Ca2+ release from heavy sarcoplasmic reticulum (SR) vesicles was induced by 2 mM caffeine, and the amount (A) and the rate constant (k) of Ca2+ release were investigated as a function of the extent of Ca2+ loading. Under both passive and active loading conditions, the A value increased monotonically in parallel to Ca2+ loading. On the other hand, k sharply increased at partial Ca2+ loading, and upon further loading, it decreased to a lower level. Since most of the intravesicular calcium appears to be bound to calsequestrin both under passive and under active loading conditions, these results suggest that the kinetic properties of induced Ca2+ release show significant variation depending upon how much calcium has been bound to calsequestrin at the time of the induction of Ca2+ release. An SR membrane segment consisting of the junctional face membrane (jfm) and attached calsequestrin (jfm-calsequestrin complex) was prepared. The covalently reacting thiol-specific conformational probe N-[7-(dimethylamino)-4-methyl-3-coumarinyl]maleimide (DACM) was incorporated into several proteins of the jfm, but not into calsequestrin. The fluorescence intensity of DACM increased with Ca2+. Upon dissociation of calsequestrin from the jfm by salt treatment, the DACM fluorescence change was abolished, while upon reassociation of calsequestrin by dilution of the salt it was partially restored. These results suggest that the events occurring in the jfm proteins are mediated via the attached calsequestrin rather than by a direct effect of Ca2+ on the jfm proteins. We propose that the [Ca2+]-dependent conformational changes of calsequestrin affect the jfm proteins and in turn regulate the Ca2+ channel functions.

The effect of H2O2 was examined to elucidate the basis of muscle injury after exercise. Exposure of single fibers to 1.5-6 mM H2O2 led to twitch potentiation followed by a marked decrease. Then, fibers contracted spontaneously. BAY K 8644 augmented twitch potentiation and slowed the decay of twitches. In 5 mM dithiothreitol (DTT), twitch potentiation and spontaneous contraction were not observed on H2O2 addition. Cytoplasmic application of 1.5-3 mM H2O2 to heavy sarcoplasmic reticulum (SR) vesicles incorporated into planar lipid bilayers increased the open probability of Ca2+ release channels, an effect reversed by DTT. We investigated oxidation of sulfhydryl groups on proteins in SR membrane by H2O2 with N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide. Pretreatment of light and heavy SR membranes with 1.5 mM H2O2 exponentially increased fluorescence intensity. The time constant of the intensity increase was increased markedly only in heavy SR in solution containing 50 microM cytoplasmic Ca2+, so Ca2+ release was associated with protein oxidation by H2O2. Thus extracellular H2O2 probably acts by oxidizing sulfhydryls of proteins at two distinct sites: the dihydropyridine receptors, oxidation of which elicits potentiation and subsequent inhibition of twitches, and Ca2+ release channels, whose oxidation elicits spontaneous contraction, resulting in muscle dysfunction.

Effects of niflumic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) on frog skeletal muscle ryanodine receptors have been studied by incorporating sarcoplasmic reticulum vesicles into planar lipid bilayers. Niflumic acid increased the mean open probability (Po) at 10 microM and decreased Po at 100 microM with no change in open time constants, unitary conductance, and reversal potential. The Po was augmented by DIDS at 5-200 microM without affecting either unitary conductance or reversal potential. DIDS induced a new third open time constant, probably contributing to a long-lived open state. Channels modified by niflumic acid or DIDS still responded to Ca2+ release channel modulators. These results provide evidence that niflumic acid and DIDS modify the gating mechanism of ryanodine receptors without affecting binding sites to the modulators and the physical pathway of the conducting pore. p-Chloromercuriphenyl sulfonic acid (pCMPS) transiently increased the Po. The channel modified by DIDS responded to pCMPS, whereas that by ryanodine did not. The long open state of the channel induced by DIDS is produced by a quite different mechanism(s) from that by ryanodine. Contrary to cardiac ryanodine receptors, Po of skeletal muscle channels was independent of voltage after DIDS modification.

No abstract

Electrical properties of colonic smooth muscle were investigated in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat, a model animal for spontaneous non-insulin-dependent diabetes mellitus (NIDDM), and the results were compared with those obtained from the Long-Evans Tokushima Otsuka (LETO) rat, a control of OLETF rat. At experiments (aged 60-80 weeks), blood glucose level was about 171 mg/dl in LETO rats and 370 mg/dl in OLETF rats. Feces in the colon were restricted to the proximal region in LETO rats and distributed widely in the whole colon in OLETF rats. In both LETO and OLETF rats , the circular smooth muscle strips of the isolated distal colon revealed two types of spontaneous electrical response, slow wave and transient hyperpolarization. The resting membrane potential was smaller in OLETF rats than in LETO rats by about 3 mV, but it was not positively related with the blood glucose level. The amplitude of hyperpolarization produced by noradrenaline (NA) was smaller in OLETF rats than in LETO rats. Transmur al nerve stimulation evoked a non-adrenergic, non-cholinergic (NANC) inhibitory junction potential (i.j.p.) in both LETO and OLETF rats ; the amplitude of the i.j.p. was smaller in OLETF rats than in LETO rats, while the latency of the i.j.p. was longer in OLETF rats than in LETO rats. Thus, in the distal colon, NIDDM may cause a depolarization of the membrane, an attenuation of NANC inhibitory transmission and a reduction in reactivity of adrenoceptors to NA. These results suggest that the constipation appearing with diabetes mellitus involves dysfunction of both the enteric autonomic nerves and the smooth muscles in the colon.