Component Of Adenylate Cyclase Research Articles

A rare case of pseudohypoparathyroidism

Pseudohypoparathyroidism (PGPT) - Albright's hereditary osteodystrophy is a sex-related disease rarely seen in medical practice. Inherited by the dominant type. It is caused by insensitivity of the kidneys and bones to parathyroid hormone (PG), which is secreted in normal or slightly elevated amounts [6, 10]. GH may not have a specific effect on cells due to pathology of GH receptors, impaired expression of information RNA synthesis by an unchanged hormone-receptor complex, the presence of pathological components of adenylate cyclase, pathological AMP-dependent protein kinase, or a defect in the synthesis of kinase substrates in the presence of circulating GH antagonists [5, 10 ].

Pathogen Sensing by Nucleotide-binding Oligomerization Domain-containing Protein 2 (NOD2) Is Mediated by Direct Binding to Muramyl Dipeptide and ATP

Nucleotide binding and oligomerization domain-containing protein 2 (NOD2/Card15) is an intracellular protein that is involved in the recognition of bacterial cell wall-derived muramyl dipeptide. Mutations in the gene encoding NOD2 are associated with inherited inflammatory disorders, including Crohn disease and Blau syndrome. NOD2 is a member of the nucleotide-binding domain and leucine-rich repeat-containing protein gene (NLR) family. Nucleotide binding is thought to play a critical role in signaling by NLR family members. However, the molecular mechanisms underlying signal transduction by these proteins remain largely unknown. Mutations in the nucleotide-binding domain of NOD2 have been shown to alter its signal transduction properties in response to muramyl dipeptide in cellular assays. Using purified recombinant protein, we now demonstrate that NOD2 binds and hydrolyzes ATP. Additionally, we have found that the purified recombinant protein is able to bind directly to muramyl dipeptide and can associate with known NOD2-interacting proteins in vitro. Binding of NOD2 to muramyl dipeptide and homo-oligomerization of NOD2 are enhanced by ATP binding, suggesting a model of the molecular mechanism for signal transduction that involves binding of nucleotide followed by binding of muramyl dipeptide and oligomerization of NOD2 into a signaling complex. These findings set the stage for further studies into the molecular mechanisms that underlie detection of muramyl dipeptide and assembly of NOD2-containing signaling complexes.

Carbaryl inhibits basal and FSH-induced progesterone biosynthesis of primary human granulosa-lutein cells

Carbaryl is known to impede female reproductive function, however, the mechanisms through which the adverse effects are mediated are not clearly elucidated. In order to get insight into the mechanisms, this study was conducted to raise fresh concerns about the potential effects of carbaryl on steroidogenesis by primary human granulosa-lutein cells (hGLCs) and explore the possible nature of this action. hGLCs were co-incubated with various concentrations of carbaryl at 0, 1, 5, 25, 125 μmol/L for 24 h to examine effects of this carbamate pesticide on progesterone accumulation. We observed that the carbaryl inhibited basal and FSH-induced progesterone production in a dose-dependent manner. We also investigated the effects of carbaryl on 22( R)-hydroxycholesterol (22 R-HC)-stimulated progesterone yield, basal and FSH-stimulated StAR gene expression and cyclic adenosine monophosphate (cAMP) production, as well as forskolin (non-specific activator of adenylyl cyclase)-induced progesterone and cAMP production of hGLCs. We found that the decreased progesterone biosynthesis was accompanied with a reduced cAMP abundance on both basal and FSH-induced condition. Furthermore, our results demonstrated that the 22 R-HC could remove the carbaryl-induced restraint of progesterone biosynthesis, suggesting that carbaryl caused a disruption of cholesterol transport across mitochondrial membranes, which was further confirmed by the observation that carbaryl inhibited the gene expression of steroidogenic acute regulatory protein (StAR). In addition, the inhibitory effects of carbaryl on progesterone and cAMP production were completely reversed by addition of forskolin to the cell culture, which indicated a repaired site on the upstream components of adenylate cyclase or adenylate cyclase per se by carbaryl in the cAMP-mediated signal pathway. All the effects mentioned above were not due to a detrimental action of carbaryl on cell viability by MTS assay. In conclusion, carbaryl may inhibit steroidogenesis, at least in part, by obstructing the delivery of cholesterol over mitochondrial membranes and attenuating cAMP generation.

