Abstract
We have examined the effects in vitro of calcitriol [1,25(OH) 2D 3], the hormonal form of vitamin D 3, on the breakdown of membrane phosphoinositides in skeletal muscle from young (3 months) and aged (24 months) rats. Calcitriol (10 −9 M) induced a rapid and transient release of IP 3/inositol phosphates and diacylglycerol (DAG) from muscle slices/membranes prelabeled with [ 3H]myo-inositol and [ 3H]arachidonate, respectively. Inositol phosphate release was maximal at 15 s and then declined. The effects of hormone specificity exhibited as the closely related derivatives of vitamin D 3, 25OHD 3, 1 αOHD 3 and 24,25(OH) 2D 3 did not alter muscle inositol phosphate levels. The stimulation of DAG was biphasic, the early phase (15 s) being abolished by neomycin (0.5 mM), an inhibitor of phosphoinositide hydrolysis, similar to IP 3 formation and consistent with a role of phospholipase C (PLC) in intracellular signal generation. Neomycin had no effect on the second DAG peak (2 min) induced by calcitriol, suggesting that the late phase of DAG formation is independent from the hydrolysis of phosphoinositides. Higher basal inositol phosphate and DAG levels were detected in muscle from aged rats thereby reducing the effects of the hormone on second messenger generation (−80 and −60% for IP 3 and DAG, respectively). Calcitriol stimulation of PLC was mimicked, in both young and old rats, by GTP γS, a non-hydrolyzable analogue of GTP, while GDP βS, a G protein inhibitor, suppressed the effect of the hormone. The early effects of calcitriol and GTP γS were not additive. Bordetella pertussis toxin abolished by 85% the effects of calcitriol on inositol phosphate release in young rats but was without effect in aged animals. These results demonstrate that calcitriol activates phosphoinositide-PLC in rat skeletal muscle by a mechanism which involves a pertussis-sensitive G protein and that the effects of the hormone are altered with ageing.
Published Version
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