Curcumin and enalapril ameliorate renal failure by antagonizing inflammation in 5/6 nephrectomized rats: role of phospholipase and cyclooxygenase.

Previously, we showed that curcumin prevents chronic kidney disease (CKD) development in ⅚ nephrectomized (Nx) rats when given within 1 wk after Nx (Ghosh SS, Massey HD, Krieg R, Fazelbhoy ZA, Ghosh S, Sica DA, Fakhry I, Gehr TW. Am J Physiol Renal Physiol 296: F1146-F1157, 2009). To better mimic the scenario for renal disease in humans, we began curcumin and enalapril therapy when proteinuria was already established. We hypothesized that curcumin, by blocking the inflammatory mediators TNF-α and IL-1ß, could also reduce cyclooxygenase (COX) and phospholipase expression in the kidney. Nx animals were divided into untreated Nx, curcumin-treated, and enalapril-treated groups. Curcumin (75 mg/kg) and enalapril (10 mg/kg) were administered for 10 wk. Renal dysfunction in the Nx group, as evidenced by elevated blood urea nitrogen, plasma creatinine, proteinuria, segmental sclerosis, and tubular dilatation, was comparably reduced by curcumin and enalapril, with only enalapril significantly lowering blood pressure. Compared with controls, Nx animals had higher plasma/kidney TNF-α and IL-1ß, which were reduced by curcumin and enalapril treatment. Nx animals had significantly elevated kidney levels of cytosolic PLA(2), calcium-independent intracellular PLA(2), COX 1, and COX 2, which were comparably reduced by curcumin and enalapril. Studies in mesangial cells and macrophages were carried out to establish that the in vivo increase in PLA(2) and COX were mediated by TNF-α and IL-1ß and that curcumin, by antagonizing the cytokines, could significantly reduce both PLA(2) and COX. We conclude that curcumin ameliorates CKD by blocking inflammatory signals even if it is given at a later stage of the disease.

Evaluation of xenobiotic impact on urban receiving waters by means of statistical methods.

Xenobiotics in urban receiving waters are an emerging problem. A sound knowledge of xenobiotic input, distribution and fate in the aquatic environment is a prerequisite for risk assessments. Methods to assess the impact of xenobiotics on urban receiving waters should address the diverse characteristics of the target compounds and the spatiotemporal variability of concentrations. Here, we present results from a one-year-monitoring program concerning concentrations of pharmaceuticals, additives from personal care products and industrial chemicals in an urban drainage catchment in untreated and treated wastewater, surface water and groundwater. Univariate and multivariate statistical methods were applied to characterize the xenobiotic concentrations. Correlation and principal component analysis revealed a pronounced pattern of xenobiotics in the surface water samples. The concentrations of several xenobiotics were characterized by a negative proportionality to the water temperature. Therefore, seasonal attenuation is assumed to be a major process influencing the measured concentrations. Moreover, dilution of xenobiotics the surface water was found to significantly influence the concentrations. These two processes control more the xenobiotic occurrence in the surface water than the less pronounced concentration pattern in the wastewater sources. For the groundwater samples, we assume that foremost attenuation processes lead to the found differentiation of xenobiotics.

Protein profiling of non-malignant and malignant ascites by SELDI-TOF MS: proof of principle.

Ascites is a common clinical symptom in liver cirrhosis, inflammatory disorders of the abdomen and a major late manifestation of metastatic malignancies. Standard cytopathological techniques and immunocytochemistry have specificities and sensitivities of approximately 95 and 60%, respectively for the presence of tumor cells. Development of faster and more accurate screening methods would be of great clinical utility. In this work we examined differential analysis of the unbound proteins in the supernatant of ascites fluid by Protein-Chip SELDI mass spectrometry. There were 21 tumor cell-positive and 34 tumor cell-negative samples. We used principal component analysis coupled with linear regression applied to the mass spectra of the samples to distinguish between the sample groups. Two sample sets for statistical analysis were created after randomization, a training set with 37 samples and a validation set with 18 samples resulting in a specificity of 93% and a sensitivity of 83% on the training set. The validation set yielded a specificity and sensitivity of 75%. This study suggests that SELDI-TOF mass spectrometry appears to have great potential as a surrogate diagnostic tool to evaluate effusion specimens.

N,N-Dialkylaminostyryl dyes: specific and highly fluorescent substrates of peroxidase and their application in histochemistry.

