Maximal Activation Of Adenylate Cyclase Research Articles

Role of protein kinase C on the acute desensitization of renal cortical adenylate cyclase to parathyroid hormone

The mechanisms of adenylate cyclase desensitization to parathyroid hormone are still unclear. Current evidence suggest that the signal generated after PTH binding to receptors results in activation of adenylate cyclase and stimulation of phospholipase C with subsequent activation of protein kinase C. Recent studies have suggested a role of protein kinase C on the regulation of the PTH-dependent receptor-adenylate cyclase system in cultured cells. Therefore, the present studies were conducted to examine the role of protein kinase C on the desensitization of canine renal cortical adenylate cyclase after an acute exposure in vivo to PTH. A group of normal dogs were treated with a single intravenous injection of 1 microgram/k of syn bPTH (1-34) or Nle bPTH (3-34). Ten minutes later, animals were subjected to bilateral nephrectomy and the kidney cortex processed for preparations of basolateral membranes for determinations of adenylate cyclase activity, as well as membrane and cytosolic fractions for analysis of protein kinase C activity. Animals not treated with PTH were used as controls. PTH administration in vivo resulted in a 46.9 +/- 9.3% decrease in maximal adenylate cyclase activity in vitro in response to syn bPTH (1-34) (P < 0.001). Likewise, PTH binding as measured with 125I-Nle8,18,Tyr34-bPTH (1-34)NH2 showed a 40 +/- 3% decrease. This alterations were associated with a marked translocation of protein kinase C from the cytosol to the membrane. Thus, protein kinase C activity in membrane fractions increased from 160.6 +/- 44.8 pmol Pi/min in controls to 500.4 +/- 123 in PTH treated dogs (P < 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)

The phorbol ester TPA potentiates cholera toxin- and isoproterenol-stimulated cyclic AMP-synthesis in primary astrocyte cultures

Cellular responses to changes in the extracellular environment are mediated by intracellular signaling systems. One of the most extensively studied systems is adenylate cyclase which generates the second messenger molecule cAMP. Another one is the phosphatidylinositol (PI) second messenger system giving rise to IP3 and diacylglycerol, the latter stimulating protein kinase C. Recently, a third potential signaling system has attracted increased scientific attention: the phospholipase A2 system which generates arachidonic acid. This substance may be used for eicosanoid synthesis or serve as a second messenger molecule. The present report gives more evidence about mechanisms how these signaling pathways interact in cultured astrocytes. Substances commonly used for stimulation of arachidonic acid release and prostaglandin synthesis in these cultures (A23187, TPA) had no influence on intracellular cAMP levels. Pertussis toxin that had previously been shown to inhibit prostaglandin synthesis, had no influence on cAMP levels either. Cholera toxin, however, raised intracellular cAMP significantly, although much less than the beta-adrenoceptor agonist isoproterenol. Cholera toxin also caused a marked change in astroglial morphology even at reduced concentrations (1-10 ng/ml). A23187 used in combination with Ctx had a moderate stimulatory effect on cAMP synthesis. In contrast, in the presence of Ctx, the PKC-activating phorbol ester TPA synergistically stimulated cAMP production, raising cAMP levels as high as isoproterenol-stimulated levels. The TPA effect was concentration-dependent. It was also dependent on an intact PKC since preincubation of cells with the phorbol ester completely abolished the synergistic effect. The synergistic effect of the phorbol ester was also observed at subthreshold concentrations of isoproterenol. The data reveal that the sole activation of most Gs molecules is a necessary but not sufficient prerequisite to achieve maximal adenylate cyclase activity. The fine-tuning of this activity apparently occurs at the catalytic subunit which is under the (partial) control of phosphorylation by PKC.

