ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 297, No. 2, September, pp. 368-376, 1992

Characterization of Polyphosphoinositide-Specific Phospholipase C in Rat Parotid Gland Membranes Yukiharu Hiramatsu, Valerie J. Horn, Bruce J. Baum, and Indu S. Ambudkar’ Clinical Investigations and Patient Care Branch, National Institute of Dental Research, National Building 10, Room 1N-l 13, 9000 Rockville Pike, Bethesda, Maryland 20892

Received February

Institutes

of Health,

12, 1992, and in revised form May 18, 1992

Hydrolysis of exogenously added, [‘Hlinositol-labeled, phosphatidylinositol4,5-bisphosphate (PIP,) by rat parotid membranes was increased, dose-dependently, by the muscarinic cholinergic agonist carbamylcholine (carbachol) in the presence of guanosine 5’-O-thiotriphosphate (GTPyS). The stimulation was inhibited by atropine and guanosine 5’-0-thiodiphosphate (GDPDS). GTPyS alone stimulated PIP2 hydrolysis, with halfmaximal activation at 0.1 PM. This was inhibited by GDP@S but not by atropine. Agonist stimulation of PIP2 hydrolysis was dependent on the presence of lipids (phosphatidylserine:phosphatidylethanolamine:PIP~ = 1:l:l). When PIP2 was added as micelles with detergent (sodium deoxycholate) only, basal hydrolysis was elevated, thus decreasing the relative stimulation by GTPyS and carbachol. The water-soluble hydrolysis products formed under either condition were 1,4,5-inositol trisphosphate, 1,4-inositol bisphosphate, and cyclic inositol trisphosphate. Hydrolysis of exogenous phosphatidylinositol (PI) was also stimulated by carbachol in the presence of GTPyS but the extent of PI hydrolysis was 44-fold lower than PIP2 hydrolysis. When [Ca2+] in the medium was increased from 100 nM to 1 MM, basal hydrolysis of both PI and PIP2 increased (9.3- and 19.2 fold, respectively). However, levels of basal and stimulated PIP2 hydrolysis were higher (37.9- and 29.6-fold, respectively) than those of PI hydrolysis. Antibodies (both polyclonal and monoclonal) raised against phospholipase C (PLC &) from bovine brain did not react with any component in either rat parotid membranes or cytosol, although a reactivity was detected in rat brain membranes. A monoclonal antibody against bovine brain PLC y1 detected a - 150-kDa protein only in the parotid cytosol, while antisera against bovine brain PLC 6i enzyme showed no reactivity with parotid membranes or cytosol. Together, these observations suggest that while there appears to be a protein similar to bovine brain PLC

1 To whom correspondence

should be addressed. Fax: (301) 402-1228.

y1 in parotid gland cytosol, the PLC which mediates PIP2 hydrolysis in rat parotid membranes and can be regulated by the muscarinic receptor via a G-protein is distinct from the well-characterized PLC enzymes yl, &, and j31. 0 1992

Academic

Press,

Inc.

Stimulation of parotid acinar cells by Ca2+ mobilizing agonists leads to intracellular Ca2+ mobilization and fluid secretion. Considerable evidence suggests that this transmembrane signaling is mediated intracellularly by the activation of phosphoinositide-specific phospholipase C (PLC)2 (l-6). This enzyme plays a central role in the process of Ca2+ signaling, not only in parotid acini but also in a variety of other cell types. Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) results in the generation of two critical intracellular messengers: inositol 1,4,5-trisphosphate UP,), which releases Ca2+ from intracellular stores, and diacylglycerol, which, in conjunction with Ca2+, activates protein kinase C. Although there are a large number of studies which demonstrate that agonist stimulation of cells leads to PLC activation and phosphoinositide hydrolysis [for reviews see Refs. (7-g)], the exact mechanisms involved in this signal-transduction process are not yet fully understood. It has been demonstrated that agonist-stimulated PIP2 hydrolysis is affected by guanine nucleotide analogs; stimulation by

’ PIP2, phosphatidylinositol4,5-b&phosphate; PIP, phosphatidylinositol4-monophosphate; PI, phosphatidylinositol; GTP-,S, guanosine 5’0-(3-thiotriphosphate); GDP@, guanosine 5’-O-(2-thiodiphosphate); 1,4,5-IPs, inositol 1,4,5-trisphosphate; 1,4-IP2, inositol 1,4-bisphosphate, 1,4,5-cIPs, cyclic inositol 1,4,5-trisphosphate; IP, inositol phosphate; PLC, phospholipase C; G-protein, guanine nucleotide binding protein; G. stimulatory guanine nucleotide binding protein; Gi, inhibitory guanine nucleotide binding protein; EGTA, ethylenebis (oxyethylenenitrilo) tetraacetic acid; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PE, phosphatidylethanolamine; PS, phosphatidylserine; DOC, sodium deoxycholate.