Fluorescent labeling of immuno-bound or endogenous peroxidase (PO) activity has been achieved to date by means of phenol derivatives with a low substitution degree. Here it is demonstrated that N,N-dialkylamino-styryl dyes can also act as fluorescent substrates of PO. They undergo enzymatically cross-linking reactions to surrounding cell constituents in an analogous manner thus permitting highly fluorescent and permanent labeling. This approach is narrowly related to the catalyzed reporter deposition (CARD) technique based on tyramine conjugates and the recently described catalytic cross-linking approach of hydroxystyryl derivatives. The substitution patterns for optimal cross-linking capability and the spectral properties of obtained specific reaction products were studied using an iterative semi-empirical approach. The best staining performance is achieved with N,N-dimethylaminoaryl derivatives. Their N,N-dialkyl homologues as well as the primary aryl amine pendants failed as PO substrates. Due to their basic character, novel substrates occasionally tend to unspecific interactions (staining nuclei, mast cells, or keratin). Centering this side specificity and repressing the staining capability of PO was achieved by chemical modification of the respective dye leading to new specific probes for keratin and cytoplasmatic RNA. In conclusion, catalytic cross-linking of heterocyclic 4-N,N-dimethylamino-styryl dyes represents a promising approach for the permanent fluorescent staining of PO in fixed cells and tissues, complementing the CARD technique. In contrast to CARD-related approaches, new substrates are characterized by a broad excitation and emission range of fluorescence and the outstanding spatial resolution of specific fluorescence signaling known so far from their 4-hydroxystyryl analogues. They currently represent the smallest fluorescent substrates of PO. Histochemical and immuno-histochemical applications share several outstanding features: High detection sensitivity, spatial resolution of fluorescence signaling, and photo stability. 4-N,N-dimethylamino-styryl substrates are compatible with their phenol and phenol-ester analogues. Their combination facilitates the trichromatic immuno-histochemical demonstration of three different targets simultaneously at one excitation wavelength in a conventional epi-fluorescence microscope.

Cerebral vasomotor responses after recombinant interleukin 2 infusion.

The effects of systemic human recombinant interleukin 2 (rIL-2) infusion upon both the vasoconstrictor effect of hypocapnia and the endothelium-dependent vasodilator effect of acetylcholine (Ach) were examined in anesthetized rats equipped with cranial windows. Prior to the functional studies, each of six animals received an i.v. infusion of rIL-2 (6 x 10(5) IU/kg) every 8 h for 3 days. At the same time, six control animals received infusions of equivalent volumes of sterile water. Eight h after the final infusion, each animal was anesthetized and equipped with a cranial window for the observation of pial arterioles overlying the left frontoparietal cortex. Pial arteriolar diameters were measured before and after the topical application of Ach which in normal cerebral arterioles elicits the release of endothelium-dependent relaxing factor, causing vasodilation. When arteriolar diameters returned to base line, they were measured again both before and during hyperventilation-induced hypocapnia. Following functional assessments, these same pial vessels were processed for study by transmission electron microscopy to determine if any observed functional changes correlated with morphological abnormality. Results of the statistical analyses suggested that normal Ach-induced endothelium-dependent vasodilation was absent in the rIL-2-infused group. Additionally, these animals exhibited reduced reactivity to the vasoconstrictive effects of arterial hypocapnia. The control group exhibited normal responsiveness to both Ach and hyperventilation. Ultrastructural studies revealed occasional morphological alterations of both vascular smooth muscle and endothelial cells in some vessels of rIL-2-infused animals but not in controls. These data suggest that repeated systemic rIL-2 infusion results in altered vasomotor responsiveness within the cerebral microcirculation. The data also suggest that the observed vasomotor changes are not always accompanied by overt morphological alterations of either endothelial or smooth muscle cells.

Characterization of bile salt-induced apoptosis in colon cancer cell lines.

