Effect of phosphodiesterase inhibitors on Sertoli cell refractoriness: reversal of the impaired androgen aromatization

Responsiveness of the Sertoli cell after FSH pretreatment was evaluated in terms of androgen aromatization. Sertoli cell cultures were preincubated with FSH for 24 h, then cells were washed free of hormone and reincubated with FSH in the presence of androstendione. The estrogen accumulated in the medium was measured by RIA. Gonadotropin pretreatment produced a marked refractory state, and a second challenge with FSH did not produce an increase in androgen aromatization. A dose-response study showed that FSH pretreatment produced three separate effects on Sertoli cell steroidogenesis: an increased basal production of estrogen; a decreased maximal response when doses of 10 ng/ml FSH or higher were employed in the preincubation; and a decreased sensitivity of the Sertoli cell to FSH. In the last case, the ED50 was reduced approximately 3- to 5-fold. Such an impaired stimulation of androgen aromatization was no longer present when cells were incubated with the phosphodiesterase inhibitors methyl-isobutyl-xanthine (MIX). In the presence of this inhibitor, refractory cells responded to FSH better than the control cells. The possibility that MIX stimulated cAMP accumulation by acting as antagonist of purine receptor was ruled out by the finding that the nonxanthine phosphodiesterase inhibitor 4-(3-butoxy-4-methoxybenzyl)2-imidazolidinone (Ro 20-1724) also reverted the refractory state. Pretreatment of the Sertoli cells with FSH produced an impaired response in the second incubation also to isoproterenol, cholera toxin, and forskolin. The response to these compounds was apparently normal when cells were incubated in the presence of MIX or Ro 20-1724. Conversely, refractory cells responded to (Bu)2cAMP in a manner indistinguishable from the fully responsive control cells. These data demonstrate that FSH induces homologous and heterologous refractory states of the Sertoli cell reflected by an impaired estrogen production. The finding that phosphodiesterase inhibitors fully restore the FSH response suggests an important role of phosphodiesterase in the induction and/or maintenance of such refractoriness.     

Mechanisms of glucagon-induced homologous and heterologous desensitization of adenylate cyclase in membranes and whole Sertoli cells of the rat

In the present work the molecular mechanisms of glucagon-induced desensitization of adenylate cyclase in cultured Sertoli cells have been studied in both whole cells and in a cell-free system. 1) Pretreatment of both whole Sertoli cells and membranes with glucagon induces a time- and dose-dependent desensitization of adenylate cyclase response that is primarily homologous and similar in the two systems. The component of heterologous desensitization, estimated by the reduced responsiveness to other hormones and NaF, accounted for only 12-20% loss of glucagon-responsive adenylate cyclase activity (P less than 0.01). 2) Glucagon-induced desensitization is ATP-dependent. Half maximal desensitization was achieved between 0.1 and 0.2 mM of ATP. 3) The typical time lag in the maximal activation of adenylate cyclase by GMPP(NH)P in the absence of hormone reappeared upon desensitization in spite of the presence of glucagon. The lag, however, was eliminated by FSH, showing that the homologous desensitization is due to a receptor-specific alteration. 4) The heterologous component of glucagon-induced desensitization is largely cAMP/protein kinase dependent. cAMP/protein kinase-induced desensitization was heterologous and caused approximately 30% loss of both hormonal and fluoride-stimulated enzyme activity. 5) Glucagon-induced desensitization is not due to altered activity of Ni since it proceeded equally well in membranes of cells pretreated with pertussis toxin (100 ng/ml) which eliminates Ni-mediated effects. It is concluded that glucagon induces both homologous and heterologous desensitization of the Sertoli cell adenylate cyclase. The locus of homologous desensitization appears to be at the level of the receptor and most probably involves a cAMP-independent phosphorylation reaction, whereas the heterologous desensitization appears to be cAMP-mediated and at least involves impaired functional activity of the Ns component.     

