Purine modulation of the hormonal response of the rat Sertoli cell in culture

To investigate the possibility that purines modulate the response of testicular cells to gonadotropin, binding of adenosine analogs and biological responses to adenosine were evaluated in Sertoli cell-enriched cultures. The adenosine analog cyclohexyladenosine bound specifically to a crude particulate fraction prepared from such cultures. Binding was saturable, and steady state studies showed the presence of a high affinity binding site (Kd = 2.1 +/- 0.3 nM; n = 4) and a receptor density of 200-300 fmol/mg protein. The bound radioactive ligand was displaced by N6-phenylisopropyladenosine (PIA), adenosine, and methylisobutylxanthine. In addition to the presence of a specific binding site, purines modulated the biological function of the Sertoli cell. The adenosine analog PIA inhibited both the FSH-dependent cAMP response and the FSH-stimulated androgen aromatization. Under all experimental conditions, the IC50 of PIA was 1-3 nM, and maximal effects were observed at 10-100 nM PIA. Adenosine itself inhibited the FSH-dependent response of the Sertoli cell, but was less potent than PIA. In addition, purine inhibition of the FSH response was antagonized by methylisobutylxanthine, while the nonxanthine phosphodiesterase inhibitor Ro 20-1724 [4-(3-butoxy-4-methoxybenzyl)2-imidazolidinone] was without effect. Purine modulation was evident not only when cells were stimulated with FSH, but also when the androgen aromatization was augmented by the beta-adrenergic agonist isoproterenol, cholera toxin, and forskolin. On the contrary, the purines had no effect when cells were stimulated with (Bu)2cAMP. The data reported are consistent with the presence of purine inhibitory receptors in Sertoli cell-enriched cultures and show that purines can regulate the response of the immature Sertoli cell in vitro.     

Inhibition of alpha 2-adrenergic agonists on AVP-induced cAMP accumulation in isolated collecting tubule of the rat kidney

A microradioimmunoassay for cAMP was developed in order to analyse the effects of alpha-adrenergic agonists on vasopressin (AVP)-induced cAMP cell accumulation in single pieces of microdissected medullary (MCT) and cortical (CCT) rat collecting tubules. Under the experimental conditions chosen (4 min of incubation in the presence of a phosphodiesterase inhibitor), no cAMP could be detected either in the bathing solution or in non-stimulating samples of tubule. In MCT, 10(-6) M AVP stimulated cAMP generation up to 128.3 +/- 9.0 (SEM) fmoles per mm of tubule per 4 min, N = 11. The response was dose-dependent with a KA value below 10(-10) M AVP. The addition of norepinephrine (NE) (10(-5) M in the presence of propranolol) suppressed the larger part of the response to AVP (from 92% with 2 X 10(-11) M AVP to 76% with 10(-6) M AVP); the addition of 10(-7) M NE still reduced by 59% the MCT response to 10(-10) M AVP (26.2 +/- 5.9 vs. 64.0 +/- 6.4 fmoles/mm, N = 3). In CCT, 10(-5) M NE reduced by 84% the cAMP generation induced by 10(-10) M AVP (8.8 +/- 2.0 vs. 54.2 +/- 3.5 fmoles/mm, N = 3). This inhibitory action of NE against the AVP effect in CCT was mimicked by 10(-7) M clonidine; in MCT it was suppressed by phentolamine and yohimbine, but not by prazosin, suggesting that alpha 2-adrenoreceptors are involved. On the other hand, the addition of the alpha-agonists to the incubation solution produced no inhibition of the cAMP cell accumulations induced by glucagon, calcitonin and isoproterenol in CCT, or glucagon in MCT, an observation demonstrating that alpha 2-adrenergic agonists selectively inhibit vasopressin-dependent cAMP generation by these nephron segments.     

Effects of adenosine analogs on glucagon-stimulated adenosine 3',5'-monophosphate formation in Sertoli cell cultures from immature rats

