Donald R. Martinson

Antisera to Vasoactive Intestinal Polypeptide Inhibit Basal Prolactin Release from Dispersed Anterior Pituitary Cells

Vasoactive intestinal polypeptide (VIP) has been identified in hypothalamic tissue, is secreted into hypophysial portal blood, and stimulates prolactin (PRL) release in vivo and in vitro. It has been proposed, therefore, that VIP is a physiologic PRL-releasing factor. In this study, we confirm that VIP stimulates PRL release from rat pituitary cells in vitro, and demonstrate that an anti-VIP antiserum blocks VIP-induced PRL secretion. Surprisingly, the anti-VIP antiserum inhibited basal PRL secretion from rat pituitary cells in 3 separate experiments. Data from these experiments were pooled, as the responses were similar, revealing basal PRL release of 10.7 +/- 1.3 ng rPRL/10(5) cells (X +/- SE), while anti-VIP antisera significantly inhibited release to 4.4 +/- 0.6 ng rPRL/10(5) cells (p less than 0.001). PRL release in incubates containing control non-immune sera did not differ from basal release, 8.1 ng rPRL/10(5) cells. A further control experiment was conducted wherein cells were incubated with an anti-ACTH antiserum, representing another hyperimmune serum, which had no effect on PRL secretion. These data suggest that VIP, in addition to its possible role as a hypothalamic-derived PRL-releasing factor, may play a role within the pituitary as a regulator of basal PRL secretion.

Running Elevates Plasma β-Endorphin Immunoreactivity and ACTH in Untrained Human Subjects

Abstract Twenty minutes of submaximal treadmill running was associated with an elevation in plasma levels of β-endorphin immunoreactivity (P < 0.02). This increase was greater in men (14.9 ± 3.4 fmole/ml) than women (2.6 ± 1.2 fmole/ml) (P < 0.05). Plasma levels of ACTH and growth hormone also increased after running. ACTH increased more in men (7.8 ± 1.1 fmole/ml) than in women (1.1 ± 0.44 fmole/ml) (P < 0.02). There was a similar growth hormone response in both sexes. No correlation can at this time be made with levels in the central nervous system. Changes in plasma levels of β-endorphin immunoreactivity may be responsible for some of the euphoria and analgesia anecdotally associated with running.

Hypothalamic prolactin: characterization by radioimmunoassay and bioassay and response to hypophysectomy and restraint stress.

Prompted by immunohistochemical reports of prolactin-like immunoreactivity in cell bodies within the rat hypothalamus, a study was undertaken to quantitate the immunologic and biologic activity of this material. Hypothalamic concentrations of prolactin-like immunoreactivity averaged 402 +/- 23 pg/mg of protein (n = 30). 97% recovery of rat prolactin standards added to homogenates of hypothalamus insured that neuronal tissue, as prepared for these studies, did not interfere with the radioimmunoassay of rat prolactin. Examination of the elution profile from Sephadex G-75 columns of the prolactin-like immunoreactivity in hypothalamic extracts showed that the majority of hypothalamic prolactin-like substance was of a larger molecular size than pituitary prolactin. While increasing amounts of brain extract progressively displaced more I125 prolactin from antibody-binding sites, the displacement curve produced by adding hypothalamic extract was not parallel to that produced by the addition of increasing amounts of anterior pituitary prolactin standards of rat origin. Hypothalamic extracts from hypophysectomized animals, analyzed for biologic activity in the Nb2 lymphoma cell assay, revealed prolactin-like bioactivity, but the bioactivity/immunoactivity (B/I) ratios for hypothalamic extracts were significantly lower than the B/I ratios for pituitary prolactin (0.71 +/- 0.04 for pituitary, vs. 0.19 +/- 0.06 in the hypothalamus; p less than 0.001). Hypophysectomy, which led to the expected fall in serum prolactin to undetectable levels, and restraint stress, which resulted in a statistically significant 4-fold rise in serum prolactin, caused no change in prolactin concentrations in the hypothalamus, indicating that brain prolactin-like substance is regulated independently of pituitary prolactin and circulating serum prolactin levels.

Extrahypothalamic brain prolactin: characterization and evidence for independence from pituitary prolactin

Prompted by reports of immunohistochemical localization of a prolactin-like immunoreactivity (PLI) within the rat brain, a study was undertaken to define the immunologic and biologic characteristics of this material in extrahypothalamic regions of the rat brain. Ninety-seven percent recovery of rat prolactin standard, added to homogenates of brain parts, insured that neuronal tissue did not interfere with the radioimmunoassay for rat prolactin. PLI was consistently found in the cerebellum, thalamus, brainstem (pons-medulla), hippocampus, cerebral cortex and caudate. Examination of the elution profile of each of the extrahypothalamic brain parts from Sephadex G-75 columns showed that, although a small amount of brain PLI elutes in the vicinity of the anterior pituitary prolactin marker, the bulk of brain-based PLI migrates with the void volume and as late eluting, low molecular weight material. While increasing amounts of brain extracts progressively displaced more 125I-prolactin from antibody binding, the displacement curve was not parallel to that produced by the addition of increasing amounts of anterior pituitary prolactin standards of rat origin. Extracts of various brain parts from hypophysectomized animals, analyzed for biologic activity in the Nb2 lymphoma cell assay, revealed prolactin-like bioactivity, but the bioactivity/immunoreactivity ratio for some of the brain parts was significantly lower than that for pituitary prolactin. Hypophysectomy, which led to the expected fall in serum prolactin to undetectable levels, and restraint stress, which resulted in a statistically significant 4-fold rise in serum prolactin, caused no change in prolactin concentrations in extrahypothalamic brain parts, indicating that brain PLI is regulated independently of pituitary prolactin and of circulating serum prolactin levels.

