Effects of short-term cortisol infusion on growth hormone-releasing hormone stimulation of growth hormone release in sheep.

Excess production or long-term administration of glucocorticoids is detrimental to longitudinal growth in people and rats. A portion of this effect is attributed to cortisol inhibition of growth hormone (GH). Glucocorticoid effects are usually studied in subjects under long-term treatment with synthetic, more potent glucocorticoids, and, to the authors' knowledge, have not been examined in domestic animals. We sought to examine the effects of cortisol infusion on GH release in sheep. Cortisol infusion into castrated, male Suffolk sheep (1 to 1.5 years old) caused a significant (P < 0.0001) increase in cortisol concentration. Basal GH release was not affected over the 4-hour period of infusion. Growth hormone-releasing hormone administration stimulated GH release in both groups (P < 0.001); however, the control group had a greater response to growth hormone-releasing hormone than did the cortisol infused group (P < 0.0001). These results were duplicated in cultured sheep pituitary cells. Cortisol inhibition of GH release may be mediated via enhanced somatostatin release, owing to a direct inhibition of somatotrope function, or a combination of both mechanisms. Because of effects of stress and disease in increasing cortisol concentration, additional study of the mechanisms for cortisol inhibition of GH release in sheep needs to be performed.

Neurotransmitter receptor agonists regulate growth hormone gene expression in cultured ovine pituitary cells.

The regulation of growth hormone (GH) secretion and GH mRNA content by the dopaminergic agonist, bromocriptine (BRO); the beta-adrenergic agonist; isoproterenol (ISO); the alpha 1-adrenergic agonist, methoxamine (MET); the alpha 2-adrenergic agonist, clonidine (CLON); the serotonergic agonist, quipazine (QUIP); somatostatin (SS) and GH-releasing hormone (GHRH) were studied using cultured ovine anterior pituitary cells. Clonidine and BRO (10(-6) M) inhibited basal and GHRH (10(-10) M)-stimulated GH release. Bromocriptine enhanced GH mRNA content and potentiated the GHRH (10(-8) M)-stimulated content of GH mRNA, while CLON had no effect on GH mRNA. Quipazine had little effect on GH secretion and no effect on GH mRNA content. Methoxamine and ISO (10(-6) M) increased basal secretion of GH and both enhanced GHRH-stimulated GH secretion. Both MET and ISO increased GH mRNA content of cultured ovine pituitary cells. Somatostatin (10(-7) M) inhibited GHRH-stimulated GH secretion and GH mRNA accumulation. These results support the hypothesis that neurotransmitters may regulate or interact to further modulate pituitary hormone release. Moreover, the data indicate that neurotransmitters may not only regulate secretion but also regulate GH mRNA content and thus affect hormone synthesis.

Cloning two isoforms of rat cyclooxygenase: differential regulation of their expression.

Two isoforms of cyclooxygenase (COX) have been identified in eukaryotic cells: COX-1 encoded by a 2.8-kb mRNA, and a mitogen-inducible COX-2 encoded by a 4-kb mRNA. We have cloned the COX-1 and COX-2 cDNAs from the cDNA library constructed from lipopolysaccharide (LPS)-stimulated rat peritoneal macrophages. The deduced amino acid sequence showed that COX-1 contained 602 amino acids, whereas COX-2 contained 604 amino acids. There is 95% conservation of the nucleotide sequence in the open reading frame of COX-1 between the rat and the mouse, while the homology of the 3' untranslated region is 68% except for a 150 bp segment adjacent to the stop codon which is nonhomologous with the mouse. Transfection of both COX cDNAs into Cos-7 cells resulted in increased COX activity. In rat vascular smooth muscle cells, interleukin-1 beta selectively increased the expression of COX-2, but not that of COX-1, as assessed by enzyme activity, immunoprecipitation of COX proteins, and mRNA analysis. Only the brain among tissues tested exhibits basal expression of COX-2 as the major form of the enzyme. However, COX-2 mRNA was expressed in vivo in the lung and kidney, but not in the heart, after systemic administration of LPS, suggesting that COX-2 but not COX-1 plays a major role in producing COX-derived products of arachidonic acid during endotoxic shock. Thus, the two COX isoforms were differentially expressed, and COX-2 was selectively induced in response to inflammatory stimuli in rats.

Selective expression of mitogen-inducible cyclooxygenase in macrophages stimulated with lipopolysaccharide.

