Na⁺/HCO₃⁻ cotransporter (NBC)e1 catalyze the electrogenic movement of 1 Na⁺:2 HCO₃⁻ into cardiomyocytes cytosol. NBC proteins associate with carbonic anhydrases (CA), CAII, and CAIV, forming a HCO₃⁻ transport metabolon. Herein, we examined the physical/functional interaction of NBCe1 and transmembrane CAIX in cardiac muscle. NBCe1 and CAIX physical association was examined by coimmunoprecipitation, using rat ventricular lysates. NBCe1 coimmunoprecipitated with anti-CAIX antibody, indicating NBCe1 and CAIX interaction in the myocardium. Glutathione- S-transferase (GST) pull-down assays with predicted extracellular loops (EC) of NBCe1 revealed that NBCe1-EC4 mediated interaction with CAIX. Functional NBCe1/CAIX interaction was examined using fluorescence measurements of BCECF in rat cardiomyocytes to monitor cytosolic pH. NBCe1 transport activity was evaluated after membrane depolarization with high extracellular K⁺ in the presence or absence of the CA inhibitors, benzolamide (BZ; 100 μM) or 6-ethoxyzolamide (ETZ; 100 μM) (* P < 0.05). This depolarization protocol produced an intracellular pH (pH_i) increase of 0.17 ± 0.01 ( n = 11), which was inhibited by BZ (0.11 ± 0.02; n = 7) or ETZ (0.06 ± 0.01; n = 6). NBCe1 activity was also measured by changes of pH_i in NBCe1-transfected human embryonic kidney 293 cells subjected to acid loads. Cotransfection of CAIX with NBCe1 increased the rate of pH_i recovery (in mM/min) by about fourfold (12.1 ± 0.8; n = 9) compared with cells expressing NBCe1 alone (3.1 ± 0.5; n = 7), which was inhibited by BZ (7.5 ± 0.3; n = 9). We demonstrated that CAIX forms a complex with EC4 of NBCe1, which activates NBCe1-mediated HCO₃⁻ influx in the myocardium. CAIX and NBCe1 have been linked to tumorigenesis and cardiac cell growth, respectively. Thus inhibition of CA activity might be useful to prevent activation of NBCe1 under these pathological conditions.

Mitochondria represent major sources of basal reactive oxygen species (ROS) production of the cardiomyocyte. The role of ROS as signaling molecules that mediate different intracellular pathways has gained increasing interest among physiologists in the last years. In our lab, we have been studying the participation of mitochondrial ROS in the intracellular pathways triggered by the renin-angiotensin II-aldosterone system (RAAS) in the myocardium during the past few years. We have demonstrated that acute activation of cardiac RAAS induces mitochondrial ATP-dependent potassium channel (mitoK_ATP) opening with the consequent enhanced production of mitochondrial ROS. These oxidant molecules, in turn, activate membrane transporters, as sodium/hydrogen exchanger (NHE-1) and sodium/bicarbonate cotransporter (NBC) via the stimulation of the ROS-sensitive MAPK cascade. The stimulation of such effectors leads to an increase in cardiac contractility. In addition, it is feasible to suggest that a sustained enhanced production of mitochondrial ROS induced by chronic cardiac RAAS, and hence, chronic NHE-1 and NBC stimulation, would also result in the development of cardiac hypertrophy.