368 All

0003-9861/92 $5.00 Copyright 0 1992 by Academic Press, Inc. rights of reproduction in any form reserved.

POLYPHOSPHOINOSITIDE-SPECIFIC

GTP$S and inhibition by GTP@S (8). Additionally, AlF;, which can directly activate guanine nucleotide binding proteins (G-proteins), stimulates PLC (8, 10-12). These studies provide convincing evidence for the involvement of G-proteins in PLC activation. Tissue-specific variations in the activity and regulation of PLC can be attributed to differences in either the Gprotein or the PLC enzyme itself; multiple forms of both proteins have been reported. In some cells, PLC activation is pertussis toxin sensitive, indicating the Gi like nature of the involved G-protein, while in others there is no effect of this toxin (13,14). We have reported that in the parotid acinar cell the G-protein which activates PLC belongs to the latter class (15). It has been demonstrated that the pertussis-toxin-sensitive G-protein from liver can activate the bovine brain PLC fll enzyme in a reconstituted system (16). Alternatively, the cytosolic polyphosphoinositide PLC, PLC yl, is stimulated and translocated to the plasma membrane by the activation of tyrosine kinasecoupled receptors without G-protein involvement (17,18). The purification and characterization of phosphoinositide-specific PLC enzymes from numerous tissues (79,17) has led to the identification of four main molecular species: PLC (Y,p, y, and 6, which are discrete gene products (17). Recently, four related PLC enzymes have also been identified, PLC & , yz, &, and a3 (19). The primary sequences of the four main PLC enzyme families have been determined, and although overall homology is low, there are two domains of significant similarity between PLC @, y, and 6, which may be the catalytic domains. However, the sequence of PLC (Y appears to be quite different. It should be noted that these enzymes have similar properties, such as specificity for phosphoinositides, like PI, PIP, and PIP2, and dependence on [Ca2’] for the hydrolysis of these phospholipids. The present studies were conducted to characterize PIP2 hydrolysis in the rat parotid gland plasma membrane. Although exocrine glands, such as the parotid gland, have been widely used to study phosphoinositide hydrolysis and intracellular signaling events (1,8), neither the PLC nor the G-protein regulating its activity in the parotid acinar cell have yet been identified. The data demonstrate the presence of a putative PIP2-specific PLC in the parotid membrane which is stimulated by the muscarinic agonist, carbachol, and GTP$S. This PLC enzyme appears to be immunologically distinct from the wellcharacterized membrane-bound PLC &. MATERIALS

AND

METHODS

Materials. Carbamylcholine chloride (carbachol), ATP, GTPyS, GDPPS, EGTA, and PIP, were purchased from Sigma. PI, phosphatidylethanolamine (PE), and phosphatidylserine (PS) were from Avanti Polar-Lipids, Inc. [3H]PIPz (1.0 Ci/mmol) and [3H]PI (17.4 Ci/mmol) were from New England Nuclear. Monoclonal antibodies against PLC pi, yi, and 6, and polyclonal antisera against PLC pi were gifts from Dr. S. G. Rhee.

