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To elucidate the role of growth hormone (GH)-releasing hormone (GRH) and somatostatin (SRIH) in the regulation of the growth hormone (GH) secretory pattern, we collected portal blood from five unanesthetized ovariectomized ewes for repeated measurements of GRH and SRIH simultaneous with those of peripheral GH. Hormones were measured at 10-min intervals for 5.5 h and their interrelationships analyzed. Mean portal GRH was 20.4±6.7 (SD) pg/ml and the estimated overall secretion rate was 13 pg/min. GRH secretion was pulsatile with peaks of 25-40 pg/ml and a mean pulse interval of 71 min. Mean portal SRIH was 72±33 pg/ml and the estimated overall secretion rate was 32 pg/min. SRIH secretion was also pulsatile with peaks of 65-160 pg/ml and a mean pulse interval of 54 min. The GH pulse interval was 62 min. A significant association was present between GRH and GH secretory peaks though not between GRH and SRIH or SRIH and GH. Insulin hypoglycemia resulted in a rapid and brief stimulation of SRIH secretion followed by a decline in GH levels. No effect was observed on GRH secretion until 90 min, when a slight increase occurred. The results suggest (a) the presence of an independent neural rhythmicity of GRH and SRIH secretion with a primary role of GRH in determining pulsatile GRH secretion, and (b) that the inhibitory effects of insulin hypoglycemia on GH in this species are attributable to a combination of enhanced SRIH secretion and possibly other factors, though without significant inhibition of GRH. (J. Clin. Invest. 1990. 86:17-24 approach the sensitivity of detection by current techniques (1). The neuroendocrine mechanism that regulates GH secretion is mediated by two hypothalamic hormones, one of which is stimulatory (GH-releasing hormone [GRH]) and the other, inhibitory (somatostatin [SRIH]). Although their effects on GH secretion have been studied extensively, there have been formidable technical difficulties in obtaining simultaneous measurements of the two neurohormones in hypothalamichypophysial portal blood and comparing their secretory patterns with those of GH in peripheral blood under physiologic conditions. Thus, the pattern of their release from the median eminence into the hypothalamic-hypophysial portal system and current knowledge of their precise interaction in regulating pulsatile GH secretion is based almost entirely on indirect evidence. Initial results of passive immunization with anti-GRH serum (2) and anti-SRIH serum (3) suggested that pulsatile GH secretion was dependent on pulses of GRH and that the role of SRIH was primarily to set the level of the GH responses to GRH. However, subsequent studies with infusions of anti-SRIH serum (4), comparison of GH responses to GRH during trough and pulse periods ofGH secretion (5), and interruption of intrahypothalamic somatostatinergic neuronal circuitry (6) suggested the presence of intermittent and possibly pulsatile secretion of SRIH as well as GRH.Attempts to measure GRH and SRIH in peripheral circulation have not clarified their hypophys...

The role of the pituitary, and in particular, of GH in GH-releasing hormone (GRH) gene expression was studied in hypophysectomized rats with and without GH treatment. Hypothalamic GRH mRNA was 6-fold greater in hypophysectomized than in control rats. Increased levels of GRH mRNA were observed at 3 days and the maximal increase was noted at 7 days, postoperatively. Administration of GH to hypox rats partially reversed the increase in GRH mRNA, suggesting a negative feedback regulation by GH of GRH gene activity at the transcript accumulation level. The overall regulation of GRH gene expression, however, appears more complex since GRH mRNA levels and GRH content exhibited discordant changes after both hypophysectomy and GH treatment, suggesting that factors other than GH are required for efficient translation of GRH mRNA.

The plasma enzyme responsible for primary proteolytic cleavage of growth hormone-releasing hormone (GRH) at the 2-3 amino acid bond was characterized. Native GRHIGRH(144)-NH2 and GRH(140)-OHI, and COOH-terminally shortened fragments [GRH(1-32)-NH2 and GRH(1-29)-NH21 were rapidly cleaved, while GRH(2-32)-NH2 was not degraded at this site. Moreover, degradation to GRH(344)-NH2 was unaffected by an aminopeptidase inhibitor, indicating that this metabolite was generated from a single step cleavage by a dipeptidylpeptidase (DPP) rather than sequential aminopeptidase cleavages. Conversion to GRH(344)-NH2 was blocked by diprotin A, a DPP type IV (DPP IV) competitive inhibitor. DAmino acid substitution at either position I or 2 also prevented hydrolysis, characteristic of DPP IV. Analysis of endogenous plasma GRH immunoreactivity from a human GRH transgenic pig revealed that the major peak coeluted with GRH(344)-NH2. Native GRH exhibited trypsin-like degradation at the 11-12 position but cleavage at the 12-13 site occurred only with GRH(1-32)-NH2 and GRH(1-29)-NH2. Formation of these metabolites was independent of prior DPP IV hydrolysis but was greatly reduced by trypsin inhibitors. Evaluation of plasma stability of potential GRH super analogues, designed to resist degradation by these enzymes, confirmed that GRH degradation in plasma occurs primarily by DPP IV, and to a lesser extent by trypsin-like enzyme(s).

