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The factors that initiate human labor are poorly understood. We have tested the hypothesis that a decline in cAMP/protein kinase A (PKA) function leads to the onset of labor. Initially, we identified myometrial cAMP/PKA-responsive genes (six up-regulated and five down-regulated genes) and assessed their expression in myometrial samples taken from different stages of pregnancy and labor. We found that the oxytocin receptor (OTR) was one of the cAMP-repressed genes, and, given the importance of OTR in the labor process, we studied the mechanisms involved in greater detail using small interfering RNA, chemical agonists, and antagonists of the cAMP effectors. We found that cAMP-repressed genes, including OTR, increased with the onset of labor. Our in vitro studies showed that cAMP acting via PKA reduced OTR expression but that in the absence of PKA, cAMP acts via exchange protein activated by cAMP (EPAC) to increase OTR expression. In early labor myometrial samples, PKA levels and activity declined and Epac1 levels increased, perhaps accounting for the increase in myometrial OTR mRNA and protein levels at this time. In vitro exposure of myometrial cells to stretch and IL-1β increased OTR levels and reduced basal and forskolin-stimulated cAMP and PKA activity, as judged by phospho-cAMP response element-binding protein levels, but neither stretch nor IL-1β had any effect on PKA or EPAC1 levels. In summary, there is a reduction in the activity of the cAMP/PKA pathway with the onset of human labor potentially playing a critical role in regulating OTR expression and the transition from myometrial quiescence to activation.

Objective: The study aims to investigate the possible mechanism of the synergistic analgesic effect of propofol-alfentanil combination., Methods: The synergistic analgesic effects of propofol-alfentanil combination in Sprague-Dawley (SD) rats were analysed through the von Frey test. Then, we examined the activity of phospholipase C (PLC) and the intracellular levels of Ca(2+) and adenosine 3', 5'cyclic monophosphate (cAMP) in primary neuronal cells of fetal SD rats. We detected the intracellular Ca(2+) concentration by fluorescence and flow cytometry. The PLC activity of the primary neuronal cells was assayed using the EnzChek(®) Direct Phospholipase C Assay Kit. The cAMP content of the cells was assayed using the cAMP Direct Immunoassay Kit (Fluorometric)., Key Findings: Both propofol and alfentanil treatments depressed cAMP levels and PLC activity, but propofol-alfentanil combination decreased these parameters to a greater extent than alfentanil treatment alone. Propofol and alfentanil both inhibited Ca(2+) channel, but propofol-alfentanil combination suppressed this channel to a greater extent than alfentanil treatment alone. Fluorescent image analysis revealed that both propofol and alfentanil reduced the intracellular levels of Ca(2+) , and propofol-alfentanil combination showed weaker signals than alfentanil alone. Propofol-alfentanil combination significantly reduced intracellular Ca(2+) level, cAMP level and PLC activity., Conclusion: Propofol and alfentanil exert synergistic analgesic effects through the adenylyl cyclase pathway., (© 2016 Royal Pharmaceutical Society.)

Hyperandrogenism is the central feature of polycystic ovary syndrome (PCOS). Due to the intricate relationship between hyperandrogenism and insulin resistance in PCOS, 50%-70% of these patients also present with hyperinsulinemia. Metformin, an insulin sensitizer, has been used to reduce insulin resistance and improve fertility in women with PCOS. In previous work, we have noted that a dietary medium-chain fatty acid, decanoic acid (DA), improves glucose tolerance and lipid profile in a mouse model of diabetes. Here, we report for the first time that DA, like metformin, inhibits androgen biosynthesis in NCI-H295R steroidogenic cells by regulating the enzyme 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase type 2 (HSD3B2). The inhibitory effect on HSD3B2 and androgen production required cAMP stimulation, suggesting a mechanistic action via the cAMP-stimulated pathway. Specifically, both DA and metformin reduced cAMP-enhanced recruitment of the orphan nuclear receptor Nur77 to the HSD3B2 promoter, coupled with decreased transcription and protein expression of HSD3B2. In a letrozole-induced PCOS rat model, treatment with DA or metformin reduced serum-free testosterone, lowered fasting insulin, and restored estrous cyclicity. In addition, DA treatment lowered serum total testosterone and decreased HSD3B2 protein expression in the adrenals and ovaries. We conclude that DA inhibits androgen biosynthesis via mechanisms resulting in the suppression of HSD3B2 expression, an effect consistently observed both in vitro and in vivo. The efficacy of DA in reversing the endocrine and metabolic abnormalities of the letrozole-induced PCOS rat model are promising, raising the possibility that diets including DA could be beneficial for the management of both hyperandrogenism and insulin resistance in PCOS.

Oocyte in vitro maturation (IVM) is an important assisted reproductive technology and research tool. The adoption of IVM into routine clinical practice has been hindered by its significantly lower success rates compared to conventional in vitro fertilization. Cyclic AMP (cAMP) modulation and follicle-stimulating hormone (FSH), independently, have long been known to improve IVM oocyte developmental competence. This study comprehensively examined the effects of FSH and cAMP/cGMP modulation, alone and in combination, on IVM oocyte metabolism and developmental outcomes. Mouse cumulus-oocyte complexes (COCs) were subjected to a 1 h prematuration phase ± the cAMP modulator forskolin and cAMP/cGMP modulator 3-isobutyl-1-methylxanthine followed by IVM ± FSH. Prematuration with these cyclic nucleotide modulators or IVM with FSH significantly improved oocyte developmental competence and reduced spindle abnormalities compared to spontaneous IVM (no treatment); however, these two treatments in combination endowed even greater developmental competence (improved subsequent blastocyst rates and quality; P < 0.05), albeit blastocyst yield and quality remained significantly lower than that of oocytes matured in vivo. A significant additive effect of combined IVM treatments was evident as increased COC lactate production and oxygen consumption and enhanced oocyte oxidative metabolism, ATP production, ATP:ADP ratio, and glutathione levels (P < 0.05). Nevertheless, IVM increased reactive oxygen species production, particularly as a consequence of FSH addition, relative to in vivo matured oocytes. In conclusion, improvements in the embryo yield following IVM is associated with increased COC oxygen consumption and oocyte oxidative metabolism, but these remain metabolically and developmentally less competent relative to in vivo derived oocytes., (© 2014 by the Society for the Study of Reproduction, Inc.)

The oviduct serves as a site for the fertilization of the ovum and the transport of the conceptus down to the uterus for implantation. In this study, we investigated the presence of adrenomedullin (ADM) and its receptor component proteins in the pig oviduct. The effect of ADM on oviductal secretion, the specific receptor, and the mechanisms involved were also investigated. The presence of ADM and its receptor component proteins in the pig oviduct were confirmed using immunostaining. Short-circuit current (I(sc)) technique was employed to study chloride ion secretion in the oviductal epithelium. ADM increased I(sc) through cAMP- and calcium-activated chloride channels, and this effect could be inhibited by the CGRP receptor antagonist, hCGRP8-37. In contrast, the nitric oxide synthase inhibitor, L-NG-nitroarginine methyl ester (L-NAME), could not block the effect of ADM on I(sc). In summary, ADM may increase oviductal fluid secretion via chloride secretion independent of the nitric oxide pathway for the transport of sperm and the conceptus.

