Hypotonic stimulation of the Na+ active transport in frog skeletal muscle: role of the cytoskeleton

Abstract

Hypotonicity produces a marked activation of the Na+ pump in frog sartorius muscle. The increase in net Na+ efflux under hypotonic conditions occurs despite the reductions in [Na+]i that are due to fibre swelling and Na+ loss. The pump density (ouabain binding) increases not only upon reduction of the medium osmotic pressure (π) from its normal value (π= 1) to one-half (π= 0.5), but also in muscles that are returned to π= 1 after equilibration in π= 2 medium. The equilibration in π= 2 medium does not affect pump density. Ouabain-binding increments cannot be ascribed to a rise in the Na+–K+ exchange rate of a fixed number of pumps: they also occurred in the continued presence of a saturating concentration of ouabain (50 μm). Under those conditions, the π= 1 ★π= 0.5 transfer produced a 43 % increase in pump sites, while the π= 2 ★π= 1 transfer induced a rise of 46 %. Actinomycin D did not alter the stimulation of Na+ extrusion elicited by hypotonicity, suggesting that de novo synthesis of pumps was not involved in the increase of the apparent number of pump sites. Disruption of microtubules by colchicine (100 μm) and intermediate filaments by acrylamide (4 mm) did not alter the hypotonic effect. Likewise, genistein (100 μm), a specific inhibitor of tyrosine kinase, did not affect significantly the hypotonic response. Microfilament-disrupting agents like cytochalasin B (5 μm) and latrunculin B (10 μm) reduced the increase in Na+ efflux induced by π= 1 ★π= 0.5 transfer by about 35 % and 72 %, respectively. Latrunculin B reduced the increases in pump density generated by π= 1 ★π= 0.5 and π= 2 ★π= 1 transfers by about 79 % and 91 %, respectively. The results suggest that the membrane stretch due to hypotonic fibre volume increase would promote a microfilament-mediated insertion of submembranous spare Na+ pumps in the sarcolemma and, consequently, the rise in active Na+ transport.