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P A Klase

Publications and source records attributed to P A Klase.

At least 19 recordsLinked to original sources
PubMed1984

Mechanisms subserving calcium's modulation of luteinizing hormone action in isolated swine granulosa cells.

We studied the mechanism(s) by which calcium ions modulate progesterone biosynthesis by isolated swine granulosa cells incubated in chemically defined medium in vitro. In selectively calcium-deficient incubations, the capacity of 8-bromo-cAMP to stimulate pregnenolone synthesis from endogenous sterol substrate was significantly impeded. This effect of calcium ions was specific, because calcium ions did not influence basal pregnenolone production or alter progesterone production in response to exogenously supplied cholesterol substrate. Moreover, calcium ions did not modify other biosynthetic processes in granulosa cells, such as de novo synthesis of cholesterol from [14C]acetate or the aromatization of testosterone to 17 beta-estradiol. The possible role of calmodulin in mediating calcium's actions in pig granulosa cells was tested by measuring the calmodulin content of these cells and assessing the functional responses to classical calmodulin antagonists. By immunoassay, swine granulosa cells contained high concentrations of calmodulin, viz. 4.21-4.88 micrograms calmodulin/mg protein. Moreover, calmodulin antagonists inhibited LH-stimulated progesterone production with the following rank order of potencies [estimated by half-maximally inhibitory concentrations (ID50)]: penfluridol (1 microM), trifluoroperazine (9 microM), chlorpromazine (95 microM), and trifluoperazine sulfoxide (greater than 300 microM). In addition, the nonphenothiazine calmodulin antagonist W7 inhibited stimulated progesterone production with an ID50 of 16.7 microM. W5 was less active. None of these antagonists significantly suppressed LH-stimulated cAMP generation at the low concentrations capable of inhibiting progesterone production. The effects of calcium ions seemed to depend upon the availability of intracellular pools of calcium, because TMB-8, an inhibitor of intracellular calcium mobilization, effectively suppressed LH-stimulated progesterone production (ID50, 18 microM). However, even 100 microM TMB-8 failed to alter basal progesterone production or suppress LH-stimulated cAMP generation in these cells. In summary, the present studies indicate that calcium ions significantly modulate LH's stimulation of pregnenolone biosynthesis from endogenous cholesterol substrate in swine ovarian cells. Calcium does not influence basal pregnenolone production, estrogen synthesis from androgen substrate, de novo biosynthesis of cholesterol from [14C]acetate, or progesterone production from exogenously supplied sterol substrate.(ABSTRACT TRUNCATED AT 400 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1983

Progesterone secretion by highly differentiated human granulosa cells isolated from preovulatory Graafian follicles induced by exogenous gonadotropins and human chorionic gonadotropin.

Human granulosa-luteal cells were harvested from preovulatory Graafian follicles at the time of oocyte retrieval for in vitro fertilization after induction of follicle maturation by sequential injections of menopausal gonadotropins and hCG. Such highly differentiated granulosa cells produced large quantities of progesterone basally (6.8 pg/cell X 2 days) in monolayer culture. Human LH significantly increased progesterone biosynthesis after 6, 12, 48, 96, or 144 h in culture, with a maximal increase of 8- to 20-fold occurring at 96 h. The stimulatory effect of LH could be observed under serum-free conditions and was maximal in the presence of 4% serum. Human granulosa-luteal cells also exhibited significant stimulatory responses to hCG, prostaglandin E2, or the cAMP effectors 8-bromo cAMP, choleratoxin, or forskolin in serum-free incubations. Concentrations of 17 beta-estradiol that are attained physiologically in ovarian follicles in vivo markedly suppressed basal and LH (or cAMP)-stimulated progesterone production in vitro (maximal suppression, greater than 90%). The nonaromatizable androgen 5 alpha-dihydrotestosterone also inhibited progesterone production, but by no more than 45-50% even at supraphysiological concentrations. Estradiol's blockade of progesterone synthesis was associated with a corresponding increase in pregnenolone accumulation. The present studies indicate that human granulosa-luteal cells isolated from preovulatory follicles induced with exogenous gonadotropins and hCG secrete large quantities of progesterone in vitro. Such cells retain stimulatory responses to human LH, hCG, prostaglandin E2, and classical cAMP effectors in serum-free incubations. Moreover, physiological concentrations of 17 beta-estradiol suppress progesterone production, probably by inhibiting cellular conversion of pregnenolone to progesterone. Thus, the present in vitro system permits an investigation of hormone action in well differentiated, human granulosa-luteal cells isolated from preovulatory Graafian follicles that have a defined endocrine history of prior gonadotropin exposure in vivo.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1982

Calcium ions modulate hormonally stimulated progesterone production in isolated ovarian cells.