In the present study, we have examined the effects of two adenosine analogs, (-)N6-(R)phenyl-isopropyl-adenosine (PIA) and 2-chloro-adenosine, on glucagon- and FSH-stimulated cAMP production in Sertoli cell cultures isolated from immature (19-day-old) rats. Both FSH and glucagon caused a 5- to 10-fold stimulation of cAMP levels in the spent media from Sertoli cell cultures during an 18-h incubation. Addition of 1 microM PIA significantly inhibited both FSH- and glucagon-stimulated cAMP levels. In the presence of a maximal concentration of glucagon (2.5 micrograms/ml), PIA caused a concentration-dependent inhibition of cAMP formation, and the concentration of PIA causing half-maximal inhibition of cAMP formation (IC50) ranged from 0.5-1 nM. When Sertoli cells were incubated with increasing concentrations of glucagon (1.28 ng/ml to 4.00 micrograms/ml) in the absence and presence of either PIA (1.0 microM) or 2-chloro-adenosine (10.0 microM), the responses to glucagon, measured as cAMP formation, were almost completely abolished. 1-Methyl-3-isobutylxanthine (MIX), a well known inhibitor of cAMP phosphodiesterase activity, is also an inhibitor of adenosine binding to receptors on the cell membrane. When Sertoli cells stimulated with glucagon (2.5 micrograms/ml) were incubated in the absence and presence of MIX (0.1 mM) and increasing concentrations of PIA (0.025-10,000 nM), the presence of MIX reduced the inhibitory activity of PIA by almost 2 orders of magnitude (IC50 without MIX, 0.5 nM; IC50 with MIX, 20 nM). Thus, the present study shows that adenosine analogs inhibit agonist-stimulated cAMP formation in cultured Sertoli cells, and that MIX reduces this effect. This indicates that cultured Sertoli cells from immature rats contain A1-receptors for adenosine mediating inhibitory effects on adenylate cyclase.     

Extensive but reversible depletion of ATP via adenylate cyclase in rat adipocytes.

In adipocytes, adenylate cyclase is positively regulated by beta-adrenergic agents and negatively regulated by adenosine. Incubation of adipocytes with adenosine deaminase relieves the inhibition of adenylate cyclase by destroying the adenosine that the cells release into the medium. When adipocytes are incubated with adenosine deaminase and the beta-adrenergic agent isoproterenol, most of their ATP is converted to AMP in 5 min. Either isoproterenol or adenosine deaminase alone has little or no effect. In the additional presence of the phosphodiesterase inhibitor 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one (Ro 20-1724) cAMP accumulates instead of AMP. Under these conditions, cAMP represents 40-50% of the total intracellular adenine nucleotides, and ATP only 5%. N6-(L-2-phenylisopropyl)adenosine, a deaminase-resistant adenosine agonist, prevents beta-adrenergic stimulation. 8-(p-Sulfophenyl)theophylline and 3-isobutyl-1-methylxanthine are both adenosine antagonists that can replace the deaminase in permitting beta-adrenergic stimulation of adenylate cyclase, but only the latter also inhibits the phosphodiesterase and causes accumulation of cAMP. When the ATP-depleted adipocytes are washed with fresh medium, the nucleoside triphosphate level can be restored within 5 min. The ATP-restored adipocytes can respond rapidly to a second dose of isoproterenol and adenosine antagonist. These findings point out the important role of adenosine in controlling adenylate cyclase activity and the possible involvement of adenylate cyclase in the control of energy flow in rat adipocytes.     

Cholinergic control of cyclic nucleotide metabolism in human thyroid cells

In the presence of Ro 20-1724, a selective inhibitor of cyclic nucleotide phosphodiesterase, carbamylcholine increases cAMP and cGMP levels in human thyroid cells in primary culture. The increase of cAMP exhibited at concentrations of carbamylcholine between 10 fM and 10 pM, is dose- and time-dependent, it is maximum after 30 min and is abolished after 60 min. At higher carbamylcholine concentration (10 microM), cAMP increases rapidly, becoming maximum after 15 min, but returns to unstimulated values after 30 min. The increase of cGMP is also dose-dependent (0.1 nM-10 microM); it reaches the maximum after 30 min and returns to unstimulated values after 120 min. A significant increase of phosphodiesterase activity is observed at 10 microM carbamylcholine. Atropine, a muscarinic receptor antagonist, blocks carbamylcholine effects on both cAMP and cGMP production without affecting the thyrotropin-induced cAMP accumulation. Hexamethonium, a nicotinic receptor antagonist does not affect the cholinergic effects. In the presence of Ro 20-1724, 10 microM carbamylcholine significantly inhibits the effect of thyrotropin on cAMP production, while the combined addition of low doses of carbamylcholine and thyrotropin (0.1 nM and 10 pM, respectively) results in an additive effect on cAMP levels. Inhibition of thyrotropin activity on cAMP production, similar to that exerted by 10 microM carbamylcholine is produced by increasing free intracellular calcium; this inhibition is relieved by using a calmodulin-sensitive phosphodiesterase inhibitor, M and B 22948 at 50 microM dose. High concentrations (10 microM) of carbamylcholine increase the adenylate cyclase activity, without any significant effect on the thyrotropin-induced activation of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of two phosphodiesterase inhibitors on fat cell metabolism

