Joao L. C. Borges

A Model System for the Study of Luteinizing Hormone (LH) Secretion: Lack of Effect of Fetal Calf Serum on Gonadotropin-Releasing Hormone Stimulated LH Release

Abstract Continuous perifusion of dispersed anterior pituitary cells recently has been applied to the study of LH secretion. We undertook a series of experiments to determine whether or not fetal calf serum(FCS) is a necessary or beneficial addition to medium perifusing cells in culture up to 43.5 hr. Male Sprague-Dawley rats provided pituitaries for two perifusion systems (columns) set up in parallel. The cells in one column were perifused on both Day 1 (first 19.5 hr) and Day 2 (subsequent 24 hr) with Medium 199, while the cells in the other column were perifused with Medium 199 on Day 1 and with Medium 199 with 2% FCS for the initial 18 hr on Day 2. On Days 1 and 2 cells in both columns were challenged with five doses of GnRH (1-100 nM) given as 1.5-min pulses in a random order. After 4-hr of perifusion, basal release of LH decreased gradually with time in a manner that fit a least-squares derived second-order polynomial function. Mean basal LH release (ng/ml/107 cells ± SEM) on Days 1 (9822 ± 2450) and 2 (7710 ± 2100) was no different (P = 0.1). Mean GnRH-stimulated LH release on Day 1 (1063 ± 144.3) was higher than on Day 2 (746 ± 143.7) (P = 0.036). GnRH-stimulated LH release on Day 2 was the same for the columns with (805.9 ± 41.9) and without (653.2 ± 190.8) FCS (P = 0.9); furthermore, the slopes of the dose-response curves were identical (P = 0.9). Thus, our results showed that, using continuously perifused dispersed rat anterior pituitary cells for up to 43.5 hr: (1) basal LH release does not change significantly; (2) GnRH-stimulated LH release becomes less with time but dose-response characteristics remain constant; and (3) preincubation with FCS appears unnecessary to maintain responsivity of cells. Furthermore, we propose that the quantitative methods used to analyze our data may prove useful to other investigators employing similar perifusion techniques, thus allowing comparison of results from various laboratories.

Mesulergine, a new dopamine agonist: Effects on anterior pituitary function and kinetics

We investigated the effects of single doses of mesulergine on basal and thyrotropin‐releasing hormone (TRH)‐stimulated serum levels of several anterior pituitary hormones in healthy men and defined its kinetics. We also compared the effects on serum prolactin (PRL) levels of three doses (0.1, 0.35, and 0.5 mg) of mesulergine to those in response to 2.5 mg bromocriptine. Secretory rates of PRL before the first dose of TRH were not affected by any dose of mesulergine or bromocriptine. TRH‐stimulated PRL secretion was not altered by 0.1 mg mesulergine but was blunted by both the 0.35‐ and 0.5‐mg doses at 10 A.M. and 1 P.M. Bromocriptine inhibited TRH‐stimulated PRL secretion at 10 A.M. and 8 P.M. When analyzed as the 8 A.M. to 8 P.M. and the 8 P.M. to 9 A.M. (day 2) intervals, PRL secretion was not changed by 0.1 or 0.35 mg mesulergine but was suppressed during both periods by the 0.5‐mg dose. A dose‐response relationship was evident, however, between mesulergine and PRL secretion during both the 8 A.M. to 8 P.M. (R2 = 0.27) and the 8 P.M. to 9 A.M (day 2; R2 = 0.18) intervals. Bromocriptine lowered PRL secretion during both intervals. Secretory rates of growth hormone during these intervals were not affected by 0.1 mg or 0.35 mg mesulergine but were increased during both intervals by the 0.5‐mg dose. Neither the secretory rates of thyrotropin in response to TRH nor those of cortisol, luteinizing hormone, or follicle‐stimulating hormone were changed by 0.1 or 0.35 mg mesulergine. Analysis of mesulergine plasma levels revealed that both AUC and peak concentrations increased in a linear fashion with increasing doses of mesulergine. Although adverse effects after both mesulergine and bromocriptine included orthostasis, nausea, nasal congestion, and headaches, frequency of orthostatic symptoms and nausea was higher after bromocriptine than after mesulergine. Mesulergine appears to inhibit PRL secretion at doses lower than those of bromocriptine and may be associated with less frequent side effects.

