Hypo-osmotic stimulation of active Na+ transport in frog muscle: apparent upregulation of Na+ pumps

The purpose of this work was to determine if hypotonicity, in addition to the stimulation of active Na+ transport (Venosa, R.A., 1978, Biochim. Biophys. Acta 510:378-383), promoted changes in (i) active K+ influx, (ii) passive Na+ and K+ fluxes, and (iii) the number of 3H-ouabain binding sites. The results indicate that a reduction of external osmotic pressure (pi) to one-half of its normal value (pi = 0.5) produced the following effects: (i) an increase in active K+ influx on the order of 160%, (ii) a 20% reduction in Na+ influx and K+ permeability (PK), and (iii) a 40% increase in the apparent density of ouabain binding sites. These data suggest that the hypotonic stimulation of the Na+ pump is not caused by an increased leak of either Na+ (inward) or K+ (outward). It is unlikely that the stimulation of active Na+ extrusion and the rise in the apparent number of pump sites produced by hypotonicity were due to a reduction of the intracellular ionic strength. It appears that, at least in part, the stimulation of active Na+ transport takes place whenever muscles are transferred from one medium to another of lower tonicity even if neither one was hypotonic (for instance pi = 2 to pi = 1 transfer). Comparison of the present results with those previously reported indicate that in addition to the number of pump sites, the cycling rate of the pump is increased by hypotonicity. Active Na+ and K+ fluxes were not significantly altered by hypertonicity (pi = 2).Facultad de Ciencias Médica

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

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.Facultad de Ciencias MédicasCentro de Investigaciones Cardiovasculare

Characteristics of Na⁺‐Ca²⁺ exchange in frog skeletal muscle

1. Fluxes studies were carried out to investigate the Na⁺-dependent outward movement of Ca²⁺ in intact frog sartorius muscle from Leptodactylus ocellatus, a preparation for which published data on the subject are sparse. 2. Under normal resting conditions the Na⁺-Ca²⁺ exchange was not readily detectable. 3. When muscles were exposed to 4 mM caffeine, the rate of fractional loss of Ca²⁺ (kCa,o) increased by about 50%. Most of this increase exhibits characteristics typical of the Na⁺-Ca²⁺ antiport working in the forward mode found in other cells. 4. The increase in kCa,o promoted by caffeine was decreased by: (a) 72% in the absence of external Na⁺ (Na⁺o); (b) 73% in Na⁺-loaded muscles ([Na⁺]i = 98 mM); (c) 70% when fibres were depolarized to -27 mV ([K⁺]o = 50 mM); and (d) 80% in the presence of 5 mM amiloride. 5. Ni²⁺ (5 mM), an inhibitor of the Na⁺-Ca²⁺ exchanger current, unexpectedly increased the caffeine-promoted rise in kCa,o. This effect of Ni²⁺ was associated with a concomitant caffeine-stimulated Ni²⁺ influx. In the absence of caffeine Ni²⁺ did not affect kCa,o. 6. It was concluded that: (a) under resting conditions the sarcolemmal Ca²⁺ pump suffices to handle the cytosolic calcium concentration ([Ca²⁺]i); (b) Na⁺-Ca²⁺ activity becomes apparent when [Ca²⁺]i is substantially increased by caffeine-induced Ca²⁺ release from the sarcoplasmic reticulum; and (c) the blocking effect of Ni²⁺ on the current generated by a Na⁺-Ca²⁺ exchange with a coupling ratio > 2 might actually represent a shift of the antiport mode toward an electroneutral 1Ni²⁺-1Ca²⁺ exchange.Facultad de Ciencias Médica

Sodium Pump in T-Tubules of Frog Muscle Fibers

It is well known that in the cytosol of most cells the high concentration of K+ ([K +] i ) and the relatively low concentration of Na+ ([Na+] i ) are kept constant in spite of their electrochemical gradients, which promotes the loss of K+ and the gain of Na+. The steadiness of [K +], and [Na+] i , is maintained by a metabolic energy-dependent active transport process first proposed by Dean(1) in skeletal muscle and known as the Na+ pump (Dean coined the name) or more properly as the Na + /K + pump.Centro de Investigaciones Cardiovasculare

Effect of caffeine on K+ efflux in frog skeletal muscle

The exposure of frog skeletal muscle to caffeine (3–4 mM) generates an increase of the K+ (42K+) efflux rate coefficient (k; K,o) which exhibits the following characteristics. First it is promoted by the rise in cytosolic Ca2+ ([Ca2+]i), because the effect is mimicked by ionomycin (1.25 µM), a Ca2+ ionophore. Second, the inhibition of caffeine-induced Ca2+ release from the sarcoplasmic reticulum (SR) by 40 µM tetracaine significantly reduced the increase in k; K,o (Δk; K,o). Third, charybdotoxin (23 nM), a blocker of the large-conductance Ca2+-dependent K+ channels (BKCa channels) reduced Δk; K,o by 22%. Fourth, apamin (10 nM), a blocker of the small-conductance Ca2+-dependent K+ channels (SKCa channels), did not affect Δk; K,o. Fifth, tolbutamide (800 µM), an inhibitor of KATP channels, reduced Δk; K,o by about 23%. Sixth, Ba2+, a blocker of most K+ channels, did not preclude the caffeine-induced Δk; K,o. Seventh, omitting Na+ from the external medium reduced Δk; K,o by about 40%. Eight, amiloride (5 mM) decreased Δk; K,o by 65%. It is concluded that the caffeine-induced rise of [Ca2+]i increases K+ efflux, through the activation of: (1) two channels (BKCa and KATP) and (2) an external Na+-dependent amiloride-sensitive process.Centro de Investigaciones Cardiovasculare