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Biomedical subjects

K Catt

Publications and source records attributed to K Catt.

At least 19 recordsLinked to original sources
PubMed1987

Hormonal control of epidermal growth factor receptors by gonadotropins during granulosa cell differentiation.

The hormonal induction of epidermal growth factor (EGF) receptor formation was analyzed during the maturation of granulosa cells obtained from diethylstilbestrol-implanted immature rats. In the absence of FSH, EGF receptors (as measured by the binding of [125I]iodo-EGF to the intact cells) rose by 50% at 6 h of culture, but then declined to about 25-40% of their initial levels at 24-96 h of culture. Scatchard analyses demonstrated the presence of high affinity EGF-binding sites in both freshly prepared cells and after FSH treatment. FSH stimulated a dose-dependent increase in the EGF receptor content of granulosa cells during a 96-h culture period. Concentrations of FSH as low as 2.5-5 ng/ml elevated EGF receptor levels 2- to 3-fold compared to those in untreated control cells, and 30 ng/ml FSH caused a maximal 15-fold rise. FSH increased EGF receptor levels approximately 2-fold in the first 6 h of culture and by up to 7-fold at 96 h compared to levels in freshly prepared cells. FSH treatment did not change the binding affinity (Kd = 5-6 X 10(-11) M) of the EGF receptor, but increased the total number of EGF-binding sites. The stimulatory effects of FSH on EGF receptor expression were mimicked by other cAMP-inducing ligands, including 8-bromo-cAMP, forskolin, and choleragen. Ligands known to inhibit granulosa cell function, including GnRH agonists and the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate, reduced the stimulation of EGF receptors by FSH. However, only 12-O-tetradecanoyl-phorbol 13-acetate suppressed the induction of EGF receptors by 8-bromo-cAMP. In granulosa cells cultured for 48 h with FSH, subsequent treatment with hCG for 24 h reduced EGF receptor content by 25%. Autoradiographic studies with [125I]iodo-EGF in ovarian thin sections demonstrated that EGF-binding sites were uniformly dispersed throughout the ovaries of diethylstilbestrol-implanted rats. Treatment with PMSG markedly increased EGF receptors in the outer walls of the growing follicles, while hCG treatment after PMSG caused a general decline in ovarian labeling. These results indicate that FSH maintains and increases the number of EGF receptors during granulosa cell differentiation, while LH/hCG reduces EGF-binding sites. Such changes in EGF receptors in the presence of endogenous growth factors may influence the number and selection of follicles destined for ovulation.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1987

Bifunctional role of transforming growth factor-beta during granulosa cell development.

Regulatory actions of transforming growth factor-beta (TGF beta) on granulosa cell function were analyzed in cells cultured from the ovaries of diethylstilbestrol-implanted rats. In the presence of a suboptimal concentration of FSH (5 ng/ml) that increased LH receptors by 100-fold during a 72-h culture, TGF beta augmented this response in a dose-dependent manner with a maximal effect at 16 pM. In contrast, the growth factor inhibited the LH receptor response to an optimal dose of FSH (50 ng) by up to 50% and was inactive in the absence of gonadotropin. TGF beta also enhanced the formation of cAMP by 5 ng FSH and partially inhibited the effects of higher FSH concentrations. However, the actions of TGF beta were more prominent on LH receptor induction than on cAMP production with either low or high amounts of FSH. In addition, TGF beta had little effect on cAMP production stimulated by cholera toxin or forskolin, but amplified the actions of these ligands as well as that of 8-bromo-cAMP on LH receptor expression. TGF beta also modulated the steroidogenic activity of the granulosa cells, with increased production of progesterone in response to 5-100 ng FSH. The bifunctional actions of TGF beta on FSH-induced LH receptor formation were observed throughout a 96-h culture period. However, the presence of the growth factor was not required for the first 24 h of culture, indicating that TGF beta alters the later events involved in LH receptor formation. TGF beta augmented the stimulatory actions of 5 ng FSH on LH receptors in the absence or presence of insulin, but its inhibitory effect on these receptors was only observed in cells treated with insulin. These results indicate that TGF beta modifies FSH action during granulosa cell development in a biphasic manner. TGF beta can exert stimulatory or inhibitory effects depending upon the concentration of FSH and the presence of insulin, and these are due to alterations in cAMP action as well as cAMP production. Autocrine and/or endocrine actions of TGF beta during granulosa cell differentiation may be involved in the processes of follicle selection and development.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1983

Epidermal growth factor and gonadotropin-releasing hormone inhibit cyclic AMP-dependent luteinizing hormone receptor formation in ovarian granulosa cells.

