Search

Introduction: This study replicates a study of interprofessional education (IPE) of physician assistant (PA) and second-year medical students conducted by Denis Oliver early in the history of the University of Iowa PA Program. Currently, there is resurgent interest in IPE in the health sciences, but sparse research evidence available on the topic. Methods: Mean exam score averages for PA and second-year medical (M2) students were compared over a recent 4-year period (August 2003–May 2007) for medical pharmacology and clinical medicine courses using de-identified existing data. Means were compared with t-tests utilizing SPSS 15. Results: Based on data gathered in 1978–1981, Oliver reported small significant differences between the groups: M2 students scored higher than PA students by 2.9 points in clinical medicine and 4.5 points in pharmacology. The current study showed less than a 1-point difference between the groups in both courses, which only reaches statistical significance in pharmacology, and which is likely of no practical significance. Discussion: The original study by Oliver validated an interprofessional approach to training PA students alongside medical students at the University of Iowa Carver College of Medicine. The current study supports the practicality of combining PA and M2 students in didactic settings, despite their disparate levels of prior academic preparation. Possible advantages include efficient use of faculty and institutional resources and the potential for fostering a team approach to medical care early in students’ professional development. This model may be most applicable to PA programs located at academic medical centers.

The Ras guanylnucleotide exchange protein SOS undergoes feedback phosphorylation and dissociation from Grb2 following insulin receptor kinase activation of Ras. To determine the serine/threonine kinase(s) responsible for SOS phosphorylation in vivo, we assessed the role of mitogen-activated, extracellular-signal-regulated protein kinase kinase (MEK), extracellular-signal-regulated protein kinase (ERK), and the c-JUN protein kinase (JNK) in this phosphorylation event. Expression of a dominant-interfering MEK mutant, in which lysine 97 was replaced with arginine (MEK/K97R), resulted in an inhibition of insulin-stimulated SOS and ERK phosphorylation, whereas expression of a constitutively active MEK mutant, in which serines 218 and 222 were replaced with glutamic acid (MEK/EE), induced basal phosphorylation of both SOS and ERK. Although expression of the mitogen-activated protein kinase-specific phosphatase (MKP-1) completely inhibited the insulin stimulation of ERK activity both in vitro and in vivo, SOS phosphorylation and the dissociation of the Grb2-SOS complex were unaffected. In addition, insulin did not activate the related protein kinase JNK, demonstrating the specificity of insulin for the ERK pathway. The insulin-stimulated and MKP-1-insensitive SOS-phosphorylating activity was reconstituted in whole-cell extracts and did not bind to a MonoQ anion-exchange column. In contrast, ERK1/2 protein was retained by the MonoQ column, eluted with approximately 200 mM NaCl, and was MKP-1 sensitive. Although MEK also does not bind to MonoQ, immunodepletion analysis demonstrated that MEK is not the insulin-stimulated SOS-phosphorylating activity. Together, these data demonstrate that at least one of the kinases responsible for SOS phosphorylation and functional dissociation of the Grb2-SOS complex is an ERK-independent but MEK-dependent insulin-stimulated protein kinase.

Concanavalin A-stimulated splenocytes secrete a factor that stimulates progesterone production in cultured rat granulosa cells. The actions of this progesterone-stimulating factor (PSF) were characterized further by purifying it by sequential chromatographies on heparin-agarose, copper chelating-Sepharose and Mono S columns. Several of its effects on granulosa cells were then compared with those of FSH. Like FSH, PSF induced accumulation of progesterone and 20 alpha-dihydroprogesterone in granulosa cell culture media; however, the time course of progesterone accumulation in response to PSF was much slower than that in response to FSH. In contrast to FSH, PSF induced little accumulation of oestrogen. Induction of other differentiated responses was compared by pretreating cultured granulosa cells with either FSH or PSF, and then challenging with LH, prolactin, epidermal growth factor or the beta-adrenergic agonist, isoproterenol. Pretreatment with FSH induced subsequent responsiveness to all four agents, whereas pretreatment with PSF induced subsequent responsiveness only to isoproterenol. These results indicate that granulosa cell responses to PSF are much more limited than are those to FSH and that these responses are characterized by an increase in progestin production.

Summary We previously showed that rat and cow blood vessels contain arginine vasopressin (AVP) of local origin. In the present study, to investigate potential roles for this locally produced peptide. we examined the effects of AVP on growth of cultured vascular endothelial and smooth muscle cells (EC, SMC). Treatment of cultured bovine aortic endothelial (BAE), murine cerebral micro-vascular endothelial (MME), or murine cerebral micro-vascular smooth muscle (MMSM) cells with AVP produced dose-dependent increases in total protein content with maximum increases of 11, 46. and 33% over vehicle-treated controls, respectively. AVP also dose-dependently increased new protein synthesis, as reflected by [3H]leucine incorporation into BAE and MME cell protein, with maximal increases of 34 and 249?, respectively. In addition, the AVP-stimulated increase in protein synthesis could be significantly attenuated in both BAE and MME cells by simultaneous addition of a specific AVP VI receptor antagonist. In contrast, AVP did not affect total cell number or DNA synthesis, as reflected by [3H]thymidine incorporation, in any cell type. Neither was cell size, as measured by forward light scatter with a fluorescence-activated cell sorter (FACS), changed by treatment with AVP. However, the density of individual cells, as measured by orthogonal light scatter with FACS, was increased by AVP. These data demonstrate that AVP stimulates protein synthesis but not proliferation of cultured vascular EC and SMC and suggest that locally produced AVP may function as a growth factor for these cells.

Progesterone accumulation in conditioned media is a frequently employed endpoint for in vitro cell culture of steroidogenic cells. Although radioimmunoassay (RIA) has been the predominant method for measurement of progesterone, a number of nonradiometric immunoassays have been described but they have not been applied to conditioned media. Here, we report the characterization of a microtitre plate enzyme immunoassay (EIA) for determination of progesterone in conditioned media. The EIA has a sensitivity of 0.3 pg per well with intraassay and interassay coefficients of variation of 7.3 and 10.2%, respectively. The specificity of the EIA is no different than that of a comparable RIA showing crossreactivities of less than 0.1% for other steroids except 5 alpha-pregnan-3,20-dione (47%) and 11 alpha-hydroxyprogesterone (18%). Progesterone levels from conditioned cell culture media of either rat or human granulosa cell cultures measured by both EIA and RIA were in close agreement (r = 0.96) and serial dilutions of culture samples in the EIA were parallel to those of the standards. Also, extraction of culture media prior to EIA was found not to be necessary. Thus, this EIA is a highly sensitive and specific assay that provides a rapid, simple, inexpensive, and non-radiometric alternative to radioimmunoassay for measurement of progesterone in conditioned cell culture media.

