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C S Rani

Publications and source records attributed to C S Rani.

At least 19 recordsLinked to original sources
PubMed1992

Carbachol-induced decrease in thyroid cell adenylyl cyclase activity is independent of calcium and phosphodiesterase activation.

The mechanism of adenylyl cyclase desensitization by carbachol, an agent that stimulates polyphosphoinositide hydrolysis, was studied in thyroid cells. Incubation of cultured dog thyroid cells with 10 microM carbachol for 2-4 hr reduced the subsequent thyrotropic hormone (TSH) stimulation of adenylyl cyclase activity of membrane preparations by approximately 40%. This inhibition was reversed by atropine, occurred even in a Ca(2+)-free medium containing ethylene glycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid, and was not reproduced by the Ca2+ ionophore A23187. The carbachol effect was not prevented by simultaneous incubation of cells with either isobutylmethylxanthine, an inhibitor of phosphodiesterase, or H-7, an inhibitor of protein kinase. Pretreatment of cells with pertussis toxin to inactivate the Gi inhibitory protein also failed to affect the carbachol inhibition. Although carbachol did not reduce the basal or the TSH-stimulated cyclase activities when added to membranes directly during the assay, exposure of cells to carbachol for 2-4 hr resulted in long lasting inhibition of TSH-stimulated cyclase activity (for at least 24 hr); recovery was seen by 48 hr after its removal. Carbachol pretreatment had no effect on 125I-TSH binding to membranes but reduced the cyclase stimulation by not only TSH but also cholera toxin, guanosine 5'-O-(3-thio)triphosphate, and forskolin; it also significantly reduced the cholera toxin-mediated AD[32P]-ribosylation of Gs in membranes. These data indicate that carbachol-induced inhibition of adenylyl cyclase occurs beyond the level of TSH receptor binding and that Gs is a possible site of its action. Thus, in dog thyroid cells, carbachol, via muscarinic receptors, can reduce the adenylyl cyclase activity by a process that does not involve Ca2+ or activation of phosphodiesterase.

Adenylate Cyclase Toxin↗
PubMed1991

Protein kinase C activation mimics but does not mediate thyrotropin-induced desensitization of adenylyl cyclase in cultured dog thyroid cells.

The mechanism and site(s) of the defect responsible for desensitization to hormone stimulation of adenylyl cyclase (AC) vary with cell type. Plasma membrane preparations were assayed after treatment of primary cultured dog thyroid cells to determine the role of the TSH receptor, stimulatory and inhibitory guanine nucleotide binding proteins (Gs and Gi), and catalytic unit in AC desensitization. Exposure of cells to TSH or the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), caused time dependent decreases in TSH-stimulated AC and [125I]TSH binding with approximately 50% decreases seen after 18 h; Bt2cAMP was unable to reproduce the TSH effect. Whereas TSH treatment caused concomitant decreases (approximately 25%) in both cyclase activity and [125I]TSH binding after 2 h, TPA treatment decreased AC activity after 6 h and binding only after 18 h. The protein kinase C inhibitor, H-7, prevented TPA-induced but not TSH-induced effects on AC and hormone binding. Membrane AC activation by cholera toxin or forskolin was not altered by 18 h pretreatment of cells with TSH or TPA, indicating that these agents had no apparent effect on intrinsic functionality of either Gs or the catalytic unit. TSH or TPA pretreatment of cells reduced subsequent toxin-mediated AD[32P]-ribosylation of Gs and Gi in isolated membranes. However, the TSH- and TPA-induced decreases in AD[32P]-ribosylation and desensitization do not appear to be due to endogenous ribosylation of G proteins, since treatment of cells with pertussis toxin, for example, to endogenously ribosylate Gi, both increased TSH-stimulated AC activity and failed to affect the ability of TSH or TPA to desensitize. Thus, in this system, although specific hormone-induced AC desensitization and receptor down-regulation conform to several aspects of classic homologous processes, similar effects are also induced by a nonreceptor (phorbol ester) pathway; desensitization, however, can precede down-regulation, possibly due to receptor-Gs uncoupling.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Inhibition of intermediary metabolism by amiodarone in dog thyroid slices.

