Stimulated cAMP Accumulation Research Articles

Glucose stimulates cAMP accumulation in the oral bacterium Actinomyces viscosus

Actinomyces viscosus T14V, a Gram-positive bacterium found in the oral cavity, was found to be insensitive to glucose-mediated catabolite repression. Basal levels of β-galactosidase (18–26 U) were observed at all phases of growth regardless of the culture conditions. Further, β-galactosidase could not be induced with lactose, or with a known inducer of the enzyme, isopropyl-β- d-thiogalactoside, or with dibutyryl cAMP. Glucose, on the other hand, stimulated cAMP accumulation in a concentration-dependent manner. Fructose and sucrose mimicked the effects of glucose on cAMP accumulation, whereas galactose, mannose and maltose had lesser stimulatory effects. Other carbon sources, i.e., lactose, α-methylglucoside, ribose, xylose and succinate were without effect. Glucose and α-methylglucoside were found to stimulate cAMP accumulation in toluene-permeabilized cells, in the presence of the phosphodiesterase inhibitor, theophylline. Glucose did not stimulate cAMP levels in other Gram-positive bacteria including Streptococcus mutans, S. sanguis and S. salivarius but did cause cAMP accumulation in other strains of A. viscosus. The results suggest that glucose effects on cAMP metabolism are independent of the induction of β-galactosidase as presently defined for Escherichia coli, and that the effects appear to be selective to the A. viscosus bacteria. The results also suggest that glucose stimulates cAMP accumulation via activation of adenylate cyclase.

Tumour necrosis factor-α enhances cAMP-induced programmed cell death in mouse thymocytes

During T-lymphocyte differentiation in the thymus, the majority of thymocytes die by apoptosis in situ. This process is characterized by internucleosomal DNA fragmentation and is induced by a number of stimuli including glucocorticoids, calcium ionophore, cAMP and 12- o-tetradecanoylphorbol 13-acetate (TPA). In this study, the effect of cytokines tumour necrosis factor-α (TNF-α) and interferon γ (IFN-γ) on the programmed cell death of thymocytes was examined by measuring DNA fragmentation and LDH release. TNF-α and IFN-γ had no effect on DNa fragmentation in control and TPA, or A23187-treated thymocytes. Both human and murine rTNF-α enhanced cAMP-induced programmed cel death dose-dependently, but IFN-γ had no effect on the process. TNF-α did not stimulate cAMP accumulation in control or 2-chloroadenosine-treated thymocytes. TPA markedly stimulated cAMP-induced DNA fragmentation as a result of 6 h incubation, whereas TNF-α did not. Thus TNF-α did not appear to activate protein kinase C directly. The effect of TNF-α did not. observed in the cell preparations from which adherent cells had been removed, suggesting that cytokines secreted by adherent cells ini response to TNF-α are not involved in the process. The enhancement of cAMP-induced DNA fragmentation was observed in CD4 +CD8 +-double positive cells, but not in CD4 +CD8 −-single positive cells. The results of the present study indicate that a physiological cytokine, TNF-α, may modulate programmed cell death in immature thymocytes in concert with cAMP.

Measurement of cAMP accumulation in Chinese hamster ovary cells transfected with the recombinant human TSH receptor (CHO-R): a new bioassay for human thyrotropin.

