Modulation of dihydropyridine-sensitive gastric mucosal calcium channels by GM1-ganglioside

Im. .I. B&hem. Vol. 24, No. 8, pp. 12891294, 1992 Printed in Great Britain. All rights reserved

0020-711X/92 $5.00 + 0.00 Copyright 0 1992 Pergamon Press Ltd

MODULATION OF DIHYDROPYRIDINE-SENSITIVE GASTRIC MUCOSAL CALCIUM CHANNELS BY GM,-GANGLIOSIDE B. L. SLOMIANY, J. LIU, Z.

FEKETE, P. YAO

and A. SLOMIANY

Research Center, New Jersey Dental School, University of Medicine and Dentistry of New Jersey, University Heights, 110 Bergen Street, Newark, NJ 07103-2400, U.S.A. [Tel. (201) 456-70521 (Received 3 December

1991)

Abstract-l. A dihydropyridine-sensitive calcium channel complex was solubilized from gastric mucosal cell membranes and purified by affinity chromatography on wheat germ agglutinin. 2. The calcium channel complex labeled with [‘HIPNZOO-110,when reconstituted into phosphatidylcholine vesicles, exhibited active “‘Ca2+ uptake into intravesicular space as evidenced by La’+ displacement and osmolarity studies. The channel complex responded in a dose-dependent manner to dihydropyridine calcium antagonist, PN200-110, which at 0.5 pM exerted maximal inhibitory effect of 66% in 45Ca2+ uptake. 3. The uptake of ?a *+ into vesicle-reconstituted gastric mucosal calcium channel complex was inhibited by GM,-ganglioside. Maximum inhibitory effect was achieved at l&l5 nM GM,, at which point a 74% decrease in 45Ca2+uptake occurred. Furthermore, GM, also inhibited dihydropyridine binding to gastric mucosal membranes, indicating the extracellular orientation of calcium channel domains for GM,. 4. The ability of GM, to modulate the intracellular calcium levels may be an important feature in gastric mucosal protection by this ganglioside.

INTRODUCTION

MATERIALS AND METHODS

The maintenance of gastric mucosal defense under adverse environment of luminal contents, which pose continuous threat to the integrity of its defense elements, is complex in nature and appears to be muiticomponential. Primary among the components of these systems are the layer of mucus and the cell membranes of gastric epithelium (Silen, 1987; Slomiany ef al., 1985, 1989). While the role of mucus gel glycoproteins and lipids in the preservation of mucosal defense perimeter is well documented (Slomiany et al., 1989; Slomiany and Slomiany, 1991; Lichtenberger et al., 1983), less is known about the way by which the constituents of gastric epithelium participate in this phenomenon. The available data, however, point towards the role of epithelial cell membrane sialoglycolipids in the maintenance of mucosal integrity (Slomiany and Slomiany, 1980; Slomiany et al., 1984, 1991; Szabo et al., 1989). As cell membrane constituents, the sialoglycosphingolipids are capable of affecting ion transport, membrane fluidity modulation, and appear to play a major role in altering the activities of various membrane associated enzymes and receptors (Yu et al., 1986; Hanai et al., 1988; Spiegel, 1989). In this report, we describe the reconstitution of the dihydropyridinesensitive gastric mucosal cell membrane calcium channels into phospholipid vesicles and show the effect of GM,-ganglioside on the dihydropyridinesensitive vesicular calcium uptake.

