Microelectrode study of K+ accumulation by tight epithelia: II. Effect of inhibiting transepithelial Na+ transport on reaccumulation following depletion

The effects of restoring serosal potassium to potassium-depleted toad urinary bladders have been re-examined using double-barrelled microelectrodes. The data confirm the existence of a time-lag phenomenon, a dissociation between potassium reaccumulation and restoration of short-circuit current. Returning serosal potassium stimulates an increase in intracellular potassium activity 21-26 min before any increase can be detected in short-circuit current. The reaccumulation of potassium has been further studied using split frog skin, a far more suitable preparation for electrophysiologic study than toad bladder. Under baseline short-circuited conditions, potassium is accumulated against an electrochemical gradient of 22 +/- 4 mV. Reaccumulation of potassium by potassium-depleted tissues can be blocked by inhibiting the Na,K-exchange pump with high concentrations of ouabain. On the other hand, blocking apical sodium entry by the addition of 10(-4) M amiloride to the outer bathing medium does not interfere with reaccumulation of potassium. The data support the concept that the time-lag phenomenon of toad bladder reflects stimulation of potassium reaccumulation by the sodium pump in exchange for the extrusion of excess cell sodium collected during the period of potassium depletion. This reaccumulation of potassium can proceed before the entry of significant added amounts of sodium across the apical plasma membrane.     

Dissociation of cellular K+ accumulation from net Na+ transport by toad urinary bladder

Summary A number of published data suggest a variable stoichiometry between the rates of cellular potassium uptake and net sodium transport (J _Na) across the urinary bladder of the toad. This problem was examined by simultaneously studying the intracellular chemical activity of potassium (a _K) with open-tip K⁺-selective microelectrodes and micropipets, and monitoringJ _Na by measuring the short-circuit current (SCC). When bathed in the short-circuited state with solutions containing ana _K of 2.7mm, the mean ±sem values for intracellulara _K were 43±0.6mm.Ouabain, at a concentration of 10^–2 m, reduced intracellulara _K by 56–67% and SCC by 96–100%. At 5×10^–4 m, ouabain reversibly reduced intracellulara _K by 40–55%, and SCC by 63–68%; the inhibition of SCC was only partly reversible during the period of observation.Removal of external potassium reduced intracellulara _K by 69–80% and SCC by 51–76%. Restoration of external potassium entirely returned intracellulara _K to its control value, but only partially reversed the inhibition of SCC during the period of study. Furthermore, recovery ofa _K began 19–43 min before that of SCC; recovery ofa _K was 90–97% complete before any increase in SCC could be measured. Although other interpretations are possible, the simplest interpretation of the data is that the processes responsible for potassium accumulation and transepithelial sodium transport are not identical. We propose the existence of a separate transfer mechanism at the basolateral cell membrane, responsible for accumulating intracellular potassium, and not directly coupled to active sodium transport.     

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

Summary 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 simultaneous 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 underestimate 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⁺.     

Stimulation of Na+ transport across the toad urinary bladder by p-chloromercuribenzene sulfonate.

The sulfhydryl reagent p-chloromercuribenzene sulfonate increased the ISC across substrate-replete toad urinary bladder when applied to the mucosal (apical) surface. This increase was accounted for by an increased mucosal to serosal net flux of Na+. In the absence of substrate, the rise in ISC was accompanied by an irreversible increase in tissue conductance which was not apparent in the replete preparation. These findings suggest that p-chloromercuribenzene sulfonate may be useful in marking mucosal functions associated with the Na+ transport apparatus.     

K+ secretion across frog skin. Induction by removal of basolateral Cl-

We examined the development of K+ secretion after removing Cl- from the basolateral surface of isolated skins of Rana temporaria using noise analysis. K+ secretion was defined by the appearance of a Lorentzian component in the power density spectrum (PDS) when Ba2+ was present in the apical bath (0.5 mM). No Lorentzians were observed when tissues were bathed in control, NaCl Ringer solution. Replacement of basolateral Cl- by gluconate, nitrate, or SO4- (0-Clb) yielded Lorentzians with corner frequencies near 25 Hz, and plateau values (So) that were used to estimate the magnitude of K+ secretion through channels in the apical cell membranes of the principal cells. The response was reversible and reproducible. In contrast, removing apical Cl- did not alter the PDS. Reduction of basolateral Cl- to 11.5 mM induced Lorentzians, but with lower values of So. Inhibition of Na+ transport with amiloride or by omitting apical Na+ depressed K+ secretion but did not prevent its appearance in response to 0-Clb. Using microelectrodes, we observed depolarization of the intracellular voltage concomitant with increased resistance of the basolateral membrane after 0-Clb. Basolateral application of Ba2+ to depolarize cells also induced K+ secretion. Because apical conductance and channel density are unchanged after 0-Clb, we conclude that K+ secretion is "induced" simply by an increase of the electrical driving force for K+ exit across this membrane. Repolarization of the apical membrane after 0-Clb eliminated K+ secretion, while further depolarization increased the magnitude of the secretory current. The cell depolarization after 0-Clb is most likely caused directly by a decrease of the basolateral membrane K+ conductance. Ba2(+)-induced Lorentzians also were elicited by basolateral hypertonic solutions but with lower values of So, indicating that cell shrinkage per se could not entirely account for the response to 0-Clb and that the effects of 0-Clb may be partly related to a fall of intracellular Cl-.     

