Association of PKC delta and active Src in PMA-treated MCF-7 human breast cancer cells.

Phorbol ester treatment of MCF-7 cells led to the tyrosine phosphorylation and activation of PKC delta. However, through Western blot analysis and in vitro immunecomplex kinase assays, we detected a differential localization of tyrosine-phosphorylated PKC delta and catalytically active PKC delta. Catalytically active PKC delta was concentrated in Triton X-100 solubilized-membrane fractions while tyrosine-phosphorylated PKC delta was localized to the cytosol fraction. Phorbol ester treatment of MCF-7 cells stimulated both the time-dependent in vivo association of Src with PKC delta, evidenced in Src immunoprecipitates by the co-immunoprecipitation of PKC delta, and activation of Src, evidenced in Src immunoprecipitates as an increase in reactivity with a Src antibody (clone 28) reactive only with active Src (dephosphorylated on residue 530) and in Src and PKC delta immunoprecipitates by an increase in Src kinase activity. While our data are consistent with reports in the literature showing the activator/stimulus-dependent tyrosine phosphorylation of PKC delta, our data show that the tyrosine phosphorylation of PKC delta is not essential for kinase activity. These results are the first to demonstrate an in vivo association between PKC delta and active Src in the absence of over-expression of either PKC delta or Src, and support the association of Src and PKC delta towards a physiological function.

Characterization of recombinant RI beta and evaluation of the presence of RI beta protein in rat brain and testicular extracts.

Based upon recent reports that the mRNA from the regulatory (R) RI beta subunit of cAMP-dependent protein kinase (PKA) was expressed in testicular extracts, we determined whether testicular extracts exhibited RI beta protein. To accomplish this goal, we initially determined the fundamental labeling and ionic characteristics of recombinant RI beta. Recombinant RI beta eluted from DEAE-cellulose with a salt concentration (of 0.075 M) equivalent to its elution position from soluble mouse brain extracts with catalytic subunit-free RI alpha. As predicted by its amino acid sequence homology to RI alpha, recombinant RI beta was not phosphorylated by PKA but was labeled specifically with 8-azido-adenosine 3':5'-[32P]monophosphate (8-N3[32P]cAMP). Additionally, RI antisera reacted equally with RI alpha (47 kDa) and recombinant RI beta (53 kDa). However, recombinant RI beta exhibited an unexpectedly basic pI of 6.65-6.85. By using a pH gradient for isoelectric focussing that allowed for clear focussing of 8-N3[32P]cAMP-labeled recombinant RI beta, 8-N3[32P]cAMP-labeled RI beta was readily detected by two-dimensional gel electrophoresis in rat brain particulate extracts and exhibited a pI equivalent to that of recombinant RI beta. The 53-kDa RI beta was undetectable either by its immunoreactivity or upon photoaffinity labeling with 8-N3[32P]cAMP by one or two-dimensional gel electrophoresis in soluble or particulate extracts of testes of 14-day-old, 45-day-old, or adult rats or in epididymal sperm. However, 8-N3[32P]cAMP-labeled RI beta was detected, albeit in very small levels, by two-dimensional electrophoresis upon separation of PKAs in testes of 14-day-old rats by DEAE-cellulose chromatography but was absent in equivalent extracts from adult rat testes. These results demonstrate that the unexpectedly basic pI of RI beta allows for its clear separation by two-dimensional electrophoresis from the RII proteins and therefore allows for its unambiguous identification. Further studies, however, are required to resolve the basis for the apparent disparity in testis RI beta mRNA and protein.

Regulation of delta protein kinase C during rat ovarian differentiation.

Studies were undertaken to classify protein kinase C (PKC) forms present in rat corpora lutea and to begin to evaluate their regulation during ovarian differentiation. Hydroxyapatite (HAP) column chromatography of rat luteal tissue revealed the presence of multiple forms of PKC (alpha, beta, delta, zeta). Identification of the PKC isoforms was based upon elution positions from HAP column chromatography and immunoreactivity. The delta PKC isoform was identified as the major Ca(2+)-independent form of PKC present in rat luteal tissue. The Ca(2+)-independent, lipid-dependent phosphorylation of the 80-kDa delta PKC was readily detectable in soluble luteal extracts and was shown to reflect autophosphorylation of delta PKC. To evaluate the regulation of PKC isoforms during ovarian differentiation, PKC protein levels were compared between preovulatory follicle-enriched ovaries and corpora lutea obtained on day 16 of pregnancy. Levels of delta PKC protein were greatly elevated in corpora lutea compared to levels in preovulatory follicles. In contrast, levels of alpha and beta PKC protein remained constant while levels of zeta PKC were slightly higher in the follicular than the luteal extract. Levels of delta PKC mRNA were also higher in corpora lutea than in preovulatory follicles. These results are the first to demonstrate the physiological regulation of delta PKC with follicular differentiation into corpora lutea and implicate a role for this prominent PKC form in the corpus luteum during pregnancy.

cAMP-dependent protein kinases in the rat testis: regulatory and catalytic subunit associations.

