Profile of phosphatidylinositol metabolism stimulated by carbachol and glutamate in primary cultures of rat cerebellar neurons

Nruropltnrmacolog~Vol. 28. No. 12.pp. 1309-1315,1989 Printed in Great Britain. All rights reserved

002%3908/89$3.00+ 0.00 Copyright 5‘ 1989Pergamoo Press plc

PROFILE OF PHOSPHATIDYLINOSITOL METABOLISM STIMULATED BY CARBACHOL AND GLUTAMATE IN PRIMARY CULTURES OF RAT CEREBELLAR NEURONS S. HYNIE,J. T. WROBLEWSKIand E. COSTA* Fidia-Georgetown

Institute for the Neurosciences, Georgetown University School of Medicine, 3900 Reservoir Rd. N.W., Washington, D.C. 20007, U.S.A. (Accepted 3 I May 1989)

Summary-The formation of inositol phosphates, after stimulation of primary cultures of cerebellar neurons of the neonatal rat, in the presence of lithium chloride, by glutamate, carbachol, norepinephrine, histamine and Mg’+-free conditions, was measured by anion exchange high-pressure liquid chromatography (HPLC) with on-line radioactivity detection. All of the above agents caused a persistent, dose-dependent and calcium-sensitive preferential accumulation of inositol-4-phosphate, while the levels of inositol-l-phosphate were virtually unaffected. Agonist stimulation produced also a transient increase of a second peak which co-eluted with the standard for inositol 1,4-bisphosphate. However, no significant accumulation of inositol-1,4,Strisphosphate and inositol-1,3,4,5-tetrakisphosphate was detected, possibly due to the fast kinetics of the metabolism of inositol phosphate. The results indicate that receptor-stimulated metabolism of inositol phosphate, in cultures of cerebellar granule cells, is due to a preferential hydrolysis of polyphosphoinositides and leads to the formation of inositol-4-phosphate through several calcium- and lithium-sensitive enzymatic steps. Key Mlords-glutamate,

carbachol,

phosphatidylinositol,

hydrolysis of inositol phospholipids in membranes plays a key role in intracellular signalling. The activation of phospholipase C leads to the formation of two intracellular messengers: diacylglycerol, which enhances the translocation and stimulation of protein kinase C and, therefore, will affect the phosphorylation of a variety of intracellular proteins and inositol I ,4,Urisphosphate [( 1,4,5)IP,] which regulates the distribution of intracellular stores of calcium ions (Berridge and Irvine, 1984; Hirasawa and Nishizuka, 1985; Berridge, 1987). Further metabolism of (1,4,5)IP, leads to a cascade of inositol phosphates (Majerus, Connolly, Deckmyn, Ross, Brass, Ishii, Bansal and Wilson, 1986) which is terminated by lithium-sensitive dephosphorylation to inositol (Drummond, 1987). Among these metabolites, 1,3,4$tetrakisphosphate [(1,3,4,5)IP,] seems to have a regulatory role in facilitating the entry of extracellular calcium ions (Irvine, 1989). In the central nervous system, the hydrolysis of phosphatidylinositol participates in signal transduction of several neurotransmitter receptors, including alpha,-noradrenergic, muscarinic cholinergic and excitatory amino acid receptors (Brown, Kendall and Nahorski, 1984; Gonzales and Crews, 1985; Sladeczek, Pin, Recasens, Bockaert and Weiss, 1985; Nicoletti, Iadarola, Wroblewski and Costa, 1986a; Nicoletti, Wroblewski, Novelli, Alho, Guidotti and The

*To whom

correspondence

should

be addressed.

inositol

phosphates,

granule

cells, HPLC.