Action of 2-aryliminothiazolidines on octopamine-sensitive adenylate cyclase in the American cockroach nerve cord and on the two-spotted spider mite Tetranychus urticae koch

The effects of 2-(2,6-diethylphenylimino)thiazolidine (HSO-783) and 2-(4-chloro-2-methylphenylimino)thiazolidine (HSO-786) were compared with those of 2-(2,6-diethylphenylimino)imidazolidine (NC-5) and 2-(4-chloro-2-methylphenylimino)oxazolidine (AC-6) in stimulating adenylate cyclase of Periplaneta americana ventral nerve cord homogenates. These activities were nonadditive with respect to the activity of a maximally effective concentration of octopamine. Washing of homogenates of ventral nerve cord incubated with NC-5, HSO-783, AC-6, and HSO-786 removed nearly all of the stimulatory activities of these agonists. Maximal stimulation of nerve cord adenylate cyclase activity by NC-5, HSO-783, AC-6, and HSO-786 was inhibited by several antagonists, including mianserin, cyproheptadine, chloromazine, and gramine. The rank-order ability of these antagonists to block the maximal adenylate cyclase activation by NC-5, HSO-783, AC-6, and HSO-786 was identical to the rank-order ability of the same antagonists to block the enzyme activation by an optimally effective concentration of octopamine. The β-adrenergic antagonist propranolol was less potent in this respect. AC-6 was a much better acaricide than HSO-783, HSO-786, and NC-5, which were much less potent octopaminergic agonists than AC-6. HSO-786 was a more potent acaricide and octopaminergic agonist than HSO-783. These observations suggest that the toxicity of NC-5, HSO-783, AC-6, and HSO-786 may be due to their octopaminergic agonist action.

Regulation of adenylate cyclase in electropermeabilized Dictyostelium discoideum cells

In Dictyostelium discoideum cells the enzyme adenylate cyclase is functionally coupled to cell surface receptors for cAMP. Coupling is known to involve one or more G-proteins. Receptor-mediated activation of adenylate cyclase is subject to adaptation. In this study we employ an electropermeabilized cell system to investigate regulation of D. discoideum adenylate cyclase. Conditions for selective permeabilization of the plasma membrane have been described by C. D. Schoen, J. C. Arents, T. Bruin, and R. Van Driel (1989, Exp. Cell Res. 181, 51–62). Only small pores are created in the membrane, allowing exchange of exclusively low molecular weight substances like nucleotides, and preventing the loss of macromolecules. Under these conditions functional protein-protein interactions are likely to remain intact. Adenylate cyclase in permeabilized cells was activated by the cAMP receptor agonist 2′-deoxy cAMP and by the nonhydrolyzable GTP-analogue GTPγS, which activates G-proteins. The time course of the adenylate cyclase reaction in permeabilized cells was similar to that of intact cells. Maximal adenylate cyclase activity was observed if cAMP receptor agonist or GTP-analogue was added just before cell permeabilization. If these activators were added after permeabilization adenylate cyclase was stimulated in a suboptimal way. The sensitivity of adenylate cyclase activity for receptor occupation was found to decay more rapidly than that for G-protein activation. Importantly, the adenylate cyclase reaction in permeabilized cells was subject to an adaptation-like process that was characterized by a time course similar to adaptation in vivo. In vitro adaptation was not affected by cAMP receptor agonists or by G-protein activation. Evidently electropermeabilized cells constitute an excellent system for investigating the positive and negative regulation of D. discoideum adenylate cyclase.

Species differences in myocardial beta-adrenergic receptor regulation in response to hyperthyroidism.

