Migrating peristaltic contractions in the mammalian upper urinary tract serve to propel urine from the kidney through the ureter to the bladder, where it is stored until micturition. In most mammals, circumferencially cut strips of the renal pelvis display spontaneous contractions which decrease in frequency as strips are taken from regions more distal of the renal calyx, the isolated ureter being quiescent in most mammals except man and pig (Constantinou et al. 1978;Constantinou, 1979). Extracellular sucrose gap recordings from the guinea-pig renal pelvis indicated that the spontaneous contractions were associated with simple oscillations in the membrane potential, analogous to the 'pacemaker' action potentials recorded in the sino-atrial node of the mammalian heart. In contrast, electrically evoked contractions in both the distal renal pelvis and ureter were associated with action potentials consisting of a rapid rising phase and a long plateau (Zawalinski et al. 1975;Constantinou et al. 1979;Santicioli & Maggi, 1997). Thus, it has often been postulated that the initiation of peristaltic contractions in the upper urinary tract involves the spontaneous generation of 'pacemaker' potentials within the proximal renal pelvis which trigger 'driven' action potentials and contraction in the usually quiescent more distal regions of the upper urinary tract (

Acetylcholine, the major excitatory neurotransmitter to the smooth muscle of mammalian intestine, is known to depolarize smooth muscle cells with an apparent increase in membrane conductance. However, the ionic mechanisms that are triggered by muscarinic receptor activation and underlie this response are poorly understood, due in part to the technical problems associated with the electrophysiological study of smooth muscle. The muscarinic action of acetylcholine in certain neurones has been shown to involve the switching off of a resting K+ current (M-current) and a similar mechanism has recently also been identified in smooth muscle of amphibian stomach. We have now applied the patch-clamp technique to single smooth muscle cells of rabbit jejunum and find that muscarinic receptor activation switches on a nonselective, voltage-sensitive inward current. In addition, acetylcholine activates and then suppresses spontaneous K+ current transients, which are probably triggered by rises in intracellular Ca2+ in these cells.

SUMMARY1. Single-channel studies were made using the patch-clamp technique of K channels in dispersed single smooth muscle cells from rabbit longitudinal jejunal muscle and guinea-pig small ( < 0-2 mm o.d.) mesenteric arteries.2. In isolated inside-out patches from these two types of smooth muscle cell there was a population of K channels which had single-channel conductances of about 100 pS in near physiological K gradients and about 200 pS with symmetrical 126 mM-K solutions. Their conductance and other properties distinguish them from a K channel of smaller conductance which we have previously described in these cells.3. The relative permeability of the channel with respect to K was 1-4 Tl: 1-0 K:0-7 Rb: < 0 05 Na: < 0 05 Cs. Cs (1 mm applied to the outside of the membrane) interfered with inward K movement when the membrane was hyperpolarized. Rb conductance of the channel when both sides of the membrane were exposed to 126 mm-Rb was 30 pS. Raising [Ca]i increased the mean duration of the (long) open state and decreased or had no effect on the duration of short, intermediate, and long mean closed states.

SUMMARY1. Membrane potential was recorded by micro-electrode in segments of small (200-500 jsm o.d.) mesenteric arteries of guinea-pig. Isotonic shortening was recorded in helical strips cut from these arteries.2. Raising the external potassium concentration, [K+]0, caused shortening and substantial depolarization. The threshold for contraction was about 30 mm which corresponded to a membrane potential of about -45 mV. Since high-potassium contractions were abolished in calcium-free solution it was suggested that they occur due to potential-sensitive calcium channels opening positive to about -45 mV.3. Noradrenaline weakly depolarized the muscle and produced contractions resistant to calcium-free conditions. It was suggested that noradrenaline contractions are mainly caused by mechanisms other than the opening of potential-sensitive calcium channels, namely entry of calcium via other channels and release of stored calcium.4. Carbachol had no effect on basal tension but inhibited shortening by noradrenaline or by raising [K+]0. The inhibitory effect of carbachol on tension under various conditions was associated with hyperpolarization or depolarization in a range negative to -45 mV, or no effect on potential, so that modulation of the number of open potential-sensitive calcium channels could not be evoked to explain its relaxant action.5. Removal or destruction of the endothelium by rubbing or by distilled water perfusion left tension responses to noradrenaline or raised [K+]. essentially unchanged. However, the inhibitory effect of carbachol on tension was attenuated and hyperpolarization of the resting artery was converted to a depolarization. 6. It was concluded that carbachol has both a strong inhibitory and a weak excitatory effect on these vascular smooth muscle cells. Membrane potential changes are not essential to its inhibitory action but may, by closing potential-sensitive calcium channels, sometimes reinforce it. Hyperpolarization by carbachol may be caused by a factor released by the action of carbachol on endothelial cells: in its absence carbachol may weakly depolarize but this alone is normally insufficient to generate tension.