BACKGROUND AND PURPOSE Na⁺/HCO₃^- co-transport (NBC) regulates intracellular pH (pH_i) in the heart. We have studied the electrogenic NBC isoform NBCe1 by examining the effect of functional antibodies to this protein. EXPERIMENTAL APPROACH We generated two antibodies against putative extracellular loop domains 3 (a-L3) and 4 (a-L4) of NBCe1 which recognized NBCe1 on immunoblots and immunostaining experiments. pH_i was monitored using epi-fluorescence measurements in cat ventricular myocytes. Transport activity of total NBC and of NBCe1 in isolation were evaluated after an ammonium ion-induced acidosis (expressed as H⁺ flux, J_H, in mmol·L⁻¹ min⁻¹ at pH_i 6.8) and during membrane depolarization with high extracellular potassium (potassium pulse, expressed as ΔpH_i) respectively. KEY RESULTS The potassium pulse produced a pH_i increase of 0.18 ± 0.006 (n= 5), which was reduced by the a-L3 antibody (0.016 ± 0.019). The a-L-3 also decreased J_H by 50%. Surprisingly, during the potassium pulse, a-L4 induced a higher pH_i increase than control,(0.25 ± 0.018) whereas the recovery of pH_i from acidosis was faster (J_H was almost double the control value). In perforated-patch experiments, a-L3 prolonged and a-L4 shortened action potential duration, consistent with blockade and stimulation of NBCe1-carried anionic current respectively. CONCLUSIONS AND IMPLICATIONS Both antibodies recognized NBCe1, but they had opposing effects on the function of this transporter, as the a-L3 was inhibitory and the a-L4 was excitatory. These antibodies could be valuable in studies on the pathophysiology of NBCe1 in cardiac tissue, opening a path for their potential clinical use.

Electroneutral (NBCn1) and electrogenic (NBCe1) isoforms of the Na⁺–HCO₃⁻ cotransporter (NBC) coexist in the heart. We studied the expression and function of these isoforms in hearts of Wistar and spontaneously hypertensive rats (SHR), elucidating the direct implication of the renin–angiotensin system in the NBC regulation. We used myocytes from Wistar, SHR, losartan-treated SHR (Los-SHR), and Angiotensin II (Ang II)-induced cardiac hypertrophy. We found an overexpression of NBCe1 and NBCn1 proteins in SHR that was prevented in Los-SHR. Hyperkalaemic-induced pH_i alkalization was used to study selective activation of NBCe1. Despite the increase in NBCe1 expression, its activity was lower in SHR than in Wistar or Los-SHR. Similar results were found in Ang II-induced hypertrophy. A specific inhibitory antibody against NBCe1 allowed the discrimination between NBCe1 and NBCn1 activity. Whereas in SHR most of the pH_i recovery was due to NBCn1 stimulation, in Wistar and Los-SHR the activity of both isoforms was equitable, suggesting that the deteriorated cardiac NBCe1 function observed in SHR is compensated by an enhanced activity of NBCn1. Using the biotin method, we observed greater level of internalized NBCe1 protein in SHR than in the non-hypertophic groups, while with immunofluorescence we localized the protein in endosomes near the nucleus only in SHR. We conclude that Ang II is responsible for the impairment of the NBCe1 in hypertrophied hearts. This is due to retained transporter protein units in early endosomes. Moreover, NBCn1 activity seems to be increased in the hypertrophic myocardium of SHR, compensating impaired function of NBCe1.

Background: Bicarbonate transport has crucial roles in regulating intracellular pH (pH_i) in a variety of cells. The purpose of this study was to evaluate its participation in the regulation of pHi in resting and stimulated human neutrophils. Methods: Freshly isolated human neutrophils acidified by an ammonium prepulse were used in this study. Results: We demonstrated that resting neutrophils have a bicarbonate transport mechanism that prevents acidification when the Na⁺/H⁺ exchanger is blocked by EIPA. Neutrophils acidified by an ammonium prepulse showed an EIPA-resistant recovery of pHi that was inhibited by the blocker of the anionic transporters SITS or the Na⁺/HCO₃^- cotransporter (NBC) selective inhibitor S0859, and abolished when sodium was removed from the extracellular medium. In western blot and RT-PCR analysis the expression of NBCe2 but not NBCe1 or NBCn1 was detected in neutrophils Acidified neutrophils increased the EIPA-insensitive pHi recovery rate when its activity was stimulated with fMLF/ cytochalasin B. This increase in the removal of acid equivalents was insensitive to the blockade of the NADPH oxidase with DPI. Conclusion: It is concluded that neutrophils have an NBC that regulates basal pH_i and is modulated by chemotactic agents.