Relationship between 5-hydroxytryptamine activation of phosphatidylinositol hydrolysis and calcium-ion entry in Calliphora salivary glands.

The salivary glands of the blowfly (Calliphora erythrocephala) are long thin tubes consisting of a single layer of homogeneous cells that secrete iso-osmotic KCI when stimulated by 5-hydroxytryptamine (Berridge, 1975). Cyclic AMP formation is accelerated by 5-hydroxytryptamine and the addition of cyclic AMP mimicks the stimulation of fluid secretion, but not the increase in transepithelial Caz+ flux owing to 5-hydroxytryptamine. 5-Hydroxytryptamine appears to independently accelerate CaZ+ entry and cyclic AMP accumulation. The increase in Ca2+ entry results in elevated anion movement, whereas cyclic AMP stimulates a potassium pump, which results in fluid secretion (Berridge, 1975). Michell et al. (1977) suggested that the breakdown of phosphatidylinositol induced by hormone-receptor complexes in a wide variety of cells is involved in the gating of CaZ+ entry into cells. The present studies were designed to determine whether an increase in phosphatidylinositol hydrolysis owing to 5-hydroxytryptamine is observed in salivary glands. In this study we examined the effects of 5-hydroxytryptamine on the breakdown of phospholipids that had been prelabelled for several hours with either [32P]P, or my0-[2-~H]inositol. In the presence of 5-hydroxytryptamine there was a preferential increase in the hydrolysis of phosphatidylinositol without any increase in the breakdown of phosphatidylcholine or phosphatidylethanolamine. Unlike [32P]Pi, which labels a wide variety of substances, the use of my0-[2-~H]inositol results in a selective labelling of phosphatidylinositol. In glands washed for 30min after a 2h incubation with 5 2 0 ~ ~ rny0-[2-~H]inositol more than 94% of the label present in the gland was in the form of phosphatidylinositol. Of the small amount of 3H present in water-soluble compounds 50 % was inositol 1 : 2-cyclic phosphate, 13 % as inositol, 28 % as glycerophosphoinositol and 9% as inositol 1-phosphate. The addition of 5-hydroxytryptamine to salivary glands in which phosphatidylinositol had been labelled by prior incubation with [3H]inositol resulted in an increase in the intracellular accumulation of inositol phosphates within 30s after the addition of hormone, which was followed by a release of labelled inositol to the medium. We could detect no release of label as any product other than inositol. 5-Hydroxytryptamine stimulated the release of inositol to both the medium and the saliva and the effect was sustained as long as the hormone was present. Low concentrations of 5-hydroxytryptamine, which stimulate fluid secretion submaximally, also stimulated phosphatidylinositol breakdown. It was not possible to find a concentration of hormone that stimulated fluid secretion without also increasing phosphatidylinositol breakdown. However, fluid secretion was maximally stimulated by 0.01 ~~-5 -hydroxytryptamine, whereas higher concentrations gave further increases in phosphatidylinositol breakdown and CaZ+ flux. Michell et al. (1976) suggested that this indicates a tight and rather direct coupling between the binding of hormone to receptors and the increase in phosphatidylinositol labelling. Similar dose-response curves are observed for the hormonal activation of adenylate cyclase in membrane preparations. The addition of 3-isobutyl-1 -methylxanthine (which presumably potentiates the effect of 5-hydroxytryptamine on cyclic AMP) increased the fluid secretion owing to 1 n~-5-hydroxytryptamine without affecting phosphatidylinositol breakdown. Cyclic AMP also increases fluid secretion, but did not affect phosphatidylinositol hydrolysis or Ca2+ flux in regular buffer. In buffer containing 4 0 n ~ K + it was possible to observe an increase in CaZ+ flux owing to cyclic AMP, but this was not accompanied by any appreciable increase in inositol release to the medium.