Hormonal regulation of 3',5'-adenosine monophosphate phosphodiesterases in cultured rat granulosa cells

The effect of gonadotropins on phosphodiesterase activity of rat granulosa cells was studied in an in vitro model. Granulosa cells were prepared from hypophysectomized or intact, estrogen-primed immature female rats and treated with FSH, hCG, or (Bu)2cAMP in vitro. Phosphodiesterase activity was determined in cell homogenates. FSH treatment for 2 days produced a marked increase in phosphodiesterase activity, while hCG was ineffective. FSH stimulation was potentiated by the addition of 1-methyl-3-isobutylxanthine, while treatment with the cAMP analog, (Bu)2cAMP by itself also markedly stimulated enzyme activity. FSH stimulated cAMP, but not cGMP, hydrolysis, suggesting that a phosphodiesterase specific for cAMP was stimulated by the gonadotropin. Time-course studies showed that an increase in phosphodiesterase activity was apparent after 1 h of incubation and was maximal at 48 h. FSH stimulation of phosphodiesterase was dose-dependent, with an ED50 of 30 ng/ml FSH and a maximal increase at 100-300 ng/ml. Treatment with cycloheximide (1 or 10 micrograms/ml) completely blocked the gonadotropin stimulation, suggesting that on-going protein synthesis is required for the FSH action. DEAE-cellulose chromatography of soluble extracts of control and FSH-treated cells indicated that two forms of phosphodiesterase were present in unstimulated granulosa cells. The first form, eluting at 0.17 M Na-acetate, hydrolyzed both cAMP and cGMP and was stimulated by Ca++ and calmodulin; the second form, eluting at 0.48 M Na-acetate, was insensitive to Ca++ or calmodulin and hydrolyzed mainly cAMP. FSH treatment markedly stimulated cAMP hydrolysis by the calmodulin-dependent first form as well as that by the second form. Double reciprocal analysis indicated that the FSH-stimulated enzymes are of high affinity for cAMP. In agreement with the data on total homogenate, the cGMP hydrolysis was not affected by the hormone treatment. These data demonstrate that FSH stimulates cAMP, but not cGMP, phosphodiesterase activity in rat granulosa cells in vitro. This stimulation might represent a mechanism for termination of the FSH primary stimulus and regulation of granulosa cell responsiveness to the gonadotropin.     

Involvement of phosphodiesterase in the refractoriness of the Sertoli cell

Gonadotropin treatment of the Sertoli cell produces a marked refractory state of the cell to subsequent hormonal stimulation. Because FSH also stimulates the phosphodiesterase activity of these cells, the possible involvement of an altered cAMP catabolism during refractoriness was investigated in an in vitro model. Sertoli cells, after 3 days of culture in a defined medium, were exposed to FSH or isoproterenol for 1-24 h. After this pretreatment, cells were stimulated for 1 h with a maximal FSH dose, and the responsiveness was measured in terms of cAMP accumulation. Sertoli cells previously treated with hormone entered a refractory state, a second exposure being ineffective in elevating intracellular or extracellular cAMP. Addition of the phosphodiesterase inhibitor 3-isobutyl-methylxanthine in the second incubation partially restored the ability of the cell to accumulate cAMP in the presence of hormone. This phosphodiesterase inhibitor also caused an apparent decrease in the potency of FSH to induce the refractory state. Such an impairment of response developed in the intact cell in 4 h, and was accompanied by a partial desensitization of the adenylate cyclase and an increase in phosphodiesterase activity. The stimulation of phosphodiesterase activity, but not the desensitization of adenylate cyclase, was inhibited by cycloheximide. The inhibition of protein synthesis also prevented the onset of the refractory state of the intact Sertoli cell. Pretreatment of the Sertoli cells with either FSH or isoproterenol rendered the cell refractory to a second stimulation with either agonist; in contrast, the adenylate cyclase desensitization in the homogenate was apparent only for the agonist employed in the preincubation. These results indicate that phosphodiesterase regulation is involved in the control of Sertoli cell responsiveness to hormone. Thus, the net decrease in cAMP production of the FSH-treated cells is the result of a decreased adenylate cyclase stimulation and an increased cAMP catabolism mediated by phosphodiesterase. The latter phenomenon appears to be the predominant cause of the partial refractoriness induced by low doses of gonadotropin.     