The present study examined the possibility that blood vessels of the rat and cow contain arginine vasopressin (AVP) and that the vascular stores of this potent vasoconstrictor are of local, rather than blood-borne, origin. Vessels from rat or cow were homogenized in acid, and the supernatants were assayed for AVP by radioimmunoassay. AVP immunoreactivity was detected in all vessels taken from the rat including aorta, mesenteric artery, renal artery, vena cava, and renal vein, as well as atria of the heart and also in cow aorta. Blood vessels from hypophysectomized and Brattleboro rats also contained AVP at levels similar to those of intact control rats. Further characterization of this immunoreactive material showed that, in both radioimmunoassay and radioreceptor assay, vascular extracts produced competition curves, which were parallel to those of synthetic AVP. Additionally, immunoreactive AVP in aortic extracts comigrated with synthetic AVP and pituitary extract on both high-pressure liquid chromatography and Sephadex G-25 chromatography. Furthermore, intravenous administration of the aortic extract produced pressor responses in conscious rats, and these responses were significantly attenuated by pretreatment with a specific AVP V1-receptor antagonist. These data demonstrate that blood vessels contain an AVP-like peptide that appears indistinguishable from authentic AVP and further suggest that this vascular peptide is of local origin.

Abstract We have reported previously that several blood vessels of the rat and cow contain immunoreactive vasopressin and further suggested that this peptide might be produced locally. To provide additional support for this hypothesis, we conducted the present study to determine whether mRNA for arginine vasopressin is also present in blood vessels. Ribonuclease protection analysis of total RNA isolated from rat hypothalamus and aorta revealed the presence of arginine vasopressin message in both tissues but not in RNA isolated from liver, a tissue devoid of vasopressin. Subsequent comparison of the autoradiographic intensities of the signals in these two tissues indicated that vasopressin message was 100- to 1000-fold lower in aorta. Additional studies showed that RNA isolated from endothelium-denuded vessels contained levels of arginine vasopressin message similar to those in intact vessels, indicating that endothelium was not a major source of this message. These data were substantiated by further studies using a vasopressin radioimmunoassay, which showed that vasopressin peptide levels in intact and endothelium-denuded vessels did not differ. Thus, the present study showed that rat aorta contains arginine vasopressin mRNA as well as the vasopressin peptide and that both the message and the peptide are contained in nonendothelial structures. However, the data do not rule out endothelium as a possible source of vasopressin. These studies add further support to the hypothesis that blood vessels are capable of producing vasopressin.

We have previously identified and purified a splenocyte-derived factor (PSF) that stimulates the accumulation of progesterone and 20α-dihydroprogesterone (20α-OH-P) in rat ovarian granulosa cells independently of FSH. In the present study, time course experiments comparing the response to PSF with that to FSH revealed that PSF-stimulated progesterone accumulation was slower than that of FSH, but PSF-stimulated 20α-OH-P accumulation had a time course similar to that of FSH. To determine the basis for the slower progesterone response to PSF, the effect of these two agents on each step of the steroidogenic pathway was assessed. First, to examine whether PSF-stimulated cholesterol mobilization was limiting, cultured granulosa cells were treated with 22( R )-hydroxycholesterol. While both FSH-and PSF-stimulated progesterone and 20α-OH-P accumulation approximately doubled, the overall time courses did not change indicating that cholesterol availability was not the factor limiting the response to PSF. Next, PSF and FSH induction of steroidogenic enzyme activities and messenger RNAs were compared. While FSH-stimulated cytochrome P450 side chain cleavage enzyme (SCC) activity rapidly increased (peaking at 2 days) and then slowly declined, PSF-stimulated SCC activity gradually increased over 5 days to approximately 35% of the maximal activity stimulated by FSH. PSF also induced slower increases in P450scc mRNA levels than did FSH. In addition, PSF stimulated 3β-hydroxysteroid dehydrogenase (3β-HSD) activity more slowly than did FSH, but after 3 days of culture, PSF-stimulated activity was significantly higher than that induced by FSH. A similar pattern was also observed with 3β-HSD mRNA levels. In contrast, FSH stimulated only minimal increases in 20α-hydroxysteroid dehydrogenase (20α-HSD), whereas PSF stimulated larger and more rapid increases in 20α-HSD activity, which were maximal by 3 days. These studies indicate that the slower onset of PSF-stimulated progesterone production is due, at least in part, to slower induction of mRNAs for P450scc and 3β-HSD leading to slower increases in the activities of their respective enzymes. Additionally, the higher 20α-OH-P levels produced by PSF may be due to greater induction of 20α-HSD activity.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a novel neuropeptide with considerable homology to vasoactive intestinal peptide (VIP) and GH-releasing hormone (GHRH). Because we have shown previously that VIP and GHRH stimulate steroidogenesis in cultured rat granulosa cells independently of FSH, the present studies evaluated whether PACAP also stimulates steroidogenesis and compared its effects to those of VIP and GHRH. Granulosa cells cultured for 2 days with PACAP-27, VIP, or GHRH (10(-9)-10(-6) M) showed dose-dependent increases in the accumulation of progesterone, 20 alpha-dihydroprogesterone, and estrogen. The rank order of potency for the three peptides was PACAPVIPGHRH. PACAP also increased cAMP accumulation and was again more potent than VIP. In addition, all three peptides synergistically augmented FSH-stimulated progesterone and 20 alpha-dihydroprogesterone accumulation with the same rank order of potency as above; however, only the highest dose of each peptide augmented estrogen accumulation. Further studies examined the effects of various androgens on these responses. PACAP-stimulated progesterone accumulation was minimal in the absence of androstenedione, but increased up to 40-fold in its presence. Other synthetic and naturally occurring androgens produced similar increases with a rank order of potency of R1881testosterone = androstenedionedihydrotestosterone. Analysis of cAMP levels indicated that by 1 h after treatment with PACAP, cAMP levels within the cells increased by 4-fold, and this response was unaltered in the presence of androstenedione; however, by 48 h, cAMP levels had markedly declined, and this response was attenuated by androstenedione. These results demonstrate that PACAP stimulates steroidogenesis and cAMP accumulation in cultured rat granulosa cells, and it is more potent than its two homologs, VIP and GHRH. Because PACAP has been shown to be present within the ovary, these data indicate that this peptide may play a role in modulating ovarian steroidogenic activity.