The coupling between myocardial beta-adrenergic receptors, adenylate cyclase activity, and the in vivo cardiac response to catecholamines is controversial in hyperthyroidism. The possibility of species differences in beta-adrenoceptor regulation after thyroxine treatment was studied in dogs and in rats. In dogs instrumented with a left ventricular (LV) pressure micromanometer, hyperthyroidism was induced by L-thyroxine (0.5 mg/kg/day i.v. for 10 days). After hyperthyroidism, heart rate was increased to 167 +/- 10 beats/min (control, 107 +/- 8 beats/min; p less than 0.005) with an increase of peak LV dP/dt from 4,243 +/- 471 to 6,105 +/- 862 mm Hg/sec (p less than 0.01). LV response to injection of increasing doses of isoproterenol and dobutamine was not significantly different before and after induction of the hyperthyroid state, as shown by the unchanged slopes of the LV peak dP/dt versus the log of the dose of isoproterenol and dobutamine. Bmax of beta-receptors measured in crude membranes using 3H-CGP 12177 and in homogenates using 125I-cyanopindolol was not increased in hyperthyroid animals as compared with a control group. Basal adenylate cyclase activity was not different in control and hyperthyroid dogs (32 +/- 3 versus 29 +/- 3 pmol/mg/min), and maximal adenylate cyclase activity response to isoproterenol was similar in control and hyperthyroid dogs. In contrast, in rats subjected to hyperthyroidism (0.5 mg/kg/day i.p. L-thyroxine for 10 days), Bmax of adrenoceptors measured using the same methods was significantly increased as compared with control (+72.5% using 3H-CGP 12177 and +41% using 125I-cyanopindolol, but adenylate cyclase activity was not increased in hyperthyroid rats. We conclude that both adenylate cyclase activity and LV response to catecholamines are not increased by thyroxine-induced hyperthyroidism in dogs and that, in contrast with rats, beta-adrenergic density is not increased in hyperthyroid dogs. This indicates a species difference in myocardial beta-adrenoceptor regulation in response to hyperthyroidism.

Evidence for regulation of human platelet adenylate cyclase by phosphorylation. Inhibition by ATP and guanosine 5′-[β-thio]diphosphate occur by distinct mechanisms

1. Incubation of human platelet membranes with guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) causes a time-dependent increase in the activation of adenylate cyclase due to Gs (the stimulatory GTP-binding protein). Forskolin enhances adenylate cyclase activity but does not interfere with the process of activation. The activation follows first-order kinetics in both the presence and the absence of the assay components. 2. ATP in the presence or the absence of an ATP-regenerating system of phosphocreatine and creatine kinase inhibits activation. 3. Hydrolysis of ATP to ADP does not lead to receptor-mediated inhibition of adenylate cyclase acting via Gi (the inhibitory GTP-binding protein). The ADP analogue adenosine 5'-[beta-thio]diphosphate (ADP[S]) does not inhibit the activation process. 4. Phosphocreatine alone inhibits adenylate cyclase activation at concentrations above 1 mM. 5. Inhibition by phosphocreatine is not due to the chelation of free Mg2+ ions. 6. Inhibition by ATP and the other assay components occurs throughout the activation process, decreasing both the rate of activation and the maximum activity obtained. 7. Maximal activation of adenylate cyclase after prolonged incubation with p[NH]ppG slowly reverses in the presence of the assay components. 8. A 10-fold excess of the GDP analogue guanosine 5'-[beta-thio]diphosphate (GDP[S]) over p[NH]ppG inhibits the activation process completely, at all stages of the time course. 9. Preincubations in the presence and absence of ATP, cyclic AMP, phosphocreatine and creatine kinase show equal sensitivity to increasing GDP[S] concentration. These data show that the inhibition observed in the presence of ATP is not due to endogenous or contaminating guanine nucleotides, and suggest that phosphoryl transfer may regulate adenylate cyclase activity.

Granulomas in murine schistosomiasis mansoni contain vasoactive intestinal peptide-responsive lymphocytes

Granulomas develop around schistosome ova in murine Schistosoma mansoni. These granulomas have eosinophils that produce VIP. It is possible that VIP participates in immunoregulation. VIP-mediated effects usually operate through a cAMP-dependent mechanism. To identify VIP-responsive inflammatory cells in murine schistosomiasis, inflammatory cells were exposed to VIP and assessed for adenylate cyclase activation and VIP binding. VIP increased adenylate cyclase activity in splenic lymphocytes from both normal and infected mice. In each case, the half-maximal stimulation was at about 5 × 10 −8 M. [ 125I]VIP bound to splenic lymphocytes specifically, with a K d of 10 −8 M. This suggested that maximal adenylate cyclase activation requires full receptor occupancy. The receptor was highly specific for VIP. Hormone analogs, that are VIP receptor antagonists in some tissues, were only weak agonists of the lymphocyte VIP receptor. Granuloma cells also bound VIP and responded with adenylate cyclase activation in a manner similar to that of spleen cells. Both splenic T and B lymphocytes responded to VIP. Deletion experiments, using anti-Thy 1.2, suggested that most of the responsive granuloma cells were T lymphocytes. Thus, VIP alters cAMP metabolism in granuloma T cells through a receptor-coupled mechanism similar to that observed for spleen cells. Binding studies on mouse intestinal epithelial cells suggested that their VIP receptor is functionally and possibly structurally different from the VIP receptor on mouse lymphocytes. Additional experiments suggested that VIP and other neuropeptides are unlikely to alter the granulomatous response through a primary interaction with the granuloma macrophages.