Study Question: Does adjudin disrupt chloride ion (Cl⁻) ion transport function in human sperm and impede sperm capacitation and fertilizing ability in vitro?, Summary Answer: In this study the results indicate that adjudin is a potent blocker of Cl⁻ channels: disrupting Cl⁻ ion transport function results in a decline in sperm capacitation and fertilizing ability in humans in vitro., What Is Known Already: Although our previous studies have demonstrated that adjudin exerts its effect by disrupting sertoli-germ cell adhesion junctions, most notably apical ectoplasmic specialization by targeting testin and actin filament bundles that disrupts the actin-based cytoskeleton in sertoli cells, it remains unclear whether adjudin impedes Cl⁻ ion transport function in the human sperm., Study Design, Size and Duration: Semen samples were obtained from 45 fertile men (aged 25-32). Spermatozoa were isolated from the semen in the human tube fluid (HTF) medium by centrifugation through a discontinuous Percoll gradient, and incubated with adjudin at 10 nM-10 µM and/or other reagents under capacitating conditions for 0-5 h., Participants/materials, Setting, Methods: We evaluated the effect of adjudin and different reagents on sperm functions with which they were incubated at 37 °C. Sperm motility and hyperactivation were analyzed by a computer-assisted sperm analysis (CASA) system. Sperm capacitation and the acrosome reaction were assessed by chlortetracycline fluorescence staining. Sperm fertilizing ability was evaluated by sperm penetration of zona-free hamster egg assay, and cellular cAMP levels in spermatozoa were quantified by the EIA kit. The proteins tyrosine, serine and threonine phosphorylation in the presence or absence of adjudin were analyzed by means of a immunodetection of spermatozoa, especially, compared the effect of adjudin on sperm hyperactivation and capacitation in the complete HTF medium with the Cl⁻-deficient HTF medium as well as the various Cl⁻ channel blockers., Main Results and the Role of Chance: Adjudin significantly inhibited sperm hyperactivation but not sperm motility. Adjudin-induced inhibition of sperm capacitation was reversible, and it was found to block the rhuZP₃β- and progesterone-induced acrosome reaction in a dose-dependent manner. Adjudin also blocked sperm penetration of zona-free hamster eggs, and significantly inhibited both forskolin-activated transmembrane adenylyl cyclase and soluble adenylyl cyclase activities leading to a significant decline in the cellular cAMP levels in human spermatozoa. Adjudin failed to reduce sperm protein tyrosine phosphorylation but it did prevent sperm serine and threonine protein phosphorylation. Interestingly, adjudin was found to exert its inhibitory effects on sperm capacitation and capacitation-associated events only in the complete Cl⁻-HTF medium but not Cl⁻-deficient medium, illustrating the likely involvement of Cl⁻. Adjudin inhibits the fertility capacity of human sperm is mediated by disrupting chloride ion and its transport function., Limitations, Reasons for Caution: This study has examined the effect of adjudin only on human sperm capacitation and fertilizing ability in vitro and thus has some limitations. Further investigations in vivo are needed to confirm adjudin is a potent male contraceptive., Wider Implications of the Findings: Our studies demonstrated that adjudin inhibition of capacitation is reversible and its toxicity is low, opening the door for the examination of adjudin as a mediator of male fertility control. Adjudin may be a safe, efficient and reversible male antifertility agent and applicable to initial clinical trials of adjudin as a male antifertility agent in humans. STUDING FUNDING/COMPETING INTEREST(S): This work was supported by the National Basic Research Program of China (2006CB504002), the Nature Science Foundation of China (Nos. 81000244 and 81170554), Zhejiang Project of Science and Technology (2011C23046), the Nature Science Fund of Zhejiang province (Nos.Y2100058 and Y2090236), the key Science and Technology Innovation Team of Zhejiang Province (No.2012R10048-07) and the National Institutes of Health (NICHD U54 HD029990 project 5), USA. The authors declare no conflict of interest.

Mast cells (MC) occur normally in the testis with a species-specific distribution, yet their precise role remains unclear. Testicular MC express histidine decarboxylase (HDC), the unique enzyme responsible for histamine (HA) generation. Evidence to date supports a role for HA as a local regulator of steroidogenesis via functional H₁ and H₂ receptor subtypes (HRH1 and HRH2, respectively) present in Leydig cells. Given that HA is a well-known modulator of physiological and pathological proliferation in many different cell types, we aimed in the present study to evaluate whether HA might contribute to the regulation of Leydig cell number as well as to the control of androgen production. Herein, we demonstrate, to our knowledge for the first time, that MA-10 Leydig tumor cells, but not normal immature Leydig cells (ILC), exhibit a proliferative response upon stimulation with HA that involves HRH2 activation, transient elevation of cAMP levels, and increased extracellular signal-regulated kinase (ERK) phosphorylation. Our results also reveal that MA-10 cells show significantly heightened HDC expression compared to normal ILC or whole-testicular lysate and that inhibition of HDC activity decreases MA-10 cell proliferation, suggesting a possible correlation between autocrine overproduction of HA and abnormally increased proliferation in Leydig cells. The facts that germ cells are also both source and target of HA and that multiple testicular cells are susceptible to HA action underline the importance of the present study, which we hope will serve as a first step for further research into regulation of non-MC-related HDC expression within the testis and its significance for testicular function.

Proper axonal targeting is fundamental to the establishment of functional neural circuits. The hippocampal mossy fibres normally project towards the CA3 region. In the hippocampi of patients with temporal lobe epilepsy and related animal models, however, mossy fibres project towards the molecular layer and produce the hyperexcitable recurrent networks. The cellular and molecular mechanisms underlying this aberrant axonal targeting, known as mossy fibre sprouting, remain unclear. Netrin-1 attracts or repels axons depending on the composition of its attraction-mediating receptor, deleted in colorectal cancer, and its repulsion-mediating receptor, uncoordinated-5, on the growth cone; but the roles of netrin-1-dependent guidance in pathological conditions are largely unknown. In this study, we examined the role of netrin-1 and its receptors in mossy fibre guidance and report that enhanced neuronal activity changes netrin-1-mediated cell targeting by the axons under hyperexcitable conditions. Netrin-1 antibody or Dcc ribonucleic acid interference attenuated mossy fibre growth towards CA3 in slice overlay assays. The axons were repelled from CA3 and ultimately innervated the molecular layer when hyperactivity was pharmacologically introduced. We first hypothesized that a reduction in netrin-1 expression in CA3 underlies the phenomenon, but found that its expression was increased. We then examined two possible activity-dependent changes in netrin-1 receptor expression: a reduction in the deleted in colorectal cancer receptor and induction of uncoordinated-5 receptor. Hyperactivity did not affect the surface expression of the deleted in colorectal cancer receptor on the growth cone, but it increased that of uncoordinated-5A, which was suppressed by blocking cyclic adenosine monophosphate signalling. In addition, Dcc knockdown did not affect hyperactivity-induced mossy fibre sprouting in the slice cultures, whereas Unc5a knockdown rescued the mistargeting. Thus, netrin-1 appears to attract mossy fibres via the deleted in colorectal cancer receptor, while it repels them via cyclic adenosine monophosphate-induced uncoordinated-5A under hyperexcitable conditions, resulting in mossy fibre sprouting.

We here report a new physiological system that governs catecholamine synthesis involving bone morphogenetic proteins (BMPs) and activin in the rat pheochromocytoma cell line, PC12. BMP type I receptors, including activin receptor-like kinase-2 (ALK-2) (also referred to as ActRIA) and ALK-3 (BMPRIA), both type II receptors, ActRII and BMPRII, as well as the ligands BMP-2, -4, and -7 and inhibin/activin subunits were expressed in PC12 cells. PC12 cells predominantly secrete dopamine, whereas noradrenaline and adrenaline production is negligible. BMP-2, -4, -6, and -7 and activin A each suppressed dopamine and cAMP synthesis in a dose-dependent fashion. The BMP ligands also decreased 3,4-dihydroxyphenylalanine decarboxylase mRNA expression, whereas activin suppressed tyrosine hydroxylase expression. BMPs induced both Smad1/5/8 phosphorylation and Tlx2-Luc activation, whereas activin stimulated 3TP-Luc activity and p38 MAPK phosphorylation. ERK signaling was not affected by BMPs or activin. Dexamethasone enhanced catecholamine synthesis, accompanying increases in tyrosine hydroxylase and 3,4-dihydroxyphenylalanine decarboxylase transcription without cAMP accumulation. In the presence of dexamethasone, BMPs and activin failed to reduce dopamine as well as cAMP production. In addition, dexamethasone modulated mitotic suppression of PC12 induced by BMPs in a ligand-dependent manner. Furthermore, intracellular BMP signaling was markedly suppressed by dexamethasone treatment and the expression of ALK-2, ALK-3, and BMPRII was significantly inhibited by dexamethasone. Collectively, the endogenous BMP/activin system plays a key role in the regulation of catecholamine production. Controlling activity of the BMP system may be critical for glucocorticoid-induced catecholamine synthesis by adrenomedullar cells.

