Biochimica et Bioph~,sicaAao, !136(1992)315-318
315
© 1992Els~-vierSciencePublishersB.V.All rightsreserved0167-4889/92/$05.00
BBAMCR13210
Gangliosides inhibit platelet-derived growth-factor-stimulated increases in intracellular calcium in Swiss 3T3 cells Zhen Guan a, Bradford T. Stokes a, James Van Brocklyn b and Allan J. Yates
b
Departments of ~ Phydologyand ~'Pathology, Collegeof Medicine, The Ohio State Unicersio; Columbus, OH (USA)
(Received18December1991) (Revisedmanusciiptreceived20 March1.o92) Keywords: Ganglioside;Growthfactor;Autophosphorylation;(Swiss3T3cell) Individual Swiss3T3 cells stimulated by platelet-derivedgrowth fac,*ordelivered by means of a picopumpdevice respond with a brisk, large, and sustained increase in intracellular cal~um concentration ([Ca2+]~).Preincubation of cells with either GMI or GTIb gaagliosidesinh~ited the proportion of respondin~ cells attd caused a dose-related diminution i~l the magnitude of the increase in [Ca2+]i. This effect of ganglinsideis probably part of the mechanismthrough whichgangliosidesexea their biological effects, includinginh$ition of platelet-derived~owth-factor-induced mitogenesis.
in~duction
Gangliosides have a variety of effects on cultured cells, including facilitation of neuritogenesis [1-6], inhibition of cell growth [7,8] and promotion of cell survival [9-14]. The molecular mechanisms through which gangliosides operate are unknown, but there is evidence from studies using 45Ca2+ that their neuritogenic effects on neuroblastoma-2A cells may involve alterations of the influx and efflus of calcium [5]. Gangliasides also prevent the delayed and prolonged increase in [Ca2+]i attendant upon a variety of receptor-mediated phenomena. Platelet-derived growth factor (PDGF) is atso known to stimulate mitogenesis of several types of murine 3,'!"3cells in a manner that involves both an immediate and prolonged elevation of [Ca2+]i [15,16]. Furthermore, ganglios[des inhibit PDGF-stimulated mitogenesis of 31"3 cells, and autophosphorylation of the PDGF recet~tor [17]. To test the above hypothesis specifically for the effects of gangliosides on PDGFmediated events, we have used the calcium-sensitive dye fura-2 and detailed calibration protocols to determine directly the effects that gang~iosides have or the responses of [Ca2+]i in individual Swiss-3T3 cells exposed to PDGF. m
Correspondenceto: B.T.Slokes,The OhioSlateUniversity,Department of Physiology,400 HamilitonHall, 1645NellAve.,Columbus, Ohio, USA. Abbrevialions:PDGF, plateleFdefivedgrowlh factor,[Ca2+ ]i,intra-
cellularfree calciumion concentration.Oangliosidenomenclatureis accordingto thai of Svennerholm[28].
Materials and Methods
Cell culture
Swiss 3T3 cells were obtained from the American Type Culture Collection. They were routinely maintained as monolayers in nulbecco's Modified Engle's medium containing 10% calf serum (Gibco, Grand Island, NY), antibiotics (80 U of penicillin G sodium, and 80 mg of streptomycin sulfate) in Coming 150-cme plastic flasks at 37°C in humidified air containing 5% CO z and subcultivated at a split ratio of 1:10 to 1:20 once per week. Trypsin (0.25%) in phosphate-buffered saline (PBS) was used as a detaching agent. For experiments, cells were seeded in the same medium onto glass cover-slips in Corning 0-well 9.6-cme plastic plates, incubated for 2 days, and the medium changed. The following morning ceils were incubated for 1 h at 3"PC in serum-free medium containing the specified concentration of gangliosides which had been quantitared with the resorcinol method on the basis of sialic acid content [18]. lntracellular calcium measurements and picospritzing