PHOSPHOLIPASE

C IN RAT

369

Preparation of plasma membrane and cytosolic fractions. Male Wistar rats, fed ad libitum, were sacrificed by ether anesthesia and subsequent cardiac puncture. Parotid glands from four rats were dissected free of fat, connective tissues, and lymph nodes. All subsequent steps were performed at 4°C. Glands were minced and homogenized for 20 s (twice, 10 s each) in 12 ml of 50 mM Tris/HCl buffer, pH 7.4, with a Polytron PT-10 homogenizer (Brinkmann Instruments) at setting 5. The homogenate was centrifuged for 10 min at 3000g. The supernatant was filtered through four layers of cheesecloth and then recentrifuged for 10 min at 40,OOOg. The resulting supernatant was used for preparing the cytosol (see below). The 40,OOOgpellet was resuspended in 20 ml of Tris/HCl buffer, rehomogenized once for 10 s and recentrifuged as above. The pellet was rehomogenized for 10 s in 10 ml Tris/HCl buffer, aliquoted, frozen in liquid nitrogen, and stored at -70°C until use. Rat brain membranes were prepared using a similar procedure. To obtain the cytosolic fraction, the supernatant from the 40,OOOg spin was centrifuged at 200,OOOg for 1 h. The resulting pellet was discarded and aliquots of the 200,OOOg supernatant were also frozen in liquid nitrogen and stored at -70°C until use. Measurement of PLC activity. PLC activity was measured by monitoring the hydrophilic products (inositol phosphates) from the hydrolysis of exogenously added [3H]inositol-labeled phosphatidylinositols according to the method described by Schwertz and Halverson (20) with minor modifications. Membrane protein (10 rg) was added to 100 ~1 (final volume) of assay medium containing 20 mM Hepes, pH 7.2, 120 mM KCl, 20 mM NaCl, 1 mM MgS04, 0.37 mM or 0.84 mM CaCl,, and 1 mM EGTA (to obtain 0.1 or 1 PM free Car+ as indicated), 10 mM LiCl, 2 mM ATP, 0.8 mM sodium deoxycholate (DOC) and 0.1 mM [sH]PIP2, unless otherwise described. The concentrations of 3H-labeled phospholipids used are indicated for individual experiments. Prior to experiments, required amounts (to give 10X solutions) of PIP?, solubilized in CHCl,/ EtOH (l/l) or PI in CHC13, were added to the respective sH-labeled compound, dried down under nitrogen gas, and resuspended in 8 mM sodium deoxycholate by sonication for 2 min. Where indicated PE and PS were added with PIP,, dried, and sonicated as described above. A bath sonicator, which was kept ice-cold, was used. The desired free [Ca’+] was obtained using Car+-EGTA buffers. Assays were performed at 30°C for 30 min in the absence or presence of various agents, unless otherwise described. When we tested the effect of GDPPS, the membranes were incubated with or without 500 pM GDP@ at 30°C for 10 min and then diluted fivefold in the assay medium. Reactions were terminated by the addition of 0.75 ml CHCl,/MeOH/HCl,,, (100/200/2) for acid extraction and CHCl,/MeOH (l/2) for neutral extraction. Samples were then mixed with 0.25 ml CHC& and 0.25 ml 0.1 N HCl, or 0.25 ml water (in the case of neutral extraction) by vortexing and centrifuged to separate the MeOH/HsO phase from the CHCls phase. Enzyme activity was determined by measuring radioactivity in the aqueous phase using a liquid scintillation counter. All assays were performed in duplicate. Where indicated, the data were statistically analyzed by the paired Students’ t test. Separation of the water-soluble inositol phosphates. HPLC separation of inositol phosphates was performed using a Varian 9010 HPLC and a Flow one/Beta liquid chromatograph equipped with a Whatmann Partisil SAX 10 anion-exchange column. The column was eluted with a linear gradient of water to 1 M (NH,),HPOI, pH 4.0, at a flow rate of 2.0 ml/min. Samples were extracted with ethyl ether (3 X 10 ml) and applied to the column. Retention times for ‘H-labeled inositol phosphate standards were: IP, 11.3 min; IP2, 17.0 min; cyclic IPs, 22.0 min; 1,3,4IPB, 23.0 min; 1,4,5-IP,, 24.0 min. Gel electrophoresis and immunoblotting. SDS-PAGE was performed according to Laemmli (21) using 7.5% acrylamide separation gels containing 0.1% SDS. Stacking gels contained 3.0% acrylamide. Electrophoresis was performed overnight at 4 V/cm in electrophoresis buffer containing 25 mM Tris/HCl, pH 8.3, 200 mM glycine, 0.1% SDS. After electrophoresis, the proteins in the gel were transferred onto nitrocellulose sheets by the method of Towbin et al. (22) using the following buffer: 25 mM Tris/HCl, pH 8.3, 200 mM glycine, 20% methanol. The

HIRAMATSU

20

10 Time

30

(min)

FIG. 1. Hydrolysis of exogenously added [3H]PIP2 in rat parotid membranes. Rat parotid gland membranes (5 Fg) were added to a reaction mixture containing 30 pM [3H]PIP, as micelles (as described under Materials and Methods), 120 mM KCl, 20 mM NaCl, 1 mM MgSO,, 1 mM EGTA, 2 mM ATP, 10 mM LiCl, 20 mM Hepes (PH. 7.2), 0.1 pM free Ca*+, and 0.8 mM DOC. The following experimental conditions were tested: no additions (open circles), 1 mM carbachol (closed circles), 10 pM GTPyS (open triangles), and 1 mM carbachol + 10 pM GTPyS (closed triangles). After incubation at 37’C for the indicated times, the reaction was terminated by the addition of chloroform and MeOH mixture, the inositol phosphates were extracted, and the radioactivity in the aqueous phase was measured. The data were obtained from three experiments; each assay was performed in duplicate. nitrocellulose sheets were blocked for 30 min at room temperature with blocking buffer, 3% BSA in 50 mM Tris/HCl, pH 7.4, and 150 mM NaCl with 0.3% NP40 detergent. Primary antibody (either monoclonal or polyclonal) in blocking buffer was added and left overnight at 4°C. The primary antibody was then removed and the blot was washed five times with blocking buffer. A secondary anti-IgG antibody coupled to horseradish peroxidase in blocking buffer was then added and left for 2 h at room temperature. 3,3’-Diaminobenzidine tetrahydrochloride dihydrate was used as the peroxidase substrate to visualize the immunoreactive proteins.