Desensitization of the cAMP system in mouse Leydig cells by hCG, cholera toxin, dibutyryl cAMP and cAMP: localization of the 'lesion' to the guanine nucleotide regulatory protein-adenylate cyclase complex

The mechanism of hCG-induced desensitization of the cAMP system was studied in Percoll-purified mouse Leydig cells. Pretreatment of Leydig cells with hCG resulted in a time- and dose-dependent decrease in the capacity of hCG-induced cAMP formation. Maximal desensitization (approximately 90%) was induced by only partial prior stimulation. Desensitization, however, was not observed without a prior increase in cAMP or testosterone production. Pretreatment of the cells with N6,O2'-dibutyryl cAMP (DBcAMP) also induced a dose- and time-dependent densensitization. cAMP was only effective in the presence of the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (MIX). Cholera toxin desensitized the hormone-induced cAMP response as drastically as hCG. Cholera toxin was unable to reverse the refractory state induced by one of the agonists. hCG-induced desensitization was not associated with a loss in [125I]hCG binding or an increase in maximal phosphodiesterase activity, and appeared not to be dependent on protein synthesis. Membranes from hCG, cholera toxin of DBcAMP-desensitized cells showed an impaired adenylate cyclase activity in response to hCG, hCG plus beta-gamma-imidoguanosine 5'-triphosphate (GPPNP) and NaF. In conclusion, hCG-induced desensitization of the adenylate cyclase system in mouse Leydig cells can be mimicked by cholera toxin, DBcAMP and cAMP, indicating a cAMP-mediated process. The site of the 'lesion' has to be localized to the guanine nucleotide regulatory protein-adenylate cyclase complex rather than to its uncoupling from the hormone receptor.     

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

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

Glucagon-stimulated cyclic AMP production and formation of estradiol in Sertoli cell cultures from immature rats

Addition of glucagon to the incubation medium of cultured Sertoli cells isolated from immature (19-day-old) rats resulted in a time- and concentration-dependent stimulation of cAMP accumulation measured both in the cells and in the medium. Maximal intracellular levels of cAMP were reached after 30 min, after which the levels decreased. In the medium cAMP levels reached a plateau after 6 h. The magnitude and kinetics of the responses were comparable to those observed with FSH in the same culture preparations. 1-Methyl-3-isobutylxanthine (MIX), a phosphodiesterase inhibitor, greatly potentiated the magnitude of the effects of glucagon and FSH. Glucagon stimulated adenylate cyclase activity in isolated membrane preparations from similar cultures, and the concentration causing half-maximal stimulation (EC50) was approximately 300 ng/ml. Glucagon also stimulated aromatization in cultured Sertoli cells to the same extent as FSH. It is concluded that cultured Sertoli cells isolated from immature rats contain receptors for glucagon, coupled to adenylate cyclase, and that glucagon also stimulates aromatization of testosterone to estradiol.     

Developmental changes in the hormonal regulation of rat testis Sertoli cell adenylyl cyclase

The stimulatory effects of FSH on Sertoli cell functions such as cAMP accumulation, protein kinase activation, and RNA and protein synthesis wane during testis maturation. However, FSH receptors increase with age and addition of cAMP stimulates these biochemical events in Sertoli cells from animals of any age. In order to determine if this loss of responsiveness to FSH was due to an inability to stimulate adenylyl cyclase, the hormonal responsiveness of this enzyme was investigated as a function of testicular development. In agreement with intact cell studies, adenylyl cyclase activity was found to be stimulated by FSH 2- to 3-fold in homogenates of testes from immature (5-20 days of age) Sertoli cell-enriched rats, while no stimulation of the enzyme by FSH was observed in similar homogenates from Sertoli cell-enriched animals 20 days of age or older. The possibility of a decrease in enzyme sensitivity to the gonadotropin as a function of maturation ws ruled out by dose-response studies. Catalytic activity of the enzyme was retained with increasing animal age as evidenced by the ability of fluoride (10 mM) to stimulate basal activity 4-fold. Hormonal responsiveness of the Sertoli cell adenylyl cyclase of mature animals could be restored, however, either by addition of the nonmetabolizable guanosine 5'-triphosphate analog, 5'-guanylyl-imidodiphosphate to homogenates or by preparation of membrane particles. We found that 5'-guanylyl-imidodiphosphate selectively potentiated FSH effects on cyclase in testicular homogenates from mature animals while having no effect on the relative degree of hormone stimulation in homogenates from immature animals, and that in contrast to homogenates, testicular membrane preparations retain their FSH responsiveness upon animal maturation.     

