Evidence for phosphatidylinositol hydrolysis in ... - Europe PMC

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Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, N.S.W. 2010, Australia. Anion-exchange h.p.l.c. was used initially to analyse the products ...
Biochem. J. (1992) 285, 541-549 (Printed in Great Britain)

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Evidence for phosphatidylinositol hydrolysis in pancreatic islets stimulated with carbamoylcholine Kinetic analysis of inositol polyphosphate metabolism Trevor J. BIDEN,* Monica L. PRUGUE and Aidan G. M. DAVISON Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, N.S.W. 2010, Australia

Anion-exchange h.p.l.c. was used initially to analyse the products formed after addition of either [3H]Ins(1,3,4,5)P4 or [3H]Ins(1,4,5)P3 to homogenates of pancreatic islets. Metabolic routes similar to those of other tissues were established: dephosphorylation of Ins(1,4,5)P3 to Ins(1,4)P2 and then Ins4P; and sequential degradation of Ins(1,3,4,5)P4 to Ins(1,3,4)P3, Ins(3,4)P2 and Ins(3 or 1)P. In addition, there was a limited conversion of Ins(1,3,4)P3 into Ins(1,3)P2. After stimulation of [3H]inositol-prelabelled islets with the muscarinic-receptor agonist carbamoylcholine (carbachol), there was a rapid (10 s) increase in Ins(1,4,5)P3, Ins(1,3,4)P3, Ins(1,4)P2 and Ins4P. In the presence of 10 mM-LiCl, Insl P was also significantly increased (P < 0.05) by 5 s, before any increase in Ins4P (10 s), Ins(1,3)P2 (60 s) or Ins(3,4)P2. When carbachol was displaced with atropine, after 1 h pre-stimulation, the maximal decreases in Ins(1,4,5)P3 and Ins 1 P from the stimulated steady state (5 s) clearly preceded those of the other metabolites. These declines were used to calculate the turnover times and rate of metabolic flux through the various inositol phosphates. These experiments confirmed the relatively minor importance of the Ins(1,3)P2 pathway (< 10% of the total flux) and demonstrated that Ins(1,4,5)P3 removal was evenly distributed through the Ins(1,4)P2 and Ins(1,3,4,5)P4 routes. They also established that flux through InsIP was 8-fold greater than that through Ins(1,4,5)P3, indicating that the former could not have been derived from PtdInsP2 hydrolysis. Similarly, in islets pretreated with neomycin, which binds to PtdInsP. with greater affinity than to Ptdlns, the increase in InsIP caused by 1 min stimulation with carbachol was not affected, despite virtual abolition of the increase in Ins4P, and an overall inhibition of PtdInsP2 hydrolysis by 67%. The results indicate that, in addition to PtdInsP2 breakdown, carbachol also promotes a rapid Ptdlns hydrolysis which becomes increasingly predominant with prolonged stimulation.

INTRODUCTION Many examples of cellular activation, brought about by the occupation of cell-surface receptors by their specific agonists, are associated with enhanced hydrolysis of PtdInsP2 and concomitant production of two intracellular messengers: Ins(1,4,5)P3, which releases Ca2+ from internal stores; and diacylglycerol (DAG), which activates protein kinase C [1-3]. It has been proposed that during prolonged stimulation phospholipase C, the enzyme which catalyses that hydrolysis, might also act on Ptdlns, a precursor of PtdInsP2 [4-7]. In this instance DAG would be formed in conjunction with InsIP, a metabolite of no known physiological function. The hydrolysis of Ptdlns would therefore be associated with a protein kinase C activation independent of Ca2+ mobilization. However, the existence of this second hydrolytic pathway has been extremely controversial [8-10]. Indeed, the interpretation of earlier supportive studies [6,7] has been severely compromised by the findings that phosphatidylcholine, as well as Ptdlns, contributes substantially to the DAG generation which is independent of PtdInsP2 hydrolysis in smooth muscle [11], and that Insi P2 can be formed independently of Ptdlns breakdown, especially in brain, via the further metabolism of Ins(1,4,5)P3 [12,13]. Ins(1,4,5)P3 is metabolized initially by either a 3-kinase, which converts it into Ins(1,3,4,5)P4, or a 5-phosphomonoesterase, which dephosphorylates it to Ins(1,4)P2. The latter enzyme also metabolizes Ins(1,3,4,5)P4 to form Ins(1,3,4)P3. This second InsP3 isomer is dephosphorylated to Ins(3,4)P2 by a 1-

phosphomonoesterase [which also generates Ins4P from Ins(1,4)P2]. Alternatively Ins(1,3,4)P3 is converted, by a 4phosphomonoesterase, into Ins(1,3)P2, which acts in turn as the precursor of Ins1P. Moreover, Ins3P, formed by the action of the 4-phosphomonoesterase on Ins(3,4)P2, is an enantiomer of InslP1, and difficult to distinguish from it [12-14]. Therefore, in order to evaluate whether accumulation of Ins(3 or 1)P represents either Ptdlns hydrolysis or Ins(1,4,5)P,3 conversion via the 3kinase route, the rates of flux through the various metabolic routes must be determined. In the two published studies where this has been undertaken, no evidence of Ptdlns breakdown was found [9,10]. However a positive conclusion was reached in a related study [15] based on the calculated fluxes through the more slowly turned-over cyclic inositol phosphates, which are formed in some cell types as by-products of the action of phospholipase C [12]. Although the regulation of Ins(1,4,5)P3 turnover has been well defined in clonal insulin-secreting cells [16,17], a more global view of inositol phosphate metabolism in either these cells or pancreatic islets is lacking. Indeed, there are only limited reports on the accumulation of inositol phosphate isomers in islets after stimulation with receptor-binding agonists, and the appropriate metabolic pathways are virtually uncharacterized in this tissue [17-20]. In the present study we have defined those pathways, calculated their relative contributions to the overall metabolism of Ins(1,4,5)PJ and, in so doing, provided strong evidence that muscarinic stimulation of islets results in a rapid hydrolysis of both Ptdlns and PtdInsP2.

