Extracellular cAMP Accumulation Research Articles

The impact of the rs8005161 polymorphism on G protein-coupled receptor GPR65 (TDAG8) pH-associated activation in intestinal inflammation

BackgroundTissue inflammation in inflammatory bowel diseases (IBD) is associated with a decrease in local pH. The gene encoding G-protein-coupled receptor 65 (GPR65) has recently been reported to be a genetic risk factor for IBD. In response to extracellular acidification, proton activation of GPR65 stimulates cAMP and Rho signalling pathways. We aimed to analyse the clinical and functional relevance of the GPR65 associated single nucleotide polymorphism (SNP) rs8005161.Methods1138 individuals from a mixed cohort of IBD patients and healthy volunteers were genotyped for SNPs associated with GPR65 (rs8005161, rs3742704) and galactosylceramidase (rs1805078) by Taqman SNP assays. 2300 patients from the Swiss IBD Cohort Study (SIBDC) were genotyped for rs8005161 by mass spectrometry based SNP genotyping. IBD patients from the SIBDC carrying rs8005161 TT, CT, CC and non-IBD controls (CC) were recruited for functional studies. Human CD14+ cells were isolated from blood samples and subjected to an extracellular acidic pH shift, cAMP accumulation and RhoA activation were measured.ResultsIn our mixed cohort, but not in SIBDC patients, the minor variant rs8005161 was significantly associated with UC. In SIBDC patients, we observed a consistent trend in increased disease severity in patients carrying the rs8005161-TT and rs8005161-CT alleles. No significant differences were observed in the pH associated activation of cAMP production between IBD (TT, CT, WT/CC) and non-IBD (WT/CC) genotype carriers upon an acidic extracellular pH shift. However, we observed significantly impaired RhoA activation after an extracellular acidic pH shift in IBD patients, irrespective of the rs8005161 allele.ConclusionsThe T allele of rs8005161 might confer a more severe disease course in IBD patients. Human monocytes from IBD patients showed impaired pH associated RhoA activation upon an acidic pH shift.

Efflux of cAMP in the exocrine pancreas: Role of secretin and atrial natriuretic factor (ANF)

We previously reported that ANF dose‐dependently reduces secretin secretory response and intracellular signaling in the exocrine pancreas through NPR‐C receptors coupled to the PLC/PKC pathway. In this study we evaluated if cAMP efflux contributed to ANF response in isolated pancreatic acini. Results showed that ANF favored the efflux of cAMP formed by secretin. The export of cAMP was also observed with secretin alone but not with isoproterenol or amthamine. Evoked‐cAMP efflux was blocked by probenecid, suggesting that multidrug resistant proteins (MRPs) were involved. RT‐PCR analysis revealed that MRP‐4 but not MRP‐5 (transport cyclic nucleotides) was expressed in the pancreas. Time course studies in the presence of phosphodiesterase (PDE) activity showed that tissue cAMP increased peaking at 3 min onset secretin stimulation and declining thereafter. With ANF, secretin‐induced cAMP augmented reaching a peak at 1 min after which it rapidly decreased. Extracellular cAMP accumulation occurred earlier and persisted throughout the experiment whereas with secretin alone it was delayed. Similar findings were observed in studies with PDE inhibition, except that cAMP content was higher and its appearance delayed in tissues and the media. Present findings suggest that cAMP export is likely mediated by MRP‐4 and further it may contribute to ANF‐induced desensitization of secretin response in the exocrine pancreas.(CONICET PIP5929/ UBACYT B079)

Cyclic AMP in Rat Ileum: Evidence for the Presence of an Extracellular Cyclic AMP-Adenosine Pathway

