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.     

Activation of cAMP-dependent protein kinase is required for heterologous desensitization of adenylyl cyclase in S49 wild-type lymphoma cells.

We report here that, contrary to previously reported findings, treatment of S49 wild-type (WT) lymphoma cells with 0-50 nM epinephrine resulted in a heterologous desensitization of adenylyl cyclase (EC 4.6.1.1)--that is, epinephrine and prostaglandin E1 (PGE1) stimulations of adenylyl cyclase were reduced. Observation of this heterologous desensitization required the assay of adenylyl cyclase with submillimolar concentrations of Mg2+ and low concentrations of epinephrine. Also, whereas previously there had been no evidence for any role of cAMP-dependent protein kinase in the desensitization of the WT beta-adrenergic receptor, our data comparing the characteristics of the desensitization in WT, kin-, and cyc- lymphoma cells [where kin- and cyc- refer to variants of S49 WT cells lacking cAMP-dependent protein kinase activity (kin-) and the alpha subunit of the stimulatory guanine nucleotide-binding regulatory protein (cyc-)] now suggest that cAMP-dependent protein kinase mediates the heterologous desensitization of adenylyl cyclase. Specifically, we found that only the WT cells exhibited epinephrine-induced heterologous desensitization. The kin- and cyc- cells exhibited only homologous desensitization, and much higher concentrations of epinephrine were required to elicit the homologous desensitization in the variants relative to the heterologous desensitization of the WT. Treatment of WT and cyc- cells with dibutyryl cAMP or treatment of WT with forskolin or PGE1 caused the heterologous desensitization of adenylyl cyclase, indicating that neither receptor occupancy nor activation of adenylyl cyclase was necessary for the heterologous desensitization.     

Lowered responsiveness of the catalyst of adenylyl cyclase to stimulation by GS in heterologous desensitization: a role for adenosine 3',5'-monophosphate-dependent phosphorylation.

Treatment of chick hepatocytes with glucagon results in homologous and heterologous desensitization of the receptor-stimulated adenylyl cyclase. The loci of postreceptor heterologous desensitization was studied. The addition of excess purified GS to glucagon-desensitized hepatocyte membranes did not fully restore fluoride stimulation of adenylyl cyclase, even though the absolute activity was increased at least 2-fold. Treatment of chick hepatocytes with 8-bromo-cAMP resulted in a similar reduction of fluoride stimulation that could not be restored by the addition of purified GS. When membranes from control and glucagon-treated hepatocytes were treated with purified catalytic subunit of protein kinase-A (PKA), fluoride stimulation was lowered in control, but not glucagon-treated, membranes. Treatment of membranes from S49 kin- lymphoma cells with PKA also resulted in decreased fluoride- and forskolin-stimulated adenylyl cyclase activity, but activity stimulated by Mn2+ was not altered. Since previous studies from our laboratory had shown that GS and G(i) are not substrates for protein kinase-A, it appears that the catalyst of adenylyl cyclase is the likely locus of modulation. To determine if both chick hepatocytes and S49 cells contain similar types of adenylyl cyclase that could account for the similar PKA regulatory properties, we used polymerase chain reaction-based techniques to identify GS-stimulated adenylyl cyclases present in these systems. The chick liver contains both type 5 and type 6 adenylyl cyclases, while S49 cells contain the type 6 enzyme. Type 5 and 6 adenylyl cyclases are members of one widely expressed subfamily of mammalian GS-responsive adenylyl cyclases and share a predicted PKA phosphorylation site in the central cytoplasmic loop. This site is not found in other known adenylyl cyclases (types 1-4), although the olfactory-specific type 3 enzyme has a predicted site nearby. These data indicate that one component of hormone-induced desensitization of the adenylyl cyclase system can be at the level of the catalyst, where PKA-mediated phosphorylation could result in lowered responsiveness. The types 5 and 6 adenylyl cyclases are likely candidates for such regulation.     