Abbreviations used: PKC, protein kinase C; DAG, 1,2-sn-diacylglycerol; PLC, phospholipase C; TCA, trichloracetic acid; carbachol, carbamoylcholine. * To whom correspondence should be addressed. Vol. 285

542 EXPERIMENTAL Islet isolation and incubation Pancreatic islets were isolated from male Wistar rats by ductal infusion of collagenase [21]. The pelleted digest was resuspended in Histopaque 1077 containing 8.3 mM-glucose, overlayed with Krebs bicarbonate buffer (see below) and centrifuged at 900 g for 20 min. Islets contained at the interface were then hand-picked under a binocular microscope. They were maintained for 72 h at 37 °C inside a modular incubation chamber equilibrated with air/CO2 (19: 1). Batches of 400-600 islets were cultured for 72 h in 4 ml of medium 199 containing 10 % (v/v) heat-inactivated calf serum, 14 mM-NaHCO3, 11.1 mm-glucose, 500 i.u. of penicillin/ml, 100 jug of streptomycin/ml, 50 ,tg of gentamycin/ml and 30 ,Ci of [2-3H]inositol. Under these conditions, islet phosphoinositide pools are labelled to steady state [19]. After culture, the islets were washed with a modified Krebs bicarbonate buffer containing 5 mM-NaHCO3, 1 mM-CaCl2, 2.8 mM-glucose, 0.5 % BSA and 10 mM-Hepes (pH 7.4). Incubations were performed with 50 islets in a final volume of 0.2 ml and terminated by addition of 0.8 ml of 100% (w/v) trichloroacetic acid [19]. In some studies LiCl was added 10 min before the stimulus. The incubation tubes were vortex-mixed immediately and placed on ice for approx. 30 min. After centrifugation, the supernatant was withdrawn and washed three times with a 5-fold excess of diethyl ether. For investigations of inositol phosphate metabolism in cellfree extracts, groups of 1200 freshly isolated islets were homogenized at 4 °C in 50 ,ul of 110 mM-KCl containing 10 mM-Hepes (pH 7.0). A 20 ,ul portion of the homogenate was added to each of two tubes containing 0.5 ml of 110 mM-KCl, 10 mM-Hepes, 10 mM-NaCl, 1 mM-MgSO , 5 mM-EGTA and CaC12 to give a final free Ca2l concentration of 100 nm (pH 7.0) [15]. Depending on the exact experimental protocol, the tubes also contained either 0.5 uM-Ins(1,4,5)P3 and 0.2 uCi of [2-3H]Ins(1,4,5)P3, or 0.5 ,#M-Ins(1,3,4,5)P4 and 0.2 #Ci of [2-3H]Ins(I,3,4,5)P4. In some investigations of Ins(I,3,4,5)P4 degradation, 10 mm LiCl was also present. The incubations were at 37 °C, and 0.1 ml samples were removed at zero time and other times as described, and quenched into 0.5 ml of 100% (w/v) trichloroacetic acid. Extracts were centrifuged and washed with diethyl ether as described above. Results are presented as means + S.E.M. Statistical analysis was by Student's t test for unpaired data.

Inositol phosphate analysis In some experiments the washed extracts were neutralized and applied to columns containing 0.6 ml of Dowex AGI-X8 (formate) resin and washed first with 5 ml of water, and then 5 ml of 5 mM-sodium tetraborate/60 mM-sodium formate. The inositol phosphates were eluted with a series of ammonium formate buffers made up in 0.1 M-formic acid: 5 ml of 0.2 M for InsP; 2 x 4 ml of 0.4 M for InsP2; 2 x 5 ml of 0.8 M for Ins(1,4,5)P3; and 2 x 3 ml of 1.05 M for Ins(1,3,4,5)P4. Samples (3 ml) of the collected fractions were mixed with 10 ml of scintillant and quantified by liquid-scintillation spectrometry. For those samples to be analysed by h.p.l.c., the washed extracts were treated for 30 min with a 0.2 ml slurry of Dowex 50 W-X8 (20-50 mesh) resin, transferred to new tubes, adjusted to pH 7.5 with Tris/HCl and made up to a final concentration of I mM-EDTA in a total volume of 1 ml. For separation of inositol phosphates, one of two h.p.l.c. protocols were used. In the first procedure, samples were loaded on to a Partisphere PAC column (12.5 cm) and eluted at 1.0 ml/min with ammonium phosphate (pH 3.8) according to the following gradient: distilled water for 10 min, 0-0.1 M over 35 min, and 0.1-0.6 M over 55 min. In the alternative method a Partisil SAX column (25 cm) was used and

T. J. Biden, M. L. Prugue and A. G. M. Davison 700

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Fig. 1. Separation of inositol phosphates by anion-exchange h.p.l.c. Results represent the time-dependent elution of radioactivity derived from experimental samples after application of an ammonium phosphate gradient to a Partisphere PAC h.p.l.c. column. For further details of the identification of the various inositol phosphate isomers see the Experimental section. Key: 1 -IP, Ins 1 P etc.; Y-IP2, an uncharacterized InsP2 isomer.

eluted at 1.0 ml/min with ammonium formate (pH 3.7). The gradient was water for 5 min, 0-0.068 M-ammonium formate over 35 min, 0.068-0.37 M over 25 min, 0.37-0.7 M over 15 min and 0.7 M over 30 min. Before sample injection, the column was pre-washed at 2.0 ml/min for 10 min with each of 0.05 M-HPG4 2 M-ammonium formate (pH 3.7 with H3PO4) and then water. In some instances a Partisil PAC (25 cm) column was alternatively used for the second procedure, with little discernible difference in separation efficiency. With either elution protocol fractions were collected every 30 s, mixed with 4 ml of scintillant, and radioactivity was assessed by liquid-scintillation spectrometry. Recovery of inositol phosphates, as assessed with radioactive standards, was always > 80 %. exact