Cells were loaded with calcium-membrane-permeant indicator fura-2/AM according to established procedures [19,20]. Briefly, 5 #M fura-2/AM was added directly to the culture medium. Cells were bathed in the loading solution for 0.5 h at 37°C, then rinr.e.d once to remo~,z excess dye, and allowed more than 2 h postit~cubation in the incubator. This provided for nearly complete hydrolysis of the ester form and
316 formation of the impermeant acid moiety. Alternatively, before loading with fura, the cells were incubated for 1 h with various amounts (25-100 pM) of GMI or GTlb ganglioside in the incubator. Coverslips (0.07 mm; 31 mm diameter) with cells were then mounted in a flow-through chamber (Biophysica Tissue Chamber) and placed on the stage of a Zciss IM microscope system. Cells in the chamber were routinely perfused ~ t h a K-H buffer containing (in mM): 116.5 NaCI, 4_5 KCI, 09 MgSO4, 1.0 NaH2PO4, 25 NaHCO 3, 1.5 CaCI 2, 5.0 pyruvate. A su~ion device was positioned on top of the chamber to allow continuous slow superfnsion (2 ml/min) of the cells; the contents (4 ~ ) of the balh are changed completely by approx. 20 s [20]. The temperature of the buffer solution in the well was monitored by a thermometer and maintained at 35-37~C by passing the experimental solutions through a temperature-controlled l:cat-ex. changer. The pH of the perfusate was adjusted to 7.35 + 0.05 with 95% 02-5% CO 2 before heating. Epifluorescence measurements were all made in single ceils isolated in the microscopic field using quartz optics and a Zeiss Plan-Neofluar (63 x ) water immersion objective for 4 rain; the peak of each response was used for c a ~ l a t i o ~ used in the figures below. Each cell was excited by fluorescence produced by a 75-W Xenon lamp as altered by the monochrometers in a PT! photometry, system at wavelengths of 340 nm and 380 nm. Emissions (420-620 nm) ,q'om the two excitation wavelengths were collected by a photomultiplier tube and then stored in the IBM-AT Deltascan storage system. To avoid photobleaching of the cell, a computer-controlled shutter was kept closed between measurements. Intracellular calcium concentrations ([Ca2+]~) were calculated from the ratio of the two excitation wavelengths using the equation, [Ca2+] =N d (R-Rmini Rm~ - R) (Fo/F~) [21]. Rmm and S~ax were also determined as before [20,22]. Appropriate adjustment was also made to correct the dissociation constant to ensure a more accurate estimation of [Ca2+]i [23]. A pneumatic picopump (WPI, Model PVS00) was used to apply PDGF (I.0 pg/ml) for 100-150 s during the recording sessions. A minimum pressure (< 4.0 PSI) was used to inject the 10-15 pl of PDGF through a 40-pro pipette tip at a distance of 100-120 pm from the cell. The low PSI was used to avoid pressure artifacts. R~ults and Discussion Control cells (untreated with ganglioside) had a resting [Ca2+]i of 128 + 10 nM (S.E.). Following the exposure of these cells to PDGF, there was a rapid increase in [Ca2+]i t o 1728 + 159 nM after a delay of about 1 min (Fig. 1). After 2 rain, the elevated [Ca'+]i
SHIS5 3T3
._. 3,(
.79~
o Lll ~,~
~IB v
(lu~i/mllPiwPe~p
Fig. !. Responseof a sillgle Swiss 31n3cell to PDGF slimulalion. At lltc ~1o~, I #g/ml ol Pi~3F was p~osp[it~d from a micropil~lle (40 /zm) at low pressure ( < 4 PSI) from a distance of approx. 100 ~n~ for 100 s. The raw data reveals a prompt and lasting increase in intracellular calciuat to lev~Is exceeding 1.0 ~tM. Ratios and free calcium calculated as in Li et al. [221 and Groden et al. I231.