Costa, 1986b; Xu and Chuang, l987a, b). In these studies, the receptor-mediated hydrolysis of phosphatidylinositol has been demonstrated by the accumulation, in the presence of lithium ions, of the total pool of inositol phosphates or of inositol monophosphates, separated by anion exchange chromatography. These methods do not distinguish between the individual isomers of inositol phosphates, although such a differentiation would provide a better insight into the chain of events leading to the formation of phosphoinositide-derived second messengers. The aim of this study was to determine the profiles of the accumulation of inositol phosphates in primary cultures of cerebellar granule cells, treated with various neurotransmitter receptor agonists in the presence of lithium ions. For this purpose, an efficient HPLC method was established which enables the separation of the isomers of inositol monophosphates and of the other main metabolites of phosphoinositide hydrolysis. METHODS

Cultures of cerebellar granule cells

Primary cultures of cerebellar granule cells were prepared from 8-day old Sprague-Dawley rats (Zivic-Miller), as described previously (Gallo, Ciotti, Coletti, Aloisi and Levi, 1982; Nicoletti et al., 1986b). Cells were plated on 35 mm culture dishes, at a density of 3.2 x IO6 cells/dish and were used for the experiments after 8-10 days in culture.

1309

1310

Measurement

S. HYNIE et

of hydroiJ,sis

qf phosphatidylinositol

Cells were incubated for 24 hr with 3 PCi of myo[‘Hlinositol in order to label the phosphoinositides of the cell membrane. Before the experiments, the cells were washed twice with prewarmed medium, containing II8 mM NaCI, 4.7 mM KCI, 1.3 mM CaC&, 35 mM NaHCO,, 1.2 mM KH2POI, 1.2 mM MgSO, and I I .7 mM glucose and equilibrated to pH 7.4 with 95% 0,/5% CO?. In some experiments magnesium was omitted from the incubation medium. Lithium chloride (IO-20 mM) was added 5 min before the addition of receptor agonists. Incubation times varied from 20 set to 60 min. The incubations were terminated by the aspiration of the medium and the addition of 1 ml of ice-cold mixture of water and methanol (I : I) with 5 mM ethyleneglycol-bis-(aaminoethyl ether)N.N’-tetraacetic acid (EGTA). The cells were harvested and the suspension was added to 0.5 ml of chloroform. After thorough mixing, the extracted phases were separated by centrifugation (1000~ for 3 min). The aqueous phase was collected and used directly for HPLC analysis. Aliquots of the organic phase were used to estimate total incorporation of radioactivity.

The HPLC

analwis

of inositol phosphates

Inositol phosphates were analyzed by anion exchange high-pressure liquid chromatography using an extended range HPLC pump with gradient mixer (Spectra-Physics), a manual injector (Rheodyne), equipped with a 2 ml loop, a 6.2 x 80 mm Zorbax SAX Bioseries column (DuPont) and an on-line radioactivity detector (Trace II, Packard). The mobile phase consisted of a linear gradient of (A) water and (B) I M ammonium phosphate, pH 3.8, according to the protocol: O-3 min 100% A, 20 min 10% B, 2540 min 80% B at a flow rate of I ml/min. After each run, the column was washed with 4ml B and equilibrated with 24ml A. This procedure separates isomers of IP, and all main inositol phosphates. The radioactive peaks were measured with 35% efficiency that was not significantly affected by the gradient of ammonium phosphate. Identification of the peaks was based on co-elution with radiolabelled standards of inositol l-phosphate [( I)IP], inositol 4-phosphate [(4)IP]. inositol I ,4-bisphosphate [( 1,4)IP& inositol 1,4.5-trisphosphate [( 1.4.5)IP,] and inositol I ,3.4.5,tetrakisphosphate [( 1,3.4.5)IP,]. Standards of [jH]glycerophosphoinositol (GPI) and its phosphorylated derivative (GPIP) were prepared from the corresponding lipids by quantitative alkaline deacylation (Downes and Michell, 1981). and [‘Hlinositol (l:2cyclic)phosphate (cIP) was obtained by enzymatic hydrolysis of [3H]phosphatidylinositol by phospholipase C (Kemp, Hubscher and Hawthorne, 1961). The recovery of all radiolabelled standards. added to the cell cultures before the extraction procedure, was greater than 80%.

al.

