In this review we discuss the properties of the Ca(2+)-activated Cl- current [ICl(Ca)] recorded in isolated smooth muscle cells with electrophysiological techniques and speculate on the possible physiological role(s) of ICl(Ca) in smooth muscle function. In particular, we concentrate on 1) the Ca2+ dependence of ICl(Ca), 2) the mechanisms that link pharmacological receptor stimulation on the cell surface membrane to activation of ICl(Ca), 3) the biophysical properties of ICl(Ca), and 4) the pharmacology of ICl(Ca). It is evident that a diverse array of pharmacological agonists can evoke ICl(Ca) in many types of smooth muscle, and it seems that the well-established G protein-phosphoinositide metabolism (inositol 1,4,5-trisphosphate)-intracellular Ca2+ store pathway couples the receptor to the membrane channels. Also, the results indicate that the biophysical and pharmacological properties of ICl(Ca) are not only similar in the various smooth muscle types studied so far but, possibly, are also similar to ICl(Ca) in non-smooth muscle tissue. Evidence is presented that the Ca(2+)-activated Cl- channel exists in two states, open and closed, with a relatively long mean open time and that some of the agents that inhibit ICl(Ca) interact directly with the open channel. It is suggested that the most likely role of ICl(Ca) in smooth muscle is to produce membrane depolarization and contraction to neurotransmitters and local mediators.

SUMMARY1. The mode of action of ACh antagonists on the parasympathetic neurones of the submandibular ganglion of the rat was studied by means of a two-micro-electrode voltage-clamp technique. The currents produced by various agonists (carbachol, ACh, suberylcholine) were studied in steady state and after voltage steps, before and after perfusion of various antagonists.2. For three antagonists (tubocurarine, hexamethonium, decamethonium) the blocking action increases with hyperpolarization. For three other antagonists (surugatoxin, trimetaphan, mecamylamine) the effects observed at low concentrations appear to be independent of membrane potential, although in some cases voltage dependence of the block was observed for mecamylamine.3. The block produced by tubocurarine, hexamethonium and decamethonium increases with the agonist concentration, an observation which supports a 'sequential' scheme in which the antagonist blocks the 'open' channel-receptor complex. The block produced by trimetaphan and mecamylamine decreases slightly with increased agonist concentration, which in turn suggests that these two compounds are competitive antagonists, preventing binding of the agonists to the closed channel-receptor complex.4. In the cases where the block is voltage dependent, voltage jumps trigger slow relaxations which are not present in control conditions. In the case of tubocurarine and hexamethonium, the relaxation following a hyperpolarizing voltage jump corresponds to a decrease in conductance. In the case of decamethonium, the slow relaxation is in the opposite direction.5. The slow relaxations observed with tubocurarine and hexamethonium are speeded by an increase of the antagonist concentration; the slow relaxations observed with decamethonium are slowed by an increase of the decamethonium concentration.6. The steady-state observations and the relaxations can be interpreted in terms of a scheme in which tubocurarine, hexamethonium and decamethonium act mainly by blocking the channels opened by the cholinergic agonists.

Angiotensin II (Ang II) is a potent vasoconstrictor with an important role in controlling blood pressure; however, there is little information on cellular mechanisms underlying Ang II-evoked vasoconstrictor responses. The aim of the present study is to investigate the effect of Ang II on cation conductances in freshly dispersed rabbit mesenteric artery myocytes at the single-channel level using patch-clamp techniques. In cell-attached patches, bath application of low concentrations of Ang II (1 nM) activated cation channel currents (I cat1 ) with conductances states of about 15, 30 and 45 pS. At relatively high concentrations, Ang II (100 nM) inhibited I cat1 but evoked another cation channel (I cat2 ) with a conductance of approximately 2 pS. Ang II-evoked I cat1 and I cat2 were inhibited by the AT 1 receptor antagonist losartan and the phospholipase C (PLC) inhibitor U73122. The diacylglycerol (DAG) lipase inhibitor RHC80267 initially induced I cat1 which was subsequently inhibited to reveal I cat2 . The DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (1 µM) activated I cat1 and I cat2 but inositol 1,4,5-trisphosphate did not evoke either conductance. The protein kinase C (PKC) inhibitor chelerythrine (3 µM) potentiated Ang II-evoked I cat1 and inhibited I cat2 whereas the PKC activator phorbol-12,13-dibutyrate (1 µM) reduced Ang II-induced I cat1 but activated I cat2 . Moreover in cell-attached patches pretreated with chelerythrine, application of 100 nM Ang II activated I cat1 . These data indicate that PKC inhibits I cat1 but stimulates I cat2 . Agents that deplete intracellular Ca 2+ stores also activated cation channel currents with similar properties to I cat2 . Bath application of anti-TRPC6 and anti-TRPC1 antibodies to inside-out patches inhibited I cat1 and I cat2 , respectively. Also flufenamic acid and zero external Ca 2+ concentration, respectively, potentiated and reduced Ang II-evoked I cat1 . Immunocytochemical studies showed TRPC6 and TRPC1 expression with TRPC6 preferentially distributed in the plasma membrane and TRPC1 expression located throughout the myocyte. These results indicate that Ang II activates two distinct cation conductances in mesenteric artery myocytes by stimulation of AT 1 receptors linked to PLC. I cat1 is activated by DAG via a PKC-independent mechanism whereas I cat2 involves DAG acting via a PKC-dependent pathway. Higher concentrations of Ang II inhibit I cat1 by activating an inhibitory effect of PKC. It is proposed that TRPC6 and TRPC1 channel proteins are important components of Ang II-induced I cat1 and I cat2 , respectively.

1 The action of niflumic acid was studied on spontaneous and evoked calcium-activated chloride (IC1(ca)) and potassium (IK(Ca)) currents in rabbit isolated portal vein cells.2 With the nystatin perforated patch technique in potassium-containing solutions at a holding potential of -77 mV (the potassium equilibrium potential), niflumic acid produced a concentrationdependent inhibition of spontaneous transient inward current (STIC, calcium-activated chloride current) amplitude. The concentration to reduce the STIC amplitude by 50% (ICM) was 3.6 x 10-6 M.3 At -77 mV holding potential, niflumic acid converted the STIC decay from a single exponential to 2 exponential components. In niflumic acid the fast component of decay was faster, and the slow component was slower than the control decay time constant. Increasing the concentration of niflumic acid enhanced the decay rate of the fast component and reduced the decay rate of the slow component. 4 The effect of niflumic acid on STIC amplitude was voltage-dependent and at -50 and + 50 mV the IC50 values were 2.3 X 10-6 M and 1.1 X 10-6 M respectively (cf. 3.6 x 10-6 M at -77 mV).5 In K-free solutions at potentials of -50 mV and + 50 mV, niflumic acid did not induce a dual exponential STIC decay but just increased the decay time constant at both potentials in a concentrationdependent manner.6 Niflumic acid, in concentrations up to 5 x 10-5 M, had no effect on spontaneous calcium-activated potassium currents.7 Niflumic acid inhibited noradrenaline-and caffeine-evoked IO(C.) with an ICM of 6.6 x 10-6 M, i.e. was less potent against evoked currents compared to spontaneous currents. In contrast niflumic acid (2 x 10-6 M-5 x 105 M) increased noradrenaline-and caffeine-induced I ). 8 The results are discussed with respect to the mechanism of block of ICl(Ca) by niflumic acid and its suitability as a pharmacological tool for assessing the role of Ic(cp) in physiological mechanisms.