The control of the synthesis and secretion of plasminogen activator by rat sertoli cells in culture

Sertoli cells in primary cultures produce plasminogen activator activity, and release it into the medium at rates greatly influenced by a variety of factors, including cell density, the presence of hormones, incubation temperature and duration of culture. In Sertoli cells maintained in culture in the presence of dibutyryl cAMP, the amounts of plasminogen activator activity secreted per cell were maximal at cell densities up to 2.5 μg DNA/cm² (350 units/μg cell DNA), and declined to 40 units/μg cell DNA at a density of 22 μg DNA/cm². Concentrations of follicle-stimulating hormone (FSH) required to elicit half-maximal stimulation of the production of plasminogen activator activity were 0.37 μg/ml for oFSH-NIH S12 and 8 ng/ml for the more purified oFSH-S1528C2. The ED₅₀ for dibutyryl cAMP was found to be 0.08 mM. Addition of an inhibitor of phosphodiesterase (3-isobutyl-l-methylxanthine) enhanced the formation of plasminogen activator by cells cultured in the presence of FSH.Addition to the culture medium of testosterone, epidermal growth factor, insulin, human chorionic gonadotropin or prostaglandins (E₁, E₂ or F_1α) did not result in increased production of PA activity by Sertoli cells. Cells in culture for as long as 14 days remained responsive to FSH or dibutyryl cAMP. Increases of cellular levels of plasminogen activator became evident within 2–4 h after addition of either FSH or dibutyryl cAMP to the medium. The stimulation by FSH or dibutyryl cAMP of the production of plasminogen activator activity was shown to be dependent upon de novo synthesis of RNA and protein. Levels of enzyme activity released by Sertoli cells maintained in culture for 48 h at 37°C were approx. 50% higher than plasminogen activator released by cells cultured at 32°C. The control of the production of plasminogen activator activity by Sertoli cells was discussed in relation to the control of plasminogen activator production by granulose cells, and the possible role of plasminogen activator in gonadal functions.     

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.     

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.     

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.     

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)     

Effects of norepinephrine on cyclic nucleotide levels in dog thyroid slices.

Addition of dibutyryl adenosine 3'-5'-cyclic monophosphate (DBcAMP) or dibutyryl guanosine 3',5'-cyclic monophosphate (DBcGMP; 1--6mM) to to enzymatically dispersed, overnight-cultured rat anterior pituitary cell preparations stimulated the release of gonadotropins (LH and FSH) from the cells into the incubation medium. Stimulation of gonadotropin release by DBcGMP was observed after only 10 min of incubation, whereas that caused by DBcAMP appeared at 180 min. Synthetic LHRH (2.5 X 10(-9) M) induced a small, transient increase in intracellular cAMP content (+16%, P less than 0.05, after 5 min coincubation), while levels of cGMP in the same cells were rapidly and markedly low for 2 h. The decrease in cGMP content was accompanied by a discharge of gonadotropins lasting for 2 h, which was detectable after 5 min in the case of LH and after 10 min in the case of FSH. These results strongly suggest that cGMP might be an intracellular mediator in the process of LHRH-stimulated release of gonadotropins.     