Bile salts have been shown to be involved in the etiology of colorectal cancer. Although there is a large body of evidence for bile salts as a cocarcinogen in azoxymethane-induced colorectal cancer, bile salt-induced apoptosis of colorectal cancer cells has not yet been studied in detail. Therefore, we investigated the effects of different bile salts on apoptosis and apoptotic signaling in colon cancer cell lines. Incubation of colorectal cancer cell lines with physiological concentrations of deoxycholic acid led to a dramatic induction of apoptosis. Caspase cleavage and caspase activation occurred as early as 30 min after the addition of deoxycholate. Caspase-2 (Ich-1, Nedd2), caspase-3 (CPP-32, YAMA, Apopain), caspase-7 (Mch-3, ICE-LAP-3), and caspase-8 (FLICE, Mach-1, Mch5) are activated in HT-29, whereas caspase-1 (ICE) remained intact. Caspase activation and cellular apoptosis induced by bile salts were reversed by broad spectrum and selective caspase inhibitors. As opposed to hepatocyte death mediated by bile acids, CD95 was not involved in deoxycholate-induced apoptosis. The cytoprotective effect of ursodeoxycholic acid in hepatocytes or other tumor cell lines, which is mediated by inhibiting the mitochondrial permeability transition, was not observed in colon cancer cell lines as well. This points to distinct intracellular functions of ursodeoxycholate in different cancer cell types. Here we describe the specificity of bile salt-induced apoptosis in colon cancer cell lines. Differences from hepatocytes are shown. Bile acid-specific caspase activation is part of the apoptotic pathway induced by bile salts in colon cancer cell lines. Furthermore, a lack of cytoprotective function of ursodeoxycholate in these cells is demonstrated. Our data raise questions as to the role of bile salts in colorectal carcinogenesis.

Modulation of prolactin binding sites in vitro by membrane fluidizers. Effects on male prostatic and female hepatic membranes in alcohol-fed rats.

The objectives of this study were (i) to determine if in vivo administration of ethanol to rats produced changes in apparent lipid fluidity and prolactin binding capacity of male prostatic and female hepatic membranes and (ii) to compare the effects of membrane fluidizers (aliphatic alcohols) in vitro on prolactin binding of prostatic and hepatic membranes in control and alcohol-fed animals. In vitro ethanol has been shown by us previously to increase prolactin receptor levels presumably by unmasking cryptic prolactin receptors. The degree of fluidization was monitored by a fluorescence polarization method using 1,6-diphenylhexatriene. Adult male and female rats were given either water or 4% ethanol as the sole source of drinking fluid for a period of 6 weeks. No significant changes in plasma prolactin were observed between control and ethanol-treated groups of either sex. However, the microviscosity parameter, inversely related to lipid fluidity, was increased approx. 34% and 40%, respectively, in male prostatic and female rat hepatic membranes after ethanol feeding. Furthermore, 125I-prolactin binding capacity was decreased approx. 30% and 26%, respectively, in prostatic and hepatic membranes of alcohol fed animals. In vitro treatment with aliphatic alcohols had no effect on either microviscosity or prolactin binding in hepatic or prostatic membranes from ethanol-fed rats, but both fluidized and increased prolactin binding in the same membrane preparations from control rats. Our observations are consistent with the direct relationship between membrane fluidity and prolactin receptor levels. The changes in prostatic and hepatic membranes after alcohol feeding, namely decreased prolactin receptor levels, decreased fluidity and increased resistance to the fluidizing effects of in vitro aliphatic alcohols may reflect a fundamental membrane defect.

Effects of intraventricular catecholamines on luteinizing hormone release in ovariectomized-steroid-primed rats.

The effects of intraventricular norepinephrine (NE) and dopamine (DA) were studied in the awake, freely behaving rat. In long-term ovariectomized, estrogen-progesterone-primed (OVE E2-P) animals, blood samples were taken via indwelling intra-atrial catheters before and after intraventricular infusion of either pH-adjusted saline, NE )5 mug, 15 mug, 20 mug), or DA (4 mug, 15 mug), and plasma LH was measured by radioimmunoassay. Under urethane anesthesia, records were made of the effects of intraventricular saline and NE on the electrical activity of the arcuate nucleus in the form of multi-unit spike activity. In unanesthetized animals, intraventricular NE caused marked changes in behavior. The typical response consisted of three phases: generalized activation (5-7 min), feeding (5-15 min), and sleep 1-2 h). DA exerted similar behavioral effects but without the marked sleep phase characteristic of the NE response. The effects of the catecholamines on LH output were significant increases in plasma LH levels for all NE doses tested (5 mug, p less than .025; 15 mug, p less than .05; 20 mug, p less than .005), while DA had no effect. The dynamics of the LH response to NEwere similar at all dosage levels, and the increase caused by 20 mug NE was found to be essentially equal to that induced by a quick intravenous infusion of 1.25 ng LHRH. Arcuate nucleus multi-unit spike activity (MUSA) showed a clear response to intraventricular NE at a dosage capable of stimulating the release of LH. In every case, the initial effect was a decrease in spike activity. These results, considered in relation to previous findings, suggest that NE may be stimulatory to neurons secreting LH-releasing hormone (LHRH). The decrease in arcuate nucleus MUSA in response to NE implies that certain elements of this nucleus are inhibited during LH release, perhaps the dopaminergic tuberoinfundibular neurons.

