the GHS receptor signal at the second messenger level. The desensitisation of the receptor may play a major role in the regulation of effect of circulating or locally produced ghrelin both in the GH and in the appetite-regulating system or in other systems where ghrelin has been shown to be active, such as the cardiovascular system or cell proliferation.     

C-type natriuretic peptide (CNP) effects in anterior pituitary cell lines: evidence for homologous desensitisation of CNP-stimulated cGMP accumulation in alpha T3-1 gonadotroph-derived cells

C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, has been found at its highest tissue concentrations in the anterior pituitary, where it is localised in gonadotrophs. Its specific guanylyl cyclase-containing receptor, GC-B, is also expressed on several anterior pituitary cell types, and CNP potently stimulates cGMP accumulation in rat pituitary cell cultures and pituitary cell lines. The mouse gonadotroph-derived alpha T3-1 cell line has been shown to express CNP as well as GC-B (but not GC-A) receptors, suggesting that CNP may well be an autocrine regulator of gonadotrophs. Comparing effects of three natriuretic peptides (atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and CNP) on cGMP accumulation in four pituitary cell lines (alpha T3-1, TtT-GF, AtT-20 and GH(3)) we find that CNP is most potent and effective in alpha T3-1 cells. In these cells, CNP-stimulated cGMP accumulation was found to desensitise during a 30 min exposure to CNP. Pretreatment with CNP for up to 6 h also caused a significant reduction in the ability of CNP to subsequently stimulate cGMP accumulation. This effect was receptor specific, because pretreatment with sodium nitroprusside (an activator of nitric oxide-sensitive guanylyl cyclase), or with ANP or BNP, did not cause desensitisation of CNP-stimulated cGMP accumulation. Protein kinase C activation with phorbol esters also inhibited CNP-stimulated cGMP accumulation and such inhibition was also seen in cells desensitised by pretreatment with CNP. Thus it appears that the endogenous GC-B receptors of alpha T3-1 cells are subject to both homologous and heterologous desensitisation, that the mechanisms underlying these forms of desensitisation are distinct, and that cGMP elevation alone is insufficient to desensitise GC-B receptors.     

Function of beta-adrenergic receptors on mononuclear cells in female patients with fibromyalgia

OBJECTIVE: To investigate the beta-adrenergic receptors (beta AR) in patients with chronic fibromyalgia syndrome (FM). These receptors are present on circulating mononuclear cells, and activation of G-protein coupled receptors like beta AR leads to an increase in the intracellular level of cyclic aminosine monophosphate (cAMP). Therefore, cAMP levels can be used to indirectly assess the functional status of the receptor. METHODS: Eight female patients with FM and 9 matched healthy female controls participated in this study. Blood samples were drawn from subjects' anterocubital vein in the morning. Mononuclear cells were isolated from the whole blood according to Boyüm's method. Basal and stimulated intracellular cAMP levels were determined by enzyme immunoassay. Aliquots of 10(6) cells were incubated with or without stimulation of beta-agonist isoproterenol for 5 min. Two different concentrations of isoproterenol (10(-3) M and 10(-5) M) were utilized. RESULTS: The basal cAMP levels in patients with FM (3.02 +/- 0.44 pmol/10(6) cells) were slightly more elevated (but not statistically different; p = 0.124, Mann-Whitney U test) than that of the control group (2.26 +/- 0.39 pmol/10(6) cells). Proterenol 10(-3) M stimulation significantly increased intracellular cAMP from the basal levels in both groups (FM group, p = 0.008; control group, p = 0.011). However, isoproterenol 10(-5) M did not increase mean intracellular cAMP levels in the FM group (p = 0.74), while a significant increase was observed in the control group (p = 0.012). CONCLUSION: These preliminary results suggest that diminished beta AR function is associated with the chronic FM state.     

