Inhibition of Voltage-Dependent Ca2+ Currents and Activation of Pertussis Toxin-Sensitive G-Proteins via Muscarinic Receptors in GH3 Cells
Stefan Offermanns, Maik Gollasch, Jurgen Hescheler, Karsten Spicher, Anke Schmidt, Giinter Schultz, and Walter Rosenthal Institut fur Pharmakologie Freie Universitat Berlin Thielallee 69/73, D-1000 Berlin 33, Germany Institut fur Physiologie Humboldt-Universitat zu Berlin (M.G.) Hessische Strafe 3/4, D-1040 Berlin, Germany
In the rat pituitary cell line GH3, carbachol inhibits PRL secretion in a pertussis toxin-sensitive manner. For elucidation of the underlying mechanisms, we studied the effect of carbachol on voltage-dependent Ca2+ currents. Under voltage-clamp conditions, carbachol inhibited whole-cell Ca2+ currents by about 25%. This inhibitory action of carbachol was not observed in cells treated with pertussis toxin, indicating the involvement of a pertussis toxin-sensitive G-protein. In membranes of GH3 cells, carbachol stimulated a pertussis toxin-sensitive highaffinity GTPase. In immunoblot experiments with peptide antisera, we identified two forms of the Gj a-subunit (41 and 40 kDa) and two forms of the Go a-subunit (40 and 39 kDa). The 40-kDa Gj a-subunit was recognized by an antibody specific for the Gi2 a-subunit, and the 39-kDa Go a-subunit was detected by an antibody specific for the Go2 a-subunit. Incubation of membranes with the photoreactive GTP analog [a-32P]GTP azidoanilide resulted in photolabeling of 40- and 39-kDa pertussis toxin substrates comigrating with G-protein a-subunits of the corresponding molecular masses. Carbachol dose-dependently stimulated incorporation of the photoreactive GTP analog into the 39-kDa pertussis toxin substrate and, to a lesser extent, into 40-kDa pertussis toxin substrates. The data indicate that muscarinic receptors of GH3 cells couple preferentially to Go, which is likely to be involved in the inhibition of secretion, possibly by conferring an inhibitory effect to voltage-dependent Ca2+ channels. (Molecular Endocrinology 5: 995-1002, 1991)
hormone-controlled secretion. Carbachol, acting through muscarinic receptors, inhibits PRL and GH secretion in these cells (1-2). The molecular mechanisms underlying the inhibition/ response are poorly understood. In membrane preparations of GH3 cells, carbachol induces inhibition of adenylyl cyclase (1, 3) and stimulation of K+ channels (4). In intact GH3 cells, carbachol decreases cytosolic Ca2+ (5). Recent studies suggest that another inhibitory receptor agonist, somatostatin, decreases Ca2+ influx through voltage-dependent Ca2+ channels by increasing a hyperpolarizing K+ conductance (6). Somatostatin also inhibits voltagedependent Ca2+ currents in pituitary AtT-20 and GH3 cells directly, i.e. without the involvement of a hyperpolarizing current (7, 8). Unlike those in other cell types, all of the effects of carbachol in GH3 cells are sensitive to pertussis toxin (4, 5, 9), indicating that carbachol acts exclusively via G-proteins that are substrates for pertussis toxin (for recent reviews on G-proteins, see Refs. 10 and 11). This assumption is supported by the finding that the stimulatory effect of acetylcholine on high-affinity GTPases in membranes of GH3 cells is abolished by pertussis toxin (12). Similar to neuronal cells, GH3 cells possess pertussis toxin-sensitive G-proteins of the Gi and Go families. Whereas the subtypes of Gj have not been characterized in this cell type, two members of the Go family have been detected, one of which corresponds to the recently identified subtype G02 (13). A Gprotein of the Gi family, Gk, which was purified from human erythrocytes and possibly corresponds to Gi3, was shown to reconstitute the stimulation of K+ channels via muscarinic receptors in isolated membrane patches of pertussis toxin-treated GH3 cells (4,14). No data exist concerning the identity of endogenous Gproteins activated by muscarinic receptors in GH3 cells. The aim of the present study was 1) to elucidate the mechanism by which carbachol lowers cytosolic Ca2+,
INTRODUCTION The rat pituitary cell line GH3 provides an informative model for studies on the molecular mechanisms of 0888-8809/91/0995-1002$03.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society
995
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 05:47 For personal use only. No other uses without permission. . All rights reserved.
Vol 5 No. 7
MOL ENDO-1991 996
B 30 -
CO
X
20 -
O
¥
10-
I Carb Con
Carb
Carb + Atr
Carb +PT
Fig. 1. Effect of Carbachol on Ca2+ Channel Currents in GH3 Cells Whole-cell Ca2+ currents were recorded under voltage-clamp conditions by depolarizing pulses from - 8 0 to 0 mV. A, Representative current traces. Con, Control currents obtained before application of agents; Carb, currents recorded during superfusion of cells with 10 HM carbachol; W, currents recorded after removal of carbachol; Carb + Atr, current obtained during superfusion of cells with 10 HM carbachol plus 50 HM atropine; PT, currents recorded after treatment of cells with pertussis toxin (25 ng/ml for 24 h). Dashed lines represent zero current. B, Reduction of Ca2+ currents, as a percentage of control peak currents. lCa, Ca2+ current. Columns represent the mean ± SEM (n = 6-16).
and 2) to examine which endogenous G-proteins are activated by muscarinic receptors in membranes of GH3 cells. Part of the data has been published in abstract form (15).