Amiodarone, an iodine-containing antiarrhythmic drug, has been reported to interfere with thyroid function and thyroid hormone metabolism. We studied the effects of amiodarone on basal and agonist [thyroid-stimulating hormone (TSH), phorbol ester, or carbachol]-stimulated glucose oxidation, 32PO4 incorporation into phospholipids, and adenosine 3',5'-cyclic monophosphate (cAMP) concentration in dog thyroid slices. Slices were preincubated with amiodarone at 37 degrees C for 1 h before the addition of agonist and the appropriate radioisotope. cAMP stimulation was measured after 20 min, glucose oxidation for 45 min, and 32PO4 incorporation into phospholipids for 2 h. Amiodarone (0.5 mM) had no effect on basal 14CO2 formation or 32PO4 incorporation into phospholipids but significantly inhibited TSH, phorbol ester, and carbachol stimulation of these parameters. It also inhibited cAMP stimulation by TSH. Inhibition of TSH-stimulated [14C]glucose oxidation was also obtained with another iodide-containing compound, iopanoic acid (0.5 mM), but not with iothalamate (up to 10 mM). Inhibition by amiodarone was still present, but to a lesser extent, when it was added at the same time as the agonist. Inhibition of stimulated [14C]glucose oxidation persisted even after the slices were incubated without amiodarone for 6 h. Inhibition by amiodarone, in contrast to that by inorganic iodide, was not prevented by 1 mM methimazole added at the same time as amiodarone. These results indicate that the inhibitory effects of amiodarone on thyroid function are not due to dissociation of iodide from the molecule.

Amiodarone↗
PubMed1990

Amiodarone effects on thyrotropin receptors and responses stimulated by thyrotropin and carbachol in cultured dog thyroid cells.

Amiodarone is an antiarrhythmic drug that often induces thyroid disorders. Its effects on several aspects of thyroid function were studied using cultured dog thyroid cells. Within 5-60 min of incubation of cell membranes with amiodarone, there were profound changes in adenylate cyclase activity and TSH receptor binding. Amiodarone specifically decreased TSH-stimulated adenylate cyclase activity, but not the basal or forskolin-stimulated activities, while it increased the binding of 125I-labeled TSH to its receptors. Significant effects were seen with 5-10 microM amiodarone, with maximal effects at 50-100 microM, when TSH-stimulated adenylate cyclase activity was completely blocked and the labeled TSH binding increased 4- to 5-fold over control. These effects of amiodarone were reversible, since membranes exposed to 50 microM amiodarone for 1 h exhibited normal binding and cyclase activities, when amiodarone was removed by washing before the assay. The above effects of amiodarone were also observed when cells, instead of membranes, were treated with the drug, although the magnitude of changes was less than in membranes. Lower concentrations of amiodarone (10-25 microM) caused significant inhibition of iodide organification, without affecting iodide uptake, while higher concentrations (50-100 microM) inhibited organification by nearly 75% and uptake by about 20%. Amiodarone (10-100 microM) also inhibited [3H]2-deoxy-glucose uptake and the increase in intracellular calcium concentration in response to TSH and carbachol. In contrast to membranes, treatment of cells with amiodarone caused persistent inhibition of TSH-stimulated cAMP formation and iodide organification even 24-48 h after removal of the drug. However, amiodarone had no effect on cell viability, as judged by trypan blue exclusion and ability to remain attached to the culture dishes. These results suggest that amiodarone has specific inhibitory effects on agonist-stimulated functions in thyroid cells, possibly by interfering with TSH-receptor interactions and also at the level of cholinergic receptors.

Adenylyl Cyclases↗
PubMed1989

Effect of iodide on glucose oxidation and 32P incorporation into phospholipids stimulated by different agents in dog thyroid slices.

