Intracellular ion activities in Malpighian tubule cells ofRhodnius prolixus: evaluation of Na+-K+-2Cl-cotransport across the basolateral membrane

SUMMARYIntracellular ion activities (aion) and basolateral membrane potential (Vbl) were measured in Malpighian tubule cells of Rhodnius prolixus using double-barrelled ion-selective microelectrodes. In saline containing 103mmoll-1Na+, 6mmoll-1 K+ and 93mmoll-1Cl-, intracellular ion activities in unstimulated upper Malpighian tubules were 21, 86 and 32mmoll-1, respectively. In serotonin-stimulated tubules, aCl was unchanged, whereas aNa increased to 33mmoll-1 and aK declined to 71mmoll-1. Vbl was -59mV and -63mV for unstimulated and stimulated tubules, respectively. Calculated electrochemical potentials(Δμ/F) favour passive movement of Na+ into the cell and passive movement of Cl- out of the cell in both unstimulated and serotonin-stimulated tubules. Passive movement of K+ out of the cell is favoured in unstimulated tubules. In stimulated tubules, Δμ/F for K+ is close to 0 mV.The thermodynamic feasibilities of Na+-K+-2Cl-, Na+-Cl-and K+-Cl- cotransporters were evaluated by calculating the net electrochemical potential (Δμnet/F) for each transporter. Our results show that a Na+-K+-2Cl- or a Na+-Cl- cotransporter but not a K+-Cl- cotransporter would permit the movement of ions into the cell in stimulated tubules. The effects of Ba2+ and ouabain on Vbl and rates of fluid and ion secretion show that net entry of K+ through ion channels or the Na+/K+-ATPase can be ruled out in stimulated tubules. Maintenance of intracellular Cl- activity was dependent upon the presence of both Na+ and K+ in the bathing saline. Bumetanide reduced the fluxes of both Na+ and K+. Taken together, the results support the involvement of a basolateral Na+-K+-2Cl- cotransporter in serotonin-stimulated fluid secretion by Rhodnius prolixus Malpighian tubules.

Effects of ouabain on intracellular ion activities of sensory neurons of the leech central nervous system

1. The intracellular K+, Na+, and Ca2+ of mechanosensory neurons in the central nervous system of the leech Hirudo medicinalis was measured using double-barreled ion-sensitive microelectrodes. 2. After inhibition of the Na(+)-K+ pump with 5 x 10(-4) M ouabain, the intracellular K+ activity (aKi) decreased, while the intracellular Na+ activity (aNai) increased. The input resistance decreased in the presence of ouabain. The intracellular Ca2+ increased more than one order of magnitude after ouabain addition. All changes in intracellular ion activities and membrane resistance were fully reversible. 3. When extracellular Na+ concentration ([Na+]o) was removed [replaced by tris(hydroxymethyl)aminomethane (Tris)], aNai decreased. In the absence of [Na+]o, aKi and aNai remained unchanged after inhibition of the Na(+)-K+ pump by reducing the extracellular K+ concentration ([K+]o) to 0.2 mM. The membrane resistance increased under these conditions. 4. The intracellular Ca2+ decreased or remained constant after removal of [Na+]o. Addition of ouabain in the absence of [Na+]o did not change intracellular Ca2+, which only increased after readdition of [Na+]o. 5. The relative K+ permeability (PK) measured as membrane potential change during a brief increase of the [K+]o from 4 to 10 mM, increased manyfold after addition of ouabain but only little if [Na+]o had been removed before adding ouabain. 6. The results suggest that the intracellular Na+ increase after inhibition of the Na(+)-K+ pump affects the intracellular Ca2+ level by stimulating a Nai(+)-Ca2+ exchange mechanism. The subsequent intracellular Ca2+ activity (aCai) rise may result in an increase of the membrane permeability to K+ ions.

Shunt resistance and ion permeabilities in normal and cystic fibrosis airway epithelia

A method for determination of shunt resistance (Rs) and absolute conductive ion permeabilities of the apical membrane in epithelia from steady-state data is described. The method assumes that the currents are satisfactorily described by the Goldman-Hodgkin-Katz regime. Its application requires measurements of standard transepithelial electrophysiological parameters and of one or more intracellular ion activities. It is applicable under both open- and short-circuit conditions. The method was tested in an electrophysiological analysis of cultured normal and cystic fibrosis (CF) human nasal epithelium. In 15 normal and 10 CF preparations with mean transepithelial resistances of 338 and 427 omega.cm2, Rs was 412 and 623 omega.cm2, respectively. The Rs values determined with the present method were strongly correlated (r = 0.94) with those obtained with another method available in the electrophysiological literature but were as a mean 20% lower. Amiloride increased Rs by 25% in CF and by 8% in normal preparations. In normal preparations, the apical Cl permeability (PCla) was 3.6 x 10(-6) cm/s, and the apical Na permeability (PNaa) was 1.6 x 10(-6) cm/s. In CF preparations, PCla was reduced to a maximum of 2.3 x 10(-7) cm/s, whereas PNaa was increased to 6.2 x 10(-6) cm/s. The apical membrane electromotive force was -1 mV in normal and 43 mV in CF preparations. It is concluded that the method can be used to calculate Rs, apical membrane ion permeabilities, and electromotive forces from steady-state electrophysiological data.

K+, Na+, and Cl- activities in ventricular myocytes isolated from rabbit heart

Intracellular K+, Na+, and Cl- activities (aiK, aiNa and aiCl) were measured in ventricular myocytes enzymatically isolated from adult rabbit heart. The activities in normal Tyrode solution containing 2.5 mM Ca2+ were the following (in mM): aiK = 100.0 +/- 3.5 (n = 9); aiNa = 8.4 +/- 1.5 (n = 6); and aiCl = 17.9 +/- 1.5 (n = 11) (mean +/- SE). Membrane potential was -81.6 +/- 0.7 mV (n = 26). These values were determined after correction for changes of junction and tip potential at the reference electrode, estimated to be 4.9 +/- 0.6 mV (n = 7) for 0.15 M KCl-filled electrodes; and intracellular interference detected by the Cl- ion-selective electrode, 11.2 +/- 0.6 mM (n = 4). Extended-tip shunting was avoided by fabricating Na+ ion-selective microelectrodes from aluminosilicate rather than borosilicate glass. These results show that isolated cardiac cells can maintain normal intracellular ion activities. Diffusion of electrolyte from the reference electrode can rapidly alter the intracellular milieu, however. After 10 min of impalement with 0.15 M KCl-filled microelectrodes (resistance approximately equal to 25 M omega), aiK increased by 8.7 +/- 2.0 mM and aiCl by 10.3 +/- 3.1 mM. In contrast, aiNa did not significantly change during the double impalement.