Direct Effects of Chronic Ethanol Exposure on β‐Adrenergic and Adenosine‐Sensitive Adenylate Cyclase Activities and Cyclic AMP Content in Primary Cerebellar Cultures

The direct effects of chronic ethanol exposure on adenylate cyclase activity and cyclic AMP content were investigated in primary cerebellar cultures. By morphological criteria these cultures mainly contain granule cells with some astrocytes, and each cell type appears to contain both beta-adrenergic and adenosine-sensitive adenylate cyclase systems. Chronic treatment of the primary cerebellar cultures with 120 mM ethanol for 6 days caused a reduction in the stimulation of cyclic AMP content by isoproterenol and by the adenosine analogue 2-chloroadenosine. Kinetic analysis indicated that the chronic ethanol treatment decreased maximal activation of adenylate cyclase, as well as increased the EC50 values for norepinephrine and 2-chloroadenosine. Activation of norepinephrine-stimulated adenylate cyclase activity by in vitro ethanol was significantly enhanced after the chronic ethanol exposure. However, the chronic treatment did not alter activation of the 2-chloroadenosine-stimulated enzyme by in vitro ethanol. A similar difference in the response to in vitro ethanol after the chronic treatment was observed when cyclic AMP content of the intact cells was measured. The present data indicate that chronic ethanol exposure causes a selective increase in the sensitivity of adenylate cyclase to ethanol in some brain cells and a more generalized desensitization of receptor-stimulated cyclic AMP production.

Mechanisms of Homologous and Heterologous Regulation of Parathyroid Hormone Receptors in the Rat Osteosarcoma Cell Line UMR-106*

We have examined the mechanisms of homologous and heterologous regulation of PTH receptor binding and receptor-mediated adenylate cyclase activity in the osteosarcoma cell line UMR-106. Pretreatment with PTH resulted in a time- and dose-dependent decrease in PTH-stimulated adenylate cyclase which was maximal after 2 h and at a concentration of 10(-8) M rat (r)PTH-(1-34). PTH pretreatment over the same dose range also diminished receptor binding of 125I-labeled rPTH-(1-34); however, maximal loss of binding required 14 h and was greater than the loss of maximal adenylate cyclase activity. After 24 h pretreatment with rPTH-(1-34), cell surface receptors were decreased from 21,000 sites per cell to 2,700 sites per cell, and these down-regulated PTH receptors could not be detected in either vesicular or cytosolic subcellular fractions. Recovery from such homologous down-regulation appeared to require new receptor synthesis. Heterologous down-regulation of PTH receptors was demonstrated when UMR-106 cells were preincubated with prostaglandin E2 or (Bu)2cAMP. Heterologous desensitization was shown to be the result of a reversible modification of the PTH receptor which decreased binding affinity and decreased PTH-stimulated adenylate cyclase. Postreceptor components were also examined, and PTH but not prostaglandin E2 pretreatment was shown to decrease guanyl nucleotide binding (G) protein-mediated adenylate cyclase stimulation. This decrease in G protein function was associated with a loss of cholera toxin-catalyzed ADP ribosylation and was also detected by immunoblotting. These results indicate that PTH responses in osteoblastic cells are modulated by diverse mechanisms involving modifications both to the receptor and to postreceptor components of adenylate cyclase.

