Cyclic Monophosphate Formation Research Articles

Adaptations of the beta-adrenoceptor-adenylyl cyclase system in rat skeletal muscle to endurance physical training.

beta-Adrenergic mechanisms may be important in the adaptation of skeletal muscle to endurance training. beta-Adrenergic signal transduction was examined in the gastrocnemius muscle of rats submitted to a progressive, 12-week treadmill running program and compared with sedentary controls. beta-Adrenoceptor density was significantly lower in exercised rats than in controls. The affinity constant for [125I]-(-) iodocyanopindolol binding was not different among the various groups. Adenosine cyclic monophosphate formation was significantly decreased in trained animals when isoproterenol plus guanosine triphosphate or forskolin plus Mn2+ were used to stimulate adenylyl cyclase. Immunoblot analyses revealed that the amount of the alpha-subunit of stimulatory guanine nucleotide-binding protein (Gs,alpha), both the small and the large isoforms, also decreased with physical exercise. Thus, the present report shows that endurance training results in alterations in beta-adrenergic receptor density, adenylyl cyclase activity and Gs protein level in rat gastrocnemius muscle.

CAMP mediates homologous downregulation of PAF receptor mRNA expression in mesangial cells.

To clarify the molecular mechanism and significance of homologous desensitization of platelet-activating factor (PAF) signaling, we examined the effect of PAF on PAF receptor mRNA expression in rat mesangial cells by Northern blot analysis. Treatment of the cells with PAF (10(-7)-10(-8) M) reduced the expression of PAF receptor mRNA in 1 h, and this reduction was recovered by pretreatment of mesangial cells with a specific PAF receptor antagonist, WEB-2086 (10(-6) M), or a cyclooxygenase inhibitor, indomethacin (10(-6) M), for 10 min. PAF-stimulated prostaglandin E2 (PGE2) and adenosine 3',5'-cyclic monophosphate (cAMP) formation was measured by radioimmunoassays specific for each substance. Reduction of PAF receptor mRNA expression was mimicked by treatment of the cells with PGE2 (10(-6) M) or dibutyryl-cAMP (10(-3) M), a cell-permeable analog of cAMP. These results, taken together, suggest that PAF receptor mRNA expression is downregulated by exposure to PAF in cultured mesangial cells. This homologous downregulation is mediated by cAMP production through PGE2 synthesis.

Vasodilation and glomerular binding of adrenomedullin in rabbit kidney are not CGRP receptor mediated.

The polypeptide adrenomedullin (ADM) was infused systemically to conscious rabbits to elucidate its actions on overall circulation and especially the renovascular bed and the formation and/or release of hormones important for body fluid homeostasis, including adrenocortical steroids. ADM lowered mean arterial pressure from 71.5 +/- 3.2 to 64.7 +/- 3.2 mmHg only at the highest dose of 25 pmol.min-1.kg-1 infused intravenously for 20 min and concomitantly induced tachycardia, possibly due to both baroreflex activation and direct cardiostimulatory effects. Renal blood flow (RBF) determined in rabbits chronically equipped with a perivascular ultrasonic flow probe increased from 55.4 +/- 2.1 to 67.4 +/- 2.7 and from 58.2 +/- 3.5 to 75.2 +/- 6.0 ml/min at ADM infusions of 5 and 25 pmol.min-1.kg-1, respectively. The elevation in RBF persisted even in the presence of the calcitonin gene-related peptide (CGRP1 receptor antagonist CGRP-(8-37). Of all osmoregulatory hormones tested, only corticosterone (Cort) plasma concentration increased in response to the highest ADM dose from 17.6 +/- 3.1 to 38.9 +/- 6.2 ng/ml, probably due to haroreflex activation. Subdepressor doses of ADM, however, caused a mild reduction in circulating Cort. Expression of functional high-affinity binding sites specific for ADM in vitro could be demonstrated for the renal artery and outer cortical glomeruli using 125I-labeled rat ADM as radioligand and determination of cellular adenosine 3',5'-cyclic monophosphate (cAMP) formation within the glomeruli. The ineffectiveness of CGRP-(8-37) to displace radiolabeled ADM from its binding sites, to inhibit ADM-induced glomerular cAMP formation, and to prevent ADM-induced renal vasodilation supports the hypothesis of ADM altering renal hemodynamics by interacting with ADM- and not CGRP-specific membrane receptors.

Modulation of gliquidone action by forskolin in the pancreas of normal and GK rats.