Based upon recent reports that the rat testis exhibits mRNAs for cAMP-dependent protein kinase (A-kinase) regulatory (R) subunits RI alpha, RI beta, RII alpha, and RII beta, this study was designed to identify R proteins present in extracts of germ cell-rich testis from adult and Sertoli cell-enriched, germ cell-poor testis from 14-15-day-old rats. Following separation by DEAE-cellulose, R subunits were identified by Mr: (a) upon labeling with 8-N3[32P]cAMP and 32P in an RII phosphorylation reaction and; (b) by Western blot analysis using R-specific antibodies on one- and two-dimensional gel electrophoresis. Elution of R subunits as catalytic (C) subunit-free dimers or in association with C subunits to form holoenzyme was determined by their sedimentation characteristics on sucrose gradient centrifugation in conjunction with their cAMP-stimulated activation characteristics on Eadie-Scatchard analysis. Soluble extracts of testes, from both adult and 14-15 day-old rats, showed the presence of a prominent type I holoenzyme containing RI alpha subunits (47 kDa, peak 1), a minor type II holoenzyme, containing RII beta subunits (52 kDa, peak 2), and a second, more abundant, type II holoenzyme peak containing predominantly RII alpha and, to a lesser extent RII beta subunits (peak 3). The 53 kDa RI beta protein predicted by mRNA studies was only tentatively identified by Western blot analysis. Testes extracts of 14-15-day-old, but not adult, rats exhibited high levels of C subunit-free RI alpha, a result not predicted by mRNA studies. This latter result may be attributable to direct RI alpha regulation or to indirect RII beta regulation at a time during testis development prior to germ cell maturation.

Calcium-independent phospholipid/diolein-dependent phosphorylation of a soluble ovarian Mr 80,000 substrate protein: biochemical characteristics.

Soluble ovarian extracts were incubated with protein kinase effectors in the presence of [gamma 32P]ATP and proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Autoradiograms revealed phosphorylation of an ovarian Mr = 80,000 substrate in the presence of EGTA ([ethylenebis(oxyethylenenitrilo)]tetraacetic acid), phosphatidylserine and 1,2-diolein. In contrast to a classical response pattern to C-kinase effectors, the ovarian Mr = 80,000 phosphorylation was inhibited by 2 x 10(-7) M or greater free Ca2+. The ovarian Mr = 80,000 substrate was distinguished from the myristoylated acidic Mr = 80,000 C-kinase substrate of brain tissue on the basis of heat stability and phosphorylative response to effectors. Phosphorylation of the exogenous substrate myelin basic protein by DEAE-resolved ovarian kinase showed the variant effector dependence, maximal in the presence of EGTA, phosphatidylserine and 1,2-diolein. Finally, the effect of Ca2+ on ovarian Mr = 80,000 [32P]phosphate content could not be accounted for by post-phosphorylation activities, or by DEAE-resolvable or hydroxylapatite-resolvable inhibitory activities.

Phosphorylation-independent desensitization of the luteinizing hormone/chorionic gonadotropin receptor in porcine follicular membranes.

Phosphorylation of several G protein-coupled receptors mediates desensitization. This study determined whether LH/CG receptor was phosphorylated under conditions that promoted human CG (hCG)-induced desensitization. Cell-free desensitization of LH/CG receptor-mediated adenylylcyclase activity in porcine follicular membranes occurred in the presence of GTP and was time- and hCG dose-dependent, reaching 36-52% upon preincubation at 30 C for 40 min with 1.0 micrograms/ml hCG. However, under conditions that promoted GTP-dependent desensitization, there was no apparent phosphorylation of LH/CG receptor (obtained via immunoprecipitation) by endogenous membrane-associated protein kinases using [gamma-32P]GTP or [gamma-32P]ATP as phosphate donor. On the other hand, LH/CG receptor (88-90 kilodaltons) from both control and hCG-incubated membranes was phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase (protein kinase A). However, protein kinase A (in the absence of exogenous GTP) did not promote LH/CG receptor desensitization. These data demonstrate that, unlike with other G protein-linked receptors, LH/CG receptor phosphorylation by endogenous follicular membrane-associated protein kinase(s) does not mediate desensitization.

Luteinizing hormone/choriogonadotropin-dependent, cholera toxin-catalyzed adenosine 5'-diphosphate (ADP)-ribosylation of the long and short forms of Gs alpha and pertussis toxin-catalyzed ADP-ribosylation of Gi alpha*.