c-Fos and c-jun are immediate early proto-oncogenes encoding proteins for the heterodimer AP-1, a DNA binding complex which regulates gene transcription. In order to investigate the presence and potential gonadotropin regulation of mRNAs for these proto-oncogenes in rat granulosa cells, we used Northern blotting of total RNA from cultured cells. Granulosa cells obtained from diethylstilbestrol (DES)-treated weanling rats were challenged with follicle-stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), dibutyryl cAMP ((Bu)2cAMP) or tetradecanoyl-13-phorbol acetate (TPA) either 2.5 h after cell isolation (day 0) or following a 2-day pretreatment with FSH (day 2). Freshly isolated cells treated with FSH exhibited 4-fold and 3-fold increases in c-fos and c-jun mRNAs, respectively, within 30 min. Two hours after FSH treatment, both c-fos and c-jun message levels diminished to near control levels. Granulosa cells pretreated for 2 days with FSH, then re-challenged with FSH, showed similar increases in both c-fos and c-jun messages. These effects were dose- and time-dependent on both day 0 and day 2. Likewise, (Bu)2cAMP also increased c-fos and c-jun mRNAs in a time- and dose-dependent manner on both day 0 and day 2. In contrast, LH or hCG minimally increased c-fos and c-jun mRNAs on day 0, but on day 2, both hormones markedly increased message levels in a manner similar to that seen with FSH. Analogous effects were observed with TPA which minimally stimulated c-fos and c-jun mRNAs on day 0, but markedly increased these messages on day 2. These studies demonstrate that c-fos and c-jun mRNAs can be induced in cultured rat granulosa cells by acute gonadotropin, (Bu)2cAMP or phorbol ester treatment and suggest that these immediate early proto-oncogenes may play a role in granulosa cell function.

The influence of cholinomimetics on follicle-stimulating hormone (FSH)-induced progestin production was studied in a primary culture of rat granulosa cells. Cells were cultured for 2 days with FSH and delta 4-androstenedione in the presence or absence of increasing concentrations of cholinergic agonists. Although ineffective as stimulators of steroidogenesis by themselves, the three nicotinic receptor-selective agonists lobeline, dimethylphenylpiperazinium iodide (DMPP), and phenyltrimethylammonium iodide (PTMA) inhibited FSH-induced progesterone and 20 alpha-hydroxypregn-4-en-3-one production in dose-dependent fashions. The rank order of inhibitory potencies was lobeline greater than DMPP greater than PTMA with IC50 values of 2 X 10(-6) M, 3 X 10(-5) M, and 3 X 10(-4) M, respectively. In contrast, the muscarinic receptor-selective agonists muscarine and bethanechol failed to inhibit steroid production. The inhibitory effect of lobeline on the time course of FSH-induced induced steroid production indicated an immediate inhibitory action; however, this inhibition was readily reversed upon removal of the drug. Further studies demonstrated that the FSH-stimulated increase in intracellular cAMP levels, as well as progesterone production induced by cholera toxin and forskolin (agents that stimulate cAMP production) and by dibutyryl cAMP (a cAMP analog), were also suppressed by lobeline. The present observations indicate that nicotinic, but not muscarinic, cholinergic agonists inhibit progesterone biosynthesis in cultured granulosa cells and suggest that endogenous acetylcholine may play a modulatory role in ovarian steroidogenesis.

As part of an assessment of endocrine influences on the actions of morphine, we have studied the effects of intracerebroventricular administration of somatostatin and thyrotropin releasing hormone (TR

Ovarian granulosa cells from small follicles have generally been considered to comprise a homogeneous cell population; however, stratified arrangements of hormone receptors have been found in antral and mural granulosa cells of Graafian follicles. Using cultured granulosa cells derived from immature, hypophysectomized, estrogen-treated rats, we have previously shown that vasoactive intestinal peptide (VIP) as well as FSH stimulates steroid production by these cells. Dose-response analysis indicated that the actions of these hormones were additive, suggesting the presence of subpopulations of granulosa cells. Using a continuous (0-30%) Metrizamide density gradient, we have identified three populations of granulosa cells with different sedimentation properties. After centrifugation for 15 min at 1500 X g, cells sedimented at Metrizamide concentrations of 13%, 18%, and 20% (peaks A, B, and C, respectively). The subpopulation with lowest density (peak A) comprised 5% of the total cells, whereas the remaining cells were distributed approximately equally in the other two peaks. The profiles of estrogen and progesterone production by these cells in response to FSH and VIP indicated that FSH preferentially stimulated steroid production in cells with the highest density (peak C), whereas VIP mainly induced steroidogenic responses in cells of intermediate density (peak B). In contrast, cells with the lowest density (peak A) were unresponsive to either hormone. Treatment with forskolin, a universal adenylate cyclase activator, induced steroid production in both subpopulations B and C. Further studies demonstrated that LH/human CG receptors were induced by FSH and forskolin in cells from peak C, whereas VIP treatment did not induce LH/human CG receptors in cells from peak B. In unfractionated cultured cells, GnRH potently antagonized FSH- but not VIP-induced steroidogenesis. Upon density-gradient fractionation, the profile of GnRH receptor content correlated well with GnRH effects since FSH-responsive cells (peak C) contained the majority of GnRH receptors. The present results demonstrate that granulosa cells from immature follicles are heterogeneous and consist of two major subpopulations of cells with differential responsiveness to FSH and VIP. These findings provide the basis for further morphological and biochemical analysis of subpopulations of granulosa cells during follicular development.