Identification of inositol phosphate isomers A typical elution profile is shown in Fig. 1 for inositol phosphates separated by the first protocol. The earliest peak was identified on the basis of co-elution with a [14C]Ins3P standard added to the injection mixture. Most of the other peaks were identified by comparison with the retention times of a series of tritiated standards run in parallel. As shown in Fig. 1, a wide separation of Ins 1 P and Ins4P was routinely obtained. However, as previously documented, it was impossible to separate the enantiomers InslP and Ins3P [22-26]: radioactivity co-eluted with the [14C]Ins3P (with which the experimental samples were always spiked) will therefore be referred to as Ins(3/1)P. Note that, in islet extracts, there was also a minor peak eluted between InsIP and Ins4P and tentatively identified as Ins2P on the basis of previously published data [26-28]. In addition to the identifiable peaks described above, an unknown InsP2 [Ins(Y)P2] was also present in the islet extracts. The concentration of neither this species nor Ins2P was ever altered during any experiment described below. By using the ammonium formate elution profile, baseline resolution of all of the isomers of interest was obtained, except for Ins(1,3)P2 and Ins(1,4)P2 which were separated by 1-2 fractions. Experimental samples were therefore spiked with [32P]Ins( 1 ,4)P2 to allow for correction of any cross-contamination of this isomer in the Ins(1,3)P2 fractions. With this gradient, the various inositol phosphates were eluted at the following times

(min): InsIP, 30; Ins2P, 32; Ins4P, 35; Ins(1,3)P2, 67.5; Ins(1,4)P2, 68.5; Ins(Y)P2, 70.5; Ins(3,4)P2, 72; Ins(1,3,4)P3, 94; Ins(1,4,5)P3, 96; Ins(1,3,4,5)P4, 108.

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Islet phosphatidylinositol hydrolysis Generation of inositol phosphate standards Radiolabelled inositol phosphate standards were generated by incubating 0.25 mCi of either [3H]Ins(1,4,5)P3 or [3H]Ins(1,3,4,5)P4, or 1 mCi of [4,5-32P]Ins(l,4,5)P3, with 6 ml of a 30 % (w/v) liver homogenate for 15-20 min in 0.5 ml of the same buffer as used for the islet experiments. It has been previously established that, when liver homogenates are employed under similar conditions, Ins(1,4,5)P3 gives rise to a mixture of Ins(1,4)P2 and Ins4P, and Ins(1,3,4,5)P4 gives a mixture of Ins(1,3,4)P3, Ins(3,4)P2, Ins(1,3)P2 and Ins3P [29,30]. The retention times of these compounds were established by h.p.l.c. analysis of samples of the various hydrolysates, run either individually or in combination. Materials All tissue-culture reagents were from Flow Laboratories, Sydney, N.S.W., Australia, except for plasticware, which was obtained from Becton and Dickinson and Co., Lincoln Park, NJ, U.S.A. Except for [4,5-32P]Ins(l,4,5)P3, which was obtained from Dupont Australia Ltd., all radionucleotides [and Ins(l,4,5)P3] were from Amersham Australia Pty. Ltd. (both of Sydney, N.S.W., Australia). Ion-exchange resins were supplied by BioRad Australia. H.p.l.c. columns were purchased from Whatman International, Maidstone, Kent, U.K., and scintillation vials and Hi-ionic fluor from Canberra Packard Pty. Ltd., Sydney, N.S.W., Australia. Collagenase was obtained from Serva Feinbiochemica G.m.b.H., Heidelberg, Germany, and Ins(l,3,4,5)P4 was from Boehringer Mannheim Australia, Sydney, N.S.W., Australia. All other biochemicals and specialized reagents were from Sigma Chemical Co., St. Louis, MO, U.S.A. RESULTS Inositol phosphate metabolism in islet homogenates As an initial step in the characterization of inositol phosphate metabolism, the rates of hydrolysis of exogenous Ins(l,4,5)P3 and Ins(1,3,4,5)P4 by islet homogenates have been compared. As shown in Fig. 2, the dephosphorylation of Ins(1,4,5)P3 was at least twice as rapid as that of Ins(1,3,4,5)P4 during the first 10 min of incubation (cf. Figs. 2a and 2b). In addition, whereas the production of InsP from Ins(1,4,5)P3 was apparent after only 10 min, and rose to 70 % conversion at 90 min, there was very little net conversion of Ins(l,3,4,5)P4 into InsP ( < 10 %) over the same time period. This appeared to be a function of the requirement for an additional dephosphorylation step, as well as the relative slowness of the individual reactions. Anion-exchange h.p.l.c. was next used to investigate further the cause of the slow accumulation of InsP. Although a small component (< 4 %) of the exogenous Ins(I,3,4,5)P4 was degraded through Ins(1,4,5)P3 and Ins(1,4)P2 (results not shown), the great majority was dephosphorylated to Ins(1,3,4)P3, and subsequently Ins(3,4)P2, where most of the radioactivity accumulated (Fig. 3a). Indeed, despite a prolonged and marked elevation, very little of the latter was converted into Ins(3/l)P (Fig. 3a insert): even after 90 min the ratio of Ins(3,4)P2 to Ins(3/ 1)P, remained greater than 12: 1. These results suggest that the 4-phosphomonoesterase, which catalyses this conversion [30,31], is relatively inactive in pancreatic islets. This conclusion is also supported by the very modest accumulation of Ins(1,3)P2 (Fig. 3a) insert) which is generated by the action of this same enzyme on Ins(1,3,4)P3. Over the entire course of the experiment, the levels of Ins(1,3)P2 remained at a relatively constant 6-12% of those found in Ins(3,4)P2 at the corresponding time point. Rapid degradation of Ins(1,3)P2 was probably not the cause of this low value, since the