The interaction of Ba2+ and TEA with Ca2+-activated K+ channels was studied in isolated membrane patches of cells from longitudinal jejunal smooth muscle of rabbit and from guinea-pig small mesenteric artery (100 micron external diameter). Ba2+ applied from the inside of the membrane did not reduce unit current, except at high concentrations, but channels failed to open for long periods (s). This effect became much stronger when the potential gradient was in a direction driving Ba2+ into the channel and was reduced by increasing K+ ion concentration on the outside of the membrane. These results are consistent with Ba2+ entering the open channel and blocking at a site most of the way through the channel bore. In contrast, TEA and procaine dose-dependently reduced unit current amplitude at all patch potentials and slightly increased mean open time. Their effects were not detectably voltage-dependent and could be explained by TEA and procaine blocking the open channel with a timecourse that was faster than the frequency response of the recording system. The lack of appreciable voltage-dependence suggests that TEA and procaine bind to a site near to the inner mouth of the channel.

2+ transient discharge suggesting that they may well be acting as 'point sources' of excitation to the TSMC layer. We speculate that ASMCs act as the primary pacemaker in the renal pelvis while ICC-LCs play a supportive role, but can take over pacemaking in the absence of the proximal pacemaker drive.

SUMMARY1. Ca2" inward current was studied using the whole-cell patch clamp technique in single smooth muscle cells enzymatically isolated from the rabbit ear artery.Currents were studied in salt solutions containing either normal (1-5 mM) Ca2`or high (110 mM) Ba2+. Outward currents were minimized by using a high-Na+ intracellular solution containing 10 mM-TEA.2. In normal-Ca2+ solution, the threshold at which inward current could be evoked was -60 mV at a holding potential of -80 mV and -48 mV at a holding potential of -60 mV. At both holding potentials, the current showed little inactivation over 500 ms near threshold, and inactivated substantially but incompletely with larger depolarizations. In high-Ba2+ solution where currents were 5-10 times larger, current threshold, maximal peak amplitude and apparent reversal potential were shifted in a positive direction along the voltage axis.3. Inward current in normal-Ca2+ solution was only fully available negative to -90 mV, was half-inactivated near -47 mV and was about 90% inactivated at -10 mV. A component of non-inactivating current was, however, present in both normal-Ca2' and high-Ba2+ solutions even after conditioning pulses of 5 s duration to + 30 mV. The inactivation-potential relationship was shifted in a positive direction in high-Ba2+ solution, and its position showed considerable variation between cells.4. The inward current from a holding potential of -70 mV was reduced in normalCa2+ solution by about 50 % by nifedipine (041 /tM) in some cells at all test potentials, although at more-negative test potentials peak current was unaffected in some cells even at 10 /iM. In some cells when the holding potential was -90 mV, the peak current amplitude was increased markedly by 10 /iM-nifedipine. In high-Ba2+ solution, from negative holding potentials (-70 to -80 mV), nifedipine augmented peak current at test potentials negative to -10 mV and shifted the current threshold in a negative direction; current at potentials positive of -10 mV was reduced. Both effects were concentration dependent. The stimulatory effect of nifedipine was abolished and its inhibitory effect was enhanced when less-negative holding potentials were employed. Bay K 8644 (0-1 or 1 /bM) approximately doubled current * Present address:

We have used homologous recombination in human embryonic stem cells (hESCs) to insert sequences encoding green fluorescent protein (GFP) into the NKX2