The actions of cholinomimetics on androgen biosynthesis were investigated using a primary culture of testicular cells derived from adult hypophysectomized rats. Testicular cells were maintained in serum-free medium and treated with hCG and/or drugs on day 8 of culture. Media were collected 2 days later for measurement of steroids and cAMP. Treatment with hCG increased testosterone production 50-fold over control values, whereas treatment with cholinomimetics alone failed to increase androgen production. Concomitant treatment of testicular cells with nicotinic cholinergic agonists (lobeline, nicotine, and dimethylphenylpiperazinium iodide) inhibited hCG-stimulated androgen biosynthesis in a dose-dependent fashion, with IC50 values of 3 X 10(-5), 1.7 X 10(-4), and greater than 10(-3) M, respectively. In contrast, two muscarinic cholinergic agonists, muscarine and bethanechol, failed to inhibit androgen production at concentrations up to 10(-3) M. A ganglionic nicotinic antagonist (hexamethonium), but not a skeletal muscle nicotinic antagonist (decamethonium), partially blocked the actions of lobeline. Lobeline (10-4) M) decreased hCG-stimulated testosterone production (50-75%) throughout the 2-day culture period; however, this inhibition was reversible upon removal of the drug. Lobeline also inhibited hCG-stimulated cAMP accumulation as well as testosterone production induced by cholera toxin (65% inhibition), forskolin (50% inhibition), or (Bu)2cAMP (70% inhibition). Lobeline inhibition of hCG-stimulated testosterone production was accompanied by decreases in medium accumulation of 17 alpha-hydroxypregnenolone (75%), 17 alpha-hydroxyprogesterone (85%), dehydroepiandrosterone (50%), and androstenedione (61%); however, the medium content of pregnenolone and progesterone were unchanged. Additional experiments demonstrated that lobeline suppressed the conversion of exogenous progesterone to testosterone, but did not affect the conversion of exogenous 17 alpha-hydroxyprogesterone to testosterone. These results indicate that nicotinic, but not muscarinic, cholinergic agonists inhibit androgen biosynthesis through selective inhibition of 17 alpha-hydroxylase activity. Thus, endogenous acetylcholine may be involved in the negative regulation of testicular steroidogenesis.

Prolactin (PRL) exerts both stimulatory and inhibitory effects upon testicular steroidogenesis in vivo. The direct effects of PRL on biosynthesis of testicular androgen were studied in primary cultures of testicular cells obtained from adult, hypophysectomized or neonatal, intact rats. In cells from adult animals, treatment with human chorionic gonadotropin (hCG) (10 ng/ml) significantly increased testosterone and progesterone production relative to their respective controls. In contrast, neither steroid was increased by treatment with rat PRL (rPRL) or ovine PRL (oPRL) alone. Upon addition of 0.1-3 ng/ml of either rPRL or oPRL to the hCG-treated cultures, testosterone production was progressively increased up to a maximum of 70% greater than with hCG alone. However, when PRL exceeded 3 ng/ml, the testosterone response began to decline and was 39 or 24% less than from cells treated with hCG alone at 300 ng/ml of rPRL or oPRL, respectively. A similar biphasic response pattern was observed in cells from neonatal animals. In contrast to the biphasic effect of PRL on production of androgen, PRL treatment enhanced hCG-stimulated production of progesterone in a dose-related manner without exerting an inhibitory effect. At 3 and 300 ng/ml, rPRL augmented hCG action by 2.5- and 8-fold, respectively. Similarly, in the presence of inhibitors of pregnenolone metabolism, rPRL also enhanced hCG-stimulated production of pregnenolone. Quantitation of steroid intermediates in the testosterone biosynthetic pathway revealed that the stimulatory effect of 3 ng/ml rPRL on testosterone production was associated with 1.3- and 2.8-fold increases in accumulation of androstenedione and 17 alpha-hydroxyprogesterone.(ABSTRACT TRUNCATED AT 250 WORDS)

We have previously demonstrated the presence of an arginine vasopressin (AVP)-like peptide and AVP receptors in rat testis. We have also shown a direct inhibitory effect of AVP on androgen biosynthesis by cultured testicular cells. This study examined the presence of testicular AVP-like peptides and AVP receptors in homozygous (di/di) Brattleboro rats, a genetic mutant known to be deficient in hypothalamic, pituitary, and circulating AVP. The supernatant of homogenized 0.1 N acetic acid-extracted testis from adult homozygous Brattleboro rats was chromatographed on a Sephadex G-25 column. The elution profile of AVP immunoreactivity, as measured by a specific RIA, showed three distinct peaks. The first peak eluted close to the column void volume, a second peak eluted at the column total volume, while a third peak coeluted with synthetic AVP. The third peak of immunoreactive material (375 pg/g tissue) behaved similarly to authentic AVP on octadecylsilica adsorption chromatography, showed a competition curve parallel to that of AVP in the RIA, and comigrated with AVP on Sephadex G-25 and reverse phase TLC. The first, but not the second, immunoreactive peak contained enzyme activity that degraded labeled AVP in a time-dependent manner. Additional studies investigated the presence of AVP receptors in testes from Brattleboro rats. Saturable and specific [3H]AVP-binding sites were present in an enriched testicular interstitial cell preparation from these animals. Scatchard analysis indicated a Kd of 5.6 X 10(-10) M and a binding capacity of 9.7 fmol AVP bound/10(6) cells. These receptors were of the vasopressor (V1) subtype, as indicated by the potencies of selective AVP analogs for competition of [3H]AVP binding. The functionality of these receptors was shown by AVP inhibition of gonadotropin-induced androgen biosynthesis in cultured testicular cells derived from Brattleboro rats. Thus, testes from Brattleboro rats contain a high amount of an AVP-like peptide even though these animals lack hypothalamic, pituitary, and circulating AVP. Also, an AVP-degrading enzyme and AVP receptors with a Kd and binding capacity similar to those of Sprague-Dawley rats are present in the testes of Brattleboro rats. These findings add further support to the hypothesis that locally produced AVP acts as an intratesticular modulator of androgen biosynthesis.

Previous experiments in our laboratory demonstrated that morphine, at doses which produced pronounced hypothermia in normal rats, not only failed to decrease but instead increased body temperature in thyroxine-treated animals. The present studies were undertaken to further investigate these initial findings. In animals treated chronically with subcutaneous thyroxine, basal body temperatures were elevated and morphine induced only hyperthermia whether given subcutaneously (10 mg/kg) or centrally (30 μg) into the anterior hypothalamus. Basal oxygen consumption, which reflects metabolic heat production, was significantly elevated when compared to controls. In response to morphine, control animals showed decreased oxygen consumption while thyroxine-treated animals showed a slight increase. In both groups of animals, changes in core temperatures reflected changes in oxygen consumption. These results indicate that hyperthyroid animals fail to decrease body temperature in response to morphine because they are unable to decrease metabolic heat production. Morphine, acting at central hypothalamic sites, reduces heat production in normal animals, but in thyroxine-treated animals morphine cannot overcome the increased thermogenesis.

