PHARMACOLOGY OF RYANODINE AND THE SR CA CHANNEL

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The goals of the experiments first, to establish the in vitro effects of ryanodine and a series of structural analogs of ryanodine on the calcium permeability and calcium binding properties of SR membranes isolated from skeletal, cardiac and vascular smooth muscles. Second, we will determine the physiological consequences of the actions of these compounds by characterizing the changes they produce in the function of intact muscle preparations. In preliminary studies we have found that two diterpine ester analogs of ryanodine, designated A and B, each cause only one of the effects of ryanodine. Therefore, these compounds may selectively increase and decrease a SR membrane calcium permeability and perhaps activate and inhibit the calcium release channel. 1. We will extract and purify the following compounds from Ryania wood: Ryanodine, dehydroryanodine, and the diterpine esters designated A, B, C1, C2 and D. In addition, ryanodine will be chemically modified to form 9-epiryanodine, isodehydroryanodine, anhydroryanodine, oxoryanodine, ryanodol and pyrrole-2-carboxylic acid. 2. In a series of in vitro investigations we will establish and compare the effects of ryanodine and its analogs on passive and active calcium fluxes across, and calcium binding by, SR membranes isolated from skeletal, cardiac and smooth muscles. We will also determine the effects of these compounds on the conductance and activation characteristics of calcium channels incorporated into lipid bilayers from membrane fractions found to be affected by the alkaloids. Ineffective compounds will be tested for their ability to antagonize the actions of those compounds found to act at one of the sites affected by ryanodine. The structure-activity information obtained from these studies will be used to identify the structural features of the ryanodine molecule which are important for each activity. 3. We will determine the ability of each compound to alter the rate of oxygen consumption, cytoplasmic calcium activity and the mechanical and electrical functions of intact skeletal, cardiac and smooth muscles. We will correlate the structure-activity relationships obtained in these in situ studies with those determined in the preceding step in order to establish the cause and effect relationships existing between the in vitro changes in SR calcium movements and the alterations in intact muscle function produced by these compounds.