began to decrease, but it typically remair,ed higher than normal until at least 3-5 min a.fie," exposure to PDGF. The absolute magnitude of the response was surprisingly large (> 1.0 p.M) and would, therefore, represent a sig~ifi~n~ stimulus to PDGF-stimulated second messenger systems. Such a respop~ was seen in 88 cells of 93 (95%) tested (Figs. 2 and 4). Similar results have been reported for Balb/c 3T3 cells, but a smaller proportion of those cells responded to PDGF, and the highest levels that the [Ca2+]i reached were less than seen in the Swiss 3'13 cells here [15,16]. Pre-incubating cells with GM1 ganglioside for 1 h before exposing them to PDGF had an inlu'bitory effect on the PDGF-stimulated increase in [Ca2+]r The de~'e¢ of inh~ition by ganglioside for both the percentage of responding cells and the levels to which [Ca2+]i increased in those cells that did respond were dose-related over the range of 25 to 100 pM of GM1. At 25 pM, 10 of 15 cells (67%) studied responded to PDGF stimulation with an elevation of [Ca2+]i, but at 100 #M only 4 out of 26 (15%) responded (Fig. 2). Following PDGF stimulation, the [Ca2+]t of PDGF-responsive cells preincubated with 25 pM GMI rose to 49% of stimulated control-cell values while those preincubated with 100 ;tM GMI rose to only 24% of controls (Fig. 3). These results are of considerab!e interest for three reasons: (1) this is the first study directly demonstrating that gangliosides alter the PDGF-mediated increase in [Ca2+]i; (2) there is a close association in 3T3 cells between elevations of [Ca2+]i and PDGF-induced mitogenesis [15,16]; and (3) GMI ganglinside inh~its PDGF-stimnlated growlh of 3T3 cells [17]. Therefore, it seems reasonable to conclude that the inhibitory effect of ganglioside on PDGFostimulated mitogenesis
317
PicoPumpPDGF SWISS3T3
PicoP~,,pPDGF SHISS3T3 150
Ii]tl~(I,~,.l)
Response Percentage 100•~oG~,~i
Response i B'-~¢rlb(~O0~l PercentageI pl~x~,~.i~bI~x~i
~ooF
(2~)
,
. . P 50 /~M.
is m~diated, at least in part, through an inhibition of the PDGF-stimulated increase in [Ca2+]~. Preincubation of celLs with GMI caused a slight increase in resting levels of [Ca2+]i from 115 to 181 riM. OTlb, however, had no effect oJ; basal levels of [Ca2+]i. How these findings relate to those of Wu et al. [5] is uncertain. "Ihose authors found that gangliosides caused small increases in both i~tfiux and efflus of Ca z+ in a neuroblastoma ceil line (Neuro2A) which responds to gangliosides with a neuritogenic effect. They concluded that such changes in flux (which were seen over a considerably longer time period than the effects we observed) is an essential component of the neuritogenic effect of gangliosides. The effect of GTIb on PDGF-stimulated changes in [Ca2+}i were qualitatively similar to those of GMI, but there were quantitative differences in the effects of the
P
315
© 1992Els~-vierSciencePublishersB.V.All rightsreserved0167-4889/92/$05.00
BBAMCR13210
Gangliosides inhibit platelet-derived growth-factor-stimulated increases in intracellular calcium in Swiss 3T3 cells Zhen Guan a, Bradford T. Stokes a, James Van Brocklyn b and Allan J. Yates
b
Departments of ~ Phydologyand ~'Pathology, Collegeof Medicine, The Ohio State Unicersio; Columbus, OH (USA)
(Received18December1991) (Revisedmanusciiptreceived20 March1.o92) Keywords: Ganglioside;Growthfactor;Autophosphorylation;(Swiss3T3cell) Individual Swiss3T3 cells stimulated by platelet-derivedgrowth fac,*ordelivered by means of a picopumpdevice respond with a brisk, large, and sustained increase in intracellular cal~um concentration ([Ca2+]~).Preincubation of cells with either GMI or GTIb gaagliosidesinh~ited the proportion of respondin~ cells attd caused a dose-related diminution i~l the magnitude of the increase in [Ca2+]i. This effect of ganglinsideis probably part of the mechanismthrough whichgangliosidesexea their biological effects, includinginh$ition of platelet-derived~owth-factor-induced mitogenesis.
in~duction
Gangliosides have a variety of effects on cultured cells, including facilitation of neuritogenesis [1-6], inhibition of cell growth [7,8] and promotion of cell survival [9-14]. The molecular mechanisms through which gangliosides operate are unknown, but there is evidence from studies using 45Ca2+ that their neuritogenic effects on neuroblastoma-2A cells may involve alterations of the influx and efflus of calcium [5]. Gangliasides also prevent the delayed and prolonged increase in [Ca2+]i attendant upon a variety of receptor-mediated phenomena. Platelet-derived growth factor (PDGF) is atso known to stimulate mitogenesis of several types of murine 3,'!"3cells in a manner that involves both an immediate and prolonged elevation of [Ca2+]i [15,16]. Furthermore, ganglios[des inhibit PDGF-stimulated mitogenesis of 31"3 cells, and autophosphorylation of the PDGF recet~tor [17]. To test the above hypothesis specifically for the effects of gangliosides on PDGFmediated events, we have used the calcium-sensitive dye fura-2 and detailed calibration protocols to determine directly the effects that gang~iosides have or the responses of [Ca2+]i in individual Swiss-3T3 cells exposed to PDGF. m
Correspondenceto: B.T.Slokes,The OhioSlateUniversity,Department of Physiology,400 HamilitonHall, 1645NellAve.,Columbus, Ohio, USA. Abbrevialions:PDGF, plateleFdefivedgrowlh factor,[Ca2+ ]i,intra-
cellularfree calciumion concentration.Oangliosidenomenclatureis accordingto thai of Svennerholm[28].