Objective: The importance of the melanin-concentrating hormone (MCH) system for regulation of energy homeostasis and body weight has been demonstrated in rodents. We analysed the human MCH receptor 1 gene (MCHR1) with respect to human obesity., Design: This consisted of genomic screening of 13.4 kb encompassing the MCHR1 in extremely obese German children and adolescents and association analyses for two coding single nucleotide polymorphisms (SNPs). To confirm initial positive association results, additional association studies and transmission disequilibrium tests in further German, Danish, French and American samples were conducted. Selected SNPs were investigated using functional in vitro studies and reporter gene assays., Methods: Single-stranded conformation polymorphism analysis, re-sequencing, PCR-restriction fragment length polymorphism analyses, tetra-primer amplification refractory mutation systems, matrix-assisted laser desorption/ionization time of flight mass spectrometry and reporter gene assays were carried out as well as measuring inositol phosphate formation, inhibition of cAMP formation and activation of p42/44 MAP kinase., Results: We identified 11 infrequent variations and two SNPs in the MCHR1 coding sequence and 18 SNPs (eight novel) in the flanking sequence. Association and transmission disequilibrium with obesity were detected for several SNPs in independent study groups of German obese children and adolescents and controls. In two German samples, encompassing 4056 and 295 individuals, trends towards association with obesity were detected. Findings in a second epidemiological German sample and in Danish, French and American samples were negative. Functional in vitro studies as well as reporter gene assays revealed no significant results., Conclusion: Our initial association of MCHR1 alleles/haplotype detected might be related to juvenile-onset obesity, conditional on a particular genetic and/or environmental background. Alternatively, we could not exclude the possibility that the initially detected association represented a false positive finding.

Cyclic 3'5'AMP is an important physiological amplifier of glucose-induced insulin secretion by the pancreatic islet beta-cell, where it is formed by the activity of adenylyl cyclase, especially in response to the incretin hormones GLP-1 (glucagon-like peptide-1) and GIP (glucose-dependent insulinotropic peptide). These hormones are secreted from the small intestine during and following a meal, and are important in producing a full insulin secretory response to nutrient stimuli. Cyclic AMP influences many steps involved in glucose-induced insulin secretion and may be important in regulating pancreatic islet beta-cell differentiation, growth and survival. Cyclic AMP (cAMP) itself is rapidly degraded in the pancreatic islet beta-cell by cyclic nucleotide phosphodiesterase (PDE) enzymes. This review discusses the possibility of targeting cAMP mechanisms in the treatment of type 2 diabetes mellitus, in which insulin release in response to glucose is impaired. This could be achieved by the use of GLP-1 or GIP to elevate cAMP in the pancreatic islet beta-cell. However, these peptides are normally rapidly degraded by dipeptidyl peptidase IV (DPP IV). Thus longer-acting analogues of GLP-1 and GIP, resistant to enzymic degradation, and orally active inhibitors of DPP IV have also been developed, and these agents were found to improve metabolic control in experimentally diabetic animals and in patients with type 2 diabetes. The use of selective inhibitors of type 3 phosphodiesterase (PDE3B), which is probably the important pancreatic islet beta-cell PDE isoform, would require their targeting to the islet beta-cell, because inhibition of PDE3B in adipocytes and hepatocytes would induce insulin resistance.

Receptor-specific agonists of the extracellular calcium-sensing receptor (CaSR) potentiate glucose-induced insulin secretion, an effect similar to that of glucagon-like peptide-1 (GLP-1). We have sequenced the full open reading frame of the CaSR from rat insulinoma (INS-1) cells and find that the predicted amino acid sequence of the receptor is identical with that of the receptor from the parathyroid gland. This receptor couples to both Gq/11 and Gi/o, and this dual coupling may partly explain the varying effects of nonspecific agonists on secretion reported previously. L-Histidine (L-His) increases the sensitivity of the CaSR to extracellular Ca2+ and potentiates glucose-dependent insulin secretion from INS-1 cells. This potentiation is partially inhibited at low extracellular [Ca2+] where the CaSR is ineffective. Coexpression of the CaSR and GLP-1 receptor (GLP-1R) produces a pertussis toxin-sensitive inhibition of GLP-1-induced cAMP production in response to elevated extracellular [Ca2+]. However, l-His potentiates cAMP response element reporter activity in INS-1 cells and in human embryonic kidney-293 cells expressing either the GLP-1R alone or the CaSR and GLP-1R. INS-1 cells express the RNA for the CaSR at a lower level than that for the GLP-1R. This difference in expression level of the receptors may explain the potentiation of insulin secretion by L-His despite coupling of the CaSR to Gi/o. In conclusion, L-His can potentiate both GLP-1R- and CaSR-activated signaling pathways, and these effects may play a role in the potentiation of glucose-induced insulin secretion in response to meals containing protein in addition to carbohydrates and fat.

Corpora lutea (CL) from Days 5, 10, and 15 after superovulation were enzymatically dispersed, and a portion of the cells were elutriated to obtain fractions enriched with small or large luteal cells. Mixed, small, and large luteal cell fractions were incubated with no treatment or with agonists or antagonists of cAMP (dbcAMP or Rp-cAMPS), protein kinase C (PKC; TPA or H-7), or calcium (A23187, EGTA, or A23187 + EGTA). The rate of contact-dependent gap junctional intercellular communication (GJIC) was evaluated by laser cytometry. Media were collected for progesterone (P(4)) radioimmunoassay, and luteal cells cultured with no treatment were fixed for immunocytochemistry or frozen for Western blot analysis. Luteal cells from each stage of the estrous cycle exhibited GJIC. The dbcAMP increased (P < 0.05) GJIC for all cell types across the estrous cycle. The Rp-cAMPS decreased (P < 0.05) GJIC for small luteal cells on Day 5 and for all cell types on Days 10 and 15. The TPA inhibited (P < 0.01), but H-7 did not affect, GJIC for all cell types across the estrous cycle. The A23187 decreased (P < 0.05) GJIC for large luteal cells touching only small or only large luteal cells, whereas A23187 + EGTA decreased (P < 0.05) GJIC for all cell types across the estrous cycle. For the mixed and large luteal cell fractions, dbcAMP increased (P < 0.05), but TPA and A23187 + EGTA decreased (P < 0.05), P(4) secretion. The A23187 alone decreased (P < 0.05) P(4) secretion by large, but not by mixed, luteal cells. For all days and cell types, the rate of GJIC and P(4) secretion were correlated (r = 0.113-0.249; P < 0.01). Connexin 43 was detected in cultured luteal cells by immunofluorescence and Western immunoblotting. Thus, intracellular regulators like cAMP, PKC, or calcium appear to regulate GJIC, which probably is an important mechanism for coordinating function of the ovine CL.

Our previous studies have identified a role for annexin 1 (also called lipocortin 1) in the regulatory actions of glucocorticoids (GCs) on the release of PRL from the rat anterior pituitary gland. In the present study we used antisense and immunoneutralization strategies to extend this work. Exposure of rat anterior pituitary tissue to corticosterone (1 nM) or dexamethasone (100 nM) in vitro induced 1) de novo annexin 1 synthesis and 2) translocation of the protein from intracellular to pericellular sites. Both responses were prevented by the inclusion in the medium of an annexin 1 antisense oligodeoxynucleotide (ODN; 50 nM), but not by the corresponding sense and scrambled ODN sequences. Unlike the GCs, 17beta-estradiol, testosterone, and aldosterone (1 nM) had no effect on either the synthesis or the cellular disposition of annexin 1; moreover, none of the steroids or ODNs tested influenced the expression of annexin 5, a protein closely related to annexin 1. The increases in PRL release induced in vitro by drugs that signal via cAMP/protein kinase A [vasoactive intestinal polypeptide (10 nM), forskolin (100 microM), 8-bromo-cAMP (0.1 microM)] or phospholipase C (TRH, 10 nM) were attenuated by preincubation of the pituitary tissue with either corticosterone (1 nM) or dexamethasone (100 nM). The inhibitory actions of the steroids on the secretory responses to vasoactive intestinal polypeptide, forskolin, and 8-bromo-cAMP were specifically quenched by inclusion in the medium of the annexin 1 antisense ODN (50 nM) or a neutralizing antiannexin 1 monoclonal antibody (antiannexin 1 mAb, diluted 1:15,000). By contrast, the ability of the GCs to suppress the TRH-induced increase in PRL release was unaffected by both the annexin 1 antisense ODN and the antiannexin 1 mAb. In vivo, interleukin-1beta (10 ng, intracerebroventricularly) produced a significant increase in the serum PRL concentration (P < 0.01), which was prevented by pretreatment of the rats with corticosterone (100 microg/100 g BW, sc). The inhibitory actions of the steroid were specifically abrogated by peripheral administration of an antiannexin 1 antiserum (200 microl, sc); by contrast, when the antiserum was given centrally (3 microl, intracerebroventricularly), it was without effect. These results support our premise that annexin contributes to the regulatory actions of GCs on PRL secretion and suggest that it acts at point distal to the formation of cAMP.