Selective formation of Gsalpha-MHC I complexes after desensitization of human platelets with iloprost.

Prolonged treatment of human platelets with the adenylate cyclase-stimulating prostacyclin analog iloprost leads to reduction in cAMP formation. Previous studies have demonstrated that this may be ascribed to modification of both receptor and Gsalpha function rather than of the catalytic component of adenylate cyclase [Mollner, S., Deppisch, H. & Pfeuffer, T. (1992) Eur. J. Biochem. 210, 539-544]. Iloprost-induced desensitization was accompanied by the formation of a Gsalpha-containing 90-kDa product in membranes treated with the bifunctional cross-linker 1,6-bismaleimidohexane. The cAMP-inducing prostanoid PGD2, which does not promote desensitization, did not cause formation of the 90-kDa species either. The long-term effect of the common G-protein activator [AlF4]- on human platelet adenylate cyclase was shown in many respects to be comparable with that of iloprost. However, [AlF4]- treatment also failed to induce the 90-kDa species, showing that different mechanisms of desensitization were operating. Treatment of the cross-linked 90-kDa complex with PNGase F demonstrated the glycoprotein nature of the Gsalpha-associated component. The 90-kDa cross-linked product was purified by consecutive immunoaffinity chromatography and preparative PAGE to apparent homogeneity. Analysis of the purified protein by MS suggested that, besides Gsalpha, the heavy chain of MHC I (HLA-A2) was part of the complex. This was confirmed by coprecipitation of Gsalpha by the monoclonal anti-(MHC I) antibody W6/32.

The biological activity of the corticotropin-releasing hormone receptor-adenylate cyclase complex in human myometrium is reduced at the end of pregnancy.

We recently suggested that placentally derived CRH might influence human parturition via specific receptor mechanisms. We identified a human myometrial CRH receptor that changes to a high affinity state in the later stages of pregnancy and becomes coupled to the adenylate cyclase system. The purpose of this study was to investigate the functional capacity of this receptor in myometrial tissue obtained from women being delivered electively by cesarian section at term (38-40 weeks gestation) and preterm (30-35 weeks gestation) before the onset of labor. Myometrial membrane suspensions were prepared by differential centrifugation, and the production of cAMP after stimulation with various test substances was measured by RIA. In preterm myometrium, both human CRH and cholera toxin stimulated cAMP production. This effect was significantly reduced in term myometrium. The adenylate cyclase was functionally active in term myometrium, as demonstrated by the use of forskolin. Furthermore, pertussis toxin pretreatment of term myometrial membranes did not increase the response to CRH. These results suggest that in human pregnant myometrium at term, there is a modification in the coupling mechanisms between CRH receptors and the catalytic component of adenylate cyclase, resulting in a reduction of CRH-stimulated cAMP production.

The G2M arrest caused by iodide is unrelated to the effects of iodide at adenylate cyclase.

