Lidia Kirsteins

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.

Autoregulation of Growth Hormone Secretion in Normal Subjects

Studies performed in animals have demonstrated fairly definitely that raised growth hormone levels exert a negative feedback or autoregulatory effect on further pituitary growth hormone release. Similar evidence was sought in normal human volunteers. Growth hormone levels were raised either by introducing a provocative stimulus, exercise or arginine, or by infusing human growth hormone at a constant rate. When circulating levels of growth hormone were raised by a measured exercise stimulus or by an arginine infusion, a second stimulus introduced in tandem failed to cause a further rise, and circulating growth hormone levels dropped to baseline. This was not due to pituitary growth hormone exhaustion, since combining the stimuli produced a far greater growth hormone release than either stimulus alone. Normal release in response to arginine or exercise could be demonstrated if a 30-min pause was allowed between tests. Similarly, when growth hormone levels were raised by injections or with a constant growth hormone infusion, no response to exercise or to arginine was seen. Thus, in man, as in animals, it appears that circulating levels of growth hormone do exert an autoregulatory influence on the secretory activity of either the hypothalamus or the pituitary. Abnormal variations in the threshold sensitivity to this feedback could account for certain disturbances of pituitary growth hormone release, in particular acromegaly.

The response of the hypothalamic-pituitary-gonadal axis to fasting is modulated by leptin.

Reproductive function is intimately related to caloric consumption. During fasting states, the hormones regulating reproduction, those of the hypothalamic‐pituitary–gonadal axis, in particular, are severely altered. With the exciting observations that the obese (ob) gene product, leptin, may also modulate neuroendocrine functions, we examined leptins ability to prevent the consequences of fasting on reproductive hormones. Two groups of male rats, aged 65 days old, were either fasted and saline‐injected or fasted and leptin‐treated for approximately three days. Another group was given free access to rat chow. Leptin was able to prevent the fasting‐induced fall of serum testosterone. Similar to testosterones dependence on leptin, leptin concentrations were somewhat dependent on testosterone. Castration accelerated the normal, age‐related increase in serum leptin. Leptin also prevented the fasting‐induced fall in luteinizing hormone (LH). The increase of β‐LH mRNA seen in the fasting state was prevented by leptin. There were no differences noted in luteinizing hormone releasing hormone (LHRH) mRNA among any of the groups. While neither fasting nor fasting plus leptin caused changes in serum prolactin, the increase in prolactin mRNA seen in fasted animals was prevented by leptin treatment. These data support the hypothesis that leptin plays a specific role in mediating the response of reproductive hormones to the nutritional status of the organism.

The effect of In Vitro Ethanol Exposure on Basal Growth Hormone Secretion

Suppressive effects of ethanol (ETOH) on in vivo serum growth hormone (GH) levels have been reported in both humans and animals. To determine whether this effect could be mediated directly at the pituitary level, we have designed a series of in vitro experiments utilizing pituitary cells from ETOH naive animals maintained in monolayer culture. We report that ETOH, in doses ranging from 50 to 400 mg%, caused a prompt and sustained reduction in basal GH secretion, as well as a significant fall in intracellular GH content. These data establish that the in vivo effects of ETOH on GH can be accounted for, at least in part, by a direct effect at the pituitary level, possibly due to reduced GH synthesis.

In Vitro Effect of Ethanol Exposure on Basal and GnRH-Stimulated LH Secretion from Pituitary Cells

The question of whether ethanols (ETOHs) known suppressive effect on serum luteinizing hormone (LH) could be mediated directly at the anterior pituitary level was addressed by examining the effects of ETOH in vitro on release of LH from cultured male rat pituitary cells. The impact of added ethanol concentrations ranging from 50 to 400 mg% on LH release was examined in the basal state and after stimulation by gonadotropin-releasing hormone (GnRH) at a dose of 5 x 10(-10) M. While ETOH did not significantly suppress basal LH release, secretion stimulated with GnRH was noted to be attenuated with higher doses of ETOH (greater than or equal to 100 mg%) compared to stimulated control cells. It is concluded that ETOH exposure in vitro alters stimulated LH secretion by acting directly on pituitary gonadotropes.

