Microprobe study of toad urinary bladder in absence of serosal K+

Abstract

The bulk of the intracellular potassium in mucosal epithelial cells from toad urinary bladder has been previously reported to exchange very slowly with the serosal medium, with a half-time of some 9 hr. This observation, based on chemical analyses of mucosal cell scrapings, has been reexamined with stimultaneous diffractive and energy dispersive electron probe X-ray microanalysis. Fifty-three intracellular sites in hydrated sections and 286 sites in dehydrated sections were studied in bladders from eight toads under baseline conditions and after removal of serosal K+ for 83-133 min, with or without 10(-2) M ouabain. The baseline data confirm and extend previous examinations of the intracellular ionic composition, and provide the most direct measure of intracellular water thus far available for this tissue. Removal of serosal K+ reduced the intracellular K+ content by 20%, increased intracellular Na+ content threefold, and slightly reduced the intracellular Cl- and water contents, qualitatively consistent with published chemical analyses. The intracellular Na+ content of mucosal origin, measured by radioactive tracers and chemical analyses of cell scrapings, has been reported to be unchanged under these conditions. Simultaneous addition of ouabain and removal of external K+ produced a dramatic fall in intracellular K+ of more than 80% in a third of the cells and reduced the mean intracellular K+ content by 60%; 20% of the cells appeared to retain K+ more effectively than the bulk of the epithelial cell population. We conclude that: (i) the low rate of net exchange of intracellular K+ with the serosal bulk solution primarily reflects recycling of K+ across the basolateral membranes, (ii) radioactive tracer and chemical measurements of the intracellular Na+ pool of mucosal origin substantially understimate the total intracellular Na+ content under certain experimental conditions, and (iii) the epithelial cells display a functional heterogeneity of response to the effects of adding ouabain and withdrawing external K+.

Electrophysiologic changes associated with potassium depletion of frog skin

“…Taken together with previously published estimates of the changes in total ionic contents induced by potassium depletion (Robinson & Macknight, 1976a), these data led to the suggestion that potassium might be accumulated by a sodium-independent mechanism. Recent electronmicroprobe analysis of toad bladder is in qualitative agreement with previous estimates based on chemical analysis of mucosal cell scrapings, but indicates that 0022-2631/80/0057-0235 $01.40 9 1980 Springer-Verlag New York Inc. more sodium is actually gained by the transporting epithelial cells than had previously been estimated; the accumulation of sodium and loss of potassium are comparable in magnitude (Civan, Hall & Gupta, 1980). Thus, given the uncertainties of the experimental techniques, and the possibility that the stoichiometry of the pump might undergo modest changes even under physiologic conditions, the dissociation between the time courses of intracellular potassium activity and short-circuit current noted by DeLong and Civan (1978) might be consistent with the operation of a single Na-K rheogenic pump at the basolateral membrane.…”

“…Initial estimates of the relative gain in sodium and loss of potassium (Robinson & Macknight, 1976a) had suggested that insufficient intracellular sodium was available following potassium depletion to sustain potassium accumulation by the Na -K exchange pump. The more recent microprobe data of Civan et al (1980) have indicated that the redistributions in sodium and potassium are comparable in magnitude, so that it is no longer necessary to invoke an additional transport mechanism responsible for cellular potassium accumulation.…”

“…Although the recycling hypothesis suggested by Civan et al (1980) accommodates certain otherwise puzzling observations, it would be desirable to test the hypothesis directly. This has not yet been done, although the model has now been found to satisfy two necessary conditions.…”

“…In part, the rate of net potassium loss is limited by the rate of apical sodium entry, leading to depolarization of the basolateral membrane in the short-circuited state. In addition, Civan et al (1980) have suggested that this slow rate of net potassium loss reflects the continued operation of the Na-K exchange pump; even in the absence of potassium from the bulk media, potassium leaking from the epithelial cells is considered to be largely reaccumulated by the pump. This hypothesis now seems consistent with the observation of Civan (1978, 1979) that restoration of potassium to the serosal medium bathing potassium-depleted tissues results in a prompt increase in the intracellular potassium activity, even before any increase in shortcircuit current can be measured.…”

“…After incubation in K +-free media for 1 89 to 2 hr, the transporting cells of toad bladder lose only some 20% of their intracellular potassium (Robinson & Macknight, 1976a;Civan et al, 1980). In part, the rate of net potassium loss is limited by the rate of apical sodium entry, leading to depolarization of the basolateral membrane in the short-circuited state.…”