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Fig. 2. Metabolism of Ins(1,4,5)P3 (a) or Ins(1,3,4,5)P4 (b) by islet homogenates Results represent the time-dependent conversion of the appropriate 3H-labelled substrate (0.5 uM) into their various metabolites. Incubations were performed in a 110 mM-KCl medium (pH 7.0) at a free Ca2l concentration of 100 nM, and started by the addition of homogenate material from 600 islets. Inositol phosphates were separated by Dowex anion-exchange chromatography. Each point is a single determination from one experiment representative of three.

summed counts in Ins(1,3)P2 and Ins(3/l)P never exceeded 15 % of those in Ins(3,4)PJ (Fig. 3a, cf. main Figure and the insert). In corresponding experiments performed in the presence of 10 mM-LiCl (Fig. 3b) there was a clear inhibition of the conversion of Ins(1,3,4)P3 into Ins(3,4)P2, such that the latter accumulated to only 25 % of the level seen previously (cf. Fig. 3a and 3b). Conversely Ins(1,3,4)P3 attained higher levels, especially at the later time points. These conditions would be expected to lead to a marked enhancement of Ins(1,3)P2 [23], and yet the summed radioactivity (c.p.m.) in Ins(1,3)P2 and Ins(3/l)P was still less than twice that seen at the corresponding time point in the absence of LiCl (cf. inserts in Figs. 3a and 3b). Under conditions similar to those described in the legend to Fig. 3(a), exogenous Ins(-1,4,5)P3 was sequentially dephosphorylated to Ins(1,4)P2 and Ins4P; formation of Ins(3/l)P accounted for < 2 % of the total InsP generated (results not shown). However, it has been previously suggested that the proportion of Ins(3/l)P formed is greater when higher concentrations of Ins(1,4)P2 are used, or when the activity of the 1phosphomonoesterase is inhibited with LiCl or the competing substrate Ins(1,3,4)P3 [9]. Therefore the degradation of [3H]Ins(1,4,5)P3 was re-investigated (Table 1) in the presence of 10 mM-LiCl, and unlabelled Ins(1,4,5)P3 and Ins(1,3,4,5)P4 (each 25 ftM). Because of the lower specific radioactivity and the presence of the competing substrate Ins(1,3,4,5)P4, degradation of Ins(1,4,5)P3 was much slower under these conditions (cf. Fig. 2a). Even so, Ins(1,4)P2 and Ins4P did accumulate in a linear fashion over the 2 h incubation. However, no evidence for the formation of Ins(3/l)P was obtained.

T. J. Biden, M. L. Prugue and A. G. M. Davison

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Table 1. Metabolism of 25 ,mM-I3HlIn(1,4,5)P3 by islet homogenates in the presence of 25 jM-Ins(1,3,4,5)P4 and 10 mM-LiCI Incubations were carried out and inositol phosphates separated and quantified as in the legend to Fig. 3. Results are single determinations.

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60 90 Time (min) Fig. 3. Metabolism of Ins(1,3,4,5)P4 by islet homogenates in the absence (a) or presence (b) of 10 mM-LiCl Islet homogenates were incubated with 0.5 ,gM-[3H]Ins(1,3,4,5)P4 in the absence (a) or presence (b) of 10 mM-LiCl. Inositol phosphates

(InsPs) were separated and quantified by anion-exchange h.p.l.c. using an ammonium formate gradient. For further details see the Experimental section. Results are averaged data from two separate experiments.

Inositol phosphate generation in stimulated islets The results described above suggest that Ins(3/1)P is derived exclusively, but rather slowly, from the sequential dephosphorylation of Ins(1,3,4,5)P4, and that therefore its accumulation in intact islets would be considerably delayed after agonist stimulation. This prediction was tested directly by quantifying the InsP isomers present in intact islets after addition of the muscarinic-receptor agonist carbachol (Fig. 4). Although Ins4P was not abundant before stimulation (approx. 10% of InslP levels), it was significantly increased 10 s after carbachol addition, and rose to 20 times the control value after I min (Fig. 4a). In contrast, the levels of Ins(3/ I)P were not significantly raised until after I min of stimulation. The levels of both isomers were maintained for at least 10 min (results not shown). As well as the effects described above, LiCl also inhibits the

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Fig. 4. Time course of generation of InslP and Ins4P in pancreatic islets stimulated with 0.5 mM-carbachol in the absence (a) or presence (b) of 10 mM-LiCI Isolated islets were prelabelled to isotopic equilibrium with [3H]inositol, incubated in Krebs-Ringer bicarbonate medium and then stimulated with carbachol for the times indicated. InsP isomers were separated by anion-exchange h.p.l.c. as described in the legend to Fig. 1. Results are expressed as a percentage of the Ins2P value derived from the same h.p.l.c. run and represent the average of six individual determinations, except for the 60 s values (n 3) and the 10 s control (n = 4). Differences which are statistically different from the appropriate time control (or 10 s control for the 5 s data) are denoted by * P < 0.05 or ** P < 0.001. Symbols: El, InslP, control; A, Ins4P, control; *, InslP, carbachol; A, Ins4P, carbachol. =

dephosphorylation of InsP [7]. Accordingly, addition of 10 mmLiCl to the islets resulted in an increase in the levels of Ins(3/ l)P at all measured time points, including both before stimulation (Fig. 4b) and 10 min after (results not shown). Most importantly, in the presence of LiCl a significant early elevation of Ins(3/1)P 1992

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Islet phosphatidylinositol hydrolysis (a)

which was most marked (5-fold) after 10 min stimulation. However, because basal levels were also increased with LiCl, a significant elevation by carbachol was not obtained until 60 s (P < 0.01). Therefore the agonist-induced rise in Ins(3/l)P, obtained within 5 s under these conditions (Fig. 4), seems too rapid to be explained by the degradation of either Ins(3,4)P2 or