Gonadotropin-releasing hormone couples to 3',5'-cyclic adenosine-5'-monophosphate pathway through novel protein kinase Cdelta and -epsilon in LbetaT2 gonadotrope cells

GnRH regulates the reproductive system by stimulating synthesis and release of gonadotropins. GnRH acts through a receptor coupled to multiple intracellular events including a rapid phosphoinositide turnover. Although the cAMP pathway is essential for gonadotrope function, the ability of GnRH to induce cAMP, as well as the coupling mechanisms involved, remain controversial. In this study, we established that GnRH increases intracellular cAMP levels in a concentration-dependent manner in LbetaT2 gonadotrope cells (maximal increase, 2.5-fold; EC(50), 0.30 nm), and this was further evidenced by GnRH activation of a cAMP-sensitive reporter gene. The GnRH effect was Ca(2+) independent, mimicked by the phorbol ester phorbol 12-myristate 13-acetate, and blocked by the protein kinase C (PKC) inhibitor bisindolylmaleimide, indicating that the GnRH effect was mediated by PKC. Pharmacological inhibition of conventional PKC isoforms with G?6976 did not prevent GnRH-induced cAMP production, whereas down-regulation of novel PKCdelta, -epsilon, and -theta by a long-term treatment with GnRH markedly reduced it. Expression of dominant-negative (DN) mutants of PKCdelta or -epsilon but not PKCtheta impaired GnRH activation of a cAMP-sensitive promoter, demonstrating that PKCdelta and -epsilon are the two endogenous isoforms mediating GnRH activation of the adenylyl cyclase (AC) pathway in LbetaT2 cells. Accordingly, we identified by RT-PCR and immunocytochemical analysis, two PKC-sensitive AC isoforms, i.e. AC5 and AC7 as potential targets for GnRH. Lastly, we showed that only sustained stimulation of GnRH receptor significantly increased cAMP, suggesting that in vivo, the cAMP signaling pathway may be selectively recruited under intense GnRH release such as the preovulatory GnRH surge.     

Homologous desensitization of the murine luteinizing hormone receptor expressed in L cells

Using a clonal cell line that stably expresses the murine luteinizing hormone receptor (LHR 11/6 cells), we studied the molecular mechanisms of agonist-induced desensitization of the luteinizing hormone/chorionic gonadotropin-responsive adenylyl cyclase. Exposure of transfected cells to human chorionic gonadotropin (hCG) resulted in a dose-dependent loss of maximal hCG-stimulable adenylyl cyclase activity without a significant shift to the right of the dose-response curve to hCG. This rapid uncoupling of the LH receptor from the cellular adenylyl cyclase system was not accompanied by internalization of receptor sites. A 6-h exposure to hCG led only to minor (ca. 25%) loss of membrane binding sites. The dose-response curve to hCG was not altered by pretreating cells with 8-Br-cAMP or prostaglandin E₁. These findings, and the observation that hCG-induced desensitization can still be monitored at Mg²⁺ concentrations in the assay as high as 10 mM, preclude a significant contribution of protein kinase A to LH receptor uncoupling. The murine LH receptor not only stimulates adenylyl cyclase but also phospholipase C and probably protein kinase C (PKC) via diacylglycerol. Activation of PKC by 4β-phorbol 12-myristate 13-acetate failed to desensitize. When PKC was down-regulated hCG could still exert a maximal desensitizing effect. It is concluded that in LHR 11/6 cells there is no evidence for a major role of PKC in homologous desensitization. Thus, it is likely that a second messenger-independent kinase, such as β-adrenergic receptor kinase, or a different, as yet unknown mechanism is involved in the agonist-induced desensitization of the LH receptor.     

Ontogeny of cardiac beta-adrenoceptor desensitization mechanisms: agonist treatment enhances receptor/G-protein transduction rather than eliciting uncoupling