Materials

Male Sprague-Dawley rats weighing 18&200 g were obtained from Taconic Farms Inc., Germantown, N.Y., [45Ca2+]CaCl, and ( + )-[methyl-3H]PN200-l 10 from New England Nuclear, Boston, Mass. Bay K8644 and egg-yolk phosphatidylcholine were supplied by Sigma. PN200-110 was generously given by Dr Houlihan, Sandoz Research Institute, E. Hanover, N.J. Wheat germ agglutinin Sepharose was obtained from Pharmacia, Piscataway, N.J., Chelex 100 (S&l00 mesh) from Bio-Rad, Rockville Centre, N.Y., GM,-ganglioside from Angio Medical Corp., N.Y. and BCA protein assay kit from Pierce, Rockford, Ill. Gastric membrane preparation The dissected stomachs were opened along the greater curvature, rinsed with ice-cold saline in 0.05 M phosphate buffer pH 7.2, and the mucosal cells were collected by scraping the mucosa with a blunt spatula on an ice-cold glass plate (Slomlany et al., 1990).Scrapings were placed in ice-cold buffer (2.5 mM Tris-HCl, pH 7.0, 250 mM sucrose, 2.5 mM EDTA, 1 mM phenyl methyl sulfonyl fluoride (PMSF), 100 TW/ml aprotinin and 1 pg/ml leupeptin, and homogenized for 1 min in a polytron tissuemizer. The homogenates were centrifuged at 400 g for 15 min at 4°C and NaCl/MgSO, was added to the supematants to form final concentrations of 0.1 mM and 0.2 mM, respectively. After centrifugation of the supematants at 40,OOOgfor 1 hr at 4”C, the pellets were resuspended in 0.1 M sodium phosphate buffer (PH 7.2) and aliquots of such prepared gastric membrane samples were stored at -70°C until use. Protein concentration of the resuspended pellets were estimated using the BCA protein assay kit.

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Solubilization and purification of dihydropyridine -sensitive calcium channels

The gastric membrane preparations were centrifuged for 20 min at 10,OOOgand 4°C. and the pellet solubilized using a buffer containing 50mM Tris-HCl, pH 7.4, 0.5 M NaCI, 20 mM 3-[(3-cholamidopropyl)dimethylammonio]-lpropanesulfonate (CHAPS), 10% glycerol, 100 TIU/ml aprotinin, 1pg/ml leupeptin and I mM PMSF. After 1 hr at 4°C the mixture was centrifuged at 105,000g for 60 min and the resulting supernatant collected, 1 nM of [rH]PN200-110 was added and incubated at 4°C for 30 min. The preparation was then applied to a column of Sepharose-bound wheat germ agglutinin. The column was washed with 3 ml of a buffer consisting of 50mM Tris-HCl, pH 7.4, containing 20 mM NaF and 0.5 M NaCl, and the channel protein was eluted with buffer containing 300 mM N-acetylglucosamine (Jay et al., 1991). 1,4-Dihydropyridine

binding

All binding studies were performed in 50 mM Tris-HCl, 0.1 mM PMSF, pH 7.4 containing 0.5% bovine serum albumin (BSA). Gastric membrane preparations containing 50-5OOpg protein were divided into two groups, one was treated with 10 nM [‘H]PN200-110 and the other with IOnM [“HIPNZOO-110 and 1 PM cold PN200-110. The mixtures were incubated at room temperature for 1 hr, and the reaction was stopped by addition of cold JOmM Tris-HCI pH 7.0 containing 0.5% BSA and centrifuged at 10,000 rpm for IOmin. The pellet was washed twice more in Tris-HCI buffer, centrifuged, dissolved in 0.1 M NaCl 0.05 M phosphate buffer, pH 7.4, and the proteinbound ['HIPNZOO-1 10 measured by scintillation spectrometry. The effect of GM,-ganglioside on the binding of PN200-110 was assessed following preincubation of membrane preparation with GM, (O-50 pg) at room temperature for 30 min. Liposomal reconstitution of calcium channel

Liposomes were prepared by the method of Mimms et al. (1981). Channel protein samples, solubilized with a buffer containing 20 mM CHAPS, 5 mM CaCI,, 50 mM Tris-HCI, pH 7.4, 10% glycerol and 5 PM Bay K8644 were mixed with 1 ml of octylglucoside containing I% egg yolk phosphatylcholine. Detergent was removed by dialysis against buffered saline for 24 hr at 4”C, thus yielding liposomes. For further purification, a suspension of liposomes was made with 45% sucrose, overlaid with 2ml of 30% sucrose and I ml of 10% sucrose and then centrifuged at 4°C for 18 hr at 45,OOOg in a Beckman SW50 rotor. The purified liposomes containing [‘HIPNZOO-110 labeled channel protein were recovered as a white band at the top of 10% sucrose layer. Measurement of 4sCa2+ uptake into vesicles