The induction of luteinizing hormone (LH) receptors was studied in granulosa cells prepared from the ovaries of hypophysectomized diethylstilbestrol-treated immature rats. Incubation of granulosa cells for 48 h with increasing concentrations of follicle-stimulating hormone (FSH) or choleragen caused parallel rises in cAMP levels and LH receptors. These observations, with the finding that 8-Bromo-cAMP also induced LH receptor formation, indicate that hormonal stimulation of LH binding sites is mediated by cAMP. Peptide hormones that inhibited FSH-stimulated cAMP production, such as epidermal growth factor (EGF) and a gonadotropin-releasing hormone agonist (GnRHa), also prevented LH receptor formation. GnRHa and EGF had negligible effects on FSH-stimulated cAMP production from 0 to 24 h of culture, but reduced cAMP accumulation by 80% and 90%, respectively, from 24 to 48 h when the majority of LH receptors appeared. FSH-sensitive adenylate cyclase activity, as measured by the conversion of (3H)-ATP to (3H)-cAMP, was inhibited by GnRHa and EGF at 48 h of culture. EGF and GnRHa also reversed the inhibiton of ectophosphodiesterase activity caused by FSH in granulosa cells between 48 and 72 h of culture. Both EGF and GnRHa inhibited induction of LH receptors by 8-Bromo-cAMP, suggesting that their effects are also on cAMP action. Addition of GnRHa, but not EGF, between 36 and 48 h of culture completely prevented further increases in LH receptors induced by 8-Bromo-cAMP, indicating that the inhibitory action of GnRHa can be initiated at later times during granulosa cell differentiation, whereas full expression of EGF action requires a longer period. These results demonstrate that EGF and GnRH inhibit FSH-induced LH receptor formation in the granulosa cell by reducing hormone-dependent cAMP production and also by impairing the ability of cAMP to stimulate LH receptor formation.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1983

Heterogeneous luteinizing hormone and follicle-stimulating hormone storage patterns in subtypes of gonadotropes separated by centrifugal elutriation.

The application of centrifugal elutriation to the separation of pituitary cells has produced a fraction that is enriched with LH and FSH gonadotropes. In this study, stains were performed on serial sections of fractions taken from six elutriation experiments with 1:10,000-1:30,000 anti-bLH beta or 1:2,000-1:8,000 anti-hFSH beta and the avidin-biotin-peroxidase complex technique. Over 900 serially sectioned gonadotropes were analyzed. In the initial cell suspensions, 59.6% of the gonadotropes contained both LH and FSH; 18% contained LH, and 23% contained FSH. Fewer than 3% contained ACTH. The elutriation fractions contained several subtypes of gonadotropes. A few small (8 micron), poorly granulated LH or FSH cells were eluted with the smallest cells (10-12%) at flow rates of 15.7 ml/min. Medium sized gonadotropes (10-10.5 micron) that eluted at flow rates of 19.8-30.5 ml/min were infrequent (3-6%) and resembled the larger gonadotrope. An analysis of serially sectioned fields showed that only 15.2%-22% of the small and medium sized gonadotropes contained both LH and FSH, whereas 30-40% contained only LH and 46-50% stored only FSH. The gonadotrope-enriched fraction was eluted at 37-39.5 ml/min; 78% of these gonadotropes (diameter, 14-15 micron) contained both LH and FSH, 10% contained only LH, and 12% contained FSH. Finally, large cells (diameter, 15-16 micron) that contained FSH only were found in the Wash fraction. These studies demonstrate that smaller gonadotropes tend to store only one of the hormones whereas most of the larger cells either store LH and FSH together or FSH alone. These heterogeneous storage patterns may correlate with studies by others who measured different responses to GnRH in gonadotropes separated by size on a unit gravity sedimentation gradient. They may also reflect different secretory phases of gonadotropes from the mixed group of cycling female rats.