Chemicals Radioactive compounds: [3H]myo-inositol (12.8 Ci/ mmol), [3H]inositol-l -phosphate (10 Ci/mmol), [-‘HIinositol-4-phosphate (I .5 Ci/mmol), [3H]inositol-l,4(1.5 Ci;‘mmol), bisphosphate [3H]inositol- I ,4,5trisphosphate (4 Ci/mmol) and [3H]inositol-l,3,4,5tetrakisphosphate (4 Ci/mmol) were purchased from New England Nuclear. All other chemicals were purchased from Sigma or Fisher.

RESULTS

The use of a strong anion exchange column allowed the separation of a wide range of products of the hydrolysis of phosphoinositide. Figure l(A) shows the elution profile of radioactive standards of inositol phosphates and glycerophosphoinositols. The procedure used provided a complete separation of the two main isomers of inositol monophosphate (l)IP and (4)IP and a sharp separation of IP,, IP, and IP,. Glycerophosphoinositol appeared as the second main peak after inositol and was followed by inositol (I :2 cyclic) phosphate. The phosphate of glycerophosphoinositol eluted in front of IP?. After the incubation of primary cultures of cerebellar granule cells with transmitter receptor agonists, the products of hydrolysis of phosphatidyl inositol were extracted from the cells using neutral conditions, which preserve cyclic inositol phosphates sensitive to acidic pH (Dawson, Freinkel, Jungawala and Clarke, 1971). The inclusion of 5 mM EGTA or ethylene diamine tetrammonium (EDTA) reduced the formation of radioactive peaks corresponding to GPI and GPIP, which accumulated during the extraction (data not shown). A typical elution profile of the products of phosphatidylinositol hydrolysis formed after I5 min of stimulation with carbachol is shown in Figure l(B). The two predominant peaks of inositol phosphates were identified as (4)IP and IP2, on the basis of the available radioactive standards. The chromatograms of extracts from stimulated cells also showed a considerable accumulation of cIP. The peaks eluting in the position of IP, and IP, were too small for reliable quantification. The chromatogram also shows a late appearing peak which may correspond to the theoretical position of inositol pentakisphosphate; however, it could not be identified due to the lack of appropriate standards. The stimulation of the turnover of phosphatidylinositol in cultures of cerebellar granule cells, by transmitter receptor agonists, led to the preferential accumulation of inositol (4)IP (Fig. 2). The greatest accumulation was observed after carbachol and glutamate, while norepinephrine and histamine produced a less pronounced increase. Corresponding increases were also observed in (I)IP; however, the extent of accumulation was much less. as compared to that of (4)IP. The accumulation of IP2 was the most pronounced with carbachol. No significant

Profile of agonist-induced PI metabolism GPICIP II) IP 14lIP

Inositol

GPIPIP,

1311 Ip4 0.a

500

=I 0.6

E

0.0 2

0.a a 9 .-I

0

io

d

20

3

tf-d40.0 40

min

Fig. I. The HPLC profile of the products of ~H]phosphatidylinositoJ hydrolysis, in a mixture of radioactive standards (A) and in samples extracted from cultures of granule cells, stimuiated for IS min with 100 p M carbachol (B). The chromatographic conditions and abbreviations used are described in Methods.

changes in the accumulation of IP, or IP, were detected (data not shown). The incubation of cell cultures in Mg?+-free conditions also led to the preferential accumulation of(4)IP (Fig. 2). This effect was additive to the stimulation obtained with 100 PM carbachol or glutamate (data not shown). The stimulation of the hydrolysis of phosphatidyl inositoi in cerebellar granule cells, caused by 10 min of incubation with carbachol or glutamate, was dosedependent in the range of 10e7 to IV4 M (Fig. 3). The threshold dose for the stimulation of the formation of (4)IP was less for carbachol (1W6 M) than for

glutamate. Moreover, a selective accumulation of this product could be obtained with carbachol, but not with glutamate. Also the accumulation of IF, appeared to be greater for carbachol than for glutamate, for every dose tested. The accumulation of (1)IP became significant only with very large doses of either compound and never compared in extent to the accumulation of (4)IP or IP,. In the concentration range tested, carbachol was more efficacious than glutamate in eliciting the accumulation of (4)IP and IP, (Fig. 3). The time course of the accumulation of inositol phosphates in cerebellar granule ceils, elicited by carbachol and glutamate, is shown in Figure 4. Both agonists stimulated the fo~atjon of (4)IP after only

