KREBS CYCLE AND CYCLIC NUCLEOTIDE CONTROL

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The investigations are aimed at assessing and defining the significance of the accelerated cyclic nucleotide metabolic flux that occurs without changes in cell cGMP or cAMP levels in association with the action of excitatory cell stimuli. This model of cyclic nucleotide involvement which contrasts with their influence mediated through changes in their cellular concentration is suggested from investigations of phototransduction and from more recent studies on contraction in smooth muscle to represent a biochemical process in excitation related to intracellular Ca2+ release. This involvement of cyclic nucleotides will be assessed with regard to excitation/contraction coupling in smooth and skeletal muscle and excitation/secretion coupling in parotid gland and platelets. The newly devised technology of monitoring cyclic nucleotide and 5' nucleotide metabolism in intact cells by the rate of incorporation of oxygen 18 from [oxygen 18] water into the Alpha-, Beta- and Gamma-phosphoryls of guanine and adenine nucleotides will be employed to assess the relationship of cyclic nucleotide metabolism to cell excitation and obtain information regarding: 1) the exclusivity of the phophodiesterase pathway in labeling cellular adenine and guanine nucleotide Alpha-phosphoryls; 2) identification of the cellular metabolic pathway that appears to derive from cAMP hydrolysis leading to rapid oxygen 18 labeling of IMP with stimulated contraction; 3) cellular high energy phosphate metabolism and Pi transport associated with excitatory and adaptive cell stimulation; 4) definition of the cellular metabolic compartments of cyclic nucleotides and 5' nucleotides affected by excitatory and adaptive stimulation; 5) relationship of intensity, frequency and duration of excitatory stimulation to cellular cGMP and cAMP metabolic flux; and 6) relationship of excitation-induced acceleration of cyclic nucleotide metabolic flux to intracellular Ca2+ release. These correlative studies will be supplemented by experiments to obtain information regarding; 7) the kinetic and regulatory characteristics of phosphodiesterases and nucleotidyl cyclases associated with cell structures in muscle with Ca2+ uptake and release capability; and 8) the relationship of cyclic nucleotide hydrolysis to the release of Ca2+ from isolated muscle triads.