Adenylyl cyclase of perifused porcine granulosa cells remains responsive to pulsatile, but not continuous stimulation with follicle-stimulating hormone

Differentiation of granulosa cells and development of ovarian follicles requires FSH for several days. The purpose of the present studies was to determine how the capacity of the adenylyl cyclase of immature porcine granulosa cells to respond to FSH depends on the dose, duration, and frequency of exposure to FSH. Cells were stimulated with various regimens of FSH and forskolin in a dynamic flow perifusion system. cAMP production during 4 h of continuous (tonic) exposure to FSH was directly related to FSH concentration (5-500 ng/ml). FSH-stimulated cAMP production declined markedly after 4 h of tonic stimulation with FSH, regardless of the FSH concentration. Preliminary experiments using a pulse interval of 3 h (perifusion medium FSH concentration, 150 ng/ml) indicated that stimulation with 15-min pulses elicited a greater cumulative cAMP response than stimulation with either 5-, 30-, or 60-min pulses. Responsiveness to FSH depended critically on the duration and frequency of stimulation and the concentration of FSH. Short pulses were more effective than long pulses in both eliciting cAMP responses of most uniform amplitude and maintaining responsiveness to a final tonic FSH stimulus. The optimal pattern of stimulation consisted of a pulse duration of 15 min, with a pulse interval of 2-3 h. A peak chamber FSH concentration of 150 ng/ml yielded the greatest cumulative cAMP production, although cells that had been perifused with FSH-free medium had the greatest response to a final tonic FSH stimulus. The attenuation of responsiveness after continuous perifusion with FSH does not appear to be due to desensitization of the cyclase itself, since 1) cells perifused with FSH continuously for 20 h still responded to forskolin (100 microM), which activates cyclase independently of the FSH receptor; and 2) cells did not become refractory to forskolin for 14 h. The transient refractoriness to FSH appears to be due to a process that alters the interaction between the FSH receptor and the guanine nucleotide regulatory component of cyclase. This refractoriness can be reversed simply by removing FSH from the perifusion medium for a critical period of time, i.e. 2-3 h.     

Regulation of follicle-stimulating hormone and dibutyryl adenosine 3',5'-monophosphate of a phosphodiesterase isoenzyme of the Sertoli cell

The regulatory role of FSH on phosphodiesterase was studied in immature Sertoli cells in culture. Cells were prepared from 15-day-old immature rats, cultured for 3 days in defined medium, and then stimulated for 24 h with gonadotropin or with other factors known to regulate Sertoli cell cAMP. All agents that increased cAMP intracellular levels also had an effect on the total phosphodiesterase activity of cell homogenates, FSH and dibutyryl cAMP being the most potent stimulators. Further, stimulation was more evident when phosphodiesterase was measured at cAMP concentrations below micromolar. With 1 microM cGMP as substrate, no modification of the activity could be detected. Reversal of phosphodiesterase activation was observed at 24 or 40 h when dibutyryl cAMP was removed from the incubation medium. Separation of the isoenzymes present in the soluble fraction of Sertoli cell made it possible to demonstrate a selective stimulation of one isoenzyme. FSH and dibutyryl cAMP increased 10- and 50-fold, respectively, the activity of a high affinity cAMP phosphodiesterase, while the Ca2+-dependent cGMP hydrolyzing enzymes were not apparently affected. The enzyme regulated by FSH and dibutyryl cAMP had an apparent Km for cAMP of 2 microM, was Ca2+ and calmodulin insensitive, and migrated on sucrose density gradients with a sedimentation coefficient of 5.5S. These results indicate that FSH, after stimulation of intracellular cAMP, induces an increase in a high affinity phosphodiesterase and, therefore, an increased cAMP turnover in the Sertoli cell. This, in turn, might be a relevant phenomenon in the control of the responsiveness of this cell to gonadotropin.     