Growth hormone (GH) secretion in the pituitary-grafted male rat: in vivo effects of GH-releasing hormone and isoproterenol and in vitro release by individual somatotropes.

Although the pituitary-grafted rat is a classic model of chronic PRL excess, the presence of somatotropes in grafted pituitary tissue indicates a potential for GH secretion. The current study was designed to investigate GH-releasing hormone (GRH)-induced GH secretion and beta-adrenergic inhibition of GH release in animals bearing ectopic pituitary tissue free from hypothalamic control. Positive findings with regard to these in vivo experiments led us to an initial determination of GH secretion by individual somatotropes from transplanted pituitary tissue. In litters of 10 30-day-old Fisher rats, 2 male animals received subcapsular renal grafts of 3 littermate pituitary glands each. Thirty-five days after grafting, 1 group received saline (SAL) followed by GRH, and the other received the beta-adrenergic agonist isoproterenol (ISO) followed by GRH. Blood samples were taken before and after SAL or ISO treatment, GRH was then infused, and sampling was continued. Plasma was assayed for GH and PRL, and the reverse hemolytic plaque assay was used to determine GH release by individual somatotropes from transplanted pituitary tissue. Plasma PRL was clearly elevated in pituitary-grafted compared to muscle-grafted animals, but there was no difference in either body weight gain or basal GH levels between the groups. As shown previously, ISO itself induced a brief release of GH due to its direct effect on the pituitary gland. The GH response to GRH was greater in pituitary-grafted animals than in muscle-grafted controls after both SAL and ISO. GRH-induced GH release was suppressed by ISO pretreatment in muscle-grafted animals, but not in pituitary-grafted animals. The reverse hemolytic plaque assay unequivocally showed that transplanted pituitary tissue was capable of tonic as well as GRH-stimulated GH release. These results demonstrate that despite similar basal GH levels, animals bearing pituitary grafts release significantly greater amounts of GH in response to GRH. The evidence for GH secretion by individual somatotropes from transplanted pituitary tissue directly shows the grafted tissue to be a source of GRH-stimulated GH. The lack of beta-adrenergic inhibition of GRH-induced GH release in pituitary-grafted animals is consistent with the hypothesis that beta-adrenergic inhibition of GRH-induced GH secretion is mediated by an effect on the hypothalamus.

Sex differences in beta-adrenergic stimulation of growth hormone secretion in vitro.

In previous in vitro studies we have shown that the amounts of GH released by pituitary cells in response to human GH-releasing factor -40 (hGRF-40) are significantly related to the sex and gonadal hormone environment of the donor animals. The present studies were designed to determine whether the beta-adrenergic stimulation of GH release is sex related and to compare the response to that observed after hGRF-40. Dispersed pituitary cells from male or female rats were exposed to sequential pulses of isoproterenol (ISO) and epinephrine (EPI), followed by a single pulse of 10 nM hGRF-40. In a second series of experiments, the cells were exposed to sequential pulses of norepinephrine (NE), followed by a single pulse of 10 nM hGRF-40. ISO and EPI stimulated GH secretion at concentrations as low as 10(-8) M, but NE required a concentration of 10(-6) M to cause significant GH release. GH release after ISO, EPI, and NE was concentration dependent, and the order of potency was ISO greater than EPI greater than NE. The amounts of GH secreted by pituitary cells from male rats were significantly greater than those from female rats, and the magnitude of the difference was directly comparable to that observed in response to hGRF-40. These results confirm the beta-adrenergic stimulation of GH release, and the order of potency is consistent with mediation by a beta 2-adrenergic receptor. The significantly greater capacity for pituitary cells from male rats to secrete GH supports the possibility that individual somatotropes in the pituitaries of male rats might have a greater responsiveness and/or sensitivity to beta-adrenergic and hGRF-40 stimulation.

Limbic system involvement in the increase in plasma prolactin following cortical spreading depression in gonadotropin-treated female rats.

