Mode of stimulation by adenosine 3':5'-cyclic monophosphate of the sodium efflux in barnacle muscle fibres

Abstract

1. Giant fibres of the barnacle Balanus nubilus have been used as a preparation for studying the mode of action of cAMP on sodium transport. 2. It is shown that a concentration of cAMP as low as 10(-6)M, when micro-injected, causes a sharp rise in the radio-Na efflux. Ouabain fails to reverse the cAMP effect. 3. The magnitude of the response of the Na efflux to cAMP is markedly reduced by pre-injecting 100 or 500 mM-EGTA solutions or by omitting Ca2+ from the bathing medium. Both together fail to bring about a greater reduction in the response. 4. The response to cAMP is greatly reduced by pre-injecting the protein inhibitor of Walsh and practically abolished by pre-injecting 500 mM-EGTA and soaking in Ca-free artificial sea water, ASW. 5. The Ca2+-independent component of the Na efflux which is also stimulated by cAMP is shown to involve Na for H exchange. The magnitude of this exchange is governed by external pH. 6. The Na efflux into Ca2+-free, Li+-ASW is shown to be markedly stimulated by injecting cAMP, an effect which is enhanced by reducing external pH. 7. The Na efflux at 0 degrees C is stimulated by injecting cAMP. This is shown to be related to activation of the protein kinase by cAMP and to depend on the presence of external Ca2+. 8 (i) Ethacrynic acid when injected reduces the ouabain-insensitive Na efflux into HEPES-Ca2+-free ASW at pH 6-3. These same fibres show a marked response to cAMP. (II) The ouabain-insensitive Na efflux into HCO3-, Ca2+-free ASW from fibres pre-treated with ethacrynic acid fails to respond to external acidification. This is interpreted as indicating that ethacrynic acid inactivates the CO2-sensitive adenyl cyclase system. These same fibres when injected with cAMP show a marked response. (iii) Stimulation of the ouabain-insensitive Na efflux into HCO-3, Ca2+-free ASW by external acidification is reversed by injecting ethacrynic acid. These fibres when injected with cAMP show a reduced response. 9. It is concluded that: (i) stimulation of the Na efflux by injected cAMP is mainly due to activation of cAMP-dependent protein kinase; (ii) the underlying exchange mechanism consists of Na:Ca and Na:H exchange. Interaction of Ca2+ with a phosphorylated membrane, thereby modifying permeability remains as a real possibility; (iii) the site of action of CO2 and ethacrynic acid is the adenyl cyclase system. 10. The implications of activation of the adenyl cyclase system by CO2 and Na:H exchange are briefly touched upon.