Extracellular adenosine plays a relevant role in regulating intestinal motility and preventing inflammatory processes. Adenosine 3',5'-cyclic monophosphate (cAMP) extruded from cells may be converted to adenosine monophosphate and then to adenosine by ecto-phosphodiesterase and CD73/ecto-5'nucleotidase, respectively, thus representing a source of adenosine. Our purpose was to assess the existence of a functional extracellular cAMP-adenosine pathway in intestinal tissue, obtaining evidence for CD73 expression and evaluating the effect of cAMP on ileum motility. The formation of cAMP metabolites in rat ileum strips incubated with exogenous cAMP or [(3)H]cAMP was monitored by high-performance liquid chromatography. CD73 was detected by immunoprecipitation and immunofluorescence. The functional activity of exogenous cAMP on ileum strips was recorded by measuring tension changes. In ileum strips, the generation of cAMP-derived adenosine monophosphate, adenosine, and inosine was time and concentration dependent and was blocked by phosphodiesterase or CD73 inhibitors in a manner consistent with exogenous cAMP being processed through the extracellular cAMP-adenosine pathway. Accordingly, [(3)H]cAMP uptake in ileum strips was negligible. Immunofluorescence revealed CD73 surface expression on intestinal smooth muscle cells and intact smooth muscle. Exogenous cAMP concentration-dependently increased ileum muscle tension partially inhibited by adenosine inactivation or receptor blockade. Forskolin-stimulated endogenous cAMP induced concentration-dependent ileum relaxations. A functioning extracellular cAMP-adenosine pathway featuring CD73 expression is present in rat ileum and affects intestinal motility. Extracellular cAMP may therefore act on intestinal muscle both directly by binding to specific smooth muscle cell membrane sites and indirectly through its degradation products.

Ligand-Directed Signaling at the β3-Adrenoceptor Produced by 3-(2-Ethylphenoxy)-1-[(1,S)-1,2,3,4-tetrahydronapth-1-ylamino]-2S-2-propanol oxalate (SR59230A) Relative to Receptor Agonists

This study examines signaling pathways activated by the mouse beta(3)-adrenoceptor (AR) expressed in Chinese hamster ovary cells at high (CHObeta(3)H) or low (CHObeta(3)L) levels. Functional responses included extracellular acidification rate (ECAR), cAMP accumulation, and p38 mitogen-activated protein kinase (MAPK) or extracellular signal-regulated protein kinase 1/2 (Erk1/2) phosphorylation. (-)-Isoproterenol and the beta(3)-AR agonist (R, R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]-propyl]1,3-benzodioxole-2,2-decarboxylate (CL316243) caused concentration-dependent increases in cAMP accumulation and ECAR in CHObeta(3)H and CHObeta(3)L cells. For cAMP accumulation, the beta(3)-AR ligand SR59230A was a partial agonist in CHObeta(3)H and an antagonist in CHObeta(3)L cells but for ECAR was an agonist at both expression levels. This suggested that SR59230A, which is normally regarded as an antagonist, can selectively activate pathways leading to ECAR. Examination of the pathways stimulated by (-)-isoproterenol, CL316243, and SR59230A for both ECAR and cAMP accumulation suggested that the cAMP pathway predominates in CHObeta(3)H cells, whereas p38 MAPK is a major contributor to ECAR in CHObeta(3)L cells and was the sole contributor to responses to SR59230A. Western blots of p38 MAPK and Erk1/2 phosphorylation confirmed that MAPKs are activated in CHObeta(3)H and CHObeta(3)L cells by CL316243 and SR59230A but that SR59230A has much higher efficacy. In addition, p38 MAPK phosphorylation displayed differences in drug potency and efficacy between CHObeta(3)H and CHObeta(3)L cells related to inhibition of the response by cAMP. Thus, CL316243 and SR59230A display reversed orders of efficacy for cAMP accumulation compared with Erk1/2 and p38 MAPK phosphorylation, providing a strong indication of ligand-directed signaling.

Change of extracellular cAMP concentration is a sensitive reporter for bacterial fitness in high-cell-density cultures of Escherichia coli.

Guanosine-3',5'-tetraphosphate (ppGpp) and sigmaS, two regulators of the starvation response of Escherichia coli, have received increasing attention for monitoring cell physiological changes in production processes, although both are difficult to quantify. The kinetics of cAMP formation and degradation were not yet investigated in such processes, although the complex regulation of cAMP by synthesis, release, and degradation in connection with straightforward methods for analysis renders it a highly informative target. Therefore, we followed the cAMP concentration in various nonrecombinant and in four different recombinant glucose-limited fed-batch processes in different production scales. The intracellular cAMP concentration increases strongly at the end of the batch phase. Most cAMP is released to the cultivation medium. The rates of accumulation and degradation of extracellular cAMP are growth-rate-dependent and show a distinct maximum at a growth rate of about 0.35 h(-1). At very low growth rates, below 0.05 h(-1), extracellular cAMP is not produced but rather degraded, independent of whether this low growth rate is caused by glucose limitation or by the high metabolic load of recombinant protein production. In contrast to intracellular cAMP, which is highly unstable, analysis of extracellular cAMP is simpler and the kinetics of accumulation and degradation reflect well the physiological situation, including unlimited growth, limitation, and severe starvation of a production host.

