Technical problems related to the analysis of the effects of inorganic phosphate on cardiac muscle

We describe how potential artifacts (due to solution composition, buffering capacity of the bathing medium, size of the skinned fiber preparation, permeability of the sarcoplasmic reticulum (SR) vesicles, and proper Kd for Ca2+ of the fluorescent indicator used to measure Ca2+ transport can be avoided in order to determine the effects of inorganic phosphate (Pi, or any other ion) on m,aximum Ca2+ activated force (Fmax) and Ca2+ sensitivity of skinned cardiac muscle fibers, and Ca2+ -ATPase activity and uptake properties of isolated cardiac SR-enriched vesicles. To maintain the ionic strengh of the bathing medium constant when adding Pi, other ions must be removed. We found that because some salts have a depressant effect on Fmax independent of increaqsed ionic strength (e.g. KCl) while others do not (e.g. Na-acetate), the salt used to adjust ionic strength influences the measured depressant effect of Pi on Fmax. The sensitivity to Ca2+ of the contractile apparatus depends on the sum of the [Na+] and [K+] in the bething medium. However, we found that the effect of Pi on Ca2+ sensitivity was not significantly influenced by the small changes in the sum of [Na+] and [K+] that were associated with the addition of Pi. The skinned fiber preparations were approximately cylindrical bundles with diameters ranging between 100 and 250 um. We found that the effect of Pi on Fmax was not influenced by diffusion limitations over this range of bundle diameters. The pH buffering capacity of the bathing solution affects Fmax at pH 6.6. We found that the buffering effect of Pi can influence the mechanical response of skinned fibers independent of a Pi on the contractile apparatus when the buffering capacity of the control solution is low. When the Ca2+-ATPase of isolated SR vesicles is activated by Ca2+ and MgATP, the vesicles accumulate Ca2+. Unless the vesicles are permeabilized with a Ca2+ ionophore (ionomycin) and the pH adequately buffered, maximum ATPase activity will be underestimated, the broadness of the curve relating Ca2+ - ATPase rate overestimated, and the sensitivity to Pi overestimated. The ionic milieu of isolated SR vesicles changes the apparent dissociation constant (Kd) of Ca2+-Fura-2, a fluorescent dye used to quantify SR Ca2+ transport rates. In order to accurately measure the inhibitory effect of Pi on Ca2+ uptake, the influence of Pi on the Kd of Fura-2 for Ca2+ must be taken into account

Effects of dichlorobenzamil, a sodium-calcium exchange inhibitor, on the calcium paradox and the sodium withdrawal contractures of frog atrial muscle

The effects of dichlorobenzamil (DCB), an amiloride derivative and potent inhibitor of Na-Ca exchange in cardiac sarcolemmal vesicles and isolated cardiac myocytes, were investigated in two paradigms involving Na-Ca exchange, namely the Ca2+ paradox and the Na + - withdrawal contractures of frog atrial muscle strips. Pretreatment with DCB (10-100 micronM) inhibited in a dose-dependent manner the contractures elicited by reexposure of the atrial strips to the control Ringer solution after a 5-20 min equilibration with a Ca2 + - free saline (Ca2 + - readmission contractures; Ca2 + paradox). These contracture were not inhibited, however, when DCB was applied after the preparation had been exposed to the Ca2 + - free saline, but before the reexposure to the control Ringer solution. DCB (10-100 micronM) did not inhibit the contractures elicited by Na + - deficient saline (Na + - withdrawal contractures) in atrial strips pretreated or not with acetylstrophantydin. This result suggests that, under our experimental conditions, DCB falied to substantially inhibit the Ca2 + influx mediated by Na-Ca exchange. The duration of the plateu of the action potentials of atrial cells equilibrated with Ca2 + - free saline was reduced from 1,42 ñ 0,27 s to 0,61 ñ 0,13 s by 50 micronM DCB (P<0.001). This was atributed to blockade of Na + currents through modified L-type Ca2 + channels. It is proposed that shortening of the Na + - dependent action potentials can account for the inhibition of the Ca2 + - readmission contractures, because these contractures have a steep dependence on the Na + influx and intracellular Na + accumulation that occurs during the Ca2 + - free period. The results of this study support the conclusion thatDCB has multiple effects on heart muscle, including a potent blockade of Ca2 + channels, and its use as a selective inhibitor of Na-Ca exchange in cellular systems in un unwarranted