Calcium fluxes in internally dialyzed giant barnacle muscle fibers

Abstract

Calcium-45 fluxes have been examined in isolated giant barnacle muscle fibers subjected to internal solute control by means of "internal dialysis". The 45Ca efflux was dependent upon the concentrations of both total and ionized internal Ca (Ca2+ buffered with EGTA). With a total Ca concentration of 2.0 mM and a 1:2 Ca/EGTA ratio (nominal [Ca2+]i =0.13 muM), the Ca efflux averaged 1.2 pmoles/cm2 sec. Under identical conditions, the mean Ca influx was only 0.36 pmoles/cm2 sec. The Ca efflux may not be attributed to leak of the CaEGTA complex, since a 2.5-fold increase in the EGTA concentration (nominal [Ca2+]i=0.032 muM) reduced the 45Ca efflux by one-third. Furthermore, when EDTA was used to buffer the internal Ca concentration (in the absence of internal Mg), the steady efflux of 14C-EDTA was only about 10% of the 45Ca efflux (in parallel experiments). The timecourse of the 45Ca fluxes also appeared anomalous in the 45Ca influx reached a steady level much more rapidly than 45Ca efflux in fibers of comparable diameters. If the muscle fibers are treated as right circular cylinders, these data imply that the apparent diffusion coefficient for inwardly-moving Ca is much larger than for outwardly-moving Ca. In contrast to Ca efflux, the outward diffusion of 22Na, 14C-EDTA and 3H2O appears to be limited primarily by the permeability of the dialysis tube wall. Some, but not all, of the anomalous behavior of the Ca fluxes can be reconciled if the deep, branched infoldings of the barnacle muscle surface membrane are taken into account.

Properties of sodium pumps in internally perfused barnacle muscle fibers.

“…The chamber was identical to the one used in our laboratory for dialysis experiments (cf. Russell and Blaustein, 1975;Blaustein, 1977). The cut (basal) end of the fiber was slipped over a glass cannula (1.0-1.5 mm o.d.)…”

“…The extracellular fluid superfused the sealed-offcentral portion of the fiber at a rate of 1.4 ml/min; 0.2 ml/min was diverted through the two guard outflows, adjacent to the vaseline seals, and discarded (See Fig. 1 in Russell and Blaustein, 1975). The remaining extracellular fluid (1.2 ml/min) passed through the main outflow: -80% (i.e., ~ 1 ml/min) was channeled through an anthracene-filled flow cell set in the well of a liquid scintillation counter (fl-Mate II, Beckman Instruments, Inc., Fullerton, Calif.), as indicated in Fig.…”

“…In microinjectcd barnacle muscle fibers, the average Na effiux (with [Na]i -19 mmol/kg wet wt) was 39 pmol/cm2.s; this flux was reduced by ~ 50% by removal of external K, and by ~ 80% when the fibers were treated with 10-30 pM ouabain (Brinley, 1968). In dialyzed barnacle muscle fibers, Na effluxes of 35 pmol/cm 2. s ([Na]i ffi 15 mM; Brinley, 1969), 30 pmol/cm 2. s ([Na]i ffi 32 mM; DiPolo and Caputo, 1977), and 23 pmol/cm 2. s ([Na]i --16 mM; Russell and Blaustein, 1975) have been reported. Removal of external K reduced the Na efflux by ~ 50%, and treatment with ouabain reduced the efflux by ~ 70% in dialyzed fibers (Brinley, 1969).…”

Properties of chloride transport in barnacle muscle fibers.

“…The procedures and the chamber have been described earlier (Russell and Blaustein, 1975). The central slot, in which external fluid bathed the dialyzed portion of the fiber, was 12 mm wide.…”

The interaction of intracellular Mg2+ and pH on Cl- fluxes associated with intracellular pH regulation in barnacle muscle fibers.

“…Individual fibers (900-1,500 um in diameter, 2-4 cm in length) were then cannulated at both ends using glass cannulae (~ 1,000 #m o.d., 800 #m i.d. ) mounted horizontally in a specially designed internal dialysis chamber (Russell and Blaustein, 1975).…”