Intralysosomal hydrolysis of thyroglobulin. II. Different fates of poorly and fully iodinated Tg and specific activation by TSH of the degradation of fully iodinated Tg

Even though adrenergic nerve terminals between and around thyroid follicles and catecholamine stimulation of thyroid adenylate cyclase have been reported, there is no uniform concept on catecholamine interaction with thyrotrophin (TSH) receptors. Therefore, the effect of catecholamines on TSH-stimulated cyclic AMP (cAMP) accumulation in human follicular thyroid cells has been investigated, to thus eliminating the extrathyroidal actions of catecholamines. Epinephrine, norepinephrine and isoproterenol appeared to be rapid and potent stimulators of intracellular cAMP accumulation, the half maximum increase doses being 4 X 10(-7)M, 1 X 10(-5)M and 5 X 10(-7)M, respectively. While propranolol (1 X 10(-5)M) prevented the stimulatory effect of catecholamines and failed to inhibit the effect of bovine TSH, phentolamine (1 X 10(-5)M) enhanced the potency of norepinephrine and bovine TSH, leaving that of epinephrine unchanged. The effects of epinephrine (2 X 10(-8)M) and isoproterenol (2 X 10(-8)M) were additive to that of bovine TSH (0.5 mU/ml), but the effect of simultaneous stimulation with norepinephrine (5 X 10(-7)M) and bovine TSH (0.5 mU/ml) was lower than expected. Prenalterol, a selective beta 1-agonist, did not stimulate cAMP accumulation, while terbutaline, a selective beta 2-agonist, exerted a potent stimulation. Metoprolol, a selective beta 1-adrenergic blocker, did not affect the response of thyroid follicular cells to isoproterenol. These results demonstrate the existence of beta-adrenergic receptors in human thyroid follicular cells, mainly of the type beta 2, apparently not correlated with TSH receptor. The existence of alpha-adrenergic receptors which counter-regulate TSH functional responses in human thyroid follicular cells is suggested.     

Establishment and characterization of steroidogenic granulosa cells expressing beta(2)-adrenergic receptor: regulation of adrenodoxin and steroidogenic acute regulatory protein by adrenergic agents

Primary granulosa cells obtained from PMSG primed immature rats were triple transfected with SV40 DNA, Ha-ras oncogene and an expression vector containing human beta(2)-adrenergic receptors resulting in granulosa cell lines constitutively expressing the beta(2)-adrenergic receptors. Isoproterenol, a potent adrenergic agent, stimulated both cAMP accumulation and progesterone production in these cells in a dose dependent manner. Responsiveness of these cells was specific only to isoproterenol, while hCG (2.4 nM) and hFSH (2.4 nM) had no effect on steroid production. ED(50) for stimulation of cAMP and progesterone in these cells by isoproterenol was 2x10(-6) M and 7x10(-6) M, respectively. Forskolin also showed a dose dependent stimulation of cAMP and progesterone with ED(50) of 1.5 and 0.35 microg/ml, respectively. Epinephrine at a dose of 10(-5) M elicited maximum response to produce cAMP and progesterone. Isoproterenol induced accumulation of cAMP and progesterone in these cells were inhibited by beta(2)-adrenergic blocker, propranolol with an ED(50) of 6x10(-8) and 7x10(-9) M, respectively, whereas the beta(1)-adrenergic blocker, metoprolol was effective only at a very high concentration (ED(50)>10(-4) and 1.9x10(-5) M for inhibiting isoproterenol induced cAMP and progesterone production, respectively). Induction of steroidogenesis by isoproterenol or forskolin involved de novo synthesis of the cytochrome P450 side chain cleavage (SCC) enzyme complex, as assessed by indirect immunofluorescence staining for adrenodoxin. Western analysis indicate that expression of adrenodoxin is upregulated by forskolin, isoproterenol and adrenalin by 7.8-, 6.9- and 10.8-fold, respectively. The presence of StAR protein was identified by Western blotting. StAR expression was elevated by 8.3-, 2.5- and 4.7-fold upon stimulation with forskolin, isoproterenol and adrenalin, respectively. Thus, this cell line could serve as a good model system to study catecholamine mediated regulation of growth and differentiation of granulosa cells and the role of oncogenes in this process.     

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

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

Adrenergic stimulation of placental progesterone production

Cells from term human placentas were maintained in culture, and progesterone production was monitored over 24 h. The beta 1-adrenergic receptor agonist dobutamine (10(-5) M) and the beta 2-adrenergic receptor agonist terbutaline (10(-5) M) increased progesterone production by 36 +/- 19% and 49 +/- 8% (+/- SE), respectively, compared with that in controls (P less than 0.001). Propranolol (10(-5) M) completely blocked the stimulatory effects of both drugs. The cAMP analog 8-bromo-cAMP (0.5 mM) also significantly altered (increased) progesterone production compared with controls (P less than 0.001), but this effect was not blocked to a significant degree by propranolol. The alpha-adrenergic receptor agonist methoxamine (10(-4) - 10(-6) M) did not significantly alter placental progesterone production compared with controls, and the stimulatory effect of terbutaline on progesterone production was not significantly affected by blockade of the alpha-adrenergic receptor with phentolamine. These data indicate that placental progesterone production can be significantly modulated by stimulation of beta-adrenergic, but not by alpha-adrenergic, receptors. This response may be mediated by increased intracellular cAMP. These findings may be important in considering other metabolic functions of the placenta as well as the treatment of preterm labor.     