For the measurements of ?a2+ uptake into protein-free vesicles (control) and vesicles containing the reconstituted calcium channels, external divalent cations were removed by application of 200 ~1 of vesicle suspension to a 3 ml Sephadex G-50 column equilibrated with a 0.34 M sucrose, 10 mM 3-(N-morpholino)-propanesulfonic acid (MOP?+ tetramethylammonium, pH 7.0, 5 PM BAY K8644 (Curtis and Catteral, 1986). Uptake into vesicles (100~1) in sucrose medium was initiated by addition of 50~1 of sucrose medium containing 0. I mM CaCI, plus 2 @i of ‘sCa2+. The mixture was incubated at 37°C for various periods of

time up to 1 hr, and the reaction was terminated with 150 mM MgCI, in 10 mM HEPESTris buffer, pH 7.4. The effect of calcium channel receptor antagonist, dihydropyridine PN200-110, on the 45Ca2+ uptake was measured following vesicles preincubation at room temperature for 20 min with PN200-110 (&I PM). The samples were then applied to a Chelex 100 column (0.5 x 5 cm) equilibrated with sucrose medium containing 1mg/ml BSA. Vesicles were eluted from the column with 1 ml of sucrose-BSA medium, and the specific 4sCa2+ uptake was determined by substracting the background uptake into the protein-free vesicles. Specificity of calcium uptake

The specificity of calcium uptake into liposome-reconstituted channel was ascertained in the experiments with changing medium osmolarity and displacement with lanthanum. For the evaluation of the effect of media osmolarity on calcium uptake, 100 ~1 aliquots of vesicles in 0.34 M sucrose, containing reconstituted calcium channels were first incubated for 20min at room temperature with increasing concentrations (O-200 mM) of mannitol. Calcium uptake was then initiated by addition of 0.1 mM CaCl, and 2 pCi 45Ca2+.The reaction was terminated after 20 min by addition of 10mM HEPES-Tris buffer, pH 7.4, containing 15OmM MgCl,. The samples were applied to a Chelex-100 column, eluted with 0.34 M sucrose-10 mM MOPS, pH 7.0, containing I mg/ml of BSA, and the 4sCa2+ counted. The effect of La’+ on calcium displacement was evaluated using aliquots (100 ~1) of vesicles preloaded for 20min at room temperature with 0.1 mM CaCl, and 2yCi “‘Ca*+. The vesicles were then diluted into incubation media containing different concentrations (O-l mM) of lanthanum oxide, incubated at room temperature for 5 min, and loaded onto Chelex-100 column. Vesicles, eluted from the column with sucrose-BSA medium, were then subjected to 4sCa2+ measurement. Effect of GM,-ganglioside on 45Ca2+ uptake into vesicles

The effect of GM,-ganglioside on the 45Ca2+uptake into protein-free vesicles and vesicles containing the reconstituted calcium channel was measured following vesicles (200 ~1) preincubation for 30min at room temperature with different concentrations of GM, (G2Opg). The external divalent cations were removed on Sephadex G-50 column (Curtis and Catteral, 1986), the vesicles (lOOj11) were suspended in 0.34 M sucrose, 1OmM MOPS-tetramethylammonium, pH 7.0, 5 PM Bay K8644, and the calcium uptake was initiated by addition of 50 ,ul of 0.1 M CaCl, plus 2 PCi of 45Ca2+.After 20 min incubation at 37”C, the reaction was terminated with I50 mM MgCl, in 10 mM N-(2-hydroxyethyl)piperazine-N’-2-ethanesulfonic acid (HEPES)-Tris buffer, pH 7.4, and the samples were passed through a Chelex 100 column, equilibrated and eluted with sucrose medium containing 1 mg/ml of BSA. The eluted vesicles were then analyzed for 4JCa2+ uptake. The specificity of calcium uptake into vesicles in the presence of GM,-ganglioside was evaluated using La3+ displacement assay. Statistical analysis

All experiments were carried out in triplicate, and the results are expressed as means f SD. Student’s t-test was used to determine significance, and P values of 0.05 or less were considered significant.

