To the Editor: We read the report of Lin et al. (1) with great interest. The authors suggest that observed increases in the Ni2+-sensitive bidirectional current after exposure of the mouse, rat, and guinea pig myocytes to isoproterenol were in fact not attributable to changes in the Na+/Ca2+ exchanger (NCX) current but to an increase in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride current. These data represent an important contribution to this relatively neglected area as well as a challenge to workers who contend that the NCX current is strongly regulated by β-adrenergic receptors and the cAMP signaling cascade.
In support of their contention, Lin et al. (1) show that superfusion of guinea pig myocytes with low-chloride solution results in a dramatic positive shift in the reversal potential and a powerful diminution of the outward component of the Ni2+- sensitive current (see Fig. 4B in Ref. 1). Those who might wish to replicate these experiments will be hampered, however, by the fact that the composition of this low-chloride solution is apparently not included in the report. Instead, the bath solution that is referenced (solution 3) contains as much chloride as other solutions (150 mmol). No doubt this represents an oversight by the authors that can be easily corrected.
Assuming that a low-chloride solution was in fact used, the experiment seems to convincingly point to a large contamination of the Ni2+-sensitive current by CFTR in guinea pig myocytes, and provides an important caveat to those working in small animal models. We did not find evidence of such contamination in myocytes from either the Yorkshire pig or mongrel dogs, instead finding that the outward component of the bidirectional current was rapidly and reversibly inhibited by Ca2+ removal consistent with Ca2+ dependence; this is inconsistent with a large contribution by CFTR (2). Furthermore, although we have not looked for expression of CFTR in the pig, this conductance has been reported to be nearly or completely absent in large animals, such as dogs or humans, but heavily represented in the guinea pig (3). Finally, Lin et al. reported the interesting result that, whereas Ni2+ blocks the CFTR current induced by isoproterenol, it did not inhibit the CFTR induced by forskolin, suggesting that Ni2+ is acting on the β-adrenergic signaling cascade rather than at the channel pore. The implication of this finding, as the authors point out, is that the NCX current measured in the presence of forskolin should not be contaminated by CFTR because Ni2+ would not inhibit this conductance and it would not contribute to the difference current. We found no difference between the magnitude of the Ni2+ sensitive current induced by forskolin, isoproterenol, or cAMP in our failing pig myocytes, arguing against a significant contribution by CFTR in our model (4).
We do not have an explanation for the lack of increase in NCX current reported by Lin et al. (5) after application of isoproterenol and forskolin despite evidence for the existence of a macromolecular complex in rat-containing PKA. Please note that the magnitude of the NCX currents in the report by Lin et al. are twice the magnitude of those we observe in the unstimulated pig myocyte. Comparing current magnitudes across species under different experimental conditions is risky, but it suggests the testable hypothesis that the NCX may be tonically phosphorylated in these species rendering them insensitive to further stimulation.
- Copyright © 2006 the American Physiological Society
To the editor: In the letter by Haigney et al., they discussed the contribution of the CFTR Cl− current in ventricular myocytes of Yorkshire pigs and mongrel dogs when recording Na+/Ca2+ exchange current as a Ni2+-sensitive current. According to their letter and abstract cited, the isoproterenol-stimulated current was not altered under free extracellular Cl− condition, but eliminated by removal of extracellular Ca2+. They found no difference between the magnitude of the Ni2+-sensitive current induced by forskolin, isoproterenol, or cAMP in failing pig myocytes. Provided that the CFTR Cl− channel is not expressed in ventricular myocytes of Yorkshire pigs and mongrel dogs, and that no other current is activated by the β-adrenergic stimulation, the augmentation of the Ni2+-sensitive current may indicate that the Na+/Ca2+ exchange is activated by the β-adrenergic stimulation. If so, we may conclude that the exchanger is regulated through β-adrenergic receptor signaling in the pig and dog, but not in guinea pig, rat, and mouse. It should be noted that ionic currents including both the CFTR Cl− current and the Na+/Ca2+ exchange currents have been extensively studied in the guinea pig and rat cardiac myocytes, and thereby the separation of ionic current are well established. Obviously, further studies of cardiac Na+/Ca2+ exchange in the larger animals are needed to validate the species difference of the Na+/Ca2+ exchange.
In our experiment using a low extracellular Cl− solution (bath solution 3), extracellular 145 mM NaCl was indeed replaced with equimolar Na-aspartate. The reduction of extracellular Cl− concentration resulted in the shift of the reversal potential of the isoproterenol-induced current (the CFTR Cl− current) to the positive potential, which was clearly different than that of the Ni2+-sensitive current before applying isoproterenol (Fig. 4). The composition of bath solution 3 listed in Table 1 of “Article in Press” version was mistyped during our revising process, and was corrected in the final published version.