Calcium-Induced Modification of the Acrosomal Matrix in Digitonin-Permeabilized Guinea Pig Spermatozoa1

In this report we describe and partially characterize a preparation of digitonin-permeabilized guinea pig spermatozoa that undergo a rapid and synchronous modification of the acrosomal matrix in response to calcium. Permeabilization of cauda epididymal spermatozoa by digitonin was monitored by using adenylate cyclase activity as an indicator. Spermatozoa (5 x 10(7) cells) treated with 0.005% digitonin for 15 s exhibited maximal adenylate cyclase activity but generally retained their structural morphology, as examined by phase-contrast and transmission electron microscopy. The ratio fo cell number to detergent concentration was the critical factor for determining both the efficiency of permeabilization and the maintenance of structural integrity. When permeabilized spermatozoa were treated with 2 mM CaCl2, the cells underwent a rapid and synchronous modification of the acrosomal matrix (AM). As observed by phase-contrast microscopy, the response to CaCl2 was characterized by events that occurred in the following temporal sequence: disruption of the sperm rouleaux, the loss of refractility by the apical segment of the sperm acrosome, and detachment of the apical segment from the spermatozoa. Transmission electron microscopy indicated that the loss of refractility from the sperm apical segment was coincident with a calcium-induced dispersion of the AM. Analysis of the proteins released during this response, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that a specific subset of sperm proteins was released from the spermatozoa, including a major = staining, 45,000 Mr protein apparently generated from a higher molecular weight precursor during the acrosome reaction.

Adenylate cyclase activity coupled to the stimulatory guanine nucleotide binding protein in patients having electrophysiologic studies and either structurally normal hearts or idiopathic myocardial disease

There has been much recent interest in transmembrane signaling involving adrenergic and other receptors both in animals and humans with congestive heart failure (CHF). In patients with idiopathic dilated cardiomyopathy and severe CHF, β-adrenoceptor density and maximal adenylate cyclase activity stimulated by (−)-isoproterenol in the presence of guanosine triphosphate (GTP) are reduced. 1 Other studies in animal models of CHF preceded by myocardial hypertrophy have revealed an increase in β-adrenoceptor density accompanied by reduced receptor affinity for agonist and a decrease in the stimulatory guanine nucleotide binding protein (Gs). 2,3 To date, there are no data reporting functional alterations in Gs in humans.

Mechanisms of glucagon-induced homologous and heterologous desensitization of adenylate cyclase in membranes and whole Sertoli cells of the rat.

In the present work the molecular mechanisms of glucagon-induced desensitization of adenylate cyclase in cultured Sertoli cells have been studied in both whole cells and in a cell-free system. 1) Pretreatment of both whole Sertoli cells and membranes with glucagon induces a time- and dose-dependent desensitization of adenylate cyclase response that is primarily homologous and similar in the two systems. The component of heterologous desensitization, estimated by the reduced responsiveness to other hormones and NaF, accounted for only 12-20% loss of glucagon-responsive adenylate cyclase activity (P less than 0.01). 2) Glucagon-induced desensitization is ATP-dependent. Half maximal desensitization was achieved between 0.1 and 0.2 mM of ATP. 3) The typical time lag in the maximal activation of adenylate cyclase by GMPP(NH)P in the absence of hormone reappeared upon desensitization in spite of the presence of glucagon. The lag, however, was eliminated by FSH, showing that the homologous desensitization is due to a receptor-specific alteration. 4) The heterologous component of glucagon-induced desensitization is largely cAMP/protein kinase dependent. cAMP/protein kinase-induced desensitization was heterologous and caused approximately 30% loss of both hormonal and fluoride-stimulated enzyme activity. 5) Glucagon-induced desensitization is not due to altered activity of Ni since it proceeded equally well in membranes of cells pretreated with pertussis toxin (100 ng/ml) which eliminates Ni-mediated effects. It is concluded that glucagon induces both homologous and heterologous desensitization of the Sertoli cell adenylate cyclase. The locus of homologous desensitization appears to be at the level of the receptor and most probably involves a cAMP-independent phosphorylation reaction, whereas the heterologous desensitization appears to be cAMP-mediated and at least involves impaired functional activity of the Ns component.