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

The stimulatory effect of noradrenaline (NA) as well as oxytocin (OT) on bovine endometrial prostaglandin (PG) F2alpha production, and the intracellular mechanisms of their actions, were investigated in cultured bovine endometrial cells (a mixture of epithelial, stromal, and glandular cells). The cells were cultured in Dulbecco's Modified Eagle's medium and Ham's F-12 medium (1:1 [v:v]) with 10% calf serum. When the cells reached confluence, the culture medium was replaced with fresh medium with 0.1% BSA and various doses of NA (10(-8)-10(-4) M). NA stimulated PGF2alpha production in a dose-dependent manner (p < 0.05). To evaluate the intracellular mechanisms of NA and OT actions, the cells were treated with forskolin (an activator of adenylate cyclase), phorbol 12-myristate 13-acetate (PMA, an activator of protein kinase [PK] C), Rp-cAMP (a competitive cAMP antagonist and an inhibitor of PKA), U-73122 (an inhibitor of phospholipase [PL] C), or anthranilic acid (ACA, an inhibitor of PLA2). Forskolin and PMA stimulated PGF2alpha production in a dose-dependent manner (p < 0.05). Rp-cAMP completely inhibited (p < 0.001) the NA-induced, but not the OT-induced, PGF2alpha production. Although U-73122 inhibited only OT-induced PGF2alpha production (p < 0.001), ACA completely stopped the actions of NA and OT. The overall results indicate that NA as well as OT is involved in the regulation of the endometrial PGF2alpha production in cattle and that the stimulatory effects of NA and OT on PGF2alpha production are mediated via the PKA and PKC pathways, respectively.

In teleosts, ovarian steroidogenesis is under the control of two gonadotropic hormones, GTH I and GTH II, that are structurally and functionally homologous to FSH and LH. The intracellular mechanisms by which GTH I and GTH II stimulate steroidogenesis in the teleost ovary are not well understood. The purpose of this study was to investigate the involvement of the cAMP/protein kinase A (PKA) and protein kinase C (PKC)/Ca2+ signaling pathways in the steroidogenic actions of GTH I and GTH II in the ovary of the brook trout (Salvelinus fontinalis). The cAMP/PKA pathway mediated the actions of GTH I before germinal vesicle breakdown (preGVBD) and GTH II after GVBD and before ovulation (preOV). Experimental increases in intracellular cAMP concentration mimicked the steroidogenic effects of GTH I and GTH II, and an antagonistic analog of cAMP partially blocked them. In addition, GTH I and GTH II stimulated the production of cAMP in preGVBD and preOV follicles, respectively. Activation of the PKC/Ca2+ pathway by a phorbol ester or a Ca2+ ionophore blocked the GTH I- and GTH II-induced steroid production, whereas inhibition of PKC by specific inhibitors potentiated the effects of GTH I. These results suggest that the cAMP/PKA signaling pathway mediates the stimulatory effects of GTH I and GTH II on steroidogenesis, and they also suggest the additional involvement of the PKC/Ca2+ signaling pathway in modulating the actions of gonadotropins in brook trout ovarian follicles.

Cellular interactions mediated by both contact-dependent and contact-independent mechanisms are probably important to maintain luteal function. The objective of the present study was to evaluate the role of cAMP in regulation of contact-dependent gap junctional intercellular communication (GJIC) of bovine luteal cells from several stages of luteal development. In experiment 1, corpora lutea (n = 5) from the mid-luteal phase of the estrous cycle were dissociated with collagenase, and cells were preincubated in a medium with serum. Then the medium was changed to serum-free media containing a cAMP agonist (dbcAMP; 1 mM) or antagonist (Rp-cAMPS; 0, 3, 10, 30, or 100 microM). In experiment 2, corpora lutea from the early (n = 7), mid- (n = 6), and late (n = 6) luteal phases of the estrous cycle were dissociated and preincubated as in experiment 1, and luteal cells were then incubated with no treatment, LH (100 ng/ml), dbcAMP (1 mM), forskolin (1 microM), Rp-cAMPS (100 microM), or LH+Rp-cAMPS. After incubation of luteal cells with treatments for 18-24 h, media were collected for determination of progesterone and cAMP concentrations. Then the rate of GJIC was evaluated for selected cells (small luteal cells in contact with small luteal cells, and large luteal cells in contact with small luteal cells) by using the fluorescence recovery after photo-bleaching technique and laser cytometry. In experiment 1, dbcAMP increased (p < 0.01) but Rp-cAMPS (p < 0.05) decreased GJIC between small luteal cells and between large and small luteal cells. In addition, dbcAMP stimulated (p < 0.01) but Rp-cAMPS did not affect progesterone secretion. In experiment 2, treatments affected (p < 0.05) GJIC and progesterone production of luteal cells from the mid- and late but not from the early luteal phase of the estrous cycle. GJIC between small luteal cells was increased (p < 0.01) by LH, dbcAMP, and forskolin. GJIC between large and small luteal cells was increased (p < 0.05) by dbcAMP and forskolin. Rp-cAMPS decreased (p < 0.01) GJIC between small luteal cells (mid-luteal phase) and between large and small luteal cells (mid- and late luteal phases). In addition, Rp-cAMPS inhibited (p < 0.05) the stimulatory effects of LH on GJIC between small luteal cells from the mid- and late luteal phases of the estrous cycle. For luteal cells from the mid- and late luteal phases, progesterone production was increased (p < 0.05) by LH, dbcAMP, forskolin, and LH+Rp-cAMPS, but was not affected by Rp-cAMPS. Across all stages of the estrous cycle, cyclic AMP accumulation in media was greater (p < 0.05) in LH- and forskolin-treated cultures than in control cultures and was greater (p < 0.01) in forskolin-treated than in LH-treated cultures. These data demonstrate that small and large luteal cells communicate with each other and that the rate of GJIC is modulated by LH and cAMP, as has been shown previously for other cell types. Thus, cAMP appears to be involved in the regulation of GJIC within the bovine corpus luteum, which probably is an important mechanism for coordinating luteal cell function.

