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Receptor-mediated net breakdown of phosphatidylinositol 4,5-bisphosphate in parotid acinar cells.

ABSTRACT: The metabolism of phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] in rat parotid acinar cells was investigated, particularly with regard to the effects of receptor-active agonists. Stimulation of cholinergic-muscarinic receptors with methacholine provoked a rapid disappearance of 40--50% of [32P]PtdIns(4,5)P2, but had no effect on PtdIns4P. Adrenaline, acting on alpha-adrenoceptors, and Substance P also stimulated net loss of PtdIns(4,5)P2. The beta-adrenoceptor agonist, isoprenaline, and the Ca2+ ionophore, ionomycin, failed to affect labelled PtdIns(4,5)P2 or PtdIns4P. By chelation of extracellular Ca2+ with excess EGTA, and by an experimental protocol that eliminates cellular Ca2+ release, it was demonstrated that the agonist-induced decrease in PtdIns(4,5)P2 is independent of both Ca2+ influx and Ca2+ release. These results may suggest that net PtdIns(4,5)P2 breakdown is an early event in the stimulus-response pathway of the parotid acinar cell and could be directly involved in the mechanism of agonist-induced Ca2+ release from the plasma membrane.

SUBMITTER: Weiss SJ

PROVIDER: S-EPMC1158623 | biostudies-other | 1982 Sep

REPOSITORIES: biostudies-other

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Project description:The relationship between receptor-induced membrane phosphatidylinositol-4'5'-bisphosphate (PIP2) hydrolysis and M-current inhibition was assessed in single-dissociated rat sympathetic neurons by simultaneous or parallel recording of membrane current and membrane-to-cytosol translocation of the fluorescent PIP2/inositol 1,4,5-trisphosphate (IP3)-binding peptide green fluorescent protein-tagged pleckstrin homology domain of phospholipase C (GFP-PLCdelta-PH). The muscarinic receptor agonist oxotremorine-M produced parallel time- and concentration-dependent M-current inhibition and GFP-PLCdelta-PH translocation; bradykinin also produced parallel time-dependent inhibition and translocation. Phosphatidylinositol-4-phosphate-5-kinase (PI5-K) overexpression reduced both M-current inhibition and GFP-PLCdelta-PH translocation by both oxotremorine-M and bradykinin. These effects were partly reversed by wortmannin, which inhibits phosphatidylinositol-4-kinase (PI4-K). PI5-K overexpression also reduced the inhibitory action of oxotremorine-M on PIP2-gated G-protein-gated inward rectifier (Kir3.1/3.2) channels; bradykinin did not inhibit these channels. Overexpression of neuronal calcium sensor-1 protein (NCS-1), which increases PI4-K activity, did not affect responses to oxotremorine-M but reduced both fluorescence translocation and M-current inhibition by bradykinin. Using an intracellular IP3 membrane fluorescence-displacement assay, initial mean concentrations of membrane [PIP2] were estimated at 261 microm (95% confidence limit; 192-381 microm), rising to 693 microm (417-1153 microm) in neurons overexpressing PI5-K. Changes in membrane [PIP2] during application of oxotremorine-M were calculated from fluorescence data. The results, taken in conjunction with previous data for KCNQ2/3 (Kv7.2/Kv7.3) channel gating by PIP2 (Zhang et al., 2003), accorded with the hypothesis that the inhibitory action of oxotremorine-M on M current resulted from depletion of PIP2. The effects of bradykinin require additional components of action, which might involve IP3-induced Ca2+ release and consequent M-channel inhibition (as proposed previously) and stimulation of PIP2 synthesis by Ca2+-dependent activation of NCS-1.

Project description:Regulation of the putative amiloride and benzamil (Bz)-insensitive TRPV1t salt taste receptor by phosphatidylinositol 4,5-bisphosphate (PIP(2)) was studied by monitoring chorda tympani (CT) taste nerve responses to 0.1 M NaCl solutions containing Bz (5 x 10(-6) M; a specific ENaC blocker) and resiniferatoxin (RTX; 0-10 x 10(-6) M; a specific TRPV1 agonist) in Sprague-Dawley rats and in wildtype (WT) and TRPV1 knockout (KO) mice. In rats and WT mice, RTX elicited a biphasic effect on the NaCl + Bz CT response, increasing the CT response between 0.25 x 10(-6) and 1 x 10(-6) M. At concentrations >1 x 10(-6) M, RTX inhibited the CT response. An increase in PIP(2) by topical lingual application of U73122 (a phospholipase C blocker) or diC8-PIP(2) (a short chain synthetic PIP(2)) inhibited the control NaCl + Bz CT response and decreased its sensitivity to RTX. A decrease in PIP(2) by topical lingual application of phenylarsine oxide (a phosphoinositide 4 kinase blocker) enhanced the control NaCl + Bz CT response, increased its sensitivity to RTX stimulation, and inhibited the desensitization of the CT response at RTX concentrations >1 x 10(-6) M. The ENaC-dependent NaCl CT responses were not altered by changes in PIP(2). An increase in PIP(2) enhanced CT responses to sweet (0.3 M sucrose) and bitter (0.01 M quinine) stimuli. RTX produced the same increase in the Bz-insensitive Na(+) response when present in salt solutions containing 0.1 M NaCl + Bz, 0.1 M monosodium glutamate + Bz, 0.1 M NaCl + Bz + 0.005 M SC45647, or 0.1 M NaCl + Bz + 0.01 M quinine. No effect of RTX was observed on CT responses in WT mice and rats in the presence of the TRPV1 blocker N-(3-methoxyphenyl)-4-chlorocinnamide (1 x 10(-6) M) or in TRPV1 KO mice. We conclude that PIP(2) is a common intracellular effector for sweet, bitter, umami, and TRPV1t-dependent salt taste, although in the last case, PIP(2) seems to directly regulate the taste receptor protein itself, i.e., the TRPV1 ion channel or its taste receptor variant, TRPV1t.

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Receptor-mediated net breakdown of phosphatidylinositol 4,5-bisphosphate in parotid acinar cells.

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