Although it is well established that activated LH/human (h) CG receptor stimulates adenylyl cyclase activity (via the heterotrimeric stimulatory guanine nucleotide-binding protein, Gs) and in some cells stimulates phospholipase C activity, there is no evidence for a direct physical interaction between the LH/CG receptor and Gs or any other G protein(s). We conducted studies using cholera toxin (CTX) and pertussis toxin (PTX) to determine which G alpha proteins were associated with the LH/CG receptor in ovarian follicular membranes. Since hormone-dependent, CTX-catalyzed ADP ribosylation (AR) constitutes evidence that a G alpha protein is specifically associated with a receptor, CTX-catalyzed AR of membrane proteins was examined both in the presence and absence of guanine nucleotides to determine which G proteins exhibit hCG-dependent labeling by [32P]NAD. Results demonstrated the time- and hCG-dependent AR of both a 45-kDa protein and a 48/50-kDa doublet as well as a 40-kDa protein that was also sensitive to AR by PTX in a time- and hCG-dependent manner. Using anti-G protein antisera to specifically immunoprecipitate photoaffinity-labeled G proteins, we were able to identify the 45- and 48/50 kDa proteins as the short and long forms of Gs alpha and the 40-kDa protein as Gi alpha. A monoclonal anti-hCG antibody immunoprecipitated the activated LH/CG receptor along with the long and short forms of Gs alpha and Gi. These results suggest that a portion of Gi along with the long and short forms of Gs alpha are associated physically with the LH/CG receptor in ovarian follicular membranes.

Separation of the complexes formed between the regulatory and catalytic subunits of cyclic adenosine monophosphate-dependent protein kinase and topoisomerase I activity in preovulatory follicle-enriched immature rat ovaries.

Our previous studies have shown that the regulatory subunits of the type II form of cAMP-dependent protein kinase (RII) present in soluble extract of immature rat ovaries elute from diethylaminoethyl-cellulose as three separate peaks of activity, based on their association with the catalytic subunit (C) of this enzyme, as R2IIC2, an apparent R2IIC, and R2II. Based upon the existence of ovarian RII in three different subunit arrangements, the large amount of C subunit-free R2II in this tissue, and a previous report which indicated that RII exhibited intrinsic topoisomerase I activity, we determined whether DNA topoisomerase I activity was associated with any of these molecular complexes of the ovarian RII subunits. Cyclic AMP-binding activities in soluble extracts of preovulatory follicle-enriched immature rat ovaries were separated by diethylaminoethyl-cellulose chromatography and sucrose density gradient centrifugation. Topoisomerase I activity cosedimented with the apparent R2IIC and R2II obtained from sucrose gradients but was not detected in fractions containing R2IIC2. Upon cAMP affinity purification of the RII derived from fractions containing R2IIC2, R2IIC, and R2II, respectively, no topoisomerase I activity could be detected in any fraction. Phosphorylation of the affinity purified RIIs by the C subunit of beef heart cAMP-dependent protein kinase did not alter this result. These data indicate that none of the RII subunits in soluble extracts of preovulatory follicle-enriched ovaries exhibit intrinsic topoisomerase I activity.

Induction of relaxin messenger RNA expression in response to prolactin receptor activation requires protein kinase C delta signaling.

The ability of PRL or rat placental lactogen (rPL)-1 to induce relaxin mRNA expression was analyzed in a luteinized rat granulosa cell culture model. PRL receptor activation induced relaxin mRNA expression in a concentration- and time-dependent manner. High concentrations of PRL receptor agonist, equivalent to those of the second half of pregnancy in rats, were required to elicit relaxin mRNA expression. A 40-fold induction of relaxin mRNA was observed in cells treated 24 h with 1 microg/ml of rPL-1. Estrogen enhanced relaxin expression induced by PRL but did not affect relaxin expression on its own. PRL/rPL-1 induction of relaxin expression was independent of the extracellular regulated kinase (ERK) members of the mitogen-activated protein kinase (MAPK) pathway, based on the inability of the ERK kinase inhibitor PD98059 to block induction of relaxin expression. PRL/rPL-1 induction of relaxin expression required protein kinase C (PKC) delta, based on the ability of the preferential PKC delta inhibitor rottlerin to abolish induction of relaxin expression. Direct activation of PKC by phorbol myristate acetate, however, was not sufficient to promote induction of relaxin mRNA expression. Stats (signal transducers and activators of transcription) 3 and 5 DNA binding activities were induced by PRL/rPL-1 treatment of luteinized granulosa cells but only Stat 3 DNA binding was reduced by rottlerin. PRL/rPL-1 treatment of luteinized granulosa cells resulted in increased phosphorylation on tyrosine-705 and serine-727 of Stat 3, and these responses were reduced and blocked, respectively, by rottlerin. Tyrosine and serine phosphorylations of Stat 3 in the corpus luteum were also increased in the second half of pregnancy when PL levels are highest. Stat 3, but not Stat 1 or 5, coimmunoprecipitated with luteal PKC delta during pregnancy; Stat 3 transiently coimmunoprecipitated with PKC delta from luteinized granulosa cells in response to PRL receptor activation; and Stat 3/PKC delta complex formation required PKC delta kinase activity. Taken together, these results show that PKC delta is obligatory for PRL/rPL-1-dependent relaxin expression, that PKC delta complexes with Stat 3 in response to PRL receptor activation, and that PKC delta is involved in the regulation of Stat 3 phosphorylation downstream of the PRL receptor. These results demonstrate that PRL/rPL-1 promotes relaxin expression in luteal cells and that this event is mediated, at least in part, via PKC delta.