Electrolyte fluxes are fundamental to normal endocrine pancreatic function. Adenosine triphosphatases (ATPases) are enzyme systems believed to modulate electrolyte movements across membranes in a number of cell types. This study was undertaken to measure cation-dependent ATPases of rat pancreatic islets. In addition, we compared effects of substances which influence endocrine pancreatic function upon ATPases in homogenates of islets and kidney, the latter being a tissue which would not be expected to have a stimulus-secretion response to substances which activate islets. Both tissues were generally similar with respect to apparent Michaelis constant (ATP) of Na(+)K(+)ATPase, Mg(++)ATPase, and Ca(++)ATPase. In islets and kidney, Na(+)K(+)ATPase specific activity was increased when the Na:K ratio was lowered from 250:1 (175:0.7 mM) to 5:1 (100:20 mM). Inhibition of Na(+)K(+)ATPase at either Na:K ratio by ouabain, an activator of secretion, and enhancement of the high-ratio Na(+)K(+)ATPase by diphenylhydantoin, an islet secretory inhibitor, were also common to both tissues. Because both inhibition and enhancement of Na(+)K(+)ATPase could be studied at the high Na:K ratio, we examined the effect of regulators of secretion upon the activity of this enzyme. Like ouabain, substances which induce or support islet secretion, glucose 16 mM or 3.3 mM, arginine 14.2 mM (with 3.3 mM glucose), or Ca(++) 1 mM, inhibited high-ratio islet Na(+)K(+)ATPase. Like diphenylhydantoin, the inhibitors of insulin secretion, diazoxide 0.22 mM, or NH(4)Cl 16 mM, enhanced this islet ATPase. Neither valine, which is non-secretogenic, nor arginine without glucose, which is a weak secretagogue, had any effect upon islet Na(+)K(+)ATPase. We examined the effect of these substances upon other cation-dependent islet ATPases. Ca(++) inhibited Mg(++)ATPase, and glucose inhibited Ca(++)ATPase. Leucine, 22.9 mM, which induces insulin secretion in the absence of glucose, suppressed islet Ca(++)ATPase and had no effect upon high-ratio Na(+)K(+)ATPase. In contrast to the observations in the islets, most substances which influence islet function had no effect on kidney ATPases, or effects which were different from those seen in islets. Except for ouabain, none of these substances influenced the three kidney ATPases in a manner similar to that seen with islets. These findings support the hypothesis that cation-dependent ATPases are involved in specificity of islet response to substances which influence endocrine pancreatic activity.

This study reports the effects of the administration of pharmacologic doses of vitamin A on multiple parameters of thyroid function. Vitamin A decreased total T4 and T3 levels. With vitamin A treatment, there was a marked increase in the percentage dialyzable T3 and T4 both in vivo and in vitro. The serum-free T3 and T4 levels as measured by dialysis were on the whole normal in vitamin A-treated rats. Following thyroidectomy, the total T4 levels were still decreased, suggesting that vitamin A produced its effects by increasing peripheral clearance of thyroxine. Vitamin A did not alter basal thyroid stimulating hormone (TSH) or its response to thyroid releasing hormone, suggesting a relatively normal hypothalamic-pituitary-thyroid axis in vitamin A-treated animals. Vitamin A may decrease tissue responsiveness to thyroid hormones as evidenced by the tendency to decreased Na-K-ATPase activity in the livers from vitamin A-treated rats and the decreased growth hormone response to T3 in GH3 pituitary cultures as shown in this study and by the decreased basal metabolic rate found after vitamin A in previous studies. Vitamin A decreased thyroid gland size and increases 125I thyroid uptake. In vitro, vitamin A enhanced T4 to T3 conversion in hepatic homogenates.

The actions of dioctanoylglycerol, a novel and specific activator of protein kinase C, on granulosa cell steroidogenesis and LH/hCG receptor induction were examined in vitro. Granulosa cells were cultured for 2 days in media containing 100 nM androstenedione. FSH treatment stimulated progesterone, 20α-hy-droxy-pregn-4-en-3-one (20α-OH-P) and estrogen production by 60- to 80-fold over basal levels. Treatment with dioctanoylglycerol increased progesterone production 3-fold over basal levels but failed to affect 20α-OH-P or estrogen production. Concomitant treatment of cells with FSH and increasing amounts of dioctanoylglycerol produced dose-dependent inhibition of FSH-stimulated progesterone, 20α-OH-P and estrogen production with IC50 values of 75, 40 and 50 μM, respectively. Dioctanoylglycerol also inhibited production of all three steroids induced by forskolin, an activator of adenylate cyclase, and dibutyryl cAMP. The effects of dioctanoylglycerol on cAMP responses to FSH were also examined. The increase in both intracellular and extracellular cAMP at l h following treatment with FSH was suppressed in the presence of dioctanoylglycerol as was the extracellular accumulation of cAMP at 48 h of culture. FSH induction of LH/hCG receptors, known to be a cAMP-mediated event, was also inhibited in the presence of dioctanoylglycerol. In contrast, a thio-derivative of dioctanoylglycerol (3-thio-1,2-dioctanoylglycerol), which does not activate protein kinase C, failed to inhibit both steroidogenesis and LH/hCG receptor induction in response to FSH. These results suggest that activation of protein kinase C inhibits gonadotropin-stimulated steroidogenesis and that this inhibition occurs at two distinct sites, the gonadotropin receptor/adenylate cyclase complex and a site distal to the generation of cAMP.