In the fetus and neonate, beta-adrenoceptor stimulation fails to produce physiological desensitization. The current study explores the mechanisms underlying the response pattern in neonatal rats. Homologous cardiac beta-adrenergic desensitization caused by isoproterenol treatment in vivo was demonstrable in adult rats by the immediate (2h) and specific loss of the ability of isoproterenol, but not glucagon, to stimulate adenylyl cyclase in vitro. Homologous desensitization was absent when the same treatment was given to neonates. By 12 h post-treatment, the adults showed heterologous desensitization (loss of the response to glucagon), an effect which was once again absent in the immature rats. The absence of desensitization in neonates did not reflect a deficiency in the activity or subcellular distribution of beta ARK1, the enzyme that initiates the phosphorylation and consequent desensitization of beta-adrenoceptors. On the other hand, neonates showed relatively poor receptor-Gs transduction as assessed by the GTP-induced shift in receptor ligand binding. Repeated isoproterenol treatment of adult rats led to uncoupling of receptor-G-protein transduction but the same treatment in neonates enhanced transduction. Furthermore, neonatal sympathectomy with 6-OHDA interfered with the ontogenetic rise in beta-adrenoceptor-Gs interactions. These results indicate that the maintenance of agonist responses in the face of neonatal adrenergic stimulation does not reflect simply an absence of the ability to elicit homologous or heterologous desensitization but rather represents an active regulatory mechanism in which neural input exerts a positive trophic role at the level of G-protein function.     

Activated protein kinase C isoforms target to cardiomyocyte caveolae : stimulation of local protein phosphorylation.

Protein kinase C (PKC) isoforms constitute an important component of the signal transduction pathway used by cardiomyocytes to respond to a variety of extracellular stimuli. Translocation to distinct intracellular sites represents an essential step in the activation of PKC isoforms, presumably as a prerequisite for stable access to substrate. Caveolae are specialized subdomains of the plasma membrane that are reported to concentrate key signaling proteins and may represent a locus for PKC action, given that PKC activators have been reported to dramatically alter caveolae morphology. Accordingly, this study examines whether PKC isoforms initiate signaling in cardiomyocyte caveolae. Phorbol ester-sensitive PKC isoforms were detected at very low levels in caveolae fractions prepared from unstimulated cardiomyocytes; phorbol 12-myristate 13-acetate (PMA) (but not 4alpha-PMA, which does not activate PKC) recruited calcium-sensitive PKCalpha and novel PKCdelta and PKCepsilon to this compartment. The subcellular localization of the phorbol ester-insensitive PKClambda isoform was not influenced by PMA. Endothelin also induced the selective translocation of PKCalpha and PKCepsilon (but not PKCdelta or PKClambda) to caveolae. Multiple components of the extracellular signal-regulated protein kinase (ERK) cascade, including A-Raf, c-Raf-1, mitogen-activated protein kinase kinase, and ERK, were detected in caveolae under resting conditions. Although levels of these proteins were not altered by PMA, translocation of phorbol ester-sensitive PKC isoforms to caveolae was associated with the activation of a local ERK cascade as well as the phosphorylation of a approximately 36-kDa substrate protein in this fraction. Finally, a minor fraction of a protein that has been designated as a receptor for activated protein kinase C resides in caveolae and (along with caveolin-3) could represent a mechanism to target PKC isoforms to cardiomyocyte caveolae. These studies identify cardiomyocyte caveolae as a meeting place for activated PKC isoforms and their downstream target substrates.     

Protein kinase Cdelta-dependent phosphorylation of syndecan-4 regulates cell migration

Endothelial cell (EC) migration is a complex process requiring exquisitely coordinated focal adhesion assembly and disassembly. Protein kinase C (PKC) is known to regulate focal adhesion formation. Because lysophosphatidylcholine (lysoPC), a major lipid constituent of oxidized low-density lipoprotein, can activate PKC and inhibit EC migration, we explored the signaling cascade responsible for this inhibition. LysoPC increased PKCdelta activity, measured by in vitro kinase activity assay, and increased PKCdelta phosphorylation. Decreasing PKCdelta activation, using pharmacological inhibitors or antisense oligonucleotides, diminished the antimigratory effect of lysoPC. LysoPC-induced PKCdelta activation was followed by increased phosphorylation of the transmembrane proteoglycan, syndecan-4, and decreased binding of PKCalpha to syndecan-4, with a concomitant decrease in PKCalpha activity. A reciprocal relationship was noted between the interaction of PKCalpha and alpha-actinin with syndecan-4. These changes were temporally related to the observed changes in cell morphology and the inhibition of migration of ECs incubated with lysoPC. The data suggested that generalized activation of PKCdelta by lysoPC initiated a cascade of events, including phosphorylation of syndecan-4, displacement and decreased activity of PKCalpha, binding of alpha-actinin to syndecan-4, and disruption of the time- and site-specific regulation of focal adhesion complex assembly and disassembly required for normal cell migration.     