The various effects of the phosphodiesterase inhibitor D-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (RO-20-1724) on fat cell metabolism were examined and compared to those obtained with 1-methyl-3-isobutylxanthine (IBMX). Like IBMX, RO-20-1724 increased the rate of lipolysis, suppressed the incorporation of glucose into fat, did not increase the basal rate of cAMP production, but induced fast linear accumulation of the nucleotide when combined with a lipolytic agent. The time scale and the effective concentrations of both reagents in exerting these effects were similar. Both inhibitors were also equally potent in inhibiting the low Michaelis-Menten constant (Km) cAMP phosphodiesterase activity in fat cell homogenate. A fundamental difference between both inhibitors however did exist. Whereas the addition of IBMX abolished the ability of insulin to inhibit lipolysis as previously reported, insulin is fully functional in inhibiting the increased rate of lipolysis in the presence of RO-20-1724. Also, RO-20-1724 does not interfere with the ability of insulin to inhibit fully cholera toxin-mediated lipolysis. It is concluded that under these conditions the antilipolytic effect of insulin may be produced without demonstrable activation of cAMP phosphodiesterase. The effects of the methylxanthines in abolishing insulin action probably relates to interference with insulin-dependent intracellular enzymic activity other than cAMP phosphodiesterase.     

Long term treatment with adenosine analogs modifies the responsiveness of immature rat Sertoli cell in culture

A1 inhibitory adenosine receptors are present in cultured Sertoli cells. Activation of these receptors by short term exposure to adenosine agonists attenuates the adenylate cyclase activity and reduces FSH stimulation of androgen aromatization to estrogen. In the present study it was investigated how long term activation of the adenosine inhibitory system affects the responsiveness of the Sertoli cell. Sertoli cells from 15- to 17-day-old Sprague-Dawley rats were incubated with medium containing adenosine deaminase (1 IU/ml) in the presence or absence of 100 nM N6-2-phenyl-isopropyl-adenosine (PIA) for 24-48 h. At the end of this pretreatment medium was changed, and cell responsiveness was measured in terms of cAMP and estrogen production. In control cells, FSH-stimulated cAMP and estradiol production were inhibited by PIA, with an EC50 of 0.70 +/- 0.13 nM. This inhibitory effect was reduced in cells that had been pretreated for 24-48 h with 100 nM PIA. The PIA concentration-response curve of pretreated cells was shifted to the right, with a 4-fold increase in the EC50. Similar effects were also evident when adenosine itself or nonmetabolizable adenosine analogs other than PIA were used in the pretreatment. In addition to these changes in the inhibitory responses, PIA pretreatment increased the response of the Sertoli cell to FSH and forskolin in terms of both cAMP accumulation and estradiol production. Potentiation of the hormonal response was due to an increase in basal and maximal stimulation without significant changes in the total stimulation. This effect was dependent on the concentration of PIA used during the pretreatment. The increase in estradiol production was also evident when cells were stimulated with (Bu)2cAMP, suggesting that adenosine analog pretreatment affects steps distal to cAMP accumulation. Moreover, the responses to both the PIA inhibitory signal and FSH stimulation were restored to control levels when pretreated cells were incubated in fresh medium in the absence of PIA for 24 h. The long term PIA effects were also blocked by pretreatment in the presence of the A1 receptor antagonist 8-[4-([([ (2-amino-ethyl)amino]carbonyl)methyl]oxy)phenyl]1,3- dipropylxanthine. These results indicate that the A1 adenosine system present in the Sertoli cell becomes refractory after prolonged exposure to adenosine analogs. Furthermore, PIA pretreatment produced a potentiation of the Sertoli cell response to stimulatory signals by affecting several steps of the cAMP-dependent pathway.     