Cortical spreading depression (SD) was induced by applying 25% KCl to the frontal cerebral cortex in female rats under continuous ether anesthesia. Three weeks previously the animals had been subjected to sham operation, bilateral surgical "deefferentation" of the amygdala or transection of the dorsal columns of the fornix. During the week prior to experiment the rats were made "pseudopregnant" by treatment with PMS and hCG. Plasma prolactin was measured by radioimmunoassay in blood samples obtained from the peripheral circulation at 20 min intervals. After two control samples had been taken, KCl was applied to the cortex and sampling continued for another 100 min; In the sham-operated group prolactin levels increased with time following the application of KCl. Fornix-cut animals showed a similar, although briefer, increase with values significantly lower than those found in sham-operated animals at 80 min. The increase in plasma prolactin observed in sham-operated and fornix groups was completely abolished in amygdala-cut animals. These results indicate that limbic structures play a significant role in the mechanisms by which cortical SD elevates plasma prolactin levels under the present experimental conditions.

Growth hormone (GH) response to GH-releasing hormone by perifused pituitary cells from male, female, and testicular feminized rats.

To determine whether a normal complement of androgen receptors is required to permit full expression of sex-related differences in pituitary GH secretion, we compared the GHRH-stimulated GH secretory responses of continuously perifused anterior pituitary cells from normal male, normal female, and androgen-resistant testicular feminized (Tfm) rats. In each experimental replicate, acutely dispersed pituitary cells were exposed to GHRH (0.03-100 nM) administered as 2.5-min pulses in random order at 30-min intervals. The eluate was collected in 5-min fractions for GH determination by RIA. Basal unstimulated secretion of GH by cells from male rats was greater than that by cells from female (P = 0.007) and Tfm (P = 0.03) rats; basal secretion by the other two groups was similar (P = 0.55). Linear concentration-response relationships between GHRH and GH release were defined for cells from male (P = 0.0002), female (P = 0.0001), and Tfm (P = 0.0002) rats. Overall GHRH-stimulated GH secretion by cells from male rats was greater (P less than 0.0001) than that by cells from female rats. Overall secretion by cells from Tfm rats was less (P less than 0.001) than that by cells from male rats but greater (P less than 0.001) than that by cells from female rats. For all experimental groups, body weight was strongly correlated with both basal (r2 = 0.42; P = 0.001) and GHRH-stimulated (r2 = 0.53; P = 0.0001) GH secretion by the dispersed pituitary cells. These data suggest that a deficiency of androgen receptors results in a diminution of the in vitro GH secretory capability of anterior pituitary cells to a level below that by cells from normal males, but not to the level in normal females. The intermediate position of cells from the Tfm rat may represent a partial masculinization or defeminization within this generally female phenotype.

Somatostatin inhibition of growth hormone secretion by somatotropes from male, female, and androgen receptor-deficient rats: evidence for differing sensitivities.

To investigate the role of somatostatin (SRIF) in regulating sexually dimorphic GH secretion, we used a reverse hemolytic plaque assay and acutely dispersed somatotropes from age-matched normal male, normal female, and androgen receptor-deficient, testicular feminized (Tfm) rats. Hemolytic plaques were developed after a 90-min incubation in the presence of GH antiserum, 10 nM GH-releasing hormone (GHRH), and the following concentrations of SRIF: 0, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 nM. Additional studies were performed with 0 or 100 nM SRIF in the absence of GHRH. The absolute number of somatotropes (x10(6); mean +/- SEM) recovered from the pituitaries of Tfm rats (1.73 +/- 0.18) was significantly greater than that from the males (1.11 +/- 0.13; P = 0.01); the number from female rats (1.30 +/- 0.15) was not different from that of either male or Tfm animals. GHRH-stimulated GH secretion, as estimated by the mean GH plaque area (micron2 x 10(4); mean +/- SEM) in the absence of SRIF, was greater for somatotropes from male rats (3.36 +/- 0.41) than that for either Tfm (2.27 +/- 0.32; P = 0.02) or female (1.78 +/- 0.24; P = 0.001) rats; values for the latter two groups did not differ. However, mean GH plaque areas for each group during maximal SRIF inhibition in either the presence or absence of GHRH were indistinguishable from each other and from mean plaque areas obtained under basal conditions. As demonstrated by a lesser EC50 value (0.04 +/- 0.02 nM; mean +/- SEM), somatotropes from female rats were more sensitive to the inhibitory effect of SRIF than were those from either male (EC50 = 1.82 +/- 0.45; P = 0.0001) or Tfm (EC50 = 0.74 +/- 0.22, P = 0.0001) rats; values for the latter two groups were indistinguishable. These observed differences suggest that gender and/or the gonadal hormone environment may be important determinants of the inhibitory effects of SRIF on GH secretion by the somatotrope. While these gender-associated differences may represent effects of the gonadal hormones directly on the somatotrope, they could reflect modulation of the secretion of hypothalamic SRIF and/or GHRH by the prevailing gonadal hormone environment. Such gender-related differences may contribute to the overall sex-dependent patterns of GH secretion in the intact animal.

