The purpose of the proposed investigation is to elucidate the role of microtubules in the intracellular mechanisms involved in PRL transformation and release in the lactating rat following suckling. Initially, we will analyze polymerized and depolymerized tubulin levels within the pituitary a) during suckling under different physiological conditions, and b) following administration of dopamine agonists or antagonists, TRH, VIP, and OT, which are known to alter the extent to which PRL is transformed and released. Next we will investigate whether the disassembly of microtubules in the anterior pituitary by drugs (colchicine, vinblastine) is accompanied by alterations in the transformation and release of PRL following suckling in vivo, or following incubation of the anterior pituitary in vitro. We will use 3H-colchicine binding, guanosine triphosphate (GTP) hydrolysis, and tubulin RIA to assess quantitative changes in anterior pituitary tubulin in these studies, and EM to confirm that tubulin disassembly has occurred. We will also use immuno-electron microscopy to investigate morphologic alterations in anterior pituitary microtubules that occur in response to suckling, and we will correlate these with physiological and biochemical indices of PRL secretion. We will test the hypothesis that cAMP mediates the effect of suckling in the regulation of tubulin polymerization-depolymerization in association with PRL transformation and release. Changes in cAMP in the pituitary following suckling or domperidone will be monitored. Also, the effects of dibutyryl cAMP, phosphodiesterase inhibitors, and forskolin, injected in vivo or added in vitro, upon pituitary tubulin will be assessed. We will use the in vitro assembly of tubulin into microtubules to assess the effect of suckling and of dopamine, TRH, VIP, and OT added in vivo and in vitro upon the rate of microtubule formation. The microtubules thus formed will be incubated with PRL granules in order to investigate, by EM and depolymerization techniques, the nature of the association between PRL and microtubules. We will develop this preparation as a model, both to establish the intracellular factors that may be required in or that influence the binding of PRL granules to microtubules, and to evaluate those factors which influence the uncoupling of PRL from microtubules.
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