RESULTS AND DISCUSSION Several experimental approaches have been used for measuring PIPz-specific PLC activity in membrane preparations (7-9). In general, these involve preparing micelles of PIP, with a detergent, like cholate or deoxycholate, either with (23,24) or without (20,25) lipids, e.g., PS and PE. In some studies the detergent has been excluded (26, 27). In this study we have assessed the effects of detergents and lipids on PIP2 hydrolysis and its activation by GTPyS and the muscarinic cholinergic agonist, carbachol, in a rat parotid gland membrane preparation. As we have previously shown, this membrane preparation is enriched in numerous receptors (e.g., muscarinic cholinergic, p-adrenergic), G-proteins (Gi and G,), and exhibits receptorregulated adenylyl cyclase activity (28-30). Coupling of PIP, Hydrolysis to G-Protein Activation and Muscarinic Cholinergic Receptor Activation in Rat Parotid Membranes Figure 1 shows the time course of PIP2 hydrolysis in rat parotid gland membranes incubated at 37°C with

ET AL.

mixed micelles of 30 PM PIP2 and 0.8 mM DOC, in the absence of phospholipids. There is a high level of basal PIP2 hydrolysis in control membranes (no additions), which increases almost linearly with time. This hydrolysis is not significantly altered when carbachol (1 mM) alone is added. Addition of GTP$S (10 PM) stimulates PIP2 hydrolysis significantly (P < 0.05), albeit by a small extent (-20% over basal). With the inclusion of carbachol with GTP$S, PIP2 hydrolysis is further increased up to -33% over basal. The rates of PIP2 hydrolysis calculated from these experiments are shown in Table I. When the membranes are incubated with the muscarinic receptor antagonist atropine (0.1 mM), carbachol stimulation of PIP2 hydrolysis is inhibited. The G-protein inhibitor, GDPPS, inhibits both carbachol and GTPyS stimulation of PIP2 hydrolysis, but not basal PIPz hydrolysis (data are not shown; see Figs. 6 and 7). To further characterize the PIP2 hydrolytic activity, the aqueous products were analyzed by HPLC. The main products detected in acid-extracted samples are 1,4,5-IP3 (-84%) and 1,4-IPZ (-12%), and in neutral extracts cIP3 (-3%) is also detected. While the relative amounts of these products are similar in basal and stimulated samples, the absolute amount of each is higher in the latter. The composition of the hydrolysis products indicates that the basal level of PIP2 hydrolysis, as well as the small but significant increase in PIP2 hydrolysis seen in the presence of carbachol and GTP-yS, results from the action of PLC. The high level of basal activity is most likely due to PLC which is “uncoupled” from the G-protein. However, it is unclear what factors are involved in coupling the PLC, G-protein, and the receptor. Importantly, the data dem-

TABLE Hydrolysis

Additions None (control) 1 mM Carbachol 10 PM GTP-yS 1 mM Carbachol + 10 ELMGTPyS

I

of Exogenously Added in Rat Parotid Membranes

[3H]PIPs

PIP? hydrolysis (nmol/mg protein/mix-i)

% Change

11.38 zk 0.66 11.22 + 0.68 13.46 + 0.76*

100 99 2 1.7 119 f 1.7

15.02 f O.sl*,t

133 + 2.9

Note. These data were obtained from 26 experiments performed with eight different membrane preparations. The assay conditions were similar to those shown in Fig. 1 and PIP2 hydrolysis after a lo-min incubation was used to calculate the rate of hydrolysis. PIPz-hydrolysis with carbachol is not significantly different from basal level (no additions). Values of PIP* hydrolysis, are given as means * SEM. Percentage change, given as means ? SEM, represents the activity compared to the control in each experiment. * PIPS-hydrolysis values significantly higher than basal values (P < 0.001). t PIP,-hydrolysis statistically greater than that obtained with GTP+ alone (P i 0.001).

POLYPHOSPHOINOSITIDE-SPECIFIC

PHOSPHOLIPASE

onstrate that there is a PIP,-PLC activity in rat parotid membranes, which is generally similar to other reported PIPs-specific PLC enzymes in that both 1,4,5-IPs and 1,4,5-cIP, are formed as products. We tried two experimental approaches to decrease the basal “uncoupled” PLC activity and more clearly resolve the stimulation by GTP-yS and carbachol. When the incubation temperature was decreased from 37 to 30°C (Table II) there is a drastic decrease in the level of basal PIP2 hydrolysis. However, its “coupling” to receptor and G-protein is not greatly enhanced; stimulation of PIP2 hydrolysis by GTPrS and carbachol is 75% over basal at 30°C as compared to 31% at 37°C. We also tried a different detergent, octylglucoside, but did not detect any enhancement of agonist stimulation of PIP:, hydrolysis (data not shown). It should be noted that in the absence of detergent basal hydrolysis was very low and we could not detect any stimulation by agonist or GTP$S.

