Basolateral proteinase-activated receptor (PAR-2) induces chloride secretion in M-1 mouse renal cortical collecting duct cells.

1. Using RT-PCR, Northern blot analysis, and immunocytochemistry, we confirmed renal expression of proteinase-activated receptor (PAR-2) and demonstrated its presence in native renal epithelial and in cultured M-1 mouse cortical collecting duct (CCD) cells. 2. We investigated the effects of a PAR-2 activating peptide (AP), corresponding to the tethered ligand that is exposed upon trypsin cleavage, and of trypsin on M-1 cells using patch-clamp, intracellular calcium (fura-2) and transepithelial short-circuit current (ISC) measurements. 3. In single M-1 cells, addition of AP elicited a concentration-dependent transient increase in the whole-cell conductance. Removal of extracellular Na+ had no effect while removal of Cl- prevented the stimulation of outward currents. The intracellular calcium concentration increased significantly upon application of AP while a Ca2+-free pipette solution completely abolished the electrical response to AP. 4. In confluent monolayers of M-1 cells, apical application of AP had no effect on ISC whereas subsequent basolateral application elicited a transient increase in ISC. This increase was not due to a stimulation of electrogenic Na+ absorption since the response was preserved in the presence of amiloride. 5. The ISC response to AP was reduced in the presence of the Cl- channel blocker diphenylamine-2-carboxylic acid on the apical side and abolished in the absence of extracellular Cl-. 6. Trypsin elicited similar responses to those to AP while application of a peptide (RP) with the reverse amino acid sequence of AP had no effect on whole-cell currents or ISC. 7. In conclusion, our data suggest that AP or trypsin stimulates Cl- secretion by Ca2+-activated Cl- channels in M-1 CCD cells by activating basolateral PAR-2.

Stimulation of a nicotinic ACh receptor causes depolarization and activation of L-type Ca2+ channels in rat pinealocytes.

1. Membrane voltage (Vm) recordings were obtained from isolated rat pinealocytes using the patch-clamp technique. In parallel to the electrophysiological experiments, intracellular Ca2+ measurements were performed using fura-2. 2. The resting Vm averaged -43 mV and replacement of extracellular NaCl by KCl completely depolarized the cells. This indicates that the resting Vm is dominated by a K+ conductance. Single-channel recordings revealed the presence of a large conductance Ca(2+)-activated charybdotoxin-sensitive K+ channel. 3. Application of ACh (100 microM) depolarized the pinealocytes on average by 16 mV. The depolarizing effect of ACh was mimicked by nicotine (50 microM) and was prevented by tubocurarine (100 microM). 4. The ACh-induced depolarization was largely abolished in the absence of extracellular Na+, but was not significantly affected by extracellular Ca2+ removal. 5. Application of ACh (100 microM) caused an increase in [Ca2+]i. This increase was completely dependent on the presence of extracellular Ca2+ and was largely reduced after extracellular Na+ removal. Nifedipine (1 microM) reduced the ACh-induced increase in [Ca2+]i by about 50%. 6. Our findings indicate that in rat pinealocytes stimulation of a nicotinic ACh receptor (nAChR) induces depolarization mainly by Na+ influx via the nAChR. The depolarization then activates L-type Ca2+ channels, which are responsible for the nifedipine-sensitive portion of the intracellular Ca2+ increase. Ca2+ influx via the nAChR probably also contributes to the observed rise in [Ca2+]i.

cAMP stimulates CFTR-like Cl- channels and inhibits amiloride-sensitive Na+ channels in mouse CCD cells.

