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

摘要

class="enumerated" style="list-style-type:decimal">We examined the effects of noradrenaline on steady-state intracellular pH (pHi) and the recovery of pHi from internal acid loads imposed by the NH4⁺ prepulse technique in hippocampal CA1 neurones acutely dissociated from adult rats.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 pHi.Noradrenaline evoked a concentration-dependent and sustained rise in steady-state pHi and increased rates of pHi 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 pHi by increasing the activity of the Na⁺-H⁺ exchanger.The effects of noradrenaline on steady-state pHi and on rates of pHi recovery from imposed acid loads were mimicked by β1- and β2-, but not α-, adrenoceptor agonists. The β-adrenoceptor antagonist propranolol blocked the ability of noradrenaline to increase both steady-state pHi and rates of pH_i recovery from acid loads.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 sa Rp-cAMPS) and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulphonamide (H-89).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.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.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 β-adrenoceptors via a pathway which involves the α-subunit of the stimulatory G-protein G_s (G_sα), adenylate cyclase, cAMP and the subsequent activation of cAMP-dependent protein kinase which, in turn, may phosphorylate the exchange mechanism.