Adenosine stimulates adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate accumulation in rat pinealocytes: evidence for a role for adenosine in pineal neurotransmission

Adenosine produces a concentration-dependent increase in pinealocyte cAMP (EC50, approximately 0.3 nM) and cGMP accumulation (EC50, approximately 0.7 nM). Maximal increases in both nucleotides are evident 10 min after treatment; 1 h later values return to pretreatment levels. Concentration-dependent effects on cAMP are also observed with N6-(L-2-phenylisopropyl)adenosine (EC50, approximately 0.75 nM), 5'-N-ethylcarboxy aminoadenosine (EC50, approximately 0.75 nM), and 2-chloroadenosine (EC50, approximately 2.0 nM); the EC50 values for stimulation of cGMP with these agents are higher by a factor of 2-10. In the case of 5'-N-ethylcarboxy amidoadenosine, the concentration-response curve is biphasic, with a significant effect evident within the range of 1-100 pM. The stimulatory nature of this response and the relative potency of the agonists tested are consistent with the involvement of an A2-like adenosine receptor. Comparison of adenosine and the selective beta-adrenergic agonist isoproterenol indicated that their maximal EC50 values were generally similar. Studies with antagonists revealed that both 8-(p-sulfophenyl)theophylline (1 microM) and the xanthine amine congener (8-[4-[[[(2-aminoethyl)carbonyl]methyl]oxy]phenyl]1,3- dipropylxanthine (1 microM) inhibited the effects of adenosine (1 nM to 1 microM), but xanthine amine congener was more potent; the latter was markedly effective at 0.1 nM, whereas 8-(p-sulfophenyl)theophylline was nearly ineffective at this concentration. It was also determined that pineal cells generate extracellular adenosine from extracellular ATP. ATP is thought to be released along with catecholamines during neurotransmission. Hence, these studies support the view that adenosine could participate in the transsynaptic regulation of pineal function.     

Cholinergic-adrenergic antagonism in the induction of tachyarrhythmia by electrical stimulation in isolated rat atria

Atrial tachyarrhythmias (AT) are the most common cardiac rhythm disturbance. In the present study, we analyzed the cholinergic-adrenergic interaction in the in vitro induction of cholinergic-dependent tachyarrhythmia by high-frequency electric stimulation. Tachyarrhythmia was evoked in isolated rat right atria by trains of electric stimuli. Atrial response was expressed as the tachyarrhythmia induction index (ATI, i.e. the fraction of applied trains that resulted in arrhythmia induction). ATI was reversibly increased by 0.6 microM carbachol (CCh), which also decreased atrial spontaneous rate (ASR). In contrast, 10 nM isoproterenol (ISO), 100 microM tyramine and the phosphodiesterase inhibitor isobutyl-methylxanthine (IBMX, 100 microM) increased ASR and decreased ATI. Amiodarone (AMI, 10 microM) reduced ATI in the presence and absence of CCh. Further CCh addition restored ATI in atria treated with either IBMX or AMI, but not when both compounds were present. Increase in ATI by CCh in atria pretreated with IBMX plus ISO was significantly attenuated by 3 mM NaF. The antagonism between cholinergic muscarinic and beta-adrenergic receptor stimulation (the former facilitating and the latter inhibiting tachyarrhythmia installation) possibly involves regulation of the phosphorylation status of adenosine cyclic 3'-5'-monophosphate (cAMP)-dependent protein kinase substrates. Additionally, cAMP-independent, AMI-sensitive mechanism stimulated by CCh (possibly muscarinic-dependent K(+) current activation) seems to contribute to AT facilitation.     

Antilipolytic action of insulin: role of cAMP phosphodiesterase activation

Exposure of 3T3-L1 adipocytes to 1 nM insulin for 10 min results in activation of particulate cAMP phosphodiesterase and suppression of lipolysis stimulated by 10 nM isoproterenol. When lipolysis was increased by cilostamide, a selective inhibitor of the particulate phosphodiesterase, the antilipolytic effect of insulin was not observed. Insulin did suppress lipolysis stimulated by Ro 20-1724, an inhibitor of soluble cAMP phosphodiesterase activity. Cilostamide did not interfere with insulin stimulation of glucose uptake, nor did it have any direct effect on cAMP-dependent protein kinase. Thus, inhibition of particulate but not soluble cAMP phosphodiesterase blocked the antilipolytic effect of insulin. Our findings support the idea that insulin inhibits lipolysis, perhaps in large part by activating particulate "low Km" cAMP phosphodiesterase, which seems to be functionally closely coupled with the hormone-sensitive lipase-regulatory system influencing primarily a pool of cAMP utilized by the relevant protein kinase.     

