Effects Of N-n-butyl Haloperidol Iodide On L-type Calcium Channel And Intracellular Free Calcium In Ventricular Cardiomyocytes From Rat

Haloperidol (Hal) is an antipsychotic agent. Our previous research showed that Hal could noncompetitively antagonize the contraction of coronary artery induced by phencyclidine, agonists of opioid receptor, and noradrenaline, etc. However, it was not used as an anti-myocardial ischemia drug due to its extrapyramidal adverse reactions. In consideration of this, we synthesized a series of quaternary ammonium salt derivatives of Hal, with polarity increased so that they do not pass through the blood-brain barrier. One of these compounds with serial number F₂ (N-n-butyl haloperidol iodide) was screened, and was found to maintain the vasodilator effects but have no side reactions. In further researches, it was found that in vitro, F₂ blocked the porcine coronary artery strips contraction induced by KCl, antagonized the reduction of coronary flow induced by pituitrin on guinea pig isolated heart. In vivo, F₂ exerted protective effects on myocardial ischemia and reperfusion injury in rat and rabbit. In the mechanism study, F₂ inhibited the L-type calcium current in ventricular cardiomyocyte of rat by using patch clamp techniques in whole-cell recording configuration.In order to confirm whether F₂ is a calcium channel blocker, and to get more insights in the kinetics of the voltage dependent calcium channel modulation, in this article, the dependence of functional state on calcium channel of F₂ was investigated to elucidate the gating mechanism of calcium channel. Moreover, the effects of F₂ on the intracellular calcium fluorescence intensity in ventricular myocytes from rat was investigated to understand the effects of F₂ on intracellular free calcium especially on the release of intramyocytic calcium using laser scanning confocal microscopy. The results are reported as follows.1. The effects of F2 on gating of L-type calcium channel in ventricular cardiomyocytes from ratEnzymatically dissociated single ventricular myocytes were used in the whole-cell patch clamp recording experiment.1.1 Inhibition of F2 on L-type calcium currentVoltage pulses were applied every 5s at holding potential of-40mV, command potential(depolarizing potential) from -40mV to +60mV, with lOmV increment. The inward calcium currents (/Ca) with time and voltage dependence were elicited. When cells were exposed to F2 at the dose of 0.1, 1, lOumol-L"1, 7Ca was blocked in dose dependent manner with a calculated EC50 of 1.19xlO'6M. The inhibitory rate of F2 on 7Ca was 40%(from 1895±462 pA to 1268±345), 72%(1781±382 pA to 486±109 pA), 74%(from 1834±443 pA to 364±117 pA) respectively at the dose of 0.1, 1, lOumol-L'Vespectively. In addition, F2 shifted the current-voltage curves of 7ca.L upward in a dose manner, without affecting the activated potential, peak amplitude potential and reversal potential of /ca.L-1.2 Effects of F2 on steady-state activation curves of 7CaSteady-state activation curve of 7Ca was studied by the application of 5s testing pulses to various voltages(from -40mV to 50mV) at the holding potential of -40mV. The magnitude of 7Ca was measured as the difference of peak inward current. Steady-state activation curve of 7ca of were described by Boltzmann functions(G/Gmax=l/{l+exp[-(V-V1/2)/s]). The results showed that F2 induced a rightward shift of the steady-state activation curves of 7Ca to positive potential with the half-activation voltage(V)/2) changing from -19.6 mV±6.3 mV to -8.3 mV± 2.7 mV(p<0.05, n = 5 cells) and the slope factor(s) from 6A ±2.2 to 7.9 + 3.3(p>0.05, n=5 cells).1.3 Effects of F2 on steady-state inactivation curves of 7CaSteady-state inactivation curve of 7ca was performed using twin-pulse protocol. 