Our aim has been to delineate the role of the major signal transduction pathways believed implicated in the control of thyroid function and growth: the cAMP-, epidermal growth factor (EGF) (tyrosine kinase)-, and protein kinase C (PKC)-mediated mechanisms. The experimental model used was our system of thyroid follicles of human origin cultured in suspension under serum-free conditions in which the follicular three-dimensional structure is retained. The phorbol ester 12-O tetradecanoylphorbol 13-acetate (TPA) time and dose dependently (10(-11)-10(-7) M) inhibited TSH-stimulated thyroid functions (cAMP formation, iodide uptake and organification, and T3 secretion). TPA also inhibited such forskolin- and 8-BrcAMP-stimulated effects, suggesting that the attenuation of the cAMP-dependent pathway occurs at steps both pre- and post-cAMP formation. The effects of TPA were mimicked by another PKC activator, phorbol 12,13-dibutyrate, but not by a phorbol ester that fails to activate PKC, 4 alpha-phorbol 12,13-didecanoate, and were reversed by staurosporine, a PKC inhibitor. The TPA actions seem, therefore, to be PKC-mediated. EGF exhibited a time- and dose-dependent (0.02-8 nM) restraining influence on the above TSH-stimulated differentiated functions, except for cAMP, which was enhanced. EGF also blunted such forskolin- and 8-BrcAMP-induced response parameters, suggesting inhibition at a post-cAMP locus. Regarding cell proliferation, during the initial stages of culture (day 2), TPA dose dependently (10(-11)-10(-7) M) attenuated cell proliferation, but subsequently (day 7 of culture) the same doses of TPA stimulated cell multiplication. The TPA-mitogenic and antimitogenic effects could not be mimicked by 4 alpha-phorbol-12,13-didecanoate and were reversed by staurosporine, thus indicating a PKC-mediated pathway for such TPA actions. EGF, on the other hand, only enhanced cell proliferation at a late stage (coincident with the TPA-mitogenic effect). TSH (0.5 U/liter) inhibited both the mitogenic and antimitogenic actions of TPA as well as the cell-proliferative influence of EGF. In conclusion, the data demonstrate mutual antagonistic interactions between the signal transduction pathways: the PKC and EGF (tyrosine kinase) pathways seem to inhibit TSH (cAMP)-mediated human thyroid cell differentiation, whereas TSH attenuates PKC-mediated thyroid cell mitogenesis and antimitogenesis as well as EGF-mediated cell proliferation.

Endogenous opioids participate in the regulation of gonadotropin secretion through an influence on the release of the hypothalamic LHRH. However, it is not clear whether opioids exert a direct effect on LHRH-producing neurons or interfere with other systems able to influence LHRH release. A neuronal LHRH-producing cell line (GT1) developed recently provides a good model to study the mechanisms controlling LHRH release. In the present study, the presence of opioid receptors on a subclone of GT1 cells (GT1-1) has been investigated. A specific and saturable binding of the 3H-labeled nonselective opioid ligand diprenorphine ([3H]DIP) was detected by a receptor binding assay on both intact GT1-1 cells and crude membrane preparations obtained from these cells. Analysis of saturation curves revealed that [3H]DIP apparently binds to a single class of sites with a Kd of 0.2 nM and a binding capacity of 125 fmol/mg protein, corresponding to approximately 20,000 sites/cell. Selective displacement of the binding of [3H]DIP to GT1-1 cells by [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin, [D-Pen2,D-Pen5]enkephalin (DPDPE), and U50488H, which are selective ligands, respectively, for mu-, delta-, and kappa-receptors, was also evaluated. Only the specific delta-ligand DPDPE produced a significant inhibition of the binding of [3H]DIP. [D-Ala2,N-Me-Phe4,Gly5-ol]Enkephalin and U50488H were totally ineffective. The inhibitory effect of the agonist DPDPE on the binding of [3H]DIP was decreased by the presence of sodium ions, a typical characteristic of the binding of agonists to opioid receptors. Finally, it has been observed that treatment with prostaglandins E1 and E2 produces a dramatic increase in cAMP accumulation in GT1-1 cells, and DPDPE is highly effective in suppressing this effect. On the basis of these results, it is possible to postulate the presence of functional delta-opioid receptors on GT1-1 cells. By extrapolation, one might suggest that endogenous opioids may affect LHRH neurons by two mechanisms: a direct one, acting via delta-receptors, and an indirect one, through the activation of neurons impinging on the LHRH system, which uses mu-receptors.

At functional luteolysis, the rat corpus luteum generates hydrogen peroxide (H2O2), which is known to rapidly inhibit gonadotropin-sensitive cAMP and progesterone production in isolated luteal cells. Lipid peroxides also increase markedly in the rat corpus luteum with the onset of functional luteolysis, and H2O2 is a potent inducer of lipid peroxidation. However, the actions of lipid peroxides on cell function are unknown. The objective of this study was to investigate the impact of typical lipid peroxides, cumene hydroperoxide (CuOOH) and 15(S)-hydroperoxyeicosatetraenoic acid, on rat luteal cells. CuOOH inhibited both LH-sensitive cAMP accumulation (ED50, 25 microM) and progesterone production (ED50, 20 microM). 15(S)-hydroperoxyeicosatetraenoic acid also dose dependently inhibited steroidogenesis. A significant reduction of LH-stimulated progesterone production was evident within 5 min of treatment with CuOOH, whereas inhibition of cAMP accumulation was not evident until 60 min. 8-Bromo-cAMP and 22-hydroxycholesterol caused partial and complete reversal of CuOOH-inhibited progesterone secretion, respectively. Preincubation of cells with o-phenanthroline completely reversed the inhibitory effects of CuOOH on cAMP accumulation and partially reversed its effects on progesterone production. Incorporation of radiolabeled amino acids into luteal proteins was significantly inhibited by CuOOH (25 microM) within 2 min of treatment and was reduced to 40 +/- 14% of control levels at 60 min. CuOOH (25 microM) maximally stimulated PGE2 production within 30 min of treatment (180 +/- 30% of control), a response that was completely blocked by aristolochic acid (100 microM), a phospholipase-A2 inhibitor, and indomethacin (1 microgram/ml), a prostaglandin (PG) synthesis inhibitor. The present results suggest that the acute inhibitory action of lipid peroxides on LH-stimulated progesterone production occurs down-stream of cAMP synthesis and appears to be due to impaired cholesterol utilization for steroidogenesis, possibly through inhibition of protein synthesis. The stimulation of PGE2 production by CuOOH appears to involve the activation of phospholipase-A2, which is a rate-limiting step in PG synthesis. Lipid peroxides as well as H2O2 may serve as mediators of functional luteolysis.

The objective of this study was to investigate the importance of cAMP during capacitation of bovine sperm. The competitive antagonist of cAMP, Rp-adenosine-3'5'-cyclic monophosphorothioate (Rp-cAMP), blocked heparin-induced capacitation (p < 0.05). The effect of Rp-cAMP on heparin-induced capacitation was reversed by 8-bromo-cAMP. The maximal inhibitory effect on capacitation occuroff when Rp-cAMP was added at the start of sperm incubation. These results support an important role for cAMP-dependent protein kinases during heparin-induced capacitation of bovine sperm. Further support for a role for protein phosphorylation during capacitation came from the use of the protein phosphatase inhibitor, okadaic acid. Okadaic acid had no affect on heparin-induced capacitation of bovine sperm (p > 0.05); however, bovine sperm were capacitated by okadaic acid in the absence of heparin (p < 0.05). The relationship of cAMP to capacitation-associated changes in sperm intracellular pH (pHi) was investigated using image analysis of bovine sperm. The pHi of sperm increased during capacitation, and Rp-cAMP did not affect the change in pHi. We conclude that heparin-induced capacitation of bovine sperm involves an increase in cAMP and a protein phosphorylation event but that these do not induce the increase in pHi associated with capacitation.

The relationships between the different circulating thyrotropin receptor antibodies (TSH-R-abs) in autoimmune thyroid disease (AITD) are complex. In order to investigate them, we have developed an assay for the simultaneous measurement of three types of TSH-R-abs: TSH-binding inhibiting immunoglobulin (TBII): thyroid-stimulating antibody (TS-ab) and TSH-stimulation blocking antibody (TSB-ab). A large number of patients with Graves' disease (GD)--untreated and treated--Hashimoto's thyroiditis (HT), primary myxedema (PM) and non-immune goiter (NIG) were investigated. In untreated Graves' patients the frequency of positive TS-ab and TBII sera was found to be 90 and 69%, respectively, the presence of TS-ab and/or TBII being detected in 98%. After long-term antithyroid treatment administered to GD patients, the frequency of positivity of both TBII and TS-ab was decreased, whether hyperthyroidism was cured or not. The TSB-ab was detected in the serum of 8% of patients with GD, and the frequency of TSB-ab did not increase following treatment and alteration in thyroid function. No significant correlation was found between TSB-ab and thyroid function in Graves' patients. Besides, we found that all the GD patients presenting positive TSB-ab were also TBII positive. A follow-up study of the three TSH-R-abs was performed in 35 patients with GD during a mean of 14.3 +/- 8.5 months (4-34 months) of antithyroid drug treatment. Ten out of 24 patients (42%) with positive TBII and 16 out of 32 (50%) with positive TS-ab turned from positive to negative during the time of follow-up. Regarding relapse in hyperthyroid GD, we found that TS-ab was positive in 80% and TBII was positive in 40% of the patients with Graves' relapse, indicating that the presence of TS-ab is a better index for relapse prediction in Graves' hyperthyroidism than TBII. The TSB-ab was found with higher frequency in HT and PM than in GD, i.e. 21%, 18% and 8%, respectively., The TSB-ab positivity was correlated significantly with TBII in our patients with AITD when TSB-ab was positive. This new simultaneous assay of the three TSH-R-abs should be very helpful for further investigation of the autoimmune aspects of AITD and it should help us to progress in a better understanding of the pathogeny of the different AITDs.