Follicle-stimulating hormone promotes histone H3 phosphorylation on serine-10.

FSH promoted the rapid phosphorylation of the nuclear protein histone H3 in immature rat ovarian granulosa cells under experimental conditions that lead to cellular differentiation and not proliferation. FSH-stimulated histone H3 phosphorylation correlated with cAMP-dependent protein kinase A (PKA) activation and translocation of the PKA catalytic subunit to a nuclear-enriched fraction and was inhibited by the PKA inhibitor H89, and histone H3 phosphorylation was stimulated in cells treated with agents that raise intracellular cAMP levels such as forskolin and 8-bromo-cAMP. FSH-stimulated histone H3 phosphorylation in granulosa cells mapped to ser-10, a site previously identified as the PKA phosphorylation site in various mitotically active cells as the mitosis-specific phosphorylation site. Injection of the FSH analog PMSG to immature rats, which is known to stimulate granulosa cell proliferation as well as differentiation, also promoted histone H3 phosphorylation on ser-10 in granulosa cells. These results establish that FSH-stimulated histone H3 phosphorylation in granulosa cells is linked not only to granulosa cell mitosis but also to granulosa cell differentiation and that FSH-stimulated histone H3 phosphorylation on ser-10 in isolated granulosa cells is mediated by PKA. These results also identify the PKA-dependent histone H3 phosphorylation as an early nuclear protein marker for FSH-stimulated differentiation of granulosa cells. Based on the recently described function of histone H3 as a coactivator of transcription, these results are consistent with the hypothesis that phosphorylated histone H3 may facilitate PKA-dependent gene transcription in granulosa cells leading to the preovulatory phenotype.

Developmental regulation of mitogen-activated protein kinase-activated kinases-2 and -3 (MAPKAPK-2/-3) in vivo during corpus luteum formation in the rat.

The current study investigates the activation in vivo and regulation of the expression of components of the p38 mitogen-activated protein kinase (MAPK) pathway during gonadotropin-induced formation and development of the rat corpus luteum, employing a sequential PMSG/human CG (hCG) treatment paradigm. We postulated that the p38 MAPK pathway could serve to promote phosphorylation of key substrates during luteal maturation, since maturing luteal cells, thought to be cAMP-nonresponsive, nevertheless maintain critical phosphoproteins. Both p38 MAPK and its upstream activator MAPK kinase-6 (MKK6) were found to be chronically activated during the luteal maturation phase, with activation detected by 24 h post hCG and maintained through 4 days post hCG. The p38 MAPK downstream protein kinase target termed MAPK-activated protein kinase-3 (MAPKAPK-3) was newly induced at both mRNA and protein levels during luteal formation and maturation, while mRNA and protein expression of the closely related MAPKAPK-2 diminished. Two potential substrates for MAPKAPKs, the small heat shock protein HSP-27 and the cAMP regulatory element binding protein CREB, were monitored in vivo for phosphorylation. HSP-27 phosphorylation was not modulated during luteal maturation. In contrast, we observed sustained luteal-phase CREB phosphorylation in vivo, consistent with upstream MKK6/p38 MAPK activation and MAPKAPK-3 induction. MAPKAPK-3-specific immune complex kinase assays provided direct evidence that MAPKAPK-3 was in an activated state during luteal maturation in vivo. Cellular inhibitor studies indicated that an intact p38 MAPK path was required for CREB phosphorylation in a cellular model of luteinization, as treatment of luteinized granulosa cells with the p38 MAPK inhibitor SB 203580 strongly inhibited CREB phosphorylation. Transient transfection studies provided direct evidence that MAPKAPK-3 was capable of signaling to activate CREB transcriptional activity, as assessed by means of GAL4-CREB fusion protein construct coexpressed with GAL4-luciferase reporter construct. Introduction of wild-type, but not kinase-dead mutant, MAPKAPK-3 cDNA, into a mouse ovarian cell line stimulated GAL4-CREB- dependent transcriptional activity approximately 3-fold. Thus MAPKAPK-3 is indeed uniquely poised to support luteal maturation through the phosphorylation and activation of the nuclear transcription factor CREB.