7Ca was studied by the application of 5s testing pulses to various voltages(from -80mV to 30mV) at the holding potential of -40mV with lOmV incrementfollowed by a test pulse to OmV. Steady-state inactivation curves of 7Ca was also fitted by the Boltzmann functions (I/Imax=l/{l+exp[-(V-Vi/2)/s]). The results showed that F2 induced a leftward shift of the steady-state inactivation curves of 7Ca to negative potential with the half-inactivation voltage(Vi/2)changing from -19.3mV±3.5 mV to -31.9 mV + 2.7 mV (p<0.05, n=5 cells)and the slope factor(s) from 9.1+4.2 mV to8.7±6.1mV (p>0.05, n=5 cells). 1.4 Effects of F2 on recovery curve from inactivation of 7CaA standard twin-pulse protocols was used to measure the rate of recovery from inactivation of /Ca. Two identical 250ms pulses from -40 to 0 mV were applied with a varying interval. The time dependence of recovery was best fit by monoexpotential function(I%=A+Bxexp(-t/T)). The results showed that F2 prolonged recovery time from inactivation with the parameter(x) of recovery curves from 82±28 ms to 134±32 ms (PO.01, n=5 cells) . 2. Effects of F2 on intracellular calcium concentration[Ca2+]jEnzymatically dissociated ventricular myocytes from rats were loaded with Fluo-3-AM, and [Ca2+]j was measured in fluorescence intensity(FI) by laser scanning confocal microscopy.2.1 Effects of F2on intracellular calcium concentration[Ca2+]j at the resting stateIn the normal Tyrode solution with extracellular Ca2+ 1.8mmol-L, the FI of [Ca2+]j in cytoplasm was not affected by F2 (0.1, 1, lOumol-L"1) without any depolarization of cell membrane.2.2 Effects of F2 on intracellular calcium concentration [Ca2+]j elevated by high K+(60mmoI-L KC1) depolarizationIn the normal Tyrode solution with extracellular Ca2+ 1.8mmol-L, the FI of [Ca2+]j in cytoplasm was markably elevated by 60mmol-L KC1. Pretreatment of myocytes with F2 at different dose of 0.1, 1, lOumol-L'1, the FI elevation was inhibited in a dose dependent manner. F2 also reduced the FI elevated by 60mmol-L KC1 in dose dependent manner. The effects of F2 were similar to that ofthe traditional calcium channel blocker, verapamil.2.3 Effects of F2 on intracellular calcium concentration[Ca2+]i elevated by caffeine which induced the ryanodine receptor operated Ca2+ channels release Ca2+In the free Ca2+ solution with EGTA 2 mmol-L, 10mmol-L caffeine caused the increase of FI of [Ca2+]j. The FI of [Ca2+]j elevated by caffeine was not inhibited by preincubation with F2 0.1, 1, lOumol-L"1, but by the pretreatment with lOmmol-L"1 procaine, an antagonist of ryanodine receptor. The FI elevated by caffeine was also not decreased by the administration of F2 0.1, 1, lOumol-L"1.2.4 Effects of F2on intracellular calcium concentration[Ca2+]j elevated by IP3 which induced the IP3 receptor operated Ca2+ channels release Ca2+In the free Ca2+ solution with EGTA 2 mmol-L, 10mmol-L IP3 caused the increase of FI of [Ca2+];. The FI of [Ca2+]i elevated by IP3 was not inhibited by preincubation with F2 0.1, 1, lOumol-L"1, but by the pretreatment by lOmmol-L"1 heparin, an antagonist of IP3 receptor. The FI elevated by IP3 was also not decreased by the administration of F2 0.1, 1, lOumol-L"1.In summary, the present study has been shown that F2 is a voltage-operated calcium channel blocker. Its inhibitory effects may be related to its combination to the open and inactivated state of calcium channel so that it induces the activation of calcium channel delay and premature appearance of inactivation of calcium channel. Besides of these, F2 possess a high affinity to the inactivated state of calcium channel with a low dissociation with calcium channel. Moreover, F2 posses no effecst on intracellular calcium release. The blockade of calcium channel appears to be the main mechanism of pharmacological effect of F2.