.J

"

CONTROL

CAR5

GLd

Mg2+-0

NE

HIS1

Fig. 2. Accumulation of [fH]inositol phosphates in cultures of cerebellar granule cells, stimulated for I5 min by neurotransmitter receptor agonists in the presence of I mM Mg2+ or by Mg*+-free conditions, All agonists were used at 100 p M concentration. Abbreviations: CARB-carbachol, GLU-glutamate. NE-norepinephrine, HIST-histamine, Mg’+ = 0-Mg’+-free medium. The values are means + SE from 4 experiments.

GLUTAMATE ILog HI

CARBACHUL (LogHI

Fig. 3. Dose-dependent accumulation of [3H]inosital phosphates in cultures of cerebellar granule cells after IO min of incubation with glutamate (A) or carbachol (B). The particular curves represent the accumulation of [‘H](I)JP (0). t3H](4)IP (A) and [‘H]IP2 (m). The points are means from 3 experiments with SE less than 10% of the mean values.

S. HYNE 6’1 01.

1312

50-A 40 30 20 10 0

0

10

20

30

40

50

60

0

MIN

10

20

30 40 MIN

50

60

Fig. 4. Time course of the accumulation of [?H]inositol phosphates in cultures of the cerebellar granule cell. after incubation with IOOpM glutamate (A) or carbachol (B). The particular curves represent the accumulation of [‘H](I)IP (O), [iH](4)IP (A) and [‘H]IP2 (a). The points are means from 3 experiments with SE less than

10% of the mean values.

1 min of incubation. A high rate of accumulation of (4)IP was maintained for 20min of incubation and then declined. The changes in the level of (I)IP elicited by carbachol or glutamate were much smaller in magnitude as compared to the formation of (4)IP. The accumulation of IP,, stimulated by glutamate, was maximal after 5 min and then declined. while the stimulation induced by carbachol persisted for up to 15 min. A slight increase in the accumulation of IP, occurred after 5-10 min of stimulation by carbachol. while glutamate was ineffective (data not shown). No changes in the content of IP, could be detected after stimulation with either agonist. Additional experiments with shorter incubation times (20- 180 set) also failed to demonstrate any changes in the accumulation of IP, (data not shown). The role of calcium ions in the formation of inositol phosphates was investigated by stimulating cultured granule cells with the transmitter receptor agonists in the absence of calcium. Figure 5 shows that, at short incubation times (20 set), the absence of calcium significantly decreased the formation of (4)IP, stimulated by both carbachol and glutamate, while no major changes could be seen in the accumu-

i3

mM

CON

CARE

GLU

CON

CARE

GLU

Fig. 5. The effect of Ca’+ ions on the accumulation of [“H](4)IP (A) and [JH]IP, (B) in cultures of cerebellar granule cells, incubated with carbachol (CARB) or glutamate (GLU). The agonist concentrations were 100 FM and the incubation time was 20 sec. Values are means f SE from 4 experiments. CON = Control.

of IP?. The accumulation in the formation of IP, by carbachol was slightly, but not significantly, decreased by the absence of calcium, while glutamate, as mentioned above, failed to cause any accumulation of IP, (data not shown). lation

