Introduction

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It is now well established that polyphosphoinositides play a vital role in cell signaling (Berridge, 1993; Hughes and Michell, 1993) and that in ASM, the phosphoinositidase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] plays a central role in pharmacomechanical coupling (Coburn and Baron, 1990; Chilvers et al., 1994a). In bovine and canine ASM, where these responses have been best studied, the increase in Ins(1,4,5)P₃ observed after the addition of spasmogens such as CCh, histamine, bradykinin and leukotriene B₄ precedes (Miller-Hance et al., 1988; Duncan et al., 1987; Chilvers et al., 1989a; Baron et al., 1989), and is responsible for (Hashimoto et al., 1985), the ensuing Ca⁺⁺ transient that is the initial trigger for ASM contraction (Taylor et al., 1989). One interesting aspect of these studies is the observation that spasmogens such as CCh, working through the M₃ muscarinic cholinoceptor (Roffel et al., 1990), induce protracted (>20 min), nondesensitizing hydrolysis of PtdIns(4,5)P₂ yet only transient (<30 sec) accumulation of Ins(1,4,5)P₃ (Chilvers et al., 1991a). This observation has been confirmed in studies examining changes in both [³H]PtdIns(4,5)P₂/[³H]Ins(1,4,5)P₃ levels (Chilvers et al., 1989a; Chilvers et al., 1990a) and PtdIns(4,5)P₂/Ins(1,4,5)P₃ mass (Chilvers et al., 1991a) and suggests that the metabolism of Ins(1,4,5)P₃ is agonist-stimulated and under tight regulatory control.

Studies in a variety of noncontractile tissues have shown that two major routes exist for metabolism of Ins(1,4,5)P₃: the Ins(1,4,5)P₃ 3-kinase and Ins(1,4,5)P₃ 5-phosphatase pathways (Majerus et al., 1988; Shears, 1989). The latter route is initiated by the removal of a phosphate group from the 5-position of the inositol ring to produce Ins(1,4)P₂, whereas the 3-kinase pathway proceeds by phosphorylation of the inositol ring on the 3-position to form Ins(1,3,4,5)P₄. Thereafter, both metabolites are subject to sequential dephosphorylation, which results in the generation of a set of distinct inositol phosphate metabolites with the eventual production of inositol, which is recycled into the phosphoinositide pool. In view of the extent and speed of agonist-stimulated removal of Ins(1,4,5)P₃ observed in muscarinic cholinergic-stimulated BTSM (Chilvers et al., 1991a; Chilvers et al., 1989b), we have attempted to exploit the difference between the Ins(P)Px isomers generated by the Ins(1,4,5)P₃ 3-kinase and 5-phosphatase pathways to assess their relative importance in Ins(1,4,5)P₃ metabolism at successive intervals after agonist addition. Besides detailing the effects of CCh and lithium ions on [³H]Ins(P)Px accumulation in BTSM, we have demonstrated that the 5-phosphatase represents the dominant pathway for Ins(1,4,5)P₃ removal at all time-points (0-30 min) after the addition of CCh. We have also identified accumulation of an [³H]InsP₂ isomer with the chromatographic characteristics of [³H]Ins(4,5)P₂, not previously identified in non-neuronal tissues and provided evidence from lithium inhibition studies that this isomer is derived from [³H]Ins(1,4,5)P₃ by the action of an inositol polyphosphate 1-phosphatase. Hence we have identified in BTSM a novel route for Ins(1,4,5)P₃ metabolism that results in the generation of the calcium-mobilizing inositol phosphate Ins(4,5)P₂.

	Materials and Methods

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Materials. The following drugs and chemicals were used in this study: myo[2-³H]inositol (17-20 Ci/mmol), myo[1-³H]inositol 1,4,5-trisphosphate (15-30 Ci/mmol) and myo[1-³H]inositol 1,3,4,5-tetrakisphosphate (15-30 Ci/mmol) (Du Pont NEN (UK) Ltd., Stevenage, UK), Dowex AG1-X8 (200-400 mesh, formate form) (Bio-Rad Laboratories, Hemel Hempstead, UK) and carbachol and flurbiproten (Sigma Chemical Co., Poole, UK). All other reagents were from Sigma or BDH (Merck Ltd., Dorset, UK) and were of the highest purity available.