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Voiding dysfunctions, including increased voiding frequency, urgency, or incontinence, are prevalent in the postmenopausal population. ␤ 3 -Adrenergic receptor (␤ 3 AR) agonists, which relax bladder smooth muscle, are being developed to treat these conditions. We utilized the rat ovariectomy (OVX) model to investigate the effect of ovarian hormone depletion on bladder function and the potential for ␤ 3 AR agonists to treat bladder hyperactivity in this setting. OVX increased voiding frequency and decreased bladder capacity by ϳ25% in awake rats and induced irregular cystometrograms in urethane-anesthetized rats. Reverse transcription-polymerase chain reaction revealed three ␤ARs subtypes (␤ 1,2,3 ) in bladder tissue, and immunostaining indicated ␤ 3 AR localization in urothelium and detrusor. Receptor expression was not different in OVX and SHAM rats. ]amino]-6,7,8,9-tetrahydro-5H-benzocyclohepten-2-yl]oxy], ethyl ester, hydrochloride] was confirmed by examining the relative potency for elevation of cAMP in CHOK1 cells overexpressing the various rat ␤ARs. Intravenous injection of each of the ␤ 3 AR agonists (0.1-500 g/kg) in anesthetized rats decreased voiding frequency, bladder pressure, and amplitude of bladder contractions. In bladder strips, ␤ 3 AR agonists (10 Ϫ12-10 Ϫ4 M) decreased baseline tone and reduced spontaneous contractions. BRL37344 (5 mg/kg) and TAK-677 (5 mg/kg) injected intraperitoneally in awake rats decreased voiding frequency by 40 to 70%. These effects were not altered by OVX. The results indicate that OVX-induced bladder dysfunction, including decreased bladder capacity and increased voiding frequency, is not associated with changes in ␤ 3 AR expression or the bladder inhibitory effects of ␤ 3 AR agonists. This suggests that ␤ 3 AR agonists should prove effective for the treatment of overactive bladder symptoms in the postmenopausal population.Lower urinary tract (LUT) dysfunctions, including increased voiding frequency, urgency, incontinence and nocturia, increase in the elderly population and following menopause (Stewart et al., 2003). These dysfunctions could result from hormonally induced changes in bladder contractile and/or relaxing mechanisms. Bladder contractions are triggered by parasympathetic nerves, which release ACh that in turn activates postjunctional muscarinic receptors (mAChRs) in the detrusor. Bladder relaxation is induced by release of norepinephrine from sympathetic nerves, which activates ␤-adrenergic receptors (␤AR) (Fowler et al., 2008

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The effects of thyroidectomy (Tx) and thyroxine replacement (T4Rx) on pituitary growth hormone (GH) secretion and hypothalamic GH-releasing hormone (GRH) concentration were compared to define the mechanism of hypothyroid-associated GH deficiency.Thyroidectomized rats exhibited a complete loss of pulsatile GH secretion with extensive reduction in GRH responsiveness and pituitary GH content. Cultured pituitary cells from Tx rats exhibited reduced GRH sensitivity, maximal GH responsiveness, and intracellular cyclic AMP accumulation to GRH, while somatostatin (SRIF) suppressive effects on GH secretion were increased. Hypothalamic GRH content was also markedly reduced. T4Rx completely restored hypothalamic GRH content and spontaneous GH secretion despite only partial recovery of pituitary GH content, GRH and SRIF sensitivity, and intracellular cyclic AMP response to GRH.The results indicate multiple effects of hypothyroidism on GH secretion and suggest that a critical role of T4 in maintaining normal GH secretion, in addition to restoring GH synthesis, is related to its effect on hypothalamic GRH.