Effect of forskolin, isoproterenol and IBMX on angiotensin converting enzyme and cyclic AMP production by cultured bovine endothelial cells

The production of angiotensin converting enzyme (ACE) is known to be increased by glucocorticoids, thyroid hormones and converting enzyme inhibitors. We have recently reported that active cAMP analogues also stimulate production of the enzyme. The effect of stimulation of adenylate cyclase in cultured endothelial cells or of phosphodiesterase inhibition on ACE production was therefore evaluated. The phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX) (10(-4) M), produced 10.5 +/- 1.3 and 1.3 +/- 0.1 (P less than 0.01 and P greater than 0.1) fold increases in extracellular and cellular cAMP levels and a 1.55 +/- 0.10 (P less than 0.0001) fold increase in ACE accumulation. The adenylate cyclase stimulator, forskolin (0.01-10 microM), acutely stimulated cellular cAMP accumulation in a dose-dependent manner, reaching a 2.8 +/- 0.1-fold increase at 10 microM. After 48 h exposure to 10 microM forskolin, significant increases in cellular (1.90 +/- 0.38-fold increase, P less than 0.0001) and extracellular cAMP (2.35 +/- 0.26-fold increase, P less than 0.0001) were also observed but ACE accumulation was unchanged (108 +/- 10% of control, P greater than 0.5). The beta-adrenoceptor agonist, isoproterenol (1-1000 nM), acutely stimulated cellular cAMP accumulation, with a threshold effect at 10 nM, an ED50 of approximately 30 nM, and a plateau effect of 2.0 +/- 0.13-fold increase by 100 nM. After 48 h exposure to isoproterenol (1 microM), extracellular cAMP levels were increased significantly (1.68 +/- 0.33-fold increase, P less than 0.01) but ACE production was slightly inhibited (83 +/- 7% of control, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine inhibits prolactin and growth hormone secretion in a clonal pituitary cell line

Although purine nucleosides have been shown to regulate the secretion of several peptide and steroid hormones, effects on pituitary hormone release have not been reported. We show here that in the clonal GH4C1 pituitary cell line maximal concentrations of adenosine (greater than or equal to 50 microM) inhibited PRL and GH secretion by 40%. Adenosine deaminase abolished the inhibitory effect of adenosine but not that of SRIF or (-)N6(R-2-phenylisopropyl)adenosine (PIA), a nonhydrolyzable adenosine analog. Furthermore, this enzyme increased basal secretion by 50%, and analysis of the incubation medium by HPLC demonstrated that the cells secreted biologically effective concentrations of adenosine. These results indicate that adenosine produced in culture tonically inhibits hormone release. In other target cells, adenosine inhibition is mediated by two types of binding sites: an extracellular Ri-site requiring an intact ribose moiety or an intracellular P-site requiring an intact purine ring. Four lines of evidence indicate that in GH4C1 cells, adenosine acts at an Ri-site. PIA, an Ri-site-specific agonist, was a potent inhibitor of hormone release (ED50 = 30 nM). Theophylline, an Ri-site antagonist, competitively inhibited the action of PIA (Ki = 2.4 microM). 3) 2'5'-Dideoxyadenosine, a P-site-specific agonist, did not inhibit PRL release even at a concentration of 1 mM. 4) Dipyridamole, an adenosine uptake inhibitor, did not reduce adenosine inhibition. In addition to its effect on basal secretion, PIA inhibited stimulation of hormone release by vasoactive intestinal peptide and TRH. PIA also reduced vasoactive intestinal peptide-stimulated cAMP accumulation by 75%, consistent with its action to inhibit adenylate cyclase via Ri receptors in other targets. Since PIA inhibition of PRL release and cAMP accumulation was not additive with the effects of SRIF and carbamyl choline, these inhibitors may act via a common rate-limiting step. Our results demonstrate that adenosine activates an Ri-type of adenosine receptor in GH4C1 cells and that the production of adenosine under normal culture conditions causes autocrine inhibition of secretion.     

Adenosine receptor-dependent modulation of inhibin secretion in cultured immature rat Sertoli cells

Inhibitory (A1) adenosine receptors that attenuate adenylate cyclase activity are present in cultured Sertoli cells. To investigate the possible effect of activating these receptors on the secretion of inhibin by the Sertoli cell, immature rat Sertoli cells were incubated for 24 h with follicle-stimulating hormone (FSH) in the absence or presence of the non-metabolizable, adenosine agonist phenyl-isopropyl-adenosine (PIA), and the accumulation of alpha-inhibin immunoreactivity was measured in the medium. Although devoid of effects when added alone, PIA inhibited the FSH-dependent secretion of alpha-inhibin in a concentration-dependent manner (ED50 = 1-1.5 nM). PIA treatment of the Sertoli cells also rendered the cells less sensitive to FSH in terms of alpha-inhibin secretion. The concentration-response curve to FSH was shifted to the right when cells were incubated in the presence of 100-1000 nM PIA. In contrast, dibutyryl cAMP stimulation of alpha-inhibin accumulation was unaffected by treatment with PIA, indicating that the site of PIA action is at the level of cAMP synthesis. These data provide experimental evidence of adenosine modulation of inhibin secretion by the Sertoli cell and suggest that adenosine may act as a local modulator within the pituitary-testicular axis.     