Two forms of cyclooxygenase are known to be present in eukaryotic organisms: a cyclooxygenase (COX-1) first purified from ram seminal vesicles encoded by a 2.8-kilobase mRNA, and a newly discovered mitogen-inducible cyclooxygenase (COX-2) encoded by a 4-kilobase mRNA. Expression of these two forms of the enzyme in rat alveolar macrophages stimulated with lipopolysaccharide was investigated by 1) determining the activity of newly synthesized enzyme after inactivating the endogenous enzyme with aspirin; 2) comparing levels of newly synthesized enzyme proteins in cells treated with or without lipopolysaccharide; and 3) assessing the expression of the mRNAs encoding COX-1 and COX-2. Levels of enzyme proteins were assessed by Western blot analysis and immunoprecipitation of 35S-labeled enzyme using two different antibodies, one specific for COX-2 and the other recognizing both forms of the enzyme but preferentially recognizing COX-1. We report here that the enhanced cyclooxygenase activity induced by the bacterial lipopolysaccharide in rat alveolar macrophages is caused by selective expression of the COX-2. Expression of COX-2 in macrophages stimulated by lipopolysaccharide was completely inhibited by dexamethasone, whereas COX-1 was unaffected. In resting unstimulated macrophages, only COX-1 but not COX-2 was detected. Levels of mRNA for the COX-2 in macrophages were increased, but those of the COX-1 were not affected by lipopolysaccharide as assessed by reverse transcription coupled with polymerase chain reaction. These results indicate that increased synthesis of prostaglandins and thromboxanes in lipopolysaccharide-stimulated macrophages results from selective expression of COX-2.

Modulation of growth hormone-releasing factor stimulated growth hormone secretion by plasma glucose and free fatty acid concentrations in sheep.

Effects of plasma glucose and free fatty acid (FFA) concentrations on bovine growth hormone-releasing factor (bGRF)-induced release of growth hormone (GH) were examined in ovariohysterectomized sheep. In experiment 1, the effects of an infusion of insulin (0.025 U/kg BW.h-1), glucose (40 mg/kg BW.h-1), insulin plus glucose or saline on the subsequent effects of bGRF on plasma GH concentrations were determined. Insulin-induced hypoglycemia inhibited GRF effects on plasma GH concentrations while glucose infusion enhanced bGRF actions. Infusing a higher glucose dose (120 mg/kg BW.h-1) had no effect on GRF actions. Subsequently, infusion of FFA (0.25 g/kg/.h-1), nicotinic acid (50 mg/kg BW) or saline for 1 h prior to bGRF injection demonstrated that FFA inhibited GRF actions but FFA depletion by nicotinic acid infusion had no effect on GRF actions. Nicotinic acid (40 mg/kg BW.h-1) infused for 2 h prior to bGRF injection significantly enhanced bGRF-stimulated GH secretion. Finally, to determine whether central nervous system glucopenia produced similar effects to insulin-induced hypoglycemia, 2-deoxyglucose (500 mg) was injected into the lateral ventricle followed in 1 by the i.v. injection of bGRF. The central glucopenia produced by 2-DG inhibited GRF-stimulated GH release. These data demonstrate that decreased peripheral or central nervous system glucose availability and exogenous administration of FFA antagonized GRF-induced release of GH. And, pharmacologic depletion of circulating FFA for at least 2 h facilitated GRF-induced release of GH.

In vitro release of PGF(2alpha) from the caprine uterus and the effect of ovarian steroids.

The purpose of the study was to determine the in vitro release of prostaglandin F(2)alpha (PGF(2alpha)) from the uterine tissues of ovariectomized goats after steroid treatment. Strips of endometrial tissues (separated by trypsin treatment) and intact uterine tissues (with endometrium and myometrium) were maintained in organ culture and exposed to estradiol-17beta(E) at 50 ng/ml and to progesterone (P) at 250 pg/ml alone or in combination. The endometrial tissues, in general, continued to release PGF(2alpha) without steroids treatment throughout the 96-h incubation period; their output was consistently higher (based on ng/gm of wet tissue) than the corresponding intact uterine tissues. However, exposure to E reversed this trend and produced a three-fold increase from the intact uterine strips at 24 h. A combined E-plus-P treatment blocked the stimulatory response, whereas P treatment alone evoked a delayed stimulatory response at 48 h. Endometrial tissue reaction was similar to that of P treatment, but it exhibited a more moderated response to E, which could not be blocked by a combined E-plus-P treatment. The results suggest that both endometrial and myometrial tissues release PGF(2alpha) and that this release is regulated by ovarian steroid hormones.