The role of the carbohydrate moiety in thyrotropin action

The relative binding affinity of deglycosylated human TSH was 6-fold higher than that of native TSH. Although deglycosylated human TSH significantly stimulated adenlyate cyclase, it was less effective than the native hormone. When deglycosylated human TSH was added with bovine TSH, however, a dose-dependent antagonism was observed. In particular, submaximal and maximal concentrations of bovine TSH and deglycosylated human TSH resulted in cAMP values much lower than the sum of activities of the individual hormones. The data suggest that although the effects of TSH deglycosylation are not as dramatic as with the gonadotropins, the carbohydrates of TSH appear to be required for maximal activation of adenylate cyclase by the hormone.

Modulation by monoamines of somatostatin-sensitive adenylate cyclase on neuronal and glial cells from the mouse brain in primary cultures.

Primary cultures of mouse embryonic neuronal or glial cells from the cerebral cortex, striatum, and mesencephalon were used to identify and determine the cellular localization of somatostatin receptors coupled to an adenylate cyclase. Somatostatin inhibited basal adenylate cyclase activity on neuronal but not on glial crude membranes in the three structures examined. The somatostatin-inhibitory effect on neuronal crude membranes was still observed in the presence of (-)-isoproterenol, 3,4-dihydroxyphenylethylamine (dopamine, DA), or 5-hydroxytryptamine (5-HT, serotonin) used at a concentration (10(-5) M) inducing maximal adenylate cyclase activation. In addition, in most cases biogenic amines modified the pattern of the somatostatin-inhibitory effect, triggering either an increase in the peptide apparent affinity for its receptors or an increase in the maximal reduction of adenylate cyclase activity or both. However, 5-HT did not modify the somatostatin-inhibitory response on striatal and cortical neuronal crude membranes. The changes in somatostatin-inhibitory responses were interpreted as a colocalization of the amine and the peptide receptors on subtypes of neuronal cell populations. Finally, somatostatin was shown to inhibit adenylate cyclase activity following its activation by (-)-isoproterenol on glial crude membranes of the striatum and the mesencephalon but not on those of the cerebral cortex.

Adenylate cyclase in the corpus luteum of the rhesus monkey. II. Sensitivity to nucleotides, gonadotropins, catecholamines, and nonhormonal activators.

The sensitivity of the adenylate cyclase of the primate corpus luteum to various nucleotides, gonadotropins, catecholamines, and nonhormonal activators was assessed in homogenates of luteal tissue obtained from rhesus monkeys at the midluteal phase of the menstrual cycle. The conversion of [alpha-32P]ATP to [32P]cAMP was used to monitor adenylate cyclase activity. GTP, the GTP analog 5'-guanylyl-imidodiphosphate, and ITP stimulated adenylate cyclase activity in the presence or absence of exogenous hormone; however CTP, UTP, GMP, and guanosine did not. The gonadotropins, human (h) LH and hCG, stimulated cAMP production in a dose-dependent manner. Maximal stimulation of adenylate cyclase was achieved at 100 nM hLH and hCG, and the activation constant was 20 nM for both hormones. The addition of GTP increased maximal activation of adenylate cyclase by hLH or hCG, but did not alter sensitivity to the hormones. Neither hFSH nor the isolated subunits of hCG stimulated cAMP production. Deglycosylated hCG (native hCG with 70% of the carbohydrate moieties removed) did not stimulate adenylate cyclase activity. However, hLH and intact hCG failed to enhance cAMP production in the presence of an equimolar amount of deglycosylated hCG. The adenylate cyclase of macaque luteal tissue did not respond to the addition of isoproterenol, epinephrine, or phenylephrine. Furthermore, these catecholamines did not affect hCG stimulation of adenylate cyclase. The nonhormonal activators of adenylate cyclase, forskolin and fluoride, stimulated cAMP production in a dose-dependent manner, with maximal stimulation at 100 microM and 10 mM, respectively. Thus, the macaque corpus luteum at the midluteal phase of the menstrual cycle contains a guanine nucleotide-regulated adenylate cyclase which is equally sensitive to the pituitary and placental gonadotropins, hLH and hCG. However, removal of carbohydrate moieties from hCG endows the molecule with gonadotropin-antagonistic properties in the primate. The adenylate cyclase system of the macaque corpus luteum was not responsive to catecholamines; thus, the primate may lack a potential mechanism for control of luteal function that is available to many nonprimate species.

