Myorelaxant action of fluorine-containing pinacidil analog, flocalin, in bladder smooth muscle is mediated by inhibition of L-type calcium channels rather than activation of KATP channels.

Flocalin (FLO) is a new ATP-sensitive K(+) (KATP) channel opener (KCO) derived from pinacidil (PIN) by adding fluorine group to the drug's structure. FLO acts as a potent cardioprotector against ischemia-reperfusion damage in isolated heart and whole animal models primarily via activating cardiac-specific Kir6.2/SUR2A KATP channels. Given that FLO also confers relaxation on several types of smooth muscles and can partially inhibit L-type Ca(2+) channels, in this study, we asked what is the mechanism of FLO action in bladder detrusor smooth muscle (DSM). The actions of FLO and PIN on contractility of rat and guinea pig DSM strips and membrane currents of isolated DSM cells were compared by tensiometry and patch clamp. Kir6 and SUR subunit expression in rat DSM was assayed by reverse transcription PCR (RT-PCR). In contrast to PIN (10 µM), FLO (10 µM) did not produce glibenclamide-sensitive DSM strips' relaxation and inhibition of spontaneous and electrically evoked contractions. However, FLO, but not PIN, inhibited contractions evoked by high K(+) depolarization. FLO (40 µM) did not change the level of isolated DSM cell's background K(+) current, but suppressed by 20 % L-type Ca(2+) current. Determining various Kir6 and SUR messenger RNA (mRNA) expressions in rat DSM by RT-PCR indicated that dominant KATP channel in rat DSM is of vascular type involving association of Kir6.1 and SUR2B subunits. Myorelaxant effects of FLO in bladder DSM are explained by partial blockade of L-type Ca(2+) channel-mediated Ca(2+) influx rather than by hyperpolarization associated with increased K(+) permeability. Thus, insertion of fluorine group in PIN's structure made the drug more discriminative between Kir6.2/SUR2A cardiac- and Kir6.1/SUR2B vascular-type KATP channels and rendered it partial L-type Ca(2+) channel-blocking potency.

Effects of the endogenous cannabinoid anandamide on voltage-dependent sodium and calcium channels in rat ventricular myocytes.

BACKGROUND AND PURPOSE: The endocannabinoid anandamide (N-arachidonoyl ethanolamide; AEA) exerts negative inotropic and antiarrhythmic effects in ventricular myocytes. EXPERIMENTAL APPROACH: Whole-cell patch-clamp technique and radioligand-binding methods were used to analyse the effects of anandamide in rat ventricular myocytes. KEY RESULTS: In the presence of 1-10 µM AEA, suppression of both Na(+) and L-type Ca(2+) channels was observed. Inhibition of Na(+) channels was voltage and Pertussis toxin (PTX) - independent. Radioligand-binding studies indicated that specific binding of [(3) H] batrachotoxin (BTX) to ventricular muscle membranes was also inhibited significantly by 10 µM metAEA, a non-metabolized AEA analogue, with a marked decrease in Bmax values but no change in Kd . Further studies on L-type Ca(2+) channels indicated that AEA potently inhibited these channels (IC50 0.1 µM) in a voltage- and PTX-independent manner. AEA inhibited maximal amplitudes without affecting the kinetics of Ba(2+) currents. MetAEA also inhibited Na(+) and L-type Ca(2+) currents. Radioligand studies indicated that specific binding of [(3) H]isradipine, was inhibited significantly by metAEA. (10 µM), changing Bmax but not Kd . CONCLUSION AND IMPLICATIONS: Results indicate that AEA inhibited the function of voltage-dependent Na(+) and L-type Ca(2+) channels in rat ventricular myocytes, independent of CB1 and CB2 receptor activation.

Effects of endogenous cannabinoid anandamide on excitation-contraction coupling in rat ventricular myocytes.

A role for anandamide (N-arachidonoyl ethanolamide; AEA), a major endocannabinoid, in the cardiovascular system in various pathological conditions has been reported in earlier reports. In the present study, the effects of AEA on contractility, Ca2+ signaling, and action potential (AP) characteristics were investigated in rat ventricular myocytes. Video edge detection was used to measure myocyte shortening. Intracellular Ca2+ was measured in cells loaded with the fluorescent indicator fura-2 AM. AEA (1 µM) caused a significant decrease in the amplitudes of electrically evoked myocyte shortening and Ca2+ transients. However, the amplitudes of caffeine-evoked Ca2+ transients and the rate of recovery of electrically evoked Ca2+ transients following caffeine application were not altered. Biochemical studies in sarcoplasmic reticulum (SR) vesicles from rat ventricles indicated that AEA affected Ca2+ -uptake and Ca2+ -ATPase activity in a biphasic manner. [3H]-ryanodine binding and passive Ca2+ release from SR vesicles were not altered by 10 µM AEA. Whole-cell patch-clamp technique was employed to investigate the effect of AEA on the characteristics of APs. AEA (1 µM) significantly decreased the duration of AP. The effect of AEA on myocyte shortening and AP characteristics was not altered in the presence of pertussis toxin (PTX, 2 µg/ml for 4 h), AM251 and SR141716 (cannabinoid type 1 receptor antagonists; 0.3 µM) or AM630 and SR 144528 (cannabinoid type 2 receptor antagonists; 0.3 µM). The results suggest that AEA depresses ventricular myocyte contractility by decreasing the action potential duration (APD) in a manner independent of CB1 and CB2 receptors.

Effects of fluorine-containing opener of ATP-sensitive potassium channels, pinacidil-derivative flocalin, on cardiac voltage-gated sodium and calcium channels.