The effect of in vitro ethanol exposure on LHRH release from perifused rat hypothalami

A variety of indirect data suggest that the luteinizing hormone (LH) lowering effects of ethanol (ETOH) are mediated at a hypothalamic level decreasing the synthesis and/or release of LH-releasing hormone (LHRH). Little direct data support this concept, however. The current study was, therefore, designed utilizing a perifusion system with frequent sampling for LHRH with and without ethanol added to determine if ethanol had a direct effect on basal or stimulated LHRH release. A variety of secretagogues, including dopamine, norepinephrine, naloxone, prostaglandin E2, and a high dose of potassium were utilized. Ethanol at a dose of 300 mg% did not alter either basal or secretagogue-stimulated LHRH release from the hypothalami of ethanol-naive male rats. Thus, ethanol did not appear to have a direct effect on LHRH in this system. Alterations in LHRH release by ethanol may occur at a suprahypothalamic level, involving neurotransmitter-LHRH interactions. Alternatively, the well-described lowering effect of ethanol on LH may be secondary to a direct pituitary locus of action, or involve a metabolic breakdown product of ethanol rather than ethanol itself.

Presence of Prolactin-Like Immunoreactivity and Bioactivity in Rat Spinal Cord

Pursuant to our identification of prolactin-like immunoreactivity (PLI), widely distributed in rat brain, the spinal cord was examined for the presence of this pituitary-hormone-like protein. PLI was present in all spinal cord extracts examined and averaged 500 +/- 53 pg/mg protein. Hypophysectomy, causing a fall in serum prolactin to undetectable levels, was not associated with any change in levels of PLI in spinal cord. Recovery of rat prolactin standards added to spinal cord homogenates was 97.6 +/- 3.9%. When increasing concentrations of spinal cord extract were assayed in a prolactin radioimmunoassay, displacement of rat 125I-Prolactin from antiserum was parallel to that displacement produced by increasing concentrations of rat anterior pituitary standards. Upon subjection to gel permeation chromatography, the elution profiles of immunoreactive prolactin from spinal cord were different from the profiles of anterior pituitary prolactin. In addition to an immunoreactive prolactin peak eluting with pituitary prolactin, spinal cord extracts showed a large void volume peak and late eluting low-molecular-weight materials not seen with anterior pituitary. In the Nb2 lymphoma cell assay, all spinal cord extracts demonstrated prolactin-like bioactivity with a bioactivity/immunoreactivity ratio of 1.05 +/- 0.13. We conclude: (1) PLI, widely distributed in rat brain, is also present in spinal cord; (2) spinal cord prolactin levels are independent of levels in pituitary and peripheral circulation; (3) this immunoreactive prolactin is bioactive, and (4) differing gel permeation chromatographic elution profiles indicate that there may be some molecular differences between pituitary and spinal cord prolactin.

The Effect of Alcohol on Quantitative and Qualitative Changes in Luteinizing Hormone (Lh) In the Female Rat

In order to study the impact of short-term ethanol feeding on pituitary luteinizing hormone (LH) levels, LH release and hormone microheterogeneity, 28 female oophorectomized rats were fed either a diet containing 36% ethanol or an isocaloric diet without ethanol for 16 days. On the 14th day of the experiment, all rats were given 50 micrograms of estradiol subcutaneously to provide a uniform steroid milieu and to induce an LH surge, and sacrificed 48 hours later. Total levels of pituitary and serum immunoreactive LH were identical between the ethanol and control groups. However, when pituitary LH was analyzed by isoelectric focusing on agarose gel a distinct difference was found. Four distinct immunoreactive peaks at pIs 3.50-4.55, 5.20-6.50, 7.00-7.50, 8.15-9.30 were noted. Ethanol-treated animals exhibited an 8-fold increase in the acidic LH subspecies in the pI range 3.50-4.55 and similarly more LH was determined at pI 7.00-7.50 compared to controls (P less than 0.01). More LH was found at the basic pIs from control pituitaries, compared to LH from the ethanol-exposed animals (P less than 0.01). In summary, short-term ethanol feeding in the female rats does not cause quantitative changes in pituitary LH levels or in the estrogen-induced LH surge. However, qualitative changes are present in isoelectric focusing patterns. A significant shift to the more acidic species of the pituitary LH isohormone was noted for ethanol-fed animals as compared to controls. These qualitative changes in the LH molecule, seen before quantitative changes could be appreciated, may help to explain why excessive exposure to alcohol results in reproductive impairments both in rats and in humans.