Swine granulosa-luteal cells incubated in Ca2+-deficient medium (5 muM final Ca2+ concentration) for short time periods produced diminished quantities of progesterone in response to lutropin. Maximally stimulating effects of prostaglandin E2 and L-adrenaline were also impaired significantly. Diminished progesterone production could not be attributed to alterations in protein synthesis or cell viability. Under Ca2+-deprived conditions, the stimulatory actions of cholera toxin, 3-isobutyl-1-methylxanthine and 8-bromo cyclic AMP were also significantly impeded. Administration of a presumptive antagonist of transmembrane Ca2+ influx (verapamil) or of EGTA to chelate extracellular Ca2+, significantly decreased the total cellular content of Ca2+, and antagonized the actions of lutropin. Micromolar concentrations of trifluoperazine mimicked the suppressive effects of Ca2+ deprivation. Conversely, the bivalent-cation ionophore, ionophore A23187, significantly augmented the stimulation of progesterone produced by lutropin. Thus the present observations implicate Ca2+ in the modulation of hormonally stimulated progesterone production in isolated ovarian cells, and suggest that Ca2+ may influence one or more processes distal to, or independent of, cyclic AMP generation. In addition, the susceptibility of progesterone biosynthesis to inhibition by trifluoperazine suggests a possible role for calmodulin in the ovary.

Animals↗
PubMed1982

Mechanisms by which calcium ions regulate the steroidogenic actions of luteinizing hormone in isolated ovarian cells in vitro.

Incubation of swine granulosa cells in chemically defined medium selectively deficient in calcium ions markedly impaired progesterone production in response to submaximal and maximally stimulating concentrations of LH. Accumulation of progesterone in response to LH was reduced significantly in both cells and medium, without a discernible shift in the time-course of progestin production. The reduction in progesterone accumulation could not be accounted by increased formation of the catabolite 20 alpha-hydroxypregn-4-en-3-one. In addition, progesterone secretion basally or in response to exogenously supplied pregnenolone was not altered in calcium-deficient incubations. Administration of verapamil or diltiazem, organic inhibitors of net transmembrane calcium uptake, also suppressed LH-stimulated progesterone production. Conversely, micromolar concentrations of the divalent cation ionophore A23187 significantly enhanced the stimulatory effects of LH. The mechanisms of calcium action were examined further in relation to the cAMP effector system. Calcium deprivation significantly suppressed the dose-dependent accumulation of cAMP in granulosa cells treated with LH but had no effect on basal levels. Omission of calcium ions from the extracellular medium also markedly impaired production of progesterone in response to 8-bromo-cAMP, cholera toxin, or 3-isobutyl-l-methylxanthine. The present studies suggest that calcium ions significantly modulate LH-stimulated progesterone biosynthesis in isolated ovarian cells in vitro. Specific regulatory actions of calcium ions in granulosa cells may be exerted at several levels, including LH-stimulated cAMP accumulation and at intracellular loci distal to the actual generation of cAMP.

Animals↗
PubMed1982

The role of estradiol as a biological amplifier of the actions of follicle-stimulating hormone: in vitro studies in swine granulosa cells.

We describe an in vitro system of swine granulosa cells which remain responsive to estradiol (E2) and FSH. In this system, we examined mechanisms by which E2 amplifies the stimulatory actions of FSH. E2 stimulated progesterone production in a dose-dependent manner and enhanced the tropic effects of FSH. The facilitative interaction between E2 and FSH could not be accounted for by mitogenic effects, by a leftward shift in the dose-response curves for FSH or E2, or by catabolism of progesterone to 20 alpha-hydroxypregn-4-en-3-one. E3 also enhanced stimulatory actions of 8-bromo-cAMP, choleratoxin, and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Lipoproteins were required for maximal interactive effects between E2 and FSH. However, estrogen significantly increased the effects of FSH even in serum- and lipoprotein-free medium. In addition, when de novo cholesterol biosynthesis by granulosa cells was suppressed by Compactin, the actions of E2 and/or FSH were not impeded. In contrast, E2 alone and FSH alone significantly augmented progesterone production in response to the oxygenated sterol 5-cholesten-3 beta, 25-diol, which is an effective substrate for cholesterol side-chain cleavage. In the presence of 5-cholesten-3 beta, 25-diol, the magnitude of the synergism between E2 and FSH was also increased markedly. We conclude that E2 augments the stimulatory actions of FSH or cAMP on steroidogenesis. This facilitative interaction is amplified by exogenous lipoproteins and does not seem to depend upon endogenous cholesterol biosynthesis. Studies with 5-cholesten-3 beta, 25-diol suggest that estrogen and FSH exert significant stimulatory effects at the level of cholesterol side-chain cleavage. These observations reveal important interactions of E2 and fSH in preparing granulosa cells for the high rates of steroidogenesis they ultimately express in the luteinized state.

