Effects of noradrenaline on intracellular pH in acutely dissociated adult rat hippocampal CA1 neurones

1. We examined the effects of noradrenaline on steady-state intracellular pH (pH(i)) and the recovery of pH(i) from internal acid loads imposed by the NH4+ prepulse technique in hippocampal CA1 neurones acutely dissociated from adult rats. 2. Under nominally HCO3--free conditions, acid extrusion was accomplished by a Na+-dependent mechanism, probably the amiloride-insensitive variant of the Na+-H+ exchanger previously characterized in both fetal and adult rat hippocampal neurones. In the presence of external HCO3-, acid extrusion appeared to be supplemented by a Na+-dependent HCO3--Cl- exchanger, the activity of which was dependent upon the absolute level of pH(i). 3. Noradrenaline evoked a concentration-dependent and sustained rise in steady-state pH(i) and increased rates of pH(i) recovery from imposed intracellular acid loads. The effects of noradrenaline were not dependent upon the presence of external HCO3- but were blocked by substituting external Na+ with N-methyl-D-glucamine, suggesting that noradrenaline acts to increase steady-state pH(i) by increasing the activity of the Na+-H+ exchanger. 4. The effects of noradrenaline on steady-state pH(i), and on rates of pHi recovery from imposed acid loads were mimicked by beta(1)(-) and beta(2)(-), but not alpha-, adrenoceptor agonists. The beta-adrenoceptor antagonist propranolol blocked the ability of noradrenaline to increase both steady-state pH(i) and rates of pH(i) recovery from acid loads. 5. The effects of noradrenaline on steady-state pH(i) and on pH(i) recovery rates following acid loads were not dependent on changes in [Ca2+](i). However, the effects of noradrenaline were blocked by pre-treatment with the adenylate cyclase inhibitor 2',5'-dideoxyadenosine and the cAMP-dependent protein kinase inhibitors R-p-adenosine-3',5'-cyclic monophosphorothioate (sodium salt; R-p-cAMPS) and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline- sulphonamide (H-89). 6. Forskolin, an activator of endogenous adenylate cyclase, and 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, mimicked the ability of noradrenaline to increase both steady-state pH(i) and rates of pH(i) recovery from imposed acid loads, as did Sp-cAMPS, a selective activator of cAMP-dependent protein kinase. The effect of forskolin on steady-state pH(i) was blocked by pre-treatment with Rp-cAMPS whereas the effect of Sp-cAMPS was enhanced by pre-treatment with the protein phosphatase inhibitor, okadaic acid. 7. Noradrenaline also increased steady-state pH(i) and rates of pH(i) recovery from imposed acid loads in cultured postnatal rat hippocampal neurones. In this preparation, the effects of noradrenaline were occluded by 18-24 h pre-treatment with cholera toxin. 8. We conclude that noradrenaline increases the activity of the Na+-H+ exchanger in rat hippocampal neurones, probably by inducing an alkaline shift in the pH(i) dependence of the antiport, thereby raising steady-state pH(i). The effects of noradrenaline are mediated by beta-adrenoceptors via a pathway which involves the alpha-subunit of the stimulatory G-protein G(s) (G(s alpha)), adenylate cyclase, cAMP and the subsequent activation of cAMP-dependent protein kinase which, in turn, may phosphorylate the exchange mechanism.