Circulating TSH bioactivity may vary in several clinical and experimental conditions. Since the reliability of the current methods for the measurement of TSH bioactivity is limited, a new bioassay based on cAMP accumulation in Chinese Hamster Ovary cells transfected with recombinant human TSH receptor (CHO-R) was set up. The sensitivity was 0.3 +/- 0.1, 0.4 +/- 0.1 and 0.01 +/- 0.01 micrograms/L for TSH IRP 80/558, recombinant human TSH and bovine TSH, respectively. Standard curves were parallel, and the intra- and inter-assay coefficients of variation were 13 +/- 1.1% and 22 +/- 1.9%, respectively. LH, FSH, CG and TSH subunits did not stimulate cAMP accumulation up to high concentrations. Circulating TSH was partially purified by immunoaffinity separation and concentrated before being bioassayed. However, plain sera with high TSH levels, such as those from primary hypothyroid patients (PH), could be directly tested in CHO-R bioassay, provided that sera were added at concentrations lower than 10%. TSH from 6 normal subjects had biological to immunological ratio (B/I) ranging from 0.6 to 2.1 (mean +/- SD = 1.4 +/- 0.5). TSH from 6 patients with PH showed bioactivity significantly lower than in normals (B/I = 0.6 +/- 0.3; p < 0.001; range = 0.3-1.1). TSH from 5 patients with central hypothyroidism of hypothalamic origin (CH) had undetectable basal bioactivity (B/I < 0.2), which normalized in only one patient after acute TRH and in all patients after chronic TRH administration. In conclusion, CHO-R cells provide an excellent tool for evaluating TSH bioactivity, owing to high sensitivity, specificity, reproducibility and feasibility of the assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Elevation of cAMP is required for down-regulation, but not agonist-induced desensitization, of endogenous dopamine D1 receptors in opossum kidney cells. Studies in cells that stably express a rat cAMP phosphodiesterase (rPDE3) cDNA

D1 dopamine receptors stimulate cAMP accumulation in opossum kidney (OK) cells, but this response is attenuated by pretreatment with dopamine. Dopamine pretreatment also causes a reduction in D1 dopamine receptor number. We transfected OK cells with a rat cAMP phosphodiesterase cDNA (rPDE3) in order to determine the contribution of elevations of cAMP to those two phenomena. Wild-type (WT) OK cells were compared to three clones (C, H, and N) which demonstrated stable expression of the rPDE3 phenotype and genotype, rPDE3 RNA expression was confirmed in clones C, H, and N (but not in WT-OK cells) by reverse transcriptase-polymerase chain reaction. A functional rPDE3 phenotype was demonstrated in that dopamine-responsive cAMP accumulation was absent in clones C, H, and N in intact cells, but could be restored by preincubation with cAMP phosphodiesterase inhibitors, or by using washed membranes from those clones. All three clones had increased cAMP phosphodiesterase activity when compared to WT-OK cells (approximately 100% increase), and blunted or absent dopamine (1 microM)-induced protein kinase A activation. After pretreatment with dopamine (1 microM) for 1 h, clones C, H, and N desensitized equally well as WT-OK cells (approximately 40-50% reduction in maximal increase in cAMP). In contrast, down-regulation of D1 dopamine receptors was blunted for clone C (20% receptor loss) and absent for clones H and N, when compared to a 45% loss of receptors for WT-OK cells. These findings suggest that in OK cells pretreated with 1 microM dopamine (i) cAMP accumulation is not necessary for dopamine-induced desensitization, but (ii) is necessary for down-regulation of D1 dopamine receptors, and (iii) that the down-regulation and desensitization processes may be differentially regulated.

Regulation of melatonin production by pineal photoreceptor cells: role of cyclic nucleotides in the trout (Oncorhynchus mykiss).

The light/dark cycle influences the rhythmic production of melatonin by the trout pineal organ through a modulation of the serotonin N-acetyltransferase (NAT) activity. In static organ culture, cyclic AMP (cAMP) levels (in darkness) and NAT activity (in darkness or light) were stimulated in the presence of forskolin, isobutylmethylxanthine, or theophylline. Analogues of cAMP, but not of cyclic GMP, induced an increase in NAT activity. Light, applied after dark adaptation, inhibited NAT activity. This inhibitory effect was partially prevented in the presence of drugs stimulating cAMP accumulation. In addition, cAMP accumulation and NAT activity increase, induced by forskolin, were temperature dependent. Finally, melatonin release, determined in superfused organs under normal conditions of illumination, was stimulated during the light period of a light/dark cycle by adding an analogue of cAMP or a phosphodiesterase inhibitor. However, no further increase in melatonin release was observed during the dark phase of this cycle in the presence of the drugs. This report shows for the first time that cAMP is a candidate as intracellular second messenger participating in the control of NAT activity and melatonin production by light and temperature.

