The Na⁺–HCO₃⁻ cotransporter (NBC) is an important sarcolemmal acid extruder in cardiac muscle. The characteristics of NBC expressed functionally in heart are controversial, with reports suggesting electroneutral (NBCn; 1HCO₃⁻ : 1Na⁺; coupling coefficient N= 1) or electrogenic forms of the transporter (NBCe; equivalent to 2HCO₃⁻ : 1Na⁺; N= 2). We have used voltage‐clamp and epifluorescence techniques to compare NBC activity in isolated ventricular myocytes from rabbit, rat and guinea pig. Depolarization (by voltage clamp or hyperkalaemia) reversibly increased steady‐state pH_i while hyperpolarization decreased it, effects seen only in CO₂/HCO₃⁻‐buffered solutions, and blocked by S0859 (cardiac NBC inhibitor). Species differences in amplitude of these pH_i changes were rat > guinea pig ≈ rabbit. Tonic depolarization (−140 mV to −0 mV) accelerated NBC‐mediated pH_i recovery from an intracellular acid load. At 0 mV, NBC‐mediated outward current at resting pH_i was +0.52 ± 0.05 pA pF⁻¹ (rat, n= 5), +0.26 ± 0.05 pA pF⁻¹ (guinea pig, n= 5) and +0.10 ± 0.03 pA pF⁻¹ (rabbit, n= 9), with reversal potentials near −100 mV, consistent with N= 2. The above results indicate a functionally active voltage‐sensitive NBCe in these species. Voltage‐clamp hyperpolarization negative to the reversal potential for NBCe failed, however, to terminate or reverse NBC‐mediated pH_i‐recovery from an acid load although it was slowed significantly, suggesting electroneutral NBC may also be operational. NBC‐mediated pH_i recovery was associated with a rise of [Na⁺]_i at a rate ∼25% of that mediated via NHE, and consistent with an apparent NBC stoichiometry between N= 1 and N= 2. In conclusion, NBCe in the ventricular myocyte displays considerable functional variation among the three species tested (greatest in rat, least in rabbit) and may coexist with some NBCn activity.

Endothelin-1 (ET-1) has been implicated in fibroblast proliferation. However, the mechanism involving ET-1 is not clear. The present study was performed to examine the role of endogenous ET-1 in ET-1–stimulated fibroblast proliferation and to investigate the regulatory mechanism of ET-1–induced ET-1 gene expression in cardiac fibroblasts. Both ET_A receptor antagonist [(hexahydro-1H-azepinyl)carbonyl-Leu-d-Trp-d-OH (BQ485)] and endothelin-converting enzyme inhibitor (phosphoramidon) inhibited the increased DNA synthesis caused by ET-1. ET-1 gene was induced by ET-1, as revealed with Northern blotting and ET-1 promoter activity assay. ET-1 increased intracellular reactive oxygen species (ROS), which were significantly inhibited by BQ485 and antioxidants. Antioxidants suppressed ET-1 gene expression and DNA synthesis stimulated by ET-1. ET-1 activated mitogen-activated protein kinases (MAPK), including extracellular signal-regulated kinase (ERK), p38 MAPK, and c-Jun N-terminal kinase, which were significantly inhibited by antioxidants. Only ERK inhibitor U0126 could inhibit ET-1–induced transcription of the ET-1 gene. Cotransfection of dominant-negative mutant of Ras, Raf, and MEK1 decreased the ET-1–induced increase in ET-1 transcription, suggesting that the Ras-Raf-ERK pathway is required for ET-1 action. Truncation and mutational analysis of the ET-1 gene promoter showed that the activator protein-1 (AP-1) binding site was an importantcis-element in ET-1–induced ET-1 gene expression. Antioxidants attenuated the ET-1–stimulated AP-1 binding activity. Our data suggest that ROS were involved in ET-1–induced fibroblast proliferation and mediated ET-1–induced activation of ERK pathways, which culminated in ET-1 gene expression.

Hoe 694 (3‐methylsulphonyl‐4‐piperidinobenzoyl, guanidine hydrochloride) is a Na⁺/H⁺ exchange (NHE) inhibitor exhibiting cardioprotective properties during ischaemia and reperfusion in animal hearts. We have (i) tested the selectivity of Hoe 694 for NHE over other pH_i‐regulating mechanisms in the myocardium, and (ii) tested if the functionally important NHE isoform contributing to intracellular pH regulation in heart is NHE‐1, as suggested from molecular biology studies of this protein. pH_i was recorded by fluorescence microscopy with carboxy SNARF‐1, AM‐loaded into single ventricular myocytes of guinea‐pig. In nominally HCO₃⁻‐free media, recovery of pH_i from an intracellular acid load is mediated by NHE, and was inhibited by Hoe 694, amiloride (an NHE inhibitor) or dimethyl amiloride (DMA, a high affinity NHE inhibitor) with potency values of 2.05, 87.3 and 1.96 μm respectively, giving the potency series: Hoe 694 ⋍ DMA > > amiloride. This potency series, and the potency values (corrected for drug competition with extracellular Na⁺) match those determined previously for cloned NHE‐1 expressed in mutant fibroblasts. In the absence of extracellular Na⁺ (to inhibit NHE), Hoe 694 had no effect on pH_i. In 5% CO₂/HCO₃⁻‐buffered solution containing DMA, pH_i recovery from acidosis is mediated by Na⁺‐HCO₃⁻ symport and was unaffected by Hoe 694. The drug also had no effect on pH_i recovery from an alkali‐load, a process largely mediated by Cl⁻‐HCO₃⁻ exchange. Finally, the fall of pH_i upon adding extracellular Na‐lactate is assisted by H⁺‐lactate symport, and this too was unaffected by Hoe 694. We conclude (i) Hoe 694 has no detectable inhibitory potency for pH‐regulating carriers in heart other than NHE. (ii) native NHE functioning during pH_i‐regulation in the cardiomyocyte is the NHE‐1 isoform. These data strengthen the case for NHE‐1 being the receptor for mediating the cardioprotective effects of Hoe 694.