RESPONSE OF CARDIAC MYOCYTES TO A RAMP INCREASE OF DIACYLGLYCEROL GENERATED BY PHOTOLYSIS OF A NOVEL CAGED DIACYLGLYCEROL

To test the responsiveness of living cells to the intracellular messen ger diacylglycerol, we developed a prototype caged diacylglycerol comp ound, xyl-2,4-dinitrobenzyl)-1,2-dioctanoyl-rac-glycerol (designated a lpha-carboxyl caged diC(8)), that produces dioctanoylglycerol (diC(8)) on photolysis. alpha-Carboxyl caged diC(8) is biologically inert towa rd diacylglycerol kinase and protein kinase C in vitro and is readily incorporated into cardiac myocyte membranes, where it has no effect be fore irradiation. Exposure to near-UV light releases biologically acti ve diC(8) in good yield (quantum efficiency = 0.2). Here we examine a cellular response to controlled elevation of diC, within single cardia c myocytes. Twitch amplitude was monitored in electrically stimulated myocytes, and a ramp increase in the concentration of diC(8) was gener ated by continuous irradiation of cells loaded with the caged compound . The myocyte response was biphasic with a positive inotropic phase (3 9% increase in twitch amplitude), followed by a large negative inotrop ic phase (>80% decrease). The time to peak inotropy for both phases de pended on the light intensity, decreasing from 376 +/- 51 s to 44 +/- 5 s (positive phase) and 422 +/- 118 s to 51 +/- 9 s (negative phase) as the light intensity was increased eightfold. Both phases were inhib ited by the protein kinase C inhibitor chelethyrine chloride. An incre ase in extracellular K+ from 5 mM to 20 mM to partially depolarize the cell membrane eliminated the positive inotropic phase, but the negati ve inotropic response was largely unaltered. The results reveal new fe atures in the response of cardiac muscle to diacylglycerol, including a positive inotropic phase and a complex responsiveness to a simple li near increase in diacylglycerol. The effects of photoreleased diC(8) w ere similar to the effects of opiate agonists selective for kappa rece ptors, consistent with a major role for diacylglycerol in these respon ses.