Evidence for activation by beta2-adrenergic receptors of adenosine 3',5'-monophosphate formation in Ehrlich ascites tumor cells.

The existence of aminergic receptors in mouse Ehrlich ascites tumor cells was studied. L-Isoproterenol in vitro stimulated the formation of cAMP in isolated Ehrlich ascites tumor cells. Stimulation by isoproterenol of cAMP formation was not significantly inhibited by practolol, a beta1-adrenoceptor antagonist-Salbutamol, a beta2-adrenoceptor agonist, markedly stimulated the formation of cAMP in Ehrlich ascites tumor cells at concentrations from 10(-8)-10(-3) M. After the addition of salbutamol, cAMP levels reached a maximum in 10 min and declined to about 2-fold of the basal level to 30 min. The stimulation by salbutamol of cAMP formation was markedly inhibited by butoxamine, a beta2-adrenoceptor antagonist, but not by practolol. Furthermore, the effect of a maximal dose of salbutamol was additive to that of prostaglandin E2. Histamine and 4-methylhistamine, a histamine H2 receptor agonist, had no significant effects. Therefore, it is suggested that a beta2-adrenergic receptor exists in the membranes of Ehrlich ascites tumor cells in terms of the adenylate cyclase-cAMP system.     

Cyclic adenosine 3',5'-monophosphate responses to parathyroid hormone, prostaglandin E2, and isoproterenol in dermal fibroblasts from patients with familial benign hypercalcemia.

Plasma concentrations of PTH are much lower for a given calcium or phosphorus level in patients with familial benign hypercalcemia (FBH, or familial hypocalciuric hypercalcemia) than in those with primary hyperparathyroidism; these and other data suggest that there might be tissue hypersensitivity to PTH in FBH. To test this hypothesis, we have used cultured dermal fibroblasts from abdominal skin biopsies of six patients with FBH and six age- and sex-matched controls as surrogate PTH-responsive tissues. Cells in 24-well plastic plates were exposed to vehicle, human PTH-(1-34) (10(-10)-10(-7) M), prostaglandin E2 (10(-6) M), or isoproterenol (10(-4) M) for 10 min in the presence of isobutylmethylxanthine, and cellular cAMP was determined by RIA. All cells responded to PTH with dose-dependent increases in cAMP, and all responded strongly to prostaglandin E2 and isoproterenol. There were no consistent or significant differences between control and FBH fibroblasts in maximal responses to the three agonists, and half-maximal stimulation was achieved with about 10(-9) M PTH in both normal and FBH cells. These data are not consistent with increased tissue sensitivity to PTH in FBH.     

Stimulation by calcitonin gene-related peptide of atrial natriuretic peptide secretion in vitro and its mechanism of action

Calcitonin gene-related peptide (CGRP) is present in nerve fibers within atrial tissue, raising the possibility that CGRP release may influence atrial natriuretic peptide (ANP) secretion. We, therefore, examined the effect of CGRP on immunoreactive ANP (ANP-IR) secretion. Isolated rat left atria paced at 2 Hz were superfused with 0.1 microM CGRP. A biphasic 2-fold increase in ANP-IR secretion occurred in response to CGRP. We next examined the mechanism of CGRP-stimulated secretion. The biphasic ANP-IR secretory response to CGRP was similar to that induced by superfusion with the beta-adrenergic agonist isoproterenol and (Bu)2cAMP, but distinct from that of the non-cAMP dependent stimuli phenylephrine, ouabain, and Bay K 8644. Superfusion with 0.1 microM CGRP for 4 min with 100 microM isobutylmethylxanthine increased atrial cAMP content from 4.29 +/- 1.21 to 10.32 +/- 2.14 pmol/mg atrial weight (P less than 0.001). Atria were next superfused with methacholine, an inhibitor of adenylyl cyclase activation. The addition of 0.1 microM isoproterenol or 0.1 microM CGRP to the superfusate containing 10 microM methacholine failed to stimulate ANP-IR secretion and lowered cAMP accumulation by 70%. Superfusion with 10 microM atropine negated the inhibitory effects of methacholine. We conclude that 1) CGRP stimulates ANP-IR secretion in vitro; and 2) CGRP-stimulated secretion appears to be mediated by cAMP. Thus, ANP-IR secretion may be modulated by atrial nerve fibers containing CGRP in vivo.     