Forskolin evokes extracellular adenosine accumulation in rat cortical cultures

In this study, the effect on extracellular adenosine concentration of direct activation of adenylyl cyclase by forskolin was investigated using rat cortical cultures. Forskolin evoked intracellular and extracellular cAMP accumulation as well as extracellular adenosine accumulation. The accumulation of adenosine in response to forskolin could be blocked by the cyclic nucleotide phosphodiesterase inhibitor 4-[(3-butoxy-4-methoxyphenyl)methyl]-2-imidazolidinone (RO 20-1724; 180 μM), but not isobutylmethylxanthine (100 μM). The accumulation of adenosine in response to forskolin could be blocked by the 5′-ectonucleotidase inhibitor guanosine 5′-monophosphate. These results demonstrate that forskolin can increase extracellular adenosine levels, and that this adenosine is ultimately derived from cAMP.

Beta-adrenergic receptor-mediated regulation of extracellular adenosine in cerebral cortex in culture

Adenosine is an important inhibitory neuromodulator in the CNS, yet the sources of extracellular adenosine have yet to be well characterized. In this study we show that beta-adrenergic stimulation of cortical cultures results in the extracellular accumulation of cAMP as well as adenosine, and that the extracellular adenosine derives from extracellular cAMP. The concentration dependence of isoproterenol in evoking cAMP secretion was determined by radioimmunoassay, and the EC50 for this effect was found to be approximately 100 nM. In order to investigate the effect of beta-adrenergic stimulation on the regulation of extracellular adenosine, the effect of isoproterenol in stimulating the extracellular accumulation of adenine-containing compounds was examined by HPLC. Isoproterenol stimulated cAMP secretion in both astrocyte cultures and astrocyte-rich mixed cultures of astrocytes and neurons. However, no extracellular cAMP was detectable in neuron-enriched astrocyte-poor cultures. Extracellular adenosine increased in response to isoproterenol in the astrocyte-rich mixed cultures, but not in the neuron-enriched astrocyte-poor cultures. After 30 min exposure to isoproterenol, the concentration of adenosine in the extracellular medium increased by 47% in 56 experiments in the mixed astrocyte-rich cultures. In order to establish whether the adenosine that accumulates in response to isoproterenol stimulation actually derives from extracellular cAMP, phosphodiesterase inhibitors were tested for their ability to block isoproterenol-stimulated adenosine accumulation. Isobutylmethylxanthine (IBMX; 100 microM), RO 20-1724 (180 microM), carbazeran (10 microM), dipyridamole (10 microM), and trifluoperazine (10 microM) had no inhibitory effect on the isoproterenol-stimulated accumulation of extracellular adenosine. However 100 microM IBMX plus 180 microM RO 20-1724 effectively blocked isoproterenol-stimulated adenosine accumulation and, as expected, increased extracellular cAMP. As a further test of the origin of isoproterenol-stimulated adenosine accumulation, we attempted to block this phenomenon by blocking cAMP secretion itself. For this purpose probenecid, a known inhibitor of cAMP secretion in many different cell types, was used. We found that probenecid at 1 mM blocked isoproterenol-stimulated adenosine accumulation. These studies suggest that one potentially important source of extracellular adenosine in the cerebral cortex is endogenous extracellular cAMP, secreted from astrocytes in response to beta-adrenergic receptor stimulation. Since the receptors of neuromodulators other than norepinephrine may also be coupled to adenylyl cyclase in the cerebral cortex, there may be several neuromodulatory systems that regulate extracellular adenosine levels by this mechanism.