The effect of proinsulin on the immunoassay of insulin and its possible relation to states of hyperinsulinemia.

The measurement of insulin or insulin-like moieties by a variety of bioassay and immunologic procedures has resulted in the designation of such nonspecific varieties as typical and atypical insulin (1), bound and unbound insulin (2), suppressible and nonsuppressible insulin (3). Insulin determined by the radioactive immunoassay and referred to as immunoreactive insulin, IRI, has been judged by most to represent an accurate appraisal of absolute insulin levels in biologic fluids (4); as such, its biologic activity, although inferred, has never been entirely certain. In the immunoassay method, the immunospecificity of antigen to specific antibody is of necessity assumed. Accordingly, if the reaction between insulin-I131, insulin antiserum, and endogenous insulin is specific, then serial dilution of serum containing IRI will yield comparable assay measurements over the range of dilutions studied. In this laboratory where a modification of the Yalow and Berson chromatographic method (4) or the dextran-coated charcoal technique (5) is used to measure radioimmunoassayable insulin, certain sera have consistently failed to show such agreement (6), and have resulted in progressively higher than predicted IRI values with serial dilution of the serum sample. In the main these sera have come from patients in whom the diagnosis of insulinoma was being entertained where unusually high levels were anticipated so that initial measurements of IRI were made at several dilutions. To date, sera from five patients with so-called reactive hypoglycemia and many samples from a patient with organic hyperinsulinism, an insulinoma, have demonstrated this phenomenon of greater than 100% increase in IRI over several-fold dilutions of serum, appropriate dilution eventually resulting in linear agreement (Fig. 1). Furthermore, when present, this phenomenon appears to be greater when absolute IRI values are higher (Fig. 2).

A dialyzable heat stable factor in acromegalic plasma associated with increased in vitro growth hormone release.

A number of reports in the recent literature have suggested the possibility of persistent hypothalamic control in acromegaly (1-3), and further that a primary pathogenetic defect may be characterized by excessive secretion of hypothalamic growth hormone releasing factor. In a previous communication from this laboratory we have shown greater in vitro release of monkey pituitary growth hormone in the presence of acromegalic plasma than in the presence of normal plasma (4). It is the purpose of this report to further characterize this observation. Methods Intact rhesus monkey pituitary glands were obtained and handled in a manner previously described (4). The incubation mixture of 10 ml included lactated ringers 7.5 ml, containing 50 μg/ml neomycin, and a glucose concentration of 100 mg/100 ml, to which 2.5 ml aliquots of plasma or dialysates of plasma with added albumin were added for study purposes. All incubations were carried out in a Dubnoff shaker at 37°, aliquots for immunoreactive growth hormone were obtained at 30 sec after the start of an incubation, during and at the conclusion of two hour incubation periods. Growth hormone was assayed using minor modifications of a solid phase radioimmunoassay (5), with highly specific rabbit anti-growth hormone antibodies, produced in our laboratory. The standard is purified human growth hormone, NIH HS-1147. Three types of experiments were carried out. In one, whole pituitaries were incubated for 2 hr periods, first either in the presence of normal or acromegalic plasma, washed and transferred to a different plasma and then finally back to the original medium. Three intact glands were started first in acromegalic plasma, transferred to normal plasma and back for the final 2 hr of incubation to acromegalic palsma. Two pituitary glands were incubated in reverse order.