The vasopressin-sensitive adenylate cyclase in collecting tubules and in thick ascending limb of Henle's loop of human and canine kidney

The major tubular effects of [8-Arg]vasopressin (AVP) in regulation of renal water excretion are initiated by stimulation of adenylate cyclase (AdC) coupled with V2 receptors. We explored whether the AVP-sensitive AdC is present in both collecting tubules and the thick ascending limb of Henle's loop of human and canine kidney. In cortical collecting tubule (CCT) and medullary collecting tubules (MCT) of human kidney, AdC was markedly stimulated by AVP [maximum change from basal level (delta), +2700%] and the the nonhormonal stimulatory agent forskolin (delta, +2000%). In human CCT, the effects of both compounds were synergistic. In contrast, AVP had no effect on AdC in either the medullary (MAL) or cortical (CAL) segment of the thick ascending limb of Henle's loop of human kidney; AVP also did not stimulate AdC in CAL or MAL in the presence of forskolin. Similar to that in the human kidney, in the canine kidney, AdC in CCT and MCT was markedly stimulated by AVP and forskolin (delta, +1000%), but AVP had no effect on AdC in CAL and MAL of the canine kidney. In intact tubules dissected from dog kidney and incubated in vitro, AVP markedly increased cAMP accumulation in MCT. AVP also elicited a small but detectable increase in cAMP accumulation in MAL. From these observations, we conclude that AVP-sensitive AdC is well developed in collecting tubules, but that AVP-sensitive AdC is absent in MAL and CAL of human kidney. Likewise, in canine nephron, the AVP-sensitive AdC of MAL and CAL is rudimentary or very labile. These findings suggest that the unresponsiveness of the AdC-cAMP system to AVP in segments of the thick ascending limb of Henle's loop may be a factor that accounts for a relatively low maximum osmotic concentration of urine which can be achieved by human or canine kidneys.     

Cyclic AMP levels of C6 glioma cells treated with cis-dichlorodiammine platinum (cis-DDP)

Rat C6 glioma cells were cultured for 3-4 days in MEM supplemented with bovine serum. After 10 min incubation of cells with 0.075, 1.0 or 7.5 micrograms ml-1 cis-DDP the basal cAMP levels (7.87 +/- 0.4 pmoles mg-1 protein) were not affected. In the presence of a phosphodiesterase inhibitor, IBMX, an increase of cAMP occurred; the later was more pronounced in cis-DDP treated cells than in the controls. This suggests that both adenylate cyclase and cAMP-phosphodiesterase were proportionally influenced at this period and that the stimulatory effect of cis-DDP on AC could be demonstrated only when increased activity of PDE had been blocked by IBMX. At later time intervals (10 h-40 h), a 5- to 17-fold elevation of cAMP levels was observed even in the absence of IBMX. Pretreatment of the cells with cis-DDP significantly potentiated cAMP accumulation in response to NE alone and to cis-DDP plus NE could be prevented to a large extent by propranolol; in cis-DDP treated cells the propranolol protection was more effective, both in the absence and the presence of IBMX. The pretreatment of cells with an alpha-blocker, Regitin, did not significantly influence cAMP accumulation. The results indicate that the cis-DDP stimulated cAMP response to NE is mediated via an interaction with beta-adrenergic receptors. The late increase in cAMP content may be a mediator of the morphological changes in these cells following exposure to cis-DDP.     

Effect of dexamethasone on adenosine 3',5'-monophosphate content and phosphodiesterase activities in 3T3-L1 adipocytes