Since iodide (I-) inhibits TSH stimulation of cAMP formation, which mediates most of the effects of the hormone, it has been assumed that this accounts for the inhibitory action of iodide on the thyroid. However, TSH stimulation of 32P incorporation into phospholipids and stimulation of thyroid metabolism by other agonists, such as carbachol, phorbol esters, and ionophore A23187, is not cAMP mediated. The present studies examined the effect of iodide on stimulation of glucose oxidation and 32P incorporation into phospholipids by TSH and other agonists to determine if the inhibition of cAMP formation was responsible for the action of iodide. Preincubation of dog thyroid slices for 1 h with iodide (10(-4) M) inhibited TSH-, (Bu)2cAMP-, carbachol-, methylene blue-, 12-O-tetradecanoyl phorbol-13-acetate-, ionophore A23187-, prostaglandin E1-, and cholera toxin-stimulated glucose oxidation. I- also inhibited the stimulation by TSH, 12-O-tetradecanoyl phorbol-13-acetate, carbachol, and ionophore A23187 of 32P incorporation into phospholipids. The inhibition was similar whether iodide was added 2 h before or simultaneously with the agonist. I- itself sometimes stimulated basal glucose oxidation, but had no effect on basal 32P incorporation into phospholipids. The effects of iodide on basal and agonist-stimulated thyroid metabolism were blocked by methimazole (10(-3) M). When dog thyroid slices were preloaded with 32PO4 or [1-14C]glucose, the iodide inhibition of agonist stimulation disappeared, suggesting that the effect of iodide involves the transport process. In conclusion, I- inhibited stimulation of glucose oxidation and 32P incorporation into phospholipids by all agonists, indicating that the effect is independent of the cAMP system and that iodide autoregulation does not only involve this system. Oxidation and organification of iodide are necessary for the inhibition. The ability of iodide to decrease glucose and 32PO4 transport may play an important role in thyroid autoregulation.

Alprostadil↗
PubMed1989

Intracellular Ca2+ mobilization by thyrotropin, carbachol, and adenosine triphosphate in dog thyroid cells.

The effect of TSH, carbachol (CC), and ATP on intracellular calcium concentration ([Ca2+]i) in primary cultures of dog thyroid cells was examined using the fluorescent Ca2+ indicator fura-2. TSH caused an increase in [Ca2+]i at 37 C, but not 22 C, while it increased cAMP formation in these cells at both 22 and 37 C. CC and ATP increased [Ca2+]i at both 22 and 37 C. The CC-induced increase in [Ca2+]i was under muscarinic receptor control, and it was biphasic, with an initial spike followed by a sustained increase at a lower level. TSH and ATP were weaker agonists compared to CC, since maximal doses of TSH (100-500 mU/ml) and ATP (100-500 microM) increased [Ca2+]i by 40-70% over basal levels, compared to a 2- to 4-fold increase in [Ca2+] induced by maximal doses of CC (10-50 microM). The TSH-induced increase in [Ca2+]i was transient, returning to basal levels within 1-2 min after application of the agonist. All three agents were able to transiently increase [Ca2+]i to be internal stores. In the presence of the inorganic Ca2+ channel blockers La3+, Ni2+, and Co2+, the peak [Ca2+]i change was little affected, while the persistent response to CC and ATP was blocked, indicating dependence of this phase on influx of Ca2+. Paradoxically, these channel blockers abolished the effect of TSH on [Ca2+]i. TSH stimulation of cAMP formation was also inhibited 80-90% by these blockers, but not in Ca2+-free/EGTA buffer. These results suggest that the Ca2+ channel blockers may have actions in addition to inhibition of Ca2+ entry in these cells. TMB-8 [8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate HCl] specifically blocked both the initial and sustained increase induced by CC, while having no effect on ATP or TSH-induced [Ca2+]i, suggesting that TMB-8 may not be a general antagonist of Ca2+ mobilization. Activators of protein kinase-C, such as phorbol esters or an analog of diacylglycerol, inhibited the [Ca2+]i rise induced by all the three agonists used, indicating a regulatory role of protein kinase-C activation on [Ca2+]i in these cells. In FRTL-5 cells, [Ca2+]i was also increased by TSH and ATP, but not by CC. ATP, however, was a more effective agonist than in dog thyroid cells, while TSH increased [Ca2+]i by a similar magnitude in both cell types. The results of the present study demonstrate that TSH, albeit of lesser potency than CC, increases [Ca2+]i by causing intracellular Ca2+ mobilization in cultured dog thyroid cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1988

Comparison of effects of thyrotropin, phorbol esters, norepinephrine, and carbachol on iodide organification in dog thyroid slices, follicles, and cultured cells.