This study aims to investigate whether agents that stimulate adenosine 3',5'-cyclic monophosphate (cAMP) formation could be used to increase insulin release evoked by hypoglycemic sulfonylureas in non-insulin-dependent diabetes mellitus. For this purpose, the effect of gliquidone (1.0 microM) on insulin and glucagon release was examined in the perfused pancreas of either normal or Goto-Kakizaki (GK) rats at a low concentration of D-glucose (2.8 mM) and in the absence or presence of forskolin (1.3 microM). In normal rats, the diterpene exerted relatively little effect on basal insulin release but markedly augmented the insulinotropic action of gliquidone. In GK rats, forskolin dramatically augmented both basal and gliquidone-stimulated insulin output. In mirror image of its effect on insulin release, forskolin augmented basal glucagon output and failed to increase the secretory response of the A cell to gliquidone, at least in normal rats. On the contrary, in GK rats, forskolin, while slightly enhancing basal glucagon output, unmasked the glucagonotropic potential of gliquidone that was otherwise not detected in the diabetic animals. These findings are interpreted in light of a dual metabolic and energy-independent response of islet cells to the forskolin-induced generation of cAMP. They document the optimalization by endogenous cAMP of the B cell secretory response to gliquidone in non-insulin-dependent diabetes.

Effect of cholestasis on regulation of cAMP synthesis by glucagon and bile acids in isolated hepatocytes.

Previously, we have reported that bile acids can directly inhibit hormone-induced adenosine 3',5'-cyclic monophosphate (cAMP) formation through a protein kinase C (PKC)-dependent mechanism [Bouscarel, B., T.W. Gettys, H. Fromm, and H. Dubner. Am. J. Physiol. 268 (Gastrointest. Liver Physiol. 31): G300-G310, 1995]. Therefore, the regulation of cAMP synthesis by glucagon and bile acids was investigated in hepatocytes isolated after 2-day ligation of the common bile duct in Golden Syrian hamsters. The bile acid concentration was increased 30-fold in the serum, whereas it was not significantly different in the bile of duct-ligated vs. sham-operated hamsters. The glycine/taurine and cholate/chenodeoxycholate ratios were significantly increased fourfold and sevenfold, respectively, only in the serum of bile duct-ligated hamsters. Ligation of the bile duct decreased the efficacy of glucagon-stimulated cAMP synthesis by 40-50% without changing its potency. This attenuation of cAMP synthesis, which was also observed with forskolin, remained in the absence of any detectable amount of bile acids in the hepatocytes. The decrease in glucagon-stimulated cAMP production was also not attributable to changes in either the affinity or the number of receptors for this hormone. The potency and efficacy of the bile acids to inhibit glucagon-induced cAMP formation was also reduced in bile duct-ligated hamsters. The inhibitory regulation of cAMP synthesis through angiotensin II was similarly diminished after bile duct ligation. Although the total expression of PKC-alpha was not affected, an increased translocation by 60% from the cytosol to the membrane fraction was observed in hepatocytes isolated after bile duct ligation. Therefore, during cholestasis and prolonged exposure of the liver to bile acids, both the stimulatory and inhibitory regulatory, mechanisms of cAMP synthesis are compromised in an irreversible manner because the effects persist even after isolation of the hepatocytes. This decreased regulation of cAMP synthesis is possibly mediated through PKC-alpha activation.

Molecular and functional identification of beta-adrenergic receptors in distal convoluted tubule cells.

Renal nerve stimulation or circulating catecholamines activate the beta-adrenergic receptors that mediate direct effects on tubular transport. Three subtypes of beta-adrenergic receptors have been characterized: beta 1, beta 2, and beta 3. beta-Adrenergic-receptor effects on Na+ and Ca2+ transport in distal convoluted tubules (DCT) have not been established. The focus of this study was to 1) identify the subtypes of beta-adrenergic receptors in DCT cells and 2) examine functional responses to beta-receptor activation on adenosine 3',5'-cyclic monophosphate (cAMP) formation and Na+ and Ca2+ entry. To determine the subtypes of beta-receptors present, RNA isolated from immortalized mouse DCT cells was reverse transcribed, and the cDNA was amplified using primers designed to reported sequences for beta 1-, beta 2-, and beta 3-receptor subtypes. Products of the appropriate sizes were obtained with beta 1- and beta 2-primers. No product was observed with primers to the beta 3 sequence. Receptor products were confirmed by sequencing and are identical to reported mouse beta 1- and beta 2-receptor sequence. Receptor binding of[3H]dihydroalprenolol was 123 +/- 13 fmol/mg protein, and a 3:1 ratio of beta 1- to beta 2-receptors was observed with DCT cell membranes. Isoproterenol, a beta-receptor agonist, increased cAMP formation 8.5-fold. Pretreatment with the antagonist propranolol abolished agonist-induced cAMP accumulation. Isoproterenol significantly increased 22Na+ uptake to 345 +/- 23 compared with a basal rate of 256 +/- 12 nmol.min-1.mg protein-1 and was blocked with propranolol and beta 1- and beta 2-selective antagonists. Isoproterenol had no effect on 45Ca2+ entry into DCT cells. In summary, DCT cells express three times more beta 1- than beta 2-receptors and express no detectable beta 3-adrenergic receptors. beta-Receptors couple to adenylyl cyclase, and activation of beta-adrenergic receptors increases Na+ but not Ca2+ entry in DCT cells.