Hormone-induced homologous and heterologous desensitization in the rat adipocyte

We examined the process of desensitization in the isolated rat adipocyte. When adipocytes were exposed to isoproterenol (10(-7) or 10(-5) M) or ACTH (250 mU/ml) for 2 h, there was a marked decline of as much as 77% in response upon restimulation by hormone, as measured by glycerol release or cAMP levels. This desensitization was both heterologous as well as homologous. Thus, for example, exposure of adipocytes to isoproterenol desensitized them to further stimulation by both isoproterenol and ACTH. The process was time dependent, since augmentation rather than desensitization was seen if cells were initially exposed to hormone for 30 min rather than 2 h. No desensitization was seen when the cells were restimulated with the nonhormonal lipolytic agent dibutyryl cAP. Similarly, no desensitization was seen when cells were first exposed to dibutyryl cAMP and then restimulated with hormone. We draw the following conclusions. First, desensitization in the adipocyte is a time- and dose-dependent process that is specific for adenylate cyclase-activating hormones. Secondly, the process is heterologous as well as homologous. Initial exposure of the adipocyte to one adenylate cyclase-activating hormone reduces its adenylate cyclase or lipolytic response upon reexposure to either the same or a different adenylate cyclase-activating hormone. Finally, the reduction in the end result of hormone activation, lipolysis, is due in part to a decrease in inducible levels of cAMP.     

Age-related and testicular regression-induced changes in adenosine 3',5'-monophosphate responses in cultured hamster Sertoli cells

Sertoli cells prepared from adult hamsters with maximally regressed testes responded to FSH with an increased accumulation of intracellular cAMP similar to that of Sertoli cells from an immature animal. That is, the age-dependent decline in responsiveness of Sertoli cells to FSH was reversed during testicular regression. To determine whether this testicular regression-induced restoration of FSH responsiveness was mechanistically a reversal of the age-dependent decline in response, the ability of cholera toxin, forskolin, catecholamines, and pertussis toxin to stimulate cAMP accumulation in Sertoli cells cultured from hamsters undergoing testicular regression was assessed and compared to the responses of Sertoli cells from 18- and 36-day-old hamsters. The age-related decline in responsiveness was evident not only with FSH but also when cells were stimulated with isoproterenol, cholera toxin, forskolin, or pertussis toxin singly or in combination. While the FSH response of Sertoli cells from regressed testes was restored to values indicative of an immature Sertoli cell, the response of the cultured cells to cholera toxin, forskolin, catecholamines, or pertussis toxin either singly or in combination suggested that the adenylate cyclase system of Sertoli cells from regressed testes was unchanged from its "adult" activity. Thus, our original hypothesis that testicular regression/recrudescence was a mirror image of sexual maturation was an oversimplification. However, since FSH is the physiological regulator of Sertoli cell function, and its response is restored to levels found in the immature animal during testicular regression and declines to adult levels during testicular recrudescence, our original hypothesis that testicular recrudescence mimics sexual maturation (i.e. the animal goes through "puberty" each time it goes through a regression/recrudescence cycle) is still tenable. However, at the molecular level, differences in mechanism exist.     

Modulation of FSH receptor phosphorylation correlates with hormone-induced coupling to the adenylate cyclase system

The authors have recently demonstrated that an inhibitor of protein phosphorylation, staurosporine (SSP), can dramatically enhance follicle-stimulating hormone (FSH) stimulated cyclic adenosine monophosphate (cAMP) accumulation in rat granulosa cell line (GFSHR-17) overexpressing about 20-fold FSH receptor than primary granulosa cells. Moreover, incubation with SSP can partially release the cells from FSH-induced desensitization. In this work, it was examined whether coupling of FSH receptor to the adenylate cyclase is correlated with the degree of receptor phosphorylation. Immunoprecipitation of FSH receptor after metabolic labeling of the cells with³²P-orthophosphate revealed that preincubation of the cells with SSP resulted in pronounced reduction in FSH receptor phosphorylation compared to control cells, concomitantly with a dramatic increase in FSH-stimulated cAMP accumulation. In contrast, incubation of the cells with saturating dose of FSH, which leads to uncoupling between the receptor and the adenylate cyclase, resulted in enhanced receptor phosphorylation. Moreover, cells preincubated with FSH could be released from desensitization by further incubation with SSP and a significant reduction in FSH receptor phosphorylation. Immunostaining of the cells with FSH receptor antibody reveal a homogeneous distribution of the receptor on the surface of SSP-treated cells. Some aggregation of the receptor was evident in control cells that were not treated with SSP. In contrast, massive clustering and capping of the receptor molecules were observed on the surface of FSH-stimulated cells. The current data suggest that phosphorylation-dephosphorylation of the receptor molecules play an important role in the degree of coupling between the receptor and the adenylate cyclase system. Moreover, desensitization to FSH stimulation that is implicated with high degree of receptor phosphorylation may lead to aggregation of the receptor molecules on the cell surface.     