Previously, we have shown that the iodide was able to inhibit TSH induced thyrocyte proliferation by arresting the cell cycle at G0G1 and G2M, suggesting that the iodide may be exerting its effects through more than the TSH-adenylate cyclase-cAMP system. To confirm the effects of iodide on the adenylate cyclase (AC) system, forskolin- and dibutyryl-cyclic-AMP (dBcAMP)-stimulated FRTL5 thyroid cells were exposed to inhibitory concentrations of iodide and the resultant effects on the cell cycle were compared to the effects observed with TSH, using flow cytometric DNA analysis. Forskolin stimulated the proliferation of FRTL5 cells in a dose-dependent manner. Cell numbers rose from baseline by 169 +/- 4% to peak at 10 microM forskolin. Interestingly, 100 microM forskolin inhibited cell proliferation, causing cell numbers to fall by approximately 50%. Iodide inhibited forskolin-induced proliferation to baseline levels. However, the pattern of cell cycle perturbation was different to that with TSH-stimulated cells. There were no differences in the proportion of cells in G0G1 between forskolin alone and forskolin + NaI, while there was a marked fall in the proportion of cells in S phase, indicating possible partial arrest at G0G1. Furthermore, there was a marked accumulation of cells in G2M over and above that found with TSH + NaI, indicating arrest at G2M. dBcAMP maximally stimulated cell numbers to rise from baseline by 125% with 1 mM dBcAMP. Again, higher concentrations of the mitogen had an inhibitory effect on proliferation. The addition of NaI inhibited dBcAMP stimulated cell proliferation.

Effects of litter removal on the lipolytic response and the regulatory components of the adenylate cyclase in adipocytes isolated from lactating rats

The effects of litter removal on the status of different components of the hormone-sensitive adenylate cyclase system were analysed in plasma membranes of rat adipocytes. These effects were correlated with the decreased lipolytic response of adipose tissue. No changes in total number of A1 adenosine receptors or their affinity were detected in response to litter removal. In contrast, beta-adrenergic receptors showed a decrease (35%) in total number of receptors, without any significant change in their affinity. The status of alpha-GS and alpha-Gi, the alpha-subunits of G proteins which mediate stimulation and inhibition respectively of adenylate cyclase, were probed by cholera- and pertussis-toxin-catalysed ADP-ribosylation respectively and by immunoblot. Associated with litter removal, decreases of 63% and 62% in the incorporation of [alpha 32P]ADP-ribose catalysed by cholera toxin and pertussis toxin into alpha-Gs and alpha-Gi respectively were detected. Immunoblotting using RM/1 (anti-alpha-Gs) and AS/7 (anti-alpha-Gi) antisera also showed decreases in the levels of alpha-Gs (52%) and alpha-Gi (55%) in adipocyte membranes from litter-removed rats compared with lactating rats. Alterations in the status of hormone-sensitive adenylate cyclase components, such as those described herein, may be biochemical mechanism(s) by which adipose tissue shows a decreased lipolytic response during recovery from lactation.

Ceratitis capitata brain adenylate cyclase and its membrane environment

Adenylate cyclase activation by GTP and octopamine as well as basal activity (in the presence of Mg 2+) have been studied as a function of membrane structure in plasma membranes from brain of the dipterous Ceratitis capitata. Benzyl alcohol and lidocaine, but not phenobarbital, inhibited the three activities to the same extent. Triton X-100-solubilized adenylate cyclase was also inhibited by benzyl alcohol and lidocaine, but not by phenobarbital. Results could be explained by an effect on the catalytic unit lipid environment, which would be maintained after solubilization, counteracting the effect of these drugs to facilitate lateral diffusion and coupling of adenylate cyclase components in the lipid bilayer. The observation that the insect adenylate cyclase is relatively insensitive to changes in bulk bilayer fluidity is strengthened by the absence of effect of phenobarbital on enzyme activities. Indeed, this compound was as active as lidocaine or benzyl alcohol in increasing bulk membrane fluidity. The response of C. capitata adenylate cyclase to changes in membrane fluidity is different from that recorded in mammalian systems. This may be functionally important and result from the fact that insects are not warm-blooded.