Inhibition of cloned adenylyl cyclases by mutant-activated Gi-alpha and specific suppression of type 2 adenylyl cyclase inhibition by phorbol ester treatment

Inhibition of the various cloned Gs-sensitive adenylyl cyclases was studied. The effect of mutant-activated Gi2-alpha on types 2, 3, and 6 adenylyl cyclases was analyzed after co-transfection into COS-7 cells. These adenylyl cyclases were chosen as representative members of the distinct families of mammalian adenylyl cyclases. COS-7 cells were transfected with cDNAs encoding (a) the required type of adenylyl cyclase, (b) luteinizing hormone receptor, and (c) Q205LGi2-alpha or Q205LGo1-alpha. Human chorionic gonadotropin (hCG)-stimulated cAMP accumulation was then measured. Transfection with Q205LGi2-alpha but not Q205LGo1-alpha resulted in 50-60% inhibition of hCG-stimulated cAMP accumulation of the type 2 adenylyl cyclase. hCG stimulation of types 3 and 6 adenylyl cyclases was also extensively inhibited by co-transfection with the Q205LGi2-alpha cDNA. Treatment with 4 beta-phorbol 12-myristate 13-acetate of cells transfected with the type 2 enzyme cDNA resulted in a 2-fold increase of the hCG-stimulated cAMP accumulation and a complete suppression of the Q205LGi2-alpha inhibition of the type 2 adenylyl cyclase. However, similar treatment did not affect Q205LGi2-alpha inhibition of the type 3 and type 6 adenylyl cyclases. These data indicate that many if not all types of adenylyl cyclases may be susceptible to inhibition by Gi2-alpha, but this inhibition can be regulated by protein kinase C in a type-specific manner.

Bradykinin inhibits cyclic AMP accumulation in D384-human astrocytoma cells via a calcium-dependent inhibition of adenylyl cyclase

Bradykinin causes a concentration-dependent, transient rise in intracellular Ca 2+ and a sustained inhibition of forskolin-, dopamine- and 5′- N-ethyl-carboxamidoadenosine (NECA)-stimulated cAMP accumulation in D384 astrocytoma cells. Chelation of intracellular calcium abolished bradykinin's inhibitory effect on cAMP accumulation. Chelating extracellular Ca 2+ did not block the initial, but eliminated the sustained inhibition of cAMP accumulation. Increasing Ca 2+ influx by calcium ionophore A23187 caused a concentration-dependent inhibition of stimulated cAMP accumulation. A hydroquinone derivative 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), which inhibits microsomal Ca 2+ sequestration, did not mimic the effect of bradykinin, although it increased [Ca 2+] i even more than A23187 did. The inhibitory effect of bradykinin was not mediated by Ca 2+/CaM-dependent stimulation of phosphodiesterase (PDE). Forskolin-stimulated adenylyl cyclase activity was inhibited by Ca 2+ (10 −7 to 10 −3 M), both in ethyleneglycol-bis-( β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid (EGTA) washed and native D384 plasma membranes. This effect was not altered by calmodulin (CaM) or CaM-antagonists. Bradykinin treatment, which attenuates cAMP accumulation in intact cells, did not do so in plasma membranes. These findings suggest that bradykinin-induced inhibition of cAMP formation in D384 cells requires mobilization of [Ca 2+] i and subsequent entry of Ca 2+ which directly interacts with a component of the adenylyl cyclase system.

Alpha1-adrenoceptor-mediated inhibition of cellular cAMP accumulation in neonatal rat ventricular myocytes

