5-Hydroxytryptamine stimulates inositol phosphate production in a cell-free system from blowfly salivary glands. Evidence for a role of GTP in coupling receptor activation to phosphoinositide breakdown.

I Litosch,C Wallis,J N Fain

doi:10.1016/s0021-9258(18)89045-7

I Litosch, C Wallis + Show 1 more

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https://doi.org/10.1016/s0021-9258(18)89045-7

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Abstract

Phosphoinositide breakdown has been linked to the receptor mechanism involved in the elevation of cytosolic Ca2+. In a cell-free system prepared from [3H] inositol-labeled blowfly salivary glands, 5-hydroxytryptamine stimulated the rapid production of inositol phosphates. Within 30 s of hormone addition, there was a 100% increase in inositol trisphosphate formation, a 70% increase in inositol bisphosphate formation, and a 90% increase in inositol monophosphate formation as compared to control homogenates incubated for the same length of time. 5-Hydroxytryptamine did not stimulate inositol or glycerol phosphoinositol formation. Half-maximal activation of inositol phosphate production was obtained with 0.33 microM 5-hydroxytryptamine. Ethylene glycol bis(beta-aminoethyl ether)-N',N',N',N'-tetraacetic acid, (EGTA) (0.3 mM) inhibited the basal formation of inositol phosphates and decreased the net accumulation of inositol bisphosphate and inositol trisphosphate due to hormone as compared to homogenates incubated in the absence of added Ca2+. EGTA, however, had little effect on the per cent stimulation of inositol phosphate production due to hormone. In homogenates, ATP, GTP or guanyl-5'-yl imidodiphosphate (Gpp(NH)p) was required for a hormone effect. Gpp(NH)p, unlike ATP or GTP, increased the basal formation of inositol phosphates. In membranes, GTP, Gpp(NH)p, or guanosine 5'-(3-O-thio)trisphosphate (GTP gamma S) sustained a hormone effect whereas ATP was ineffective. GTP did not affect production while Gpp(NH)p and GTP gamma S increased inositol phosphate production. Half-maximal effects of Gpp(NH)p and GTP gamma S on hormone-stimulated inositol phosphate formation occurred at 10 microM and 100 nM, respectively. In the presence of 1 microM GTP gamma S, 5-methyltryptamine stimulated inositol phosphate formation within 2 s in membranes. These results indicate that in a cell-free system, GTP is involved in mediating the effects of Ca2+-mobilizing hormones on phosphoinositide breakdown.

Highlights

Phosphoinositide breakdown has been linked to the ration into phosphatidylinositol and its precursor phosphareceptor mechanism involved in the elevationof cyto- tidic acid
Pharmacotryptamine stimulated the rapid productiofninositol logical studies have shown that activation of phosphatidyliphosphates.Within 30 s of hormone addition, there nositol turnover is associated with a class of receptors includwas a 100% increase in inositol trisphosphate formation, a 70% increase in inositol bisphosphate formation, and a 90% increase in inositolmonophosphate formation as compared to control homogenates incubated for the same length of time. 5-Hydroxytryptamine did not stimulate inositol golrycerol phosphoinositol formation
Inhibited the basal formatoiofninositol phosphates and P2).In hepatocytes [4,5,6], parotid [7, 8], GH, cells (9, lo), decreased thenetaccumulation of inositolbisphos- platelets [11, 12], and blowfly salivary glands [13, 14], horphate and inositol trisphosphate due to hormone as mone-stimulated phosphoinositide breakdown occurred in compared to homogenates incubated in the absencoef seconds

Summary

MATERIALS AND METHODS

Salivary glands were isolated from adult blowflies (Calliphora erythrocephala) 6-10 days post-emergence from the pupal stage. After 2.5 h, the salivary glands wereremoved from the labeling medium and rinsed with 50 pl of ice-cold homogenization buffer containing 320 mM sucrose, 0.5 mM Na2EDTA, 25 mM LiC1, 100 p~ Na2ATP, and 10 mM Tris/HCl (pH 7.8 a t 5 "C). Theentire aqueous phase was incubated with 0.5 ml of a resin slurry (AGl-X8 in formate form) for 15 min to allow for adsorption of the inositol phosphates. Release of adsorbed water-soluble products was initiated by the addition of 2 X 4 mlof 5 mM disodium tetraborate, 60 mM sodium formate (glycerol phosphoinositol and cyclic phosphoinositol); 100 mM formic acid, 200 mM ammonium formate (inositol monophosphate); 100 mM formic acid, 400 mM ammonium formate (inositol bisphosphate); and 100 mM formic acid, 1000 mM ammonium formate (inositol trisphosphate) This procedure was initially characterized by the collection of 10 drop aliquots of each elution buffer to determine the distribution of radioactivity. Statistical analysis of the data was performed by the Student's t test using paired analysis

RESULTS

There was no detectable lag in the production of inositol

Studies with intact blowfly salivary glands have shown that

DISCUSSION