Evaluation of the rate of basal oxygen consumption in the isolated frog skin and toad bladder.

In the study of active transport it is important to distinguish between oxygen consumption sustaining transepithelial transport and that responsible for other tissue functions (basal metabolism). Since amiloride blocks transepithelial active sodium transport and the associated oxygen consumption in the frog skin and toad bladder, we and others have employed this agent to evaluate the rate of basal metabolism. This technique has recently been criticized in a report that amiloride (and ouabain) increased oxygen consumption when no sodium was available for transport. We have been unable to corroborate these observations. With magnesium-Ringer as external bathing solutions, amiloride and ouabain failed to stimulate oxygen consumption. With sodium-Ringer as external bathing solution amiloride reduced oxygen consumption about 30%, to a level indistinguishable from that found on external substitution of magnesium-Ringer for sodium-Ringer. We conclude that the use of amiloride permits evaluation of the rate of basal metabolism with acceptable accuracy; a possible slight depressant effect of ouabain on basal metabolism remains to be investigated.     

Effects of parathyroid hormone on H+ and NH+4 excretion in toad urinary bladder.

The urinary bladder of Bufo marinus excretes H+ and NH+4, and the H+ excretion is increased after the animal is placed in metabolic acidosis. The present study was done to determine if parathyroid hormone could stimulate the bladder to increase the excretion of H+ and/or NH+4. Parathyroid hormone added to the serosal solution in a final concentration of 10 mug/ml was found to increase H+ excretion by 50 per cent above the control hemibladders, while there was no effect on NH+4 excretion. Parathyroid hormone had no effect on H+ excretion when added to the mucosal solution. We also performed experiments utilizing theophylline and dibutyryl cyclic AMP which mimicked those of the parathyroid hormone experiments. A dose-response analysis was performed and the results indicate that 1 mug/ml of parathyroid hormone was the minimal effective dose. These results suggest that parathyroid hormone can stimulate H+ excretion in the toad urinary bladder and this effect seems to be mediated by cyclic AMP. In addition, it was found that parathyroid hormone has no effect on NH+4 excretion.     

Role of endogenous opiates and extracellular K+ accumulation in the inhibition of frog spinal reflexes by electrical skin stimulation

Electrical skin stimulation of the hind limb (10-100 Hz, 30 s-5 min) at the intensity which leads only to the excitation of low threshold afferents depressed (for 1-30 min) the flexor reflex evoked in spinal frogs by nociceptive stimuli. The inhibition, which lasted for longer than 5 min was blocked by naloxone. Short-term poststimulation effects were associated with an increase of extracellular K+ concentration (delta [K]e) and were not blocked by naloxone. Enkephalins or morphine applied to the spinal cord surface increased the threshold for flexor reflexes while naloxone decrease their threshold. The stimulation was followed by short-term hyperpolarization of primary afferents (PAH; 1-5 min) and by depression of dorsal root potentials (DPRs) which had a similar time course to the delta [K]e, and were not blocked by naloxone. This period was frequently followed by longlasting PAH and enhancement of DRPs (5-30 min), which were abolished by naloxone. Superfusion of the isolated spinal cord with opioids produced PAH and enhanced DRPs evoked by nociceptive stimuli, while naloxone or increase of [K] in Ringer solution depolarized primary afferents and depressed DRPs. It is suggested that the antinociceptive effects of electrical stimulation of low threshold cutaneous afferents in spinal frogs involves at least two mechanisms. The short-term effect may result from delta [K]e, especially at high stimulus strength and is equally effective against noxious and non-noxious stimuli. The longlasting effects selectively affecting nociceptive transmission appear to be produced by endogenous opioids.     