We studied adrenergic regulation of cellular cAMP in neonatal rat ventricular myocytes. Since cAMP content depends on synthesis, breakdown and egress, the contribution of each of these mechanisms was assessed. In the presence of the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine, cAMP accumulation stimulated by the beta-adrenoceptor agonist (-)-isoprenaline was diminished when the mixed alpha + beta adrenoceptor agonist (-)-noradrenaline was coincubated with (-)-isoprenaline. Moreover, adenylyl cyclase activation stimulated by (-)-isoprenaline was decreased by (-)-noradrenaline and by the selective alpha 1-adrenoceptor agonists (-)-phenylephrine and methoxamine, suggesting that alpha-adrenoceptor agonism regulates cAMP metabolism through its effect on the synthetic pathway. Evidence for alpha 1-adrenoceptor mediation of this response was enhancement of (-)-noradrenaline-induced cAMP generation by the selective alpha 1-adrenoceptor antagonist terazosin (10 nmol/l). The selective alpha 2-adrenoceptor antagonist yohimbine (10 nmol/l) had no effect. The alpha 1-adrenoceptor mediated depression of (-)-isoprenaline-stimulated cAMP generation and adenylyl cyclase activation was prevented by terazosin and in separate experiments markedly enhanced by pertussis toxin pretreatment, suggesting involvement of a guanine-nucleotide regulatory protein in this process. Occupation of the alpha 1-adrenoceptor by (-)-noradrenaline did not accelerate the rate of cAMP breakdown in the absence of phosphodiesterase inhibition. Furthermore, there was no enhancement of total phosphodiesterase activity by (-)-noradrenaline in the presence of (-)-propranolol. By contrast, pertussis toxin pretreatment augmented phosphodiesterase activity. Neither pertussis toxin nor (-)-noradrenaline increased cAMP egress. We conclude that in rat neonatal cardiac myocytes agonist occupation of the alpha 1-adrenoceptor inhibits beta-adrenoceptor stimulated cAMP accumulation most likely by coupling to a guanine nucleotide inhibitory protein.

Transfected Go1 alpha inhibits the calcium dependence of beta-adrenergic stimulated cAMP accumulation in C6 glioma cells.

Increasing evidence indicates that heterotrimeric G proteins, and in particular Go, regulate ionic channel activities. In order to investigate the role of Go proteins in the modulation of the Ca2+ influx, C6 glioma cells were stably transfected with alpha o1 cDNA. Expression of the Go1 alpha protein was checked by Bordetella pertussis toxin-catalyzed ADP-ribosylation and Western blots using one- and two-dimensional gel analyses. Three clones were selected based on their degree of Go1 alpha expression. In alpha o1-transfected cells, cAMP accumulations, in response to isoproterenol or forskolin, were lower than in control cells. This inhibitory effect was a function of the amount of expressed Go1 alpha. In contrast, Go1 alpha expression was not followed by a significant inhibition of isoproterenol- or forskolin-stimulated adenylyl cyclase activities in particulate fractions. In C6 parental cells, 50-60% of the isoproterenol-induced cAMP accumulation was dependent on external Ca2+ concentration. This Ca(2+)-dependent cAMP accumulation was related to an induced transient Ca2+ influx. In transfected cells, expression of Go1 alpha inhibited the Ca2+ influx and the Ca(2+)-dependent component of isoproterenol-induced cAMP accumulation. In conclusion, beta-adrenergic agonists stimulate an entry of Ca2+ which exerts a positive feedback on cAMP production, and Go1 alpha blocks this positive feedback by inhibiting the Ca2+ influx.

Divergent actions of prostaglandins-E2 and -F2 alpha on the regulation of insulin-like growth factor-binding protein-3 in luteinized granulosa cells.

Insulin-like growth factor (IGF)-I synergizes with gonadotropin to further stimulate ovarian steroidogenesis. In contrast, the IGF-binding proteins (IGFBPs) produced by granulosa cells have been shown to antagonize the stimulatory actions of the IGFs and gonadotropins. The purpose of the present study was to examine the effects on IGFBP-3 production of prostaglandin (PG)-E2, a compound known to stimulate luteal function and prevent/delay luteal regression (a luteotropic compound), and PGF2 alpha, a compound known to be luteolytic. PGF2 alpha significantly stimulated IGFBP-3 production to 2.6-fold of control (P < 0.05) while PGE2 attenuated its production to half of control (P < 0.05). In contrast to the effects of IGFBP-3, PGE2 stimulated progesterone production to 8-fold of control (P < 0.05) while PGF2 alpha had no effect. Possible mechanisms of action of PGE2 and PGF2 alpha were also examined. PGE2, but not PGF2 alpha, stimulated cAMP accumulation which has been previously shown to inhibit IGFBP-3 production. PGF2 alpha is suspected to act via activation of protein kinase-C. However, a phorbol ester did not mimic PGF2 alpha's action toward IGFBP-3. This study demonstrated that PGE2 and PGF2 alpha conversely modulate IGFBP-3 production. Since IGFBPs have been shown to antagonize gonadotropin and IGF actions, this action of the prostaglandins may impact on the synergism between IGFs and gonadotropin necessary for follicular growth and luteal function.