Preparation, [³H]inositol labeling and extraction of BTSM slices. BTSM slices (300 × 300 µm) were prepared using a McIlwain tissue chopper as described previously (Chilvers et al., 1989a) and preincubated in bulk for 60 min in a shaking water bath at 37°C in 100 ml of oxygenated Krebs-Henseleit buffer (KH buffer) containing 1.3 mM CaCl₂ and 1 µM flurbiprofen. This preparation consists of more than 95% ASM cells; connective tissue and adipose tissue are the major nonmuscle components. Three-milliliter portions of gravity-packed slices were transferred to 25-ml Erlenmeyer flasks containing 9 ml of KH buffer, 100 µCi myo[³H]inositol and 1 µM CCh and incubated at 37°C for a further 60 min. This short-term agonist-stimulated labeling protocol has previously been shown in this tissue to give steady-state radiolabeling of the agonist-sensitive phosphoinositide pool and, of critical importance for [³H]Ins(P)Px flux measurements, does not result in inositol penta- or hexakisphosphate labeling (Chilvers et al., 1989a). After extensive washing with CCh-free KH buffer over 30 min, 50-µl samples of gravity-packed BTSM slices were transferred to flat-bottomed 5-ml polypropylene insert vials containing 230 µl of KH buffer replenished with 2.5 µCi myo[³H]inositol. After a further 10-min incubation in the presence or absence of 0.01 to 30 mM LiCl, CCh (0.1 mM) or KH buffer (control) was added to the slices. Reactions were terminated, at the time-points indicated, by addition of an equal volume (300 µl) of ice-cold 1 M TCA. Samples were allowed to extract on ice for 20 min, vortex mixed and centrifuged at 3000 × g for 20 min at 4°C. Supernatants were decanted, added to 125 µl of 10 mM EDTA (pH 7.0) and neutralized with 1,1,2-trichlorotrifluoroethane/tri-n-octylamine (1:1, v/v) as detailed previously (Chilvers et al., 1989a). Samples were adjusted finally to pH 7.0 by using NaHCO₃ and were stored at 4°C before chromatographic separation.

Metabolism of [³H]Ins(1,4,5)P₃ and [³H]Ins(1,3,4,5)P₄ by BTSM cell-free extracts. BTSM was dissected free from overlying epithelium and connective tissue, chopped with scissors, washed four times in 3 volumes of buffer A (10 mM Tris maleate-HCl, pH 7.5, 150 mM sucrose) and homogenized in 3 volumes of buffer A using a Polytron tissue homogenizer (maximal speed, 30 sec). Tissue homogenate was centrifuged at 5000 × g for 10 min at 4°C, and after removal of a surface fatty layer, either the pellet and supernatant were rehomogenized to generate a "BTSM homogenate" or the supernatant was respun at 48,400 × g (90 min, 4°C) to generate "BTSM cytosol." Both cytosol and homogenate preparations were aliquoted and stored at 80°C for later use.

Separation of [³H]inositol (poly)phosphates. Pooled triplicates of neutralized TCA extracts either were analyzed by open-column Dowex anion-exchange chromatography for assessment of total [³H]InsP1, [³H]InsP₂, [³H]InsP₃ or [³H]InsP₄ (Chilvers et al., 1990a) or were spiked with 2.5 µM adenosine- and guanosine- mono-, di-, tri- and tetraphosphate nucleotides and separated by HPLC using a Partisphere 5 SAX column (Whatman International Ltd., Maidstone, UK) as described previously (Batty et al., 1989). The positions of [³H]Ins1P, [³H]Ins4P, [³H]Ins(1,4)P₂, [³H]Ins(1,4,5)P₃ and [³H]Ins(1,3,4,5)P₄ peaks were confirmed by coelution with commercial standards [Du Pont NEN (UK) Ltd., Stevenage, UK].

