Pinacidil, a KATP channel opener stimulates a cardiac Na⁺/Ca²⁺ exchanger function through NO/cGMP/PKG pathways in guinea-pig cardiac myocytes

Abstract

Pinacidil, a nonspecific ATP-sensitive K⁺ (KATP) channel opener, has cardioprotective effects against hypertension, ischemia/reperfusion injury and arrhythmia. Pinacidil was reported to inhibit ouabain-induced delayed afterdepolarizations (DADs), which are caused by the inward Na⁺/Ca²⁺ exchange current (I_NCX), the causal relationship between pinacidil and cardiac Na⁺/Ca²⁺ exchanger (NCX1) is still elusive. Thus, we investigated the effect of pinacidil on the NCX1.

The isolated ventricular myocytes were obtained from male guinea-pig (200 ～ 250 g) by enzymatic dissociation. To evaluate the effect of pinacidil on NCX1, we estimated I_NCX with the whole-cell patch-clamp technique and intracellular Ca²⁺ with Fura-2/AM (Ca²⁺-indicator). L-NAME [an endothelial NO synthase (eNOS) inhibitor], ODQ [a soluble guanylyl cyclase (sGC) inhibitor], KT5823 [a cGMP-dependent protein kinase (PKG) inhibitor], glibenclamide [a plasma membrane KATP channel inhibitor] and 5-HD [a mitochondrial KATP channel inhibitor] were applied to ensure the contribution of each pathway with NCX1 activation.

In patch-clamp study, I_NCX was induced by 20 mM Na⁺ and 226 nM of free Ca²⁺ in the pipette solution and 140 mM Na⁺ and 1mM Ca²⁺ in the external solution. Pinacidil enhanced I_NCX in a concentration-dependent manner. The EC₅₀ values were 23.5 μM and 23.0 μM for the outward and inward components of I_NCX, and the Hill coefficients were 1.1, respectively. We also confirmed the effect of pinacidil on increasing intracellular Ca²⁺ through cardiac I_NCX with changing from 140 mM Na⁺ external solution to Na⁺-free external solution by using Fura-2/AM method in isolated guinea-pig ventricular myocytes. Pinacidil (30 μM) enhanced intracellular Ca²⁺ concentration ([Ca²⁺]_i). NOS/cGMP/PKG signaling pathway inhibitors (L-NAME, ODQ and KT-5823) suppressed the pinacidil-induced [Ca²⁺]_i increase by the outward components of NCX1, whereas the inhibitors of plasma membrane and mitochondrial KATP channel (glibenclamide and 5-HD) did not blocked, respectively.

In conclusion, enhancement of the NCX1 function by pinacidil may be contributed to KATP channel-independent NOS/cGMP/PKG signaling pathways, not through plasma membrane and mitochondrial KATP channels opening.