Separation of two Cl^– Currents in Cultured Human and Murine Mesangial Cells: Biophysical and Pharmacological Characteristics of I_Cl.vol and I_Cl.Ca

Abstract

Mesangial cells, a combined smooth muscle- and fibroblast-like phenotype, are important regulators of renal function. These cells exist in a region of variable osmolarity and may require Cl^– channels for volume regulation. Additionally, Ca²⁺-activated Cl^– channels in these cells may participate in Ca²⁺-dependent contractile responses to vasoactive agonists. Relatively little, however, is known about mesangial cell Cl^– currents (I_Cl); including the biophysical description and pharmacological characterization. We used whole-cell patch clamp to study I_Cl in cultured human and SV40-transformed murine mesangial cells. I_Cl was measured in cells dialyzed and bathed with symmetrical N-methyl-D-glucamine chloride solutions to minimize cation currents. Dialysis with buffers to control intracellular Ca²⁺ ([Ca²⁺]_i), extracellular solutions of varied osmolarity, and manipulation of the transmembrane Cl^– gradient were used to separate two currents: I_Cl.vol (volume-sensitive), and I_Cl.Ca (Ca²⁺-activated). In symmetrical Cl^– with low [Ca²⁺]_i, I_Cl.vol was outwardly rectifying and modulated by osmolarity. I_Cl.vol demonstrated slight time- and voltage-dependent inactivation. In symmetrical Cl^– with elevated [Ca²⁺]_i and hypertonic bath, I_Cl.Ca was linear, but in asymmetrical Cl^– (low [Cl^–]_i) was outwardly rectifying and demonstrated time- and voltage-dependent activation. Permeability sequences for both I_Cl.vol and I_Cl.Ca were I^– > Br^– > Cl^– > F^–; however, there were differences in the relative magnitudes. Tamoxifen inhibited I_Cl.vol more potently than I_Cl.Ca, whereas niflumic acid inhibited I_Cl.Ca more potently than I_Cl.vol. We have separated and characterized two types of I_Cl in cultured human and murine mesangial cells. I_Cl.Ca and I_Cl.vol have different biophysical and pharmacological characteristics. These observations on I_Cl.Ca and I_Cl.vol may provide insight into mesangial cell reactivity and volume regulation.

Myogenic tone in mouse mesenteric arteries: evidence for P2Y receptor-mediated, Na+, K+, 2Cl− cotransport-dependent signaling

“…Second, the apparent affinity of sustained contractile responses was higher for UDP compared to UTP (Fig. 3B), which is consistent with the rank-order potency for P2Y 6 rather than for other UTP-and UDP-sensitive receptors, such as P2Y 2 and P2Y 4 [40,43]. Third, UTP-induced constrictions were sharply suppressed in the presence of 10 μM MRS2567 (Fig.…”

Ion Channels in Mesangial Cells: Function, Malfunction, or Fiction

“…A study using the whole cell patch-clamp configuration in both human MC and a cell line from SV40transformed mice provided evidence for a I Cl.vol that is distinct from I Cl.Ca (12). These investigators found that buffers that prevented an increase in intracellular Ca 2+ uncovered an outward-rectifying Clcurrent that was dependent on osmolarity (12). This volume-sensitive Clcurrent may have been the same Ca 2+ -independent Clcurrent that was identified (under both permissive and nonpermissive growth conditions) in MC harvested from H-2K b -tsA58 transgenic mice in an earlier study (7).…”

“…This volume-sensitive Clcurrent may have been the same Ca 2+ -independent Clcurrent that was identified (under both permissive and nonpermissive growth conditions) in MC harvested from H-2K b -tsA58 transgenic mice in an earlier study (7). Pharmacologically, the I Cl.vol could be distinguished from I Cl.Ca by its sensitivity to block by tamoxifen, an estrogen receptor agonist, with an IC 50 of 1 M for I Cl.vol and an IC 50 of 30 M for I Cl.Ca (12). The I Cl.Ca could be blocked more specifically with niflumic acid with an IC 50 of 0.2 M, compared with an IC 50 of 20 M against I Cl.vol .…”

“…A study using the whole cell patch-clamp configuration in both human MC and a cell line from SV40transformed mice provided evidence for a I Cl.vol that is distinct from I Cl.Ca (12). These investigators found that buffers that prevented an increase in intracellular Ca 2+ uncovered an outward-rectifying Clcurrent that was dependent on osmolarity (12).…”

Identification, discrimination and heterogeneity of fibroblasts

“…In the kidney glomerulus (Fig. 3A ), mesangial cells display intermediate features between those of pericytes and fibroblasts, VSMC and myofibroblasts 86 . Mesangial cells have indeed been proposed to be a specialized type of pericytes 87 , 88 , a notion supported by their lineage-dependence on PDGFB-PDGFRB signaling 89 – 91 , which is shared with other mural cell lineages.…”