Increased gonadotrophin releasing hormone receptors on pituitary gonadotrophs: effect on subsequent LH secretion

GnRH, high potassium concentrations, and cAMP derivatives have been previously shown to increase GnRH receptor levels (GnRH-R) in cultured rat pituitary cells. However, the effect of these changes in receptor number on subsequent stimulated LH release has not been investigated. In this study pretreatment of pituitary cells with either 1 nM GnRH, 58 mM KCl, or 1 mM dibutyryl cAMP (dbcAMP) resulted in a 70-100% increase in GnRH-R 7-10 h later. Subsequent LH responses to GnRH in those cells pretreated with GnRH and KCl were markedly reduced and the dose-response characteristics altered such that the curves were non-sigmoidal. When corrected for depletion of cellular LH during the pretreatment period these GnRH response curves were similar to control, implying that hormone depletion was the explanation for apparent desensitisation. By contrast, dbcAMP and low-dose calcium ionophore (0.1 microM A23187) pretreatment, which did not deplete cellular LH, neither enhanced nor decreased subsequent sensitivity to GnRH. Thus, 4 agents which all, under these conditions, increased GnRH receptors did not sensitise gonadotrophs to GnRH. By contrast, pretreatment with 10(-9) and 10(-8) M GnRH for either 12 or 16 h rendered cells completely or partially refractory to further GnRH stimulation, despite an increase in GnRH receptors. This desensitisation could not be explained by cellular LH depletion, and was specific to the homologous ligand since dose-responses to the Ca2+ ionophore A23187 and KCl were normal when corrected for LH depletion. Non-receptor-mediated depletion of cellular LH during A23187 pretreatment (10 microM for 10 h) did not alter subsequent GnRH dose-responses, after correction for LH content. These data indicate that, under these in vitro conditions, the increased GnRH receptors are not functionally linked to the secretory apparatus of the gonadotroph. Furthermore, homologous ligand-induced desensitisation is both time- and concentration-dependent and is mediated largely by post-receptor cellular events independent of cellular LH content. Therefore, post-receptor cellular processes may be more important than changes in GnRH receptors in regulating gonadotrophin secretion. It is suggested that an increase in GnRH receptors may represent a cellular response to generalised gonadotroph activation by a variety of agents, and does not necessarily signify enhanced responsiveness to GnRH.     

Studies on desensitization of adrenergic receptors of human adipocytes

The studies described here were undertaken to determine whether or not desensitization of human adipocyte beta and alpha-2 adrenergic receptors could be demonstrated. Cells, isolated from peritoneal adipose tissue obtained from patients undergoing elective abdominal surgery, were preincubated for 3 hr in buffer alone or in the presence of isoproterenol, 10-5M. Cells in both sets of flasks were then washed and exposed to isoproterenol for 1/2 hr; cyclic AMP was then measured as an end point of beta receptor activation. Cells which had had no prior exposure to isoproterenol responded significantly greater to isoproterenol than did cells that had had prior exposure to the catecholamine, The beta receptor characteristics of cells undergoing beta desensitization were assessed using [3H] dihydroalprenolol. Compared to control cells, adipocytes exposed to isoproterenol had a reduction in Bmax while KD values were the same. Thus desensitization of beta adrenergic receptors of human adipocytes occurs and is associated with down regulation in the number of beta receptors. In comparable studies, preincubation with epinephrine 10-5M did not affect the response of cells to a subsequent exposure to this catecholamine. In alpha-2 receptor binding assays, there was a decreased number of [3H]p-aminoclonidine binding sites, but the level of [3H]yohimbine binding was not altered following the incubation with epinephrine. Thus, desensitization of alpha-2 receptors was not demonstrated.     