The effect of TSH, phorbol ester, norepinephrine (NE), and carbachol, agents known to influence thyroid metabolism, was compared on iodide organification in dog thyroid slices, freshly isolated follicles, and cultured cells. TSH stimulated iodide organification in all three types of preparations, and this effect was mimicked by (Bu)2cAMP. In contrast, the phorbol ester, tetradecanoyl phorbol acetate (TPA) stimulated iodide organification in slices and follicles but inhibited it in cells. The dose and time required for these divergent effects were similar. Other stimulators of protein kinase C such as aplysiatoxin and teleocidin mimicked the effects of TPA, and these effects were partially reversed by H-7, an inhibitor of protein kinase C. Pretreatment of cells with TPA for 4 h did not affect TSH-stimulated cAMP production, but TPA inhibited iodide organification in cells even in the presence of TSH or (Bu)2cAMP. Similarly NE and carbachol stimulated iodide organification in follicles but inhibited it in cells under basal as well as TSH-stimulated conditions. These effects of NE and carbachol were via alpha 2-adrenergic and muscarinic cholinergic receptors, respectively. However, NE and carbachol inhibited TSH-stimulated cAMP production in both follicles and cells. In thyroid cells, carbachol inhibited uptake and increased efflux of iodide within minutes. TPA also produced similar effects after longer periods of incubation, where an inhibition of uptake was seen by 1 h and an increase in efflux by 2 h. NE had a marginal inhibitory effect on uptake and had no effect on efflux of iodide. In contrast to these agents, TSH increased uptake but not efflux of iodide. The present results suggest that the response of the freshly isolated tissue to phorbol esters, NE and carbachol differs from that of cells in culture with respect to an important metabolic function of the thyroid gland. These agents seem to have direct effects on iodide transport and organification unrelated to their effects on cAMP production.

1-Methyl-3-isobutylxanthine↗
PubMed1988

Effects of thyrotropin, carbachol, and protein kinase-C stimulators on glucose transport and glucose oxidation by primary cultures of dog thyroid cells.

Thyroid glucose metabolism can provide NADPH and H2O2 for thyroid hormone synthesis. Several agents stimulate glucose oxidation in thyroid slices, but little is known about glucose transport in this tissue. In the present study, various thyroid stimulators were tested on glucose transport and oxidation using primary cultures of dog thyroid cells. After preincubating the cells with the agonists, glucose uptake was measured by adding 2-deoxy-D-[1-3H]glucose [( 3H]2-DOG) for 5 min, and glucose oxidation was assessed during a single 60-min incubation with agonist and D-[1-14C]glucose. TSH (0.1-10 mU/ml), 12-O-tetradecanoyl phorbol-13-acetate (TPA; 10(-8)-10(-6) M), and carbachol 10(-6)-10(-2) M) stimulated [3H]2-DOG transport and glucose oxidation in a dose-dependent manner. The effect of TSH appears to be mediated by cAMP, since N6,2'-O-dibutyryl cAMP, 8-bromo-cAMP, cholera toxin, and isobutylmethylxanthine also stimulated [3H]2-DOG transport. Norepinephrine, which had no effect by itself on either transport or oxidation, inhibited TSH stimulation of [3H]2-DOG transport via an alpha 2-adrenergic receptor. The mechanism of the TPA and carbachol effect does not involve cAMP. A combination of maximal amounts of TSH or bromo-cAMP and carbachol or TPA produced additive effects on transport, while addition of TPA with carbachol produced no such additive effect. Kinetic analysis of 2-DOG transport indicated that all three agonists reduced the Km and increased the maximum velocity. Basal 2-DOG transport was increased in Ca2+-free medium, with or without EGTA, or in the presence of calcium channel blockers such as La3+ or Mn2+. In the presence of such increased basal glucose transport, TSH further stimulated it when Mn2+ was used, had no effect in Ca2+-free buffer plus EGTA, and caused an inhibition with La3+. Such inhibition was not caused when N6,2'-O-dibutyryl cAMP was used in the presence of La3+. Carbachol and TPA did not stimulate transport when the Ca2+ channel blockers were used, but a small increase was seen in Ca2+-free buffer containing EGTA. TSH stimulation of cAMP production was also diminished in the presence of La3+, but enhanced in the presence of Mn2+. The calmodulin inhibitor W-7 and the intracellular Ca2+ blocker 8-N,N-diethylamino octyl-3,4,5-trimethoxybenzoate hydrochloride diminished the stimulation of [3H]2-DOG transport and glucose oxidation induced by TSH, carbachol, and TPA. These data indicate that thyroid glucose transport and glucose oxidation are regulated by both cAMP-dependent and cAMP-independent systems.