Impaired parathyroid hormone receptor-adenylate cyclase system in the postobstructed canine kidney.

The present studies examine the initial events in the action of PTH, namely receptor binding and adenylate cyclase activation in the postobstructed canine kidney. Both kidneys were removed from five mongrel dogs 2 h after relief of unilateral ureteral obstruction (UUO) of 42 h duration. Basolateral membranes (BLM) were prepared from both the UUO and contralateral (control) kidneys. Maximal activation of adenylate cyclase by PTH was 35% lower (P less than 0.01) in BLM from UUO than control kidneys (3869 +/- 200 vs. 5978 +/- 425 pmol cAMP/mg protein X 30 min). The concentration of PTH required for half-maximal activation of the enzyme was unchanged. Addition of 1 mM GTP failed to correct the decreased enzyme activity in response to PTH of BLM from kidneys with UUO. NaF activation, a measure of interaction of the nucleotide regulatory component (G/F) with the catalytic unit of the adenylate cyclase was similar in BLM from UUO and control kidneys. Similarly, activation of the catalytic unit of the enzyme by Mn2+ was not different. Specific binding of [125I]Nle8, Nle8, Tyr34-bovine PTH NH2 was markedly reduced (P less than 0.01) in BLM from UUO vs. control (6.37 +/- 1.20 vs. 2.43 +/- 0.09 fmole bound/microgram protein). There was no change in hormone affinity for the binding site. These data indicate that there is decreased activation of adenylate cyclase by PTH as a consequence of apparent loss of receptors for the hormone in the BLMs of renal tubular cells of postobstructed kidneys. These abnormalities may play a role in the abnormal regulation of phosphorus excretion after ureteral obstruction.

Identification of glucagon receptors in rat brain.

The binding of radiolabeled glucagon to rat brain membranes was investigated. Regional distribution studies indicate higher specific binding of 125I-labeled monoiodoglucagon to olfactory tubercule, hippocampus, anterior pituitary, and amygdala membranes, with somewhat lower binding to membranes from septum, medulla, thalamus, olfactory bulb, and hypothalamus. 125I-labeled glucagon bound to rat brain synaptic plasma membrane fractions with high affinity (KD = 2.24 nM). Specific binding was greater to synaptosomal membrane fractions relative to myelin, mitochondrial nuclear, or microsomal fractions. Inclusion of 0.1 mM GTP in the binding assay reduced the glucagon binding affinity (KD = 44.5 nM). Several neuropeptides and other neuroactive substances tested did not affect binding of labeled glucagon to brain membranes. Three different glucagon analogs inhibited labeled glucagon binding. Synthetic human pancreatic growth hormone-releasing factor, hpGRF-44, also inhibited binding, although the concentration required for half-maximal displacement was 100-fold higher than for native glucagon. Addition of glucagon to brain membranes resulted in approximately equal to 3-fold maximal activation of adenylate cyclase over basal levels. Glucagon at a concentration of 4.74 nM was required for half-maximal activation of pituitary membrane adenylate cyclase. These findings provide evidence for rat brain binding sites that respond to the pancreatic form of glucagon and can transduce this binding into the activation of adenylate cyclase.

Requirement for both choleragen and pertussis toxin to obtain maximal activation of adenylate cyclase in cultured cells

NG108-15 cells contain both the inhibitory and stimulatory guanyl nucleotide-binding regulatory proteins of the cyclase system. Choleragen activates cyclase directly by ADP-ribosylating the stimulatory guanyl nucleotide-binding protein; prostaglandin E1 does not further increase activity of cells treated with maximally effective concentrations of choleragen. Including pertussis toxin during incubation with this concentration of choleragen, however, further augments both cyclase activity and cAMP accumulation by intact cells. These observations suggest that the inhibitory guanyl nucleotide-binding protein exerts basal inhibition on catalytic activity which cannot be overcome by maximally effective concentrations of choleragen, stimulatory hormones, or both.

