KREBS CYCLE AND CYCLIC NUCLEOTIDE CONTROL

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DESCRIPTION (Adapted from the applicant's abstract): The planned experimentation has 2 major goals: to define the biological importance and operational mechanism of a non-second messenger, cyclic nucleotide (CN) metabolic system, designated here as "excitatory," that exhibits the distinctive characteristic of markedly accelerated rates of turnover with no change in cellular CN concentration in response to cell signals and ionophores that promote increases in intracellular Ca2+ concentration; and to utilize a uniquely effective analytical procedure based on measuring rates of appearance of 18-0 in the phosphoryls of metabolic intermediates to define the dynamic behavior in intact cells of the enzymic pathways that function in high energy phosphoryl transfer. In the studies planned high energy phosphate metabolism will be characterized in subcellular compartments of chromaffin cells to determine requirements for and cellular locales of energy-consuming events. The possible operation of a phosphorylcreatine shuttle and an adenylate kinase phosphoryl transfer system operating to maintain high energy phosphate status of actively metabolizing granules in chromaffin cells will be defined. The excitatory CN metabolic system will be characterized in several ways. First, regarding its intracellular operation by determining its metabolic behavior in relation to Ca2+ transients and excitation/secretion coupling induced by dissimilar Ca2+-linked signals; second, by assessing if the mechanism of its regulation involves calmodulin (CM by determining its metabolic behavior in relation to the in situ kinetics of phospho-diesterase (PDE) activation by Ca2+/CM using conformation-specific antibodies to detect rates of Ca2+/CM PDE complex formation; third, by assessing the requirement for a "down-stream" G-protein in regulating or coupling the excitatory CN system to secretion by determining if botulinum toxin inhibition of secretion beyond release of intracellular Ca2+ interferes with activation of this CN metabolic system through the ADP ribosylation of a 22 kDa G-protein toxin target; and fourth, by determining whether a mechanism for the functional utility of this excitatory CN system derives from an intrinsic property of CN PDE to act as an effector of high conductance Ca2+-activated K+ channels "opened" in close correspondence with the Ca2+-linked stimulation of the excitatory CN system in parotid. This proposed action of PDE would coincide with its very recently demonstrated direct effect on photoreceptor cation channels which predicts that rates of CN hydrolysis determine channel open time.