Purified human α-fetoprotein inhibits follicle-stimulating hormone-stimulated estradiol production by porcine granulosa cells in culture

We have investigated the effects of purified α-fetoprotein (AFP) on follicle-stimulating hormone (FSH)-stimulated estradiol production by porcine granulosa cells in monolayer culture. Granulosa cells isolated from small follicles of prepubertal pigs were cultured for 2 days in 5% fetal bovine serum for attachment and incubated for 3 days in medium containing androstenedione and various treatments. The media were then collected and assayed for estradiol by radioimmunoassay. Human AFP significantly ( P < 0.05) and dose-dependently inhibited FSH-stimulated estradiol production with 313 ng/ml AFP returning FSH-stimulated estradiol to basal levels; human serum albumin was without effect. AFP purified from either term cord blood or midtrimester amniotic fluid dose-dependently inhibited estradiol production stimulated by the combination of FSH and insulin-like growth factor-I. Furthermore, 125 ng/ml AFP inhibited estradiol production stimulated by cholera toxin, forskolin and cAMP. In contrast, extracellular accumulation of cAMP and progesterone production was not inhibited by AFP. These data indicate that physiological concentrations of purified AFP significantly and dose-dependently inhibit FSH-stimulated estradiol production by porcine granulosa cells in culture. Since AFP is known to augment growth factor-mediated cell growth, these data suggest that AFP inhibits differentiated functions (such as aromatase) while enhancing the proliferation of porcine granulosa cells.

Lysophosphatidic acid is a chemoattractant for Dictyostelium discoideum amoebae.

The naturally occurring phospholipid lysophosphatidic acid (LPA) can induce a number of physiological responses in vertebrate cells, including platelet aggregation, smooth muscle contraction, and fibroblast proliferation. LPA is thought to activate a specific G-protein-coupled receptor, thereby triggering classic second messenger pathways such as stimulation of phospholipase C and inhibition of adenylate cyclase. Here we report that 1-oleoyl-LPA, at submicromolar concentrations, evokes a chemotactic response in amoebae of the cellular slime mold Dictyostelium discoideum. LPA-induced chemotaxis is specific in that other lysophospholipids, phosphatidic acid, and monoacylglycerol have no effect. We show that the response to LPA is not secondary to the accumulation of extracellular cAMP, a well-established chemoattractant for nutrient-starved D. discoideum. Compared with cAMP-induced chemotaxis, LPA-induced chemotaxis has a somewhat lower efficiency and is not accompanied by the characteristic cellular elongation and orientation along the gradient. These results indicate that LPA has a previously unsuspected role as a chemoattractant for D. discoideum and imply that its biological function as a "first messenger" is not restricted to vertebrate cells.

Expression of recombinant human follicle-stimulating hormone receptor: species-specific ligand binding, signal transduction, and identification of multiple ovarian messenger ribonucleic acid transcripts.

The ligand specificity and biochemical properties of the human (h) FSH receptor are poorly characterized due to the low abundance of these receptors and the limited availability of human tissues. Using a fragment of rat FSH receptor cDNA, we screened a human testicular cDNA library and obtained a FSH receptor cDNA covering the entire amino acid-coding region. After transfection of a human fetal kidney cell line (293) with the hFSH receptor cDNA, radioligand receptor analysis revealed the presence of high affinity (Kd, 1.7 x 10(-9) M) FSH-binding sites on the plasma membrane. Both recombinant and wild-type hFSH displaced [125I]hFSH binding, with ED50 values of 25 and 70 ng/ml, respectively, whereas hLH, hCG, and hTSH were ineffective. Although human, rat(r), and ovine FSH as well as equine CG competed for rat testicular FSH receptor binding, only hFSH and rFSH interacted effectively with the recombinant hFSH receptor, suggesting that species-specific ligand recognition exists between human and rodent receptors. After incubation of transfected cells with hFSH, but not recombinant hLH or hCG, a dose-dependent increase (ED50, 10 ng/ml) in extracellular cAMP accumulation was observed, indicating a functional coupling of the expressed human receptor with the endogenous adenyl cyclase. In cells cotransfected with the FSH receptor expression plasmid and a luciferase reporter gene driven by the promoter of a cAMP-responsive gene, treatment with hFSH, but not hCG, resulted in a dose-dependent increase in luciferase activity. Northern blot analysis using a cRNA probe derived from the human receptor cDNA indicated the presence of multiple FSH receptor mRNA transcripts (7.0, 4.2, and 2.5 kilobases) in RNA prepared from human follicular phase ovary, but not from human corpus luteum or placenta. Additionally, two FSH-binding sites of 76 and 112 kilodaltons were detected in transfected 293 cells after ligand/receptor cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. These results demonstrate the expression of functional hFSH receptor with unique ligand specificity and provide new data on the biochemical properties of the human receptor at the mRNA and protein levels.