1

400 -

300 200 -

100

None (control) Carbachol, 1 mM GTPrS 1 PM lo /.iM

Carbachol, 1 PM lo PM

PIP2 hydrolysis (nmol/mg protein/min)

45

60

(min)

FIG. 2. Effects of lipids on agonist stimulation of PIP2 hydrolysis. Membranes (10 pg) were incubated with mixed micelles of 100 yM [3H]PIP,, with PE and PS (PIP,:PE:PS = l:l:l), and DOC at 30°C. All the other assay conditions were the same as those described for Fig. 1. The following experimental conditions were tested: no additions (open circles), 1 mM carbachol (closed circles), 1 pM GTPyS (open triangles), and 1 mM carbachol + 1 pM GTPyS (closed triangles). The data were obtained from three experiments; each assay was performed in duplicate. PIPz-hydrolysis values obtained with GTPyS alone were significantly higher than those either with carbachol alone or without any addition. *PIP,-hydrolysis values obtained with carbachol + GTP-&S that are statistically greater than those obtained with GTPyS alone (P < 0.05).

The water-soluble PIP, hydrolysis products from these assay samples were analyzed by HPLC and the data are given in Fig. 3 and Table III. The composition of the inositol phosphates released during [H3]PIP2 hydrolysis under basal and stimulated conditions are similar (Table III) and compare well with that released in the absence of lipids. However, consistent with the observed stimu-

II

[3H]PIPz

in Rat Parotid

-Phospholipids

Additions

30

15

Time

TABLE Added

.

00

We examined the effect of exogenous lipids, PE and PS, in the micellar preparation (PS:PE:PIP2 = 1:l:l) and the data are given in Fig. 2 and Tables II and III. In the presence of phospholipids the basal level of PIP2 hydrolysis is further decreased, compared to hydrolysis at 30°C in the absence of exogenous lipids. However, under this condition the stimulation of PIP2 hydrolysis, either by GTPyS (1 PM) alone or GTP$S in the presence of 1 mM carbachol, is markedly amplified (-3.6- and -6.2-fold over basal, respectively; see Table II) and a more or less linear increase in hydrolysis is seen with time (up to 60 min, shown in Fig. 2).

of Exogenously

I /.

Effect of Lipids on Agonist Stimulation of PIP, Hydrolysis

Hydrolysis

371

C IN RAT

Membranes +Phospholipids

% Change

2.01 f 0.69 Not done

100

3.28 + 0.94* Not done 3.51 f 1.21* Not done

PIP? hydrolysis (nmol/mg protein/min)

% Change

0.95 f 0.29 0.98 f 0.31

100 102 f

167 f 11

3.06 5~0.65* 3.71 + 0.68*

363 t 74 455 * 113

175+

4.98 f 0.81* 5.62 f 0.73*~7

621 k 166 726 1?I229

2

1 mM + GTPyS 1

Note. These data were calculated from two to three experiments with individual preparations. The assay conditions were generally similar to those shown in Fig. 1, except that the temperature was maintained at 30°C during the assay and incubations were for 30 min. PE and PS were added (PIP,:PS:PE = 1:l:l) where indicated (+ phospholipids). PIP*-hydrolysis with carbachol is not significantly different from basal level (no additions). Values of PIP* hydrolysis are given as mean f SEM. Percentage change, given as mean f SEM, is the activity compared to control in each experiment. * PIP,-hydrolysis with GTPyS alone or carbachol + GTPyS is significantly different from basal (P < 0.001). t PIP,-hydrolysis with carbachol + GTPyS is statistically greater than that obtained with GTPyS alone (P < 0.001).

372

HIRAMATSU TABLE HPLC

III

Analysis of Products of PIP, following Acidic Extraction

ET AL.

5000

n

Hydrolysis

1,4-IP2

4000 3000 -

1,4,5-IP3

Addition

CPM

% Total

CPM

% Total

None 1 mM carbachol 1 /iM GTPyS 1 mM carbachol + 1 /.tM GTPyS

841 843 1685

63.9 67.7 59.7

473 401 1133

36.1 32.3 40.3

4.

/

0

2000 -

1000

-

0

is!/.1.

