Coupling of histamine H3 receptors to neuronal Na+/H+ exchange: a novel protective mechanism in myocardial ischemia

Abstract

In myocardial ischemia, adrenergic nerves release excessive amounts of norepinephrine (NE), causing dysfunction and arrhythmias. With anoxia and the concomitant ATP depletion, vesicular storage of NE is impaired, resulting in accumulation of free NE in the axoplasm of sympathetic nerves. Intraneuronal acidosis activates the Na(+)/H(+) exchanger (NHE), leading to increased Na(+) entry in the nerve terminals. These conditions favor availability of the NE transporter to the axoplasmic side of the membrane, causing massive carrier-mediated efflux of free NE. Neuronal NHE activation is pivotal in this process; NHE inhibitors attenuate carrier-mediated NE release. We previously reported that activation of histamine H(3) receptors (H(3)R) on cardiac sympathetic nerves also reduces carrier-mediated NE release and alleviates arrhythmias. Thus, H(3)R activation may be negatively coupled to NHE. We tested this hypothesis in individual human SKNMC neuroblastoma cells stably transfected with H(3)R cDNA, loaded with the intracellular pH (pH(i)) indicator BCECF. These cells possess amiloride-sensitive NHE. NHE activity was measured as the rate of Na(+)-dependent pH(i) recovery in response to an acute acid pulse (NH(4)Cl). We found that the selective H(3)R-agonist imetit markedly diminished NHE activity, and so did the amiloride derivative EIPA. The selective H(3)R antagonist thioperamide abolished the imetit-induced NHE attenuation. Thus, our results provide a link between H(3)R and NHE, which may limit the excessive release of NE during protracted myocardial ischemia. Our previous and present findings uncover a novel mechanism of cardioprotection: NHE inhibition in cardiac adrenergic neurons as a means to prevent ischemic arrhythmias associated with carrier-mediated NE release.

Figure 1

Representative experimental traces from individual SKNMC-H₃ cells showing the Na⁺-dependent pH_i recovery after acute exposure to an NH₄Cl acid pulse. (A) A control cell: The y axis represents the pH_i as determined from the intracellular calibration of the dye in this cell. The cell was initially superfused with Na⁺-Ringer's solution (NaR), then with 20 mM NH₄Cl. Acute exposure to NH₄Cl resulted in acidification of the cytosol to ≈pH_i 6.2 after its removal. In the absence of extracellular Na (0 Na), there was no measurable pH_i recovery. With the reintroduction of extracellular Na⁺ (NaR), pH_i increased with intracellular alkalinization occurring at a rate of 0.094 pH_i units/min (dotted line). The final pH_i, achieved in the cell shown in this trace, was higher than the starting pH_i (6.8 vs. 6.6). This overshoot of the final pH_i relative to the initial resting pH_i was seen in the majority of control cells studied and is shown in the bar graph accompanying A. For the 221 control cells studied, the initial pH_i was 6.69 ± 0.01 and the final pH_i was significantly higher at 6.76 ± 0.01 (P < 0.001). (B) Effect of imetit: The y axis represents pH_i as determined from the intracellular calibration of the dye in this cell. Imetit (100 nM) was present in the superfusate from the addition of NH₄Cl until the end of the protocol as shown. Acute exposure to NH₄Cl resulted in acidification of the cytosol to ≈6.2 after its removal. In the absence of extracellular Na⁺ (0 Na), there was no measurable pH_i recovery. With the reintroduction of extracellular Na⁺ (NaR), pH_i started to increase with intracellular alkalinization occurring at a rate of 0.050 pH_i units/min. The mean rate of Na⁺-dependent pH_i recovery was significantly less than that observed in the control cells (P < 0.0001) (Figs. 2 and 3). The final pH_i achieved in the cell shown in this trace was lower than the starting pH_i (6.7 vs. 6.5). This pH_i undershoot relative to the starting pH_i was seen in the majority of cells exposed to imetit, as shown in the bar graph of B. Unlike control cells, the final pH_i of the 277 imetit-treated cells was significantly lower than the initial pH_i (6.61 ± 0.01 vs. 6.74 ± 0.01; P < 0.0001).

Figure 2

Histogram showing variability of Na⁺-dependent pH_i recovery rates (NHE activity) in control (A) and imetit-treated SKNMC-H₃ cells (B). The ordinates represent the number of cells and the abscissae the Na⁺-dependent pH_i recovery rates binned in 0.09 pH_i unit/min increments. A illustrates the variability of the response of 221 control cells studied from five coverslips, and B, the response of 277 cells from six coverslips exposed to imetit (100 nM).

Figure 3

Comparison of NHE activity in the absence and presence of imetit, EIPA, thioperamide, and imetit plus thioperamide in SKNMC-H₃ cells. The mean control Na⁺-dependent pH_i recovery rate or NHE activity (pH_i units/min) is compared with rates measured in cells exposed to imetit (100 nM), EIPA (10 μM), thioperamide (300 nM), and thioperamide in combination with imetit. *, Significantly different (P < 0.0001) from control NHE activity. Values are means ± SEM, and n refers to the number of cells studied. Imetit, thioperamide, and EIPA were present in the superfusate from the NH₄Cl pulse to the end of the experiment. In the experiments with imetit and thioperamide in combination, thioperamide was first introduced in the NH₄Cl solution, and, 5 min later, imetit was added to the Na⁺-free solution, for an additional 5 min, before the reintroduction of Na⁺ into the superfusate.