1 The effect of the bradycardic agent S 16257 on the main ionic mechanisms of diastolic depolarization in sinoatrial node cells isolated from rabbit heart, was investigated by the patch-clamp technique in whole-cell and macro-patch recordings. 4 A high concentration of S 16257 (10 yM) had no detectable effect on T-type calcium current and slightly decreased L-type calcium current (-18.12+0.66%), without significant use-dependent blockade. 5 S 16257 had no effect on the delayed outward potassium current IK at 3 pM and slightly decreased it only at high concentrations, -16.3+ 1.2% at 10 gM. In contrast, zatebradine, another bradycardic agent, reduced IK by 20.3+2.5% at 3 gM. 6 In conclusion, S 16257 may lower heart rate without significant negative inotropic action. In comparison with zatebradine, S 16257 had less effect on IK suggesting less prolongation of repolarization time.

Epoxyeicosatrienoic acids (EETs) are produced from arachidonic acid via the cytochrome P-450 epoxygenase pathway. EETs are able to modulate smooth muscle tone by increasing K(+) conductance, hence generating hyperpolarization of the tissues. However, the molecular mechanisms by which EETs induce smooth muscle relaxation are not fully understood. In the present study, the effects of EETs on airway smooth muscle (ASM) were investigated using three electrophysiological techniques. 8,9-EET and 14,15-EET induced concentration-dependent relaxations of the ASM precontracted with a muscarinc agonist (carbamylcholine chloride), and these relaxations were partly inhibited by 10 nM iberiotoxin (IbTX), a specific large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel blocker. Moreover, 3 microM 8,9- or 14,15-EET induced hyperpolarizations of -12 +/- 3.5 and -16 +/- 3 mV, with EC(50) values of 0.13 and 0.14 microM, respectively, which were either reversed or blocked on addition of 10 nM IbTX. These results indicate that BK(Ca) channels are involved in hyperpolarization and participate in the relaxation of ASM. In addition, complementary experiments demonstrated that 8,9- and 14,15-EET activate reconstituted BK(Ca) channels at low free Ca(2+) concentrations without affecting their unitary conductance. These increases in channel activity were IbTX sensitive and correlated well with the IbTX-sensitive hyperpolarization and relaxation of ASM. Together these results support the view that, in ASM, the EETs act through an epithelium-derived hyperpolarizing factorlike effect.

The action of the two diastereometric phosphorothioate derivatives of cAMP, Rp‐cAMPs and Sp‐cAMPs, was investigated on hyperpolarization‐activated ‘pacemaker’ current (if) recorded in inside‐out macropatches from rabbit sino‐atrial (SA) node myocytes. When superfused on the intracellular side of f‐channels at the concentration of 10 μm, both cAMP derivatives accelerated if activation; their action was moderately less pronounced than that due to the same concentration of cAMP. The measurement of the if conductance‐voltage relation by voltage ramp protocols indicated that both cAMP analogues shift the activation curve of if to more positive voltages with no change in maximal (fully activated) conductance. Dose–response relationships of the shift of the if activation curve showed that both Rp‐cAMPs and Sp‐cAMPs act as agonists in the cAMP‐dependent direct f‐channel activation. Fitting data to the Hill equation resulted in maximal shifts of 9.6 and 9.5 mV, apparent dissociation constants of 0.82 and 5.4 μm, and Hill coefficients of 0.82 and 1.12 for Sp‐cAMPs and Rp‐cAMPs, respectively. The activating action of Rp‐cAMPs, a known antagonist of cAMP in the activation of cAMP‐dependent protein kinase, confirms previously established evidence that f‐channel activation does not involve phosphorylation. These results also suggest that the cAMP binding site of f‐channels may be structurally similar to the cyclic nucleotide binding site of olfactory receptor channels.

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The effect of external ATP on intracellular pH (pHi) was investigated using a pH imaging system in a human bronchial epithelial cell line (16HBE14o‐) loaded with BCECF‐AM. The steady‐state pHi of 16HBE14o‐ epithelial monolayers was 7.137 ± 0.027 (n= 46). Apical addition of ATP (10−4m) to epithelial monolayers induced a rapid and sustained pHi decrease of 0.164 ± 0.024 pH units (n= 17; P < 0.001). The intracellular acidification was rapidly reversed upon removal of external ATP. In contrast, the non‐hydrolysable ATP analogue AMP‐PNP did not produce any significant change in pHi. Inhibition of purinoreceptors by suramin did not affect the acidification induced by apical ATP. Inhibition of Na+‐H+ exchange by apical Na+ removal or addition of amiloride (0.5 mm) reduced the apical ATP‐induced pHi decrease, suggesting the involvement of a Na+‐H+ exchanger or surface pH effects on the ATP‐induced pHi response. Inhibitors of proton channels such as ZnCl2 (10−4m) also partially inhibited the ATP response. The pHi response to ATP was dependent on the external pH (pHo), with increasing acidification produced at lower pHo values. Neither the basal pHi nor the ATP‐induced intracellular acidification was affected by thapsigargin (a Ca2+‐ATPase inhibitor), chelerythrine chloride (a protein kinase C (PKC) inhibitor), RpcAMP (a protein kinase A (PKA) inhibitor) or PMA (a PKC activator). Therefore, the intracellular acidification of human bronchial epithelial cells induced by apical ATP does not involve signalling via Ca2+, PKC or PKA nor binding to a purinoreceptor. We interpret the effect of ATP to produce an intracellular acidification as a three step process: activation of H+ channels, inhibition of Na+‐H+ exchange and influx of protonated ATP.

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