Proteinase-activated receptors (PARs) are a new family of G-protein-coupled membrane receptors for serine proteases (D ery et al. 1998). Proteases cleave within the extracellular N-terminus of PARs, exposing tethered ligand domains that bind and activate the cleaved receptors. Thrombin activates

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.

1. Membrane voltage (Vm) recordings were obtained from isolated rat pinealocytes using the patch-clamp technique. In parallel to the electrophysiological experiments, intracellular Ca2P measurements were performed using fura-2. The mammalian pineal organ transduces information about the ambient photoperiod into a neuroendocrine message by rhythmic synthesis and secretion of melatonin (for review see Arendt, 1995). In mammals the sympathetic innervation of the pineal organ is mandatory to maintain the lightsynchronized circadian rhythm of melatonin production that is highest during the second half of the night. In rats, noradrenaline acts upon melatonin biosynthesis by stimulating both al-and /,3-adrenergic receptors. Stimulation of /,3-receptors increases intracellular cAMP, which mediates the activation of serotonin-N-acetyl-transferase, the rate-limiting enzyme of melatonin biosynthesis.Activation of az1-receptors potentiates the /,3-adrenergic effect via an increase in intracellular calcium concentration ([Ca2+]1) (for review see Klein, 1985). Thus, the essential role of noradrenaline for stimulation of melatonin biosynthesis has been firmly established. In contrast, it is still unclear whether and how neuronal pathways other than the noradrenergic sympathetic innervation (for review see Korf, 1996) are involved in the regulation of melatonin biosynthesis and pineal functions in mammals. Several morphological investigations point toward the existence of a parasympathetic innervation of the mammalian pineal gland, which may originate from the pterygopalatine ganglion and employ acetylcholine as primary neurotransmitter (for review see M0ller, 1992). Further evidence for a cholinergic innervation of the pineal organ has been obtained by recent immunocytochemical studies (E. Weihe, M. K.-H.

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.