A new technique, based on the property of phosphodiesterases to promote P-O bond cleavage during the hydrolysis of cyclic 3 feet, 5 feet nucleotides and the insertion of 18-O from [18-O]water into the phosphoryls of the 5 feet-nucleotide product will be used to monitor the dynamics of cAMP and cGMP metabolism in intact sea urchin sperm. By this new analytical approach information has recently been obtained indicating that in addition to the allosteric mode of cyclic nucleotide action involving protein kinase activation, there may be an influence on certain cell functions that are related to the hydrolysis of cyclic nucleotides. This relationship was uncoverd in the visual retina where it was found that light stimuli over a 4 log range of intensities promote incremental increases in cGMP hydrolytic flux. These light-induced increases in cGMP metabolic flux were not associated with any changes in cellular cGMP steady state levels, indicating that the increased rate of cGMP hydrolysis is tightly coupled to an equal increase in cGMP synthesis. This increase in cGMP metabolic flux has been postulated to be integral to a mechanism for releasing bound calcium. One of the intensions of the experiments described in this proposal is to determine if the modification of certain metabolic and functional events in sperm are also associated with this newly disclosed modification of cyclic nucleotide metabolism that can only be uncovered by the type of metabolic measurements that the 18-O labeling procedure provides. Obtaining information regarding the fundamental dynamics of cGMP and cAMP metabolism in sperm represents the primary goal of the studies to be conducted. Several biological processes in sea urchin sperm including the acrosome reaction, respiration, motility and ion transport are activated or elicited by specific factors derived from sea urchin eggs. The actions of these egg factors have been found to be associated with alterations in cyclic nucleotide metabolism but the precise involvement of cyclic nucleotides in influencing sperm events is unclear. The information that will be obtained from the 18-O labeling studies proposed should help to provide some new insights into the role that cyclic nucleotides and alterations in their metabolism may play in modulating and/or promoting certain of the cellular events in sperm. The measurement of 18-O labeling of adenine and guanine nucleotide Alpha, Beta, and Lambda-phosphoryls within the intact cells also provides the potential for examining overall nucleotide and energy metabolism in relation to the processes that can be promoted in these cells.
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