1. In the stretch receptor neurones of the crayfish Astacus astacus, the intracellular pH (pHi), the intracellular Na+ concentration ([Na+]i) and the membrane potential (Em) were measured simultaneously using ion-selective and conventional microelectrodes. Normal Astacus saline (NAS), and salines containing varying amounts of Ca2+ (Ca2+-NAS) but of constant ionic strength, with Na+, Mg2+ or Ba2+ as substituting ions, were used to investigate the effects of extracellular Ca2+ concentration ([Ca2+]o) on pHi and pHi regulation, on [Na+]i and on Em. The maximum rate of pHi recovery was used as a measure of pHi regulation. Acid loads were imposed using the NH4+/NH3 rebound technique. 2. [Ca2+]o affected pHi, pHi regulation, [Na+]i and Em. The magnitudes of the effects were inversely related to [Ca2+]o and were specific to the ion used for [Ca2+]o substitution. 3. Compared with controls, increasing [Ca2+]o threefold (in exchange for Na+) elicited some alkalization, a 7 % faster maximum rate of pHi recovery and generally lower values of [Na+]i. 4. In low-Ca2+ or Ca2+-free NAS (substitutions by Na+ or Mg2+), pHi became more acid, the maximum rate of pHi recovery was reduced by up to 50 % and [Na+]i was generally higher. The effects were faster and larger at lower [Ca2+]o, and stronger with Na+ than with Mg2+ as the substituting ion. 5. In Ca2+-free NAS (Ca2+ substituted for by Ba2+), the effects on pHi, on the maximum rate of pHi recovery and on [Na+]i were generally small. In this respect, Ba2+ had similar physiological properties to Ca2+ and was almost equally effective. 6. Changes in Em, including rapid depolarizations and occasional burst activity in Ca2+-free NAS, indicate that alterations in the properties of the membrane, such as a change in its permeability or selectivity, are occurring. Measurements of [Na+]i support this view. In addition, Ba2+ per se induced a (small) depolarization, as shown when Ba2+ was present in NAS or in low-Ca2+ NAS. 7. Changes in [Ca2+]o affected [Na+]i. *[Na+]i is defined as [Na+]i determined at the onset of the maximum rate of pHi recovery, and the ratio *[Na+]i/[Na+]o at that instant was calculated. A linear relationship between the maximum rate of pHi recovery and the *[Na+]i/[Na+]o ratio was found, irrespective of the amount and of the ion species used for [Ca2+]o substitution. This is strong evidence that pHi and pHi regulation were indirectly affected by [Ca2+]o, which altered membrane properties and thus caused a change in [Na+]i. We could find no evidence for a direct contribution of [Ca2+]o to acid extrusion or to a direct modulatory action on the transport protein of the Na+/H+ antiporter.
Regulation of intracellular pH (pHi) following acidosis induced by NH4+/NH3 exposures was re-investigated in a crayfish stretch receptor neurone using H+- and Na+-selective microelectrodes. All experiments were performed in nominally HCO3-/CO2-free salines. From studies in Na+-free saline and from electrochemical calculations, we concluded that pHi regulation was dependent on extracellular Na+ concentration ([Na+]o). The half-maximal rate of pHi recovery had an apparent Michaelis-Menten constant of 21 mmol l-1 [Na+]o. The use of this experimental approach and an improved technique enabled us to observe pHi regulation even in Cl-- free saline, in contrast to earlier findings. In addition, amiloride (2 mmol l-1) inhibited the maximum rate of pHi recovery by about 80 %, SITS (1 mmol l- 1) by about 20 %. The results strongly suggest the operation of two separate pHi-regulating mechanisms, a Na+-dependent HCO3-/Cl- antiporter (probably the Na+/H+/HCO3-/Cl- antiporter described earlier) and a Na+/H+ antiporter. Both mechanisms have been described in crayfish ganglion cells and muscle fibres, but the individual contribution to pHi regulation varies considerably in these preparations. Functional aspects of the pHi-regulating mechanisms in relation to ionic changes during the moulting cycle are discussed.
1. An NH 4 + -selective membrane for microelectrodes (NH 4 ISMs) was tested under ‘biological conditions’ in normal Astacus saline (NAS), two simulated intracellular salines (SIS) and the sensory neurone of the crayfish stretch receptor. 2. The effects of several physiological variables on intracellular NH 4 + measurements were tested in vitro . Changes in the background K + and Na + concentrations, the ratio K + /Na + , pH, ionic strength, osmotic pressure and volume were examined. 3. Phenomena specific for NH 4 ISMs, such as a positive potential shift, an undershoot and a difference between pre- and postcalibration curves are described and discussed. We propose to consider the values of intracellular NH 4 + concentration as apparent. 4. The detection limit of the NH 4 ISM is closely related to background K + concentration. It is in the region of 0.1 mmoll −1 NH 4 + in NAS (at 5.4mmol l −1 K + ) and about 5mmoll −1 in SIS (at 194mmol l −1 K + ). 5. When comparing levels of intracellular NH 4 + , either measured directly by NH 4 ISM or calculated, according to Boron and de Weer ( J. gen. Physiol. 67 , 91–112), from simultaneously recorded pHi, we found that [NH 4 + ] i obtained by direct measurement differed quantitatively from that of the Boron and de Weer model, but that some of the qualitative and temporal aspects of the model agreed with our results. The quantitative difference in [NH 4 + ] i determined by the two methods cannot be attributed to temporal and/or quantitative limitations of the NH 4 ISM.