Regulation of protein kinase D activity in adult myocardium: novel counter-regulatory roles for protein kinase Cepsilon and protein kinase A

Protein kinase D (PKD) is activated downstream of protein kinase C (PKC) in many cell types, although little is known about the mechanisms that regulate PKD in adult myocardium. Exposure of cultured adult rat ventricular myocytes (ARVM) to phorbol 12-myristate 13-acetate (PMA; 100 nM for 5 min) activated PKD, as evidenced by significantly increased phosphorylation at Ser744/8 (PKC phosphorylation sites) and Ser916 (autophosphorylation site). PKD activation occurred concomitantly with translocation of the enzyme from the cytosolic to the particulate fraction. The role of PKC was confirmed by pretreatment (15 min) of ARVM with the PKC inhibitors GF109203X (1 microM) and Ro31-8220 (1 microM), both of which prevented PKD phosphorylation on subsequent exposure to PMA. Exposure of ARVM to endothelin-1 (ET1; 100 nM for 10 min) also activated PKD by a PKC-dependent mechanism. To determine the PKC isoform(s) involved in the ET1-induced PKD activation, ARVM were infected with adenoviral vectors encoding dominant-negative (DN) mutants of PKCalpha, PKCdelta and PKCepsilon. Expression of DN-PKCalpha and DN-PKCdelta had little effect on ET1-induced PKD activation, whilst this was significantly attenuated by expression of DN-PKCepsilon, indicating that PKCepsilon plays a predominant role in the pertinent ET1 signaling pathway. Intriguingly, prior exposure to the adenylyl cyclase activator forskolin (1 microM for 5 min) or the beta-adrenergic agonist isoprenaline (100 nM for 5 min) markedly attenuated ET1-induced PKD activation, but not PMA-induced PKD activation. The ET1-induced response was rescued when protein kinase A (PKA) was inhibited (H89, 10 microM) before exposure to isoprenaline. These results show that ET1-induced PKD activation in ARVM is mediated by PKC, primarily the PKCepsilon isoform, and is suppressed by PKA activation.     

Endometrial cancer cell survival and apoptosis is regulated by protein kinase C alpha and delta

Endometrial cancer is the most common invasive gynecologic malignancy but the molecular mechanisms underlying its onset and progression are poorly understood. Paradoxically, endometrial tumors exhibit increased apoptosis, correlating with disease progression and poor patient prognosis. Endometrial tumors also show altered activity and expression of protein kinase C (PKC) isoforms, implicated in the regulation of programmed cell death; however, PKC modulation of apoptosis in endometrial cancer cells has not been investigated. We detected nine out of ten PKC isoforms in Ishikawa endometrial cancer cell lines, and demonstrated expression of both PKCalpha and delta in human endometrial tumors. To determine the functional roles of PKCalpha and delta in apoptosis in endometrial cancer, Ishikawa cells were treated with selective PKC inhibitors or adenoviral constructs encoding wild-type or isoform-specific, dominant-negative mutants. Apoptosis was assessed by DNA fragmentation and caspase-mediated poly-(ADP-ribose)-polymerase cleavage. The inhibition of PKCdelta suppressed etoposide-induced apoptosis, while overexpression of PKCdelta enhanced it. In contrast, inhibition of PKCalpha elevated basal levels of apoptosis and potentiated etoposide-induced cell death. Etoposide treatment also selectively activated PKCdelta, but resulted in both cytosolic translocation and decreased activity of PKCalpha. A fraction of PKCdelta also underwent caspase-dependent cleavage, in response to etoposide. Our results suggest that changes in apoptosis and PKC expression in endometrial cancer are mechanistically linked, such that PKCdelta is required for DNA damage-induced apoptosis, while PKCalpha mediates a survival response. Thus, PKCalpha and delta expression and signaling may be important in endometrial tumorigenesis and could serve as potential prognostic indicators and/or novel targets for therapeutic intervention.     