1-Methyl-3-isobutylxanthine↗
PubMed1986

Adenosine 3',5'-monophosphate-mediated induction of 17 alpha-hydroxylase and C 17-20 lyase activities in cultured mouse Leydig cells is enhanced by inhibition of steroid biosynthesis.

We recently reported that treatment of mouse Leydig cell cultures for 5 days with LH or cAMP caused an induction of the microsomal cytochrome P-450 activities 17 alpha-hydroxylase and C17-20 lyase. We also have shown that the microsomal P-450s are very sensitive to oxygen-mediated loss of these activities and that this decrease is enhanced by steroids produced during acute cAMP stimulation of Leydig cells. In the present study, we investigated whether steroids produced during chronic cAMP treatment of Leydig cells limit the extent of induction of 17 alpha-hydroxylase and C17-20 lyase. Treatment of Leydig cell cultures with 8-bromo-cAMP in the presence of aminoglutethimide, an inhibitor of cholesterol side-chain cleavage, resulted in a 4- to 7-fold enhancement of cAMP-mediated induction of the 17 alpha-hydroxylase and C17-20 lyase activities and, after 11 days of treatment, completely restored the activities to those found in freshly isolated Leydig cells. Treatment with aminoglutethimide in the absence of cAMP had no effect on these enzyme activities. Addition of the steroid products androstenedione and/or testosterone (5 microM) to cAMP-plus amino-glutethimide-treated cultures caused a significant reduction in cAMP-mediated induction of microsomal P-450, while addition of estradiol (50 nM) had no significant effect. 3 beta-Hydroxysteroid dehydrogenase-isomerase, another microsomal enzyme that is not a P-450 enzyme, was not induced by cAMP in either the presence or absence of aminoglutethimide. The data suggest that Leydig cell microsomal P-450 activities are maintained in vivo by a balance between two processes: cAMP-mediated induction and steroid product-induced degradation.

Aldehyde-Lyases↗
PubMed1986

Effects of phorbol esters on metabolic variables in the thyroid.

Since 12-O-tetradecanoyl-phorbol-13-acetate (TPA) reproduced some of the effects of TSH on phosphorylation of polypeptides in the thyroid, its effects on several thyroid metabolic variables were investigated. Like TSH, TPA stimulated glucose oxidation, iodide organification, and 32P incorporation into phospholipids in thyroid slices. However, in contrast to TSH, it did not augment cAMP accumulation. An inactive phorbol ester, 4 alpha-phorbol, did not reproduce any of the effects of TPA. An initial incubation of thyroid slices with TPA decreased the stimulation of cAMP, glucose oxidation, and colloid droplet formation induced by TSH. However, an initial incubation with TPA did not modify the subsequent stimulation of glucose oxidation induced by (Bu)2 cAMP. TPA potentiated the ability of TSH to desensitize the adenylate cyclase system. Although both TPA and TSH increased 32P incorporation into phospholipids, the patterns were different when individual phospholipids were examined. These results indicate another regulatory mechanism for thyroid cell functions independent of cAMP.

Animals↗
PubMed1985

Thyroid cell responses to thyrotropin and 12-O-tetradecanoyl-phorbol-13-acetate: translocation of protein kinase C and phosphorylation of thyroid cell polypeptide substrates.

