SUMMARY1. Whole-cell membrane currents in voltage-clamped single isolated cells of longitudinal smooth muscle of guinea-pig ileum were studied at room temperature using patch pipettes filled with either high-K+ solution or high-Cs' solution, to suppress K+ outward current, and containing 0 3 mM-EGTA.2. In the presence of high-K+ solution in the pipette, membrane depolarization from the holding potential of -50 mV evoked an initial inward calcium current (ICa) followed by a large initial transient outward current and a sustained outward current with spontaneous oscillations superimposed. Prolonged depolarization above -20 mV produced a late transient outward current which reached a maximum (up to several nanoamps at +10 mV) within approximately 1 s and lasted several seconds.3. The late outward current (ILTO) was voltage dependent and reversed at the EK (potassium equilibrium potential) in cells exposed to high-K+ external solution. It was blocked by TEA+ (tetraethylammonium) or Ba2+ applied externally (calculated Kd (dissociation constant) values were 0-67 and 4-43 mm, respectively) or by high-Cs+ solution perfusing the cell. The removal of extracellular Ca2+, application of Ca2+ channel blockers (3 mM-Co2+, 0-2 mM-Cd2+ or 1 4uM-nifedipine) or perfusion of 5 mm-EGTA inside the cell also abolished the current. Thus, the current seems to be a Ca2+_ activated K+ current.4. There is a great discrepancy between the time course of the ICa and that of the late ILTO' which suggests that Ca2+ release from intracellular storage sites may contribute to the generation of the ILTO* 5. Bath application of caffeine (10 mM

1 Whole-cell patch clamp methods were used to analyse voltage-dependent calcium currents in cultured myenteric neurones enzymatically isolated from adult guinea-pig small intestine. 2 Activation of G-proteins by intracellular administration of GTP-y-S (100-200 gM in pipette) decreased the amplitude of high voltage activated Ca21 current (Ica) by more than 50%. Residual ICa was activated more slowly and was non-inactivating during 500 ms test pulses when GTP-y-S was included in the pipette solution. 3 Inclusion of 500 giM GDP-,B-S in the patch pipettes increased the amplitude of Ia by over 30% without altering the voltage-dependency. 4 Extracellular application of 2-chloroadenosine suppressed ICa dose-dependently by reducing both transient and sustained components of the current. 5 Pretreatment of the neurones with cholera toxin or forskolin did not alter the actions of GTP-y-S or GDP-,B-S or 2-chloroadenosine. 6 The results suggest that high threshold calcium channels in myenteric neurones are influenced by Gproteins and that the inhibitory action of 2-chloroadenosine on ICa involves G-protein coupling of the adenosine receptors to the Ca2" channel.

Fura 2 microfluorimetry was used to test the hypothesis that ATP acts at P1 and P2 purinoceptors to elevate cytosolic free Ca2+ concentrations [Ca2+]i) in calbindin-immunoreactive cultured myenteric neurons from adult guinea pig small intestine. Local "micro-puff" application of ATP or ATP(gamma)S caused an increase in [Ca2+]i in 99% of 200 multipolar neurons. The potency profile of agonists for the rise in [Ca2+]i was ATP(gamma)S = ATP >> ADP >> AMP, adenosine, 5'-(N-ethylcarboxamido)adenosine, and 2-chloro-N(6)-cyclopentyladenosine. Tetrodotoxin-sensitive synaptic transmission could contribute as much as 25% to the ATP response. The P1 antagonist 8-cyclopentyl-1,3-dipropylxanthine blocked 50% of the peakATP Ca2+ response. P2 antagonists blocked the ATP response: pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid > reactive blue 2 > suramin. Suramin enhanced the ATP response in 27.5% of neurons. Some neurons (<15%) displayed distinct multiphasic Ca2+ signatures. About 54% of ATP-responsive neurons expressed calbindin. The data support the following hypotheses: 1) two distinct P2 purinoceptors are linked to the rise in [Ca2+]i in myenteric neurons; 2) purinergic Ca2+ signaling is not restricted to one neuronal phenotype; and 3) intraneuronal Ca2+ is not involved in adenosinergic hyperpolarization in AH/type 2 neurons.

The patch-clamp method has been used to investigate the action of caffeine on the calcium current (ICa) in single isolated smooth muscle cells of the guinea-pig ileum. Caffeine (10 mM) substantially inhibited ICa. This effect occurred in a biphasic manner and it was not due either to activation of additional ionic currents of opposite direction nor to inhibition of phosphodiesterase activity. It strongly depended upon the ethylenebis-(oxonitrilo)tetraacetate (EGTA) concentration in the pipette solution. When there was K+ in the pipette solution, application of caffeine evoked a transient Ca-dependent K+ current and an abrupt and transient increase in the frequency of channel openings. Such well-known blockers of Ca release as procaine and ruthenium red strongly decreased ICa. Ryanodine had only little effect on ICa, but application of caffeine in the presence of ryanodine led to a complete and irreversible inhibition of ICa. The results of experiments involving different EGTA concentrations and comparison of the time courses of all caffeine-induced phenomena clearly indicated that only the initial, transient component of the ICa inhibition by caffeine was related to a Ca-dependent inactivation of Ca channels, evoked as a result of Ca release from intracellular stores. The tonic component of ICa inhibition was probably due to a direct blocking action of caffeine on Ca channels.

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