Hormonal regulation of inhibin production by cultured Sertoli cells

The hormonal regulation of inhibin production by cultured rat Sertoli cells was examined using a specific radioimmunoassay (RIA) which detects the N-terminal portion of the porcine inhibin alpha chain. FSH, but not hCG or prolactin caused a dose-dependent increase in inhibin production (EC50 for FSH = 2.4 ng/ml); both secreted and intracellular levels of inhibin were increased, but the secreted form represented one-half to two-thirds of the total. The FSH-stimulated production of inhibin was augmented by addition of a phosphodiesterase inhibitor, and could be mimicked by cholera toxin, forskolin, or dibutyryl cAMP, all of which are known to increase intracellular cAMP levels. Inclusion of either dihydrotestosterone or estradiol in the cultures had no effect on inhibin production, both in the presence and absence of FSH. Examination of the conditioned media from forskolin-treated Sertoli cells by gel filtration chromatography revealed a single peak of bioactive and immunoreactive inhibin, at a molecular weight of approximately 32,000, similar to that observed for the porcine and bovine follicular fluid inhibins. Thus, FSH activated the cAMP pathway to stimulate Sertoli cell production of inhibin which in turn suppresses pituitary FSH release to form a closed-loop feedback system.     

Follicle-stimulating hormone activation of glycogen phosphorylase in the Sertoli cell-enriched rat testis

The potential role of glycogen phosphorylase in providing energy for the Sertoli cell-enriched testis has been investigated. This enzyme is detectable in testes from rats 6-54 days of age. Glycogen phosphorylase in isolated Sertoli cell-enriched testes is specifically stimulated by FSH. Maximal activation (2-fold) is obtained within 10 min after adding 0.5 micrograms FSH/ml to isolated immature testes (16 days old). There is only a 1.1-fold activation by FSH in testes from mature (34 days old) animals. The sensitivity to the gonadotropin can be restored by adding 1-methyl-3-isobutylxanthine, a phosphodiesterase inhibitor, with the FSH. Phosphorylase can be activated by effectors that mimic the actions of the two proposed mediators of FSH action, cAMP and Ca+2. Phosphorylase from testis of either age is maximally activated by an analog of cAMP, 8-bromo-cAMP. While phosphorylase is rapidly activated 1.4-fold by incubating isolated testis for 2 min with A23187, a Ca+2 ionophore, the age, time, and dose dependence of FSH activation are consistent with conversion mediated by cAMP. Phosphorylase was localized in cultured Sertoli cells by indirect immunofluorescence microscopy. Affinity-purified antiphosphorylase decorated cytoskeletal structures that resemble stress fibers, suggesting that phosphorylase may function in Sertoli cells to provide energy for cytoskeletal motility.     

Follicle-stimulating hormone regulates in vivo testicular phosphodiesterase

The effect of FSH on testicular phosphodiesterase was studied in immature rats in order to verify that the regulation of response to hormone in the gonad involves an increased cyclic AMP catabolism. Hydrolysis of cyclic AMP and cyclic GMP was measured in the homogenates of seminiferous tubules and interstitium of control animals and animals injected i.p. with 50 micrograms ovine FSH twice, 24 h and 12 h before necroscopy. After hormonal treatment, cyclic AMP phosphodiesterase activity in the seminiferous tubules was markedly increased whether results were expressed per testis or per mg protein, while cyclic GMP phosphodiesterase present in the same compartment was apparently unaffected. In the interstitium, cyclic nucleotide hydrolysis was usually decreased after FSH injection. The stimulation of phosphodiesterase, a slow process reaching maximal stimulation after 12 h, was dependent on the dose of FSH injected. In addition, DEAE-cellulose chromatography of cytosol prepared from control and treated seminiferous tubules confirmed that stimulation was restricted to a cyclic AMP hydrolysing enzyme while the activity of the cyclic GMP hydrolysing form was not modified. Thus it is demonstrated that testicular phosphodiesterase is under FSH control. It is proposed that this in vivo regulation is a relevant phenomenon in the modulation of Sertoli cell function and contributes to the refractoriness that follows gonadotropin treatment.     

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.     

Gonadotropin stimulation of porcine ovarian ornithine decarboxylase in vitro: the role of 3',5'-adenosine monophosphate.