Ultradian endocrine rhythms are altered by a circadian mutation in the Syrian hamster.

A single gene defect of the circadian clock (tau mutation) has recently been described that results in a shortening of the circadian activity cycle of the Syrian hamster. In the homozygous animal, free running activity is shortened by 4 h, resulting in a circadian period of approximately 20 h. Here, we examine the effect of the tau mutation on noncircadian oscillators by comparing the frequency of episodic secretion of LH and cortisol in normal period wild-type (approximately 24-h circadian rhythm) and tau mutant (approximately 20-h circadian rhythm) castrate females. Animals were ovariectomized at 14 weeks of age and maintained thereafter under conditions of constant illumination. Wheel-running records were obtained, and only those animals exhibiting clear single bouts of circadian activity were used in the experiment. Two days after intraatrial cannulation, blood samples were collected for a 5-h period every 5 min during the subjective day at the same relative phase of the circadian cycle. Deconvolution analysis revealed that LH pulse frequency was significantly reduced in the tau mutant females (33.3 +/- 2.25- and 28.7 +/- 2.0-min interpulse intervals for tau and normal period females, respectively). Cortisol pulse frequency also exhibited significant differences, with a reduced pulse frequency (32.8 +/- 3.6- and 27.8 +/- 1.4-min interpulse intervals for tau and wild-type females, respectively). There were no significant differences with respect to secretory pulse amplitude, hormone half-life or estimated burst amplitude, or mass of hormone secreted per burst for either hormone. We conclude that a genetic defect that affects the circadian clock located in the suprachiasmatic nucleus may have a more general effect on neural oscillators, including those controlling episodic hormone secretion.

Growth hormone secretion by individual somatotropes of the testicular feminized rat.

To investigate the cellular mechanisms underlying the unique GH secretory apparatus of the androgen-resistant testicular feminized (Tfm) rat we employed a reverse hemolytic plaque assay to assess GH secretion by individual cells from normal male, normal female, and Tfm rats. Acutely dispersed pituitary cells were incubated for 90 min with GH anti-serum in the presence of medium alone, 0.01, 0.1, 1, 10, or 100 nM GHRH, or 3 microM forskolin after which hemolytic plaques were developed over an additional 30 min. Body weights of the Tfm rats [318 +/- 7 g (mean +/- SEM)] were intermediate between intact males (372 +/- 18 g) and females (218 +/- 7 g). The total number of cells recovered from dispersion of Tfm rat pituitaries [3.20 +/- 0.42 X 10(6) (mean +/- SEM)] was greater than that from males (1.43 +/- 0.12 X 10(6); P = 0.001), but not distinguishable from that from females (2.31 +/- 0.30 X 10(6); P = 0.06). However, the absolute population of recovered somatotropes from the Tfm animals (1.24 +/- 0.22 X 10(6) exceeded both male (0.56 +/- 0.10 X 10(6); P = 0.002) and female (0.80 +/- 0.14 X 10(6); P = 0.046) values. Mean basal and maximal GH plaque areas were greater for cells from male rats than for those from either female or Tfm rats (P less than 0.05) regardless of whether GHRH or forskolin was used as the secretagogue. Plaque areas from female and Tfm cells were indistinguishable under all study conditions. These data suggest that a deficiency of androgen receptors prevents establishment of the greater GH secretory capacity of individual somatotropes characteristic of the adult male rat. This androgen receptor-dependent modulation of GH secretory capacity appears to occur at a step distal to the GHRH receptor. The data also suggest that an increase in the absolute population of somatotropes is an additional consequence of androgen receptor deficiency. This combination of individual somatotropes, each possessing a GH secretory capacity similar to that of cells from normal females, but present in greater absolute numbers, may explain the intermediate values found during previous studies of the Tfm rat GH axis which were based on assessment of large mixed populations of pituitary cells.

Short-term adult exposure to estradiol feminizes the male pattern of spontaneous and growth hormone-releasing factor-stimulated growth hormone secretion in the rat.