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In the absence of LiCl, both before stimulation and at all time points thereafter, Ins(3,4)P2 predominated (P < 0.01) over Ins(1,4)P2 (by about 3-fold) and Ins(1,3)P2 (approx. 10-fold) (Fig. 5a). Indeed, throughout the time course of stimulation, Ins(1,3)P2 never represented more than 6% of the total InsP2 fraction in the absence of LiCl, or 15 % in its presence. These results suggest that, with time and provided that flux through Ins(3,4)P2 was virtually abolished, dephosphorylation of Ins(1,3,4)P3 to Ins(1,3)P2 did occur in stimulated islets, but not sufficiently rapidly to prevent a 2-4-fold build-up of Ins(1,3,4)P3 as compared with corresponding levels attained in the absence of LiCl. However, conclusions such as this remain tentative unless independently supported by direct measurements of metabolic flux. This was addressed in the next series of experiments (Fig. 6). Islets were stimulated for 1 h with carbachol to allow the relevant inositol phosphates to reach steady-state concentrations (results not shown). At this time carbachol was displaced from its receptor with the muscarinic antagonist atropine, thereby abolishing the stimulation of PLC. A statistically significant (P < 0.05) fall in Ins(1,4,5)P3 was seen within 5 s of atropine addition. Decreases (all P < 0.05) were not apparent until 15 s for Ins(1,3,4)P3, 30 s for Ins(1,4)P2, Ins4P and Ins(3,4)P2, and 1 min for Ins(1,3)P2. These results confirm that Ins(1,4,5)P3 is the

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was now revealed at 5 and 10 s after addition of carbachol. Indeed, this increase actually preceded the rise in Ins4P under these conditions. However, comparing data obtained in the presence and the absence of LiCl, the accumulation of Ins4P was enhanced at all time points except 1 min after stimulation (cf. Figs. 4a and 4b). Further time-course experiments were undertaken to establish whether the accumulation of the two InsP isomers was consistent with their sequential derivation from InsP2s and InsP3s (Fig. 5). For clarity of presentation, and because similar data have already been published [19], the Ins(1,4,5)P3 results are not shown. However this compound was significantly increased (P < 0.05) at 10 s after carbachol addition, rose to a maximum of approximately double pre-stimulatory values at 60 s, and thereafter declined. As shown in Fig. 5(a), Ins(1,3,4)P3 and Ins(1,4)P2 were also increased, by at least 2.5-fold (P < 0.005), within 10 s. In contrast, there was a lag before any significant elevation of either Ins(3,4)P2 or Ins(1,3)P2 (both non-significant 10 s, and P < 0.001 at 30 s). Addition of 10 mM-LiCl to the islets did not significantly alter the accumulation of Ins(1,4,5)P3 (results not shown). The most obvious effect of the cation was to enhance the stimulated levels of Ins(1,4)P2 and Ins(1,3,4)P3, but these were also significantly increased (P < 0.005) even before agonist addition, by approx. 3and 4-fold respectively (Fig. 5b). LiCl also completely blocked the agonist-induced rise in Ins(3,4)P2 and conversely potentiated the levels of Ins(1,3)P2 accumulating at all time points, an effect

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Time (s) Fig. 6. Time course of inositol phosphate disappearance after blockade of the muscarinic receptor Prelabelled islets were incubated for 1 h with carbachol (0.5 mM) under conditions identical with those described in the legend to Fig. 4. After 1 h, atropine (0.1 mM) was added and the incubation was terminated with 10 % trichloroacetic acid at the indicated times after atropine addition. Inositol phosphates (InsPs) were extracted, separated and quantified as described in the legend to Fig. 1. Results are triplicate determinations of a single experiment typical of three. Radioactivity in the individual metabolites has been normalized with respect to that in Ins2P, which never changed during the courses of the experiments and averaged 209 + 8 c.p.m. (n = 15).

T. J. Biden, M. L. Prugue and A. G. M. Davison

546 Table 2. Kinetics of inositol phosphate turnover Data from Fig. 6 and two similar experiments were replotted as the natural logarithm of the inositol phosphate/Ins2P ratio. The initial slopes, calculated by regression analysis of the linear portion of the graphs, were used to determine a single rate constant (k) for each metabolite in each individual experiment. These were used to calculate a value for flux (60 x k x the initial concentration) and halflife (ti, calculated as 0.693/k). The portions of the curves used were: 0-5 s for Ins(1,4,5)P3 and Ins lP; 5-30 s for Ins(1,3,4)P3 and Ins(1,4)P2; 15-60 s for Ins(3,4)Ps and Ins4P; and 30-60 s for Ins(1,3)P2 (cf. Fig. 6). Results are mean values derived from the three experiments. Flux (A inositol phosphate/

Inositol phosphate

InslP Ins4P

Ins(1,3)P2 Ins(1,4)P2 Ins(3,4)P2 Ins(l ,3,4)P3 Ins(1,4,5)P3

Ins4P+Ins(3,4)P2 Ins(1,3)P2+ Ins(3,4)P2

tl (s)

Ins2P min-')

27.8+9.2 62.7+ 11.1 61.8 + 8.9 39.4+ 3.4 84.3 +2.1 33.9 + 16.8 12.9+2.3

11.5+2.5 1.65 +0.34 0.115 +0.010 0.777 +0.086 0.870+0.200 0.636 + 0.150 1.44+0.16 2.52 +0.39 0.985 +0.200 1.413 +0.39 1.76 + 0.22

-

Ins(l,3,4)P3+ Ins(1,4)P2

-

Ins(1,3)P2 + Ins(1,4)P2 + Ins(3,4)P2

-

Table 3. Effect of neomycin pretreatment on the generation of InsP (a) or inositol phosphates (b) in islets Islets were preincubated for 20 min in the absence or presence of 5 mM-neomycin, and then for a further 1 min without or with (0.5 mM) carbachol. Inositol phosphates were extracted and separated as described in the legend to Fig. 1. Results in (a) are means of 10-11 individual determinations and are expressed as a percentage of the respective control; ** P < 0.001, * P < 0.05 versus control. Results in part (b) represent the increment above control after all results were calculated initially as percentage of Ins2P. Each value is the mean of triplicate determinations from a single experiment; ns, not significant. InsIP