Not all of the effects of thyroid-stimulating hormone (TSH) on the thyroid are mediated by activation of the adenylate cyclase-cyclic AMP system, indicating that other control systems must also exist. Although a calcium-phospholipid-dependent protein kinase (protein kinase C) and specific substrates had been identified in thyroid tissue, their responsiveness to TSH and other stimulators has not been determined. In thyroid cells which had been preloaded with [32P]orthophosphate, TSH and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) increased the phosphorylation of a 33K polypeptide substrate within 5 min in a dose-dependent fashion. The effect was observed with 1 mU/ml TSH and 3 nM TPA and was maximal with 100 mU/ml TSH and 100 nM TPA. The biologically inactive analog of TPA, 4 alpha-phorbol, had no effect. Isobutylmethylxanthine (IBMX) decreased the phosphorylation of the 33K polypeptide and inhibited the effect of TSH and TPA, indicating that the phosphorylation is not mediated by cyclic AMP. TSH and IBMX, but not TPA, augmented phosphorylation of a 38K polypeptide, suggesting involvement of cyclic AMP. In contrast TPA, but not TSH, increased the phosphorylation of 58K and 28K polypeptides. TSH, but not TPA or 4 alpha-phorbol, elevated the cyclic AMP level of thyroid slices. Incubation of thyroid slices with TSH or TPA significantly decreased protein kinase C activity in the 100,000g cytosol fraction and increased it in an extract of plasma membranes. The effect was present within 5 min and was maximal by 30 min. The effect was observed with 100 mU/ml TSH or 1 nM TPA. The stimulation by TSH or TPA of protein kinase C and its translocation from the cytosol to the plasma membranes of thyroid tissue may provide another mechanism for control of thyroid cell metabolism.

1-Methyl-3-isobutylxanthine↗
PubMed1985

Differences in the behavior of luteinizing hormones of various species at the rat gonadal cell receptor site.

The ability of different LH-like hormones, such as hCG, PMSG/equine (e) CG, ovine (o) LH, eLH, and rat (r) LH, to bind to and stimulate steroidogenesis in two types of rat gonadal cells was studied under the same experimental conditions. In both Leydig and granulosa cells, the maximal steroidogenic responses elicited by optimal doses of different LHs present during a 2-h incubation were comparable. However, if the cells were exposed to the different LHs for a brief period and then subjected to interference with hormone action by removing the unbound hormone from the medium by washing or adding specific antisera, differences were observed in the amount of steroid produced during subsequent incubation in hormone-free medium. Thus, in the case of hCG, either of these procedures carried out at 15 or 30 min of incubation had little inhibitory effect on the amount of steroid produced at 2 h, the latter being similar to that produced by cells incubated in the continued presence of hCG for 2 h. With eCG and rLH, the effect was dramatic, in that there was a total inhibition of subsequent steroidogenic response. In cells exposed to eLH and oLH, inhibition of subsequent steroidogenesis due to either removal of the free-hormone or addition of specific antisera at 15 or 30 min was only partial. Although all of the antisera used were equally effective in inhibiting the steroidogenic response to respective gonadotropins when added along with hormones at the beginning of incubation, differences were observed in the degree of inhibition of this response when the same antisera were added at later times of incubation. Thus, when antisera were added 60 min after the hormone, the inhibition of steroidogenesis was total (100%) for eCG, partial (10-40%) for eLH and oLH, and totally lacking in cells treated with hCG. From this, it appears that hCG bound to the receptor probably becomes unavailable for binding to its antibody with time, while in the case of eCG and other LHs used, the antibody can still inhibit the biological activity of the hormone. Studies with 125I-labeled hormones further supported the conclusion that hCG differs from all other LHs in being most tightly bound and, hence, least dissociable, while eCG and rLH dissociate most readily; oLH and eLH can be placed in between these hormones in the extent of their dissociability.

Animals↗
PubMed1983

Development of catecholamine responsiveness in granulosa cells from preovulatory rat follicles--dependence on preovulatory luteinizing hormone surge.