Steady-state levels of inhibin-alpha and -beta B mRNAs are higher in stages II-VI of the seminiferous epithelial cycle than in stages VII-VIII. We investigated follicle-stimulating hormone (FSH) regulation of inhibin alpha-subunit mRNA in stages II-VI and VII-VIII to study whether the stage specificity is due to differential hormonal regulation by FSH. Follicle-stimulating hormone caused a significant increase of inhibin-alpha mRNA levels in both stages during a 20-h incubation. The mechanism of the FSH effect was studied further in stages VII-VIII. Maximal stimulation of the inhibin-alpha mRNA level was achieved with 100 micrograms/l FSH, dibutyryl-3',5'-cyclic adenosine monophosphate (db-cAMP, 0.2 mmol/l) and Sp-adenosine-3',5'-monophosphothionate (Sp-cAMPS, 10 mumol/l) (a cAMP agonist). The presence of Rp-cAMPS (200 mumol/l) (a cAMP antagonist) abolished the stimulation, Rp-cAMPS alone had no effect. Inhibin-beta B mRNA levels in stages VII-VIII were not affected by FSH, db-cAMP, Sp-cAMPS or Rp-cAMPS. Phorbol 12-myristate 13-acetate (100 nmol/l) had no effect on inhibin-alpha or -beta B mRNA levels. Actinomycin D abolished the stimulatory effect of FSH on inhibin-alpha mRNA expression. In conclusion, FSH stimulated inhibin-alpha mRNA expression similarly both in stages II-VI and VII-VIII of the seminiferous epithelial cycle and the stimulation in stages VII-VIII was cAMP-mediated.

FSH signal transduction in Sertoli cells involves the generation of cAMP and calcium as second messengers; however, the relationship between these two signals is not clear. In order to determine whether these were serial or parallel signals, we studied cytosolic calcium levels in freshly isolated rat Sertoli cells using maneuvers to dissociate generation of endogenous cAMP from cytosolic calcium. Pretreatment with 1 mM MDL 12,330A, an adenylate cyclase inhibitor, reduced by greater than 90% increases in cytosolic calcium induced by FSH (97 +/- 6 vs. 213 +/- 16 nM), whereas, despite adenylate cyclase blockade, 1 mM (Bu)2cAMP continued to elevate cytosolic calcium (from 87 +/- 6 to 182 +/- 23 nM), indicating the involvement of adenylate cyclase in the FSH-induced rise of cytosolic calcium. A cAMP antagonist, 1 mM Rp-cAMP, reduced by 75% the FSH-induced rise of cytosolic calcium (115 +/- 14 vs. 213 +/- 16 nM), suggesting that endogenous cAMP levels generated by FSH are sufficient to activate the cytosolic calcium response to FSH. Pretreatment with pertussis toxin (1 mg/liter) to dissociate the FSH-receptor interaction from its G-protein-mediated linkage to adenylate cyclase also suppressed the FSH-induced rise in cytosolic calcium (97 +/- 11 vs. 213 +/- 16 nM). Sertoli cells preincubated with 1 mM staurosporine, an inhibitor of protein kinases, exhibited a reduced calcium response to FSH (125 +/- 14 vs. 213 +/- 16 nM), suggesting that FSH-induced calcium flux might be mediated by protein kinase, presumably cAMP-dependent protein kinase A. The present findings therefore strengthen the premise that the cytosolic calcium response to FSH in Sertoli cells is predominantly attributable to serial signaling after the generation of endogenous cAMP.

Corticotropin releasing factor (CRF), a key neuroregulator of the hypothalamic-pituitary-adrenal cortical axis, also displays a broad range of effects on the endocrine, central nervous and immune systems. Having recently characterized the human pituitary CRF receptor by expression cloning of cDNA from a human Cushing's corticotropic adenoma, we report here the structure of the cDNA for a rat brain CRF receptor (rCRF-R) which was cloned by hybridization from a rat brain cDNA library. The sequence of the rCRF-R encodes a 415 amino acid protein comprising seven membrane spanning domains. The rCRF-R is 97% identical at the amino acid level to the human pituitary tumor CRF receptor, differing by only 12 amino acids. When expressed in COSM6 cells, the rCRF-R binds CRF with high affinity (Kd = 1.7 (0.8-3.8)nM). The receptor transduces a CRF stimulated accumulation of intracellular cAMP which is inhibited by the CRF antagonist, alpha helCRF(9-41). These results suggest that the brain expresses a CRF receptor similar to that in the pituitary.

Insulin-like growth factor-1 (IGF-I), an endocrine and autocrine/paracrine factor that enhances collagen synthesis and bone matrix formation by osteoblasts, has been implicated in the coupling of bone formation with bone resorption. We have found, using SaOS-2 osteoblastic cells, that IGF-I inhibits PTH-stimulated cAMP production. Pretreatment of SaOS-2 cells with IGF-I for 24 h inhibited cAMP production stimulated by PTH with an IC50 of 1 nM and maximal inhibition to 10-20% of control values at an IGF-I concentration of 10 nM. Pretreatment with IGF-I had no effect on vasoactive intestinal peptide-stimulated cAMP production, but it enhanced cAMP production stimulated by prostaglandin E2 (PGE2) by as much as 5-fold at a concentration of 10 nM and with an EC50 of 1 nM. Pretreatment of SaOS-2 cells with IGF-I did not affect cholera toxin- or forskolin-stimulated cAMP accumulation. Taken together, these findings indicated that IGF-I did not affect Gs alpha, coupling of Gs alpha to adenylate cyclase, or adenylate cyclase itself. Binding experiments using [125I] chicken PTH-related peptide (PTHrP)-(1-36)-[Tyr36]NH2 demonstrated that IGF-I reduced PTH/PTHrP receptor number to 25% of the control value without affecting receptor affinity. IGF-I and the related growth factors, insulin and IGF-II, inhibited PTH-stimulated cAMP production with a rank order of potency of IGF-I > or = IGF-II > insulin, indicating that the actions of IGF-I on SaOS-2 cells were probably mediated by the IGF-I receptor. We conclude that physiologically relevant concentrations of IGF-I specifically inhibited PTH-stimulated and enhanced PGE2-stimulated production of cAMP by an action at the level of PTH and PGE2 receptors and/or coupling of the receptors to Gs alpha.

Sustained elevations in cytosolic calcium concentrations ([Ca2+]i) have been shown to render insulin target cells resistant to insulin action. In this study we examined the mechanisms of the detrimental effect of high levels of [Ca2+]i on insulin-induced 2-deoxyglucose (2-DOG) uptake. To elevate [Ca2+]i, we incubated rat adipocytes with either 40 mM potassium (K+) or 20 ng/ml PTH for 1 h for in vitro experiments and injected rats with PTH (injections of 50 micrograms, ip, every hour for 3 h) for in vivo studies. Adipocytes with elevated [Ca2+]i demonstrated a 30% decrease in insulin-stimulated 2-DOG uptake. A calcium channel blocker (nitrendipine) and a cAMP antagonist (RpcAMP) each partially restored insulin-stimulated glucose transport, but together they completely restored 2-DOG uptake. Concomitantly, we found a significant increase in phosphorylation of GLUT-4 in adipocytes with elevated [Ca2+]i. This change in GLUT-4 phosphorylation was also attenuated by nitrendipine and RpcAMP. These observations confirm that elevated [Ca2+]i diminishes insulin-stimulated glucose transport and suggest that increased phosphorylation of GLUT-4 in adipocytes with high [Ca2+]i may alter its intrinsic activity.