Adrenocorticotropic Hormone↗
PubMed1978

Angiotensin II receptors and aldosterone production in rat adrenal glomerulosa cells.

Specific receptors for angiotensin II (A II) were demonstrated in membrane fractions and collagenase-dispersed cells from the zona glomerulosa of the rat adrenal gland. The equilibrium association constant (Ka) of the A II binding sites was similar in particulate fractions (2.0 +/- 0.4 (SE) X 10(9) M-1) and intact glomerulosa cells (1.8 +/- 0.3 X 10(9) M-1). Specific binding of [125I]iodo-A II was enhanced by increasing sodium concentration, and in the presence of dithiothreitol, EDTA, and EGTA. Plasma membrane fractions prepared by density gradient centrifugation showed increased binding of [125I]iodo-A II, and were correspondingly enriched in adenylate cyclase and sodium-potassium-dependent ATPase. Steroid production by collagenase-dispersed adrenal glomerulosa cells was highly responsive to A II and ACTH. Significant increases in aldosterone and corticosterone production were elicited by A II concentrations as low as 3 X 10(-11) M, equivalent to normal blood levels of A II in rats (5 X 10(-11) M). The maximum increase in aldosterone production, of 6--7 times the basal value, was obtained at 10(-9) M A II. Dispersed capsular cells were also highly sensitive to ACTH, responding to concentrations down to 3 X 10(-12) M with increased aldosterone production, reaching a maximum aldosterone response of 20-fold above the basal value. The magnitudes of the aldosterone and corticosterone responses to A II in capsular and fasciculata-reticularis cells were commensurate with the distribution of A II receptors, which were 11-fold more concentrated in capsular cells. The ability of A II to evoke aldosterone production at physiological concentrations, and the correspondence between A II binding and steroidogenesis in capsular cells, demonstrate the functional importance of A II receptor sites in the zona glomerulosa of the rat adrenal cortex.

Adrenal Glands↗
PubMed1978

Formation of DES-ASP1-angiotensin II is not an obligatory step in the steroidogenic action of angiotensin II in the canine adrenal.

A comparison of the receptor binding properties and in vitro metabolism of angiotensin II and [des-Asp1]-angiotensin II was performed in collagenase-dispersed glomerulosa cells from dog adrenal glands. Each peptide was bound rapidly and reversibly to isolated glomerulosa cells, with similar equilibrium and kinetic constants of association and dissociation. The labeled peptide that was bound to glomerulosa cells after incubation with 125I- and 3H-angiotensin II at 37C for 30-60 minutes was predominantly angiotensin II, with about 15% [des-Asp1]-angiotensin II. These findings demonstrate that local formation of the heptapeptide is not an obligatory step in the action of angiotensin II on aldosterone production in the canine adrenals.

Adrenal Glands↗
PubMed1978

An examination of possible mechanisms of angiotensin II-stimulated steroidogenesis.

Dog and rat adrenal glomerulosa cells and subcellular fractions have been utilized to evaluate the mechanism of angiotensin II- and angiotensin III-induced aldosterone production. The effects of angiotensin, ACTH, and potassium have been compared on cyclic AMP and cyclic GMP in isolated glomerulosa cells and adenylate cyclase activity in subcellular fractions. The effect of angiotensin II has also been assessed on Na+-K+-activated ATPase of plasma membrane enriched fractions of dog and rat adrenals. We have demonstrated no effect of angiotensin II or angiotensin III on either adenylate cyclase, cyclic AMP, cyclic GMP, or Na+-K+-dependent ATPase activity over a wide range of concentrations. Potassium ion in concentrations that stimulate significant aldosterone production was also without effect. The negative effects of angiotensin and potassium were contrasted against a positive correlation between an ACTH-induced effect on aldosterone production, adenylate cyclase, and cyclic AMP accumulation. These studies have served to demonstrate that neither adenylate cyclase, cyclic AMP, cyclic GMP, or Na+-K+-activated ATPase seem to be directly involved in the mechanism of action of angiotensins on aldosterone production in the rat and dog adrenal glomerulosa.