(a) Condition Control Carbachol Neomycin Neomycin/carbachol

100+ 7 162+4** 115 + 8 154 + 9**

100+ 13 1310+93** 153 + 16* 318 + 22**

Stimulation

(Alnositol phosphate/Ins2P) Decrease

(b) Inositol phosphate

InslP

precursor of the other metabolites. However, this was clearly not the case for Ins I P, the levels of which were significantly decreased at 5 s, well before those of any of the metabolites postulated to be its immediate precursor. Since at steady state the rates of formation of individual inositol phosphates must equal their rates of degradation, the data from Fig. 6 and two similar experiments were used to quantify the rates of flux through the individual metabolites. This is most straightforward for Ins(1,4,5)P3: its initial rate of degradation, as measured by the downward slope during the first 5 s after addition of atropine, must very closely approximate to its rate of formation from PtdInsP2 during the stimulated steady state. It should be stressed that the formation of Ins(1,4,5)P3 alone would be abolished immediately by atropine; the more distal metabolites would still be formed, albeit at an everdiminishing rate, until their immediate precursors declined towards pre-stimulatory levels. For this reason, we have not included the lag periods for calculation of the slopes, and hence rates of metabolic flux (see the legend to Table 2). If the measured inositol phosphates are truly metabolites of Ins(1,4,5)P3, then the rates of flux through them cannot exceed that through Ins(1,4,5)P3. The calculated rates are shown in Table 2 and lead to several important conclusions. Firstly, except for Ins(3/l)P (see below), the rate of flux through any particular metabolite was not significantly greater than that through any of its putative precursors. Moreover, the combined flux through Ins(1,3)P2 plus Ins(3,4)P2 did not significantly exceed that through Ins(1,3,4)P3, nor were the summed values of either Ins4P plus Ins(3,4)P2, or Ins(1,3,4)P3 plus Ins(1,4)P2, significantly greater than that for Ins(1,4,5)P3. These findings confirm the product/ precursor relationships established in islet homogenates. Secondly, Ins(1,4,5)P3 disposal was evenly distributed through the 3kinase and 5-phosphomonoesterase pathways, since flux through Ins(1,3,4)P3 did not differ significantly from that through Ins(1,4)P2. Thirdly, the rate of conversion of Ins(1,3,4)P3 into Ins(1,3)P2 was only 10-20 %/1 (P < 0.02) of that metabolized via Ins(3,4)P2. Fourthly, the combined flux through all of the InsP2 isomers was not significantly greater than that through

Ins4P

(% of control) (q° of control)

Ins4P Ins(1,3)P2 Ins(1,4)P2

Ins(3,4)P2 Ins(l ,3,4)P3 Ins(l,4,5)P3 Total -Ins 1 P

Control

Neomycin

(MO)

327 + 23 744+27 83.5 + 2.8 148 +6 473 + 11 311 + 26 90.0+ 7.0 1850+46

237 + 57 124 + 18 26.4+4.6 82+4 207 + 16 127 +9 47.6+4.9 617 + 63

ns 83 68

45 56 59 47 67

Ins(1,4,5)P3. This rules out any major contribution of PtdlnsP hydrolysis to Ins(1 ,4)P2 formation. Fifthly, the flux through Ins(3/1)P was significantly greater (P < 0.02) than that through any other metabolite or combination of metabolites shown in Table 2. Most importantly, it was approx. 8 times larger (P < 0.01) than the flux through Ins(1,4,5)P3. This finding is incompatable with Ins(1,4,5)P3 being the precursor of Ins(3/l)P and suggests, rather, that Ptdlns was being hydrolysed under these conditions and, indeed, being hydrolysed considerably more rapidly than PtdInsP2. Consistent with this conclusion is the low half-life of Ins(3/l)P, which, despite its much higher steady-state concentration, was only double that of Ins(1,4,5)P3. The most slowly turned-over intermediate was Ins(3,4)P2, although the half-life of Ins( 1,3)P2 was also high for a compound present at such low levels. In order to provide independent evidence for Ptdlns hydrolysis, and to investigate whether it occurred over shorter periods of stimulation, a further approach was adopted. This involved preincubation of the islets with neomycin, which, by virtue of its greater affinity for more negatively charged phospholipids, has been previously used as a selective inhibitor of PtdInsP2 hydrolysis [32]. In pretreated islets, which were subsequently stimulated for I min with carbachol, the rise in Ins4P was inhibited by approx. 85 %, compared with untreated controls, whereas the increase in Ins(3/1)P was not significantly affected (Table 3a). This could not be explained by a selective inhibition of the 5phosphomonoesterase route, since the increases in metabolites such as Ins(1,3,4)P3, Ins(3,4)P2 and Ins(1,3)P2, which are generated via the 3-kinase pathway, were also markedly attenuated (Table 3b). Indeed, by summing the increments in the individual