	Results

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To examine the effects of CCh and lithium ions on [³H]Ins(P)Px accumulation and to explore the fate of [³H]Ins(1,4,5)P₃ in this tissue, we utilized an HPLC system that allows optimal separation, identification and quantification of the individual metabolic products of [³H]Ins(1,4,5)P₃ (fig. 1), the only exception being nonresolution of the enantiomers [³H]Ins1P and [³H]Ins3P. These experiments were performed specifically under conditions of steady-state phosphoinositide labeling where we have previously demonstrated minimal time- or agonist-induced changes in specific radioactivity over the time course employed (Batty et al., 1989) and where [³H]InsP₅ and [³H]InsP₆ accumulation is not observed (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   HPLC separation of [3H]inositol polyphosphates present in extracts from CCh-stimulated BTSM slices by HPLC. Neutralized TCA extracts from BTSM slices, prelabeled with [3H]inositol and stimulated with CCh (0.1 mM) for 30 min were analyzed for [3H]inositol-labeled inositol polyphosphate isomers by HPLC, utilizing (NH4) H2PO4 gradients and a Partisphere 5 SAX column as described in Batty et al., 1989. A single representative trace is shown, and isomers are identified in brackets.

Effects of CCh on [³H]inositol (poly)phosphate accumulation. Accumulation of [³H]Ins(P)Px isomers over the initial 0 to 5-min incubation period with 0.1 mM CCh in the presence of 5 mM LiCl is shown in figure 2, and steady-state (30 min) accumulation values in the presence and in the absence of LiCl are presented in table 1. This concentration of CCh has previously been shown to be maximally effective for M₃ muscarinic cholinoceptor-mediated PLC activation in this tissue (Chilvers et al., 1989a; Chilvers et al., 1991a). The major findings of these experiments were first, confirmation of rapid and transient accumulation of [³H]Ins(1,4,5)P₃ (fig. 2C), second, a delayed and sustained accumulation of [³H]Ins(1,3,4)P₃, [³H]Ins(1,3)P₂, [³H]Ins(3,4)P₂ and [³H]Ins1/3P (fig. 2, a-c) and third, a rapid and major increase in [³H]Ins(1,4)P₂ and [³H]Ins4P (fig. 3a), which were the most dominant [³H]InsP₂ and [³H]InsP isomers to accumulate. The identification, accumulation and lithium ion sensitivity of a late-running [³H]InsP₂ peak shown in figure 1 are discussed below.

View larger version (32K): [in this window] [in a new window]   Fig. 2.   [3H]Inositol polyphosphate accumulation in CCh-stimulated BTSM slices. BTSM slices were labeled with [3H]inositol as described in "Materials and Methods" and preincubated in the presence of LiCl (5 mM) before stimulation with CCh (0.1 mM) for the times indicated. [3H]Inositol polyphosphates were extracted and analyzed by HPLC as described previously. Accumulation of individual [3H]inositol polyphosphate isomers was as detailed in panels a through d. Results are expressed as mean dpm/50 µl BTSM slices ± S.E.M. from n = 3 experiments, all performed in triplicate.

View this table: [in this window] [in a new window]   TABLE 1 Effect of CCh on [3H]inositol (poly)phosphate accumulation BTSM slices were labeled with [3H]inositol as described in "Materials and Methods." Inositol polyphosphates were analyzed by HPLC after extraction from tissue samples preincubated with (+) or without () LiCl (5 mM) for 10 min, under control conditions or stimulated with CCh (0.1 mM) for 30 min as indicated. HPLC separation was carried out as described in Batty et al., 1989 and identification of isomers was by coelution with commercial standards. Results are expressed as mean % ± S.E.M. of total radioactivity per sample, from n = 5 separate experiments.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Effect of LiCl on CCh-stimulated [3H]inositol monophosphate accumulation in BTSM slices. [3H]inositol-labeled BTSM slices were preincubated with varying concentrations of LiCl, as indicated, for 10 min before stimulation with CCh (0.1 mM) for 30 min. Analysis of [3H]inositol monophosphates was by HPLC as described previously. Isomers are indicated in panels a and b, and results are expressed as mean % increase over control ± S.E.M. from n = 3 experiments, all performed in triplicate.