The role of cAMP as a mediator of gonadotropin stimulation of ovarian ornithine decarboxylase (ODC) activity was studied in granulosa cells isolated from small (1--2 mm) porcine ovarian follicles. These cells responded to both FSH and LH with significant increases in intracellular concentration of cAMP. At concentrations of gonadotropins which were saturating for the induction of ODC activity, FSH was a more potent stimulator of both cAMP production and ODC activity than LH. N,O'-Dibutyryl cAMP (1.0--10.0 mM) caused a dose-dependent stimulation of ODC activity which equaled the maximal effect of LH but was significantly less effective than the saturating dose of FSH. 8-Bromo-cAMP was more potent than N,O'-dibutyryl cAMP and as effective as FSH as an inducer of ODC activity. Addition of theophylline, a phosphodiesterase inhibitor, to the incubation medium resulted in a dose-dependent inhibition of ODC activity in both control and gonadotropin-stimulated cells. In contrast, 1-methyl,3-isobutyl xanthine, another phosphodiesterase inhibitor, potentiated effects of both submaximal and maximal effective doses of gonadotropins while producing no effect on basal ODC activity of these cells. The results of this study are consistent with the concept that cAMP can mediate gonadotropin stimulation of ODC in porcine granulosa cells. In addition, this study shows the importance of proper selection of cAMP analogs and phosphodiesterase inhibitors, and their concentration in studying such effects.     

Secretion of plasminogen activator by Sertoli cell enriched cultures.

Conditions were established to assay plasminogen activator (PA) levels in tissue culture medium in which Sertoli cell aggregates, prepared from testes of immature rats, had been cultured. PA activity was measured by determining ¹²⁵I fibrinolysis dependent upon added plasminogen. The PA levels were higher in medium taken from Sertoli cell-enriched cultures treated with follicle-stimulating hormone (FSH) or dibutyryl cAMP. In addition, the fibrin-agar overlay technique was employed to detect plasminogen-dependent lysis zones around Sertoli cell aggregates in culture. Lytic zones appeared earlier and developed faster around FSH-treated Sertoli cell aggregates. Peritubular myoid cells had no detectable PA activity when tested by either technique. The possible physiological role of PA in the testis is discussed.     

Follicle-stimulating hormone amplifies insulin-like growth factor I-mediated activation of AKT/protein kinase B signaling in immature rat Sertoli cells

FSH and IGF-I are both important determinants of testicular development and Sertoli cell function. The present studies were performed to determine the actions of FSH and IGF-I on PI3K/AKT protein kinase signaling in immature rat Sertoli cells. Primary cultures of rat Sertoli cells were prepared from 10-d-old rats. After 7 d in culture, Sertoli cells were treated with IGF-I, FSH, or IGF-I plus FSH. In some experiments cultures were treated with 8-bromo-cAMP (40 microM), (Bu)(2)cAMP (40 microM), or forskolin (10 microM). After treatments, cell lysates were prepared, and the activation state of AKT and cAMP response element-binding protein (CREB) was determined by Western blot analysis using phosphorylation site-specific antibodies. IGF-I had little effect on CREB phosphorylation, but rapidly increased the phosphorylation of AKT in a concentration-dependent manner. Maximal stimulatory effects of IGF-I were observed at 10-20 ng/ml. Treatment with FSH (0.9 IU/ml) or forskolin for 20 min increased CREB phosphorylation, but had little effect on AKT phosphorylation. However, FSH caused a concentration-dependent increase in IGF-I-induced AKT phosphorylation. Longer incubations (1-4 h) with FSH alone resulted in the elevation of AKT phosphorylation concomitant with an increased secretion of IGF-I and decreased production of IGF-binding protein-3, implicating endogenous IGF-I in the action of FSH on AKT phosphorylation. IGF-I- and FSH-dependent AKT phosphorylation was inhibited by LY29400 (10 microM), a PI3K inhibitor, and by IGF-binding protein 3, but not by a PKA inhibitor (H89). The present study demonstrates that immature rat Sertoli cells possess multiple protein kinase signaling cascades that are regulated by FSH. Furthermore, FSH amplifies IGF-I-mediated PI3K/AKT signaling in Sertoli cells. The results provide evidence for intracellular signaling mechanisms that may be required for the proliferation and differentiation of Sertoli cells.     

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.     