1992

547

Islet phosphatidylinositol hydrolysis

metabolites, it can be calculated that stimulated PtdInsP2 hydrolysis was inhibited by approx. 66% after neomycin pretreatment. The fact that Insi P was not significantly affected under these conditions argues strongly against its formation from PtdInsP2, but rather suggests that it is derived almost completely from, and represents a direct measure of, Ptdlns hydrolysis. Since the rise in Ins(3/l)P amounted to approx. 200% of the summed increases in all metabolites derived from PtdInsP2, it might be concluded that Ptdlns hydrolysis represented only a minor source of DAG in islets after stimulation with carbachol for I min. Although this conclusion is probably correct, it should be noted that total Ptdlns hydrolysis is probably underestimated according to this calculation, because it does not take into account Ins(3/l)P degradation. DISCUSSION Pathways of inositol phosphate metabolism in islets In the present study, we have focused on the major metabolic routes involved in the rapid removal of Ins(1,4,5)P3. Although the levels of other metabolites, such as cyclic inositol phosphates [12] or the more highly phosphorylated derivatives of Ins(1,3,4)P3 [13,23], are also changed during cellular activation, these are turned over slowly and so have not been studied here. The major routes involved in the rapid metabolism of Ins(1,4,5)P3 in islets are similar to those described for other cell types [12-14] and are shown in Scheme 1. Evidence consistent with these pathways includes the sequential conversion of either [3H]Ins(1,3,4,5)P4 or [3H]Ins(1,4,5)P3 into appropriate intermediates in islet homogenates, the time courses of inositol phosphate accumulation in islets stimulated with carbachol, the relative lags before these metabolites declined from their stimulated steady-state levels after displacement of carbachol from its receptor, and direct measurements of flux through the individual metabolites. Although not shown in Scheme 1, some dephosphorylation of Ins(1,3,4)P3 to Ins(1,3)P2 also occurred in islets. However, this was a very minor pathway, as indicated by the very low rates of Ins(1,3)P2 accumulation in islet homogenates, and the direct demonstration that Ins(1,3)P2 accounted for < 100% of total Ins(1,4,5)P3 disposal after 60 min stimulation. Formation of Ins(1,3)P2 in homogenates in intact cells and cell-free systems has been well documented [23-31], but there is no other report on its turnover. However, from the ratio of Ins(1,3)P2 to Ins(3,4)P2 formed in stimulated cells, it would appear that the Ins(1,3)P2 pathway displays an activity which is also low in cardiac myocytes [25], vascular endothelial cells [33] and neutrophils [22], in-

| Ptdt DAG

:

If

InslP Ins4P -0-

Inositol

:

InWA,4P2 e

ns(1,4,5)P3

Ins(1,3,4,5)P4

Ins(1,3,4)P3 Ins(3,4)P2 4 Ins3P *Scheme 1. Major routes of inositol phosphate metabolism in pancreatic islets after 1 h stimulation with 0.5 mM-carbachol The arrow thickness approximates to the magnitude of flux. Minor pathways (accounting for < 100% of disposal) have been omitted. These include the back-conversion of Ins(1,3,4,5)P4 into Ins(1,4,5)P3, and the small production of Ins(1,3)P2 from Ins(1,3,4)P3.

Vol. 285

termediate in adrenal glomerulosa [23], but predominant in rat cerebral cortex [26] and bovine brain [34]. The slow rate of flux through Ins(1,3)P2 in islets might be explained by the high level of Ins(3,4)P2, which would compete with Ins(1,3,4)P3 as a substrate for the 4-phosphomonoesterase [35]. Flux measurements were consistent with Ins(1,4,5)P3 disposal being evenly divided between the 3-kinase and 5-phosphomonoesterase routes, at least after 60 min stimulation. The same conclusion probably holds true both for earlier times and for the unstimulated state, since LiCl augmented the levels of Ins(1,3,4)P3 and Ins(1,4)P2 to similar extents, under either basal or stimulated conditions. A similar analysis has been used as an indirect quantification of the two pathways in adrenal glomerulosa [36] and assumes that the 1-phosphomonoesterase enzyme is equally sensitive to LiCl inhibition, irrespective of whether Ins(1,3,4)P3 or Ins(1,4)P2 is the substrate. Available evidence [34,37] suggests that inhibition of Ins(1,3,4)P3 dephosphorylation is a little more complete with LiCl, meaning that the contribution of the Ins(1,4)P2 route might be slightly underestimated according to this interpretation. In any event, it is apparent that the Ins(1,4,5)P3 3-kinase pathway does not greatly predominate. However, the results presented here do not preclude our previous suggestion [17] that 3-kinase activity predominates in the very early seconds after agonist addition. At that time the transient rise in intracellular [Ca2+] would be maximal and exert its most pronounced stimulation on the of the calmodulin-regulated 3-kinase [16,38]. An unusual aspect of inositol phosphate metabolism in pancreatic islets is its high basal rate. This is evident from the effect of LiCl to promote severalfold increases in the basal concentrations of a number of metabolites, in addition to markedly potentiating their accumulation after addition of carbachol. Although the potentiatory effect after agonist stimulation is well documented in other tissues, with the exception [27] of a 40 % rise in Ins(1,4)P2 observed in GH3 cells there have been no reported increases in basal inositol phosphates due to LiCl. A high basal rate of inositol phosphate turnover in islets might also explain the predominance of Ins(1,3,4)P3 over Ins(1,4,5)P3, a condition usually associated in other cell systems with agonist stimulation. It would also be consistent with the fact that PLC is highly sensitive to Ca2+ in insulin-secreting cells and so might be partially activated at resting intracellular [Ca2+] [19,39]. Leakiness of the islet-cell plasma membrane to Ca2+ is not a contributing factor, since the effects of LiCl on basal inositol phosphates were also apparent when using islets incubated in the presence of EGTA (T. J. Biden, unpublished work). Another novel finding was the fairly constant 3-fold excess of Ins(3,4)P2 over Ins(1,4)P2 both before and after agonist addition. In contrast, repeated investigations using a wide variety of tissues have shown a vast excess of Ins(1,4)P2 over Ins(3,4)P2 [17,22,23,26,27,33,36]. In several studies this was accompanied by markedly elevated Ins(1,3,4)P3 levels, attesting to a high activity of Ins(1,4,5)P3 3-kinase [17,23,27]. Moreover, in rat parotid cells Ins(3,4)P2 was the least elevated on the InsPJs, despite the fact that, as in islets, the rates of metabolic flux through Ins(1,3,4)P3 and Ins(1,4)P2 were very similar [9]. Therefore this disproportionate accumulation of Ins(3,4)P2 in islets cannot be explained by an elevated Ins(1,4,5)P3 kinase pathway. It is more likely to be explained by the fact that its half-life was double that of Ins(1,4)P2 and it is therefore turned over much more slowly. It would be of interest to compare this half-life ratio with that in tissues in which Ins(1,4)P2 predominates, but this information is unavailable. However it is noteworthy that Pirotton et al. [33], commenting on indirect evidence of slow turnover of Ins(3,4)P2 in vascular endothelial cells, speculate that the Ins(3,4)P2 4-phosphomonoesterase might be subjected to