Effects of Mono(2-Ethylhexyl) Phthalate, a Testicular Toxicant, on Follicle-Stimulating Hormone Binding to Membranes from Cultured Rat Sertoli Cells1

The widely used plasticizer di(2-ethylhexyl) phthalate (DEHP) is a male reproductive toxicant. Its toxicity has been shown to be due primarily to the action of its metabolite mono(2-ethylhexyl) phthalate (MEHP) on Sertoli cells. We have previously shown that at least one of the sites of action of MEHP on the Sertoli cell is the cAMP second messenger system. MEHP inhibits the ability of FSH but not isoproterenol, forskolin, or cholera toxin to stimulate cAMP accumulation in cultured Sertoli cells in a dose- and time-dependent manner. To further characterize this effect of MHP, we prepared a light membrane fraction from control and MEHP-treated Sertoli cells cultured from 18-day-old Fischer 344 rats and measured FSH binding in a radioligand receptor assay using 125I-labeled human FSH (125I-hFSH). MEHP inhibited FSH binding when preincubated with Sertoli cells in culture but not when added simultaneously with 125I-hFSH to the purified membrane preparation. Attenuation of FSH binding was evident after a 3-h preincubation with 100 microM MEHP (18%) and was maximal after 15-24 h of preincubation (70-90%). Preincubation of Sertoli cells for 24 h with 100 microM DEHP had no effect on FSH binding. Half-maximal inhibition occurred at approximately 0.1 microM MEHP. Scatchard analysis indicated a four-fold decrease in FSH affinity with no change in receptor concentration. Exposure of Sertoli cells to MEHP amplified the attenuating effect of guanosine triphosphate (GTP) on FSH binding, suggesting that the action of MEHP may be at the level of the GTP-binding protein that couples the FSH receptor to the adenylate cyclase catalytic subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasoactive properties of a parathyroid hormone-related protein in the rat aorta

Parathyroid hormone-related protein (PTHrP) or its mRNA transcripts have been documented in a variety of normal tissues, including vascular smooth muscle. A synthetic amino-terminal fragment of PTHrP, PTHrP(1–34), was tested a) for its vasoactive properties in a perfused rat aorta and b) its ability to stimulate cAMP accumulation in cultured rat aorta smooth muscle cells; PTHrP(1–34) was a potent vasodilator and increased cAMP accumulation. The results confirm the potent vasorelaxant properties of PTHrP(1–34), and show that the peptide stimulates adenylate cyclase in vascular smooth muscle cells per se, suggesting, but not proving, linkage of cAMP accumulation to vasorelaxation.

Okadaic acid induces both augmentation and inhibition of beta 2-adrenergic stimulation of cAMP accumulation in S49 lymphoma cells.

To address the role of protein phosphatases in regulating hormonal responses in mammalian cells, we investigated the effects of okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A, on epinephrine and prostaglandin E1 stimulation of cAMP accumulation and adenylyl cyclase in S49 WT and kin- lymphoma cells. Depending on the dose and time of okadaic acid pretreatment of both cell lines, there were two distinguishable effects on cAMP accumulation, an augmentation and an inhibition. The augmentation occurred rapidly (t1/2 < 1 min), was maximal with 3 microM okadaic acid, and was observed with concentrations of okadaic acid as low as 0.3 microM. Prolonged (t1/2 of 5-15 min) pretreatment of cells with okadaic acid caused an inhibition of epinephrine-stimulated cAMP accumulation, which was characterized by a 2-3-fold increase in the EC50 for the response to epinephrine. The EC50 for the okadaic acid-mediated inhibition was similar to that for the augmentation. In assays of adenylyl cyclase in membrane fractions prepared from okadaic acid-pretreated cells the inhibitory, but not the stimulatory, effects of okadaic acid pretreatment were observed. The data demonstrate that protein phosphatases play an important role in regulating adenylyl cyclase and suggest that cAMP-dependent protein kinase is not involved in either of its actions.