Transforming growth factor β enhances parathyroid hormone stimulation of adenylate cyclase in clonal osteoblast‐like cells

The effects of transforming growth factor beta (TGF beta) on parathyroid hormone (PTH)-responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR-106) with the osteoblast phenotype. Purified TGF beta incubated with UMR-106 cells for 48 hr produced a concentration-dependent increase in PTH stimulation of adenylate cyclase, with maximal increase in PTH response (37%) occurring at 1 ng/ml TGF beta. TGF beta also enhanced receptor-mediated activation of adenylate cyclase by isoproterenol and prostaglandin E2 (PGE2) and nonreceptor-mediated enzyme activation by cholera toxin and forskolin. In cells in which PTH-stimulated adenylate cyclase activity was augmented by treatment with pertussis toxin, the incremental increase in PTH response produced by TGF beta was reduced by 33%. However, TGF beta neither mimicked nor altered the ability of pertussis toxin to catalyze the ADP-ribosylation of a 41,000-Da protein, presumably the alpha subunit of the inhibitory guanine nucleotide-binding regulatory component (Gi) of adenylate cyclase, in cholate-extracted UMR-106 cell membranes. TGF beta also had no effect on the levels of alpha or beta subunits of Gi, as assessed by immunotransfer blotting. In time course studies, brief (less than or equal to 30 min) exposure of cells to TGF beta during early culture was sufficient to increase PTH response but only after exposed cells were subsequently allowed to grow for prolonged periods. TGF beta enhancement of PTH and isoproterenol responses was blocked by prior treatment of cells with cycloheximide but not indomethacin. The results suggest that TGF beta enhances PTH response in osteoblast-like cells by action(s) exerted at nonreceptor components of adenylate cyclase. The effect of TGF beta may involve Gi, although in a manner unrelated to either pertussis toxin-catalyzed ADP-ribosylation of the alpha subunit of Gi or changes in levels of Gi subunits. The regulatory action of TGF beta on adenylate cyclase is likely to be mediated by the rapid generation of cellular signals excluding prostaglandins, followed by a prolonged sequence of events involving protein synthesis. These observations suggest a mechanism by which TGF beta may regulate osteoblast responses to systemic hormones.

1,25-dihydroxyvitamin D-3 and 24,25-dihydroxyvitamin D-3 affect parathormone (PTH) - sensitive adenylate cyclase activity and alkaline phosphatase secretion of osteoblastic cells through different mechanisms of action

In UMR 106 rat osteosarcoma cells, parathormone (1–34hPTH) and calcitonin (sCT) stimulated adenylate cyclase (AC) activity 5.5-and 2.8-fold, respectively. AC in osteoblasts (OB) from collagenase-treated calvaria of 3-day-old rats responded similarly to 1–34hPTH. In contrast, fibroblasts (mouse fibroblastomas) displayed a marginal 1–34hPTH sensitive AC. Osteoclasts (OC) of collagenase-treated rat calvariae, rat monocytes and mouse macrophages did not demonstrate 1–34hPTH inducable AC activity. Physiological concentrations of 24,25-dihydroxyvitamin D-3 attenuated PTH-sensitive AC in OB and UMR 106 cells within 20 min, while 1,25-dihydroxyvitamin D-3 showed no such immediate effect. In contrast, the AC response to Gpp(NH)p was unaffected by 24,25-(OH) 2D 3, indicating that 24,25-(OH) 2D 3 interrupts the coupling of the PTH receptor to the GTP binding protein G s. OB and UMR 106 cells were also subjected to long-term (48 h) incubation with vitamin D-3 metabolites, 1–34hPTH or 20% serum from patients with secondary hyperparathyroidism (sHPT-serum), respectively. PTH-sensitive AC was markedly attenuated by pre-exposure to both 1–34hPTH and 1,25-(OH) 2D 3, while minimally affected by corresponding 24,25-(OH) 2D 3 and 20% sHPT-serum treatment. The secretion of alkaline phosphatase (Alphos) from the two cell types was strongly increased by 1–34hPTH, the effect being abolished by the presence of 24,25-(OH) 2D 3. Iliac crest biopsies of normal individuals exhibited a clear negative correlation between PTH-sensitive AC and corresponding serum 24,25-(OH) 2D 3 levels. Basal AC activity was, however, negatively correlated to serum 1,25-(OH) 2D 3 concentrations. In summary, the results show that 24,25-(OH) 2D 3 reduces PTH-stimulated AC activity in and Alphos secretion from osteoblastic bone cells by rapidly and directly interfering with the plasma membrane. These data reinforce the probable in vivo significance of 24,25-(OH) 2D 3. Moreover, the negative correlation between basal AC activity and serum 1,25-(OH) 2D 3 levels indicates a possible role for 1,25-(OH) 2D 3 in regulating bone cell synthesis of AC components in vivo.

