[Effect of compound 48/80 on masseter muscle sarcoplasmic reticulum calcium transport system].

To define the role of calmodulin in Ca2+ fluxes behavior of canine masseter muscle sarcoplasmic reticulum (SR) vesicles, the effect of condensation product of N-methyl-p-methoxy-phenethylamine with formaldehyde (compound 48/80), a selective and powerful inhibitor of calmodulin-regulated function, on Ca(2+)-ATPase activity, oxalate-supported Ca2+ uptake velocity, and on interaction with Ca2+ permeability and Ca2+ loading at steady-state were evaluated. Compound 48/80, at concentrations of 10 to 100 micrograms/ml, reduced oxalate-supported Ca2+ uptake velocity without affecting Ca(2+)-ATPase activity. In the presence of 10 micrograms/ml compound 48/80, there was a shift of pH- or temperature-response curve of oxalate-supported Ca2+ uptake velocity, but not of Ca(2+)-ATPase activity, down. It was found that Arrhenius plots of the Ca(2+)-ATPase activity show a break at about 21 degrees C in the presence or absence of 10 micrograms/ml compound 48/80, and that compound 48/80 has no effect on Arrhenius plots of the oxalate-supported Ca2+ uptake velocity. Furthermore, Ca2+ loading at steady-state, but not passive Ca2+ permeability, was decreased by compound 48/80 at low concentrations (1-2 micrograms/ml). The results of this study suggest that calmodulin-dependent process plays a functional role in the coupling of ATP hydrolysis and Ca2+ accumulation, perhaps through regulation of Ca2+ release channels in masseter muscle SR membrane. Calmodulin-dependent component of Ca2+ fluxes in the SR vesicles may be directly modified by compound 48/80, thereby diminishing Ca2+ accumulation without affecting the Ca2+ uptake mechanism.

Free radicals-induced changes in mesenteric microvascular dimensions in the anesthetized cat.

Effect of free radicals on microvascular dimensions was studied in anesthetized cat intestinal mesentery. Generation of free radicals by xanthine oxidase, acting on endogenous substrates (e.g., xanthine, hypoxanthine), produced sustained vasodilation at pH 7.4 or at pH 6.6. These effects were reversed by superoxide dismutase (SOD), a superoxide radical (.O2-) scavenger, at pH 7.4, and by SOD plus hydroxyl radical (.OH) scavenger d-mannitol at pH 6.6. These findings suggest that the sustained vasodilation is caused by .O2- and by .OH derived from .O2- at low pH.

Characterization of the effect of pH on the excitation-contraction coupling system of canine masseter muscle.

The effect of pH on the excitation-contraction coupling system of canine masseter muscle was studied by evaluating the functional integrity of the sarcoplasmic reticulum (SR) and myofibrils. Increasing proton concentration (pH 7.0-5.8) significantly reduced oxalate supported SR calcium uptake velocity, while Ca2+-stimulated, Mg2+-dependent ATPase activity was unaffected by pH. The efficiency ratio of calcium transport, or the coupling ratio (mumoles Ca2+ transported/mumoles ATP hydrolyzed), decreased from 1.094 +/- 0.042 at pH 7.0 to 0.946 +/- 0.036 at pH 6.0 (P less than 0.05) and to 0.780 +/- 0.024 at pH 5.8 (P less than 0.01). Myofibrillar pCa (-log [free Ca2+] )-ATPase activity was unaffected between pH 7.0 and pH 6.5. At pH 6.0, increasing Ca2+ concentration inhibited myofibrillar ATPase activity, and this inhibitory phenomenon was accentuated at pH 5.8. Kinetic analysis of the myofibrillar pCa-ATPase data, utilizing double-reciprocal plots, demonstrated an increase in Km at low pH. It is concluded that acidosis significantly uncouples calcium transport from ATP hydrolysis in the SR of masseter muscle and significantly alters myofibrillar ATPase activity. It is hypothesized that these defects may explain an observed depression in skeletal muscle cell function during ischemia.

The effect of oxygen free radicals on calcium permeability and calcium loading at steady state in cardiac sarcoplasmic reticulum.

It has been proposed that oxygen free radical production is an important mediator of the myocardial dysfunction during the course of acute ischemia. We tested this hypothesis by characterizing the pathway of calcium efflux across sarcoplasmic reticulum (SR) membranes affected by oxygen free radicals. The effect of oxygen free radicals on the steady state calcium load, calcium permeability, and Ca,Mg-ATPase activity of isolated canine cardiac SR vesicles was investigated at pH 7.0. In vitro generation of oxygen free radicals by xanthine oxidase (0.09 units/ml), acting on xanthine in doses up to 50 microM as a substrate, increased the permeability of the SR vesicles to calcium, determined by measuring net efflux of calcium after stopping pump-mediated fluxes, and decreased total intravesicular calcium and free intravesicular calcium with no effect on Ca,Mg-ATPase activity. The effect of oxygen free radicals on calcium permeability was calcium gradient-dependent. Xanthine alone or xanthine plus denatured xanthine oxidase had no effect on this system. Superoxide dismutase (SOD, 56 units/ml), but not denatured SOD, significantly inhibited the effect of xanthine-xanthine oxidase reaction. The calcium permeability of the SR membrane decreased with decreasing calcium load. In addition, inasmuch as extravesicular calcium exerts only a slight effect on calcium permeability, the decrease in the permeability with calcium load is specifically related to the calcium load. Oxygen free radical-induced increase in calcium permeability was unaffected by Mg concentration between 2.1 and 21 mM. In summary, our data reveal that .O2- can produce a diminished level of accumulated calcium, which is reflected by the decreased calcium load and an increase in passive calcium permeability, and that the decreased calcium accumulation in the presence of the xanthine-xanthine oxidase system may not be mainly due to an inhibited calcium pump but due to an increased calcium permeability. Our results also suggest that increased SR membrane passive calcium permeability induced by oxygen free radicals is not carrier mediated. It is postulated that, with the oxygen free radical-mediated progressive increase in calcium permeability, free cytosolic calcium concentrations would increase in ischemic myocardium.

Free radical damage to sarcoplasmic reticulum of masseter muscle by arachidonic acid and prostaglandin G2.

In vitro generation of free radicals by xanthine oxidase acting on hypoxanthine as a substrate induced a decreased calcium uptake velocity and reduced calcium-dependent ATPase activity of isolated sarcoplasmic reticulum (SR) vesicles from canine masseter muscle at pH 7.0. At pH 5.5 calcium uptake velocity was also reduced but ATPase activity was unaffected. Application of arachidonic acid or prostaglandin G2 induced the depression of both calcium uptake velocity and ATPase activity. The effect of arachidonic acid and prostaglandin G2 on ATPase activity depended on the pH. At pH 7.0, ATPase activity was decreased, but at pH 5.5 it was unchanged. These effects were reversed by superoxide dismutase (SOD) at pH 7.0, and by SOD plus mannitol at pH 5.5. Prostaglandin H2, prostaglandin E2 and 11,14,17-eicosatrienoic acid had no effect on calcium uptake velocity and ATPase activity at both pH 7.0 and pH 5.5. These results suggest that damage to the masseter muscle is caused by a free radical superoxide anion generated as a result of increased prostaglandins synthesis, and by the production of more lethal hydroxyl radical switched from the production of superoxide anion at low pH.