Adrenal Glands↗
PubMed1975

Dependence of gonadotropin-induced steroidogenesis upon RNA and protein synthesis in the interstitial cells of the rat testis.

The effects of inhibitors of RNA and protein synthesis upon gonadotropic stimulation of testosterone and cyclic AMP production by the Leydig cellwere investigated in vitro with enzyme-dispersed interstitial cells of the rat testis. The testosterone response to human chorionic gonadotropin was abolished by cycloheximide and puromycin, and was markedly reduced by actinomycin D and cordycepin. During 3-h time studies, cycloheximide caused complete inhibition of subsequent steroid production when added at times up to 90 min after the commencement of incubation with human chorionic gonadotropin. Actinomycin D did not completely abolish the testosterone response when added at zero time, and became progressively less effective when added at later times during the incubation period. The stimulation of steroidogenesis by dibutyryl cyclic AMP was also completely abolished by cycloheximide and puromycin, and was significantly reduced by acinomycin D and cordycepin. By contrast with the marked inhibition of steroid production by cycloheximide and actinomycin D, the formation of cyclic AMP during human chorionic gonadotropin stimulation was relatively unaffected by either inhibitor. These results indicate that the stimulation of testosterone production by the Leydig cell in response to gonadotropins and dibutyryl cyclic AMP is dependent upon the synthesis of new RNA and protein moleucles. Thus, effects of gonadotropin and cyclic AMP upon both transcriptional and translational processes appear to be essential intermediate steps in the activation of testicular steroidogenesis. The rapid and complete abolition of subsequent steroid synthesis following addition of cycloheximide or puromycin to human chorionic gonadotropin-stimulated interstitial cells suggests that a relatively labile protein is formed during gonadotropin action.

Animals↗
PubMed1975

Gonadotropin binding and stimulation of cyclic adenosine 3':5'-monophosphate and testosterone production in isolated Leydig cells.

Gonadotropin binding and stimulation of cyclic adenosine 3':5'-monophosphate (cyclic AMP) formation and testosterone synthesis were studied in collagenase-dispersed interstitial cells from the adult rat testis. Binding of 125I-human chorionic gonadotropin (hCG) by isolated Leydig cells was of high affinity (Ka = 10(10) M-1) and low capacity, equivalent to approximately 6000 sites/cell. The binding data were consistent with the presence of a single order of receptors, with no interaction between binding sites. Stimulation of testosterone synthesis by increasing concentrations of hCG was completely dissociated from changes in cyclic AMP formation, and maximum activation of steroidogenesis was induced by hCG concentrations which had no effect upon cyclic AMP production. Kinetic analysis of gonadotropin-induced responses in dispersed Leydig cells also showed a marked dissociation between steroidogenesis and cyclic nucleotide formation. Low concentrations of hCG caused maximum stimulation of testosterone production which was not accompanied by a rise in cyclic AMP formation at any time after addition of gonadotropin. Higher concentrations of hCG caused marked elevations of cyclic AMP at progressively earlier time intervals, but did not alter the 20 to 30 min lag period required for induction of testosterone synthesis. These observations indicated that occupancy of gonadotropin receptors occurs over a much wider range of hCG concentration than that required for maximum steroidogenesis.

Animals↗
PubMed1967

Solid-phase radioimmunoassay in antibody-coated tubes.

The adsorption of antibody to polymeric surfaces has been used to develop a new method of solid-phase radioimmunoassay. Incubation is performed in antibody-coated, disposable tubes that are finally washed-out with water and counted for quantitation of the bound tracer. The method is simple, rapid, inexpensive, and suitable for automation.

Adsorption↗