1-Methyl-3-isobutylxanthine↗
PubMed1982

Facilitative interactions between estradiol and luteinizing hormone in the regulation of progesterone production by cultured swine granulosa cells: relation to cellular cholesterol metabolism.

Well differentiated swine granulosa cells in monolayer culture were employed to investigate the mechanisms by which estradiol amplifies the stimulatory actions of LH in the later stages of follicular maturation. The facilitative interaction between estradiol and LH could not be attributed to altered rates of catabolism of progesterone to 20 alpha-hydroxypregn-4-en-3-one. Moreover, estradiol, LH, and estradiol combined with LH clearly stimulated pregnenolone production, measured in the presence of trilostane, to inhibit 3 beta-hydroxysteroid dehydrogenase-delta-5-4-isomerase activity. Thus, at least one component of the synergism between estradiol and LH must reside at or proximal to the cholesterol side-chain cleavage system. In the absence of lipoproteins, the magnitude of the synergism between estradiol and LH was significantly reduced. However, the facilitative interaction between estradiol and LH could still be observed in lipoprotein-deficient and serum-free medium and after the administration of ML-236B to suppress the de novo biosynthesis of cholesterol. In contrast, estradiol alone and LH alone significantly augmented progesterone production in the presence of the oxygenated sterol 5-cholesten-3 beta,25-diol, which can serve as an effective substrate for cholesterol side-chain cleavage. In addition, in the presence of 5-cholesten-3 beta,25-diol, the magnitude of the synergism between estradiol and LH was increased markedly. Thus, the present studies demonstrate that estradiol and LH significantly stimulate progesterone production, at least in part, by augmenting pregnenolone biosynthesis, but not by inhibiting progesterone catabolism. Studies with 5-cholesten-3 beta-25-diol further suggest that estradiol and LH increase cholesterol side-chain cleavage activity. These observations delineate important mechanisms by which estradiol and LH prepare well differentiated granulosa cells for the high rates of steroidogenesis that are ultimately required in the corpus luteum.

20-alpha-Dihydroprogesterone↗
PubMed1981

Urethane anesthesia in rats. Altered ability to regulate hydration.

Anesthesia in rats produced by urethane administered intraperitoneally caused (1) peritoneal fluid accumulation; (2) inability to undergo a renal response to NaCl or water loading, and (3) pronounced hyperosmolality of body fluids without affecting plasma [Na+]. The impairment of the renal function appears not to be due to anesthesia per se, angiotensin, aldosterone, vasopressin or renal nerves. It probably is attributable to osmotoxicity of the mesenteric vasculature. By contrast, urethane administered intravenously evokes a brisk osmotic diuresis without fluid leakage into the peritoneum. Plasma osmolality is still increased. The osmotic toxicity to the mesenteric vasculature, poor renal function and altered composition of body fluids that occur after intraperitoneal urethane may complicate the interpretation of data obtained in rats anesthetized in this manner.

Aldosterone↗
PubMed1981

Sex steroids modulate prolactin action in spontaneously luteinizing porcine granulosa cells in vitro.

Prolactin (20-1000 ng/ml) stimulated progesterone secretion in monolayer cultures of granulosa cells isolated from mature (greater than 8 mm) porcine follicles. The action of 17 beta-estradiol alone and the interaction between estradiol and prolactin in controlling steroidogenesis were critically dependent upon the duration of hormone treatment. Acute administration (within 36 h) of 17 beta-estradiol, but not 17 alpha-estradiol, suppressed both spontaneous and prolactin-stimulated progesterone production. Continuation of estrogen treatment beyond 48 h markedly increased progesterone secretion per cell and with prolactin produced synergistic enhancement of progesterone accumulation. However, 5 alpha-dihydrotestosterone also significantly augmented progesterone production after both acute and prolonged administration. The steroidogenic effect of prolactin was greater in the presence of 5 alpha-dihydrotestosterone, but did not elicit true synergism. Measurements of sex-steroids levels in porcine follicular fluids indicated that estrogen concentrations that were biologically active in vitro are similar to those attained in vivo. These observations suggest a major role for sex-steroid hormones both in the control of basal steroidogenesis and in the modulation of prolactin action in mature spontaneously luteinizing granulosa cells.