Direct effects of bromocriptine on the steroidogenic capability of porcine granulosa cells.

The direct effects of bromocriptine on steroidogenesis were examined in cultured porcine granulosa cells. The following observations were made with bromocriptine: (1) It significantly increased the basal or FSH-stimulated secretion of progesterone in cultured porcine granulosa cells at concentrations exceeding 10(-7) mol/l; (2) its inhibitory effect on basal estradiol secretion was demonstrated; (3) it did not influence cell number in cultured porcine granulosa cells; (4) it increased the extracellular accumulation of cAMP in a concentration-dependent manner; and (5) it did not induce a change in cytosolic free Ca2+ concentration. These findings suggest that bromocriptine exerts a direct effect on steroidogenesis in ovarian granulosa cells.

Modulation of differentiation of rat granulosa cells in vitro by interferon-gamma.

The effects of recombinant rat interferon-gamma (rRaIFN-gamma) and rat IFN (RaIFN, a mixture of IFN-gamma and -alpha) on basal and FSH-induced ovarian granulosa cell function were studied. Granulosa cells were harvested from diethylstilboestrol-treated immature rats and cultured (2 x 10(5) viable cells/well per 0.5 ml) in serum-free medium with or without treatment for 48 h. In the presence of FSH (20 ng/ml), rRaIFN-gamma (10-1000 U/ml) significantly inhibited FSH-stimulated aromatase activity (76.4 +/- 2.3% maximum inhibition compared with FSH treatment alone), inhibin (40.4 +/- 3.7%), progesterone (47.7 +/- 8.6%) and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OHP) (51.8 +/- 1.7%) production in a dose-dependent manner. Furthermore, rRaIFN-gamma inhibited FSH- and forskolin (FSK; 30 mumol/l)-induced extracellular cAMP accumulation (46.0 +/- 6.6% and 29.1 +/- 7.3% respectively). The inhibitory effect of rRaIFN-gamma on FSK-induced cAMP was accompanied by decreased FSK-induced aromatase activity, inhibin, progesterone and 20 alpha-OHP production. rRaIFN-gamma had no detectable effect on aromatase activity, progesterone production and 20 alpha-OHP production in the absence of FSH, but significantly stimulated basal inhibin production by 1.5-fold. rRaIFN-gamma alone also caused a small but significant increase in basal levels of cAMP. The time-course studies showed that FSH-induced aromatase activity and inhibin production were consistently suppressed by rRaIFN-gamma, FSH-induced progesterone and 20 alpha-OHP were inhibited at 1 and 2 days and then stimulated on days 3, 4 and 5 relative to FSH alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyrotoxicosis and hyperemesis gravidarum associated with a serum activity which stimulates human thyroid cells in vitro.

To investigate in-vitro thyroid stimulatory activity in the serum of patients with hyperemesis gravidarum and thyrotoxicosis. Serum from hyperthyroid patients was incubated with cultures of human thyroid cells. Attempts were made to neutralize stimulatory activity with antisera to hCG. Five patients presenting in early pregnancy with hyperemesis and thyrotoxicosis. Serum concentrations of thyroid hormones (total and free), TSH and hCG. Accumulation of extracellular cAMP in response to serum. All five patients had biochemical hyperthyroidism with no evidence of an underlying autoimmune disease. The mean cAMP accumulation over 4 hours with sera from 12 non-pregnant controls was 130.6 (121.8-142.8), from 12 pregnant controls 132.4 (118.1-143.8), compared with values of 144.7, 159.1, 166.2, 178.9 and 320.5 for the thyrotoxic patients. The stimulatory activity could not be neutralized by addition of anti-hCG or by depleting the sera of hCG. Thyrotoxicosis may present with hyperemesis in early pregnancy. Clinical and biochemical features may be masked by the pregnancy or by the intercurrent illness. The hypothesis that hCG is a thyroid stimulator in patients with hyperemesis gravidarum is not supported by the immuno-neutralization data in this study.