Vm.x

T. J. Biden, M. L. Prugue and A. G. M. Davison

548

end-product inhibition by Ins3P. An analogous effect of the enantiomer Ins I P might explain the relatively high accumulation of Ins(3,4)P2 in islets. Ptdlns hydrolysis The present study raises several compelling arguments for the occurrence of Ptdlns hydrolysis in pancreatic islets stimulated with carbachol. First, significant early increases were seen in Ins(3/1)P which, in the presence of LiCl, preceded those in Ins4P. This implies that although 5 s was too soon for Ins(1,4,5)P3 to be dephosphorylated to Ins4P, it was sufficient time for Ins(3/l)P to be generated by the 3-kinase route. This seems highly improbable, since significant increases in Ins(1,3)P2 or

Ins(3,4)P2

did not occur before 60 s under these conditions.

Moreover, there was no evidence to suggest that Ins(3/ 1)P might be formed in the necessary quantities by an alternative route, such as dephosphorylation of Ins(1,4)P2. Thus it would appear very unlikely that the early increase in Ins(3/1)P could be explained by anything other than Ptdlns hydrolysis. To our knowledge there is no other report in which a stimulated rise in Ins(3/l)P has been shown to precede that of Ins4P. In the great majority of cell systems, including pituitary gonadotrophs [40], neutrophils [22], GH3 cells [28], adrenal glomerulosa [23], vascular endothelium [33] and neuroblastoma cells [10], the increase in Ins(3/l)P owing to agonist addition is slower in onset, and less substantial, than the rise in Ins4P. The stimulation profiles of the two InsPs are more similar in a second study performed with GH3 cells [27], in adrenal glomerulosa stimulated with endothelin [36], and with platelets stimulated with thrombin [41]. The second and most compelling argument in favour of PtdIns hydrolysis was a rapid disappearance of Ins(3/l)P after the

following increases in intracellular [Ca2"] [12] or alterations in intracellular pH [42]. The results presented here are consistent with that scenario, in that PtdIns breakdown appeared to predominate greatly over PtdInsP2 hydrolysis after 1 h of stimulation, but appeared much less important after 1 min. However, this could be explained by a down-regulation of the PtdInsP2 response [5], rather than a delayed stimulation of Ptdlns hydrolysis, since the latter clearly does occur within 1 min of carbachol addition. Furthermore, the rapidity of both the generation of InslP (5 s in the presence of LiCl), and its decline from the stimulated steady state (maximal over 5 s), suggest a more direct coupling to the muscarinic receptor in islets than has been proposed in previous studies. Indeed, the very existence of Ptdlns hydrolysis in other tissues has been controversial, and probably does not occur to any significant extent in parotid [9] or neuroblastoma cells [10]. The compelling evidence in favour of a substantial PtdIns breakdown in islets raises questions as to whether DAG from this source fulfils the same cellular function, during insulin secretion, as that derived from PtdInsP2, or whether it serves merely as a means of regulating protein kinase C activity independently of Ca21 mobilization. In this context it is noteworthy that hydrolysis of phosphatidylcholine, usually considered an alternative source of DAG in most tissues, does not occur in islets stimulated with carbachol [43]. This work was supported by a Research Grant from the Juvenile Diabetes Foundation International and, in part, by an Apex/Diabetes Australia Research and Education Grant and a Queen Elizabeth II National Research Fellowship. T. J. B. is the recipient of a Juvenile Diabetes Foundation International Career Development Award. We are extremely grateful to Dr. Don Chisholm for his support and advice, to Nick Oakes and Arthur Jenkins for helpful discussions, and to Gilbert Meunier for technical assistance.

abolition of carbachol stimulation. The onset of this decrease

preceded that of any immediate precursor, and its rate was too great for Ins(3/l)P to have been formed solely as a result of Ins(1,4,5)P3 metabolism. In previous studies using parotid [9] or neuroblastoma cells [10], in which a similar kinetic approach was adopted, no evidence for Ptdlns hydrolysis was obtained, since the calculated rates of flux through Ins(3/l)P were not significantly greater than that through Ins(1,4,5)P3. The discrepancy with the present results is probably due to specific tissue differences. For example, the half-life of Ins(3/l)P in the neuroblastoma cells [10] was 119 s, as opposed to 28 s in islets, despite very similar turnover times for the other metabolites measured. In the other study [9] a half-time for Ins(3/1)P could not be measured, and the metabolic flux through it was calculated indirectly. However, in a third investigation, based on a kinetic analysis of the turnover of cyclic inositol phosphates, it was concluded that Ptdlns breakdown did occur in the exocrine pancreas [15]. The third indication of PtdIns hydrolysis was the insensitivity of the carbachol-stimulated rise in Ins(3/1)P to pretreatment with neomycin. Under these conditions PtdInsP2 hydrolysis was markedly attenuated. Neomycin has previously been used in permeabilized 3T3 cells, in which it was shown to abolish the direct activation of PtdInsP2 hydrolysis (as measured by InsP3 levels), to inhibit partially the rise in InsP2, but to be without effect on InsP [32]. Since individual isomers were not measured in that study, it cannot be certain whether the InsP came from Ptdlns or PtdInsP2. In generating these three complementary lines of evidence, we have been able to present a fuller characterization of Ptdlns hydrolysis in islets than is probably available for any other tissue. Hitherto, it has been generally thought that Ptdlns hydrolysis served chiefly as a source of DAG during prolonged stimulation and, accordingly, was initiated as a secondary phenomenon

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