Confluent M-1 mouse cortical collecting duct (CCD) cells express highly selective low-conductance amiloride-sensitive Na+ channels (B. Letz, A. Ackermann, C. M. Canessa, B. C. Rossier, and C. Korbmacher, J. Membr. Biol. 148: 129-143, 1995). Here we investigated the effect of forskolin on membrane voltage and whole cell currents of confluent M-1 cells using the patch-clamp technique. Forskolin (1 microM) reduced the hyperpolarization in response to amiloride (10 microM) from 17 to 4 mV and decreased the amiloride-sensitive Na+ inward currents from 81 to 26 pA. Furthermore, forskolin increased the hyperpolarization caused by changing from an apical low-Cl- solution (9 mM) to a high-Cl- solution (149 mM) from 11 to 30 mV and increased the magnitude of the inward current changes induced by alternating between high-Cl- and low-Cl- solutions from 25 to 138 pA. This demonstrates that forskolin stimulates an apical Cl- conductance. Anion substitution experiments revealed a permeability sequence SCN- > Br- > Cl- > I- >> gluconate. This suggests that the stimulated channels are cystic fibrosis transmembrane conductance regulator (CFTR)-like Cl- channels. 3-Isobutyl-1-methylxanthine and 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate mimicked the effects of forskolin, whereas 1,9-dideoxyforskolin had no effect. We conclude that, in addition to amiloride-sensitive Na+ channels, CFTR-like Cl- channels are present in the apical membrane of confluent M-1 cells. An increase in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) activates these Cl- channels and concurrently reduces the activity of the Na+ channels. This reciprocal regulation by cAMP suggests that the channels are functionally coupled.

Amiloride-sensitive sodium channels in confluent M-1 mouse cortical collecting duct cells.

Confluent M-1 cells show electrogenic Na+ absorption and possess an amiloride-sensitive Na(+)-conductance (Korbmacher et al., J. Gen. Physiol. 102:761-793, 1993). In the present study, we further characterized this conductance and identified the underlying single channels using conventional patch clamp technique. Moreover, we isolated poly(A)+ RNA from M-1 cells to express the channels in Xenopus laevis oocytes, and to check for the presence of transcripts related to the epithelial Na+ channel recently cloned from rat colon (Canessa et al., Nature 361:467-470, 1993). Patch clamp experiments were performed in 6-13-day-old confluent M-1 cells at 37 degrees C. In whole-cell experiments application of 10(-5) M amiloride caused a hyperpolarization of 24.9, SEM +/- 2.2 mV (n = 35) and a reduction of the inward current by 107 +/- 10 pA (n = 51) at a holding potential of -60 mV. Complete removal of bath Na+ had similar effects, indicating that the amiloride-sensitive component of the inward current is a Na+ current. The effect of amiloride was concentration-dependent with half-inhibition at 0.22 microM. The Na+ current saturated with increasing extracellular Na+ concentrations with an apparent Km of 24 mM. Na+ replacement for Li+ demonstrated a higher apical membrane conductance for Li+ than for Na+. In excised inside-out (i/o) or outside-out (o/o) patches from the apical membrane, we observed single-channels which showed slow kinetics and were reversibly inhibited by amiloride. Their average conductance for Na+ was 6.8 +/- 0.5 pS (n = 15) and for Li+ 11.2 +/- 1.0 pS (n = 14). They had no measurable conductance for K+. In o/o patches, channel activity was slightly voltage dependent with an open probability (NPo) of 0.46 +/- 0.14 and 0.16 +/- 0.05 at a holding potential of -100 and 0 mV, respectively (n = 8, P < 0.05). Using the two-microelectrode voltage-clamp technique, we assayed defolliculated stage V-VI Xenopus oocytes for an amiloride-sensitive inward current 1-6 days after injection with H2O or with 20-50 ng of M-1 poly(A)+ RNA. In poly(A)+ RNA-injected oocytes held at -60 or -100 mV application of amiloride (2 microM) reduced the Na-inward current by 25.5 +/- 4.6 nA (n = 25) while it had no effect in H2O-injected oocytes (n = 19). Northern blot analysis of M-1 poly(A+) RNA revealed the presence of transcripts related to the three known subunits of the rat colon Na+ channel (Canessa et al., Nature 367:463-467, 1994). We conclude that the channel in M-1 cells is closely related to the amiloride-sensitive epithelial Na+ channel in the rat colon and that the M-1 cell line provides a useful tool to investigate the biophysical and molecular properties of the corresponding channel in the cortical collecting duct.