Immunohistochemical studies have identified immunoreactive prolactin (IR-PRL) in the hypothalamus and other areas of the rat brain. However, immunocytochemical techniques make it difficult to quantify the amount of antigen localized in a specific region. In this study, IR-PRL was extracted from selected regions of the rat brain, consisting of the median eminence, dorsal and ventral hypothalamus, thalamus, amygdalae, cerebellum, cortex, hippocampus, septum, pons-medulla, and olfactory lobes, and the concentrations of IR-PRL were determined by radioimmunoassay. Whereas IR-PRL was detected in all brain regions in both the male and the female rat brain, the concentrations of IR-PRL in the female rat were significantly greater than those measured in the corresponding region of the male rat brain. In the female rat, hypophysectomy significantly reduced, but did not eliminate, the concentration of IR-PRL in hypothalamus, amygdala, thalamus, and pons-medulla. In contrast, hypophysectomy did not affect the concentration of IR-PRL in any brain regions of the male rat. Injection of colchicine into the lateral ventricle decreased the concentration of IR-PRL in the median eminence and increased the concentration of IR-PRL in the ventral hypothalamus in male and female rats. In addition, extracted hypothalamic and pituitary IR-PRL displayed similar dilution curves in the PRL assay and elution patterns on Sephadex G-100. These data indicate that both the male and the female rat brain contains an IR-PRL-like material with physicochemical properties similar to those of pituitary PRL. This material is differentially distributed in the male and female brain and is found in greater concentrations in the female brain. Finally, the maintenance of IR-PRL in the brain in hypophysectomized rats and the decrease in median eminence IR-PRL in colchicine-treated rats suggest that this IR-PRL-like material is synthesized in the central nervous system.

Immunocytochemical studies have identified immunoreactive prolactin (IR-PRL) in the hypothalamus and other areas of the rat brain. However, neither the release of IR-PRL from the hypothalamus nor its subcellular localization have been demonstrated. In this study, the release of IR-PRL from hypothalami obtained from female rats was examined using hypothalamic units incubated in vitro in Krebs-Ringer bicarbonate-glucose buffer. Hypothalamic tissue spontaneously released IR-PRL, and this release was increased by depolarizing concentrations of potassium by a calcium-dependent mechanism. Hypothalamic IR-PRL was also released from hypothalamic tissue obtained from hypophysectomized rats (14 days). The subcellular localization of IR-PRL was investigated using equilibrium-density centrifugation. Tissue homogenates from intact or hypophysectomized rats were centrifuged at 150 g at 4 °C for 10 min, and the supernatants were layered onto continuous sucrose gradients (1.00–1.27 g/ml) and centrifuged at 100,000 g (max.) for 16 h. IR-PRL in pituitary supernatants showed a high equilibrium-density peak with a modal density of 1.23 g/ml. Fractionation of the supernatant from ventral or dorsal hypothalamic tissue resulted in two high-equilibrium density peaks, a primary peak with a modal density of 1.23 g/ml and a smaller peak with a modal density of 1.10 g/ml. Both high-density peaks were maintained in tissue obtained from hypophysectomized rats and were disrupted by homogenization in hypo-osmotic medium. Together, these data suggest that hypothalamic IR-PRL is stored in membrane-bound particles which have densities similar to those of secretory granules and is released by a calcium-dependent mechanism when the tissue is depolarized.

These experiments characterized behaviorally two strains of rat that have been selectively bred for blood pressure differences. Avoidance performances of the spontaneously hypertensive rat (SHR) and normotensive rat (WKYN) were examined under two conditions: a discrete-trial two-way shuttle box avoidance procedure and a discrete-trial lever-press avoidance procedure. The WKYN strain reached a higher level of acquisition in the two-way shuttle box task than did the SHR strain, but this difference was confounded by an initial difference in pretest avoidance rate. In contrast, the SHR strain was superior in the lever-press avoidance procedure under all conditions employed. These data are interpreted as being consistent with a hypothesis offered by Satinder that strains selectively bred for some behavioral feature may also differ in central arousal which will interact with task difficulty to determine performance differences. The data clearly indicate that the selective breeding for high and low blood pressure has simultaneously influenced the behavioral properties of these two strains.Exposure to a stressful environment has often been postulated to play a role in the etiology of hypertension (Cobb & Rose, 1973), and many diverse research strategies have been utilized to clarify the influence of chronic stress on blood pressure (Henry & Oassel, 1969). Since animal models allow direct assessment of hemodynamic responses to stress, they have been frequently employed in physiological (Folkow, Hall-