Adenylate Cyclase System in Fetal Rat Keratinizing Epidermal Cells (FRSK Cells) and SV40‐transformed Human Keratinocytes

The adenylate cyclase system of FRSK cells, a cultured cell line of fetal rat epidermal keratinocytes, and SV40-transformed human keratinocytes was investigated. Stimulators of the human epidermal adenylate cyclase, epinephrine, adenosine, and prostaglandin E2 increased cyclic AMP levels of these cells. There were marked differences in the stimulatory effects; while epinephrine revealed a much stronger effect than the other stimulators in FRSK cells, epinephrine and prostaglandin E2 revealed similarly marked effects in SV40-transformed cells. Histamine had little or only slight effect on the cyclic AMP levels of these cells. Cholera toxin and forskolin, which work on the stimulatory guanine nucleotide binding protein (Gs) and the catalytic component of adenylate cyclase, respectively, also increased cyclic AMP levels. Northern blot hybridization analysis revealed that both FRSK cells and SV40-transformed human keratinocytes express mRNAs for the beta 2-adrenergic receptor, as well as the stimulatory and inhibitory guanine nucleotide binding proteins (Gs and Gi, respectively). The presence of Gs as well as Gi were confirmed by cholera toxin-, and pertussis toxin (IAP)-induced ADP-ribosylation of membranous proteins of these cells. Our results indicate that both FRSK cells and SV40-transformed human keratinocytes express the fundamental components of the adenylate cyclase system. These cell lines might be useful tools for the analysis of the adenylate cyclase system in epidermal keratinocytes.

Effects of follicle stimulating hormone, cholera toxin, pertussis toxin and forskolin on adenosine cyclic 3',5'-monophosphate output by granulosa cells from Booroola ewes with or without the F gene

The cAMP outputs by granulosa cells from 3-4.5 mm diameter (medium) follicles of Booroola FF ewes were similar to those by cells from greater than or equal to 5 mm diameter (large) follicles of ++ ewes with respect to time or dose of FSH, cholera toxin or forskolin. Likewise, the cAMP outputs by cells from 1-2.5 mm diameter (small) FF follicles were similar to those by cells from small and medium ++ follicles with respect to time or dose of FSH, cholera toxin or forskolin. At FSH, cholera toxin or forskolin doses of 1 microgram/ml, 0.5 microgram/ml and 10(-4) M respectively, the granulosa cell cAMP outputs of medium FF or large ++ follicles were approximately 2-fold (P less than 0.05) higher than in the respective small FF and medium ++ follicles. The effects of cholera toxin plus forskolin or FSH plus forskolin were additive irrespective of genotype or follicle size, with significant differences (P less than 0.05) observed between follicle sizes but not genotype. No differences were noted between cholera toxin plus forskolin or FSH plus forskolin on granulosa cell cAMP output. For the FSH and forskolin treatments, increased mean cAMP outputs were evident after 10 min, whereas after cholera toxin treatment they were not evident until after 20 min incubation. For all treatments the rate of cAMP production tended to slow down after 40-60 min. Pre-incubation of granulosa cells with pertussis toxin subsequently resulted in a significantly greater (P less than 0.05) FSH-induced output of cAMP relative to the untreated controls irrespective of follicle size. However, no gene-specific differences were noted when the cAMP outputs of cells from medium or small FF follicles were compared with cells from large or small-medium ++ follicles respectively. These results indicate that the activity (or composition) of the regulatory and catalytic components of adenylate cyclase in the FF granulosa cells change in a manner similar to those observed in ++ cells with the only difference being that the increases in cyclase in FF ewes occurs as follicles enlarge from 1-2.5 to 3-4.5 mm in diameter, whereas in ++ ewes they occur as follicles enlarge from 3-4.5 to greater than or equal to 5 mm in diameter. No evidence was found to link the F gene to the granulosa cell cAMP response independently of follicle size. It is suggested that the association between the F gene and the size-specific difference in follicle maturation may be unrelated to the FSH receptor/cAMP generating system.