Effect of uncoupling NO/cGMP pathways on carbachol- and CCK-stimulated Ca2+ entry and amylase secretion from the rat pancreas.

Nitric oxide (NO) production reportedly regulates guanosine 3', 5'-cyclic monophosphate (cGMP) formation and Ca2+ influx in pancreatic acini. We have investigated the functional roles of the NO/cGMP messenger system in rat pancreatic acini. In dispersed acini, the levels of amylase secretion, cytosolic [Ca2+]([Ca2+]i), NO synthase, and cGMP were measured. The NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 0.01-100 microM) had no effect on amylase secretion induced by various concentrations of carbachol, cholecystokinin octapeptide (CCK-8) or the high affinity CCK agonist, JMV-180. Similarly, L-NAME up to 100 microM did not affect the changes in Ca2+ spiking evoked by these secretagogues; nor was Ca2+ entry, refilling or oscillation altered by L-NAME. Sub- and supramaximal concentrations of these secretagogues did not change NO synthase activities compared with basal levels. While sodium nitroprusside (SNP), a NO donor, caused a 9.4-fold increase in cGMP levels compared with basal levels, carbachol, CCK-8 and JMV-180 had no effect. In addition, the guanylate cyclase inhibitor LY 83583 (10 nM to 10 microM) altered neither amylase secretion nor Ca2+ signaling induced by these secretagogues. These findings indicate that the stimulatory action of carbachol or CCK-8 is not mediated by NO or cGMP. To investigate whether cGMP stimulates pancreatic secretion we showed that both SNP and a cell-permeant cGMP analog at 0.1-1 mM stimulated amylase secretion and Ca2+ transients to a level equal to 10-15% and 13-24%, respectively, of those observed with maximal concentrations of secretagogues. The guanylate cyclase activator guanylin (1-10 microM), which increased cGMP levels 2.4-fold compared with basal levels, elicited a small amount of amylase secretion and a small Ca2+ transient. In conclusion, exogenous NO is capable of increasing endogenous cGMP, which results in a modest increase in the [Ca2+]i transient and pancreatic amylase secretion. However, the NO/cGMP system does not appear to be involved significantly in the mediation of Ca2+ signaling and amylase secretion stimulated by carbachol and CCK-8.

Adenylate cyclase-coupled ATP receptor and surfactant secretion in type II pneumocytes from newborn rats.

ATP stimulation of surfactant secretion in type II cells is mediated by both a P2Y2 receptor coupled to phospholipase C and a receptor coupled to adenylate cyclase. UTP also activates the P2Y2 receptor but does not stimulate adenosine 3',5'-cyclic monophosphate (cAMP) formation. We have examined surfactant secretion and signaling parameters in response to ATP and UTP in type II cells from newborn rats. There was a developmental increase in the response to both agonists. However, whereas ATP increased secretion as early as day 1, the effect of UTP did not become significant until 4 days after birth. ATP increased cAMP formation as early as day 1 but did not promote diacylglycerol formation or phospholipase D activation until day 4. Thus the adenylate cyclase-coupled ATP signaling mechanism is functional early in development but the P2Y2 pathway is not. We therefore used type II cells from 1- to 2-day-old rats to investigate the adenylate cyclase-coupled mechanism in the absence of interactions with the P2Y2 system. Effects of ATP and 5'-(N-ethylcarboxamido)adenosine (NECA) on surfactant secretion and cAMP formation were not additive, and their effects on secretion were antagonized by the same adenosine receptor antagonists. Overnight culture of the cells with NECA almost completely abolished the subsequent increase in cAMP formation in response to NECA, adenosine, and ATP but not to terbutaline. These data suggest that ATP, NECA, and adenosine activate the same receptor. Effects of ATP were not decreased by adenosine deaminase, showing that they are not mediated by adenosine acting directly at adenosine receptors. We suggest that ATP directly activates an adenosine receptor on the type II cell.

