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Volume 2 Supplement 1

Neural Control of Breathing

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CO2chemotransduction in central neurons: role of intracellular pH (pHi) and extracellular pH (pHo)

We have been studying the role of changes of pHi as part of the signaling pathway of hypercapnia in neonatal rat brainstem neurons from both chemosensitive (NTS-nucleus tractus solitarius, VLM-ventrolateral medulla, LC-locus coeruleus) and non-chemosensitive (IO-inferior olive, Hyp-hypoglossal) regions. We are testing the model of chemotransduction that proposes that hypercapnia acidifies neurons, which leads to inhibition of K+ channels, resulting in neuronal depolarization and increased firing rate. We measure pHi in individual neurons within brain slices using fluorescence imaging microscopy [1,2]. We developed a new technique to measure pHi and Vmsimultaneously by loading pH-sensitive fluorescent dyes using perforated patch (amphotericin B) pipettes in LC neurons [3]. All studies were done at 37°C.

Neurons from all brainstem regions have high intrinsic buffering power, around 45 mM/pH unit for NTS, VLM, IO and Hyp neurons [1] and about 90 mM/pH unit for LC neurons [3]. The only pH-regulating transporter available for recovery from an acid load in these neurons is Na+/H+ exchange (NHE) [1,2]. The only HCO3-dependent transporter present is the acidifying Cl-/HCO3 - exchanger, which has been found in VLM, IO, Hyp and some NTS neurons [1]. Interestingly, the NHE in neurons from chemosensitive regions (eg, NTS and VLM) is more sensitive to inhibition by decreased pHo than neurons from non-chemosensitive regions (eg, IO and Hyp) [1].

We observed different responses to hypercapnia in neurons from chemosensitive vs. non-chemosensitive regions. Hypercapnic acidosis (HA–10% CO2, pHo 7.15) resulted in a maintained fall of pHi in neurons from chemosensitive areas whereas pHi recovery from acidosis was evident in neurons from non-chemosensitive areas [2]. Under conditions of isohydric hypercapnia (IH–10% CO2, 52 mM HCO3-, pHo 7.45), pHi recovery was seen in neurons from all regions [2], indicating that pH-regulating mechanisms are present in all these neurons but that they are inhibited more fully by decreased pHo in neurons from chemosensitive regions. The same pHi responses to HA (15% CO2, pHo 6.8) and IH (15% CO2, 77 mM HCO3-, pHo 7.45) were seen in LC neurons where pHi and Vm were measured simultaneously. The changes of pHi ccurred before the changes in neuronal firing rate [3]. In LC neurons, HA resulted in a larger fall of pHi (0.27 vs. 0.15 pH unit) and a larger increase in firing rate (1.31 vs. 1.06 Hz) than seen with IH. Notably, increased firing rate was accompanied by membrane depolarization (2.5 mV) in response to HA but by membrane hyperpolarization (2.3 mV) in response to IH. Other acid challenges involving decreased pHo (acidified HEPES; isocapnic acidosis–5% CO2, 7 mM HCO3-, pHo 6.9) resulted in increased firing rate with depolarization while acid challenges involving constant pHo (50 mM propionate, pHo 7.45) resulted in slightly increased firing rate with membrane hyperpolarization.

In summary, the response of Vm to a fall of pHi is dependent on whether changes or not and is not well correlated with pHo increased firing rate in LC neurons. The parameter that best correlates with increased firing rate in response to an acid challenge is the change of pHi, indicating that pHi is likely involved as part of the chemosensitive signaling pathway in LC neurons.


  1. Ritucci NA, Chambers-Kersh L, Dean JB, Putnam RW: Intracellular pH regulation in neurons from chemosensitive and non-chemosensitive areas of the medulla. Am J Physiol. 1998, 275: R1152-1163.

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  2. Ritucci NA, Dean JB, Putnam RW: Intracellular pH response to hypercapnia in neurons from chemosensitive areas of the medulla. Am J Physiol. 1997, 273: R433-441.

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  3. Filosa JA, Dean JB, Putnam RW: Intracellular pH as the proximal signal in the chemosensitive response of locus coeruleus neurons. FASEB J. 2001, 15: A151-

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This work was funded by NIH Grant R01-HL56683.

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Putnam, R. CO2chemotransduction in central neurons: role of intracellular pH (pHi) and extracellular pH (pHo). Respir Res 2 (Suppl 1), 6.4 (2001).

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