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.

Effects of treatment with a lithium-imipramine combination on components of adenylate cyclase in the cerebral cortex of the rat

This study was aimed at investigating the effects of treatment with a lithium-imipramine combination on the activity of adenylate cyclase in membranes from the cerebral cortex of the rat. Treatment with (1) lithium for 2 weeks, yielding a level of lithium in serum of 0.54 ± 0.12 mmol/ l, (2) imipramine for 4 weeks (10mg/kg i.p. twice per day) and (3) a combination of the two drugs reduced isoprenaline-induced stimulation of adenylate cyclase by GTP, with a greater decrement with the combined treatment. None of the treatments exerted any effect on the activity of the enzyme stimulated by GTP alone. Lithium ex vivo inhibited the calcium (Ca 2+)- and Gpp(NH)p-stimulated activity of adenylate cyclase, but imipramine ex vivo did not affect the activity of adenylate cyclase, stimulated by these activators. The lithium-imipramine treatment reduced Ca 2+- and Gpp(NH)p-stimulated activity of adenylate cyclase, but this was not different from that observed in the lithium-treated group. In conclusion, the β-adrenoceptor-stimulated adenylate cyclase was affected markedly by administration of lithium and imipramine together. In contrast to lithium ex vivo, imipramine ex vivo did not impair the activity of either the guanine nucleotide regulatory protein or the catalytic subunit, since no change in activity was observed in the presence of β,γ-imidoguanosine-5' triphosphate (Gpp(NH)p) or Ca 2+. Furthermore, lithium ex vivo exerted its post-receptor effects on the adenylate cyclase, independent of imipramine. The decrement in activity of β-adrenergic adenylate cyclase, induced by administration of the two drugs together may partly be involved in the therapeutic action of the augmentation of antidepressants by lithium in refractory depression.

The activation of rabbit intestinal adenylate cyclase by cholera toxin

Brush-border and basal-lateral membranes were prepared from rabbit intestinal epithelial cells by differential centrifugation and MgCl 2 precipitation. The ADP-ribosylation of proteins in these fractions when incubated with [[ adenylate- 32P]NAD + and cholera toxin was investigated. Three proteins of molecular mass 45, 40 and 37 kDa were labelled in a toxin-dependent manner in each membrane fraction. The incorporation of 32P-labelled ADP-ribose was 18-fold greater in brush-border membranes than in basal-lateral membranes, comparable to the enrichment of sucrase (marker enzyme for the brush border) in these membranes. There was a 20% release of the 40 and 45 kDa proteins from the brush-border membrane following this ADP-ribosylation. Activation of adenylate cyclase by both cholera toxin and sodium fluoride was 2.7- and 2.3-fold greater, respectively, in basal-lateral membranes than in brush-border membranes, comparable to the enrichment of Na +/K +-ATPase (marker enzyme for the basal-lateral membrane) in these membranes. The effect of sodium fluoride on membranes pretreated with cholera toxin revealed no increase in adenylate cyclaseactivity above that due to the toxin. This presumably means that both toxin and fluoride activate adenylate cyclase by the same regulatory protein. The results show that cholera toxin catalyzes the ADP-ribosylation of regulatory proteins in the brush-border membrane, and these proteins then migrate to the basal-lateral membrane where they activate the catalytic component of adenylate cyclase.