Rp-cAMPS, the Rp-diastereomer of adenosine 3',5'-phosphorothioate, is often referred to as a cAMP antagonist, since it binds to the regulatory subunit of cAMP-dependent protein kinase without dissociation of free catalytic subunits. To evaluate the role of cAMP-dependent protein kinase in amylase exocytosis, we examined the effect of Rp-cAMPS on amylase release from rat parotid acini. Rp-cAMPS did not stimulate amylase release from saponin-permeabilized parotid acini, whereas its Sp-isomer strongly evoked amylase release. Rp-cAMPS dose-dependently inhibited amylase release stimulated by Sp-cAMPS. In the presence of Rp-cAMPS, the dose-response curve of Sp-cAMPS was shifted to the right. The inhibitory effect of Rp-cAMPS on isoproterenol-induced amylase release was not detected in intact acini, but was clearly observed in the permeabilized ones. Rp-cAMPS markedly inhibited protein phosphorylation evoked by Sp-cAMPS, indicating that Rp-cAMPS prevents the dissociation of cAMP-dependent protein kinase. These results, taken together with synergistic increase in amylase release by the combination of site-selective cAMP analogues [T. Takuma (1990) J. Biochem. 108, 99-102], suggest that cAMP-dependent protein kinase is involved in the exocytosis of amylase from parotid acini.

The kidney and parathyroid gland play key roles in calcium (Ca++) homeostasis. Recent data suggest that the kidney, in addition to being a primary target for PTH, also recognizes changes in the concentration of extracellular Ca++, thereby modulating hormone-dependent cAMP production, 1,25-dihydroxyvitamin D synthesis, and renin secretion. In this study, we examined: 1) the effects of varying concentration of divalent cations on PTH-dependent cAMP production in renal proximal tubular cells; and 2) the mechanisms by which extracellular Ca++ exerts its inhibitory effects on cAMP production. Single cell suspensions composed of 80-90% proximal tubular cells were prepared from cortical homogenates by collagenase digestion and sieving. In the presence of 1 mM isobutylmethylxanthine, cAMP content was measured by RIA in 5-15 min incubations and showed a 5- to 6-fold increase in response to PTH (10(-11) -10(-6) M). Increasing extracellular Ca++ and magnesium (Mg++) from 0 and 0.5 mM, respectively, to 5.0 mM inhibited PTH-dependent (3 x 10(-9) M) cAMP production by 54 +/- 4% and 47 +/- 6%, respectively. The half maximal inhibitory concentration for both Ca++ and Mg++ was 0.9 mM. In addition, increasing extracellular barium (Ba++) or strontium (Sr++) from 0-10 mM inhibited PTH-dependent (3 x 10(-9) M) production by 54 +/- 7% and 62 +/- 6% with half of the maximal observed inhibition at 2.2 and 2.7 mM, respectively. The inhibition of PTH-dependent cAMP production by 2.5 mM Ca++ was not reversed by the calcium channel blockers diltiazem or verapamil (10(-4) M). However, changes in intracellular calcium may play some role in the inhibitory effects of Ca++ on cAMP production, since ionomycin (10(-6)-10(-5) M) lowered PTH-dependent cAMP production by 25-36%. Our data suggest that the proximal tubular cell can sense physiologically relevant changes in Ca++, providing a potential mechanism for the modulation of 1,25-dihydroxyvitamin D production or other tubular functions relevant to fluid and mineral homeostasis.

We determined whether tumor-promoting factor phorbol ester modulates cellular cAMP production and the cellular free calcium concentration ([Ca2+]i) in response to arginine vasopressin (AVP) in rat renal papillary collecting tubule cells in culture. In the presence of 5 x 10(-4) M 3-isobutyl-1-methylxanthine, AVP increased cellular cAMP production in a dose-dependent manner. A 1-h exposure to 3 x 10(-7)-3 x 10(-6) M phorbol-12-myristate-13-acetate (PMA) significantly attenuated the cAMP response to AVP (1 x 10(-9) M AVP; 474.9 +/- 24.8 vs. 368.1 +/- 22.8 fmol/microgram protein; P less than 0.01). The dose-response relation with AVP thus shifted to the right. Such an inhibition was totally reversed in the presence of 2 x 10(-6) M 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), an inhibitor of protein kinase-C. Also, 1 x 10(-7) M AVP produced an increase in [Ca2+]i from 99.4 +/- 3.3 to 200.0 +/- 8.6 nM. When cells were preexposed to 1 x 10(-6) M PMA, an increase in [Ca2+]i in response to 1 x 10(-7) M AVP was significantly diminished (75.5 +/- 4.6 to 101.4 +/- 4.3 nM). The inhibition by PMA of AVP-induced increment in [Ca2+]i was significantly attenuated in the presence of 2 x 10(-5) M H-7 compared to that in its absence. Prolonged exposure to PMA did not alter the AVP-induced increases in cAMP production and [Ca2+]i. These results indicate that phorbol ester inhibits the cellular action of AVP mediated through the activation of protein kinase-C and suggest that there is an interaction between cAMP and phosphatidylinositol systems in modulating the AVP action in renal papillary collecting tubule cells.

Clonal cell lines are ideal model systems for the study of monoamine receptors. Cell lines that express dopamine and serotonin receptors have been isolated from pituitary tumor 7315a. The SUP1 cell line contains mRNA for the dopamine D-2 receptor and expresses D-2 receptors coupled to the inhibition of cAMP production and to the inhibition of prolactin secretion. The P11 cell line expresses 5-HT2 receptors coupled to the stimulation of phosphoinositide hydrolysis. These cell lines will be used to investigate manipulations that affect the expression and function of dopamine and serotonin receptors.

Extracellular calcium (Ca2+) is the major physiological regulator of parathyroid function; high Ca2+ decreases PTH secretion as well as reduces cAMP accumulation. There is an increasing body of evidence suggesting the presence of a receptor-like mechanism at the surface of the parathyroid cell which mediates these and other actions of Ca2+. In the present studies we used the lectin Concanavalin-A (Con-A) to investigate the possible role of carbohydrate moieties in the regulation of cAMP metabolism by Ca2+ in bovine parathyroid cells, which is thought to involve inhibition of adenylate cyclase via activation of the guanine nucleotide regulatory protein Gi. Pretreatment of parathyroid cells with Con-A for 15-60 min significantly reversed the inhibitory effect of high Ca2+ on dopamine-stimulated cAMP accumulation, reducing the inhibition at 3 mM Ca2+ from 70 +/- 3% to 30 +/- 3%. This effect was also observed in the absence of preincubation and with concentrations of Con-A as low as 40 micrograms/ml and was reversed by alpha-methyl-D-glucoside, a specific antagonist of the lectin. The lectin also reversed the inhibitory effects of Ca2+ (2-3 mM) on cAMP accumulation stimulated by isoproterenol and forskolin to a comparable extent. Prostaglandin F2 alpha-induced inhibition of cAMP accumulation (likewise mediated by Gi) was, however, not reversed by Con-A, suggesting that the lectin did not have a generalized effect on the cell surface or on receptors inhibiting adenylate cyclase. Moreover, fluoride-induced inhibition of cAMP accumulation was not reversed by Con-A, providing additional evidence that the lectin did not act at or distal to Gi (i.e. modulate Gi, adenylate cyclase, and/or phosphodiesterase). The present study suggests that Con-A may modulate the actions of extracellular Ca2+ on parathyroid secretion, possibly modifying the interaction of Ca2+ with the cell surface by affecting carbohydrate moieties that seem to be important in the Ca2(+)-sensing process. The structural element involved in Ca2+ sensing in the parathyroid cell may be a glycoprotein or closely associated with glycoproteins with carbohydrate chains containing alpha-methyl-D-glycoside.

