Inhibition by P1075 and pinacidil of a calcium‐independent chloride conductance in conditionally‐immortal renal glomerular mesangial cells

Abstract

1 Depolarization of mesangial cells has been shown to occur following an outward movement of chloride ions from the cell. We have shown previously that mesangial cells from the H-2K'-tsA58 transgenic mouse possess a significant whole-cell chloride conductance and consequently are a suitable preparation for the study of potential chloride channel inhibitors. 2 The effects on the whole-cell chloride conductance of the chloride channel inhibitor, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and the potassium channel openers, (KCOs) P1075 and pinacidil were investigated in mesangial cells from the H-2Kb-tsA58 transgenic mouse cultured in permissive conditions (at 33°C in the presence of 50 u ml-' murine y-interferon).3 In symmetrical solutions of 140 mM tetramethylammonium chloride (TMACl) the whole-cell chloride conductance was 1.08 + 0.05 nS (n = 63) and this could be reversibly inhibited by 5 x 10-5 M NPPB.4 Both P1075 and pinacidil inhibited the whole-cell chloride conductance. This inhibition was not reversible after drug washout and was demonstrated only when drugs were applied to the extracellular surface of the cells. Very low concentrations of the drugs were found to reduce the chloride conductance after 16 h incubation but under no circumstances studied was the conductance totally inhibited, leaving a mean residual current of 0.33 + 0.03 nS (n = 12). 5 The effects of different peptide calcium concentrations on the magnitude of the residual current in the presence of the drugs were investigated. The residual current was reduced with 10-8 M calcium in the pipette and increased with 10-3 M pipette calcium. Therefore, these data suggest that P1075 and pinacidil selectively inhibit a calcium-independent chloride conductance in mesangial cells from the H-2K"-tsA58 transgenic mouse.

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

“…Alternatively, the volume sensitivity of the Ca 2+ -activated conductance described previously may represent a species-specific difference or a mixed contribution of Ca 2+ -activated and -independent Cl -channels to the whole-cell current. The nature of the Ca 2+ -independent Cl -current in mesangial cells has been less clear, but it is inhibited by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and the K + channel openers pinacidil and P1075 [31]. The volume sensitivity of the current in previous studies was not assessed; however, the NPPB-sensitive current is likely to be I Cl.vol , as NPPB inhibits I Cl.vol in renal epithelia [26,27].…”

Immortalized CNS pericytes are quiescent smooth muscle actin-negative and pluripotent

“…The Immortomouse® harbors a stably incorporated conditionally expressed transgene (Jat et al, 1991; Noble et al, 1992; Barber and Henderson, 1996; Dennis and Caplan, 1996b; Kanda et al, 1996; Walther et al, 1996), which encodes a variant of the tumor antigen (T-Ag) from the simian virus 40 A (SV40). A missense mutation of T-Ag (TsA) renders this protein functional at 33°C but inactive at body temperature (TsA58).…”

“…The Immortomouse® has been a source for conditionally immortalized cells from the endothelium (Kanda et al, 1996), renal mesangial cells (Barber and Henderson, 1996), Sertoli cells (Walther et al, 1996) mesenchymal cells (Dennis and Caplan, 1996b), cochlear hair cells (Kachar et al, 1986), skeletal muscle (Morgan et al, 1994; Ehler et al, 1995), epithelial cells (Whitehead et al, 1993; Kershaw et al, 1994; Whitehead and Joseph, 1994; Paradis et al, 1995), glial cells (Groves et al, 1993), and osteoclasts (Chambers et al, 1993). These immortalized cells continue to replicate at 33°C in the presence of IFNγ and do not exhibit differentiated phenotypes (Holley and Lawlor, 1997).…”

Binding and effects of P1075, an opener of ATP-sensitive K+ channels, in the aorta from streptozotocin-treated diabetic rats

“…This suggests that these effects of P1075 are indeed due the opening of K ATP channels; in addition, 3 H-P1075 binding in segments of rat aorta is inhibited with IC 50 ≈400 nM (Bray and Quast 1992). It has been reported that P1075 also inhibits swelling-activated Clchannels (Holevinski et al 1994;Barber and Henderson 1996); however, the latter effect is not sensitive to inhibition by glibenclamide (Holevinski et al 1994). The aorta, as a large conduit vessel, is not ideally suited to reflect the changes in vascular structure and reactivity important in the pathology of diabetes (Tomlinson et al 1992).…”