Disappearance of β- Adrenergic Response of Human Myometrial Adenylate Cyclase at the End of Pregnancy*

The density of beta-adrenergic receptors in both the outer and inner layers of the human myometrium decreases during the last weeks of pregnancy. Although in preterm myometrium (32-35 weeks of pregnancy) beta-adrenergic receptors are positively coupled to adenylate cyclase, we found that isoproterenol, epinephrine, and norepinephrine did not stimulate the enzyme in the inner or outer myometrial layer at term (39-40 weeks of pregnancy). At this stage, the addition of 10(-4) mol/L guanyl-5'-imidodiphosphate increased (from 10(-8) to 10(-4) mol/L) basal adenylate cyclase activity in a dose-dependent manner, indicating that the catalytic component of the enzyme remains linked to the stimulatory guanyl nucleotide binding protein (Gs). Compared to the preterm response, at term the myometrial adenylate cyclase response to 10(-4) mol/L guanyl-5'-imidodiphosphate was decreased, which may reflect a decrease in the amount of functional Gs. Altogether these changes are consistent with reduced Gs coupling to the catalytic component. However, the similarity of the responses of preterm and term myometrium to forskolin excluded the possibility of an alteration of the catalytic component of adenylate cyclase during the last weeks of pregnancy. The fact that a stimulatory effect of isoproterenol on adenylate cyclase was found after islet-activating protein pretreatment indicates that human term myometrium contains a functional inhibitory guanyl nucleotide binding protein which is involved in the modulation of the beta-adrenergic adenylate cyclase response. Our data suggest that modifications in the coupling mechanisms between receptors and the catalytic component are implicated in the loss of beta-adrenergic adenylate cyclase stimulation in the myometrium at the end of pregnancy.

Stimulatory and inhibitory effects of forskolin on adenylate cyclase in rat normal hepatocytes and hepatoma cells.

Forskolin synergistically potentiated adenosine 3',5'-cyclic monophosphate formation by prostaglandin E1 (PGE1) in rat normal hepatocytes freshly prepared by collagenase digestion and rat ascites hepatoma AH66 cells, but dose-dependently inhibited the accumulation by PGE1 in AH66F cells. Forskolin activated adenylate cyclase in a dose-dependent manner in homogenates of all cell lines. In normal hepatocytes and AH66 cells, simultaneous addition of forskolin and other adenylate cyclase activators [isoproterenol (IPN), PGE1, guanosine 5'-triphosphate sodium salt (GTP), 5'-guanylylimidodiphosphate sodium salt (Gpp (NH)p), NaF, cholera toxin, islet activating protein and MnCl2] gave greater than additive responses. On the other hand, in AH66F cells, the effect of forskolin on adenylate cyclase was hardly influenced by GTP, but forskolin diminished the activities induced by high concentrations of GTP to that by the diterpene alone. Forskolin also significantly inhibited the PGE1-stimulated and the guanine nucleotide binding regulatory protein-stimulated activities. Because AH66F cells were insensitive to IPN, the combination with forskolin and IPN gave similar activity to that obtained with the diterpene alone. The effect of forskolin on the activation by manganese ion was neither synergistic nor inhibitory but was additive in AH66F cells. These results suggest that forskolin promotes the interaction between the stimulatory guanine nucleotide binding regulatory protein and the catalytic unit in normal hepatocytes and AH66 cells, but in AH66F cells forskolin interferes with the coupling of the two components of adenylate cyclase.

Adenylate cyclase-modulating mediators of insulin.

Plasma membranes prepared from pig liver incubated with insulin (50-300 microU/ml) resulted in the release of at least two insulin chemical mediators. They appeared to modulate the activity of adenylate cyclase in liver plasma membranes of pig. One of them was fraction 1 of insulin mediator (M. W. about 3700-4000 dalton) which markedly stimulated the activity of the enzyme, the other was fraction 2 of insulin mediator (M. W. about 1000 dalton) which inhibited the enzyme activity. The results showed that the inhibitor of fraction 2 generated was significantly higher than that of fraction 1 when the membranes were incubated with insulin of low concentration (50-100 microU/ml). On the other hand, the generation of stimulator of fraction 1 from plasma membranes incubated with insulin of high concentration (200 microU/ml) was higher than that of fraction 2. So the ratio of yield between two mediators produced from the membranes was dependent on the concentration of insulin added. The results also showed that the effect of fraction 1 of insulin mediator on adenylate cyclase activity in liver cell plasma membranes was biphasic while fraction 2 of insulin mediator showed an inhibitory effect only even though it was at very high concentration. The results that both mediators combined with Gpp(NH)p and forskolin to affect the enzyme activity show that the action of insulin mediators likely resides in the GTP regulatory component of adenylate cyclase.