Fluorine-containing pinacidil-derivative flocalin is an effective adenosine triphosphate-sensitive potassium (K(ATP))-channel opener with pronounced vasodilatory, cardioprotective effects and low general toxicity. By activating cardiac K(ATP) channels, flocalin hyperpolarizes cardiac myocytes, decreases their excitability, reduces Ca(2+) entry, and inhibits Ca(2+)-dependent signalling processes. Since our previous studies indicated that the drug also influences the rate of rise and amplitude of the cardiomyocyte's action potential, here we have investigated its possible actions on depolarizing inward currents through voltage-gated sodium (VGSC) and L-type calcium (VGCC) channels. Experiments were conducted on cultured cardiac myocytes prepared from the whole hearts of neonatal rats and maintained in culture for 1-3 days using whole-cell patch-clamp technique with no distinction of myocyte's type. Flocalin concentration dependently inhibited the Na(+) inward current through VGSCs with IC(50) = 17.4 µM and a maximal extent of 0.54, slowed down its inactivation kinetics, and hyperpolarized steady-state inactivation by 5.6 mV. The drug also inhibited calcium current through L-type VGCCs with IC(50) = 24.1 µM and a maximal block of 0.38, without affecting its inactivation but producing 5.3-mV hyperpolarization shifting of steady-state activation. Inhibition of both depolarizing currents by flocalin in addition to its ability to open K(ATP) channels enhances the suppressive action of the drug on cardiac excitability and broadens its pharmacological effects. Since, according to our previous data, cardiac K(ATP)-channel opening by flocalin occurs with ЕC(50) = 8 µM, the possibility of partial blockade of VGSC and L-type VGCCs should be considered when determining the therapeutic concentrations of the compound during its use as a cardioprotector.

Modulation of excitability, membrane currents and survival of cardiac myocytes by N-acylethanolamines.

N-acylethanolamines (NAE) are endogenously produced lipids playing important roles in a diverse range of physiological and pathological conditions. In the present study, using whole-cell patch clamp technique, we have for the first time investigated the effects of the most abundantly produced NAEs, N-stearoylethanolamine (SEA) and N-oleoylethanolamine (OEA), on electric excitability and membrane currents in cardiomyocytes isolated from endocardial, epicardial, and atrial regions of neonatal rat heart. SEA and OEA (1-10µM) attenuated electrical activity of the myocytes from all regions of the cardiac muscle by hyperpolarizing resting potential, reducing amplitude, and shortening the duration of the action potential. However, the magnitudes of these effects varied significantly depending on the type of cardiac myocyte (i.e., endocardial, epicardial, atrial) with OEA being generally more potent. OEA and to a lesser extent SEA suppressed in a concentration-dependent manner currents through voltage-gated Na(+) (VGSC) and L-type Ca(2+) (VGCC) channels, but induced variable cardiac myocyte type-dependent effects on background K(+) and Cl(-) conductance. The mechanisms of inhibitory action of OEA on cardiac VGSCs and VGCCs involved influence on channels' activation/inactivation gating and partial blockade of ion permeation. OEA also enhanced the viability of cardiac myocytes by reducing necrosis without a significant effect on apoptosis. We conclude that SEA and OEA attenuate the excitability of cardiac myocytes mainly through inhibition of VGSCs and VGCC-mediated Ca(2+) entry. Since NAEs are known to increase during tissue ischemia and infarction, these effects of NAEs may mediate some of their cardioprotective actions during these pathological conditions.

Sarcolemmal cardiac K(ATP) channels as a target for the cardioprotective effects of the fluorine-containing pinacidil analogue, flocalin.

BACKGROUND AND PURPOSE: A class of drugs known as K(ATP) -channel openers induce cardioprotection. This study examined the effects of the novel K(ATP) -channel opener, the fluorine-containing pinacidil derivative, flocalin, on cardiac-specific K(ATP) -channels, excitability of native cardiac myocytes and on the ischaemic heart. EXPERIMENTAL APPROACH: The action of flocalin was investigated on: (i) membrane currents through cardiac-specific K(ATP) -channels (I(KATP) ) formed by K(IR) 6.2/SUR2A heterologously expressed in HEK-293 cells (HEK-293(6.2/2A) ); (ii) excitability and intracellular Ca²(+) ([Ca²(+) ](i) ) transients of cultured rat neonatal cardiac myocytes; and (iii) functional and ultrastructural characteristics of isolated guinea-pig hearts subjected to ischaemia-reperfusion. KEY RESULTS: Flocalin concentration-dependently activated a glibenclamide-sensitive I(KATP) in HEK-293(6.2/2A) cells with an EC50= 8.1 ± 0.4 µM. In cardiac myocytes, flocalin (5 µM) hyperpolarized resting potential by 3-5 mV, markedly shortened action potential duration, reduced the amplitude of [Ca²(+) ](i) transients by 2-3-fold and suppressed contraction. The magnitude and extent of reversibility of these effects depended on the type of cardiac myocytes. In isolated hearts, perfusion with 5 µmol·L⁻¹ flocalin, before inducing ischaemia, facilitated restoration of contraction during reperfusion, decreased the number of extrasystoles, prevented the appearance of coronary vasoconstriction and reduced damage to the cardiac tissue at the ultrastructural level (state of myofibrils, membrane integrity, mitochondrial cristae structure). CONCLUSION AND IMPLICATIONS: Flocalin induced potent cardioprotection by activating cardiac-type K(ATP) -channels with all the benefits of the presence of fluorine group in the drug structure: higher lipophilicity, decreased toxicity, resistance to oxidation and thermal degradation, decreased metabolism in the organism and prolonged therapeutic action.