Experimental evidence indicates that the neonatal gonadal steroid environment is an important determinant of the sexual dimorphism of GH secretion and body growth. However, the influence of the sex steroids in GH control during adult life and their mechanism/site of action are largely unknown. In the present study we examined the effects of adult gonadectomy (GNX) and short term adult exposure to 17 beta-estradiol (E2) on both spontaneous and GRF-stimulated GH release in free-moving adult male rats. The rate of body weight gain was also monitored. GNX (3 weeks postoperatively) resulted in a 2-fold reduction in GH pulse amplitude compared to that in sham-operated control rats, but did not significantly alter the GH nadir or the interpeak interval. Exposure to E2 (sc implants) for 4 days markedly disrupted the spontaneous GH secretory profile of both sham-operated and GNX rats; E2-treated animals exhibited a striking elevation (4- to 20-fold) of GH trough levels and a significant decrease in GH interpeak interval, remarkably similar to the typical female rat GH secretory profile. The augmentation in both GH nadir and GH pulse frequency was evident as early as 12 h after a single sc injection of E2 valerate. In sham and GNX rats bearing control implants, the GH response to 1 micrograms rat GRF-(1-29)NH2, iv, was significantly greater when GRF was administered at peak (1100 h) than at trough (1300 h) times of GH secretion; the latter is known to be due to antagonism by the cyclical increased release of endogenous somatostatin (SRIF) in the male rat. Treatment with E2 abolished this time-dependent difference in both groups and produced a regular pattern of GH responsiveness to GRF similar to that typically observed in the female rat, thus suggesting that E2 has altered the pattern of hypothalamic SRIF secretion from a cyclic to a more continuous mode of release. Chronic exposure to E2 for 2 weeks resulted in an almost 6-fold inhibition of the rate of body weight gain in sham-operated male rats to levels comparable to those in normal adult female rats. Taken together, these results demonstrate that short term exposure to E2 during adult life can profoundly feminize the male pattern of spontaneous and GRF-stimulated GH secretion, as well as rate of somatic growth.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of gonadal steroids on somatotroph function in the rat: analysis by the reverse hemolytic plaque assay.

The mechanism by which gonadal steroids modulate GH secretion is not known. We have used the reverse hemolytic plaque assay to examine whether gonadal steroid-induced modulation of GH secretion is effected by changes in the population of somatotrophs and/or alterations in their secretory properties. Two groups of Sprague-Dawley rats were studied: group 1 (n = 6) comprised male (M), castrate (Cx), and testosterone-replaced castrate male (Cx + T) rats and group 2 (n = 5) consisted of male (M), female (F), and 17 beta-estradiol-replaced castrate male (Cx + E) rats. The number of plaque-forming cells (expressed as both absolute number and a percentage of all cells) was determined, and secretory status was assessed by measuring plaque areas in response to 0, 0.01, 0.1, 1, 10, and 100 nM GHRH. While mean basal GH plaque areas were similar among the treatment groups of group 1, the maximal GH plaque area was significantly decreased in Cx [16.8 +/- 2.4 vs. 26.4 +/- 3.9 X 10(6) microns2 (mean +/- SEM); P less than 0.05], but not in Cx + T (27.5 +/- 4.1 microns2) rats. The GHRH EC50 was unaffected by castration or T replacement. The percentage and absolute population of somatotrophs were reduced in Cx, but not in Cx + T, rats, while the numbers of lactotrophs remained unchanged in these treatment groups. For group 2, the mean peak GH plaque area was reduced in Cx + E (16.5 +/- 2.9 microns2; P less than 0.001) compared to that in M rats (36.2 +/- 2.3 microns2), but was not significantly different from that in F (13.0 +/- 1.5 microns2) rats. The EC50 was significantly (P less than 0.025) greater in Cx + E (10.9 +/- 2.3 nM) and F (7.9 +/- 1.6 nM) compared to M rats (2.8 +/- 0.7 nM). The absolute somatotroph and lactotroph populations were increased in Cx + E compared to M and F rats, as were the populations of other pituitary cell types. Testosterone enhances GH secretion by increasing the secretory capacity, but not the sensitivity, of somatotrophs to GHRH and by recruiting the function of a subpopulation of somatotrophs. Estradiol reduces the secretory capacity and sensitivity of somatotrophs to GHRH, but increases the population of somatotrophs, lactotrophs, and non-GH- and non-PRL-secreting cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Gonadotropin-releasing hormone-stimulated luteinizing hormone secretion by perifused pituitary cells from normal, gonadectomized, and testicular feminized rats.