RESULTS
c
9 8 7 6 carbachol (M,-log)
Fig. 2. Effect of Carbachol on High Affinity GTPase in Membranes of GH3 Cells Membranes (5 Mg protein) of control cells (O) and pertussis toxin-treated cells (•) were employed, c, Control (without hormone). Shown are mean values of triplicates (SD < 5% of values).
Voltage-dependent Ca2+ currents were measured in the whole-cell configuration of the patch-clamp technique (16). Ba2+ was used as charge carrier, and Cs+ was employed to block K+ outward currents. GH3 cells were kept at a holding potential of - 8 0 mV. Ca2+ currents were activated by 200-msec pulses to 0 mV. The Ca2+ channel blocker (-t-)isradipine (1 ^M) reversibly inhibited, and the Ca2+ channel agonist Bay K 8644 (1 HM) reversibly stimulated Ca2+ currents by 93 ± 3% (n = 7) and 62 ± 9% (n = 8), respectively (means ± SEM). Ca2+ currents were not sensitive to the N-type Ca2+ channel blocker w-conotoxin (10 HM; n = 3). Within 1020 sec after the application of carbachol (10 HM) to the extracellular solution, Ca2+ currents were reduced by 27.4 ± 2.6% (mean ± SEM; n = 16; Fig. 1). After removal of carbachol, currents increased immediately, and within 20-30 sec, they reached an amplitude close to that of control currents. The inhibitory effect of car-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 05:47 For personal use only. No other uses without permission. . All rights reserved.
Carbachol-lnduced Inhibition of Ca2+ Currents in GH3 Cells
997
9467-
-94 -67
67
•
43-
-43
43
•
30DF-
-30 — DF
CXj2 CXjC ( X c CX0C&02
> >1 o H
~0
>
1
CX0c(
o H
/
untreated
PT-treated
Fig. 3. G-Protein a-Subunits in Membranes of GH3 Cells Membrane proteins of untreated and pertussis toxin (PT)treated cells were photolabeled with [a-32P]GTP azidoanilide, separated electrophoretically, and transferred onto nitrocellulose filters. The filters were autoradiographed (AA-GTP) and subsequently incubated with the indicated antibodies («c, acommon-peptide antibody; aic, aicommon-peptide antibody; aa, ai2peptide antibody; ax, aocOmmon-peptide antibody; ao2, «02-peptide antibody). Bound antibodies were visualized by a color reaction. Values on the left and right margins indicate molecular masses of marker proteins (kilodaltons). DF, Dye front. In contrast to other experiments (see Figs. 4-8), photolabeling was performed in the absence of GDP. Under this condition, the 40-kDa proteins were predominantly labeled.
bachol (10 MM) was not observed in the presence of the muscarinic receptor antagonist atropine (50 ^ M ; n = 6) or after treatment of cells with pertussis toxin (n = 10). The data indicate that muscarinic receptors inhibit dihydropyridine-sensitive Ca2+ currents via pertussis toxinsensitive G-proteins. In membranes of GH3 cells, carbachol dose-dependently stimulated a high-affinity GTPase, the enzymatic activity of G-proteins (Fig. 2). The stimulation was halfmaximal and maximal at carbachol concentrations of about 0.3 and 1 HM, respectively. In membranes prepared from pertussis toxin-treated cells, the basal GTPase activity was decreased, and the effect of carbachol was abolished. The data show that the membrane preparation is suitable to study the interaction of muscarinic receptors and G-proteins. To identify the pertussis toxin-sensitive G-proteins activated by muscarinic receptors, we probed membranes of GH3 cells with the photoreactive GTP analog [a-32P]GTP azidoanilide and with antibodies directed against different G-protein a-subunits. For identication
— 67
c -8 -7 -6 -5 -4 carbachol (log M) Fig. 4. Influence of Carbachol on Photolabeling of Proteins in Membranes of GH3 Cells Membrane proteins were photolabeled at increasing concentrations of carbachol (abscissa). Shown is an autoradiogram of a SDS-gel. Values on the left and right margins indicate the molecular masses of marker proteins (kilodaltons). c, Control (without carbachol); DF, dye front.