Parathyroid hormone-induced calcium efflux from cultured bone is mediated by protein kinase C translocation.

Activation of adenylate cyclase is believed to be the major intracellular mediator of bone resorption induced by parathyroid hormone (PTH), prostaglandins, and various bone resorbing cytokines. Studies have demonstrated a dissociation between PTH-induced bone resorption and adenosine 3',5'-cyclic monophosphate (cAMP) formation, as well as suggested a role of protein kinase C (PKC) in mediating in part the actions of PTH. We therefore investigated the relative contribution of the adenylate cyclase or PKC signal transduction pathways in mediating the PTH-induced net calcium release from cultured neonatal calvariae, an in vitro model of bone resorption. PTH (10(-11) to 10(-7) M) caused a dose-dependent increase in calcium efflux from cultured bone and activated both cAMP and PKC. To determine the role of each of these second messengers in mediating PTH-induced calcium release from bone, calvariae were preincubated with either the adenylate cyclase inhibitor SQ-22536 (10(-5) to 10(-4) M) or the PKC inhibitor staurosporine (10(-7) M) before coincubation with PTH. Compared with control, PTH caused a significant calcium efflux, whereas preincubation with SQ-22536 had no effect on basal calcium efflux and partially inhibited the calcium efflux caused by PTH. In contrast preincubation with staurosporine completely obliterated the PTH-induced calcium efflux. PTH is a potent stimulator of calcium release and activates both the cAMP and PKC signal transduction pathways in cultured bone. Inhibition of PTH-stimulated PKC activity completely abolished the PTH-induced calcium efflux from calvariae, whereas PTH-induced calcium efflux persisted despite adenylate cyclase inhibition. Thus the bone resorbing effect of PTH appears to be dependent predominantly on activation of PKC.

Inhibition of purified soluble guanylyl cyclase by copper ions

The aim of the present study was to investigate the effect of Cu(II) ions on soluble guanylyl cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2; sGC] and to test for a possible physiological role of this putative cofactor of the enzyme [Gerzer et al., FEBS Lett. 132: 71–74, 1981]. CuSO 4 was found to inhibit NO-stimulated sGC with an IC 50 of 2.2 ± 0.3 μM. Virtually complete inhibition of guanosine-3′,5′-cyclic monophosphate (cGMP) formation was observed at 10 μM of the copper salt. Presence of CuSO 4 (2 μM) did not significantly affect the potency of 2,2-diethyl-1-nitroso-oxyhydrazine (DEA/NO) but did markedly decrease maximal cyclase activity from 3.71 ± 0.2 μmol cGMP × mg −1 × min −1 to 1.75 ± 0.2 μmol cGMP × mg −1 × min −1. The nonstimulated enzyme was also sensitive to CuSO 4 (IC 50 of 6.2 ± 1.2 μM). Addition of glutathione, which potently complexes Cu(I) ions, induced a pronounced rightward shift of the concentrationresponse curves for inhibition by CuSO 4 of both DEA/NO-stimulated and nonstimulated guanylyl cyclase. The inhibitory effect of CuSO 4 was completely antagonized by the specific Cu(I) chelator neocuproine, with a half-maximal effect at 5.9 ± 0.2 μM. In contrast, the Cu(II) chelator cuprizone and several thiols, which do not form stable Cu(I) complexes, were far less protective. Our results suggest that inhibition of soluble guanylyl cyclase by CuSO 4 is unrelated to heme-mediated enzyme stimulation and may arise from the reversible high affinity binding of Cu(I) ions to a site of the protein that is critically involved in enzyme catalysis.