To elucidate further the manner in which gonadal steroids influence the secretion of LH, we examined the effects of gonadectomy and the absence of functional androgen receptors on GnRH-induced LH release from dispersed rat anterior pituitary cells. Intact and gonadectomized (GNX) normal rats and androgen-resistant, testicular feminized (Tfm) animals from the King x Holtzman strain (a mutant strain that possesses defective androgen receptors) were used. Dispersed pituitary cells were perifused with Medium 199 during a 4-h equilibration period and then subjected to eight 2.5-min pulses of GnRH introduced at 30-min intervals at concentrations ranging from 0.03-100 nM. Basal LH secretion by cells from intact male and female rats was indistinguishable (P = 0.79) and was substantially lower (P less than 0.0001) than that by cells from GNX male and female animals. Basal LH secretion by cells from Tfm rats was significantly higher (P less than 0.01) than that by cells from intact animals, but lower (P less than 0.005) than that by cells from GNX animals. In response to GnRH, perifused pituitary cells from animals representing all experimental groups demonstrated concentration-dependent LH release. Pituitary cells from intact female rats showed an overall greater (P less than 0.05) response to GnRH than cells from intact male rats. Pituitary cells from Tfm rats demonstrated a greater GnRH-stimulated LH mean response than cells from intact male (P less than 0.0001) or intact female (P less than 0.0001) rats. Gonadectomy of male rats resulted in an overall GnRH-stimulated LH release similar to that exhibited by cells from gonadectomized female rats (P = 0.61). Cells from Tfm animals released more LH in response to GnRH than those from gonadectomized male and female rats (P less than 0.001). These data demonstrate that the release of LH in response to GnRH by pituitary cells from intact male rats (i.e. in the presence of androgen and functional androgen receptors) is less than that seen by cells from intact females rats. Since circulating levels of testosterone and estradiol are known to be elevated in the testicular feminized rat, the heightened GnRH-stimulated LH release by cells from such animals may reflect either the long term lack of androgenic influence and/or the combined effects of androgen resistance and elevated levels of circulating estrogens.

Beta-adrenergic stimulation of growth hormone (GH) release in vivo, and subsequent inhibition of GH-releasing factor-induced GH secretion.

In vivo and in vitro studies of beta-adrenergic influences on GH secretion have produced apparently conflicting data in which the in vivo effect seems to be inhibitory and the in vitro effect to be stimulatory. The present studies were designed to observe the in vivo effect of isoproterenol (ISO), a beta-adrenergic agonist, on 1) GH release during a brief interval after intraatrial infusion, and 2) GH release in response to GRF infused 10 min after ISO. ISO was found to stimulate GH release in both intact and hypothalamus-lesioned animals within 2 min after infusion, but GH returned to control levels within 10 min. ISO also profoundly inhibited the release of GH in response to GRF. Pretreatment of animals with somatostatin (SRIF) antiserum prevented the inhibitory action of ISO on GRF-induced GH release. No change in peripheral levels of SRIF was detected. Also, there was no suppression of GRF-induced GH release by ISO when the treatments were applied in vitro to dispersed perifused pituitary cells. These data show that beta-adrenergic systems can stimulate a rapid but brief release of GH in vivo, and that the subsequent inhibitory action on GRF-induced GH release might be by means of SRIF release.

Augmented hypothalamic proopiomelanocortin gene expression with pubertal development in the male rat: evidence for an androgen receptor-independent action.

To investigate the mechanism(s) during pubertal development by which androgens alter hypothalamic proopiomelanocortin (POMC) gene expression and beta-endorphin content, we used the technique of in situ hybridization histochemistry and the androgen-insensitive testicular feminized (Tfm) rat. We evaluated POMC mRNA levels in the arcuate nuclei and periarcuate regions of 12 coronal brain slices from prepubertal (age, 30 days) and adult (age, 60 days) normal male and Tfm rats (n = 4 for each group). Hybridizations were performed using an 35S-radiolabeled oligonucleotide probe complementary to a 30-base sequence within POMC mRNA. The tissue sections were sequentially exposed to x-ray film and photographic emulsion with subsequent analysis by both densitometry and computer-assisted grain counting. beta-Endorphin was measured in hypothalamic tissue blocks from similar animals in each of the four experimental groups. The results of densitometry and grain counting were consistent and revealed an increase in POMC mRNA with pubertal development in both the male and Tfm animals. The concentration of hypothalamic beta-endorphin was greater for the adult Tfm animals than for all other groups, which did not differ from each other. These results suggest that androgens may stimulate POMC gene transcription by their action through estrogen receptors after conversion by aromatase. Alternatively, additional pubertal factors may be responsible for act directly through their respective receptors to alter translation, posttranslational processing, or secretion of beta-endorphin, resulting in diminished intracellular hypothalamic peptide concentration.