We have previously demonstrated the suppression of Leydig cell steroidogenesis by arginine vasopressin (AVP) in vitro. Since the circulating level of AVP is too low to mediate a testicular action of this peptide, we have conducted studies to identify testicular AVP-like substances. The supernatant of homogenized, acid-extracted rat testes was found to contain AVP immunoreactivity which showed parallelism with synthetic AVP in a specific radioimmunoassay for AVP. Chromatography of this extract on a Sephadex G-25 column produced three peaks of AVP immunoreactivity. The largest peak eluted close to the column void volume, a second smaller peak eluted at the total column volume, while a third peak co-eluted with synthetic AVP. Following acetone precipitation, ether extraction, and octadecylsilica (C18) adsorption chromatography of the acid extract, the third peak of AVP immunoreactivity (about 600 pg/testis) was fully retained by C18 chromatography and showed parallelism with synthetic AVP in both radioimmuno- and radioreceptor assays. This substance also co-migrated with synthetic AVP on both Sephadex G-25 and reverse-phase thin layer chromatography (RPTLC). The second peak was only partially retained by C18 adsorption chromatography, dilution curves were not parallel with synthetic AVP in radioimmuno- or radioreceptor assay, and this material failed to co-migrate with synthetic AVP on Sephadex G-25 and RPTLC. The first peak of apparent AVP immunoreactivity was associated with an enzyme(s) that degraded labeled AVP. This enzymatic activity, as well as the immunoreactivity, could be eliminated by heating the extract to 90 degrees C. These results demonstrate, by a number of independent criteria, that rat testes contain a substance which behaves like authentic AVP. Due to its high concentration, the AVP-like peptide may be synthesized or concentrated by testis cells. In addition, testis tissue contains enzymatic activity which degrades AVP and could represent a site of regulation of AVP action. Coupled with the previously demonstrated testis action of AVP, these results suggest a paracrine or autocrine role of the neurohypophysial hormone at the testis level.

Animals with impaired immune function show numerous reproductive disorders. To determine whether immune factors might play a direct role in the regulation of ovarian function, we examined the effects of Concanavalin-A-stimulated lymphocyte supernates (CAS) on steroidogenesis by cultured rat granulosa cells. Granulosa cells from immature estrogen-primed rats were incubated for 48 h with increasing doses of CAS in the presence or absence of FSH. In the absence of FSH, CAS produced a dose-dependent increase in progestin (progesterone and 20 alpha-hydroxyprogesterone) production to a maximum of 190-fold greater than untreated controls, but did not stimulate estrogen production. In the presence of FSH, the stimulatory effect of CAS on progestin production was additive with that of FSH. In contrast, CAS inhibited FSH-stimulated estrogen production in a dose-dependent manner. The maximum inhibition was greater than 90%. Addition of the phosphodiesterase inhibitor isobutylmethylxanthine to CAS-containing cultures significantly enhanced the stimulatory effect of CAS on progesterone production, suggesting that this action may be exerted through a cAMP-mediated pathway. The stimulatory component of CAS was heat labile, acid stable, and required a trypsin-sensitive cell surface recognition site, whereas the inhibitory component was both heat and acid stable. Neither the stimulatory nor the inhibitory actions of CAS were mimicked by treating granulosa cells with supernates from Concanavalin-A-stimulated neonatal cardiac or hepatic cell cultures. Thus, the present studies demonstrate that secretory products of lymphocytes (collectively termed lymphokines) can affect steroidogenesis in cultured rat granulosa cells. These data imply that immune cell factors may play a significant role in the differentiation and maturation of granulosa cells.

Domperidone, a dopamine receptor antagonist which apparently does not penetrate the blood-brain barrier in rats was administered to adult males. Domperidone 500 micrograms and 100 micrograms, given through intracarotid cannula, significantly elevated plasma beta-endorphin-immunoreactivity (beta-EP-I) at +15 min. To show that only a peripheral site(s) of action is implicated, domperidone was given to rats by cannulae implanted into both lateral ventricles. Plasma beta-EP-I was unaffected by this route of administration. These results suggest that plasma beta-EP-I is tonically inhibited by dopamine acting at site(s) outside of the blood-brain barrier.

Vasoactive Intestinal Peptide (VIP), originally considered to be a gut hormone, has recently been found to increase estrogen and progesterone production by ovarian granulosa and luteal cells. Because several studies indicate that granulosa cells and oocytes are capable of producing plasminogen activators, we have studied the effects of VIP on plasminogen activator activity in cultured granulosa cells and cumulus-oocyte complexes collected from the ovaries of hypophysectomized, estrogen-treated immature rats. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by a fibrin overlay technique to assess plasminogen activator activity, we observed that treatment with VIP stimulated the secretion of tissue-type plasminogen activator (tPA), but not urinary-type plasminogen activator (uPA), in a dose-dependent manner by cultured granulosa cells as well as by cumulus-oocyte complexes, but not by denuded oocytes. However, preparation of cumulus-free oocytes from cumulus-oocyte complexes which had previously been treated with VIP indicated substantial increases in tPA activity within the oocyte. The actions of VIP on tPA activity in granulosa cells were specific, because other closely related peptides (PHM-27 and glucagon) were ineffective. These effects of VIP, in addition to the previously observed effects on steroidogenesis, suggest that VIP may be an important regulator of ovarian function.

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

A growing body of evidence indicates that factors secreted by cells of the immune system can directly affect a variety of endocrine phenomena. In the present study we examined the direct effects of the cytokine, interferon (IFN), on FSH-stimulated steroidogenesis and LH/hCG receptor induction in rat granulosa cells. We show that gamma-IFN, but not alpha-IFN, inhibits FSH-stimulated progesterone, 20 alpha-hydroxypregn-4-en-3-one and estrogen production as well as gonadotropin-induced LH/hCG receptor formation in a dose-dependent manner. The results suggest that gamma-IFN may play a role in the maturation and differentiation of granulosa cells and thus may serve as a regulatory link between the immune and reproductive endocrine systems.

SummarySerum globulin and IgG levels were measured by radial immunodiffusion from 3 to 75 days after birth in rats neo-natally treated with 1 mg of cortisol acetate. In controls, globulins rose steadily during suckling, peaked at 11 days, and remained high until weaning, when they dropped considerably. Thereafter, they rose steadily until 75 days. In treated animals, levels increased slowly during development and were significantly lower than controls until 30 days. Serum IgG followed a similar pattern. The most severe immunological deficit in runts began on Day 12. The most marked mortality in treated rats also began at this age and in part may be due to the severe immunological impairment. No difference was detected in absorption of IgG from the gastrointestinal tract of the two groups. However, dramatic deficits were demonstrated in the ability of treated rats to synthesize antibody when sensitized with a t-dependent antigen. Thus, the difference in immunological competence is due to endogenous deficit...