Factors responsible for development of catecholamine (CA) responsiveness in granulosa cells (Gc) of preovulatory follicles from immature rats injected with 10 IU of pregnant mare's serum gonadotropin (PMSG) on Day 26 were studied. CA did not stimulate cyclic AMP (cAMP) production in whole follicles isolated before (morning) or after (evening) the preovulatory gonadotropin surge on Day 28, while newly formed corpora lutea found on Day 29 responded to CA. Gc from the preovulatory follicles did not respond to CA when tested immediately after isolation. Gc were cultured for various periods in Eagle's MEM without serum and subsequently tested for a possible stimulation of cAMP and steroidogenic responses by CA. In Gc from follicles isolated in the morning (AM-Gc) and cultured for 12 or 24 h, no response to CA was found, while the Gc isolated in the evening (PM-Gc) and cultured similarly for 12 h showed a marked response to CA both in stimulation of cAMP and progesterone production. By a total or partial elimination of the gonadotropin surge, using pentobarbital in combination with luteinizing hormone (LH) or using specific antisera to LH or follicle-stimulating hormone (FSH), it was found that previous exposure to LH was necessary for the PM-Gc to develop CA responsiveness during culture. Further, it was possible to induce CA responsiveness in AM-Gc by treatment of rats with LH for a short period in vivo followed by a period of culture. The appearance of CA responsiveness in PM-Gc cultured for 12 h was abolished when cycloheximide (5 micrograms/ml) was present during culture. It appears that the following three conditions have to be satisfied for the isolated Gc to develop CA responsiveness: 1) exposure to LH in vivo, 2) culture for a short period, and 3) an active protein synthesis. It is concluded that under physiological conditions the process of luteinization is associated with acquisition of responsiveness to CA and this process depends on the LH component of the preovulatory gonadotropin surge.

Animals↗
PubMed1983

Studies on corticotropin-induced desensitization of normal rat adrenocortical cells.

The effects of prior exposure of normal rat adrenocortical cells to ACTH on the responsiveness of the cells to subsequent stimulation with the hormone have been studied. ACTH induces a time- and concentration-dependent refractoriness of both cAMP formation and steroidogenesis. Desensitization of either response was observed only upon activation of the response. Thus, both ACTH and 8-Br-cAMP caused desensitization of the steroidogenic response. The ACTH-induced desensitization of steroidogenesis, however, was completely prevented by blocking the steroidogenic action of ACTH with aminoglutethimide during exposure of cells to the hormone. Aminoglutethimide had no effect on ACTH-induced desensitization of the cAMP response. Studies with analogs of the hormone also confirmed that induction of desensitization of the steroidogenic response is independent of the desensitization of the cAMP response. Binding studies showed that the insignificant decrease in ACTH receptors could not account for the large changes in the responsiveness induced by prior exposure of cells to ACTH. Desensitization of the steroidogenic response appears to result from a defect in the rate-limiting first step of the steroidogenic pathway, namely conversion of cholesterol to pregnenolone.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1981

Follicle-stimulating hormone induction of luteinizing hormone receptor in cultured rat granulosa cells: an examination of the need for steroids in the induction process.

The induction of LH receptors by FSH in cultured rat granulosa cells and the effects of ovarian steroids on this process were examined. Granulosa cells were isolated from the ovaries of untreated immature rats (25 days old) and cultured with highly purified FSH (Sairam; 250 ng/ml). After culture (48-96 h in chemically defined media), both the binding of [125I]hCG and the responsiveness (cAMP and progesterone production) to an acute LH stimulus (100 ng/ml; NIH B8) were measured. The appearance of LH/hCG-binding sites and LH responsiveness indicates the presence of functional LH receptors. The induction of LH receptors by FSH requires a lag period of 24-48 h. After 48 h, the concentration of LH receptors in cultured granulosa cells continues to increase with time in culture with FSH; the continuous presence of FSH is required to maintain the induction process. If granulosa cells are cultured without hormone for 24-48 h before FSH is added, no induction of LH receptors occurs. However, if 17 beta-estradiol (5 X 10(-7) M) is added during this initial period, then the cells are responsive to the later addition of FSH. This maintenance of FSH responsiveness is not observed when dihydrotestosterone or progesterone is substituted for 17 beta-estradiol in the initial culture period. The FSH-dependent induction of LH receptors in cultured rat granulosa cells can be blocked if an inhibitor of steroidogenesis, such as aminoglutethimide phosphate (AGP; 1 mM), is added along with FSH at the initiation of the culture. The inclusion of progesterone, dihydrotestosterone, or 17 beta-estradiol (but not 20 alpha-dihydroprogesterone) in the culture together with FSH and AGP will overcome the inhibition by AGP and restore the induction of LH receptors. The results suggest that steroids produced by the developing follicle can modulate the FSH-dependent induction of LH receptors, and this may play a role in the development of follicular responsiveness to LH.

Animals↗