After incubation with 0.5 mM isobutylmethylxanthine, 1 microM dexamethasone, and 1 microM insulin for 72 h, 3T3-L1 cells acquire the phenotypic characteristics of mature adipocytes, including a hormone-sensitive particulate cAMP phosphodiesterase activity. In addition, adipocytes contain soluble cAMP and calmodulin-sensitive and -insensitive cGMP phosphodiesterase activities. After exposure of the differentiated cells to 1 microM epinephrine, cAMP content increased, reaching a maximum in 2-4 min, and then declined to the control level by 20 min. After incubation of adipocytes with 10 nM dexamethasone for 72 h, the initial increment in cAMP produced by epinephrine was not altered, but the decline in cellular cAMP to basal levels was delayed. Treatment with 10 nM dexamethasone prevented hormonal activation of particulate cAMP phosphodiesterase activity without altering basal activity (11). Soluble cAMP and calmodulin-sensitive and -insensitive cGMP phosphodiesterase activities were also reduced by exposure to 10 nM dexamethasone; higher concentrations were required to decrease basal particulate phosphodiesterase activities. Estradiol did not alter phosphodiesterase activities. Incubation of either undifferentiated (fibroblasts) or differentiated (adipocytes) 3T3-L1 cells with 1 microM dexamethasone for 48 h reduced cAMP and cGMP phosphodiesterase activities. After removal of dexamethasone, phosphodiesterase activities were restored to control levels in 4-6 days. The effects of dexamethasone on phosphodiesterase activities could in part account for the observed alterations in hormone-induced accumulation of cAMP in steroid-treated cells and for the permissive effects of glucocorticoids on certain cAMP-mediated processes.     

Adenosine inhibition of the hormonal response in the Sertoli cell is reversed by pertussis toxin

The rat Sertoli cell in culture expresses A1 inhibitory adenosine receptors. In this study, we have used pertussis toxin as a tool to characterize the mechanism of action of adenosine on these cells. Cells were preincubated for 18-24 h with pertussis toxin, and the responses to FSH and to the adenosine analog phenylisopropyladenosine (PIA) were measured by assaying cAMP accumulation. The effect of toxin on adenosine receptors was also evaluated by measuring binding of the adenosine agonist cyclohexyladenosine (CHA). The total number of specific CHA-binding sites was reduced 60-70% in membranes prepared from cells cultured for 24 h in the presence of pertussis toxin; the binding sites remaining after treatment displayed no apparent change in affinity for [3H]CHA. The effect of guanine nucleotides on CHA binding was also reduced after toxin pretreatment, but not abolished. PIA inhibited FSH-stimulated cAMP accumulation by 70-80%. Maximal inhibition was observed at a concentration of 10 nM PIA, and the ED50 of the dose-response curve was 1 nM. Pretreatment of the Sertoli cell with pertussis toxin completely blocked the PIA inhibition. The pertussis toxin effect was time and dose dependent. Reversal of the inhibition was observed after 6 h of treatment with a maximal dose of toxin (100 ng/ml). The dose of toxin producing a half-maximal effect was 10-30 ng/ml. In addition to this blockade of purine nucleotide inhibitory effects, exposure of the Sertoli cell to pertussis toxin concentrations ranging from 1-400 ng/ml consistently led to a potentiation of the FSH response measured as cAMP accumulation. In cell-free preparations (crude particulate fraction of the Sertoli cells, or sucrose gradient-purified plasma membranes), pertussis toxin catalyzed the incorporation of [32P]ADP ribose into a polypeptide with a molecular mass of 40-41 K. This peptide had electrophoretic mobility similar to that of a partially purified guanine nucleotide-binding protein (Gi). These data indicate that adenosine A1 inhibitory receptors are coupled to an inhibitory component (Gi) of adenylate cyclase. In the Sertoli cell, inhibitory and stimulatory signals interact in a bimodal regulation of adenylate cyclase and intracellular cAMP.     

Prostaglandin-E2 enhances EPO-mediated STAT5 transcriptional activity by serine phosphorylation of CREB

Erythroid colony formation in response to erythropoietin (EPO) stimulation is enhanced by costimulating the cells with prostaglandin-E2 (PGE2). The present study further analyzed the underlying mechanisms and demonstrated that EPO-mediated STAT5 transactivation in the erythroid AS-E2 cell line was enhanced 6-fold by PGE2 (10 microM), without affecting the STAT5 tyrosine phosphorylation or STAT5-DNA binding. Moreover, the PGE2-enhancing effect was independent of STAT5 serine phosphorylation. In AS-E2 cells STAT5 is constitutively phosphorylated on Ser780 (STAT5A) and EPO-dependently phosphorylated on Ser726/731 (STAT5A/STAT5B), but overexpression of STAT5 serine mutants did not affect STAT5 transactivation. In addition, PGE2 did not affect STAT5 serine phosphorylation. Instead, the stimulatory effect of PGE2 on STAT5 signaling could be mimicked by dibutyryl-cyclic adenosine monophosphate (cAMP) and the phosphodiesterase inhibitor IBMX, suggesting that the effect was mediated by cAMP. Activation of the cAMP pathway resulted in cAMP-response element binding protein (CREB) phosphorylation, which was sustained in the presence of EPO plus PGE2 and transient on EPO stimulation alone. The costimulatory effect of PGE2 on EPO-mediated STAT5 transactivation was inhibited by overexpression of serine-dead CREB or protein kinase A (PKA) inhibitor (PKI), in contrast to EPO-mediated transactivation, which was PKA independent. Furthermore, CREB-binding protein (CBP)/p300 was shown to be involved in EPO-mediated STAT5 transactivation, and a CBP mutant with increased affinity for CREB resulted in an additional enhancement of the PGE2 effect. Finally, we demonstrated that the STAT5 target genes Bcl-X, SOCS2, and SOCS3 were up-regulated by costimulation with PGE2. In summary, these studies demonstrate that PGE2 enhancement of EPO-induced STAT5 transactivation is mediated by the cAMP/PKA/CREB pathway.     

