THE EFFECT OF PH ON THE CA2-C IN SKINNED MUSCLE-FIBERS( AFFINITY OF THE CA2+ REGULATORY SITES OF SKELETAL AND CARDIAC TROPONIN)

It is known that intracellular pH drops rapidly after the onset of isc hemia in cardiac muscle and may play some role in the rapid drop in fo rce that ensues. It is also known that alpha(1)-adrenoceptor agonists alkalinize intracellular pH by stimulating Na+/H+ exchange and may rep resent a mechanism which facilitates recovery of intracellular pH from acidosis. Lowering or raising pH shifts the Ca2+ dependence of force development in muscle fibres to higher or lower free Ca2+ concentratio ns, respectively, yet the precise mechanism is unknown. To investigate this phenomenon we have used skinned skeletal or cardiac muscle fibre s whose endogenous troponin C (TnC) has been replaced with chicken ske letal TnC labelled with DANZ (STnC(DANZ)) or recombinant cardiac TnC l abelled with IAANS (CTnC3(C84)(IAANS)), respectively. The fluorescence of the STnC(DANZ) or CTnC3(C84)(IAANS) was enhanced by Ca2+ binding t o the Ca2+-specific (regulatory) site(s) of STnC or CTnC when incorpor ated into skinned fibres, and was measured simultaneously with force. When the pH was changed from 7.0 to 6.5 or 7.5 the shift in the Ca2+ d ependence of force paralleled the shift in fluorescence. Since the for ce and fluorescence shift in parallel as the pH is lowered or raised, it can be concluded that these changes in Ca2+ sensitivity are caused by a decrease or increase, respectively, in the Ca2+ affinity of the C a2+-specific site(s) of TnC. Since lowering or raising the pH also res ulted. in lower or higher, respectively, maximal Ca2+ activated force while maximal fluorescence remained unchanged, it is possible that Hmay act indirectly, as well, by reducing or increasing, respectively, the number or type of crossbridges attached to actin and thereby alter the crossbridge induced depression or elevation, respectively of the observed TnC Ca2+ affinity. Experiments with 2,3-butanedione monoxime, however, where force-generating crossbridges were greatly reduced, in dicated that the pH effect may be primarily related to a direct change in the Ca2+ affinity to the regulatory sites of TnC.