Eicosanoids as pluripotential modulators of pancreatic islet function.

Eicosanoids both negatively and positively modulate glucose-induced insulin secretion. Although the identity of the positive modulator is uncertain, the negative modulator appears to be prostaglandin E2 (PGE2), because 1) glucose stimulates PGE2 synthesis from islet cells; 2) exogenous PGE2 inhibits glucose-induced insulin secretion; 3) inhibition of beta-cell PGE2 synthesis increases glucose-induced insulin secretion, and this increase is reversed by exogenous PGE2; and 4) PGE2 binds to specific beta-cell receptors that are coupled to inhibitory regulatory components of adenylate cyclase whose activation decreases cAMP levels. Other possible regulatory effects of eicosanoids on islet function include modulation of islet blood flow and its immune responsiveness. From these considerations, the perspective is offered that eicosanoids are pluripotential modulators of islet function.

Inhibition of adenylate cyclase from luteinized rat ovary by monovalent cations: Roles of the stimulatory guanine nucleotide-binding regulatory component and stimulatory hormone receptor

Sodium and other monovalent cations (added as chloride salts) inhibited adenylate cyclase of luteinized rat ovary. Sodium chloride (150 m m) inhibited basal enzyme activity by 20%. Sodium chloride inhibition was enhanced to 34–54% under conditions of enzyme stimulation by guanine nucleotides (GTP and its nonhydrolyzable analog 5′-guanylyl imidodiphosphate), fluoride anion, and agonists (ovine luteinizing hormone (oLH) and the β-adrenergic catecholamine isoproterenol) acting at stimulatory receptors linked to adenylate cyclase. Sodium chloride inhibition was dependent on salt concentration over a wide range (25–800 m m) as well as the concentrations of GTP and oLH. Inhibition by NaCl was of rapid onset and appeared to be reversible. The order of inhibitory potency of monovalent cations was Li + > Na + > K +. The role of individual components of adenylate cyclase in the inhibitory action of monovalent cations was examined. Exotoxins of Vibrio cholerae and Bordetella pertussis were used to determine respectively the involvement of the stimulatory and inhibitory guanine nucleotide-binding regulatory components (N s and N i) in NaCl inhibition. Sodium chloride inhibited cholera toxin-activated adenylate cyclase activity by 29%, N i did not appear to mediate cation inhibition of adenylate cyclase because pertussis toxin did not attenuate inhibition by NaCl. Enzyme stimulation by agents (forskolin and Mn 2+) thought to activate the catalytic component directly was not inhibited by NaCl but was instead significantly enhanced. Sodium chloride (150 m m) increased both the K d for high-affinity binding of oLH to 125I-human chorionic gonadotropin binding sites and the K act for oLH stimulation of adenylate cyclase by sevenfold. In contrast, NaCl had no appreciable effect on either isoproterenol binding to (−)-[ 125I]iodopindolol binding sites or the K act for isoproterenol stimulation of adenylate cyclase. The results suggest that in luteinized rat ovary monovalent cations uncouple, or dissociate, N s from the catalytic component and, in a distinct action, reduce gonadotropin receptor affinity for hormone. Dissociation of the inhibitory influence of N i from direct catalytic activation could account for NaCl enhancement of forskolin- and Mn 2+-associated activities. On the basis of these results, the spectrum of divergent stimulatory and inhibitory effects of monovalent cations on adenylate cyclase activities in a variety of tissues may be interpreted in terms of differential enzyme susceptibilities to cation-induced uncoupling of N and catalytic component functions.