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.     

Evidence that organic iodine attenuates the adenosine 3',5'-monophosphate response to thyrotropin stimulation in thyroid tissue by an action at or near the adenylate cyclase catalytic unit

Studies were conducted to define more clearly the site in the thyroid adenylate cyclase complex at which iodine exerts its inhibitory effect on activation of this enzyme by TSH. Iodine- and TSH-induced desensitization were additive. Dissociation was observed between the rates of recovery from TSH- and iodine-induced desensitization. Cycloheximide (10(-4) M) prevented recovery from the inhibitory effect of iodine on thyroid adenylate cyclase activation. Preincubation of freshly isolated dog thyroid follicles in 10(-4) M iodide decreased the subsequent cAMP response to cholera toxin (0.5 micrograms/ml) stimulation. This effect of iodide was prevented by 3 mM methimazole. Thyroid adenylate cyclase regulatory protein (Ns) activity was assessed by the ability of detergent extracts of thyroid plasma membranes to reconstitute adenylate cyclase responsiveness to isoproterenol in N-deficient S49 cyc- plasma membranes. Thyroid Ns activities were similar in control and iodide-pretreated thyroid cells. The inhibitory effect of iodine on TSH activation of thyroid cAMP generation was additive to that of inhibition via the alpha 2- adrenergic pathway and also additive to inhibition by 2',5'-dideoxyadenosine (an adenosine P-site agonist). Preincubation of freshly dispersed dog thyroid cells in 10(-4) M NaI reduced the cAMP response to stimulation by 100 microM forskolin. These data provide evidence that in iodine-induced TSH desensitization in the thyroid; 1) TSH receptor function is normal, 2) the regulatory protein (Ns) in the adenylate cyclase stimulatory pathway is functionally unaltered, 3) iodine does not exert its effect via the regulatory protein (Ni) in the pathway that inhibits adenylate cyclase activation, 4) iodine does not act via the adenosine P-site inhibitory pathway, 5) the action of iodine is at or near the adenylate cyclase catalytic unit, and 6) new protein synthesis is necessary for recovery from iodine desensitization.     

Stimulation of Adenosine 3′:5′-Cyclic Monophosphate Accumulation in Anterior Pituitary Gland In Vitro by Synthetic Luteinizing Hormone-Releasing Hormone

A near-maximal dose (20 ng/ml) of synthetic luteinizing hormone(LH)-releasing hormone/follicle-stimulating hormone(FSH)-releasing hormone added to incubated anterior pituitary tissue of male rats leads to concomitant increases of intracellular concentrations of adenosine 3':5'-monophosphate and of release of both LH and FSH. The stimulatory effect of LH-releasing hormone/FSH-releasing hormone is observed after a lag period of about 90 min and is progressive at later time intervals; a 3-fold stimulation of cAMP accumulation over control is seen after 210 min of incubation. Half-maximal stimulation of cAMP accumulation is observed between 0.1 and 1.0 ng/ml (0.1-1 nM) of LH-releasing hormone/FSH-releasing hormone. In the presence of 10 mM theophylline, the stimulatory effect of LH-releasing hormone/FSH-releasing hormone on cAMP accumulation is similar to that observed in the absence of the inhibitor of cyclic nucleotide phosphodiesterase, indicating that the releasing hormone exerts its effect by specific activation of adenylate cyclase in LH- and FSH-secreting cells rather than by inhibition of cyclic nucleotide phosphodiesterase. Since the release of growth hormone, thyrotropin, prolactin, and adrenocorticotropic hormone is not affected by LH-releasing hormone/FSH-releasing hormone, and since cAMP stimulates the release of all six adenohypophyseal hormones. the observed changes of cAMP concentrations indicate specific stimulation of adenylate cyclase activity in LH-and FSH-secreting cells of the adenohypophysis.     