Role of chloride ions in progesterone production by chicken granulosa cells

The importance of chloride ions in luteinizing hormone (LH)-stimulated progesterone production by chicken granulosa cells from the two largest preovulatory follicles was investigated in vitro. Reduction of the extracellular chloride concentration from 147.8 mM to 2.8 mM, by substitution with equimolar concentrations of non-permeant glutamate and aspartate, inhibited the ability of LH to stimulate progesterone production and cAMP accumulation during a 4 h incubation. LH-stimulated granulosa cell progesterone production was also suppressed in a concentration-dependent manner by the chloride channel blockers 4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonic acid (SITS; 10 −8−5 × 10 −5 M) or 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid (DIDS; 10 −8−5 × 10 −5 M). The inhibitory effect was observed within 30 min of the addition of the blockers and was irreversible. DIDS appeared to act at a site(s) proximal to the generation of cAMP, since concentrations of DIDS (10 −8−10 −6 M) which inhibited LH- and human chorionic gonadotropin-stimulated progesterone production, did not affect progesterone production stimulated by dibutyryl cAMP, 8-bromo cAMP or forskolin. In addition, concentrations of DIDS (10 −8−10 −6 M) which attenuated LH-stimulated progesterone production also reduced the accumulation of extracellular cAMP. These studies suggest that chloride ions may play an important role in the stimulatory action of LH on chicken granulosa cell progesterone production.

Neuropeptide regulation of interleukin-6 production from the pituitary: stimulation by pituitary adenylate cyclase activating polypeptide and calcitonin gene-related peptide.

Interleukin 6 (IL-6) production was shown to be stimulated by vasoactive intestinal peptide via cAMP dependent signal transduction pathway in the pituitary. We were interested in whether other hypothalamic neuropeptides, which activate adenylate cyclase in the pituitary, also stimulate pituitary IL-6 production. Whereas vasoactive intestinal peptide was effective in stimulating pituitary IL-6 production only at concentrations of 10(-6) M or higher, pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38) and calcitonin gene-related peptide (CGRP) at concentrations from 10(-10) to 10(-9) M significantly stimulated IL-6 production. Similar effective concentrations of each peptide were required for activating adenylate cyclase, as measured by extracellular cAMP accumulation. H89, a specific inhibitor of cAMP dependent protein kinase (protein kinase A), inhibited IL-6 production stimulated by PACAP38, CGRP, and (Bu)2cAMP. However, H89 failed to inhibit the IL-6 production stimulated by lipopolysaccharide, a ligand which enhanced IL-6 production in the absence of cAMP accumulation. Two other peptides which are known to activate pituitary adenylate cyclase, corticotropin-releasing factor and GRF failed to stimulate IL-6 production in pituitary cells. Using discontinuous Percoll gradients to fractionate the pituitary cells, the greatest PACAP38-stimulated IL-6 secretion was observed in the low density fraction 1 (F1). This fraction also contained the highest percentage of folliculo-stellate (FS) cells, one of the nonhormone secreting pituitary cells. However, the largest PACAP38-induced accumulation of cAMP was observed in F4. These results suggest that the production of IL-6 stimulated by PACAP and CGRP is mediated by the adenylate cyclase/protein kinase A signal transduction system. FS cells appear to be the most likely target cell type for PACAP-induced IL-6 production. However, IL-6 producing FS cells may not be an exclusive target for PACAP in the pituitary.

Follicle-stimulating hormone signal transduction: Role of carbohydrate in aromatase induction in immature rat Sertoli cells