Effect of LiCl on [³H]inositol (poly)phosphate accumulation. In keeping with the known effects of lithium as a potent uncompetitive inhibitor of inositol monophosphatase, inclusion of 5 mM LiCl during CCh time course studies resulted in 8.8-fold and 13-fold increases in [³H]Ins1/3P and [³H]Ins4P accumulation, respectively, at 30 min (table 1). This effect of lithium ions monitored at 30 min was concentration-dependent (fig. 3) with EC₅₀ values of 1.3 mM and 1.1 mM for [³H]Ins1/3P and [³H]Ins4P accumulation, respectively. As anticipated from the relative insensitivity of the inositol polyphosphate 1-, 3- and 4-phosphatases to lithium ions reported in other tissues (Shears, 1989), LiCl (0.01-30 mM) had only a minor effect on CCh-stimulated [³H]Ins(3,4)P₂ and [³H]Ins(1,3,4)P₃ accumulation over 30 min (fig. 4; table 1) and had no effect on [³H]Ins(1,3)P₂ accumulation (fig. 4; table 1). A significant effect of LiCl on [³H]Ins(1,4)P₂ accumulation was observed only at very high inhibitor concentrations (30 mM) where the dpm loss in [³H]Ins4P observed mirrored precisely the increase in [³H]Ins(1,4)P₂ accumulation.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Effect of LiCl on CCh-stimulated [3H]inositol bisphosphate accumulation in BTSM slices. [3H]inositol-labeled BTSM slices were preincubated and stimulated, and [3H]inositol bisphosphates were extracted and analyzed as detailed in the legend to figure 3. [3H]inositol phosphate isomers are indicated in panels a through d. Results are expressed as mean % increase over control ± S.E.M. from n = 3 experiments, all performed in triplicate.

[³H]Ins(4,5)P₂ accumulation. Four peaks of radioactivity were detected in the [³H]InsP₂ region of the initial CCh-stimulated samples analyzed by HPLC (figs. 1; fig. 5c). Identification of three of these as [³H]Ins(1,3)P₂, [³H]Ins(1,4)P₂ and [³H]Ins(3,4)P₂ was based on their coelution with, or relationship to, authentic [³H]Ins(1,4)P₂ and the nucleotides ADP and GDP (fig. 5a). This identification has been validated by previous alditol analysis of identically positioned [³H]InsP₂ peaks obtained by using the same HPLC column and gradient system (Batty et al., 1989). Identification of the later-running peak as [³H]Ins(4,5)P₂ was based on the generation of an [³H]InsP₂ isomer with identical chromatographic properties after alkaline hydrolysis of authentic [³H]PtdIns(4,5)P₂, a reaction that generates [³H]Ins(1,4,5)P₃, [³H]Ins(2,4,5)P₃ and [³H]Ins(4,5)P₂ (Chilvers et al., 1991a) (fig. 5b), and on the nearly perfect match between our elution profiles and those obtained by Jenkinson and co-workers (1992) when analyzing both rat cerebral cortex extracts and alkaline phosphatase products of [³H]Ins(1,4,5)P₃. The neglible accumulation of [³H]Ins(4,5)P₂ observed in unstimulated BTSM slices (less than 10 dpm/50 µl of gravity-packed BTSM slices) also indicates that this [³H]InsP₂ isomer does not represent a lipid breakdown product generated during the extraction procedure.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Identification of [3H]inositol bisphosphate isomer accumulation in CCh-stimulated BTSM slices. HPLC analysis was used a) Retention time of commercial [3H]Ins(1,4)P2 in relation to ADP and GDP. b) Retention time of a [3H]inositol bisphosphate derived from alkaline hydrolysis of [3H]PtdIns(4,5)P2, which is known to be [3H]Ins(4,5)P2. c) The separation of [3H]inositol bisphosphate isomers that accumulate under control (open symbols) and CCh-stimulated (closed symbols) conditions in the presence of 5 mM LiCl in BTSM slices.

Metabolic fate of [³H]Ins(1,4,5)P₃ in CCh-stimulated BTSM slices. Data from the initial time course experiments were used to estimate the relative proportion of [³H]Ins(1,4,5)P₃ metabolized via the inositol polyphosphate 3-kinase and 5-phosphatase pathways at different time intervals after CCh stimulation. The major assumptions involved in such calculations are 1) that all accumulating [³H]Ins(P)Px isomers formed are derived from breakdown of [³H]Ins(1,4,5)P₃, i.e., that there is no major contribution to [³H]Ins(P)Px accumulation aside from PLC-mediated [³H]PtdIns(4,5)₂ hydrolysis, 2) that the resulting [³H]Ins(P)Px isomer products of the 3-kinase and 5-phosphatase pathways are distinct and can be fully resolved and 3) that the lithium trap preventing [³H]inositol monophosphate breakdown is complete or, if not absolute, allows recycling of [³H]Ins1/3P and [³H]Ins4P only in strict proportion to their ambient concentrations. This latter point is addressed in "Materials and Methods."