Kinetic and pharmacological characterization of vasopressin membrane receptors from human kidney medulla: Relation to adenylate cyclase activation

Membranes from the medullopapillary portions of three human kidneys unsuitable for transplantation were prepared and stored in liquid nitrogen. Specific vasopressin binding sites were detected on these membranes and characterized using tritiated lysine vasopressin. The biological activities of vasopressin and of structurally related peptides were estimated on the same membrane preparations by measuring dose-dependent adenylate cyclase activation. The population of vasopressin binding sites was homogeneous and displayed the following characteristics: equilibrium dissociation constant K D 27 ± 7 nM (for lysine vasopressin); maximal specific binding capacity 0.7±0.1 pmol [ 3H]LVP bound/mg protein. Maximal adenylate cyclase activation by lysine vasopressin was 5.4 times the basal value of 50 ± 4 pmol cAMP formed/5 min per mg protein. Half-maximal activation (K act) was obtained at a concentration of 34 ± 5 nM. Both lysine vasopressin binding and hormone-induced adenylate cyclase activation were sensitive to guanyl nucleotides. 5′-Guanylyl imidodiphosphate reduced K act by a factor of 3 and increased K D by a factor of about 8. K D and K act were determined for a series of peptides including natural vasopressins, oxytocin and their structural analogues which were found to have enhanced or reduced antidiuretic activities in the rat. The concentration range of the K D and K act values covered a concentration range of more than three orders of magnitude. The most active peptide was the natural arginine vasopressin (K D 4.4 ± 0.7 nM; K act 3.1 ± 1.6 nM). There was a highly significant correlation between paired K D and K act values. The two vasopressin structural analogues [1-(β- mercapto-β, β-cyclopentamethylene propionic acid), 4-valine, 8-D-arginine]vasopressin and [1-β-mercapto-β, β-cyclopenthamethylene propionic acid), 2-O-ethyltyrosine, 4-valine]arginine vasopressin, behaved like competitive inhibitors of vasopressin-induced adenylate cyclase activation (K i = 65 and 59 nM respectively). Freezing the membranes in liquid nitrogen reduced the affinity of vasopressin receptors but fully preserved their selectivity towards vasopressin structural analogues. It is concluded that the vasopressin binding and/or adenylate cyclase assays might be useful for estimating several aspects of the pharmacological activities of vasopressin structural analogues in man.

Calcium inhibition of adenylate cyclase: Studies in turkey erythrocyte and S49 CYC − cell membranes

We studied the mechanism of calcium inhibition of adenylate cyclase using partially purified components of the enzyme complex and computer analysis of free metal and substrate concentrations. A sigmoidal relationship was observed between percentage maximal adenylate cyclase activity with 1-isoproterenol/guanylyl-β,γ-imidodiphosphate and the calculated free calcium. Fifty percent inhibition occurred at 2.5 × 10 −4 m free calcium. This inhibition appeared to be independent of calmodulin. Calcium inhibited the holocatalytic enzyme in a manner indentical to that of the native enzyme, but did not affect the ability of 1-isoproterenol and guanylyl-β,γ-imidodiphosphate to promote the formation of the holocatalytic state. There was no effect of calcium on the conformation of the activated G unit nor on the holocatalytic enzyme as determined by sedimentation velocity analysis. Calcium did not cause detectable dissociation of the activated G unit from the catalytic unit, nor convert activated G unit to an inactive form. Calcium inhibition of the catalytic unit of adenylate cyclase was studied in S49 CYC − lymphoma cell membranes. High concentrations of calcium inhibited manganese-stimulated CYC − enzyme, but this could be explained by competition between calcium and manganese for ATP. With addition of forskolin, CYC − adenylate cyclase utilized MgATP 2− as substrate and was shown to have a separate binding site for free magnesium. Calcium inhibited forskolin-stimulated CYC − enzyme by competing with free magnesium for its regulatory site. Calcium inhibition was noncompetitive with respect to MgATP 2−. We conclude that calcium inhibits adenylate cyclase by direct competition with magnesium for a regulatory site on the catalytic unit.