ETA and ETB receptors cooperate in DNA synthesis via opposing regulations of cAMP in human lung cell line

We investigated the contribution of endothelin type A (ETA) and ETB receptors on ET-induced DNA synthesis in CCD-18Lu cells, a human lung cell line possessing both ETA and ETB (ETA/ETB ratio: 9:1). ET-1 (0.05-2 nM) potently induced [3H]thymidine incorporation by 2- to 14-fold over the basal level. An ETA-selective antagonist, FR139317, inhibited 0.2 nM ET-1-induced DNA synthesis dose dependently, showing complete inhibition at 1 microM. ET-3 was inactive up to 2 nM. In contrast, ETB-selective antagonists, 100 nM of BQ-788 or IRL 2500, partially (30-60%) inhibited 0.2 nM ET-1-induced DNA synthesis. Stimulation of either ETA or ETB evoked the increases in intracellular Ca2+ concentration ([Ca2+]i). ETB-mediated but not ET-1-induced [Ca2+]i increase was pertussis toxin (PTX) sensitive. Adenosine 3',5'-cyclic monophosphate (cAMP) formation via ETA was observed in PTX-treated cells, whereas the inhibition of isoproterenol-stimulated cAMP formation via ETB was observed in PTX-untreated cells. Like the ETB-selective antagonists, PTX treatment or dibutyryl cAMP partially (50-70%) inhibited ET-1-induced DNA synthesis. These data suggest that 1) ET-1 induces DNA synthesis predominantly through ETA, via PTX-insensitive G protein; 2) ETA-mediated cAMP formation inhibits DNA synthesis; and 3) stimulation of ETB coupling to Gi protein modulates ETA-mediated DNA synthesis by inhibiting cAMP formation.

Diurnal and seasonal changes in sympathetic signal transduction in cardiac ventricles of European hamsters.

This investigation of the relationship between cardiac beta-adrenoceptors and adenosine 3',5'-cyclic monophosphate (cAMP) formation in cardiac ventricles of the nocturnally active European hamster both during euthermia under a 12:12-h dark-light cycle and during hibernation under constant-darkness conditions showed that neither the densities, affinities, nor distribution of the beta 1- and beta 2-receptor subtypes differed between the dark phase, light phase, and hibernation. Basal formation of cAMP by the cardiac adenylyl cyclase of euthermic hamsters was higher in ventricles obtained at night [core temperature (Tcore) = 37.8 degrees C] than in ventricles obtained during the day (Tcore = 36.4 degrees C). Basal formation of cAMP was also significantly lower in hibernating hamsters (Tcore = 7.0 degrees C) than in euthermic hamsters. When adenylyl cyclase activity was stimulated by isoprenaline, guanylylimidodiphosphate [Gpp(NH)p], or forskolin, the rank order of potency was the same in euthermic hamsters and hibernating hamsters: isoprenaline < Gpp(NH)p < forskolin. Functional competition curves indicated that in the euthermic hamsters beta 1-receptors were responsible for 67% of the response to isoprenaline at night and 62% of the response during the day. In hibernating hamsters, in contrast, most of the response to isoprenaline (58%) was mediated via beta 2-receptors. This shift in the relative importance of the receptor subtypes may facilitate arousal from hibernation by making the heart more sensitive to circulating epinephrine.

Polarized distribution of renal natriuretic peptide receptors in normal physiology and ischemia.

The polarized expression of guanylyl cyclase-coupled natriuretic peptide receptors, types A (GC-A) and B (GC-B), was measured in inner medullary collecting ducts (IMCD) of normal and ischemic rat kidneys, as well as in IMCD cells. Exposure of normal rat kidney medulla to an anti-GC-A antibody demonstrated a propensity of receptor staining on the cellular basal membrane. The polarization of GC-A receptors was lost in the ischemic kidney. The maximal binding capacity of 125I-atrial natriuretic factor (ANF) to the basal membrane of the inner medullary cell line mIMCD-K2 was five times greater than that to the apical membrane. ANF or C-type natriuretic peptide (CNP) added to the basal side of cultured cells resulted in guanosine 3',5'-cyclic monophosphate formation that was greater than when applied to the apical side. Depletion of ATP stores in cultured cells was followed by an increase of 125I-ANF binding to apical cellular membranes. Similar results were obtained when receptor guanylyl cyclase activity was assayed. In conclusion, these results suggest that functional GC-A and GC-B receptors are present predominantly on the basal membrane of IMCD. However, depletion of cellular ATP stores such as in ischemia is followed by a partial loss of polarization.