Epinephrine-sensitive adenylyl cyclase activity in rabbit ovarian tissues.

Epinephrine-sensitive adenylyl cyclase (AC) activity was investigated in homogenates of rabbit ovarian tissues. Optimal epinephrine-stimulated AC activity in corpora lutea homogenates was achieved with millimolar concentrations of ATP (1-4 mM) and Mg2+ (2-12 mM) in the absence of exogenously added Ca2+. Epinephrine stimulated the luteal AC under these conditions in a dose-dependent manner, with an ED50 of 0.45 micrograms/ml. The AC of preovulatory follicle homogenates was found to be unresponsive to epinephrine stimulation. Epinephrine responsiveness of the AC slowly appeared in late preovulatory and early postovulatory follicles in response to the gonadotropin surge induced by coitus. By 18 h after mating, epinephrine-stimulated AC activity had doubled, and maximal activities in the newly formed corpora lutea were reached within 72 h. Thereafter, epinephrine-stimulated AC activity gradually declined to a relatively stable value that was maintained throughout the remainder of gestation, except for two transient increases in activity on days 14 and 19 of pregnancy. With parturition, luteal epinephrine-stimulated AC activity fell to basal levels. In corpora lutea of pseudopregnancy, epinephrine-stimulated AC activity was very similar to that of corpora lutea of pregnancy during the first 16 days but fell to basal levels on day 17 with the termination of pseudopregnancy. Neither corpora albicans nor interstitial tissue exhibited any epinephrine-stimulated AC activity. The presence of epinephrine-stimulated AC activity only in corpora lutea of pseudopregnancy and pregnancy and not in preovulatory Graafian follicles, corpora albicans, or interstitial tissue suggests a functional to role for catecholamines in luteal tissue. Moreover, these data indicate that the presence of an epinephrine-sensitive AC may be used as a functional marker for luteinization, at least in rabbit ovarian tissues.

Unique properties of the follicle-stimulating hormone- and cholera toxin-sensitive adenylyl cyclase of immature granulosa cells.

Previous studies have shown that the adenylyl cyclase of intact granulosa cells from immature porcine follicles has a uniquely prolonged responsiveness to FSH and an atypically poor responsiveness to cholera toxin relative to other cells. The present studies were designed to determine if these characteristics were due to unique regulation of the adenylyl cyclase activation process by Mg2+ and guanine nucleotides. GTP and Mg2+ enhanced adenylyl cyclase activity in the absence and presence of FSH in a manner similar to that in other systems. For example, GTP and Mg2+ increased the maximal velocity rather than the sensitivity of the cyclase to GTP and Mg2+. However, several unique properties distinguish the FSH-sensitive adenylyl cyclase from other hormonally activated adenylyl cyclases. First, FSH did not increase the sensitivity of the enzyme to Mg2+; thus the apparent Km for Mg2+ remained nonphysiologically high in the presence of FSH. Second, FSH was only one fourth as effective as NaF in activating the enzyme. Third, maximal activity attained in the presence of NaF was very low relative to that in other cells. Fourth, even in the presence of exogenous NAD+, cholera toxin activated adenylyl cyclase only as well as FSH rather than as well as NaF. Fifth, instead of causing maximal activation in the absence of stimulator, guanyl-5'-ylimidodiphosphate enhanced by almost 2-fold basal, FSH-activated, and cholera toxin-activated adenylyl cyclase. The inability of either guanyl-5'-ylimidodiphosphate or cholera toxin alone to cause maximal activation of the guanine nucleotide-binding protein, as reflected by cyclase activity, indicates that guanine nucleotide binding in the absence of hormone or cholera toxin is limiting.

Rabbit ovarian protein kinases. II. Effect of an ovulatory dose of human chorionic gonadotropin or luteinizing hormone on the multiplicity of follicular and luteal protein kinases.