Calcium channels and GM,

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RESULTS

PuriJication of gastric mucosal sensitive calcium channels

dihydropyridine -

Gastric mucosal epithelial cell membrane preparation was employed for dihydropyridine-sensitive calcium channel isolation. The solubilized membranes were prelabeled with [3H]PN200-1 10, subjected to affinity chromatography on Sepharosebound wheat germ agglutinin, and the labeled channel-receptor complex was eluted with medium buffer containing 300 mM N-acetylglucosamine. Following rechromatography, this fraction was used for dihydropyridine binding activity assays and for the evaluation of the effect of GM,-ganglioside on this binding. Figure 1 shows the effect of purified calcium channel protein on the binding of dihydropyridine calcium channel antagonist, PN200-110. The results revealed a concentration-dependent binding of PN200-110 with increased protein concentration, attaining maximum value of 139.1 fmol/mg at 400 pg protein. The specific binding of PN200-110 to the calcium channel antagonist receptor, was inhibited by GM,ganglioside, as preincubation of membrane preparation prior to the binding assay with increasing doses of GM, caused a concentration dependent decrease in 1,4_dihydroxypyridine binding, which reached maximum inhibition value of 62% (Fig. 2). 4SCaz+ uptake mediated by vesicle reconstituted calcium channels

0 0

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conditions, the reconstituted gastric mucosal calcium channel protein complex exhibited about 6-fold greater 45CaZ+ uptake into vesicles as compared to that of protein-free vesicles (Fig. 3). The 4sCa2+ uptake into vesicles containing membrane protein was proportional to the time of incubation and reached maximum at 30 min. The vesicles containing

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Fig. 1. Specific binding of [‘H]PN200- 110 to gastric mucosal membrane preparations as a function of protein concentration. Binding assays were conducted as described in Materials and Methods, except that the amounts of protein added were varied. Values represent the means f SD of 5 separate experiments performed in triplicate.

50

Fig. 2. Effect of GM,-ganglioside on specific binding of [3H]PN200-l 10 to gastric mucosal membrane preparations. Assays were conducted as described in Materials and Methods, except that membranes prior to binding assay were preincubated at room temperature for 30 min with (O-50 pg) GM,. Values represent the means f SD of 5 separate experiments performed in triplicate.

The role of calcium antagonist receptor in calcium uptake was evaluated following the incorporation of [3H]PN200-1 10 labeled channel protein complex into phosphatidylcholine vesicles which conform the structure of membrane bound protein. After purification on sucrose gradient, the isolated vesicles were used to measure 4sCa2+ uptake. Under the assay

0 f

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Fig. 3. Uptake of 4sCa2+into phospholipid vesicles containing the reconstituted gastric mucosal calcium channel protein complex. Purified dihydropyridine-sensitive calcium channels, labeled with tracer amount of [3H]PN200-l10, was reconstituted into vesicles, and loaded with 4sCa2+for 20 min at 37°C. Aliquots of vesicles were then diluted into an incubation media containing lanthanum oxide (0.7 mM), incubated at room temperature for 5 min, and subjected to Chelex- 100 column and intravesicular 4%Za2+determination. Uptake of ‘Ca*+ into vesicles free of membrane protein in the absence (A) and the presence (B) of La3+ treatment. Uptake of 4sCa2+ into vesicles containing the reconstituted calcium antagonist receptor in the absence (C) and the presence (D) of La’+ treatment. Values represent the means f SD of 6 separate experiments performed in triplicate.

