Steven C. Sansom

Electrophysiological properties of cellular and paracellular conductive pathways of the rabbit cortical collecting duct

SummaryMicroelectrode techniques were applied to the rabbit isolated perfused cortical collecting duct to provide an initial quantitation and characterization of the cell membrane and tight junction conductances. Initial studies demonstrated that the fractional resistance (ratio of the resistance of the apical cell membrane to the sum of the resistances of the apical and basolateral membranes) was usually independent of the point along the tubule of microelectrode impalement—implicating little cell-to-cell coupling—supporting the application of quantitative techniques to the cortical collecting duct. It was demonstrated that in the presence of amiloride, either reduction in the luminal pH or the addition of barium to the perfusate selectively reduced the apical membrane potassium conductance. From the changes inGte and fractional resistance upon reducing the luminal pH or addition of barium to the perfusate, the transepithelial, apical membrane, basolateral membrane and tight junction conductances were estimated to be 9.3, 6.7, 8.1 and 6.0 mS cm−2, respectively. Ninety to ninety-five percent of the apical membrane conductance reflected the barium-sensitive potassium conductance in the presence of amiloride with an estimated potassium permeability of 1.1×10−4 cm sec−1. Reduction in the perfusate pH to 4.0 caused a 70% decrease in the apical membrane potassium conductance, implying a blocking site with an acidic group having a pKa near 4.4. It is concluded that both the transcellular and paracellular pathways of the cortical collecting tubule have high ionic conductances, and that the apical membrane conductance primarily reffects a high potassium conductance. Furthermore, both reduction in the perfusate pH and addition of barium to the perfusate selectively block the apical potassium channels, although the site of inhibition likely differs since the two ions display markedly different voltage-dependent blocks of the channel.

Physiological role of large, Ca2+-activated K+ channels in human glomerular mesangial cells.

1 Contraction assays and patch clamp methods were used to determine the role of K+ channels in the regulation of contractile tone of human mesangial cells (MC) in culture. 2 MC contraction was induced by vasoconstrictor agents, such as angiotensin II (AngII; 100nmol/L) and glybenclamide (Glyb), but not by iberiotoxin (IbTX), a blocker of large Ca2+‐activated K+ channels (BK(Ca)). These results suggest that Glyb‐sensitive K+ channels, but not BK(Ca) channels, were active at rest. 3 In the presence of 100nmol/L IbTX, contraction by Angll was slightly, but not significantly, enhanced, indicating that BK(Ca) has a minimal role as a negative feedback regulator of contraction. Nitroprusside (NP; 100μmol/L), a nitric oxide (NO) donor, atrial natriuretic peptide (ANP; 1.0 μmol/L) and db‐cGMP (10μmol/L) attenuated AngII‐induced contraction in the absence, but not in the presence, of IbTX, suggesting that BK(Ca) channels were activated by cGMP. 4 In patch clamp experiments, three distinct K+‐selective channels of 9, 65 and 150 pS (outward currents) were found in excised, inside‐out patches. The 150 pS channel was completely inhibited by 100nmol/L IbTX and displayed ***voltage‐ and calcium‐dependent gating qualitatively similar to BK(Ca) in other cell types. 5 In cell attached (CA) patches, the response of BK(Ca) to bath AngII (100nmol/L) was relatively minor in control solutions, but was considerably greater in the presence of db‐cGMP. 6 In excised patches, Mg‐ATP (1 mmol/L) plus db‐cGMP (1 μmol/L) activated BK(Ca) in the absence, but not the presence, of the non‐specific kinase inhibitor, staurosporine. 7 Separate experiments showed that BK(Ca) were also activated by arachidonic acid and high ambient glucose concentrations. 8 These results indicate that: (i) resting MC tone is sensitive to glybenclamide and apamin; and (ii) the role of BK(Ca) as a negative feedback regulator of contraction is minimal under normal conditions but is markedly enhanced by cGMP‐stimulating relaxants and arachidonic acid.

Activation by methylene blue of large Ca2+-activated K+ channels

Using the patch-clamp method, we found that methylene blue (MB), a free radical inhibitor of guanylyl cyclase, activated large, Ca(2+)-activated K+ channels (BKCa) in either cell attached or excised, inside-out patches of human mesangial cells in culture. Since BKCa are important feedback regulators of contraction of smooth muscle and mesangial cells, these results indicate that MB may be an important opener of BKCa channels and a regulator of vascular volume and resistance.