8’

0

20

30

0

2335

58.0

1685

42.0

Note. Rat parotid gland membranes (10 pg) were added to a reaction mixture similar to that described for Fig. 2. At the end of incubation at 30°C for 30 min, the reaction was terminated by the addition of chloroform-methanol mixture with HCl (as described under Materials and Methods) and the aqueous fraction was extracted and analyzed by HPLC. Two peaks corresponding to 1,4-IPz and 1,4,5-IP, were detected and the radioactivity determined (mean) from duplicate experiments is given.

latory effect of lipids on the hydrolysis of PIP2, the amount of products generated by stimulation with GTPyS and carbachol is greater than that in the absence of lipids. 1,4,5-IPs, 1,4-IPs, and cIP3 all increase with time in stimulated membranes (Fig. 3). Under our experimental conditions, while the inclusion of lipids alone with PIP2 induces agonist stimulation of hydrolysis, the extent of hydrolysis is much greater when DOC (0.8 mM) is also included in the micellar preparation. It should be noted that at lower [DOC] (co.8 mM) both basal hydrolysis and agonist stimulation are greatly reduced while at higher [DOC] (>l mM), basal activity is greatly increased and agonist stimulation is relatively decreased (these data are not presented here). Thus, it appears that the inclusion of exogenous phospholipids increases the coupling of the putative PIP,-PLC activity in rat parotid membranes to receptor activation. Conversely, it has been demonstrated in plasma membranes from rat brain that carbachol + GTPyS stimulation of PIP2 hydrolysis can be clearly detected in the presence of DOC and PIP2 alone without the addition of any other phospholipid (25). Unless otherwise specified, all further experiments were carried out at 30°C in the presence of lipids. Characteristics of Stimulation of PIP, Hydrolysis GTPyS and Carbachol in Parotid Membranes

by

In the absence of GTP$S, carbachol does not stimulate PIP2 hydrolysis, even at concentrations as high as 1 mM (Fig. 4). In the presence of GTP$S, the rate of PIP2 hydrolysis is increased with increasing concentrations of carbachol and above 10 PM carbachol there is a significant increase in the rate compared to that obtained with GTPyS alone.

4 0

10 Time

(min)

FIG. 3. Time course of generation of water soluble PIP* hydrolysis products following carbachol + GTP-yS stimulation. Rat parotid gland membranes (10 pg) were added to a reaction mixture similar to that described for Fig. 2. After incubation at 30°C for the indicated period, the reaction was terminated by the addition of chloroform-methanol mixture without HCl (as described under Materials and Methods). The aqueous fraction of each sample was analyzed by HPLC. The hydrolysis products of exogenously added PIP, by rat parotid membranes, in the presence (closed symbols) or absence (open symbols) of 1 mM carbachol + 1 PM GTP-yS, were separated into two major peaks corresponding to 1,4-IPz (circles), and 1,4,5-IP3 (squares) and one minor peak, 1,4,5-cIP, (triangles). All products were increased by the stimulation of the memin the presence of 1 mM carbachol. The data branes with 1 pM GTP+ represent average values from two experiments.

Figure 5 shows the effect of increasing concentrations of GTP$S on PIP2 hydrolysis. The rate of PIP* hydrolysis is significantly increased with increasing concentrations of GTPrS, up to about 10 /IM, and at higher concentra-

‘OL

I

-0-o-o

o--fro-o 0

7

6 -Log[CCh]

5

4

3

(M)

FIG. 4. Effect of carbachol on hydrolysis of exogenously added [3H]PIP2 in rat parotid membranes. Membranes were incubated at 30°C in a medium similar to that described for Fig. 2 and the indicated concentrations of carbachol either with (closed circles) or without (open circles) 1 pM GTP$S. After 30 min, the reaction was terminated as described in the legend to Fig. 1. The data shown (mean + SEM) were obtained from three experiments. *PIP*-hydrolysis values that are statistically higher than those obtained in the presence of GTPyS alone (P < 0.05).

POLYPHOSPHOINOSITIDE-SPECIFIC

0

a

7 -Log[GTPTS]

5

6

4

(U)

FIG. 5. Effect of GTPyS on hydrolysis of exogenously added [sH]PIPz in rat parotid membranes. Membranes were incubated at 30°C in a medium similar to that described for Fig. 2 and the indicated concentrations of GTPyS either with (closed circles) or without (open circles) 1 mM carbachol. After 30 min, the reaction was terminated as described in the legend to Fig. 1. The data shown (mean + SEM) were obtained from three experiments. *PIPz-hydrolysis values that are statistically higher than those obtained in the presence of carbachol alone (P < 0.05).