Androstenedione↗
PubMed1981

Effects of prolactin on DNA synthesis and growth of the accessory sex organs in male mice.

The role of PRL in the development of male accessory sex organs remains unresolved. In the present studies, the influence of PRL and testosterone upon DNA synthesis and cell proliferation was examined in the anterior prostate gland (AP) and the seminal vesicles (SV) of mice. Hormonal effects on DNA synthesis were evaluated by examining the in vitro incorporation of [3H]thymidine into DNA in relation to temporal alterations in tissue DNA content. In intact mice, PRL (300 IU/kg daily) produced peak stimulation of [3H]thymidine incorporation into DNA by SV after 3 days of treatment. In contrast, PRL failed to alter [3H]thymidine incorporation by the AP. Only in SV did PRL injections lead to elevated levels of tissue DNA. Injections of testosterone (0.75 or 7.5 mg/kg daily for 3 days) to castrated mice also produced significant stimulation of labeled thymidine incorporation into DNA by AP and SV. Concomitant injections of PRL (150 IU/kg) and testosterone (0.75 or 7.5 mg/kg) enhanced the stimulatory effects of this androgen on DNA synthesis and DNA content in the SV, but not in the AP. When PRL alone was administered to castrated mice, it failed to affect either [3H]thymidine incorporation into DNA by AP and SV or accessory sex organ weights and DNA contents. The results of these studies suggest that PRL enhances the effects of androgens upon DNA synthesis and growth of the seminal vesicles, but not of the anterior prostate glands of mice.

Animals↗
PubMed1981

Direct actions of 17 beta-estradiol on progesterone production by highly differentiated porcine granulosa cells in vitro. II. Regulatory interactions of estradiol with luteinizing hormone and cyclic nucleotides.

We investigated direct actions of 17 beta-estradiol and LH in the coordinate control of progesterone production by highly differentiated porcine granulosa cells maintained in monolayer culture. The administration of estradiol acutely suppressed both basal and LH-stimulated progesterone synthesis in vitro, i.e within the first 24-36 h of estrogen treatment. In contrast, continuation of estradiol administration alone beyond 48 h significantly augmented progesterone production per 10(5) granulosa cells. Among 12 independent experiments, the absolute stimulatory effects of estradiol were highly correlated (r = 0.991) with basal progesterone production by granulosa cells at the outset of culture, i.e. when steroid synthesis presumably reflected the degree of prior cytodifferentiation attained in vivo. Notably, estrogens also facilitated the dose-dependent actions of LH in a synergistic fashion. Synergism occurred during periods of both maximal and spontaneously declining steroidogenesis in vitro, and could be impeded by specific inhibitors of steroid biosynthesis (10 microM cyanoketone and 50 microM trilostane). In experiments designed to assess granulosa cell responsivity to delayed hormone rechallenge, there was a critical bihormonal requirement for both estradiol and LH in order to sustain maximal long term progesterone secretion. Further investigation of the biochemical mechanisms subserving synergistic effects demonstrated that estradiol was capable of augmenting the stimulatory actions of either exogenously supplied or endogenously generated cAMP. In particular, estradiol markedly enhanced the effects of potent phosphodiesterase resistant analogs of cAMP, 8-bromo-cAMP (0.1 mM), dibutyryl cAMP (2 mM) or 8-thio-cAMP (1 mM). Estradiol also significantly facilitated the stimulatory effects of agents that putatively increase or sustain intracellular pools of cAMP by various well defined mechanisms, i.e. choleratoxin (10 microgram/ml), guanyl-5'-imido-diphosphate (1.0 mM) or 3-isobutyl-1-methylxanthine (0.25 mM). Thus, the current in vitro studies delineate directly major interactions between estradiol and LH in the control of progesterone synthesis by highly differentiated granulosa cells. The present data further indicate that the synergistic stimulation of progesterone production by LH and estradiol is mediated in part by intracellular mechanisms operating distal to LH-stimulated cAMP production. These in vitro observations using physiological concentrations of hormones suggest a critically bihormonal role for estradiol and LH in the facilitation of progesterone secretion in vivo during late follicular phase differentiation of granulosa cells.