Microelectrode studies in toad urinary bladder epithelium. effects of Na concentration changes in the mucosal solution on equivalent electromotive forces

Microelectrode techniques were employed to measure membrane potentials, the electrical resistance of the cell membranes, and the shunt pathway, and to compute the equivalent electromotive forces (EMF) at both cell borders in toad urinary bladder epithelium before and after reductions in mucosal sodium concentration. Basal electrical parameters were not significantly different from those obtained with impalements from the serosal side, indicating that mucosal impalements do not produce significant leaks in the apical membrane. A decrease in mucosal Na concentration caused the cellular resistance to increase and both apical and basolateral EMF to depolarize. When Na was reduced from 112 to 2.4 mM in bladders with spontaneously different baseline values of transepithelial potential difference (Vms), a direct relationship was found between the change in Vms brought about by the Na reduction and the base-line Vms before the change. A direct relationship was also found by plotting the change in EMF at the apical or basolateral border caused by a mucosal Na reduction with the corresponding base-line EMF before the change. These results indicate that resting apical membrane EMF (and, therefore, resting apical membrane potential) is determined by the Na selectivity of the apical membrane, whereas basolateral EMF is at least in part the result of rheogenic Na transport. These results are consistent with data of others that suggested a link between the activity of the basolateral Na pump and apical Na conductance.     

Evaluation of the rate of basal oxygen consumption in the isolated frog skin and toad bladder

In the study of active transport it is important to distinguish between oxygen consumption sustaining transepithelial transport and that responsible for other tissue functions (basal metabolism). Since amiloride blocks transepithelial active sodium transport and the associated oxygen consumption in the frog skin and toad bladder, we and others have employed this agent to evaluate the rate of basal metabolism. This technique has recently been criticized in a report that amiloride (and ouabain) increased oxygen consumption when no sodium was available for transport. We have been unable to corroborate these observations.With magnesium-Ringer as external bathing solutions, amiloride and ouabain failed to stimulate oxygen consumption. With sodium-Ringer as external bathing solution amiloride reduced oxygen consumption about 30%, to a level indistinguishable from that found on external substitution of magnesium-Ringer for sodium-Ringer. We conclude that the use of amiloride permits evaluation of the rate of basal metabolism with acceptable accuracy; a possible slight depressant effect of ouabain on basal metabolism remains to be investigated.     

Effects of tetracyclines on aldosterone- and insulin-mediated Na+ transport in the toad urinary bladder.

The effect of oxytetracycline and demethylchlortetracycline on aldosterone- and insulin-mediated Na+ transport (short-circuit current) were examined in toad urinary bladders mounted in modified Ussing chambers. Oxytetracycline had little or no effect on either basal or aldosterone-mediated Na+ transport. In contrast, demethylchlortetracycline markedly inhibited both basal and aldosterone-mediated Na+ transport. Furthermore, demethylchlortetracycline inhibited the aldosterone response significantly out of proportion to its effects on basal Na+ transport. Neither of the drugs had an effect on insulin-mediated Na+ transport. Consequently, the natriuresis observed in certain patients treated with demethylchlortetracyline may be related to drug-induced renal resistance to the effects of aldosterone.     

Effects of parathyroid hormone on H+ and NH 4 + excretion in toad urinary bladder

Summary The urinary bladder ofBufo marinus excretes H⁺ and NH ₄ ⁺ , and the H⁺ excretion is increased after the animal is placed in metabolic acidosis. The present study was done to determine if parathyroid hormone could stimulate the bladder to increase the excretion of H⁺ and/or NH ₄ ⁺ . Parathyroid hormone added to the serosal solution in a final concentration of 10 g/ml was found to increase H⁺ excretion by 50% above the control hemibladders, while there was no effect on NH ₄ ⁺ excretion. Parathyroid hormone had no effect on H⁺ excretion when added to the mucosal solution. We also performed experiments utilizing theophylline and dibutyryl cyclic AMP which mimicked those of the parathyroid hormone experiments. A dose-response analysis was performed and the results indicate that 1 g/ml of parathyroid hormone was the minimal effective dose. These results suggest that parathyroid hormone can stimulate H⁺ excretion in the toad urinary bladder and this effect seems to be mediated by cyclic AMP. In addition, it was found that parathyroid hormone has no effect on NH ₄ ⁺ excretion.     

The effect of catecholamines on H+ and NH+4 excretion in toad urinary bladder

The catecholamines epinephrine and norepinephrine, when placed on the toad urinary bladder in vitro, at a final concentration of 50 microM, caused a significant increase in H+ and NH+4 excretion by the bladder. Isoprenaline in a final concentration of 50 microM also increased H+ and NH+4 excretion in the bladder. Propranolol at a concentration of 50 microM blocked the stimulation of H+ excretion by isoprenaline but propranolol at 100 microM was required to block the stimulation of NH+4 by isoprenaline. The dose-response analysis indicates that the concentration of epinephrine used (50 microM) is at or near the maximal effective dose. These findings indicate that catecholamines stimulate H+ and NH+4 excretion in the toad urinary bladder and evidence suggests this may be mediated via the beta receptor mechanism.