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Fig. 4. Effect of PN200-110 on the uptake of 45CaZ+into vesicles containing the reconstituted gastric mucosal calcium antagonist receptor. Uptake assays were conducted as described in Materials and Methods using vesicles preincubated for 20min at room temperature with different concentrations (O-l PM) of PN200-110. Values represent the means f SD of 5 separate experiments performed in

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( mM )

Fig. 5. Effect of media osmolarity on 45Ca2+ uptake into vesicles containing the reconstituted dihydropyridine-sensitive gastric mucosal calcium channels. Uptake assays were conducted as described in Materials and Methods using vesicles preincubated for 20 min at room temperature with different concentrations (O-200 nM) of mannitol. Values represent the means + SD of 4 experiments performed in triplicate.

triplicate. reconstituted calcium channels responded in a concentrated dependent manner to PN200-110, a dihydropyridine calcium antagonist. The maximal inhibitory effect was attained at 0.5 PM PN200-110 at which concentration of antagonist a 66% decrease in 4sCa2+ uptake occurred (Fig. 4). This level of inhibition suggests that the 45Ca2+ uptake in reconstituted vesicles is mediated by functional calcium channels with the pharmacological properties of the dihydropyridine-sensitive calcium channel. 4SCa’+ uptake specificity

To ascertain whether the calcium uptake by the vesicles represent transport into osmotically active space or mere binding to the vesicular surface, two types of experiments were conducted. In one, 45Ca2+preloaded vesicles were treated with La’+ to displace the calcium from the vesicle surface, while in the other, 45Ca2+uptake assays were carried out with the vesicles exposed to different media osmolarity. The data obtained revealed that lanthanum at its optical concentration (0.7 mM/ml) caused 4sCa2+ displacement of about 11% (Fig. 3). This indicated that majority of calcium was present in the intravesicular space and hence inaccessible to La3+ displacement. Osmolarity studies, depicted in Fig. 5, showed nearly linear relationship (I = 0.98) between decrease in 45CaZ+uptake and the increase of medium osmolarity, which is indicative of calcium uptake into the intravesicular space. GM,-ganglioside effect on 45Ca2f vesicular uptake

The effect of GM,-ganglioside on the vesicular uptake of 45Ca2+is shown in Fig. 6. The data revealed that preincubation of the vesicle-reconstituted gastric

mucosal calcium channels with the ganglioside led to a concentration-dependent inhibition of 4sCa2+ uptake. The inhibitory effect was proportional to GM, concentration and reached a maximum at 10-15 nM ganglioside. The La3+ displacement assays indicated a GM, concentration dependent increase in 4sCa2C binding to the vesicular surface which reached a maximum value of 12% at 15 nM ganglioside. Based on the corrected values for this nonspecific effect (Fig. 6), GM,-ganglioside at its optimal concentration exerted a 74.5% inhibition in the intravesicular 4sCa2+ uptake.

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Fig. 6. Effect of GM,-ganglioside on 45Ca2+ uptake into vesicles containing the reconstituted dihydropyridine-sensitive gastric mucosal calcium channels. Uptake assays were conducted as described in Materials and Methods using vehicle preincubated for 30min at room temperature with different concentrations (O-20 pg) of GM,. Values represent the means + SD of 5 experiments performed in triplicate.

Calcium channels and GM, DISCUSSION

Among the factor implicated in the preservation of gastric mucosal integrity is the maintenance of intracellular calcium level. Studies indicate that calcium is an important regulatory element for many cellular processes, including contraction, cell differentiation and secretion (Hosey and Lazdunski, 1988; Kass et al., 1990), and that its influx potentiates mucosal injury evoked by ethanol (Tarnawski et al., 1990).

Under normal physiological conditions, calcium entry in most excitatory and secretory cells occurs through carefully controlled processes involving specific voltage or receptor-dependent channels (Hosey and Lazdunski, 1988). The channels present in majority of different types of cells are those known as L-type or dihydropyridine-sensitive calcium channels. These channels are characterized by their sensitivity to 1,4-dihydropyridine derivatives which by binding to the receptors located on the channel protein are capable of calcium transport modulation (Hosey and Lazdunski, 1988; Regulla et al., 1991). The results of our study presented herein, provide evidence for the presence of dihydropyridine-sensitive calcium channels in gastric epithelial membrane and show that GM,-ganglioside, an indigenous cell membrane component, is capable of calcium uptake inhibition. The 1,4-dihydropyridine-sensitive calcium channel complex was solubilized from gastric mucosal membranes with CHAPS as detergent. The calcium antagonist receptor site was then labeled with trace amounts of [3H]PN200-1 10, saturated with calcium channel activator BAY K8644, and purified by

affinity chromatography on wheat germ agglutinin. Elution of the column with 300mM N-acetylglucosamine yielded [3H]PN200- 110 labeled calcium antagonist-receptor complex which when incorporated into the phosphatidylcholine vesicles exhibited active 45ca2+