Receptor activated adenylate cyclase acts as a major transmembrane signalling system. It is widely accepted that upon binding to its receptor, follicle-stimulating hormone (FSH) activates the cAMP-dependent pathway which in turn mediates FSH-induced estradiol production in Sertoli cells. Studies utilizing several chemically derived variants of FSH have demonstrated that these variants bind to the FSH receptors with equal avidity but differ in their ability to activate cAMP-dependent pathways. Since cAMP is believed to be the second messenger responsible for FSH signal transduction, we tested two hypotheses: (1) that the effects of different oFSH variants on cAMP production and aromatase induction (as measured by estradiol production) would be in parallel; and (2) that deglycosylated ovine FSH (DG-oFSH) would antagonize the ability of intact oFSH to stimulate aromatase induction, similar to its reported antagonistic effect on cAMP production. Immature rat (7- to 10-day-old) Sertoli cells were cultured and the effects of several different oFSH variants on cAMP production and/or aromatase induction were tested. The variants tested were native oFSH, DG-oFSH, asialo oFSH (AS-oFSH), a recombinant of intact LHα and FSHβ (α + β) and a recombinant of deglycosylated LHα and intact FSHβ (DGα + β). Both native oFSH and α + β recombinant at relatively large doses (10 ng) elicited a significant increase in extracellular cAMP accumulation as well as total cAMP production. In contrast, DG-oFSH did not produce an increase in cAMP even at 10-fold higher doses than native oFSH. Intracellular cAMP concentrations did not increase following stimulation with native oFSH, DG-oFSH or DGα + β. In contrast to the divergent effects of oFSH and DG-oFSH on cAMP production all variants of oFSH stimulated estradiol production from Sertoli cells albeit with varying potencies. The sensitivity (minimal effective dose) and ED 50 (dose at which half maximal response is achieved) of the estradiol (E 2) response curve to increasing concentrations of native oFSH were 0.025 ± 0.01 and 0.33 ± 0.05 ng, respectively. Asialo-oFSH (AS-oFSH) increased E 2 production with a potency (comparative dose required for effect) similar to that of native oFSH. In contrast, there was a 10-fold reduction in potency of DG-oFSH (ED 50 of 3.27 ± 1.7 ng). The efficacy (maximum effect on aromatase induction) of native, DG and AS-oFSH were equipotent. Coincubation of DG-oFSH (0.005–100 ng) with oFSH (0.01–0.625 ng) demonstrated the inductive effects to be additive. In contrast to the reduced potency of DG-oFSH, DGα — β recombinants (0.156–160 ng) increased E 2 production with a potency similar to that of the native oFSH. Our data suggest: (1) the effects of oFSH variants on cAMP production and aromatase induction are not always parallel; (2) DG-oFSH is a weak agonist and not an antagonist of oFSH-mediated aromatase induction; (3) the reduced potency of DG-oFSH must involve oligosaccharides internal to sialic acid; and (4) depleting the carbohydrate moiety on the α subunit alone may not be adequate to reduce the potency of oFSH to induce aromatase. Since DG-oFSH does not increase cAMP accumulation, but is able to induce aromatase, the FSH-mediated induction of aromatase may be transduced through other signal pathways. Alternatively, the maximal steroidogenic response may be induced by small, but undetectable, increases in intracellular cAMP concentrations or availability of segregated cAMP pools.

Interactions between activin and follicle-stimulating hormone-suppressing protein and their mechanisms of action on cultured rat granulosa cells

Direct roles of follicle-stimulating hormone (FSH)-suppressing protein (FSP) and activin in regulation of ovarian granulosa cell differentiation have been reported recently. The present study further investigated the effects of these peptides on steroidogenesis and inhibin production as well as cAMP generation in cultured granulosa cells from immature, diethylstilbestrol (DES)-treated rats. In the presence of FSH (20 ng/ml) and activin (30 ng/ml), which enhanced FSH-induced aromatase activity, progesterone production and inhibin production, FSP (1–100 ng/ml) reversed the stimulating activities of activin in a dose-dependent manner. In addition, activin reversed the inhibitory effects of FSP on FSH-induced aromatase activity and inhibin production. In the presence of FSH, activin enhanced FSH-stimulated extracellular cAMP accumulation, and FSP caused a reduction in extracellular cAMP. Activin but not FSP also stimulated basal cAMP level. In the presence of forskolin, a potent stimulant of adenyl cyclase activity which stimulated extracellular cAMP, aromatase activity, progesterone production and inhibin production, activin augmented the effect of forskolin on all four parameters, whereas FSP significantly enhanced progesterone production without changing the other three parameters. Our findings suggest that activin action on rat granulosa cells may be mediated via regulation of cAMP generation. The action of FSP and FSH and/or activin-dependent, consistent with either an action as an activin binding protein or by a direct action of FSP on the granulosa cells.