Introduction THE NEUROHYPOPHYSIAL hormones constitute a family of nonapeptides best known for their roles in the conservation of body water (antidiuretic effect) and the contraction of uterine (oxytocic effect) and vascular (pressor effect) smooth muscle. All the known naturally occurring neurohypophysial hormones of vertebrates (see Table 1) are endowed with a disulfide bridge (position 1–6) and display phylogenetic constancy for amino acids at positions 1, 5, 6, 7, and 9. In contrast, substitutions have occurred in the remaining amino acids thereby modifying biological activity and potency. While none of the neurohypophysial peptides is entirely selective in terms of its biological properties, one activity tends to predominate. Thus, peptides 1–5 (see Table 1) are predominantly antidiuretic and pressor active while peptides 6–11 are primarily oxytocic. Most species possess a characteristic pair of neurohypophysial peptides regulating antidiuretic and oxytocic activities. In most mammals, this pair is re...

Vasoactive intestinal peptide (VIP) was originally isolated from porcine duodenum and considered to be a gut hormone. Recent evidence indicates that it may also be involved in reproductive functions. In this study, a possible action of VIP on steroidogenesis by cultured testicular cells was investigated. Neonatal testicular cells were treated in vitro with hormones for 3 days and medium steroid or cAMP content was measured by radioimmunoassay. Treatment of cultured cells with VIP (10−9 to 10−6 M) increased the production of testosterone, progesterone, and pregnenolone in a dose-dependent fashion. Testosterone production in response to 10−6 M VIP was about 5–10% of that maximally induced by LH. Addition of methyl-isobutyl-xanthine, a phosphodiesterase inhibitor, to the VIP-containing cultures significantly enhanced production of testosterone by 13-fold, of progesterone by 9-fold, and of pregnenolone by 2.5-fold as compared to treatment with VIP alone. Additional experiments also showed a dose-dependent stimulation of cAMP production by VIP. The VIP-related hormones PHM-27, secretin, and glucagon also stimulated progesterone and testosterone production with a potency order (PHM-27 > secretin > glucagon) consistent with that observed for other VIP receptor-mediated actions. A direct stimulatory effect of VIP on Leydig cells was indicated in studies on steroidogenesis by testicular cells separated on a metrizamide density gradient. In these studies, VIP stimulated androgen production in an LH-responsive subpopulation of testis cells but failed to affect steroid production in non-LH-responsive cells. Thus, these experiments demonstrate that VIP, independent of LH, stimulates steroidogenesis in cultured neonatal testicular cells and suggest a possible stimulatory role for VIP in testicular steroidogenesis by the developing testis.

Previous studies have implicated arginine vasopressin (AVP) as a negative intratesticular modulator of androgen production. The present studies were undertaken to further define the testicular AVP system by investigating potential influences of pituitary hormones on testicular receptors for AVP. Binding of [3H]AVP to testicular membranes was time, temperature, and pH dependent, with optimum binding at 90 min, 22 C, and pH 8.0. Addition of divalent cations to the incubation mixture significantly increased binding by 4-, 10-, 15-, and 35-fold with Ca2+, Zn2+, Mg2+, and Mn2+, respectively. The influence of pituitary hormones on testicular AVP receptors was initially assessed by comparing binding parameters obtained by Scatchard analysis of saturation binding data between membranes prepared from intact or hypophysectomized rats. Testicular membranes from intact animals bound 66 +/- 2 fmol AVP/mg protein, whereas membranes from animals hypophysectomized 10 days previously showed a significantly lower binding capacity of 40 +/- 3 fmol AVP/mg protein; however, the affinity of the receptors for AVP did not differ between these two groups. A similar decrease was observed when binding was compared between purified Leydig cells isolated from intact and hypophysectomized animals by Metrizamide density gradients. Time-course analysis of this effect showed a continuous decline in total testicular AVP-binding capacity for the first 2 weeks after hypophysectomy and a slight increase at 3 weeks. Treatment of hypophysectomized animals with pituitary hormones beginning on the third day after hypophysectomy and continuing for either 4 or 7 days showed that LH and GH were able to dose-dependently restore testicular AVP-binding capacity to levels found in intact animals, whereas FSH and PRL were ineffective. These data indicate that testicular AVP receptors are under the control of LH and GH from the pituitary and suggest that pituitary hormones may impact on the efficacy of the testicular AVP system by regulating AVP receptor levels.

In addition to the well-known growth stimulating effects of insulin-like growth factors (IGFs), recent studies suggest that these peptides may also modulate the differentiated functions of endocrine cells. Thus, in the present studies, we have investigated the actions of IGFs on androgen biosynthesis by cultured testicular cells. Treatment of cells obtained from adult hypophysectomized rats with LH (1 ng/ml) stimulated testosterone production 60-fold over basal levels. In contrast, treatment with either synthetic human IGF-I or IGF-II failed to stimulate androgen production. However, concomitant treatment of the LH-containing cultures with increasing doses of IGF-I (10–500 ng/ml) augmented testosterone production up to 70% over that seen with LH alone (ED50 = 67 ng/ml). Similar effects were obtained with IGF-II but this peptide was about 10-fold less potent than IGF-I. In addition, these peptides also stimulated the accumulation of pregnenolone and progesterone in the culture medium. Additional studies demonstrated specific binding of [125I]iodo-IGF-I to testicular cells. This binding was competed by IGF-related peptides with the potency order IGF-II = IGF-I > insulin whereas unrelated peptides did not compete. The cellular localization of these receptors was examined in testicular cells separated on a metrizamide density gradient. IGF-I receptors were evenly distributed between two cell peaks containing subpopulations of Leydig cells whereas much less binding was found in other testicular cell types. Coupled with recent findings indicating testicular production of IGF-I, the present results suggest that this peptide may act as a positive intratesticular modulator of Leydig cell differentiation.

Ca2+-dependent ATPase (Ca2+-dependent ATP phosphohydrolase, EC 3.6.1.3) present in a subcellular fraction derived from rat pancreatic islet homogenates was examined to determine kinetic parameters and responses to various substances with known effects upon insulin secretion. Experiments demonstrated the presence of a Ca2+-ATPase with a Km ATP of 7 · 10−5 M and two Km Ca of 1.3 · 10−7 M and 5.7 · 10−6 M. The enzyme had little activity in acidic media while retaining considerable activity in basic media. Optimal activity was obtained at pH 7.5. The enzyme was relatively temperature insensitive (Q10 = 1.49), since activity decreased less than 50% with a 15°C decrease in temperature. Studies on the stability of enzyme activity upon storage at −20°C indicated that for intact islets activity was stable for 3 weeks, while in homogenates activity was stable for only 1 week, after which activity rapidly declined in both cases. Certain substances known to either stimulate or inhibit insulin secretion were tested for their ability to alter enzyme activity. Potassium, glibenclamide and cyclic AMP had no effects upon activity. Mannoheptulose significantly suppressed enzyme activity while 2-deoxyglucose did not alter activity. These observations are consistent with the hypothesis that a Ca2+-ATPase present in pancreatic islets may act as a modulator of pancreatic islet β cell activity.