1-Methyl-3-isobutylxanthine↗
PubMed1981

Dissociated regulation of cellular progesterone secretion and cytosolic ornithine decarboxylase activity in isolated ovarian cells in vitro.

We have utilized isolated, highly differentiated porcine granulosa cells maintained under chemically defined conditions in vitro to examine directly the relationship between ornithine decarboxylase (ODC) activity and progesterone secretion in ovarian cells. The administration of saturating concentrations of LH, prostaglandin E2, L-epinephrine, or 8-bromo-cAMP each elicited highly significant increases in progesterone production, which correlated closely with corresponding stimulated levels of ODC activity. Highly purified preparations of FSH elicited no enzymatic or steroidogenic response. ODC activity and progesterone production also correlated closely after administration of various inhibitors of protein or RNA synthesis [cycloheximide (10 microgram/ml), actinomycin D (1 microgram/ml), or alpha-amanitin (1 microgram/ml)]. However, in time-course experiments, progesterone secretion increased significantly before the rise of ODC activity. Furthermore, a direct irreversible catalytic inhibitor of ODC activity, DL-alpha-difluoromethylornithine, and three indirect inhibitors of ODC that putatively induce intracellular antizyme, produced profound suppression of enzymatic activity, without altering maximal progesterone production in response to LH. Thus, the current studies close parallelism between hormonal regulation of ODC activity and progesterone production in ovarian cells in vitro. However, our ability to demonstrate apparent dissociation between these two processes suggests that hormonal stimulation of steroidogenesis may not be obligatorily coupled to the rapidly turning-over cytosolic protein ODC.

Animals↗
PubMed1981

Interaction between prolactin and androgens in the accessory sex organs of male mice.

Treatment of adult male mice with varying doses of prolactin increased the weights of the seminal vesicles and the anterior prostate gland. Only in the seminal vesicles were these increases in organ weights associated with increased levels of DNA. In castrated mice, prolactin alone failed to alter the weights of the accessory sex organs or DNA content. However, the simultaneous administration of prolactin and testosterone resulted in enhanced androgenic stimulation of seminal vesicle weights and their DNA content. Prostatic weights, but not DNA content, were augmented by treatment with prolactin and testosterone. Although the kidneys exhibited androgen sensitivity, prolactin failed to enhance the effect of testosterone upon the kidney. Augmentation of androgen action in the accessory sex organs was observed only after treatment with prolactin or growth hormone. Prolactin also enhanced the effects of dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol, but not of 5 alpha-androstane-3 beta, 17 beta-diol. These studies revealed that prolactin enhances the proliferative actions of androgens in mouse seminal vesicles, but not in the anterior prostate glands or the kidneys.

Animals↗
PubMed1978

Consummatory behavior and urine production after cerebroventricular injection of vasopressin and vasopressin antiserum.

Vasopressin, or vasopressin antiserum, was injected into a lateral cerebral ventricle of conscious rats. These rats were normally hydrated, cellular dehydrated (NaCl loading) or hypovolemic (polyethylene glycol model). Elevation or reduction of vasopressin in cerebrospinal fluid produced no consistent change in consummatory behavior, urine volume or sodium and potassium excretion. These results show vasopressin in cerebrospinal fluid not to be an absolute requirement for maintenance of hydration or for response to acute volume and osmotic stimuli.

Animals↗
PubMed1977

Drinking behavior in water deprived rats after angiotensin receptor blockade.

Angiotensin II is a peptide normally present in the bloodstream and central nervous system. Exogenous angiotensin induces drinking which is inhibited by saralasin, a specific receptor antagonist. Administration of saralasin does not reduce endogenously stimulated drinking. Angiotensin is dipsogenic after intravenous or intracerebroventricular infusion, raising the possibility of multiple access routes to the brain. Water deprived rats were given saralasin by both routes simultaneously to block the access of endogenous angiotensin to recentors reached from blood and ventricular cerebrospinal fluid (CSF). Water deprivation increased plasma (Na+), hematocrit, vasopressin content and renin activity but saralasin treatment did not reduce water intake after 30 or 60 min. Therefore, blood or CSF-bore angiotensin does not appear to be an absolute requirement for water deprivation drinking behavior.

Angiotensin II↗