Human pancreatic tumor GH-releasing factor

SummaryWithin the past year, three similar peptides with specific growth hormone (GH) releasing effects have been extracted from human tissue, identified, and synthesized. Human pancreatic tumor GH releasing factor (1–40)-OH (hpGRF-40) was the sole hpGRF isolated from the pancreatic tumor of a patient in Charlottesville and was the predominant peptide isolated from the pancreatic tumor of a patient in Lyon. The Lyon tumor also contained hpGRF(1–37)-OH and hpGRF(1–44)-NH2. Both immunological and biochemical data suggest that hpGRF-40 and hpGRF-44 are present in the human hypothalamus and may be the human GH releasing hormone(s) (GHRH).In cultures of rat pituitary cells, hpGRF stimulates GH but affects neither basal and dopamine-inhibited prolactin release nor basal and gonadotropin releasing hormone (GnRH)-stimulated luteinizing hormone (LH) release. hpGRF stimulates cyclic AMP production within seconds, an effect which is blocked by somatostatin. In contrast, while hpGRF stimulates phosphatidylinositol turnover in the pituitary, the effect is not inhibited by somatostatin.In the human, hpGRF-40 (1 μg/kg) given intravenously (i.v.) stimulates GH release within 5 minutes. hpGRF-40 does not elevate serum prolactin levels, thyrotropin (TSH), LH, or corticotropin (measured indirectly through plasma cortisol), or blood glucose or plasma concentrations of insulin, glucagon, pancreatic polypeptide, cholecystokinin, gastrin, gastric inhibitory peptide, motilin, or somatostatin. When graded doses of hpGRF (0.1–10 μg/kg) are given i.v., no differences are noted in the maximal levels of serum GH achieved. Doses of 1, 3.3 and 10 μg/kg hpGRF-40 elicits a prolonged and biphasic pattern of GH release. Twenty-four hours after hpGRF-40 administration, serum somatomedin C is increased in 66% of subjects tested. Side effects including a feeling of warmth and facial flushing are observed in 66% (3.3 gmg/kg) and 100% (10 μg/kg) of men given hpGRF-40. hpGRF-40 (3.3 μg/kg, i.v.) selectively stimulates GH release and somatomedin C production in normal women, although no differences are found in GH responsivity during the menstrual cycle. hpGRF-40 given intranasally to normal men (30 μg/kg) stimulates GH release within 30 minutes. The calculated metabolic clearance rate for hpGRF-40 is 194±17.51/m2/d; the disappearance rate occurs as two phases: an initial equilibration phase (7.6±1.2 minutes) and a subsequent elimination phase (51.8±5.4 minutes). hpGRF-40 administered i.v. stimulates the release of GH in some adult patients with GH deficiency documented in childhood. Serum somatomedin C concentrations may increase in patients in whom hpGRF-40 fails to stimulate GH release. If patients with GH deficiency who do not respond to hpGRF-40 administration (10 μg/kg i. v.) are given the peptide (0.33 μg/kg i. v. every 3 hours) for five days, some will respond to a subsequent 10 μg/kg challenge. Of those who do respond initially, the response to the subsequent challenge may be greater. Serum somatomedin C increases significantly following the 5 days of intermittent administration of hpGRF-40.hpGRF-40 and/or hpGRF-44 may be the long sought GHRH. Clinical studies with hpGRF suggest that GH deficiency may often result from hypothalamic GHRH deficiency rather than pituitary disease. hpGRF and its analogues and antagonists may find therapeutic application in the treatment of GH deficiency and in other disorders in which an increase or decrease in the secretion of GH would be beneficial.

Actions of calcium ions and a calcium-influx blocker on basal and TRH- and GnRH-stimulated hormone release in patients with pituitary adenomas

We investigated the influence of calcium ions on the secretion of anterior pituitary hormones basally and in response to exogenous hypothalamic releasing factors in 6 men with pituitary tumors. To this end, concentrations of LH, FSH, TSH, growth hormone and prolactin were measured in blood collected at 10-min intervals basally and during a continuous infusion of combined TRH (2 μg/min) and GnRH (1 μg/min). Study sessions were randomized to iv saline, calcium, or diltiazem infusions or oral diltiazem administration. Our results indicate that in contrast to responses in normal men, iv calcium injections do not suppress circulating prolactin concentrations in patients with prolactin-secreting pituitary tumors. Moreover, neither oral diltiazem administration for one week nor acute iv diltiazem infusion suppressed the hyperprolactinemia of tumor patients. However, there were significant effects of drug and calcium treatments on serum concentrations of FSH, GH and testosterone, but not LH or TSH. Moreover, during GnRH-TRH stimulation, there were significant differences in LH, TSH, and testosterone responses in tumor patients compared to normal men. In summary, iv calcium infusion was associated with invariant basal release of anterior pituitary tumoral hormones in patients with pituitary adenomas. However, there were significant differences in the GnRH/TRH-stimulated release of certain anterior pituitary hormones in tumor patients compared to normal men in response to iv calcium and the calcium-channel antagonist, diltiazem.