The antinociceptive and temperature responses to morphine were compared in male and female rats from two different strains. Males of both the Sprague-Dawley and Wistar-Furth strains were slightly more responsive to the acute actions of morphine than were females of the same strain. However, Wistar-Furth animals required approximately twice the dose of morphine to display equivalent antinociceptive responses and four times the dose to display equivalent hypothermic responses when compared with Sprague-Dawley animals. During chronic morphine treatment, the development of tolerance was slightly more rapid in males than in females and in Sprague-Dawley animals than in Wistar-Furth animals. Gonadal hormones also influenced morphine responses. Ovariectomized rats were significantly more responsive acutely to morphine and developed tolerance less rapidly than estradiol-treated females. However, alterations of gonadal hormones in males did not affect morphine responses. These results indicate that morphine responses vary considerably between strains of animals and are influenced by gonadal hormones of females, but not of males.

A growing body of evidence indicates that substances released by activated immune cells can directly influence the functions of various endocrine cells. In the present study, the direct in vitro effects of interleukins (IL) 1, 2, and 3 on follicle-stimulating hormone (FSH)-stimulated steroidogenesis and luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor induction in granulosa cells were examined. In the absence of FSH, none of the interleukins stimulated steroid production or LH/hCG receptor induction during a 2-day culture period. However, in the presence of FSH, both IL-1 alpha and IL-1 beta inhibited, in a dose-dependent manner, progesterone and estrogen production as well as LH/hCG receptor induction in response to FSH. In contrast, both agents augmented 20 alpha-hydroxy-4-pregnen-3-one (20 alpha-OH-P) production stimulated by FSH. In all cases, less IL-1 beta than IL-1 alpha was required to produce a comparable effect. IL-2 slightly, but significantly, enhanced both FSH-stimulated progesterone and 20 alpha-OH-P production but had no effect on FSH-stimulated estrogen production or LH/hCG receptor induction. IL-3 potentiated the 20 alpha-OH-P response to FSH by up to 65% but had no effect on FSH-stimulated progesterone or estrogen production or LH/hCG receptor induction. These data suggest that the interleukins, which are key mediators of immune responses, may affect mechanisms crucial for the maturation and differentiation of granulosa cells and thus may also play a regulatory role in reproductive function.

Although central administration of arginine vasopressin (AVP) has been reported to increase arterial pressure mediated by activation of the sympathetic system, we found that peripheral blockade of sympathetic transmission did not attenuate this pressor response. To elucidate the mechanism, rats were pretreated with either phentolamine (3 mg/kg), chlorisondamine (2.5 mg/kg), a vasopressin V1 receptor antagonist d(CH2)5Tyr(Me)AVP (AVP-X) (10 micrograms/kg), or the combinations of phentolamine and AVP-X or chlorisondamine and AVP-X. The pressor response to intracerebroventricular injection of AVP in unrestrained conscious rats was reduced but not significantly altered by intravenous injection of phentolamine or AVP-X; however, combined treatment with these agents abolished the response. To determine that the amount of central AVP leaked to the periphery did not contribute to the pressor effect, tritiated AVP and AVP (100 ng total) were injected intracerebroventricularly. Blood samples collected at 0, 3, and 30 minutes after injection showed that radioactivity in plasma was primarily metabolites and that the amount of intact AVP estimated to leak from the brain was too low to produce a pressor effect. Comparative regional hemodynamic studies between intracerebroventricular and intravenous injection of AVP performed in conscious rats instrumented with Doppler flow probes demonstrated a qualitatively similar pattern of increased resistance in the renal, mesenteric, and hindquarters beds. These data suggest that central pressor action of AVP is mediated by both activation of the sympathetic system and release of AVP.

Although insulin-like growth factor-I (IGF-I) and insulin have been shown to augment rat granulosa cell differentiation, their mechanism(s) of action has not yet been elucidated. In the present study, we have examined granulosa cells obtained from immature hypophysectomized estrogen-treated rats for specific IGF-binding sites that might mediate the effects of the insulin-like peptides. Using synthetic [125I]iodo-IGF-I, we have found specific high affinity, low capacity (Kd = 1.36 +/- 0.131 nM; 3250 +/- 662 sites/cell) IGF-I-binding sites that have lower affinities for the related peptides IGF-II and insulin (potency ratio, 1:9:700 for IGF-I, IGF-II, and insulin). We have also found specific binding sites for [125I]iodo-IGF-II, a newly available synthetic peptide. The IGF-II-preferring sites were of a single class (Kd = 1.54 +/- 0.32 nM; 4728 sites/cell) and exhibited a rank competition order of IGF-II greater than IGF-I much greater than insulin. To study the functional correlates of these binding activities, granulosa cells were cultured for 2 days in serum-free medium in the presence of FSH, with or without increasing concentrations of IGF-I, IGF-II, or insulin. Medium steroids were then determined by specific RIA, and cellular LH/hCG receptors were measured by specific [125I]iodo-hCG binding. Treatment with FSH increased estrogen and progestin production and induced the formation of LH/hCG receptors. Concomitant treatment with the three peptides dose-dependently enhanced both FSH-stimulated steroidogenesis and LH/hCG receptor induction, with a rank order of potency of IGF-I greater than IGF-II greater than insulin (potency ratio, 1:8:36). This rank order of potency of the insulin-like peptides was more closely correlated with their ability to compete for IGF-I binding rather than IGF-II binding, suggesting the preferential involvement of IGF-I receptors in the ovarian actions of the IGFs, although the involvement of IGF-II and insulin receptors cannot be dismissed. Our results demonstrate, for the first time, a biological action of synthetic IGF-II in granulosa cells and further show a novel insulin effect, enhancement of LH/hCG receptor induction. These findings also indicate that rat granulosa cells possess specific IGF-I and IGF-II-binding sites that may mediate the gonadotropin-enhancing actions of the insulin-like peptides. Since IGF-I appears to be the most biologically potent peptide, it is likely to be the most important insulin-like peptide involved in granulosa cell differentiation in vivo.