Prostaglandin-Dependent in Vitro Stimulation of Adrenocortical Steroidogenesis by Interleukins*

The effects of interleukins on adrenal steroidogenesis and their mode of action were studied using cultured rat adrenal cells. The addition of rat interleukin-1 alpha (IL-1 alpha) or rat IL-2 increased corticosterone levels in the medium in a concentration-dependent manner during 24 h of incubation. The minimum, half-maximum, and maximum effective concentrations of both rat IL-1 alpha and rat IL-2 were almost same (approximately 3, 10, and 100 U/ml, respectively). After a latent period, the effect became apparent after 12 h of incubation. Human IL-1 beta and human IL-6 also showed a stimulatory effect on corticosterone production, whereas human IL-2 was inactive in this system. To clarify the cellular mechanism of these stimulatory effects, we measured the levels of prostaglandin E2 (PGE2) and cAMP in the cells and media as well as the corticosterone levels. Corticosterone production stimulated by IL-1 alpha or IL-2 was accompanied by intracellular and extracellular cAMP and PGE2 accumulation. Although the stimulation of both cAMP and corticosterone was observed only after 12 h of incubation, PGE2 levels increased during the first 4 h of incubation. Corticosterone, cAMP, and PGE2 production stimulated by ILs was almost completely blocked by the addition of 0.1 mM aspirin, a cyclooxygenase inhibitor. Lipoxygenase inhibitors, i.e. AA-861, nordihydroguaiaretic acid, and 5,8,11,14-eicosatetrynoic acid, did not abolish corticosterone production stimulated by ILs. Submaximal doses of IL-1 alpha and IL-2 synergistically stimulated PGE2 production, but did not have even additional effects on cAMP and corticosterone levels. On the other hand, submaximal doses of ACTH, which did not significantly affect PGE2 levels, acted synergistically with IL to increase cAMP and corticosterone levels in these cells. These results indicate that 1) IL-1 alpha and IL-2 directly stimulate glucocorticoid synthesis in a dose- and time-dependent manner; 2) a half-maximum effective concentration of ACTH acts synergistically with IL in stimulating glucocorticoidogenesis; 3) the stimulatory process initially requires PGs, followed by the activation of the adenylate cyclase system; 4) although the profiles of steroidogenic action of IL-1 alpha and IL-2 are quite similar, they may exert their effects through different mechanisms in their early steps of PGE2 production; and 5) the low effective concentrations of both cytokines suggest possible physiological or pathophysiological roles of circulating cytokines in the glucocorticoidogenesis under certain conditions.

Epidermal growth factor potentiates cyclic AMP accumulation in A-431 cells.

Epidermal growth factor (EGF) treatment of A-431 cells potentiates up to 5-fold the intracellular cyclic AMP (cAMP) accumulation induced by isoproterenol, cholera toxin, forskolin, or 3-isobutyl-1-methylxanthine (IBMX). EGF potentiates cAMP accumulation in several epithelial cell lines which overexpress the EGF receptor including A-431 cells, HSC-1 cells, and MDA-468 cells, and in the A-431-29S clone which expresses a normal complement of EGF receptors. Although EGF potentiates cAMP accumulation, EGF by itself does not measurably alter the basal level of cAMP. EGF rapidly enhances cAMP accumulation (within 1 to 3 min) in A-431 cells treated with these cAMP-elevating agents. EGF potentiation of cAMP accumulation does not reflect enhancement of beta-adrenergic receptor activation and is not a consequence of intracellular cAMP elevation or the concomitant activation of cAMP-dependent protein kinase. Since EGF potentiates accumulation of both intracellular and extracellular cAMP in isoproterenol-treated A-431 cells, EGF does not potentiate intracellular cAMP accumulation by inhibition of cAMP export. EGF potentiation of cAMP accumulation is pertussis toxin-insensitive and does not result from EGF inhibition of cAMP degradation in A-431 cells. These results demonstrate that EGF transmembrane signaling includes an interaction with a component of the adenylate cyclase system and that this interaction stimulates cAMP synthesis resulting in enhancement of cAMP accumulation.

Catecholamines inhibit early steps of lymphocyte activation

In this study, the effect on extracellular adenosine concentration of direct activation of adenylyl cyclase by forskolin was investigated using rat cortical cultures. Forskolin evoked intracellular and extracellular cAMP accumulation as well as extracellular adenosine accumulation. The accumulation of adenosine in response to forskolin could be blocked by the cyclic nucleotide phosphodiesterase inhibitor 4-[(3-butoxy-4-methoxyphenyl)methyl]-2-imidazolidinone (RO 20-1724; 180 μM), but not isobutylmethylxanthine (100 μM). The accumulation of adenosine in response to forskolin could be blocked by the 5′-ectonucleotidase inhibitor guanosine 5′-monophosphate. These results demonstrate that forskolin can increase extracellular adenosine levels, and that this adenosine is ultimately derived from cAMP.