DEAE-cellulose chromatography of the 105,000 X g supernatant fraction (cytosol) obtained from popped estrous rabbit follicles revealed the presence of a single form of cAMP-dependent protein kinase, designated protein kinase 3. The iv injection of an ovulatory dose of hCG to estrous rabbits promoted the appearance of a second, transient peak of cytosol cAMP-dependent protein kinase, protein kinase 1. Protein kinase 1 was detected within 10 min of hCG administration but had regressed to undetectable levels by 24 h in corpora lutea (CL) of pseudopregnancy and by 72 h in CL of pregnancy. Ovulation and subsequent CL formation were accompanied by the appearance of a third form of cAMP-dependent protein kinase, designated protein kinase 2. Protein kinase 2 was present within 2 h after hCG administration and persisted as a major form of cytosol cAMP-dependent protein kinase throughout the life span of CL. All three forms of protein kinase were inhibited by the heat-stable protein kinase inhibitor from rabbit skeletal muscle, possessed cAMP-binding activity, and were markedly stimulated by 10(-7) M cAMP. The activity of protein kinase 3 in CL of pregnancy, in corpora albicantia, and in interstitial tissue was markedly greater than that in follicles or in CL of pseudopregnancy, while the activity of protein kinase 2 remained relatively constant throughout the luteal life span. The iv injection of a luteolytic dose of hCG to 4-day pseudopregnant rabbits promoted no alterations of the protein kinase elution profile upon DEAE-cellulose chromatography of the luteal cytosol obtained 10 min to 3 days post-hCG injection. However, with dedifferentiation of corpora albicantia into interstitial tissue, the cAMP dependency of protein kinase 2 was reduced. The results indicate that the enzymatic activity and multiplicity of cAMP-dependent protein kinases in the cytosol of ovarian structures are subject to regulation by LH (hCG) and depend upon the various reproductive stages of the rabbit.

Progesterone production by ectopic corpora lutea is independent of luteinizing hormone-stimulated adenylyl cyclase during early luteal development in the rabbit.

Although estradiol is luteotropic in rabbits, corpora lutea develop and secrete progesterone in the absence of estradiol for 4-5 days. This period of estradiol independence is associated with high levels of LH-activated adenylyl cyclase and low levels of estradiol receptor. To determine if progesterone secretion is dependent on LH-stimulated adenylyl cyclase during this period of development, ectopic corpora lutea were established in ovariectomized rabbits. Rabbits were injected daily or twice daily with saline or 50, 100, or 150 IU hCG to desensitize LH-responsive adenylyl cyclase. On day 3, 4, 5, or 7, adenylyl cyclase activity was measured in luteal homogenates, and serum progesterone levels were determined by RIA. Basal and LH-stimulated adenylyl cyclase levels rose through day 5 of pseudopregnancy. Daily treatment with 50 or 100 IU hCG did not alter basal activity, but decreased LH-stimulated adenylyl cyclase activity by approximately 50% on day 3, by 80-90% on day 4, and by 90-95% on days 5 and 7. Serum progesterone levels were not different in hCG- and saline-treated rabbits. When LH-stimulated adenylyl cyclase was completely desensitized on days 3 and 4 of pseudopregnancy with twice daily injections of 150 IU hCG, serum progesterone levels were again, not different in saline- and hCG-treated rabbits. These results demonstrate that progesterone production by ectopic corpora lutea is not altered when LH-stimulatable adenylyl cyclase is desensitized and suggest that this early period of luteal development is independent of LH as well as estradiol.

Studies on the mechanism of luteinizing hormone-induced desensitization of the rabbit follicular adenylyl cyclase system in vitro.

In vitro studies were conducted to evaluate the possible mechanisms of LH-induced desensitization of the rabbit follicular adenylyl cyclase (AC) system. We tested the effects of cAMP, dibutyryl cAMP, and inhibitors of various cellular functions on LH-stimulated AC activity as well as the reversibility of AC desensitization. Refractoriness of the AC to LH was induced by a1 1- or 2-h incubation of Graafian follicles with 10 microgram/ml LH. We fund that the initial 60-min phase of AC desensitization to LH in Graafian follicles was not prevented by a 60-min preincubation of follicles with 11 microM puromycin, 30 microM cycloheximide, 8 microM actinomycin D, 5 microgram/ml cytochalasin B, 50 microM colchicine, or 1 or 10 mM trinitrophenol or by a 90-min preincubation of follicles with 50 microM colchicine. We evaluated the effects of cAMP and dibutyryl cAMP on LH-stimulated AC activity by incubating Graafian follicles with these nucleotides for 30-min to 4 h. While LH-stimulated AC activity was not significantly reduced in follicles which had been incubated 30-min or 1-h with either nucleotide, 2 h incubations resulted in significant reductions in LH-stimulated AC activity, and 4-h incubations promoted a complete refractoriness of the LH-stimulable AC. cAMP also caused desensitization of the FSH-stimulable AC in 4-h incubations, but not in the 1-h incubations. Lastly, once the follicular AC was desensitized to LH, neither 4-guanylyl imidodiphosphate nor ATP could reverse desensitization. These results indicate that AC desensitization in rabbit Graafian follicles is biphasic event. The initial 60-min phase is not mediated by cAMP, RNA, or protein synthetic events, by energy-requiring events inhibited by trinitrophenol, or by microtubule- or micro-filament-associated processes. A secondary phase occurs within 2-h and appears to be mediated, at least in part, by cAMP.