Subcellular distribution of high-affinity type IV cyclic AMP phosphodiesterase activity in rabbit ventricular myocardium: relations to the effects of cardiotonic drugs

Rabbit ventricular myocardium contains distinct cytosolic and particulate cyclic AMP (cAMP) phosphodiesterase activities that exhibit characteristics ascribed to a high-affinity type IV cAMP phosphodiesterase activity found in several tissues. The particulate activity associated with sarcoplasmic reticulum vesicles has an apparent Km for cAMP of about 0.3 microM and a Vmax of 2.45 +/- 0.55 nmol/min/mg. Cyclic GMP (cGMP) inhibits hydrolysis measured at 0.25 microM cAMP with an IC50 value of 0.28 microM. In comparison, a ventricular cytosolic high-affinity cAMP phosphodiesterase activity obtained by anion exchange chromatography (Peak III) has an apparent Km of 0.93 microM and a Vmax of 17 +/- 1 nmol/min/mg. Hydrolysis of 0.25 microM cAMP by this cytosolic activity is weakly inhibited by cGMP with an IC50 value of 142 microM. Particulate enzyme activity is 60-fold more sensitive to inhibition by milrinone than is the cytosolic form (Ki = 0.18 versus 11 microM, respectively); the pyridazinone imazodan is a 12-fold more potent inhibitor of the particulate activity than of the cytosolic form (Ki = 1.5 versus 18 microM, respectively). Inhibition of both cytosolic and particulate enzyme activities appears competitive in nature. Solubilization of particulate activity did not significantly alter its affinity for substrate or sensitivity to inhibition by cGMP. In the presence of a submaximally activating concentration of forskolin (0.4 microM), selective phosphodiesterase inhibitors potentiated the activation of protein kinase in isolated ventricular septal slices. Under these conditions, changes in cAMP-dependent protein kinase activity ratios correlated more closely with contractile responses than did changes in intracellular content of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Follicle-stimulating hormone-induced, adenosine 3',5'-monophosphate-mediated movement of immature rat sertoli cells in primary culture.

Sertoli cells dissociated from 10-day-old rat testes form colonies in primary culture in response to FSH. FSH and dibutyryl cAMP stimulated the attachment of Sertoli cells to an equal extent; however, FSH-treated cultures contained a small number of large colonies while dibutyryl cAMP-treated cultures contained a large number of small colonies. This relationship was not altered by the addition of a number of other peptide or steroid hormones. Extracellular cAMP levels and colony density were negatively correlated. Colony size at 24 h of culture was diminished in FSH-treated cultures by the addition of a cAMP antibody at 6 or 12 h of incubation. The addition of cAMP at 24 h to FSH-treated cultures caused a dose-dependent stimulation of colony size but not colony density at 48 h of culture. A point source of cAMP (4 x 10(-3) M in agar) inhibited migration of cells toward the agar spot. An agar spot on the dish substratum containing Sepharose-bound FSH exhibited a halo of cells next to the spot, with a zone lacking cells distal to it. Radioautographs of [125I]iodo-FSH-treated cultures exhibited a nonhomogeneous distribution of silver grains; colony size increased faster than the number of labeled cells. Taken together, the results suggest that 1) FSH is stimulating the attachment of Sertoli cells through an increase in intracellular cAMP, 2) FSH is promoting active aggregation of Sertoli cells in culture through a modulation of extracellular cAMP, and 3) cells with a large amount of bound FSH are acting as centers for aggregation.     

Cellular distribution of phosphodiesterase isoforms in rat cardiac tissue.