Parathyroid hormone (PTH) stimulates adrenocorticotropin release in AtT-20 cells stably expressing a common receptor for PTH and PTH-related peptide.

Complementary DNA encoding a rat bone PTH/PTHrP receptor was stably expressed in the murine corticotroph cell line, AtT-20. Several clones, expressing variable numbers of PTH/PTHrP receptors, were developed. In contrast to the relatively low binding affinity (apparent Kd = 15 nM) observed in COS-7 cells transiently expressing the PTH/PTHrP receptor, all AtT-20 stable transfectants bound [Nle8,18,Tyr34]bPTH(1-34)NH2 (NlePTH) with an affinity that was indistinguishable from that observed in ROS 17/2.8 cells expressing native PTH/PTHrP receptors. Additionally, NlePTH dramatically increased cAMP accumulation and ACTH release in AtT-20 cells expressing the PTH/PTHrP receptor with an ED50 of 0.6 +/- 0.3 and 0.3 +/- 0.1 nM, respectively. The high binding affinity and the high efficacy of NlePTH in stimulating cAMP accumulation and ACTH release indicate that the PTH/PTHrP receptor is efficiently coupled to the intracellular signalling system responsible for stimulation of ACTH release in AtT-20 cells. No additivity of cAMP accumulation or of ACTH release was observed when these cells were treated with maximally active concentrations of both NlePTH and CRF. This suggests that the receptors for both of these hormones share the same intracellular effectors, and that intracellular signaling in AtT-20 cells is not compartmentalized. Additionally, the ability of NlePTH to stimulate ACTH release in AtT-20 cells, a function that is normally performed by CRF, demonstrates promiscuity between activated receptors and distal biological functions.

Interleukin-1 activates protein kinase A and stimulates adrenocorticotropic hormone release from AtT-20 cells.

Studies from this and other laboratories have shown that interleukin-1 (IL-1) stimulates ACTH secretion directly from AtT-20 cells. The present studies were conducted to determine the signal transduction mechanisms activated by IL-1 to stimulate ACTH release. IL-1 significantly (P < 0.05) elevated ACTH release after incubation periods of 4, 8, and 24 h. IL-1-induced ACTH release was not additive to that of CRF, cholera toxin, 8-bromo-cAMP, or forskolin. In contrast, IL-1 and the phorbol ester phorbol 12-myristate 13-acetate together produced a greater increase (P < 0.05) in ACTH release than either agent alone. IL-1 did not stimulate cAMP accumulation at any time period between 5 min and 24 h and did not affect cAMP accumulation induced by CRF, cholera toxin, or forskolin. The lack of additivity between IL-1 and CRF, cholera toxin, 8-bromo-cAMP, and forskolin suggests that IL-1 stimulates ACTH release by a pathway that shares some common step(s) with CRF. Because IL-1 did not affect cAMP accumulation, the effect of IL-1 on protein kinase A (PKA) was investigated. IL-1 began to increase (P < 0.05) PKA activity at 15 min and remained elevated for 2 h before returning to control levels. IL-1 stimulation of PKA and the lack of additivity between IL-1 and CRF, forskolin, and cholera toxin indicate that PKA is the intracellular mediator used by IL-1 to stimulate ACTH release in AtT-20 cells.

Deglycosylated human chorionic gonadotropin (hCG) antagonizes hCG stimulation of 3',5'-cyclic adenosine monophosphate accumulation through a noncompetitive interaction with recombinant human luteinizing hormone receptors.