Acute signaling by the LH receptor is independent of protein kinase C activation

LH receptor activation leads to the phosphorylation/activation of p42/44 MAPK in preovulatory granulosa cells. As the LH receptor can activate both adenylyl cyclase and phospholipase C, we hypothesized that the LH receptor could elicit phosphorylation of p42/44 MAPK through activation of protein kinase A (PKA) and/or protein kinase C (PKC). Preovulatory granulosa cells in serum-free primary cultures were treated with ovulatory concentrations of human chorionic gonadotropin (hCG), an LH receptor agonist, with or without various inhibitors. The PKA inhibitor H89 as well as the myristoylated PKA inhibitor peptide PKI strongly inhibited hCG-stimulated p42/44 MAPK phosphorylation, whereas the PKC inhibitor GF109203X had no effect on p42/44 MAPK phosphorylation. LH receptor-stimulated phosphorylation of cAMP response element-binding protein (CREB), histone H3, and MAPK kinase (MEK) was also strongly inhibited by H89 and not by GF109203X. The extent of PKC activation was assessed in preovulatory granulosa cells using three criteria: translocation of PKC isoforms to the membrane fraction, phosphorylation of a known PKC substrate, and autophosphorylation of PKC delta on an activation-related site. By all three criteria PKCs were partially activated before hCG stimulation, and hCG treatment failed to elicit further PKC activation, in vitro or in vivo. Taken together, these results indicate that, under primary culture conditions where physiological levels of signaling proteins are present, hCG signals to activate MEK, p42/44 MAPK, CREB, and histone H3 in a predominantly PKA-dependent and PKC-independent manner. Unexpectedly, PKCs were partially activated in the absence of LH receptor activation, and LH receptor activation did not elicit further detectable PKC activation.     

Altered action of glucagon on human liver in Type 2 (non-insulin-dependent) diabetes mellitus

Summary Glucagon may play a role in the metabolic derangements of overt Type 2 (non-insulin-dependent) diabetes mellitus. We therefore have evaluated the early steps in glucagon action by investigating the hormone-sensitive adenylyl cyclase system in liver membranes from seven Type 2 diabetic patients with fasting hyperglycaemia and two-fold elevations in plasma glucagon. The comparison was made with seven control subjects matched for age, sex and body weight. Glucagon receptor binding was almost identical in the two groups. There were, however, marked alterations in the adenylyl cyclase activity in membranes from the diabetic patients. This activity was reduced by 35–50% when compared to control activity. Basal cyclase activity, as well as the activity after stimulation with glucagon or with agents (i. e., sodium fluoride and forskolin) that act beyond the glucagon receptor, was significantly decreased (p<0.05, p<0.001 respectively). In conclusion, uncontrolled Type 2 diabetes in associated with an over-all loss of responsiveness of the hormone-sensitive adenylyl cyclase in human liver, which apparently results from post-receptor alterations. This change may provide a mechanism for reducing the effect of hyperglycagonaemia in Type 2 diabetes mellitus.     

Catecholamine-induced heterologous desensitization of rabbit luteal adenylyl cyclase: loss of luteinizing hormone responsiveness is associated with impaired G-protein function

The effects of injecting epinephrine into pseudopregnant rabbits on the luteal adenylyl cyclase system were analyzed. Epinephrine-induced desensitization was heterologous and associated with a reduced response to isoproterenol, LH, NaF, and forskolin. Epinephrine-induced desensitization was rapid in onset, with a maximum decrease in responsiveness 6 h after treatment and responsiveness returning to control levels within 24 h of treatment. The changes in beta-adrenergic receptor content paralleled changes in catecholamine responsiveness. The affinity of the beta-receptors from treated animals decreased 1.5- to 2-fold before down-regulation. LH receptor number was not altered by epinephrine treatment, although responsiveness to LH was depressed. LH receptor affinity, however, was reduced about 2-fold by epinephrine treatment. Epinephrine treatment also altered G-protein function in corpora lutea, as assessed by reconstitution of adenylyl cyclase activity in S49 cyc- membranes and ADP ribosylation by cholera and pertussis toxins. NaF- and isoproterenol-reconstituting activities of luteal Gs (the stimulatory G-protein of adenylyl cyclase) were depressed for the first 6 h after treatment. The ability of cholera toxin to ADP ribosylate alpha s 46 and alpha s 45 was reduced 1.5-6 h and 3-12 h, respectively, after epinephrine treatment. The reduced ability of cholera toxin to ADP ribosylate alpha s 45 was associated with the decrease in LH receptor affinity after treatment. This supports the contention that alpha s 45 preferentially interacts with the LH receptor. These studies demonstrate that the loss of LH responsiveness upon epinephrine-induced heterologous desensitization is associated with altered G-protein function.     