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Metabolism of [3H]Ins(1,4,5)P3 by cell-free preparations from BTSM. BTSM cytosol was prepared as described in "Materials and Methods," and [3H]Ins(1,4,5)P3 metabolism was assessed for the indicated times at 37°C in the absence of ATP. a) The time course of [3H]Ins(1,4,5)P3 metabolism and formation of [3H]Ins(P)Px products as analyzed by Dowex anion-exchange chromatography. Results are expressed as mean % radioactivity ± S.E.M. from n = 3 experiments performed in duplicate. b) A typical HPLC analysis of [3H]Ins(P)Px isomers formed after the incubation of [3H]Ins(1,4,5)P3 with active BTSM cytosol preparations (filled symbols) or heat-inactivated BTSM cytosol preparations (open symbols) for 10 min.

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Metabolism of [3H]Ins(1,3,4,5)P4 by cell-free preparations from BTSM. BTSM homogenate was prepared as described in "Materials and Methods," and [3H]Ins(1,3,4,5)P4 metabolism was assessed for the indicated times at 37°C. a) The time course of [3H]Ins(1,3,4,5)P4 metabolism and formation of [3H]Ins(P)Px products as analyzed by Dowex anion-exchange chromatography. Results are expressed as mean % radioactivity ± S.E.M. from n = 3 experiments performed in duplicate. b) A typical HPLC analysis of [3H]Ins(P)Px isomers formed after the incubation of [3H]Ins(1,3,4,5)P4 with active BTSM homogenate preparations (filled symbols) or heat-inactivated BTSM homogenate preparations (open symbols) for 10 min.

View larger version (89K): [in this window] [in a new window]   Fig. 8.   Relative contribution of the Ins(1,4,5)P3 3-kinase and 5-phosphatase pathways to [3H]Ins(1,4,5)P3 metabolism after CCh stimulation in BTSM slices. [3H]Inositol-prelabeled BTSM slices were incubated with 0.1 mM CCh in the presence of 5 mM LiCl in a final volume of 300 µl. Reactions were terminated after 0 sec, 5 sec, 30 sec, 1 min, 5 min and 30 min by the addition of 300 µl of 1 M TCA, and the individual [3H]Ins(P)P isomers present in pooled triplicate neutralized extracts were separated and quantified by HPLC. The individual [3H]Ins(P)Ps accumulating over the individual time intervals shown were calculated, and the radioactivity associated with the [3H]Ins(1,4,5)P3 3-kinase and 5-phosphatase metabolites was determined and expressed as a % of total metabolism occurring via the 3-kinase (bottom filled bars) and 5-phosphatase (top stippled bars) pathways. Results are expressed as mean ± S.E.M. from n = 5 separate experiments. * P < .05 compared with 0 to 5-sec values (Student's t test).

	Discussion

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In this study, we have sought to characterize the accumulation of [³H]inositol polyphosphates after muscarinic cholinoceptor stimulation of BTSM and to assess the routes of metabolism of Ins(1,4,5)P₃ at sequential intervals after the addition of agonist. We have verified that the 5-phosphatase and 3-kinase pathways yield mutually exclusive products in BTSM and have been unable to demonstrate directly the hydrolysis of [³H]PtdIns or [³H]PtdIns4P under the conditions employed. This indicates that the [³H]Ins(P)Px accumulation observed was a consequence of the sequential dephosphorylation of Ins(1,4,5)P₃ and its primary metabolite Ins(1,3,4,5)P₄. The short-term labeling protocol used in these studies has been extensively validated to provide steady-state phosphoinositide labeling and prevent agonist-stimulated changes in [³H]PtdIns(4,5)P₂-specific radioactivity (Chilvers et al., 1989a) or labeling of higher inositol phosphates such as Ins(1,3,4,5,6)P₅ and InsP₆. It hence avoids many of the problems inherent in attempting to follow Ins(1,4,5)P₃ metabolism in cells labeled to true isotopic equilibrium over a period of many days. The BTSM preparation described therefore provides an ideal model for the study of [³H]Ins(1,4,5)P₃ metabolism; in particular, it makes possible analysis of the relative contributions of the 3-kinase and 5-phosphatase pathways.