Opposing effects of ethanol on pig ovarian adenylyl cyclase desensitized by human choriogonadotropin or isoproterenol.

Pig ovarian follicular membranes contain a gonadotropin-responsive adenylyl cyclase, which becomes partially desensitized (approximately 40%) upon a 40-min incubation with a saturating concentration of human (h) CG. This in vitro desensitization is time and hormone dependent and also requires the presence of micromolar concentrations of GTP. In this report we show that 10% ethanol present during the desensitization phase of the incubation increases the extent of hCG-induced desensitization of adenylyl cyclase by 2-fold. Ethanol shortened the time necessary to reach maximal hCG-induced desensitization from 20 to 10 min, but had no effect on the dose dependency for GTP. In addition, ethanol had no effect on the affinity of the LH/hCG receptor for 125I-hCG but did cause an increase in the ED50 of hCG for inducing desensitization from 0.25 to 0.75 nM. Interestingly, ethanol decreased the apparent number of LH/hCG-receptor sites by 55%, yet the control hCG-sensitive adenylyl cyclase activity was not reduced. The "hyperdesensitized" state achieved in the presence of ethanol could not be reversed by washing the membranes and incubating them in ethanol-free medium. NaF-sensitive adenylyl cyclase was also not impaired in hCG-desensitized membranes from control or ethanol-treated samples. Thus, hCG-induced desensitization was not due to a defect in the functioning of the stimulatory guanine nucleotide-binding regulatory protein (G8) or catalytic subunits, but rather was caused by an impairment of the coupling of the lutropin (LH)/hCG receptor with G8, which was exacerbated further by ethanol. In spite of the effect of ethanol on hCG-induced desensitization, this agent had an inhibitory effect on isoproterenol-induced desensitization of isoproterenol-responsive luteal adenylyl cyclase. These results indicate that membrane fluidity is important in modulating the structure and functional interaction of the LH/hCG receptor with G8 because ethanol is a well known lipid-fluidizing agent. The resistance to ethanol potentiation of desensitization of the isoproterenol-sensitive adenylyl cyclase suggests that there are differences between the LH/hCG- and beta-adrenergic receptors in factors controlling their structures and or interactions with G proteins, and that there is a fundamental difference in their mechanisms of desensitization.

Comparison of the luteinizing hormone-sensitive adenylyl cyclase of the pig ovarian follicle and corpus luteum and its susceptibility to in vitro hormone-dependent desensitization.

The hormone responsiveness of the adenylyl cyclase of pig ovarian follicles or corpora lutea was examined. Adenylyl cyclase activity was assayed in 10,000 x g membrane fractions that had been prepared with or without (control) a urea extraction. In control luteal membranes there was little stimulation (less than 2-fold) or adenylyl cyclase by saturating ovine (o) LH, hCG, or (-)isoproterenol in the absence or presence of 10 microM GTP. However, in urea-treated luteal membranes, a 2- to 3-fold stimulation of adenylyl cyclase was caused by saturating oLH or hCG, and a 4- to 5-fold stimulation by (-)isoproterenol; the marked stimulation by the gonadotropins was only observed if 10 microM GTP was added. In follicular membranes, a 3- to 4-fold stimulation of adenylyl cyclase by gonadotropins was observed regardless of whether GTP was added or the membranes had been urea extracted. Stimulation of adenylyl cyclase by (-)isoproterenol was always less than 2-fold in follicular membranes. The binding affinity for [125I]hCG was similar in control follicular and luteal membranes, but there were approximately 10-fold more [125I]hCG-binding sites in follicular compared with luteal membranes. The binding affinities and number of receptor sites were not significantly changed by urea treatment. The ED50 values for hCG or (-)isoproterenol were the same in follicular and luteal membranes and were uneffected by the addition of 10 microM GTP, but the ED50 for oLH was 3-fold lower in follicular than in luteal membranes. GTP caused a dose-dependent increase in adenylyl cyclase activity in luteal and follicular membranes, and both tissues had the same ED50. A saturating hormone concentration resulted in an approximately 2-fold decrease in the ED50 for GTP. In vitro hCG-induced desensitization of the hCG-responsive adenylyl cyclase was 31% in follicular membranes, but only 11-15% in luteal membranes. Hormone-induced desensitization was not increased in incubations of luteal homogenate or membranes plus cytosol. These results establish the existence of a LH/hCG-sensitive adenylyl cyclase in the pig corpus luteum and indicate that the G-protein and catalytic moieties of the follicular and luteal adenylyl cyclase complex are functionally the same, but some difference exists in the way the LH/hCG-receptor in the two tissues interacts with the G-protein/catalytic complex.