Differential effects of jaundice and cirrhosis on beta-adrenoceptor signaling in three rat models of cirrhotic cardiomyopathy

BACKGROUND/AIMS: Attenuated cardiac function has been reported in cirrhosis as well as in jaundice, but the mechanisms remain unclear. This study aimed to explore the differential effects of jaundice and cirrhosis on the heart. METHODS: Three rat models of cirrhosis were studied: chronic bile duct ligation, bile duct ligation followed by choledochojejunostomy to relieve jaundice, and a less jaundiced model induced by thioacetamide administration. Controls underwent a sham operation. Cardiac function was assessed by measuring isolated ventricular papillary muscle contractility. Cardiac beta-adrenergic receptor signaling was studied by measuring cAMP production stimulated at the receptor, G-protein, and adenylyl cyclase levels in the signaling pathway, using isoproterenol, aluminum fluoride and forskolin, respectively. RESULTS: Serum bilirubin and bile salt levels were markedly elevated in the bile duct-ligated group, moderately increased in the thioacetamide rats, and normal in the choledochojejunostomy and sham-operated controls. Papillary muscle contractile force after maximal beta-adrenergic receptor stimulation was decreased to a similar extent in all three cirrhotic models. In the bile duct-ligated and thioacetamide-induced cirrhotic rats, production of cAMP by all three drugs was significantly attenuated. However, the cAMP production in the choledochojejunostomy group was blunted only with isoproterenol and fluoride, and remained intact with forskolin stimulation. CONCLUSIONS: These results demonstrate that cirrhosis per se impairs cardiac function by attenuating the portion of the beta-adrenergic receptor signaling pathway upstream of adenylyl cyclase. Furthermore, significant jaundice and/or cholemia can inhibit adenylyl cyclase, which may contribute to blunted cardiac contractility in jaundiced patients.     

Human chorionic gonadotropin-induced heterologous desensitization of rabbit luteal adenylyl cyclase is associated with altered receptor and G-protein function

We injected hCG into pseudopregnant rabbits on day 7 of pseudopregnancy and analyzed changes in the components of luteal adenylyl cyclase system in order to determine which components are responsible for altered hormonal responsiveness upon desensitization. hCG-induced desensitization was homologous (loss of responsiveness to LH) early (first 6 h), then became heterologous (partial loss of responsiveness to catecholamines) later (12-48 h). The total number of LH receptors was reduced approximately 30% 3 h after treatment at a time when LH stimulation of adenylyl cyclase activity was not altered. Total LH receptors remained at this level until 24 h, when total receptors were reduced by 88%. While total LH receptor number remained constant, LH-stimulated adenylyl cyclase activity was declining to 57% of the control value at 12 h. Available unoccupied LH receptors were reduced by 96% at 12 h. The affinity of the occupied receptors was reduced 4-fold before down-regulation. The changes in beta-adrenergic receptor number paralleled the changes in catecholamine responsiveness. hCG treatment also altered luteal G-protein function, as assessed by reconstitution of adenylyl cyclase activity in S49 cyc- lymphoma membranes and ADP ribosylation by cholera and pertussis toxins. Isoproterenol (ISO)-reconstituting activities of luteal Gs (the stimulatory G-protein of adenylyl cyclase) were depressed by 65% 12-48 h after hCG treatment, the same time as reduced catecholamine responsiveness. In contrast, NaF-reconstituting activities were at control levels at 12 h and reduced by 55% at 24 and 48 h. Pertussis toxin's ability to ADP ribosylate alpha i 40 was increased 3 and 6 h after treatment, while cholera toxin's ability to ADP ribosylate alpha s 45 was reduced throughout the study period. These studies demonstrate that hCG-induced heterologous desensitization results in a complex series of changes in beta-adrenergic and LH receptors as well as G-protein function, which account for the altered hormonal responsiveness.     