Coexpression of 5-HT 2A and 5-HT 4 receptors coupled to distinct signaling pathways in human intestinal muscle cells

Background & Aims: The type and function of 5-hydroxytryptamine (5-HT) receptors on intestinal muscle cells in humans are not known. 5-HT receptors were characterized pharmacologically and by radioligand binding. Methods: Contraction, relaxation, inositol 1,4,5-triphosphate (IP 3) and adenosine 3′,5′-cyclic monophosphate (cAMP) formation, and 5-HT binding were measured in dispersed muscle cells and in cells in which only one receptor type was preserved by selective receptor protection. Results: 5-HT binding was completely inhibited by 5-HT and partially by 5-HT 2A (ketanserin), 5-HT 4 (SDZ-205,557), and 5-HT 1p ( N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide; 5-HTP-DP) receptor antagonists. 5-HT caused contraction that was inhibited by ketanserin and augmented by SDZ-205,557 and 5-HTP-DP. In the presence of ketanserin, 5-HT caused relaxation of cholecystokinin-contracted cells that was inhibited by SDZ-205,557 and 5-HTP-DP. 5-HT increased IP 3, which was inhibited by ketanserin, and cAMP, which was inhibited by SDZ-205,557 and 5-HTP-DP. In cells with only 5-HT 2A receptors, 5-HT caused contraction only, and residual binding was inhibited by ketanserin. In cells with only 5-HT 4 5-HT 1p receptors, 5-HT caused only relaxation and residual binding was inhibited by SDZ-205,557 and 5-HTP-DP. Conclusions: 5-HT 2A receptors mediating contraction and 5-HT 4 receptors mediating relaxation coexist on human intestinal muscle cells. The 5-HT 4 receptors are closely similar or identical to 5-HT 1p receptors.

Effects of LDL on intracellular free calcium and nitric oxide-dependent cGMP formation in porcine endothelial cells

Nitric oxide (NO)-mediated, endothelium-dependent vasodilation is reduced in atherosclerotic arteries. A number of in vivo studies suggest that infusion of the substrate of NO synthase, L-arginine, partly counteracts this effect. We studied the potential inhibitory effects of native and of oxidized low density lipoproteins (n-LDL, ox-LDL) on NO-dependent cyclic guanidine monophosphate (cGMP) formation in porcine aortic endothelial cells and the role of extracellular L-arginine in counteracting this process. NO-dependent cGMP production in the cells (passage 1; preincubated in L-arginine-free medium for 24 h) was stimulated for 3 min with bradykinin (BK 1 nM) or the calcium-ionophore A23187 (100 nM) or by a 20 min incubation with L-arginine (L-Arg 0.1 mM, 20 min). The endothelium-independent NO-donor, sodium nitroprusside (SNP 1 μM) was used as control stimulus. Experiments were performed in the presence of LDL which was kept as much as possible under antioxidative conditions, further referred to as n-LDL (1 mg/ml), or LDL which was oxidized by incubation with copper (ox-LDL 0.1 mg/ml). The N G-nitro-L-arginine (L-NNA, inhibitor of NO-synthase)-sensitive intracellular cGMP concentration was taken as a measure of endothelial NO formation and determined by radioimmunoassay. BK-induced changes of intracellular free Ca ++ were measured immediately after washout of LDL using the FURA-2 method. n-LDL reduced the cGMP-levels in unstimulated cells as well as the cGMP increase in response to bradykinin (−10%) and the calcium-ionophore A23187 (−80%). The SNP-induced cGMP-increase was, however, not affected. L-arginine increased the intracellular cGMP concentration under both conditions by a similar amount, without affecting intracellular free calcium. Uptake of 3H-L-arginine was not significantly altered by n-LDL treatment. Ox-LDL significantly reduced SNP-induced cGMP-increases but did not alter bradykinin-induced cGMP increases. The BK-induced increase of intracellular free calcium was even enhanced after exposure of the cells to ox-LDL. L-arginine increased cGMP by a similar amount as in untreated cells. It is concluded that both n-LDL and ox-LDL can reduce the NO-dependent cGMP formation in cultured endothelial cells, albeit by different mechanisms. However, a limitation of the uptake or availability of extracellularly applied L-arginine does not appear to be a causal factor, at least not after 2 h exposure.

Dual regulation of cGMP formation by calcium in pancreatic acinar cells.