In the rat, the antagonistic properties of deglycosylated (dg) gonadotropins in vitro are characterized by high affinity receptor binding but impaired ability to stimulate cAMP accumulation. In human, the functional role of N-linked sugars in human CG (hCG) action is unclear because of the unavailability of totally deglycosylated hCG and because of the difficulty involved in obtaining human gonadal tissues. We have recently prepared completely deglycosylated hCG using site-directed mutagenesis and expressed functional human LH (hLH) receptors using cloned complementary DNA. Since hLH receptor shows distinct ligand specificity from that of rat LH receptor, we examined binding kinetics and signal transduction of recombinant dg-hCG using recombinant hLH receptors. In embryonic human kidney cells (293) transfected with hLH receptor complementary DNA, 125I-hCG binding to its receptor was studied in the presence of varying amounts of unlabeled dg-hCG or wild type (WT)-hCG. Lineweaver-Burk analysis of the binding kinetics showed that the displacement of 125I-hCG by dg-hCG was noncompetitive whereas that seen for WT-hCG was competitive. The noncompetitive nature of dg-hCG binding was further confirmed using rat LH receptors present in testis membrane preparations. After preincubation of LH receptor-expressing 293 cells with WT-hCG, inclusion of 125I-hCG competitively displaced WT-hCG. In contrast, preincubation with dg-hCG prevented subsequent 125I-hCG binding to human LH receptor for at least 46 h. WT-hCG caused a dose-dependent increase in cAMP accumulation in the 293 cells with an ED50 of 10 ng/ml. However, dg-hCG was ineffective in inducing cAMP production with a maximal effect of only 12% of that stimulated by WT-hCG. In the presence of increasing doses of dg-hCG, stimulation of cAMP by WT-hCG was antagonized in a dose-dependent manner. In contrast, forskolin stimulation of cAMP was not antagonized by dg-hCG, indicating receptor-mediation of dg-hCG action. Similar to binding studies, preincubation with dg-hCG also dose-dependently blocked the subsequent stimulatory effect of WT-hCG on cAMP production. Thus, the noncompetitive binding of dg-hCG to hLH receptors and its antagonism of hCG stimulation of cAMP accumulation suggest that dg-hCG is an irreversible receptor blocker with unique antagonistic properties.

Hydrogen peroxide evokes antisteroidogenic and antigonadotropic actions in human granulosa luteal cells.

The nature of the luteolysin in humans is unknown. Hydrogen peroxide (H2O2), notably released by activated leukocytes, is generated in the rat corpus luteum at luteolysis and evokes luteolytic-like effects in rat luteal cells. We, therefore, evaluated the actions of H2O2 in human luteinized granulosa cells. After 2 days of preculture with low levels of hCG, human granulosa luteal cells were placed in suspension culture for 1 h in the presence of isobutylmethylxanthine (100 microM). A 60-min challenge with hCG evoked dose-dependent stimulation of cAMP and progesterone production. H2O2 dose-dependently inhibited progesterone production (ED50, 50-100 microM) in the absence or presence of hCG and blocked hCG-stimulated cAMP accumulation. Inhibition of progesterone synthesis by H2O2 was near maximal within 5 min, whereas inhibition of cAMP accumulation was not evident until 60 min. Cell viability was unaffected by H2O2, and inhibition of cAMP was reversible, but inhibition of steroidogenesis was long-lasting. Progesterone production stimulated by 8-bromo-cAMP, 22-hydroxycholesterol, and pregnenolone was inhibited by H2O2 as was androstenedione-dependent estradiol production. These findings indicate that H2O2 blocked progesterone synthesis by inhibition of cholesterol side-chain cleavage cytochrome P450, 3 beta-hydroxysteroid dehydrogenase, aromatase, and/or 17 beta-hydroxysteroid dehydrogenase. While H2O2 blocked stimulation of cAMP accumulation in response to hCG and cholera toxin, this same response produced by forskolin or aluminum fluoride was unaffected by H2O2. Thus, H2O2 appears to uncouple LH (hCG) receptors by interruption of G-protein-dependent activation of adenylate cyclase. In summary, H2O2 evokes effects in isolated human granulosa luteal cells that are associated with luteal regression, which raises the interesting possibility that H2O2 may serve a role as a mediator of this process like that in the rat.

