(Ca 2+ + Mg 2+)-ATPase activity associated with the maintenance of a Ca 2+ gradient by sarcoplasmic reticulum at submicromolar external [Ca 2+]. The effect of hypothyroidism

Abstract

The formation and maintenance of Ca 2+-filling levels by sarcoplasmic reticulum vesicles from euthyroid (control) and hypothyroid skeletal muscle were investigated using the Ca 2+-indicator quin-2, at [Ca 2+] in the medium ([Ca o 2+]) of 0.05 – 0.3 μM. Rapid ATP-dependent Ca 2+ uptake resulted in a steady-state Ca 2+-filling level, Ca i 2+, within one minute. This Ca 2+ gradient was maintained for at least three minutes, during which < 20% of the ATP was consumed. Ca i 2+ was maximal (120 nmol/mg) for [Ca o 2+] > 0.3 μM and decreased to 40 nmol/mg at [Ca o 2+] of 0.05 μM. Preparations from both experimental groups showed qualitatively and quantitatively the same relationship between Ca i 2+ and [Ca o 2+] at steady state, despite a significantly lower Ca 2+-pump content of hypothyroid sarcoplasmic reticulum, which resulted in a 25% lower maximal (Ca 2+ +Mg 2+)-ATPase activity. Maintenance of the steady state, at all levels of Ca i 2+, was associated with net ATP consumption by the Ca 2+ pump and cycling of Ca 2+, which processes were 30% slower in the hypothyroid group as compared to the control group. Determination of the passive efflux of Ca 2+, as well as the fraction of leaky or unsealed sarcoplasmic reticulum fragments, excluded either of these possibilities as an explanation for the relatively high (Ca 2+ + Mg 2+)-ATPase rates at steady state. On the basis of these and previously reported results, it is concluded that the maintenance of a Ca 2+ gradient by sarcoplasmic reticulum under physiological conditions with respect to external [Ca 2+] and the concentrations of ATP, ADP and P i, is associated with the cycling of Ca 2+ coupled to net ATP hydrolysis. Using the obtained data it is calculated that the sarcoplasmic reticulum may account for 20% of the resting metabolic rate in skeletal muscle. Consequently, together with the previously reported lower sarcoplasmic reticulum content of skeletal muscle in hypothyroidism, we calculate that about one third of the decrease in basal metabolic rate in this thyroid state can be related to the alterations of the sarcoplasmic reticulum.