Regulation of guanosine 3',5'-cyclic monophosphate (cGMP) formation by calcium and calcium-binding proteins was studied at the levels of nitric oxide synthase (NOS) and guanylyl cyclase (GC) in dispersed pancreatic acini isolated from guinea pig. In intact cells, in the cytosol, and on diethylaminoethyl fractions from cytosolic proteins, GC activity was negatively regulated by Ca2+. An increase in Ca2+ concentration ([Ca2+]) from 25 to 950 nM suppressed cGMP formation by 85%. On the other hand, NOS was stimulated by agents increasing cytosolic [Ca2+] and inhibited by intracellular Ca2+ chelators. Thus Ca2+ regulates cGMP production in opposite directions by activating NOS and inhibiting GC. Calmodulin antagonists W-7, trifluoperazine, and R-24571 inhibited NOS, suggesting that the enzyme is regulated by calmodulin as in other cell types. Calmodulin antagonists appeared to inhibit GC. In particular, 200 microM W-7 completely abolished the cGMP rise evoked by the nitric oxide donor, nitroprusside. The effect was not reversed by addition of excess calmodulin. The findings suggest that the negative regulation of GC by Ca2+ is due to factors other than calmodulin but affected by calmodulin antagonists.

Heparin‐insensitive calcium release from intracellular stores triggered by the recombinant human parathyroid hormone receptor

1. In the present study we have characterized the parathyroid hormone (PTH)-induced calcium signalling in 293 cells stably transfected with the human PTH receptor cDNA. In these cells, human PTH-1(1-38) strongly stimulates adenosine 3':5'-cyclic monophosphate (cyclic AMP) formation (EC50 = 0.39 nM) but fails to activate phosphoinositide (PI) turnover. The latter pathway is strongly activated, however, by carbachol (CCh) acting through endogenous M3-muscarinic receptors. 2. Despite the lack of detectable inositol phosphate (IP) formation, hPTH-(1-38) elicited calcium transients (EC50 = 11.2 nM) which were comparable to the signals evoked by CCh. These signals are independent of cyclic AMP generation as cyclic AMP elevating agents did not mimic or modify the PTH response. 3. The PTH-stimulated calcium signal still occurred in calcium-free medium but was absent in cells pretreated with thapsigargin, an inhibitor of the calcium pump of the endoplasmic reticulum (ER). hPTH-(1-38) did not accelerate Mn(2+)-influx through the plasma membrane. These data indicate that PTH releases calcium from intracellular stores. 4. Using heparin, an inhibitor of the IP3-activated calcium release channel of the ER, we tested whether the formation of a low amount of IP3, escaping detection by our biochemical assay, might be the origin of the PTH-induced calcium response. However, intracellular infusion of heparin through patch pipettes in voltage clamp experiments failed to block hPTH-(1-38)-induced calcium signals, whereas it abolished the CCh response. 5. The PTH response, like the CCh response, was insensitive to micromolar concentrations of ryanodine and ruthenium red, eliminating the possibility that hPTH-(1-38) stimulates calcium-induced calcium release through ryanodine receptors.6. We conclude that the recombinant human PTH receptor stimulates calcium release from intracellular stores through a novel pathway not involving IP3- or ryanodine receptors.

Cardiac beta-adrenoceptor-effector coupling in portal vein-stenosed rats.

Cardiac beta-adrenergic signal transduction was examined in chronic portal vein-stenosed rats. Basal tension and maximum rate of tension development were significantly depressed in left ventricular papillary muscles (0.21 +/- 0.03 N/cm2 and 8.2 +/- 1.7 N.s-1.cm-2, respectively) compared with sham-operated controls (0.51 +/- 0.05 N/cm2 and 19.9 +/- 4.4 N.s-1.cm-2, respectively). The positive inotropic response to isoproterenol was also attenuated. Adenosine 3',5'-cyclic monophosphate formation was decreased significantly when GTP (-41.9%), isoproterenol with GTP (-45.3%), or guanosine 5'-O-(3-thiotriphosphate) (-52.4%) was used to stimulate adenylyl cyclase, but not when Mn2+ or forskolin was used. Beta-Adrenoceptor density (sham operated 24.6 +/- 2.0 fmol/mg; portal vein stenosed 26.4 +/- 2.1 fmol/mg) and the apparent dissociation constant (sham operated 0.26 +/- 0.04 nM; portal vein stenosed 0.29 +/- 0.04 nM) were unaffected. Portal venous hypertension did not alter beta-adrenergic receptor affinity for isoproterenol. However, it was necessary for isoproterenol to occupy three times the number of receptors in papillary muscles from stenosed animals to produce an equal increase in force generation. These data suggest that although portal vein stenosis does not alter cardiac beta-adrenoceptor density or affinity for ligands, transduction of the signal between the receptor and adenylyl cyclase is adversely influenced and may be responsible for the diminished responsiveness of beta-adrenoceptors in the myocardium.