Prostaglandins Stimulate Serotonin Acetylation in Chick Pineal Cells: Involvement of Cyclic AMP‐Dependent and Calcium/Calmodulin‐Dependent Mechanisms

The effects of prostaglandins (PGs) on the activity of the rate-limiting enzyme of melatonin biosynthesis, arylalkylamine-N-acetyltransferase (NAT) were investigated on primary cultures of dispersed chick pineal cells. In indomethacin-treated cells, PGs caused a four-fold increase in NAT activity. This response was associated with an eight-fold increase in cyclic AMP (cAMP) levels. The potency order of PGs was the same for NAT and for cAMP responses (PGE1 > PGE2 > PGF2 alpha >> cloprostenol). However, each PG tested was 30- to 200-fold more potent to increase NAT activity than to stimulate cAMP accumulation. As a result, half-maximal stimulation of NAT by PGs was not associated with an increase in cAMP levels. Half-maximal stimulation of NAT by PGE1 was highly sensitive to inhibition by a calcium/calmodulin antagonist (W-7). In contrast, maximal stimulation of NAT by PGE1 as well as stimulations evoked by either forskolin or 8-bromo-cAMP were poorly sensitive to inhibition by W-7. These results indicate that an increase in cAMP levels may be responsible for the maximal stimulation of NAT evoked by PGs, whereas half-maximal stimulation of NAT by PGs would rely principally on a calcium/calmodulin-dependent mechanism.

Epidermal Growth Factor Produces Inotropic and Chronotropic Effects in Rat Hearts by Increasing Cyclic AMP Accumulation

Previously we have shown that epidermal growth factor (EGF) stimulates cardiac adenylyl cyclase and increases cAMP accumulation in the rat heart (Nair et al., Biochem. J. 264, 563-571, 1989). Moreover, we have shown that the stimulation of adenylyl cyclase by EGF in heart is mediated via activation of the stimulatory GTP binding regulatory protein Gs alpha (Nair et al., J. Biol. Chem. 265, 21317-21322, 1990). Since cAMP increases the beating rate of hearts, studies were performed to investigate the effects of EGF on mechanical function of the heart and the role of cAMP in mediating the cardiac effects of EGF. In isolated perfused rat hearts EGF (15 nM) decreased perfusion pressure, increased ventricular contractility and heart rate in a manner similar to that observed with the beta-adrenergic receptor agonist isoproterenol (10 nM). In the presence of the adenosine A1 receptor agonist (-)-N6-(R-phenylisopropyl)-adenosine (PIA, 100 nM) which via activation of the inhibitory GTP binding protein Gi inhibits adenylyl cyclase, the effects of EGF on cAMP accumulation in the heart were markedly attenuated. PIA also decreased the ability of EGF and isoproterenol to alter cardiac contractility and beating rate. However, PIA did not attenuate the increase in heart rate and contractility induced by the alpha-adrenergic agonist phenylephrine which does not stimulate cAMP accumulation in the heart. These data suggest that EGF alters cardiac function by increasing cellular cAMP accumulation.

Intracellular Signaling Events Associated with the Induction of DNA Synthesis in Human B Lymphocytes: II. Different Pathways Triggered by IL-2 and IL-4

In attempts to detect associations between early signaling events triggered by interleukins and the induction of DNA synthesis, inhibitors of various second messenger pathways were tested for their effects on IL-2- and IL-4-elicited mitogenesis in preactivated human B lymphocytes. Inhibitors of phosphoinositidase C and of InsP 3-induced calcium release suppressed IL-4- but not IL-2-mediated proliferation. The response to both lymphokines was also impaired by an inhibitor of the calcium/calmodulin complex and was modulated by variations of the [Ca 2+] i. PKC inhibitors and PK-C depletion did not significantly alter the response to IL-2 and IL-4. The response to IL-2, but not to IL-4, was inhibited by cAMP analogues or by agents that raise cAMP. In contrast, IL-4, but not IL-2, stimulated cAMP accumulation in activated B cells. Taken together, these observations indicate that IL-2 and IL-4 use different signaling pathways to induce the G1 → S transition in these cells and suggest that the IL-4 inhibition of the B cell response to IL-2 may result from its effect on cAMP generation.