Cellular tolerance to adenosine receptor-mediated inhibition of lipolysis: altered adenosine 3',5'-monophosphate metabolism and protein kinase activation

Prolonged exposure of many types of cells to drugs or hormones that inhibit the activity of the enzyme adenylate cyclase, such as narcotics and alpha 2-adrenergic agonists, leads to enhanced accumulation of cAMP upon removal of the inhibitory drug. We have found previously that chronic infusion of the adenosine A1 receptor agonist phenylisopropyladenosine (PIA), an inhibitor of adenylate cyclase, into rats leads to enhanced isoproterenol-stimulated cAMP accumulation in adipocytes isolated from these animals. The enhanced cAMP accumulation was associated with an impaired ability of PIA to inhibit lipolysis in these cells. In the present study we have investigated the mechanism of the enhanced cAMP accumulation in adipocytes from PIA-infused rats and the relationship of these changes to the impaired antilipolytic action of the drug. The enhanced isoproterenol-stimulated cAMP accumulation in adipocytes prepared from PIA-infused rats was due to both an increased rate of cAMP synthesis and a decreased rate of cAMP metabolism at high concentrations of cAMP without a change in phosphodiesterase activity. There was heterologous desensitization of the ability of PIA, prostaglandin E1, and nicotinic acid to inhibit cAMP accumulation in the adipocytes from PIA-infused rats; there was an increase in the EC50 of each of these agonists, although maximal inhibition of cAMP accumulation was similar. The relationship between the activation of cAMP-dependent kinase and extent of lipolysis was similar in the two groups of cells. We demonstrated that the explanation for the impaired ability of PIA to decrease the rate of isoproterenol (10(-7) M)-stimulated lipolysis in the cells from the PIA-infused rats was due to the markedly increased concentrations of cAMP in these cells, which led to sufficient activation of the kinase to maintain a high rate of lipolysis even in the presence of PIA. In addition, we found that the changes induced by the PIA infusion were largely reversible over a 2-day period after discontinuing the PIA infusion. These results demonstrate that adipocytes from PIA-infused rats provide an interesting model to investigate the mechanisms of tolerance to inhibitory drugs.     

Effects of parathyroid hormone on cyclic-AMP concentrations of in vitro Necturus maculosus gastric antrum

The effects of bovine parathyroid hormone (bPTH1-84) on the stimulation of intracellular cyclic-AMP [cAMP] were investigated in an in vitro preparation of Necturus maculosus antral mucosa. When the antrum was exposed to 1, 5, 10, or 100 nM bPTH1-84, there was an approximately 2-fold nonlinear increase in tissue [cAMP] over basal values. The pretreatment of the antral mucosa with 1 mM isobutylmethylxanthine (IBMX, a phosphodiesterase inhibitor) increased with detectability of mucosal [cAMP]. The addition of 1, 5, 10, or 100 nM bPTH1-84 to tissues pretreated with IBMX resulted in an approximately 3.5-fold linear increase in mucosal [cAMP] over basal values. The time course of the generation of mucosal cAMP to 10 nM bPTH1-84 resulted in a small but significant transient increase at 2.5 min after the addition of bPTH1-84 but no change in the medium [cAMP]. In tissues pretreated with 1 mM IBMX the response to 10 nM bPTH1-84 was a large biphasic increase of [cAMP] at 2.5 min that progressively declined to near basal values by 15 min. There was also a significant sustained increase in the [cAMP] in the bathing medium at 2.5 min of tissues pretreated with IBMX followed by 10 nM bPTH1-84. These results suggest the presence of an adenylate cyclase that can be activated by a mammalian bPTH1-84 in elevating intracellular cAMP levels in the N. maculosus antral mucosa.