Materials and Methods
Cell culture
Swiss 3T3 cells were obtained from the American Type Culture Collection. They were routinely maintained as monolayers in nulbecco's Modified Engle's medium containing 10% calf serum (Gibco, Grand Island, NY), antibiotics (80 U of penicillin G sodium, and 80 mg of streptomycin sulfate) in Coming 150-cme plastic flasks at 37°C in humidified air containing 5% CO z and subcultivated at a split ratio of 1:10 to 1:20 once per week. Trypsin (0.25%) in phosphate-buffered saline (PBS) was used as a detaching agent. For experiments, cells were seeded in the same medium onto glass cover-slips in Corning 0-well 9.6-cme plastic plates, incubated for 2 days, and the medium changed. The following morning ceils were incubated for 1 h at 3"PC in serum-free medium containing the specified concentration of gangliosides which had been quantitared with the resorcinol method on the basis of sialic acid content [18]. lntracellular calcium measurements and picospritzing
Cells were loaded with calcium-membrane-permeant indicator fura-2/AM according to established procedures [19,20]. Briefly, 5 #M fura-2/AM was added directly to the culture medium. Cells were bathed in the loading solution for 0.5 h at 37°C, then rinr.e.d once to remo~,z excess dye, and allowed more than 2 h postit~cubation in the incubator. This provided for nearly complete hydrolysis of the ester form and
316 formation of the impermeant acid moiety. Alternatively, before loading with fura, the cells were incubated for 1 h with various amounts (25-100 pM) of GMI or GTlb ganglioside in the incubator. Coverslips (0.07 mm; 31 mm diameter) with cells were then mounted in a flow-through chamber (Biophysica Tissue Chamber) and placed on the stage of a Zciss IM microscope system. Cells in the chamber were routinely perfused ~ t h a K-H buffer containing (in mM): 116.5 NaCI, 4_5 KCI, 09 MgSO4, 1.0 NaH2PO4, 25 NaHCO 3, 1.5 CaCI 2, 5.0 pyruvate. A su~ion device was positioned on top of the chamber to allow continuous slow superfnsion (2 ml/min) of the cells; the contents (4 ~ ) of the balh are changed completely by approx. 20 s [20]. The temperature of the buffer solution in the well was monitored by a thermometer and maintained at 35-37~C by passing the experimental solutions through a temperature-controlled l:cat-ex. changer. The pH of the perfusate was adjusted to 7.35 + 0.05 with 95% 02-5% CO 2 before heating. Epifluorescence measurements were all made in single ceils isolated in the microscopic field using quartz optics and a Zeiss Plan-Neofluar (63 x ) water immersion objective for 4 rain; the peak of each response was used for c a ~ l a t i o ~ used in the figures below. Each cell was excited by fluorescence produced by a 75-W Xenon lamp as altered by the monochrometers in a PT! photometry, system at wavelengths of 340 nm and 380 nm. Emissions (420-620 nm) ,q'om the two excitation wavelengths were collected by a photomultiplier tube and then stored in the IBM-AT Deltascan storage system. To avoid photobleaching of the cell, a computer-controlled shutter was kept closed between measurements. Intracellular calcium concentrations ([Ca2+]~) were calculated from the ratio of the two excitation wavelengths using the equation, [Ca2+] =N d (R-Rmini Rm~ - R) (Fo/F~) [21]. Rmm and S~ax were also determined as before [20,22]. Appropriate adjustment was also made to correct the dissociation constant to ensure a more accurate estimation of [Ca2+]i [23]. A pneumatic picopump (WPI, Model PVS00) was used to apply PDGF (I.0 pg/ml) for 100-150 s during the recording sessions. A minimum pressure (< 4.0 PSI) was used to inject the 10-15 pl of PDGF through a 40-pro pipette tip at a distance of 100-120 pm from the cell. The low PSI was used to avoid pressure artifacts. R~ults and Discussion Control cells (untreated with ganglioside) had a resting [Ca2+]i of 128 + 10 nM (S.E.). Following the exposure of these cells to PDGF, there was a rapid increase in [Ca2+]i t o 1728 + 159 nM after a delay of about 1 min (Fig. 1). After 2 rain, the elevated [Ca'+]i
SHIS5 3T3
._. 3,(
.79~
o Lll ~,~
~IB v
(lu~i/mllPiwPe~p
Fig. !. Responseof a sillgle Swiss 31n3cell to PDGF slimulalion. At lltc ~1o~, I #g/ml ol Pi~3F was p~osp[it~d from a micropil~lle (40 /zm) at low pressure ( < 4 PSI) from a distance of approx. 100 ~n~ for 100 s. The raw data reveals a prompt and lasting increase in intracellular calciuat to lev~Is exceeding 1.0 ~tM. Ratios and free calcium calculated as in Li et al. [221 and Groden et al. I231.