Adrenergic and serotonergic regulation of skeletal muscle metabolism in the rat: specificity of the serotonin- and isoproterenol-stimulable adenylyl cyclase in sarcolemma

The specificity and kinetics of rat sarcolemmal serotonin- and isoproterenol-stimulable adenylyl cyclases were studied. The stimulation of adenylyl cyclase by serotonin was less than that by isoproterenol and required low concentrations of ATP (0.1 mM), 10 microM GTP, and free Mg ion concentrations of 10-20 microM. The isoproterenol-stimulated activity was readily detectable over a much wider range of MgCl2 (1.0-5.0 mM) and ATP (0.1-3.0 mM). (+/-)-Oxprenolol and l(-)-propranolol inhibited isoproterenol stimulation with KB values of 6.3 X 10(-9) M and 1.9 X 10(-9) M, respectively. Interference by (+/-)-cyproheptadine, (+/-)-methysergide and d(+)-propranolol was not strictly competitive. However, these were much more potent inhibitors of serotonin stimulation than were oxprenolol and l-propranolol. The concentrations of antagonists producing 50% inhibition (IC50) of the maximal adenylyl cyclase stimulation by serotonin were compared to the IC50 obtained with isoproterenol; the ratios were 0.02 for oxprenolol, 0.50 for l-propranolol, 295 for d-propranolol, 2438 for cyproheptadine, 578 for methysergide and greater than 125,000 for lysergic acid diethylamide (LSD). These data indicate that rat skeletal muscle adenylyl is stimulated by discrete serotonergic and adrenergic receptors, and that there may be distinct conditions for optional stimulation.     

Stimulatory effect of beta-adrenergic agonists on ileal L cell secretion and modulation by alpha-adrenergic activation.

Postprandial release of peptide YY (PYY) and glucagon-like peptide-1 (GLP-1) from L cells results from both nutrient transit in the ileal lumen and neural drive of endocrine cells. The adrenosympathetic system and its effectors have been shown to induce secretion of L cells in vivo or in vitro. Because these transmitters act through three receptors, beta, alpha1, alpha2, coupled to different intracellular pathways, we evaluated the responses of L cells to specific agonists, using the model of isolated vascularly perfused rat ileum. General stimulation of adrenergic receptors with epinephrine (10(-7) M) induced significant GLP-1 and PYY secretions (94+/-38 and 257+/-59 fmol/8 min respectively) which were abolished upon propranolol (10(-7) M) pretreatment and strongly decreased upon infusion with 10(-8) M prazosin. Blockade of alpha2-receptors with idazoxan (10(-8) M) did not alter epinephrine-induced peptide secretion. The beta-adrenergic agonist isoproterenol (10(-6) M) infused for 30 min induced a transient release of GLP-1 and PYY (integrated release over the 8 min of the peak secretion: 38+/-16 and 214+/-69 fmol for GLP-1 and PYY respectively, P<0.05). Because terbutaline but not dobutamine or BRL 37,344 (10(-5) M) induced significant GLP-1 and PYY secretions (135+/-30 and 305+/-39 fmol/8 min respectively), isoproterenol-induced secretions are suggested to result mainly from stimulation of the beta2-isoreceptor type. In contrast, the alpha1-agonist phenylephrine (10(-7) M) did not stimulate peptide release. When co-infused with 10(-6) M or 10(-7) M isoproterenol, 10(-7) M phenylephrine raised GLP-1 release to 174+/-53 and 108+/-28 fmol/8 min respectively (vs 38+/-16 and 35+/-10 fmol/8 min for isoproterenol alone, P<0.05) whereas PYY secretion was not significantly increased. Clonidine (10(-7) M), an alpha2-agonist, induced a moderate and delayed increase of GLP-1 and PYY but abolished the isoproterenol-induced peptide secretion. Our results showed that general stimulation of adrenergic receptors stimulates the secretory activity of ileal endocrine L cells. The net peptide secretion results from the activation of the beta2-isoreceptor type. Additionally, GLP-1 and PYY secretions are positively modulated by alpha1-receptor stimulation and inhibited by alpha2-receptor activation upon beta-receptor occupation.     

Characterization and regulation of high affinity calcitonin gene-related peptide receptors in cultured neonatal rat cardiac myocytes.