Self-limitation of intravenous tocolysis with beta2-adrenergic agonists is mediated through receptor G protein uncoupling

Tocolysis with a beta-adrenergic receptor agonist is the most common approach to premature labor management after the 25th wk of pregnancy. However, prolonged treatment is associated with a marked loss of efficacy. The biochemical mechanisms involved remain unclear. This study was undertaken to investigate the effect of fenoterol on beta-adrenergic receptor signal transduction in human myometrium. Myometrial biopsy specimens were obtained from 40 women at cesarean section between the 25th and 34th wk of pregnancy. Nineteen patients had received no tocolysis (controls, group I) and 21 had been treated with fenoterol (<48 h in 10, group II; > or = 48 h in 11, group III). As methods we used membrane preparation, adenylyl cyclase assay and cAMP RIA. Adenylyl cyclase activity was determined by the measurement of cAMP levels to evaluate signal transduction after stimulation of beta-adrenergic receptors with isoproterenol, G protein with GTP, and adenylyl cyclase with forskolin. The functional activity of GTP-binding regulatory proteins (G(s)) and adenylyl cyclase was not altered by fenoterol treatment. In the control group, the increase in adenylyl cyclase activity in response to GTP plus isoproterenol was greater than in response to GTP alone. The increase was reduced by 50% in group II and was insignificant in group III. There was no correlation between gestational age and basal adenylyl cyclase activity. Intravenous tocolysis with the beta2-adrenergic receptor agonist fenoterol leads to complete desensitization of the beta-adrenergic receptor system. In addition to the known reduction in receptor number (down-regulation) as underlying mechanism, uncoupling of the receptor from the stimulatory G protein G(s) was identified.     

Mechanism of action of des-His1-[Glu9]glucagon amide, a peptide antagonist of the glucagon receptor system.

We have investigated the mechanisms through which des-His1-[Glu9]glucagon amide functions as a peptide antagonist of the glucagon receptor/adenylyl cyclase system. Studies with radiolabeled peptides identified that (i) the antagonist bound to intact hepatocytes according to a single first-order process, whereas the rate of association of glucagon with the same preparation could be described only by the sum of two first-order processes; (ii) the interaction of the antagonist with saponin-permeabilized hepatocytes was not affected by the addition of GTP to the incubation medium or by the elimination of Mg2+, whereas the interaction of glucagon with the same cell preparation was modified significantly by the presence of the nucleotide or by the absence of the divalent metal ion; (iii) the dissociation of antagonist from intact hepatocytes incubated in buffer was complete, whereas that of agonist was not; and (iv) the antagonist bound to intact hepatocytes at steady state according to a single binding isotherm (as did both agonist and antagonist in permeabilized hepatocytes), whereas glucagon bound to the intact cell system with two clearly defined apparent dissociation constants. A model is presented for the mechanism of action of the glucagon antagonist in which the analog binds to glucagon receptors in a Mg(2+)- and GTP-independent fashion and in which resulting ligand-receptor complexes fail to undergo sequential adjustments necessary for the stimulation of adenylyl cyclase.     