began to decrease, but it typically remair,ed higher than normal until at least 3-5 min a.fie," exposure to PDGF. The absolute magnitude of the response was surprisingly large (> 1.0 p.M) and would, therefore, represent a sig~ifi~n~ stimulus to PDGF-stimulated second messenger systems. Such a respop~ was seen in 88 cells of 93 (95%) tested (Figs. 2 and 4). Similar results have been reported for Balb/c 3T3 cells, but a smaller proportion of those cells responded to PDGF, and the highest levels that the [Ca2+]i reached were less than seen in the Swiss 3'13 cells here [15,16]. Pre-incubating cells with GM1 ganglioside for 1 h before exposing them to PDGF had an inlu'bitory effect on the PDGF-stimulated increase in [Ca2+]r The de~'e¢ of inh~ition by ganglioside for both the percentage of responding cells and the levels to which [Ca2+]i increased in those cells that did respond were dose-related over the range of 25 to 100 pM of GM1. At 25 pM, 10 of 15 cells (67%) studied responded to PDGF stimulation with an elevation of [Ca2+]i, but at 100 #M only 4 out of 26 (15%) responded (Fig. 2). Following PDGF stimulation, the [Ca2+]t of PDGF-responsive cells preincubated with 25 pM GMI rose to 49% of stimulated control-cell values while those preincubated with 100 ;tM GMI rose to only 24% of controls (Fig. 3). These results are of considerab!e interest for three reasons: (1) this is the first study directly demonstrating that gangliosides alter the PDGF-mediated increase in [Ca2+]i; (2) there is a close association in 3T3 cells between elevations of [Ca2+]i and PDGF-induced mitogenesis [15,16]; and (3) GMI ganglinside inh~its PDGF-stimnlated growlh of 3T3 cells [17]. Therefore, it seems reasonable to conclude that the inhibitory effect of ganglioside on PDGFostimulated mitogenesis
317
PicoPumpPDGF SWISS3T3
PicoP~,,pPDGF SHISS3T3 150
Ii]tl~(I,~,.l)
Response Percentage 100•~oG~,~i
Response i B'-~¢rlb(~O0~l PercentageI pl~x~,~.i~bI~x~i
~ooF
(2~)
,
. . P 50 /~M.
is m~diated, at least in part, through an inhibition of the PDGF-stimulated increase in [Ca2+]~. Preincubation of celLs with GMI caused a slight increase in resting levels of [Ca2+]i from 115 to 181 riM. OTlb, however, had no effect oJ; basal levels of [Ca2+]i. How these findings relate to those of Wu et al. [5] is uncertain. "Ihose authors found that gangliosides caused small increases in both i~tfiux and efflus of Ca z+ in a neuroblastoma ceil line (Neuro2A) which responds to gangliosides with a neuritogenic effect. They concluded that such changes in flux (which were seen over a considerably longer time period than the effects we observed) is an essential component of the neuritogenic effect of gangliosides. The effect of GTIb on PDGF-stimulated changes in [Ca2+}i were qualitatively similar to those of GMI, but there were quantitative differences in the effects of the
P