DlSCL’SSlOFi

In cerebellar granule cells. the hydrolysis of phosphatidylinositol (PI) can be enhanced by several neurotransmitter receptors agonists, including glutamate, carbachol, serotonin, norepinephrine and histamine (Nicoletti et al., 1986b; Xu and Chuang. 1987a. b). The chohnergic receptor, present in the granule cells, is of the muscarinic type, and is blocked by atropine and other M, receptor antagonists (Xu and Chuang, 1987a). Glutamate enhances the hydrolysis or PI in granule cells. acting at two distinct metabolotropic receptors, selectively activated by Nmethyl-p-aspartate (NMDA) and quisqualate, respectively. In contrast to the NMDA and muscarinic receptors, the quisqualate-sensitive receptor is coupled to phosphohpase C, through a pertussis toxinsensitive guanosine triphosphate (GTP) binding protein (Nicoletti, Wroblewski, Fadda and Costa, 1988). Moreover, NMDA-stimulated hydrolysis of PI is strongly inhibited by phencyclidine (Wroblewski, Nicoletti. Fadda and Costa, 1987) and by magnesium ions, and its activity can be detected in granule cells incubated in magnesium-free conditions, due to the presence of endogenous glutamate (Nicoletti, Wroblewski and Costa, 1987). The present study has demonstrated that the agonists of all of the above described receptors, when added to cultures of granule cells in the presence of lithium ions, increased preferentially the accumulation of inositol 4-phosphate. while inositol I-phosphate accumulated in small amounts and was less dependent on the concentration of the agonists. The efficacy of glutamate and carbachol was greater than that of norepinephrine and histamine. The use of magnesium-free conditions in the incubation medium

Profile of agonist-induced PI metabolism made it possible to distinguish between the actions of the two metabolotropic glutamate receptors, and the results indicate that both NMDG and quisqualatesensitive receptors cause a preferential a~umulation of (4)IP. In addition to (4)IP, the stimulation by agonists caused the appearance of two other radioactive peaks co-eluting, respectively, with cIP and (1,4)IP, standards. However, the identity of this latter peak could not be confirmed, due to the lack of appropriate standards of other IP, isomers. The accumulation of (4)IF confirmed earlier sug gestions that agonist-stjmulated phospholipase C prefe~ntially hydrolyzes polyphosp~oinositides, PIP and PIP,, and not phosphatidylinositol (Berridge, 1983; Irvine, Letcher and Dawson, 1984; Nakanishi, Nomura, Kikkawa, Kishimoto and Nishizuka, i985). However, the possiblity that ia oivo, in lithium-treated rats, PI may be the main substrate for phospholipase C in brain has been suggested (Ackermann, Gish, Honchar and Sherman, 1987). In granule cells, stimulated with carbachol or glutamate, the present authors failed to observe a sizeabie ae~umulat~on of IP?, or IF,, as has been reported in slices and homugena~es from brain tissue (Batty, Nahorski and Irvine, 1985; Irvine, Letcher, Heslop and Berridge, 1986). It could be that a very rapid metaboi~sm of inositol phosphates made it impo~ible to detect IPj or IP,, even in the shortest periods of incubation used in this study (20 se& An alternative possibility is that, in the system used, the major substrate for pbospholipase C is PIP and not PIP,, which leads to a direct formation of fl+4)fF,, as has previously been reported for ~bospholipa~ C in human platelets ~Ritt~nhouse* 1983) and in the seminal vesicles of sheep (Wilson, Bross, Hoffman~ and Majerus, 1984). The decreased a~cnmulat~on of (4)IF, in the absence of extra~ellular calcium ions indicates the participation of this ion in the metabolism of inositol phosphates. However, it does not seem to reelect a decrease in the activity of phospholipase C. It has been shown that in the brain of rat, the hydrolysis of polyphosphoinos~tol becomes calcium-sensitive oniy at very small (below 100 nM) concentrations of calcium (Irvine et af., 1984), a condition which can only be achieved by the use of cell-free preparations and CaZf/EGTA buffers. In intact cells, the intracellular concentrations of calcium are sufilcient to maintain the activity of the enzyme and i~cnb~tions without extracellular calcium fail to affect the agoniststimulated hydrolysis of polyphosp~oinos~t~~es and the production of (1 ,4,S)IP3. In contrast, the changes in extracellular calcium may influence the subsequent steps of the metabolism of inositot phosphates; (1,4,.S)IF, can be metaboiized by two Ca2+ -sensitive routes that include the pkosphorylation to (1,3,4,5)IP, by a 3-kinase and the dephosphorylation to (1,4)IF, by a S-phosphomonoesterase (SPME) (Connolly, Bansal, Bross, Irvine and Majerus, 1987). In RINmSF cells, IP, 3-kinase is stimulated by calcium and calmodulin (Biden,