there is no further increase. In the absence of carbachol, half-maximal stimulation is seen at 0.1 @M GTP$S. In the presence of 1 mM carbachol there is further hydrolysis of PIP2 and this effect is more pronounced at GTPyS > 0.01 PM. However, between 0.1 and 100 PM GTP+, a similar stimulation of PIP2 hydrolysis by carbachol is observed (-2.1 nmol/mg protein/min, 2.2-fold of basal, P < 0.001). The data in Fig. 6 show that the muscarinic antagonist atropine blocks the carbachol-stimulated increase in PIP2 hydrolysis, but not that stimulated by GTP$S alone. Thus, the stimulatory effect of carbachol is mediated via the muscarinic receptor, while that of GTPyS is likely via a direct effect on the G-protein coupled to the PLC. This is more directly demonstrated by the observation that GDP@ can block the GTP$S as well as the GTPyS + carbachol stimulation of PIP2 hydrolysis (Fig. 7). These stimulatory effects of GTPyS and muscarinic agonist are consistent with an earlier report by Taylor et al. (31) showing that in electrically permeabilized parotid acini nonhydrolyzable GTP analogs and AlF; can stimulate the hydrolysis of endogenously labeled [3H]inositol lipids. We have also shown earlier that AlF; can stimulate phosphoinositide hydrolysis in intact parotid acini (11). Together, these data are consistent with the involvement of a G-protein in the regulation of PIP2 hydrolysis following the stimulation of the muscarinic cholinergic receptor.

tions

Substrate Specificity

of Parotid Membrane PLC

The results in Fig. 8 demonstrate the substrate specificity of the PLC activity in rat parotid membranes. The

PHOSPHOLIPASE

AtFGiine GTPrS

373

C IN RAT

- -+-+ - -

-

-

-I- +

++++ -+-+ ----I-+

FIG. 6. Inhibition of PIP* hydrolysis by atropine in rat parotid membranes. All experimental conditions were similar to those described for Fig. 2. Atropine (100 pM) was included in the assay mixture where indicated. The values (mean + SEM) were obtained from three experiments for each condition (with each assay performed in duplicate). *Significantly different (P < 0.05) from unmarked values and values marked ** but not significantly different from other values marked with *.

basal rate of PIP2 hydrolysis (Fig. 8A) does not increase significantly with increase in substrate concentration. However, carbachol- and GTPyS-stimulated PLC activity increases in a dose-dependent manner with increasing concentration of PIP2, with maximum stimulatory effect at 100 /*M PIP2 (6.7 t 0.3 nmol/mg protein/min, 4.9-fold over basal, P < 0.001). At higher concentrations there is a decrease in G-protein-mediated stimulation of hydrolysis. The reason for this decrease is not clear. However,

G%P ----+-+ GTPrS

--++

++++

-+-+ --++

FIG. 7. Inhibition of PIPp hydrolysis by GDP&S in rat parotid membranes. All experimental conditions were similar to those described for Fig. 2. Membranes were preincubated with 500 yM GDP@ at 30°C for 10 min and then added to the reaction mixture (final concentration of GDP@ was 100 PM). The values (mean + SEM) were obtained from three experiments for each condition (with each assay performed in duplicate). *Significantly different (P < 0.05) from values marked ** and from unmarked values. Unmarked values are not significantly different from each other.

374

HIRAMATSU 30 1

A

4.5

4.0

3.5

-LKlIPIP21 (M)

yg- 0.6

E Ii> o *jj 0.6 !: 2 -2 P 8 I”\

ET AL.

beled rat parotid acinar cells is not affected by [Ca2+]i (32). Figure 8 also shows that at the higher [Ca2+], basal hydrolysis rates of both PI and PIP2 are increased. However, while agonist stimulation of PI hydrolysis is observed at either [Ca2+], agonist stimulation of PIP2 hydrolysis is not detected at 1 pM Ca2+. Additionally, even at 1 PM Ca2+, basal and stimulated PIP2 hydrolysis (with 100 PM PIP2) are 37.9- and 29.6-fold higher, respectively, than that of 100 pM PI. Thus, the putative PLC in rat parotid membrane appears to have a high selectivity for PIP2, which is maintained even in the presence of elevated [Ca2+]. It has been reported that the hydrolysis rates of PIP2 are higher than PI in membranes isolated from rat heart ventricles, platelets, rat liver, and turkey erythrocytes (20, 23, 24, 33). In addition, a soluble PLC in human epidermal cells has also been reported to be more selective for PIP2 compared to PI (27). However, soluble forms of PLC in platelets and rat ventricles exhibit similar rates of PIP2 and PI hydrolysis, and the purified PLC fil from bovine brain membrane has also been shown to hydrolyze PI and PIP2 with the same efficiency (34).

E” 0.4

Immunological Characteristics Membrane PLC

E 5 0.2

0.0 5.0

4.5

4.0

3.5

FIG. 8.

