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Evidence for lithium-sensitive inositol 4,5-bisphosphate accumulation in muscarinic cholinoceptor-stimulated cerebral-cortex slices.

ABSTRACT: Stimulation of [3H]inositol-prelabelled rat cerebral-cortex slices with carbachol results in the accumulation of four [3H]inositol bisphosphate isomeric species, Ins(1,3)P2, Ins(1,4)P2, Ins(3,4)P2 and Ins(4,5)P2. Although the last isomer ran as a minor peak on h.p.l.c., its accumulation was dramatically enhanced in the presence of Li+ (1 mM), such that at 30 min it represented almost 35% of the total bisphosphate fraction. The accumulation of Ins(4,5)P2 appeared to be very sensitive to Li+ (EC50 = 94 +/- 3 microM), strongly implicating a Li(+)-sensitive metabolism. Evidence for this is provided from the rapid but Li(+)-sensitive decay of Ins(4,5)P2 when muscarinic-receptor stimulation is antagonized by atropine at a time when accumulations have reached a new steady state. Manipulation of phospholipase D by activators and inhibitors of protein kinase C did not suggest a role for phospholipase D hydrolysis of PtdInsP2 in the formation of Ins(4,5)P2. Attempts to reveal Ins(4,5)P2 metabolism, or indeed its synthesis from Ins(1,4,5)P3, were not successful with broken cell preparations and strongly suggest discrete compartmentation of inositol phosphate metabolism in the intact cell.

SUBMITTER: Jenkinson S

PROVIDER: S-EPMC1133184 | biostudies-other | 1992 Oct

REPOSITORIES: biostudies-other

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Project description:1. Basal and carbachol-stimulated accumulations of isomeric [3H]inositol mono-, bis-, tris- and tetrakis-phosphates were examined in rat cerebral-cortex slices labelled with myo-[2-3H]inositol. 2. In control samples the major [3H]inositol phosphates detected were co-eluted on h.p.l.c. with Ins(1)P, Ins(4)P (inositol 1- and 4-monophosphate respectively), Ins(1,4)P2 (inositol 1,4-bisphosphate), Ins(1,4,5)P3 (inositol 1,4,5-tris-phosphate) and Ins(1,3,4,5)P4 (inositol 1,3,4,5-tetrakisphosphate). 3. After stimulation to steady state with carbachol, accumulation of each of these products was markedly increased. 4. Agonist stimulation, however, also evoked much more dramatic increased accumulations of a second [3H]inositol trisphosphate, which was co-eluted on h.p.l.c. with authentic Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate) and of three further [3H]inositol bisphosphates ([3H]InsP2(s]. 5. Examination of the latter by chemical degradation by periodate oxidation and/or h.p.l.c. allowed identification of these as [3H]Ins(1,3)P2, [3H]Ins(3,4)P2 and [3H]Ins(4,5)P2 (inositol 1,3-, 3,4- and 4,5-bisphosphates respectively), which respectively accounted for about 22%, 8% and 3% of total [3H]InsP2 in extracts from stimulated tissue slices. 6. By using a h.p.l.c. method which clearly resolves Ins(1,3,4,5)P4 and Ins(1,3,4,6)P4 (inositol 1,3,4,6-tetrakisphosphate), only the former isomer could be detected in extracts from either control or stimulated tissue slices. Similarly, [3H]inositol pentakis- and hexakis-phosphates were not detectable either in the presence or absence of carbachol under the radiolabelling conditions described. 7. The catabolism of [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4)P3 by cell-free preparations from cerebral cortex was also studied. 8. In the presence of Mg2+, [3H]Ins(1,4,5)P3 was specifically dephosphorylated via [3H]Ins(1,4)P2 and [3H]Ins(4)P to free [3H]inositol, whereas [3H]Ins(1,3,4)P3 was degraded via [3H]Ins(3,4)P2 and, to a lesser extent, via [3H]Ins(1,3)P2 to D- and/or L-[3H]Ins(1)P and [3H]inositol. 9. In the presence of EDTA, hydrolysis of [3H]Ins(1,4,5)P3 was greater than or equal to 95% inhibited, whereas [3H]Ins(1,3,4)P3 was still degraded, but yielded only a single [3H]InsP2 identified as [3H]Ins(1,3)P2. 10. The significance of these observations with cell-free preparations is discussed in relation to the proportions of the separate isomeric [3H]inositol phosphates measured in stimulated tissue slices.

Project description:Kv7 K(+)-channel subunits differ in their apparent affinity for PIP(2) and are differentially expressed in nerve, muscle, and epithelia in accord with their physiological roles in those tissues. To investigate how PIP(2) affinity affects the response to physiological stimuli such as receptor stimulation, we exposed homomeric and heteromeric Kv7.2, 7.3, and 7.4 channels to a range of concentrations of the muscarinic receptor agonist oxotremorine-M (oxo-M) in a heterologous expression system. Activation of M(1) receptors by oxo-M leads to PIP(2) depletion through G(q) and phospholipase C (PLC). Chinese hamster ovary cells were transiently transfected with Kv7 subunits and M(1) receptors and studied under perforated-patch voltage clamp. For Kv7.2/7.3 heteromers, the EC(50) for current suppression was 0.44 +/- 0.08 microM, and the maximal inhibition (Inhib(max)) was 74 +/- 3% (n = 5-7). When tonic PIP(2) abundance was increased by overexpression of PIP 5-kinase, the EC(50) was shifted threefold to the right (1.2 +/- 0.1 microM), but without a significant change in Inhib(max) (73 +/- 4%, n = 5). To investigate the muscarinic sensitivity of Kv7.3 homomers, we used the A315T pore mutant (Kv7.3(T)) that increases whole-cell currents by 30-fold without any change in apparent PIP(2) affinity. Kv7.3(T) currents had a slightly right-shifted EC(50) as compared with Kv7.2/7.3 heteromers (1.0 +/- 0.8 microM) and a strongly reduced Inhib(max) (39 +/- 3%). In contrast, the dose-response curve of homomeric Kv7.4 channels was shifted considerably to the left (66 +/- 8 nM), and Inhib(max) was slightly increased (81 +/- 6%, n = 3-4). We then studied several Kv7.2 mutants with altered apparent affinities for PIP(2) by coexpressing them with Kv7.3(T) subunits to boost current amplitudes. For the lower affinity (Kv7.2 (R463Q)/Kv7.3(T)) or higher affinity (Kv7.2 (R463E)/Kv7.3(T)) channels, the EC(50) and Inhib(max) were similar to Kv7.4 or Kv7.3(T) homomers (0.12 +/- 0.08 microM and 79 +/- 6% [n = 3-4] and 0.58 +/- 0.07 microM and 27 +/- 3% [n = 3-4], respectively). The very low-affinity Kv7.2 (R452E, R459E, and R461E) triple mutant was also coexpressed with Kv7.3(T). The resulting heteromer displayed a very low EC(50) for inhibition (32 +/- 8 nM) and a slightly increased Inhib(max) (83 +/- 3%, n = 3-4). We then constructed a cellular model that incorporates PLC activation by oxo-M, PIP(2) hydrolysis, PIP(2) binding to Kv7-channel subunits, and K(+) current through Kv7 tetramers. We were able to fully reproduce our data and extract a consistent set of PIP(2) affinities.

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Evidence for lithium-sensitive inositol 4,5-bisphosphate accumulation in muscarinic cholinoceptor-stimulated cerebral-cortex slices.

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