The dominant route of [³H]Ins(1,4,5)P₃ metabolism in BTSM, both at early and late time-points after CCh addition, appears to be via the Ins(1,4,5)P₃ 5-phosphatase pathway, though a small increase in the relative contribution of the 3-kinase pathway is observed at later times. Despite continued formation, [³H]Ins(1,4,5)P₃ accumulates only transiently after CCh stimulation (Chilvers et al., 1991a). The underlying reason for this apparent tight metabolic control of Ins(1,4,5)P₃ accumulation in BTSM remains uncertain, and it is in marked contrast to the situation in a number of other tissues, such as rat cerebral cortex, where Ins(1,4,5)P₃ levels remain elevated for a prolonged period after agonist stimulation (Challiss et al., 1988). One potential explanation for such a difference may be related to the Ca⁺⁺ sensitivity of Ins(1,4,5)P₃ receptor binding in brain, which, in contrast to BTSM (Chilvers et al., 1990b), provides a distal negative feedback mechanism to curtail Ins(1,4,5)P₃-mediated Ca⁺⁺ release. It is also possible, however, that other recently described functions of Ins(1,4,5)P₃, such as its effects on plasmalemmal Ca⁺⁺ channel function and tyrosine phosphatase activity (Stader and Hofer, 1992) underlie a need for differential regulation of Ins(1,4,5)P₃ metabolism between tissues. Hence it may be more important, for reasons aside from acute Ca⁺⁺ homeostasis, that the Ins(1,4,5)P₃ response in BTSM be rapidly curtailed.

A second bifurcation point in inositol polyphosphate metabolism is seen with the breakdown of Ins(1,3,4)P₃, which is converted either to Ins(1,3)P₂ or to Ins(3,4)P₂ or, under certain circumstances, is also phosphorylated to Ins(1,3,4,6)P₄. In these studies, a 3-fold greater accumulation of [³H]Ins(1,3)P₂ relative to [³H]Ins(3,4)P₂ was observed in both the presence and the absence of LiCl. This pattern of [³H]Ins(1,3,4)P₃ metabolism is in agreement with the results obtained by Batty and co-workers in CCh-stimulated rat cerebral cortex slices (Batty et al., 1989) and by Wreggett and Irvine (1993) in thrombin- and histamine-stimulated human umbilical vein endothelial cells. One explanation for the greater accumulation of [³H]Ins(1,3)P₂ than of [³H]Ins(3,4)P₂ is competition between [³H]Ins(1,3,4)P₃ and the much larger concentration of [³H]Ins(1,4)P₂ for the inositol polyphosphatase 1-phosphatase (Inhorn and Majerus, 1988).

Although the 3-kinase enzyme contributes significantly to Ins(1,4,5)P₃ breakdown under the conditions specified, metabolism via the 5-phosphatase appears to dominate, especially at early time-points after agonist stimulation when accumulation of Ins(1,4,5)P₃ remains elevated over control values. Of interest was our finding that the 3-kinase pathway becomes an increasingly important route for Ins(1,4,5)P₃ metabolism at later time-points after agonist stimulation. This could be related to the Ca⁺⁺/calmodulin regulation of the 3-kinase, because increases in [Ca⁺⁺]_i cause a modest increase in the V_max value of this enzyme (Biden et al., 1988; Rosenberg et al., 1991). In contrast, the activity of the 5-phosphatase has been found in most studies to be unaffected by changes in Ca⁺⁺ over a physiological concentration range (Biden et al., 1988; Connolly et al., 1985). A notable exception to this is the 5-phosphatase from porcine coronary artery smooth muscle, whose activity is enhanced over a range of free-Ca⁺⁺ concentrations of 10⁷ to 10⁶ M. Regulation of Ins(1,4,5)P₃-metabolizing enzymes by PKC has also been demonstrated. For example, in human platelets, PKC-mediated phosphorylation activated 5-phosphatase (Connolly et al., 1986), whereas PKC-mediated serine phosphorylation caused inhibition of rat brain 3-kinase activity (Sim et al., 1990). From the kinetic parameters reported for the Ins(1,4,5)P₃ 5-phosphatase and 3-kinase enzymes in rabbit ASM (Rosenberg et al., 1991), where K_m values were calculated as 95 µM and 5 µM, respectively, it would be anticipated from the high basal Ins(1,4,5)P₃ concentrations present in BTSM [approximately 3 µM (Chilvers et al., 1989b)] that this second messenger would be metabolized preferentially by the 3-kinase enzyme. It is important to note, however, that these estimates indicate that Ins(1,4,5)P₃ is likely to be compartmentalized within the cell and therefore may be present in higher localized concentrations at its immediate site of production.