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.     

In vitro effects of germ cells on the secretory activity of Sertoli cells recovered from rats of different ages

Indirect (hypotonically treated culture) and direct (coculture) approaches were used to study the influence of germ cells on androgen-binding protein (ABP) and 17 beta-estradiol secretion by Sertoli cells prepared from 10-, 15-, 20-, and 45-day-old rats. Using these approaches, the mechanisms that govern Sertoli cell-germ cell membrane recognition were shown to be unaltered by the hypotonic treatment, to vary with the age of the Sertoli cell donors, and to be FSH/(Bu)2cAMP dependent in the younger animals (10-20 days old). Hypotonic treatment had no effect on estradiol levels at all ages studied, but resulted in a marked fall of ABP production by Sertoli cells from 20 days onward. However, the relative stimulation of ABP induced by FSH/(Bu)2cAMP (ABP levels in FSH/(Bu)2cAMP/hypotonically treated Sertoli cell cultures vs. ABP in hypotonically treated cultures) was markedly increased at 20 and 45 days, indicating that germ cells influence Sertoli cell responsiveness to FSH/(Bu)2cAMP. The addition of crude germ cell preparation to the Sertoli cell cultures stimulated ABP and inhibited estradiol levels at all ages studied. Moreover, the addition of germ cells to hypotonically treated cultures induced a decrease in the relative ABP response to FSH and (Bu)2cAMP, thus confirming (see above) that germ cells may be involved in the mechanism of Sertoli cell refractoriness to FSH that occurs with advancing age in the rat. Germ cell-stimulated ABP production was higher in adult Sertoli cells than in immature cells. That Sertoli cell responsiveness to germ cells varies with age is further supported by the data showing that whereas spermatocytes stimulated ABP and inhibited estradiol productions at all ages studied, early spermatids only influenced these parameters from 20 days onward. It is concluded that germ cell control over Sertoli cell function in vitro may be of important physiological significance during Sertoli cell maturation in mammals.     

Regulation of protein kinase inhibitor by follicle-stimulating hormone in Sertoli cells in vitro

The heat-stable protein inhibitor of cAMP-dependent protein kinase is specifically regulated by hormones in cultures of rat Sertoli cells maintained under completely defined conditions. Hormones that are known to elevate Sertoli cell cAMP concentrations, namely FSH and isoproterenol, produce a 4- to 5-fold increase in the specific activity of protein kinase inhibitor, whereas testosterone and LH have no effect. The stimulatory effects of FSH or isoproterenol on protein kinase inhibitor are completely mimicked by dibutyryl cAMP. The ability of FSH to stimulate protein kinase inhibitor is dependent upon the age of the animals used as a source for the Sertoli cells. FSH stimulates protein kinase inhibitor activity in cells from 9- and 16-day-old animals, but not in cells from 32-day-old animals. On the other hand, isoproterenol or dibutyryl cAMP will stimulate protein kinase inhibitor in cells from both young and old animals. FSH can stimulate protein kinase inhibitor activity in older cells only in the added presence of the phosphodiesterase inhibitor, 1-methyl-3-isobutyl xanthine. Using specific antibodies to protein kinase inhibitor, we have shown that this protein is regulated by hormones via preferential stimulation of de novo synthesis of the inhibitor. (Endocrinology 108: 427, 1981)     

Forskolin effects on the cAMP system and steroidogenesis in the immature rat ovary