Fibroblast growth factors (FGF) are osteoblast mitogens, but their effects on bone formation are not clearly understood. Most in vitro studies examining the effects of FGFs on osteoblasts have been performed only during the initial proliferative stage of osteoblast culture. In these studies, we examined the consequential effect of acidic FGF in cultures of rat fetal diploid osteoblasts that undergo a developmental differentiation program producing a mineralized bone-like matrix. During the initial growth period (days 1-10), addition of acidic FGF (100 micrograms/ml) to actively proliferating cells increased (P < 0.05) 3H-thymidine uptake (2,515 +/- 137, mean +/- SEM vs. 5,884 +/- 818 cpm/10(4) cells). During the second stage of maturation (days 10-15), osteoblasts form multilayered nodules of cells and accumulate matrix, followed by mineralization (stage 3, days 16-29). Addition of acidic FGF to the osteoblast cultures from days 7 to 15 completely blocked nodule formation. Furthermore, addition of acidic FGF after nodule formation (days 14-29) inhibited matrix mineralization, which was associated with a marked increase in collagenase gene expression, and resulted in a progressive change in the morphology of the nodules, with only a few remnants of nonmineralized nodules present by day 29. Histochemical and biochemical analyses revealed a decrease in alkaline phosphatase and mineral content, confirming the acidic FGF-induced inhibition of nodule and matrix formation. To identify mechanisms contributing to these changes, we examined expression of cell growth and bone phenotypic markers. Addition of acidic FGF during the proliferative phase (days 7-8) enhanced histone H4, osteopontin, type I collagen, and TGF-beta mRNA levels, which are coupled to proliferating osteoblasts, and blocked the normal developmental increase in alkaline phosphatase and osteocalcin gene expression and calcium accumulation. Addition of acidic FGF to the cultures during matrix maturation (days 14-15) reactivated H4, osteopontin, type I collagen, and TGF-beta gene expression, and decreased alkaline phosphatase and osteocalcin gene expression. In an in vivo experiment, rats were treated with up to 60 micrograms/kg/day acidic FGF intravenously for 30 days. Proliferation of osteoblasts and deposition of bone occurred in the marrow space of the diaphysis of the femur in a dose-related fashion. The metaphyseal areas were unaffected by treatment. In conclusion, our data suggest that acidic FGF is a potent mitogen for early stage osteoblasts which leads to modifications in the formation of the extracellular matrix; increases in TGF-beta and collagenase are functionally implicated in abrogating competency for nodule formation. Persistence of proliferation prevented expression of alkaline phosphatase and osteocalcin, also contributing to the block in the progression of the osteoblast developmental sequence.

PRL has been reported to activate cell cycle-specific enzyme markers in nonreproductive tissues. To determine if PRL stimulates cell cycle-specific markers and cell growth in the central nervous system, the effect of PRL on cellular proliferation was examined in cultured astrocytes. Astrocytes from confluent cultures were plated onto glass slides at a density of 2 x 10(4) cell/ml 24 h before use. In some experiments, cells were serum deprived for 24 h (Go-arrested) after plating. Cell proliferation was examined directly by an increase in cell number and in individual cells by immunofluorescent detection of proliferating cell nuclear antigen (PCNA) and the incorporation of 5-bromo-2'-deoxyuridine (BrUd). When incubated with 1% serum, a small percentage (less than 5%) of the cells expressed PCNA. In cells cultured with rat PRL (10(-10)-10(-7) M) for 18 h, staining of PCNA increased in a dose-dependent manner, with maximal expression occurring at 10(-9) M PRL. At concentrations above 10(-9) M, PCNA staining decreased. To examine the specificity of the PRL-induced increase in PCNA, cells were incubated in the presences of 10(-9) M rat (r) or bovine (b) GH. Whereas incubation of astrocytes with rPRL and bPRL activated PCNA, cells incubated with either rGH or bGH showed only a slight increase in the number of cells expressing PCNA. Further, incubation of astrocytes with 1 nM PRL in the presence of 100 nM cyclosporine, an immunosuppressive agent that specifically displaces PRL from its receptor, decreased the percentage of nuclei stained for PCNA to that observed in non-PRL-stimulated controls. Transient exposure of cells to 10(-9) M PRL for 30 min resulted in an increase in the number of cells expressing PCNA when cultured for 18 h in the presence of 1% serum. In the presence of 1% serum, 10(-9) M PRL increased the incorporation of BrUd and resulted in a 3-fold increase in the doubling rate. In cells incubated in serum-free medium, only a few PCNA-positive cells could be detected. Treatment of Go-arrested astrocytes with PRL (10(-10)-10(-7) M) for 18 h resulted in a dose-dependent increase in the expression of PCNA. PCNA-positive cells were detected in cultures incubated with 10(-11) M PRL, with maximal expression at 10(-9) M.(ABSTRACT TRUNCATED AT 400 WORDS)