The proestrous LH surge is largely due to a major increase in the ability of the anterior pituitary to secrete LH which, in turn, reflects an interaction of LHRH and estrogen feedback at the pituitary. Hemolytic plaque assay studies suggest that this change in responsiveness is mediated by 1. a progressive increase in sensitivity to LHRH and 2. an abrupt increase in capacity to release LH. The molecular steps involved are poorly understood. The applicants hypothesize that these two processes are independently regulated: while sensitivity is regulated by (Ca2+)i,, capacity is a function of the """"""""readily releasable pool"""""""" of LH. The proposed mechanisms are as follows: LHRH receptor activation stimulates phospholipase C to generate equivalent amounts of IP3 and diacylglycerol at each stage of the estrous cycle; IP3 mobilizes different amounts of (Ca2+)i during the estrous cycle; diacylglycerol 3 activates protein kinase C via a process which is modulated by (Ca2+)i - i.e., the greater the (Ca2+)i the lower the concentration of diacylglycerol required to activate the enzyme. Protein kinase C effects the release of LH. It is proposed that variations in the amounts of calcium mobilized by IP3 dictate sensitivity to LHRH. Utilizing the hemolytic plaque assay in combination with techniques to measure (Ca2+)i in single gonadotropes with the calcium sensitive dye fura-2, the following questions will be addressed: 1. Is gonadotrope IP3 generation in response to LHRH invariant during the estrous cycle? 2. Does IP3 mobilize calcium in varying amount at different stages of the estrous cycle? 3. Is the calcium response to LHRH digital in mode? 4. Does alteration of (Ca2+)i in the gonadotrope affect gonadotrope sensitivity (in respect to LH secretion) to LHRH? 5. What is the significance of gonadotrope shape changes in response to LHRH? 6. What is the relationship between calcium and the other messenger pathways? If the hypothesis is correct a role for digital signals at the single cell level will have been defined. This model may facilitate our understanding of the pathophysiology of gonadotropin secretion disorders, allow for the design of novel methods of contraception, and find application in delineating mechanisms subserving other endocrine systems.
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