The diterpene forskolin was found to activate the adenylate cyclase system in intact tissue and membrane preparations of the immature rat ovary. The cyclic AMP (cAMP) response reached a maximal level after 5 min and no decline was observed even after 4 h of incubation. Forskolin stimulated production of both progesterone and testosterone in a pattern similar to that produced by luteinizing hormone (LH) or dibutyryl-cAMP (dbcAMP). In combination with LH, follicle-stimulating hormone (FSH) or prostaglandin E2 (PGE2), forskolin potentiated the hormone effects on adenylate cyclase activity in membrane preparations. Pretreatment with LH or PGE2 desensitized the cells to further hormone stimulation, while the forskolin response was unaffected. Pre-exposure to forskolin did not desensitize the cells to a subsequent stimulation by LH or PGE2. The presence of 8-bromo-cAMP (brcAMP) in the preincubation medium reduced the subsequent hormone response. These results demonstrate a rapid and sustained activation of the adenylate cyclase system by forskolin in the rat ovary. The steroidogenic response was similar to that of known stimulators of ovarian cells (LH, dbcAMP). The inability of forskolin to induce desensitization of the adenylate cyclase system demonstrates, however, important differences between hormone and non-hormone activation. Consequently, forskolin can be a useful tool for investigation of the mechanisms involved in the desensitization process.     

Desensitization of the stimulatory A2 adenosine receptor-adenylate cyclase system in vascular smooth muscle cells from rat aorta

We have previously shown that adenylate cyclase present in rat aorta vascular smooth muscle cells can be stimulated by adenosine, its analogs and other agonists. In the present studies, we have examined the effect of preexposure of aorta vascular smooth muscle cells to N-ethylcarboxamide adenosine (NECA) on adenylate cyclase activity stimulated by NECA and other agonists. The vascular smooth muscle cells, when exposed to NECA, resulted in a concentration- and time-dependent loss of NECA-stimulated adenylate cyclase activity. NECA stimulated adenylate cyclase activity by about 120% in control cells, which was decreased to 20% in cells pretreated with 50 microM NECA for 30 min at 37 degrees C. However, GTP-, isoproterenol-, and forskolin-sensitive adenylate cyclase activities were not affected by such treatment, suggesting that NECA treatment of the cells resulted in homologous desensitization. Similarly, the exposure of the cells to isoproterenol resulted in the desensitization of isoproterenol-stimulated adenylate cyclase activity without affecting the NECA-stimulated adenylate cyclase activity. Furthermore, when NECA-treated cells were washed free of agonist, the desensitized state was reversed and the cells regained about 75% responsiveness to NECA stimulation of adenylate cyclase.     

Diphtheria toxin prevents catecholamine desensitization of A431 human epidermoid carcinoma cells.

We proposed that a rapidly turning over protein, induced in response to catecholamine stimulation of C6-2B rat astrocytoma cells, inhibits subsequent hormonal activation of adenylate cyclase. Studies upon which our hypothesis is based and confirmatory work in a variety of other cell lines and in vivo have utilized actinomycin D and cycloheximide to inhibit RNA and protein synthesis, respectively. These inhibitors, however, are not specific and have been reported also to interfere with other cellular processes. Diphtheria toxin is a specific protein synthesis inhibitor that acts only by ADP-ribosylating elongation factor 2, thus preventing peptide chain elongation. We thus tested whether diphtheria toxin could prevent catecholamine-induced desensitization in A431 human epidermoid carcinoma cells. The toxin inhibited protein synthesis and altered the time course of isoproterenol-stimulated cAMP accumulation as did the less-specific protein synthesis inhibitor cycloheximide. Cellular cAMP content after a 30-min exposure to isoproterenol was similar in control and in toxin-treated cells. However, after 4 hr of treatment with isoproterenol, toxin-treated cells accumulated up to six times more cAMP than controls. When cells or cell-free adenylate cyclase preparations were rechallenged with agonists, toxin-mediated inhibition of protein synthesis prevented desensitization. These results show that diphtheria toxin, a specific inhibitor of protein synthesis, can interfere with the normal physiological regulation of cAMP metabolism in eukaryotic cells and provide compelling evidence that catecholamine stimulation of adenylate cyclase promotes the synthesis of a protein(s) that, in some way, inhibits hormone-stimulated adenylate cyclase.