One additional finding of particular interest in this study was the ability of CCh to stimulate the accumulation of [³H]Ins(4,5)P₂. This compound has been shown to be as effective as Ins(1,4,5)P₃ in mobilizing intracellular Ca⁺⁺ (though with lower potency) (Burgess et al., 1984), and its accumulation cannot be readily accounted for by conventional routes of Ins(P)Px metabolism. Its agonist-stimulated formation, which has previously been demonstrated only in neuronal cells (Jenkinson et al., 1992) suggests that it, or perhaps one of its immediate precursors or metabolites, may play an important role in signaling in this tissue. Our results are in broad agreement with the above study, which reported a highly lithium-sensitive accumulation of this isomer in rat cerebral cortex slices (EC₅₀ = 94 µM), a result that suggests that the flux of the inositol headgroup through Ins(4,5)P₂ may be substantial. These workers demonstrated a similar bell-shaped LiCl concentration-response curve for CCh-stimulated [³H]Ins(4,5)P₂ accumulation in their model and predicted that at therapeutic LiCl concentrations, Ins(4,5)P₂ may accumulate to a significant degree. In agreement with these authors, we were unable to demonstrate conversion of [³H]Ins(1,4,5)P₃ to [³H]Ins(4,5)P₂ in cell-free experiments (data not shown). However, the precise match observed between the increase in [³H]Ins(1,4)P₂ and the decrease in [³H]Ins4P accumulation seen between 10 and 30 mM LiCl, and the very unusual pattern and tight matching of the [³H]Ins4P and [³H]Ins(4,5)P₂ LiCl inhibition data, point strongly to the possibility that [³H]Ins(4,5)P₂ is derived from [³H]Ins(1,4,5)P₃, as occurs in Dictyostelium discoideum (Van Lookeren Campagne et al., 1988). The fact that LiCl at high concentrations does not have a similar effect to enhance Ins(1,4,5)P₃ accumulation (data not shown) probably reflects the presence of two alternative LiCl-insensitive pathways for Ins(1,4,5)P₃ metabolism. The lack of [³H]Ins(4,5)P₂ accumulation after ionomycin or phorbol ester treatment (Chilvers et al., 1994b) and the observation that agonist-stimulated phospholipase activation by CCh in this tissue occurs to a very limited extent and is very transient³ count for the delayed and progressive accumulation of [³H]Ins(4,5)P₂ that was observed. Regarding the further metabolism of [³H]Ins(4,5)P₂, this isomer has been shown to be a weak substrate for the Ins(1,4,5)P₃/Ins(1,3,4,5)P₄ 5-phosphatase (Mitchell et al., 1989). However, this enzyme is not lithium-sensitive; conversely, the involvement of a 4-phosphatase in Ins(4,5)P₂ metabolism is supported by evidence of Ins5P accumulation in rat cerebral cortex (Ackermann et al., 1987). These data therefore represent the first demonstration of agonist-stimulated [³H]Ins(4,5)P₂ accumulation in non-neuronal tissue and point to a novel, though relatively minor, route for Ins(1,4,5)P₃ metabolism. Whether accumulation of Ins(4,5)P₂ at later times induces Ca⁺⁺ release in this tissue via interaction with the Ins(1,4,5)P₃ receptor remains to be determined.

This study confirms that agonist-stimulated accumulation of [³H]Ins(1,4,5)P₃ in BTSM appears to be under tight metabolic control and demonstrates that although the 3-kinase pathway contributes significantly to [³H]Ins(1,4,5)P₃ removal, the 5-phosphatase pathway dominates as the major enzymatic route for [³H]Ins(1,4,5)P₃ metabolism. Furthermore, the accumulation of a novel [³H]InsP₂ isomer in this tissue, namely [³H]Ins(4,5)P₂, points to the presence of an active intracellular Ins(1,4,5)P₃ 1-phosphatase in BTSM and hence to an additional route for Ins(1,4,5)P₃ metabolism.