Beta-adrenergic regulation of Sertoli cell adenylyl cyclase: desensitization by homologous hormone

Incubation of Sertoli cell-enriched cultures with D,L-isoproterenol caused a time- and concentration-dependent, homologous desensitization of isoproterenol-responsive adenyl cyclase, whereas the response to FSH was unaffected. Half-maximal desensitization was achieved within 1 h of preincubation, after which a more gradual loss of response was observed. Preincubation of Sertoli cells for 24 h with increasing concentrations of D,L-isoproterenol demonstrated that the concentration required to obtain half-maximal densensitization was approximately 10-fold lower than the Km for activation of adenylyl cyclase. The function of the guanine nucleotide regulatory component (N-component) of the adenylyl cyclase complex in hormonally desensitized Sertoli cells, as evaluated by activation of adenylyl cyclase by GTP, GMPP(NH)P, fluoride and Mg2+, was not affected by the hormone pretreatment. Preincubation of Sertoli cells with a high concentration of dbcAMP (10(-3) M) for 24 h was associated with a 45% reduction in adenylyl cyclase activation by both FSH and isoproterenol. Also in this case fluoride- and GTP-stimulated adenylyl cyclase activities were normal. However, the effects of dibutyryl cyclic AMP occurred much more slowly than agonist-induced desensitization, indicating that cAMP may not be the primary mediator of homologous desensitization of Sertoli cell adenylyl cyclase by isoproterenol.     

Modulation of ANF-R2/ANP-C receptors by angiotensin II in vascular smooth muscle cells

In the present studies, we have investigated the modulation of atrial natriuretic factor (ANF) receptor of R2 subtype (ANF-R2) coupled to adenylyl cyclase/cAMP signal transduction system by angiotensin II (AII). C-ANF_4–23 [C-ANF_4–23, [des (Gln¹⁸, Ser¹⁹, Gln²⁰, Leu²¹, Gly²²) ANF_4–23NH₂] and AII inhibited adenylyl cyclase activity in control vascular smooth muscle cells (VSMC A-10) by about 40% and 30% respectively. Pretreatment of the cells with AII resulted in the attenuation of both C-ANF_4–23- and AII-mediated inhibition of adenylyl cyclase. Losartan treatment of the cells was able to partially block (∼50%) the AII- as well as C-ANF_4–23-mediated inhibitions of adenylyl cyclase that are completely lost by AII pretreatment. The pretreatment of the cells with AII alone or with losartan did not affect the [¹²⁵I]-ANF binding to ANF receptors. However, AII treatment resulted in the augmentation of the levels of Giα2 and Giα3. On the other hand, staurosporine (a protein kinase C [PKC] inhibitor) treatment of cells before AII treatment was able to prevent the attenuation of both C-ANF_4–23 as well as AII-mediated inhibition of adenylyl cyclase elicited by AII. These results indicate that the AII modulation of ANF-R2 receptor-mediated inhibition of adenylyl cyclase is independent of ANF-R2 receptor density or the levels of Gi regulatory protein and may be due to the uncoupling of the ANF-R2 receptor from the Gi protein. This uncoupling may be associated with the phosphorylation of the Gi protein by PKC activated by AII.     

Protective effects of ursodeoxycholic acid on chenodeoxycholic acid-induced liver injury in hamsters

AIM To investigate the effects of ursodeoxycholic acid (UDCA) on chenodeoxycholic acid (CDCA)-induced liver injury in hamsters, and to elucidate a correlation between liver injury and bile acid profiles in the liver.METHODS Liver injury was induced in hamsters by administration of 0.5% (w/w) CDCA in their feed for 7 d.UDCA (50 mg/kg and 150 mg/kg) was administered for the last 3 d of the experiment.RESULTS At the end of the experiment, serum alanine aminotransferase (ALT) increased more than 10 times and the presence of liver injury was confirmed histologically. Marked increase in bile acids was observed in the liver. The amount of total bile acids increased approximately three-fold and was accompanied by the increase in hydrophobic bile acids, CDCA and lithocholic acid (LCA). UDCA (50 mg/kg and 150 mg/kg) improved liver histology, with a significant decrease (679.3 ±77.5 U/L vs 333.6 ± 50.4 U/L and 254.3 ± 35.5 U/L, respectively, P ＜ 0.01) in serum ALT level. UDCA decreased the concentrations of the hydrophobic bile acids, and as a result, a decrease in the total bile acid level in the liver was achieved.CONCLUSION The results show that UDCA improves oral CDCA-induced liver damage in hamsters. The protective effects of UDCA appear to result from a decrease in the concentration of hydrophobic bile acids, CDCA and LCA, which accumulate and show the cytotoxicity in the liver.