1313

Comte, Cox and Wollheim, 1987). A similar calciumdependent conversion of IFI to IP, has been reported in human leukemic HI&O cells (Lew, Monod, Krause, Waldvo~el, Biden and Schlegel, 1986) and in brain slices of the rat (Baird and Nahorski, 1986). It has been also reported that the activity of SPME in human platelets can be enhanced by the addition of phorbol esters, activators of protein kinase C (Molina y Vedia and Lapetina, 1986). Furthermore, in brain, 5PME was found to be phasphorylated by protein kinase C, with an increase in its activity (Connolly, Lawing and ~aj~r~s, 1986). The present results indicate that in cerebellar granule cells the aEcumul~tion of IPz was more persistent after stimulation by carbachol than by glutamate. Since, in cerebellar granule cells, glutamate has the ability to open channels permeable to calcium ions (Wroblewski, Nicoletti and Costa, 1985) and, in contrast to earbachol, induces the transiocation and activation of protein kinase C {Vaccarino, Guidotti and Costa, 19873, one may expect that the stimulation by glutamate will lead to a more rapid metabolism of inositol phosphates than the stimulation by earbachol. As previously reported, lithium ions amplify the responses of agonists by causing an accumulation of inositol phosphates, due to the blockade of the final step of their hydrolysis to inositoi (Berridge, Downes and Hanley, 1982; Batty and Nahorski, 1985). The preferential accumulation of (4)IF in granule cells after the stimulation by ~euro~ra~smitter receptor agonists was measured in presence of lithium ions, This raises the issue of the ability of lithium ions to inhibit the various phosphomonoesterases (PME) that may participate in the metaboiism of inositol phosphates~ The rote of SPME has been already discussed. The activity of this enzyme which is insensitive to ~nhibit~o~ by lithium (Storey, Shears, Kirk and Michell, 1984; Seyfred, Farrell and Wells, 1984), wilt yield (1,4)IP, or (1,3,4)IP, depending on whether the substrate was (1,4,5)IP, or (1,3,4,5)iF+ These metaboiites may be, in turn, hydrolyzed by specific I-, 3- or 4-PME. It has been reported that in liver, the 1PME that hydrolyzes (1,3,4)IP, to (3,4)IP, is not sensitive to lithium (Shears, Storey, Morris, Cubit& Parry, M&hell and Kirk, 1987), which is in contrast to the 1FME that hydrolyzes (l)IF, to yield inositol ~~ailcher and Sherman, 1980; Storey et al., 1984). Thus, under the conditions of this study, (3,4)IP2 couJd be the metabolite co-eluting with (1,4)IP, in the same ~~romatog~aphi~ peak, It has not been resolved whether a single enzyme is responsible for the hydrolysis of (l,4)IFz and the inositol I- and 4-monophosphates, In the liver (1,4)IPz is hydrolyzed by a 4PME which is inhibited by lithium and by a lithi~m~i~se~sitive 1PME (Storey et al., 1984), which may explain the accumulation of (4)IP. However, if in neuronai cells, ( 1,4)IPz and (I)IP are both hydrolyzed by the same lithium-sensitive lFME, then, the acc~mul~ti~~ of (4)IF would indicate that the

1314

S. HYNIE et al.

metabolism of inositol phosphates after stimulation of a neurotransmitter receptor stimulation proceeds through the (1,4,5)IP,-( I ,3,4,5)IP,-(l,3,4)IP,(3,4)IPZ-(4)IP route. In conclusion, it has been demonstrated that in the cerebellar granule cell, the stimulation by glutamate, carbachol and other neurotransmitter receptor agonists, results in the hydrolysis of polyphosphoinositides which, in the presence of lithium ions, leads to a preferential accumulation of (4)IP. This study indicates that the receptor-mediated metabolism of inositol phosphates in neurons may undergo a complex regulation by multiple calcium- and lithiumdependent enzymatic steps and may reflect the integrative properties of inositol phosphates in intracellular signalhng.

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