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R. R. GALA
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SUMMARY

A pituitary was obtained from a patient with metastatic breast cancer and cultured for 81 days in Medium 199. The medium was changed every 3 days. The prolactin activity of the medium, as estimated by the local crop gland proliferation assay, was significantly lower in the first 3-day culture period than in subsequent periods. Prolactin activity was similar between days 6 and 21 of culture, decreasing to the limits of the bioassay system on the 27th day of culture. Analytical disc electrophoresis of the medium revealed the presence of a protein component which migrated behind human growth hormone (HGH). A densitometric analysis of the bands in disc electrophoresis showed that the protein concentration of the component migrating behind HGH correlated with the prolactin activity of the medium for later periods of culture. A calculation of the specific activity of this component gave a value of 125 i.u. prolactin/mg protein.

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D. R. PIEPER
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R. R. GALA
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SUMMARY

The role of the pineal gland, olfactory bulbs and photoperiod in the regulation of the two daily surges of plasma prolactin in the pseudopregnant rat has been investigated. Pinealectomy had no effect on the surges of prolactin in pseudopregnant rats maintained on either a long (14 h light: 10 h darkness; 14L: 10D) or a short (2L: 22D) photoperiod, but olfactory bulbectomy decreased the nocturnal surge in animals maintained on 14L: 10D. This effect of bulbectomy was eliminated if the rats maintained on 14L: 10D were also pinealectomized. After cervical stimulation, bulbectomized rats maintained on a 2L: 22D photoperiod had nocturnal-type prolactin surges similar to those of intact rats maintained on the same photoperiod. These results indicate that the pineal gland and length of photoperiod are not involved in the regulation of the surges of plasma prolactin in pseudopregnant rats but that the olfactory bulbs may enhance the nocturnal surge.

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D. M. LAWSON
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R. R. GALA
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SUMMARY

Plasma levels of prolactin were determined, by radioimmunoassay, in ovariectomized, oestrogen-treated rats after administration of ether, sodium pentobarbitone, urethane, chloral hydrate or ketamine. These anaesthetics, when administered alone, induced sustained increases in the plasma level of prolactin (continuous ether inhalation), no change in prolactin secretion (urethane), or depressions in the level of prolactin (sodium pentobarbitone, chloral hydrate and ketamine). These same anaesthetics when given before perphenazine failed to alter the stimulatory effect of this phenothiazine on prolactin secretion. Sodium pentobarbitone did not alter the normal increase in prolactin concentration after intra-arterial administration of synthetic thyrotrophin releasing hormone (TRH). These results indicated that anaesthetics do not affect the response of either the central nervous system or the anterior pituitary to perphenazine or TRH although they affect prolactin secretion when administered alone. The site of action of anaesthetics must, therefore, be different from that of perphenazine or perphenazine must be capable of overcoming their influence by direct action on the pituitary.

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D. M. LAWSON
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R. R. GALA
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SUMMARY

Plasma prolactin was measured by double antibody radioimmunoassay in ovariectomized rats bearing indwelling aortic catheters. Samples were obtained during recovery from ovariectomy and catheterization, at various times during the day, after blood volume reduction, and after administration of various anaesthetics. Prolactin levels were stabilized by day 3 after the operation but not by day 1. There was a significant (P < 0·01) increase in plasma prolactin during the afternoon in both ovariectomized rats and ovariectomized rats treated with 1 mg polyoestradiol phosphate. The magnitude of the response was more pronounced in the animals treated with oestrogen. Withdrawal of blood samples without subsequent replacement of fluid volume induced a significant (P < 0·05) decrease in plasma prolactin concentration after removal of only 1·2 ml blood within 20 min. No change was observed, however, when blood was replaced with saline. Ether and the intraperitoneal injection of sodium pentobarbitone produced prolonged increases in plasma prolactin whereas the intra-arterial administration of sodium pentobarbitone produced no change. Urethane and chloral hydrate caused only transient increases in plasma levels of prolactin regardless of the route of administration. Ketamine did not significantly alter plasma levels of prolactin.

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S. W. SMITH
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R. R. GALA
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The effect of restraint on plasma prolactin and corticosterone concentrations was investigated in chronically catheterized, ovariectomized (OVX) or ovariectomized, oestrogen-treated (OVX-PEP) rats. Restraint was induced by tying the hind legs together. In OVX rats, prolactin levels were unchanged following restraint, either during the morning (10.00 h) or afternoon (14.00 h). Prolactin levels increased in OVX-PEP animals when restraint was initiated in the morning; when restraint was initiated in the afternoon the prolactin response depended upon the level of prolactin before restraint. If levels were low (pre-surge) the response to restraint was similar to that observed in the morning; if prolactin levels were high (surge) the response to restraint was reversed and the prolactin level declined. The morning prolactin response to restraint was significantly inhibited and the afternoon surge was retarded in adrenalectomized OVX-PEP (OVX-PEP-ADX) rats; however, in OVX-PEP animals maintained on 0·9% NaCl drinking solution, the morning prolactin response to restraint was also blunted, although the afternoon surge was normal. In OVX-PEP-ADX animals injected with either vehicle alone, or 2 or 4 mg corticosterone for 4 days, and sampled on the morning of day 5, the prolactin response to restraint was absent. Furthermore, when OVX-PEP animals were injected daily with either vehicle or 4 mg corticosterone/day, they showed no increase in prolactin in response to restraint when values were compared with those of uninjected animals. Corticosterone levels after restraint were higher than initial values in all of the above experimental conditions.

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D. M. Lawson
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D. J. Haisenleder
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R. R. Gala
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J. A. Moy
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ABSTRACT

The objectives of this study were to determine (1) whether pre-release transformation (depletion) of pituitary prolactin occurs as the result of suckling to the same extent in several strains of lactating rats, (2) the molecular nature of the transformed hormone, (3) whether the quantity of transformed (depleted) prolactin recovered is dependent upon the type of homogenization buffer used and (4) whether the method of assay influences the extent to which transformed prolactin is detected. During the course of these experiments other factors such as the methods of handling and storing pituitaries and homogenates were also found to influence the amount of prolactin recovered.

The results indicated that transformation of prolactin is a very labile event which is affected by many factors. Strain and supplier of rats was critical to the observation of suckling-induced depletion of prolactin, with Spartan- and Holtzman-derived Sprague–Dawley strains exhibiting the most consistent responses. When transformation was observed, it mattered little which buffer was used for homogenization; however, alkaline or acidic buffers extracted more prolactin than did neutral buffers. Triton X-100 also significantly enhanced the efficiency of extraction by neutral buffers. Maintaining pituitaries on dry ice immediately upon removal from the animal increased the amount of prolactin recovered, as did freezing the homogenate for 1–5 weeks before assay. The addition of the protease inhibitor, benzamidine hydrochloride, did not affect the pituitary content of prolactin. Assay of prolactin by polyacrylamide electrophoresis and densitometry yielded more prolactin than either radioimmunoassay or the Nb2 lymphoma bioassay. The molecular nature of pituitary prolactin, extracted at neutral pH, as judged by gel filtration was altered slightly but consistently by suckling, such that large molecular forms increased at the expense of the smallest molecular form.

We conclude from these studies that great care must be exercised when attempting to characterize dynamic changes in pituitary prolactin content in lactating rats. Strain and supplier of rats, methods of handling and storing pituitaries, types of buffers used for homogenization and methods of assay all influence the amount of prolactin recovered and can influence the extent to which rapid changes in pituitary prolactin are detected.

J. Endocr. (1987) 113, 71–80

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