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cosal calcium antagonist receptor. The data obtained revealed that the ganglioside caused a decrease in 1,4_dihydropyridine binding which reached the maximum value of 62% inhibition at 30 nM GM,. Furthermore, GM,-ganglioside also effectively blocked the uptake of 45Ca2+ into vesicles containing the reconstituted calcium antagonist receptor, indicating the extracellular orientation of calcium channel receptor antagonist binding domain for GM,. The maximal inhibition (74%) was observed at IO-15 nM GM,, values which compare favorably with the inhibitory (66%) effect obtained with 0.5 PM PN200110, an established dihydropyridine calcium antagonist (Curtis and Catterall, 1985). Thus, the inhibition of calcium uptake in the reconstituted vesicles by GM,-ganglioside shows properties typical to functional calcium channels with the pharmacological properties of dihydropyridine-sensitive calcium channel (Hosey and Lazdunski, 1988). The data on the distribution of GM,-ganglioside indicate that this sialoglycolipid is present in most cell membranes and occurs in particularly large quantities in excitatory cells, such as nerve where the regulation of calcium entry is particularly important for neurotransmitter release (Rapport and Gorio, 1981; Yu et al., 1986). The gastric epithelial cell membrane GM, level in man is in the range of 100-150 nmol l/g wet tissue (Slomiany and Slomiany, 1991). These concentrations of GM, are well within the levels shown in this study to affect the calcium uptake by vesicle reconstituted gastric mucosal membrane channel complex. Hence, the identified GM, effect on calcium uptake may be of physiological relevance to the ability of gastric mucosa to preserve its integrity under the adverse environment of luminal content. Indeed, the ability of GM, ganglioside to modulate the function of calcium channels in gastric mucosa may be viewed as one of the important features of the multicomponential aspect of mucosal protection against injury.

uptake and responded in a concentration dependent manner to PN200-110, a dihydropyridine calcium antagonist. Thus, the isolated and reconstituted calcium antagonist-receptor complex exhibited properties typical of a membrane embedded protein and bears close similarity to dihydropyridine calcium antagonist receptors from other tissues (Curtis and Catterall, 1986; Hosey and Lazdunski, 1988; Nunoki er al., 1989; Knaus et al., 1990). Furthermore, like

Acknowledgements-This work was supported by USPHS Grant DK21684-15 from the National Institute of Diabetes and Digestive and Kidney Diseases, and Grant AA05858-10 from the National Institute on Alcohol Abuse and Alcoholism, NIH.

reconstituted calcium antagonist receptors from these tissues, the vesicles containing gastric mucosal receptor showed greater (6-fold) rate of 45Ca2f uptake than that into protein-free vesicles. Since GM,-ganglioside has been demonstrated to possess the ability to protect cellular integrity from calcium imbalance (Harris and Bruno, 1985; Shah and Pant, 1988), and since its intragastric administration provides gastroprotection against chemically induced injury (Szabo et al., 1989; Slomiany et al., 1991), experiments were conducted on the effect of GM, on PN200-I 10 binding to purified gastric mu-

Curtis B. M. and Catterall W. A. (1986) Reconstitution of the voltage-sensitive calcium channel purified from skeletal muscle transverse tubules. Biochemistry 25, 3077-3083. Hanai N., Nores G. A., MacLeod C., Torres-Mendez C. R. and Hakomori S. L. (1988) Ganglioside-mediated modulation of cell growth. J. biol. Gem. 263, 10915-10921. Harris R. A. and Bruno P. (1985) Membrane disordering by anesthetic drugs: relationship to synaptosomal sodium and calcium fluxes. J. Neurochem. 44, 12741281. Hosey M. M. and Lazdunski M. (1988) Calcium channels: pharmacology, structure and regulation. J. Membr. Biol. 104, 81-105.

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