Melatonin's transduction mechanisms were investigated using in vitro cultured anterior hemipituitaries. Melatonin inhibited cAMP and 3',5'-cyclic GMP accumulation in neonatal rat anterior pituitary stimulated with LHRH. Maximal inhibitory effect was reached within 25 min and persisted for at least 20 min. Inhibition of cAMP accumulation is specific for melatonin because its analogs N-acetylserotonin and 5-methoxytryptamine are 1000 times less potent. Melatonin effect is age- and time-dependent. Marked inhibition was observed in 5-, 10-, and 14-day-old rats but not in 29-day-old ones. Melatonin was significantly more potent when applied at the end of the light period as compared with the first half of the day. Melatonin's effects on cAMP correlate with its effect on reproductive functions and on LH release. Cyclic nucleotides may thus serve as second messengers transducing the effect of melatonin on cellular level.

To elucidate the mechanisms of the inhibitory effect of interleukin-1 (IL-1) on LH-stimulated progesterone secretion by cultured porcine granulosa cells, we examined which steps of the LH-stimulated, cAMP-mediated, progesterone biosynthetic pathway were affected by IL-1. Pretreatment of the cells for 48 h with IL-1 reduced intra- and extracellular cAMP accumulation in response to LH by 73% and 83%, respectively. The inhibitory effects of IL-1 were time and concentration dependent. Significant inhibition was observed at as low as 50-250 pg/ml, and the effect was maximal at 100 ng/ml (ID50; 2 ng/ml). The lowest concentration of IL-1 used (0.5 pg/ml), in contrast, showed a tendency to stimulate LH-induced cAMP accumulation. Effect of IL-1 on the specific binding of [125I]LH to granulosa cells was then examined, which showed that IL-1 (100 ng/ml) significantly reduced the specific binding by 36%. IL-1 also significantly reduced intra- and extracellular cAMP accumulation by the cells in response to forskolin (50, 150 microM) by 28-46%, indicating that IL-1 can directly inhibit adenylate cyclase activity. Contrary to these inhibitory actions of IL-1 on LH-stimulated cAMP generation, IL-1 did not significantly reduce progesterone secretion induced by (Bu)2cAMP. These results indicate that the inhibitory effect of IL-1 on LH-stimulated progesterone secretion is due to its actions at at least two different sites along the LH-stimulated, cAMP-mediated, progesterone biosynthetic pathway, the LH receptor level and adenylate cyclase systems. The post-cAMP steps of progesterone production, in contrast, did not seem to be affected by IL-1.

Elderly humans demonstrate decreased responsiveness in several hormone-receptor systems, including adrenergic receptors. Studies of the beta-adrenergic receptor (beta-AR) system have shown that reduced beta-adrenergic sensitivity in the elderly may be due to reduced beta-AR affinity for agonists. To determine the mechanisms underlying altered alpha-adrenergic sensitivity in the elderly, we assessed the relationships between age and platelet membrane alpha 2-adrenergic receptor (alpha 2-AR)-binding properties, receptor-linked adenylate cyclase (AC) activity, and the affinity of the alpha 2-AR-AC complex for agonists in 18 young (mean age, 24 yr; range 19-34) and 13 elderly (mean age, 69 yr; range, 63-85) normal subjects. In platelet membrane preparations from elderly compared to young subjects, we found similar antagonist-binding properties and similar activity of the catalytic unit of platelet AC, as indicated by the cAMP response to sodium fluoride stimulation. However, mean epinephrine-mediated inhibition of sodium fluoride-stimulated platelet AC activity was less in the elderly [20 +/- 4% (+/- SEM) vs. 31 +/- 2% inhibition; P less than 0.005). In addition, platelet alpha 2-AR affinity for agonist was lower in the elderly, as indicated by the higher concentration of epinephrine needed to inhibit 50% of specific [3H]yohimbine binding (IC50, 3.2 +/- 0.6 vs. 1.4 +/- 0.3 microM; P less than 0.02). These data provide evidence that platelet membranes from elderly humans have decreased responsiveness to alpha-adrenergic stimulation, which can be attributed to reduced alpha 2-AR-AC affinity for agonists. Similarly to reported age-related alterations in beta-adrenergic receptor function, these results suggest that there is also functional uncoupling of the alpha 2-AR-AC complex in elderly humans.

We examined the mechanisms involved in the relaxation of rat vascular smooth muscle by PTH. PTH increased intracellular cAMP 10-fold in cultured vascular smooth muscle cells from rat aorta. Forskolin, methylisobutylxanthine, and papaverine all potentiated PTH action. The cAMP responses to PTH were not altered by concurrent addition of propranolol, phentolamine, atropine, or [Sar1,Ile8]angiotensin II. Only the synthetic PTH antagonist analog [Nle8,Nle18,Tyr34] bovine PTH-(3-34) inhibited the cAMP and vascular relaxation responses to PTH. Isoproterenol produced increases in intracellular cAMP and adenylate cyclase activity which were additive to those produced by PTH. In contracted rat aortic strips, PTH caused a dose-dependent relaxation which was not altered by removal of the vessel intima or treatment with nordihydroguaiaretic acid. Also, membrane preparations from intact aortas or aortas with the endothelium or adventitia removed displayed identical PTH-stimulated adenylate cyclase activities. These findings indicate that the relaxant action of PTH in rat aorta does not require an intact endothelium and results from a direct effect on the vessel medial layer. Relaxation appears to be mediated by a receptor unique for PTH which is linked to the adenylate cyclase of vascular smooth muscle cells.

Cyclic AMP, glucose and cortisol in plasma were measured in three groups of patients undergoing hysterectomy. The operations were performed under general anaesthesia, under general anaesthesia combined with epidural analgesia and under epidural analgesia alone. Surgery elicited a significant rise in plasma cyclic AMP, glucose and cortisol when performed under general anaesthesia alone. Epidural analgesia extending from T4-6 to S5 combined with general anaesthesia abolished the rise in cyclic AMP and reduced the increase in glucose and cortisol and epidural analgesia alone extending from T4 to S5 blocked the rise in glucose and cortisol as well as that in cyclic AMP. The results support the theory that afferent nerve impulses from the area of trauma are of major importance for the catabolic state induced by surgical procedures and indicate that anaesthetic management which includes blockade of afferent nerve impulses which includes blockade of afferent nerve impulses from the area of trauma can be reduce the catabolic response to surgery. These observations could be of value in the operative management of patients with diabetes mellitus and possibly in other groups by patients with a high surgical morbidity.

Epinephrine was shown to inhibit TSH-stimulated cAMP formation in cat and pig thyroid slices and isolated rat and hamster thyroid lobes. In contrast, no such inhibitory action could be demonstrated in sheep or beef thyroid slices or mouse thyroid-trachea preparations. The inhibitory effect of epinephrine on TSH-stimulated cAMP formation in pig thyroid slices was blocked by 10 microM yohimbine, but not by 10 microM prazosin, suggestive of mediation through an alpha 2-catecholamine receptor mechanism. In cat thyroid slices, the inhibitory effect of epinephrine was blocked by both yohimbine and prazosin, suggestive of a mixed alpha-adrenoceptor mechanism. Meclofenamate and indomethacin attenuated the epinephrine response in cat, but not pig, thyroid slices, but other prostaglandin inhibitors were ineffective. Beef thyroid slices responded to epinephrine with an increase in iodide organification that is mediated through an alpha-adrenergic mechanism not blocked by propranolol. This stimulatory effect of epinephrine was abolished by both 10 microM prazosin and 10 microM yohimbine. In contrast to the inhibitory effect of the catecholamines on TSH-stimulated cAMP formation in cats, the stimulatory response of beef iodide organification to epinephrine was not modified by meclofenamate, indomethacin, or verapamil. These findings suggest that the receptor mechanisms mediating the inhibitory effect of catecholamines on cat thyroid and the stimulatory effect of catecholamines in beef thyroid slices may well be mediated by separate and as yet undefined receptor mechanisms. In contrast, the inhibitory effect of epinephrine on TSH-stimulated cAMP formation in pigs is most likely mediated through an alpha 2-adrenoceptor mechanism. These findings further document the multiplicity of catecholamine actions on thyroid function as well as the diversity observed among various species.