Effects Of N-n-butyl Haloperidol Iodide (F₂) On Inwardly Rectifying Potassium Channels Of Cardiac Myocytes

Haloperidol (Hal) is a typical antipsychotic agent. Our previous research showed that Hal had clear vasodilatory and anti-myocardial ischemia effects. However, it cannot be used as an anti-myocardial ischemia drug due to its adverse effects on extrapyramidal system. In consideration of this, we have synthesized a series of polarity-increased quaternary ammonium salt derivatives of Hal. Thus, they do not pass through the blood-brain barrier, eliminating the side effects of Hal on the central nervous system (CNS) while preserving the beneficial cardiac and vascular effects. N-n-butyl haloperidol iodide ( F₂ ) was screened from a series of quaternary ammonium salt derivatives of Hal. Researches have shown that F₂ does not pass the blood-brain barrier, hat no side effects on CNS but maintains a vasodilating effects. Further studies performed on the whole-body, organ and cellular level have established that F₂ can antagonize the reduction of coronary flow induced by various substances, and has protective effects on myocardial ischemia reperfusion injury. Patch clamp and laser scanning confocal microscopy have established that F₂ could inhibit I_ca of vascular smooth muscle cells and ventricular myocytes by blocking L-type calcium channel. Also, F₂ canevoke potassium channel of vascular smooth muscle cells and block I_t0 of ventricular myocytes. Moreover, experiment results indicated that F2 attenuates cardiac dysfunctoin associated with ischemia/reperfusion without affecting the heart rate. This is in contrast to the control drug Verapamil, which clearly reduces the heart rate. The parasympathetic transmitter acetylcholine slows the heart rate by activating the inwardly rectifying muscarinic potassium channels via a membrane delimited pathway. A signalling system characteristic of supraventricular tissue is represented by the muscarinic K+-current (/?(ACh))- This current in supraventricular cells is a major target of vagal regulation of cardiac frequency [21). Gating of /k(ach) channels is controlled by a pertusssis-toxin sensitive G-protein, most likely Gi. There is growing evidence that the G protein By-subunit released by the activated M2 muscarinic acetylcholine receptor (M2 AChR) acts directly on the muscarinic potassium channel to cause channel activation. In the present study, cell culture and the patch-clamp technique in whole cell configuration were used. The objective of experiment was to determine the effects of quaternary ammonium salt derivative of haloperidol (F2) on muscarinic K+ current in adult guinea-pig atrial myocytes and on classical inwardly rectifzing K+ channel in adult rat ventricular myocytes.1. Effect of /V-n-butyl haloperidol iodide on muscarinic K+ channel (Vcn)) in guinea-pig atrial myocytesMETHODS:I. Culture of atrial myocytesSingle atrial myocytes from adult guinea-pig hearts of either sex were enzymatically isolated cultured, as described earlier1251. These atrial cells can bekept in culture for several days, while they maintain their myojor physiological properties. Cells were plated several thousand cells/dish on 36-mm culture dishes. The culture medium was fetal-calf-serum-free bicabonate-buffered M199 containing gentamicin (1Ojug / ml) and kanamycin (10/L/g / ml). The Medium was changed 24 h after plating and then every second day. Myocytes were used experimentally from day 0 until day 8 after isolation in culture. No effects of time in culture were found for the key experiments.II. Current measurementMembrane currents were measured using the whole cell mode patch clamp (Hamill). Experiments were performed at ambient temperature (22-24°C). k and expediat inactivation of )- F2 canresult in a reduction of acetylcholine concentration curve.B. F2 caused the reversible inhibition of the inward current (/k(ACh>) in a concentration-dependent manner with an IC50 of 29.15 /iM.C. When myocytes were loaded with GTP-y-S in pipette filling solution, F2 induced inhibition of /k(Ach) in reversible and concentration-dependent.D. Similarly, it is also found that Intracellular F2 failed to inhibit /k(ACh) ?E. A staturating concentration of acetylcholine (ACh) elicited muscarinic K+ current (/k since (i) inhibition was independent of the nature of the activating receptor, (ii) F2 acted from the outside only. The components of the signalling pathway accessible from the outside are, apart from the receptors, the channel proteins, (iii) Intracelluar F2 failed to inhibit /?(ACh) , interference of F2 with Gey-GIRK interaction can be excluded as a mechanism underlying its inhibition of /?(ACh). On the ather hand, F2 has the differential selectivity in different inwardly rectifying potassium channels.