We have resolved multiple forms of cyclic nucleotide phosphodiesterase (PDE) in whole rat ventricle and in isolated rat ventricular myocytes by use of anion-exchange high-performance liquid chromatography. One major form, the soluble calmodulin-stimulated PDE, is apparently absent from isolated myocytes. We discern four peaks of PDE activity (designated A-D in the order of their elution) in a soluble fraction obtained from whole rat ventricle. Peak A is stimulated twofold to threefold by the addition of calcium and calmodulin (Ca2+/CalM) and preferentially hydrolyzes cGMP over cAMP (in the presence of Ca2+/CalM, KmcGMP = 1.5 microM, KmcAMP = 17 microM). Peak B has similar affinities for both cAMP and cGMP (half-maximum velocities achieved at 30 microM substrate) and demonstrates positive cooperativity with cAMP but not with cGMP. The hydrolysis of cAMP by peak B is stimulated by cGMP at substrate concentrations up to 20 microM; the maximum effect is seen at 1 microM cAMP (25-fold stimulation by 3 microM cGMP). This pattern of stimulation by cGMP results from two kinetic changes: an increase in the enzyme's apparent affinity for cAMP (apparent KmcAMP decreases from 33 to 11 microM) and the abolition of positive cooperativity. Peaks C and D selectively hydrolyze cAMP, are not stimulated by Ca2+/CalM or cGMP, and differ in their affinities for substrate (peak C, apparent KmcAMP = 7.2 microM; peak D, 0.44 microM). In addition, peak D is much more sensitive than peak C to inhibition by cGMP, cilostamide, rolipram, and milrinone. Ro20-1724 is a slightly more potent inhibitor of peak D than of peak C. Peak D appears to consist of two different enzyme activities, one inhibited by cGMP, cilostamide, and cardiotonic drugs and the other potently inhibited by rolipram. In contrast to whole ventricle, the soluble fraction of isolated rat ventricular myocytes lacks peak A. Three major peaks in myocytes are entirely analogous to peaks B, C, and D of whole ventricle in terms of the NaCl concentration at which they elute, substrate affinities, and stimulation or inhibition by various drugs and effectors. We conclude that the soluble Ca2+/CalM-stimulated PDE in whole rat ventricle is present in nonmyocyte cells.     

Transforming growth factor beta 1 inhibits gonadotropin action in cultured porcine Sertoli cells.

In the present study, we have tested the effects of transforming growth factor beta 1 (TGF beta 1) on FSH action toward aromatase activity and lactate production in cultured Sertoli cells isolated from immature porcine testes. Whereas treatment of Sertoli cells with FSH resulted in a dose-dependent increase (about 7-fold) in aromatase activity (conversion of testosterone into estradiol) (ED50 = 80 ng/ml FSH), the addition of TGF beta 1 reduced this gonadotropin action. The inhibitory effect of TGF beta 1 on FSH aromatase activity was dose dependent (ED50 = 0.1 ng/ml, 4 pM TGF beta 1) with a maximal decrease (about 40%) observed after a long term (48-h) treatment. TGF beta 1 exerted its inhibitory effect on FSH action at the level(s) of cAMP accumulation, exerting no apparent effect on the gonadotropin receptor or at a site(s) related to cAMP action. TGF beta 1 (2 ng/ml) significantly (P less than 0.002) reduced (52% decrease) FSH-stimulated cAMP levels in cultured porcine Sertoli cells. However, such an inhibitory effect of the growth factor was no longer observed when stimulation of cAMP accumulation with FSH occurred in the presence of methyl isobutyl xanthine (0.5 mM), an inhibitor of cAMP-phosphodiesterase activity. This observation suggests that TGF beta 1 decreased cAMP levels by increasing catabolism of the cyclic nucleotide through an enhancement of cAMP-phosphodiesterase activity. The inhibitory effect of TGF beta 1 was not limited to the action of FSH on aromatase activity but also extended to the gonadotropin action (mediated by cAMP) on lactate production. As for the inhibitory effect of TGF beta 1 on FSH-induced aromatase activity, the inhibitory effect of the growth factor on FSH-stimulated lactate production was dose and time dependent with a maximal decrease (about 30%) observed in the picomolar range (1 ng/ml, 40 pM) after 48 h treatment with TGF beta 1. In conclusion, the present study demonstrates that TGF beta 1 attenuates FSH action on Sertoli cell activity and that such inhibitory action is potentially exerted through a decrease in cAMP levels. Because of the local production of TGF beta 1, it is suggested that the effects of the growth factor reported here might be exerted in the context of the testicular paracrine mechanisms.