PH Regulating Transporters in Neurons From Various Chemosensitive Brainstem Regions in Neonatal Rats

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hypercapnia, locus coeruleus, Na+/H+ exchange, HCO3 transport, NTS, retrotrapezoid nucleus

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We studied the membrane transporters that mediate intracellular pH (pHi) recovery from acidification in brainstem neurons from chemosensitive regions of neonatal rats. Individual neurons within brainstem slices from the retrotrapezoid nucleus (RTN), the nucleus tractus solitarii (NTS), and the locus coeruleus (LC) were studied using a pH-sensitive fluorescent dye and fluorescence imaging microscopy. The rate of pHi recovery from an NH4Cl-induced acidification was measured, and the effects of inhibitors of various pH-regulating transporters determined. Hypercapnia (15% CO2) resulted in a maintained acidification in neurons from all three regions. Recovery in RTN neurons was nearly entirely eliminated by amiloride, an inhibitor of Na+/H+ exchange (NHE). Recovery in RTN neurons was blocked ∼50% by inhibitors of isoform 1 of NHE (NHE-1) but very little by an inhibitor of NHE-3 or by DIDS (an inhibitor of HCO3-dependent transport). In NTS neurons, amiloride blocked over 80% of the recovery, which was also blocked ∼65% by inhibitors of NHE-1 and 26% blocked by an inhibitor of NHE-3. Recovery in LC neurons, in contrast, was unaffected by amiloride or blockers of NHE isoforms but was dependent on Na+ and increased by external HCO3−. On the basis of these findings, pHi recovery from acidification appears to be largely mediated by NHE-1 in RTN neurons, by NHE-1 and NHE-3 in NTS neurons, and by a Na- and HCO3-dependent transporter in LC neurons. Thus, pHi recovery is mediated by different pH-regulating transporters in neurons from different chemosensitive regions, but recovery is suppressed by hypercapnia in all of the neurons.

the response to hypercapnia of brainstem neurons located in various chemosensitive regions is of major interest in the study of the control of respiration. Chemosensitive neurons have been defined as neurons that respond to changes of CO2/H+ and that are found in brainstem areas shown to alter ventilation when exposed to focal acidification (38). A maintained fall of intracellular pH (pHi), with no pHi recovery, has been shown to be an important component of the signaling pathway of hypercapnia in neurons from chemosensitive brainstem areas (17, 19, 38, 42, 51). However, these neurons do exhibit pHi recovery from acidification when external pH is held constant (17, 19, 42). This recovery can be mediated by several different membrane transport proteins including Na+/H+ exchangers (NHE) (33), Na-driven Cl−/HCO3− exchangers (NDCBE), and Na+-HCO3− cotransporters (NBC-both electrogenic and electroneutral) (12, 37, 45). To our knowledge, there is no evidence for any functional HCO3-dependent transporter in neurons from any central chemosensitive region.

Previous studies have suggested that NHE is the predominant transport protein mediating pHi recovery from acidification in brainstem neurons (32, 42, 53). Under conditions of extracellular acidification, as with hypercapnic acidosis, NHE is inhibited and chemosensitive neurons do not exhibit pHi recovery from acidification (41, 42). Several isoforms of the NHE protein exist and are found to be active in different parts of the body. Of the nine isoforms, only five are thought to be present in rat brains (12). NHE-1 is the ubiquitous isoform, is very sensitive to amiloride inhibition (37), and is completely inhibited by cariporide (HOE642). NHE-2 and NHE-3 are found predominantly in the gastrointestinal tract (3) and the kidney (37) but are also present in the brain. NHE-3 has a relatively specific inhibitor, S1611 (53, 58). The isoform NHE-4 is found in the hippocampus (7) and is thus not of interest in this study. NHE-5 is found in several regions of the rat brain, is closely related to NHE-3, and has no specific inhibitor (2).

Knowing that there are several isoforms of NHE present in the brain, the next issue to address is the question of which isoform is mediating pHi recovery in neurons from various chemosensitive brainstem regions. We have evidence that amiloride inhibits recovery from acidification in neurons from the nucleus of the solitary tract (NTS) and the ventrolateral medulla (VLM) (42). Studies have also been conducted in the VLM showing that NHE-3 mediates pHi regulation as well. Expression of mRNA for NHE-3 was found in some areas of the brain involved in respiratory control (23). When looking at cultured VLM neurons, exposure to NHE-3-specific inhibitors resulted in neuronal acidification and increased firing rate (58). In in vivo studies, specific NHE-3 inhibitors have been shown to stimulate the central respiratory system (22), and studies in which the brain-permeant NHE-3 inhibitor S8218 was applied to live rabbits showed that the drug caused an increase in ventilation (23). These studies suggest that NHE-3 is important in pHi regulation in neurons from chemosensitive brainstem regions.

The aim of our study was to determine which pH-regulating transporters mediate recovery from acidification in neurons from three chemosensitive regions of the brainstem: the retrotrapezoid nucleus (RTN), the NTS, and the locus coeruleus (LC). We particularly wanted to find evidence for any functional HCO3-dependent transporters and which isoforms of NHE are active in the neurons from the various regions. We found that NHE-1 predominates in NTS and RTN neurons, with a smaller possible contribution from NHE-3, while a Na- and HCO3-dependent transporter predominates in LC neurons.

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American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, v. 297, issue 5, p. R1409-R1420