Adenylyl cyclase activities in ovarian tissues. III. Regulation of responsiveness to LH, FSH, and PGE1 in the prepubertal, cycling, pregnant, and pseudopregnant rat.

In the ovaries of prepubertal rats, responsiveness of adenylyl cyclase (AC) to LH, FSH, and PGE1 is acquired at day 10, coinciding with the appearance of the ability to produce steroids in response to gonadotrophins. The activity and responsiveness of AC on day 11 were similar to those at puberty on day 40 and relatively stable in between. In the follicles of the cycle, the responsiveness of AC to LH and FSH was poor (ca 2-fold stimulation) on metestrus and diestrus, and became high (ca 10-fold stimulation) between 1000 h of diestrus and 1000 h of proestrus. Thereafter, the system slowly became desensitized to LH and FSH, being unresponsive by the morning of estrus. Nembutal injected at 1230 h and again at 1500 h on proestrus blocked ovulation and prevented the decline in LH- and FSH-stimulated AC activity. In the CL of the cycle, the AC was unresponsive to LH on day 1 (estrus), became responsive by the morning of day 2 (metestrus), and maintained responsiveness throughout that day. Thereafter, the responsiveness and basal AC activity declined rapidly. In the CL of pregnancy, LH-stimulated AC was indistinguishable from that of the CL of the cycle during days 1 and 2, then increased steadily until day 9, showing a transient decrease on days 10 and 11, followed by a sharp rise to maximal activity on days 15 and 16. Thereafter, activity declined as parturition approached. In the CL of pseudopregnancy (PSP), LH-stimulated AC was very similar to that of the CL of pregnancy during the first 11 days. Thereafter, it decreased coincident with the termination of PSP. Injections of PRL (100 mug SC twice daily, from metestrus through estrus, and from proestrus through proestrus) or estradiol-17 beta (20 mug, SC at 1230 h on metestrus) resulted in "rescue" of the CL-AC system, which remained at metestrus levels when measured on the days of expected proestrus or estrus. Injections of pregnant mare serum gonadotrophin into prepubertal rats at day 26 (3 IU, iv), induced by day 28 a highly responsive AC system in follicles, with activities equivalent to those found in Graafian follicles on proestrus. By day 29, synchronous ovulation had occurred with a concomitant loss of LH-stimulated AC such as seen in the 1-day-old CL of mature rats. Our results suggest that the LH-sensitive AC may be indicative of the final development of ovulability of the follicles, and that it may correlate with the functional capacity of CL during various reproductive stages of the rat.

Adenylyl cyclase activities in ovarian tissues. IV. Gonadotrophin-induced desensitization of the luteal adenylyl cyclase throughout pregnancy and pseudopregnancy in the rabbit and the rat.

We measured the adenylyl cyclase (AC) activity in dissected CL and the responsiveness of the AC system to LH, FSH, and prostaglandin (PG)E1 at different times following the administration of high doses of hCG or hLH to pseudopregnant and pregnant rats and rabbits. In rabbits, ovulatory doses of hCG promoted desensitization of the AC system in both CL of pregnancy and CL of pseudopregnancy (PSP), but at varying rates. At least a 50% decline in the LH-stimulated AC system was demonstrable 2 h after the hCG injection in CL obtained during PSP and the first 18 days of pregnancy. However, after day 21, AC activity was unaltered at 2 or 24 h after hCG injection, necessitating as much as 72 h for the AC system to become desensitized to LH. It seems that CL in the last third of pregnancy are afforded partial protection from the desensitizing effects of hCG. This protective effect was found not to be conferred upon follicles contained in ovaries after day 21 of pregnancy or upon newly, hCG-induced 3-day-old CL in 24-day pregnant rabbit ovaries. hCG-induced desensitization of CL adenylyl cyclase in rabbits was prevented neither by cauterization of tertiary follicles not by the continued administration of estradiol-17beta (1.5 mug SC twice daily), suggesting that this effect of hCG is due to a direct interaction with the CL, and not due to interruption of the follicular estrogen supply. In rats, the injection of an ovulatory dose of hCG (50 IU SC into prepubertal rats; 50 IU ip plus 50 IU SC into mature rats) also induced desensitization of the AC system in ovaries of superovulated prepubertal rats and in CL of pseudopregnant and pregnant rats. Desensitization of the AC system was not detectable at 2 h, was 30% of total by 6 h, and was complete at 24 h after hCG injection. Both regression of the CL and desensitization of the AC system in CL are induced only by doses of hCG which are ovulatory and not subovulatory. Desensitization of AC appears to precede functional luteolysis, at least in the pseudopreganant rabbit. Thus, the apparent close association between hCG-induced luteolysis and the desensitization of the adenylyl cyclase system in CL would suggest that desensitization may be a marker for luteal regression.