Previous studies have shown that stimulation of cultured beating cardiac myocytes with calcitonin gene-related peptide (CGRP) produces increased beating frequency, increased cellular cAMP concentration, and a homologous desensitization of the cAMP-elevating action of CGRP. In the present study, the characteristics and regulation of [125I]CGRP binding sites in cultured cardiac myocytes were investigated. Binding of [125I] CGRP to membranes prepared from these cells was selective, saturable, and of high affinity. Scatchard transformation of the saturation isotherm generated a linear plot suggesting the existence of a homogeneous population of binding sites with an equilibrium binding constant of 41 +/- 7 pM and maximum binding capacity of 31 +/- 5 fmol/mg protein. Binding of [125I]CGRP to membranes was inhibited completely by guanosine 5'-(3-O-thio)triphosphate (250 microM), suggesting association of the binding sites with a G protein. Consistent with the saturation binding data, association kinetic studies indicated that [125I]CGRP associated with a single population of binding sites. Dissociation kinetic data, in contrast, indicated that [125I]CGRP dissociated from two affinity component sites on membranes, suggesting the existence of multiple affinity states of the G protein-coupled forms of the CGRP receptor. Nonequilibrium dissociation kinetic experiments revealed a time-dependent conversion of [125I] CGRP binding sites from a fast- to a slow-dissociating state. Desensitization of cells to CGRP by prior exposure to CGRP (10 nM) for 5 min reduced the maximal cAMP response of cells to further CGRP challenge and the number of [125I]CGRP binding sites in membranes prepared from these cells approximately 90% and 80%, respectively. These results demonstrate the existence of high affinity CGRP receptors in cardiac myocytes which appear coupled to G proteins and which undergo ligand-induced affinity alterations and desensitization-induced loss of receptor activity. The present findings also suggest the existence of multiple affinity states of the CGRP:receptor:G protein ternary complex.     

The effects of trimetoquinol on the intact rabbit heart and myocardial adenylate cyclase activity: evidence for spare myocardial beta receptors

We have compared and contrasted the actions of (-)isoproterenol and (+/-) trimetoquinol on rabbit heart preparations. In the presence of either GTP or Gpp[NH]p (guanosine-5'-(beta, gamma imino) triphosphate), trimetoquinol displayed partial agonist activity in stimulating adenylate cyclase activity in a particulate rabbit heart preparation. Trimetoquinol enhanced adenylate cyclase activity 20% or 65% of the maximum obtainable by isoproterenol in the presence of GTP or Gpp[NH]p respectively. In the presence of GTP, concentrations of catecholamines required to enhance cyclase activity 15% of the maximum obtainable with isoproterenol (EC15) were 2.0 X 10(-7) M and 5.5 X 10(-8) M for trimetoquinol and isoproterenol, respectively. In the presence of Gpp[NH]p EC30 values were 2.0 X 10(-7) and 3.5 X 10(-8) M for trimetoquinol and isoproterenol respectively. Trimetoquinol also displayed partial agonist activity for the ability to increase cAMP levels in the isolated perfused rabbit heart. By contrast trimetoquinol was equieffective to isoproterenol at increasing tension development and rate of contraction of the isolated perfused heart. Concentrations of catecholamines required to increase tension and rate of contraction 50% of the maximum obtainable with isoproterenol were 1.5 X 10(-7) M and 1.7 X 10(-8) M for trimetoquinol and isoproterenol, respectively. These data show that only a partial stimulation of adenylate cyclase activity and cAMP levels by trimetoquinol is sufficient to produce maximal changes in mechanical activity of the heart.(ABSTRACT TRUNCATED AT 250 WORDS)     

DOPAMINE INHIBITS Na,H-EXCHANGER VIA D1-LIKE RECEPTOR-MEDIATED STIMULATION OF PROTEIN KINASE A IN RENAL PROXIMAL TUBULES

Dopamine causes natriuresis and diuresis via activation of D₁-like receptors located in the renal proximal tubules. It is reported that this response to dopamine results from the inhibition of Na,H-exchanger and Na,K-ATPase. Earlier studies have suggested a role of protein kinase A (PKA) in the inhibition of Na,H-exchanger, however, the effect of dopamine or the dopamine receptor subtype responsible for the stimulation of PKA has not been reported. Present study was designed to examine the effect of dopamine and D₁-like receptor agonist, SKF 38393, on the stimulation of PKA activity in rat renal proximal tubules. Dopamine and SKF 38393 (1 nM – 1 μM) caused stimulation of PKA activity, an effect which was antagonized by a D₁-like receptor antagonist, SCH 23390 (10 μM). Stimulation of PKA activity was also seen with forskolin and di-butyryl cAMP. We also observed that dopamine and SKF 38393 inhibited Na,H-exchanger activity in the proximal tubules. This response was blocked by SCH 23390 and Rp-cAMPS triethylamine, a selective inhibitor of PKA. Similarly, forskolin and di-butyryl cAMP inhibited Na,H-exchanger activity. The data provide direct evidence showing that dopamine, through the activation of D₁-like receptors stimulates PKA activity which in turn inhibits Na,H-exchanger in the proximal tubules.