Human muscarinic receptors expressed in A9L and CHO cells: activation by full and partial agonists

1. A comparative study of receptor activation by ten full and partial muscarinic agonists was undertaken on the five subtypes of human muscarinic receptors expressed at similar receptor densities in Chinese hamster ovary (CHO-K1) cells. In addition, m1, m2 and m3 receptors were expressed in mouse fibroblast A9L cells in order to compare the influences of cell type on agonist activation of these receptors. 2. Receptor-effector coupling efficiencies were greater in CHO than A9L cells and agonists displayed greater potencies and similar or greater intrinsic activities at CHOm1 and CHOm3 than A9Lm1 and A9Lm3 receptors. Although m2 receptor density was 6 fold higher in A9L than CHO cells, carbachol elicited significantly greater inhibition of adenosine 3':5'-cyclic monophosphate (cyclic AMP) formation in CHOm2 cells. These data suggest that not only receptor density but receptor-effector coupling and/or coupling efficiencies play significant roles in agonist-induced responses. 3. In CHO cells, receptor-effector coupling efficiencies were m3 = m1 > m5. Although CHOm5 receptors were the least efficiently coupled, some partial agonists displayed higher intrinsic efficacies at m5 than m3 receptors suggesting that, in CHO cells, m5 and m3 receptors may activate different G proteins and/or effectors to stimulate inositol monophosphate (IP1) formation. 4. McN-A-343 was a functionally selective m4 agonist. It had little or no agonist activity at m3 receptors expressed in either A9L or CHO cells. The slopes of McN-A-343 concentration-response curves inCHOm2 cells were significantly lower than the slopes obtained with this compound in CHOm4 cells suggesting that the mode of activation by McN-A-343 differed between the two muscarinic receptors negatively coupled to adenylyl cyclase.5. Cloned receptors provide valuable tools for the study of agonist-receptor interaction and agonist receptor activation but caution should be applied in assuming that the results are valid for all cell types or for tissue-expressed receptors.

Antiproliferative effects of A02011‐1, an adenylyl cyclase activator, in cultured vascular smooth muscle cells of rat

1. The effects of A02011-1, a pyrazole derivative, on the proliferation of rat vascular smooth muscle cells (VSMCs) were examined. 2. A02011-1 (1-100 microM) concentration-dependently inhibited [3H]-thymidine incorporation into DNA in rat VSMCs that were synchronized by 48 h serum depletion and then re-stimulated by addition of foetal calf serum (FCS, 10%), platelet-derived growth factor (PDGF, 10 ng ml-1), 5-hydroxytryptamine (10 microM) or ADP (10 microM). The inhibitory effect of A02011-1 was fully reversible. However, FCS-induced [3H]-thymidine incorporation into rat endothelial cells was unaffected by A02011-1. 3. The concentration of A02011-1 necessary for inhibition of the FCS-induced proliferation was similar to that necessary for adenosine 3':5'-cyclic monophosphate (cyclic AMP) formation. Adenylyl cyclase activity was increased in A02011-1-treated VSMCs, whereas cyclic AMP-specific phosphodiesterase activity was unchanged. 4. A02011-1 was equipotent with forskolin but was more potent than 8-bromo-cyclic AMP against FCS (10%)-induced proliferation. 5. The antiproliferative action of A02011-1 was mimicked by 8-bromo-cyclic AMP, a membrane-permeable cyclic AMP analogue and was antagonized by 2',5'-dideoxyadenosine, an adenylyl cyclase inhibitor and by Rp-cyclic AMPS, a competitive inhibitor of cyclic AMP-dependent protein kinase (PKA) type I and II. 3-Isobutyl-1-methylxanthine (IBMX) caused significant potentiation of the antiproliferative activity of A02011-1. However, Rp-8-bromo-cyclic GMPS and staurosporine did not affect the antiproliferative activity of A02011-1. 6. A02011-1 still inhibited the FCS-induced DNA synthesis even when added 10-18h after restimulation of the serum-starved VSMCs with 10% FCS. Flow cytometry in synchronized cells revealed an acute blockade of FCS-inducible cell cycle progression at a point in the G,/S phase in A02011-1-treated cells. The inhibition of proliferation by A0201 1-1 was shown to be independent of cell damage,as documented by several criteria of cell viability.7. These results indicate that A0201 1-1 inhibition of VSMC proliferation was mediated by cyclic AMP and was due to a delay in the progression from the G1 into S phase of the cell cycle. A02011-1 did not cause cell toxicity and may thus hold promising potential for the prevention of atherosclerosis or vascular diseases.