Effect of glucose on the growth hormone response to L-dopa in normal and diabetic subjects.

The effect of hyperglycemia on the growth hormone response to oral L-dopa (500 mg.) was assessed in eight normal and eight insulin-dependent diabetic subjects. A peak growth hormone response of 21.0 ±4.0 ng./ml. (mean / S.E.M.), significantly above baseline (p <0.01), was achieved in the normal group following oral L-dopa. Glucose concentrations did not change and were approximately 80 mg./100 ml. throughout. Administration of 100 gm. oral glucose with the L-dopa, or thirty minutes thereafter, totally suppressed the growth hormone response in all eight and six of the subjects, respectively. A peak growth hormone response of 20.0 /1.7 ng./ml. (mean / S.E.M.), significantly above baseline (p <0.001), was obtained in eight nonobese, insulin-dependent diabetics, in spite of prevailing hyperglycemia (mean plasma glucose 243-258 mg./100 ml.) throughout the test. Endogenous hyperglycemia was achieved in these patients by lessening the usual strict adherence to plasma glucose control for the purpose of the study. These results suggest an abnormality in the hypothalamus or pituitary of diabetic subjects allowing growth hormone responsiveness in spite of hyperglycemia.

DYNAMICS OF PROLACTIN SECRETION FROM DIETHYLSTILBESTROL-INDUCED RAT PROLACTINOMA TISSUE IN VITRO

Abstract Experiments were performed to determine whether PRL secretion in the rat diethylstilbestrol (DES)-induced prolactinoma model is affected by the addition of thyrotropin-releasing hormone (TRH) and/or immunoneutralization of intrapituitary vasoactive intestinal polypeptide (VIP) in vitro. Male Fischer 344 rats were implanted with either a 10 mg DES or placebo pellet 30 days prior to obtaining the anterior pituitary glands for perifusion. The anterior pituitaries were quartered and used in three different perifusion experiments. In Experiment I, placebo-treated tissue channels were perifused for 2 baseline hr followed consecutively by a 30-min exposure to 1:100 nonimmune rabbit serum (NRS), a 30-min wash, and a final 30-min exposure to 10-5 M TRH. Additional placebo channels were run as above except 1:100 VIP antiserum (AVIP) was substituted for NRS and AVIP was added to the TRH. In Experiment II, the same perifusion protocol was used as in Experiment I, except DES-Induced tumor tissue was used instead of placebo tissue. Results from Experiment I and II reveal that AVIP significantly decreased PRL secretory rate in both DES and placebo groups. In the tumor group, both TRH alone and in the presence of AVIP significantly increased the PRL secretory rate. In Experiment III DES-Induced tumor tissue channels were perifused with a similar protocol, except the concentrations of NRS and AVIP were increased to 1:10. Both NRS and AVIP significantly decreased PRL secretory rate; however, AVIP had a significantly greater effect than NRS. In this experiment, 1:10 AVIP overcame the stimulatory effect of TRH. In conclusion, AVIP decreases and TRH increases, even in the presence of AVIP, PRL release in DES-induced prolactinoma tissue in vitro. Increasing the AVIP concentration 10-fold diminished the PRL-releasing action of TRH in the tumor tissue. These data suggest that PRL secretion is not autonomous in these prolactinomas and can be affected by exogenous TRH and partial immunoneutralization of endogenous VIP.

Circulating corticotropin releasing factor-like activity in man.

Summary In order to investigate the possible existence of corticotropin releasing factor (CRF)-like activity in human plasma, methanol extracts of plasma were incubated with rat anterior pituitary tissue in vitro and ACTH release was determined. The extracts were reconstituted so that 1.0 ml was equivalent to 20 ml of original plasma, and the following doses in 5.0-ml incubation volumes elicited a stepwise increase in ACTH release: 100, 250, 500, and 750 μ1. The 250-, 500-, and 750-μl doses resulted in a linear log dose-response. Parallel experiments with 0.5, 1, 2, 5, and 10 rat hypothalamic equivalents in 5.0-ml incubation volumes resulted in a similar stepwise increase in ACTH release. Control experiments indicated a significantly greater ACTH release when pituitary tissue was incubated with methanol extracts of rat hy-pothalami in comparison to hypothalami added directly in vitro, validating the methanol extraction technique with a known source of CRF. Ultrafiltration experiments with the human plasma CRF-like activity suggest a molecular weight larger than 10,000 daltons.

Vasoactive Intestinal Polypeptide Antiserum Affects Rat Prolactin mRNA in 40-Day but Not 110-Day Diethylstilbestrol-Induced Prolactinoma Tissue

Experiments were designed to examine whether vasoactive intestinal polypeptide (VIP), a known stimulator of basal prolactin (PRL) secretion, regulates PRL gene expression in the rat diethylstilbestrol (DES)-induced prolactinoma model. The VIP-induced increase in PRL release could result from increased PRL synthesis and/or decreased PRL degradation. Male Fischer 344 rats were implanted with 10 mg DES pellets 40 or 110 days prior to obtaining the anterior pituitary glands for cell dispersal. Cells were incubated in 1:10 normal rabbit serum or VIP antiserum (AVIP). After incubation, cells were pelleted, washed, and pooled for total nucleic acid extraction. The rat PRL (rPRL) mRNA abundance was quantitated using a solution hybridization/ribonuclease protection assay. Supernatant was collected and analyzed for PRL content using radioimmunoassay. Results from this experiment reveal partial immunoneutralization of intrapituitary VIP significantly decreased PRL secretory rate by rapid reduction in rRPL mRNA in the 40-day tumors. However, in the 110-day tumors the rPRL mRNA steady-state levels were unchanged but the basal release of PRL continued to be decreased by AVIP. These results indicate VIP exerts its effects on PRL secretion through at least two mechanisms.