Selective inhibition of the sodium proton exchanger subtype 3 (NHE3) as a tool to study central chemosensitivity

In vitro studies on organotypic medullary cultures of new born rats (OMC) have shown that CO₂/H ⁺-sensitive neurons of the ventrolateral medulla oblongata (VLNcs) increase their bioelectric activity upon a decrease of intracellular pH (pHi), while changes of extracellular pH are of minor importance [1]. Therefore, acid extrusion of VLNcs appears to be a key element for the control of steady state pHi and hence neuronal activity. To extrude H⁺, ventrolateral medullary neurons use sodium proton exchange but not Na⁺-dependent Cl^-/HCO₃ ^- exchange [2]. Our previous studies have shown that NHE3 is expressed in ventrolateral parts of OMC (obex level). Furthermore, the NHE3 inhibitor S1611 (1 μmol/l) decreased pHi and mimicked the bioelectric response of VLNcs to hypercapnia [3]. Due to these findings we hypothesized that NHE3 as a membrane transporter plays an important role for the control of chemosensitivity and breathing. Aim of this study was to test a new brain permeant NHE3 inhibitor (S8218) for effects on pHi, bioelectric activity and phrenic nerve activity in vivo.

For in vitro studies OMC were prepared and kept in R16 medium for 2–3 weeks [3]. Fluorimetric measurements of pHi were carried out after loading neurons with BCECF AM as described [3]. Membrane potentials were recorded with sharp microelectrodes. S8218 at concentrations of 1–2 μmol/l lowered the steady state pHi of a subset of ventrolateral neurons (the IC50 of S8218 for NHE3 = 0.81 μmol/l). In these cells pHi regulation after an ammonium prepulse (20 mmol NH₄Cl/l, 3 min) was also impaired by S8218. In contrast, cells whose steady state pHi was not altered by S8218 showed normal pHi recovery after ammonium prepulse. This finding strongly suggests that NHE3 expressing cells in culture use the NHE3 for maintainance of their steady state pHi. Accordingly, S8218 also changed the bioelectric activity of VLNcs: firing rates increased by up to 200% and periodic discharges appeared. Changes of the bioelectric activity resembled the neuronal responses to hypercapnia. In general, the effect was smilar to the effects of S1611 and S3226 [cf. 3].

For in vivo studies anaesthetized, ventilated and vagotomized rabbits were used. By means of RT-PCR, NHE3 mRNA was detected in homogenates of the rabbits' brainstems. Indirect immunocytochemistry showed that NHE3 immunoreactive protein was expressed in ventrolateral areas of the brainstem. Cumulative doses of S8218 (9.2 ± 1.1 mg/kg) resulting in plasma concentrations of 0.9 ± 0.2 μg/ml increased integrated phrenic nerve activity (IPNA, measured as IPNA^*f_R) by 51 ± 6.4% (n = 7, P < 0.0001). Phrenic nerve responses to increased PaCO₂ were also changed: at a plasma concentration of 0.3 μg S8218/ml the PaCO₂ at the apneic threshold was lowered by 0.43 ± 0.1 kPa (n = 7, P < 0.01). In 4 out of 7 animals even strong hyperventilation failed to suppress phrenic nerve activity completely suggesting an ongoing activity at least of parts of the respiratory network. In the range of PaCO₂ being 1–6 kPa above the apneic threshold we observed a 38 ± 8.5% increase of IPNA^*f_R (35 measurements in 7 animals).

The data underline the role of NHE3 for central chemosensitivity both in vitro and in vivo. We suggest that also in vivo a decrease in intracellular pH of NHE3 expressing neurons precedes the augmentation of the respiratory drive. Since NHE3-inhibitors act on central chemoreception of rats, rabbits and also piglets [cf. 4], these drugs may become useful tools to study mechanisms of central chemosensitivity.