Effect of [Ca’+] on the hydrolysis of exogenously added [3H]PIP, or [3H]PI by isolated rat parotid membranes. Membranes were incubated at 30°C for 30 min in a reaction mixture similar to that described for Fig. 2, with the indicated concentrations of [3H]PIPz (A) or [3H]PI (B), 0.1 pM (circles) or 1 FM [Ca”‘] (triangles), either with no addition (open symbols) or with 1 mM carbachol + 1 pM GTP-yS (closed symbols). The data shown (mean f SEM) were obtained from two to three experiments.

it should be noted that while the ratio of PE, PS, and PIP2 was kept constant, the concentration of DOC was not increased. It is possible that this could result in variations in the nature of the micelles formed with higher amounts of lipid. [3H]PI is also hydrolyzed by the parotid membrane preparations (Fig. 8B) and under similar experimental conditions (100 nM Ca2+, 1 PM GTPyS, and 1 mM carbachol) the hydrolysis is stimulated by carbachol and GTP+. However, [3H]PI is hydrolyzed very poorly (0.15 ? 0.03 nmol/mg protein/min, 45-fold less than PIP2 hydrolysis). Similar results were obtained in the absence of added lipids (data not shown). In a number of studies it has been shown that the efficiency of hydrolysis of various exogenously added phosphoinositide substrates by PLC is dependent on [ Ca2+], although an earlier report suggests that inositol trisphosphate generation in [3H]inositol-la-

of Parotid

We have used monoclonal antibodies against the bovine brain PLCs &, yl, and 6i (Fig. 9A) and a polyclonal antibody against bovine brain PLC & (Fig. 9B) to examine the immunological reactivity of the rat parotid plasma membrane PLC component. The monoclonal antibody against PLC y1 reacts with a -150-kDa protein in the parotid cytosol. This is similar to the reported molecular weight for PLC yi. No reactivity is observed in parotid membranes with this antibody. The monoclonal antibody against PLC 6i does not react with either the membrane or the cytosolic fractions. Both monoclonal and polyclonal antibodies against PLC & were tested and despite high amounts of protein on the gel (100 pg) there is no reactivity of either antibody with the parotid plasma membrane fraction. As a positive control 100 pg of rat brain membrane was loaded onto the same gel and a 150-kDa protein was clearly detected (Fig. 9B). Also, it should be noted that under our experimental conditions, parotid gland membranes exhibited PIP,-hydrolytic activity twofold higher than that of brain membranes (data not shown). Differences in the nature of PLC enzymes in various types of cells have been described earlier. For example, the PLC enzyme in platelets appears to be immunologically different from those in brain (34). In addition, a PIP,-PLC isolated from turkey erythrocytes also shows no reactivity with antisera against the brain enzymes (23). Our data show that there are at least two forms of PLC in the rat parotid acinar cell, which are distinctly localized in the cytosol and membrane. Consistent with our data,

POLYPHOSPHOINOSITIDE-SPECIFIC

PHOSPHOLIPASE

375

C IN RAT

B 200 KDa A

97 KDa 97 KDa --+ 69 KDa -

67 KDa -

46 KDa --w

45 KDa MC

MC

B

MC

P

FIG. 9. Immunoblotting of PLC by several antibodies. In A membrane protein, M (3000-40,00Ogpellet, 25 pg), and cytosolic protein, C (200,OOOg supernatant, 25 pg), were treated with sample buffer and loaded onto gels as indicated. In B, lane B contained 100 ng of brain membranes and lane P contained 100 ng of parotid membranes. SDS-PAGE was performed as described under Materials and Methods. After electrophoresis the protein was transferred to nitrocellulose and blotted with (A) monoclonal antibody against either PLC /3, , yi, or 6i or (B) polyclonal antibody against PLC pi. We also used an enhanced chemiluminescence system to detect the immunoblot and had similar observations (data not shown).

it has been recently reported that a PLC yi like enzyme from the parotid cytosol migrates to the plasma membrane when the cells are treated with epidermal growth factor (35). The PLC activity in rat parotid gland plasma membranes, which we have described here, appears to be PIP2specific and regulated by the muscarinic-cholinergic receptor, via a G-protein-dependent mechanism. We have shown that PIP2 hydrolysis is stimulated, dose-dependently, by the muscarinic-cholinergic receptor agonist, carbachol, in the presence of GTPyS, and also directly by the activation of G-proteins by GTP+yS. In general the characteristics of hydrolysis of exogenously added PIP2 by rat parotid gland membranes are similar to those observed when endogenously labeled lipids are hydrolyzed; e.g. in both cases agonists and G-proteins stimulate hydrolysis and yield the same products, 1,4,5-IP3, 1,4-IP2, and cIP, (31, 36). Based on these data, we suggest that this putative membrane-bound PLC enzyme is most likely involved in initiating the Ca2+ mobilization response of rat parotid acini when the muscarinic-cholinergic receptor is stimulated. In order to establish conclusively whether or not this enzyme is related to the well-characterized PLC enzyme families it will be essential to purify this enzyme and obtain the sequence of gene encoding the protein. ACKNOWLEDGMENTS We thank Dr. Sue Goo Rhee for the gift of the phospholipase C antibodies used in these studies and for his helpful comments and suggestions during the course of this work. We are also grateful to Dr. M. Humphreys-Beher for providing us with a copy of Ref. (35).

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