of photolabeled proteins, membranes of GH3 cells were incubated with [a-32P]GTP azidoanilide, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto nitrocellulose filters. The filters were first autoradiographed and then incubated with antibodies; filter-bound antibodies were visualized by a color reaction. In membranes of control cells (Fig. 3, left panels), the aCOmmon-peptide antibody, which recognizes a-subunits of Gi and Go, reacted with proteins of 39, 40, and 41 kDa. The aOCommon-peptide antibody detected Go a-subunits with molecular masses of 39 and 40 kDa; by the use of a subtype-specific antibody (13), the former was identified as the recently described G02 «subunit (17, 18). Since only two a-subunits of Go proteins are known, i.e. Goi and Go2 a-subunits (19), the 40-kDa species is likely to represent the Goi a-subunit. In addition, two G, a-subunits were detected by the aicommon-peptide antibody, of which the 40-kDa form was identified as the Gi2 a-subunit. The 41-kDa Gi a-subunit may represent the a-subunit of one of the other Gi subtpyes, Gn or Gi3. The identity of immunoreactive proteins with molecular masses greater than 41 kDa is not known. In particular, the ai2- and ao2-peptide antibodies, which, in contrast to the other employed antibodies, were not affinity-purified (see Materials and Methods), reacted with a number of unknown high molecular mass proteins. [a-32P]GTP azidoanilide was incorporated mainly into proteins of 40 and 39 kDa, comigrating with the 40-kDa a-subunits of Gi and Go and the 39-kDa a-subunit of G02, respectively. Photo-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 05:47 For personal use only. No other uses without permission. . All rights reserved.
Vol 5 No. 7
MOL ENDO-1991 998
o o> TJ-
CO
T T
O 0>
Stefan Offermanns, Maik Gollasch, Jurgen Hescheler, Karsten Spicher, Anke Schmidt, Giinter Schultz, and Walter Rosenthal Institut fur Pharmakologie Freie Universitat Berlin Thielallee 69/73, D-1000 Berlin 33, Germany Institut fur Physiologie Humboldt-Universitat zu Berlin (M.G.) Hessische Strafe 3/4, D-1040 Berlin, Germany
In the rat pituitary cell line GH3, carbachol inhibits PRL secretion in a pertussis toxin-sensitive manner. For elucidation of the underlying mechanisms, we studied the effect of carbachol on voltage-dependent Ca2+ currents. Under voltage-clamp conditions, carbachol inhibited whole-cell Ca2+ currents by about 25%. This inhibitory action of carbachol was not observed in cells treated with pertussis toxin, indicating the involvement of a pertussis toxin-sensitive G-protein. In membranes of GH3 cells, carbachol stimulated a pertussis toxin-sensitive highaffinity GTPase. In immunoblot experiments with peptide antisera, we identified two forms of the Gj a-subunit (41 and 40 kDa) and two forms of the Go a-subunit (40 and 39 kDa). The 40-kDa Gj a-subunit was recognized by an antibody specific for the Gi2 a-subunit, and the 39-kDa Go a-subunit was detected by an antibody specific for the Go2 a-subunit. Incubation of membranes with the photoreactive GTP analog [a-32P]GTP azidoanilide resulted in photolabeling of 40- and 39-kDa pertussis toxin substrates comigrating with G-protein a-subunits of the corresponding molecular masses. Carbachol dose-dependently stimulated incorporation of the photoreactive GTP analog into the 39-kDa pertussis toxin substrate and, to a lesser extent, into 40-kDa pertussis toxin substrates. The data indicate that muscarinic receptors of GH3 cells couple preferentially to Go, which is likely to be involved in the inhibition of secretion, possibly by conferring an inhibitory effect to voltage-dependent Ca2+ channels. (Molecular Endocrinology 5: 995-1002, 1991)
hormone-controlled secretion. Carbachol, acting through muscarinic receptors, inhibits PRL and GH secretion in these cells (1-2). The molecular mechanisms underlying the inhibition/ response are poorly understood. In membrane preparations of GH3 cells, carbachol induces inhibition of adenylyl cyclase (1, 3) and stimulation of K+ channels (4). In intact GH3 cells, carbachol decreases cytosolic Ca2+ (5). Recent studies suggest that another inhibitory receptor agonist, somatostatin, decreases Ca2+ influx through voltage-dependent Ca2+ channels by increasing a hyperpolarizing K+ conductance (6). Somatostatin also inhibits voltagedependent Ca2+ currents in pituitary AtT-20 and GH3 cells directly, i.e. without the involvement of a hyperpolarizing current (7, 8). Unlike those in other cell types, all of the effects of carbachol in GH3 cells are sensitive to pertussis toxin (4, 5, 9), indicating that carbachol acts exclusively via G-proteins that are substrates for pertussis toxin (for recent reviews on G-proteins, see Refs. 10 and 11). This assumption is supported by the finding that the stimulatory effect of acetylcholine on high-affinity GTPases in membranes of GH3 cells is abolished by pertussis toxin (12). Similar to neuronal cells, GH3 cells possess pertussis toxin-sensitive G-proteins of the Gi and Go families. Whereas the subtypes of Gj have not been characterized in this cell type, two members of the Go family have been detected, one of which corresponds to the recently identified subtype G02 (13). A Gprotein of the Gi family, Gk, which was purified from human erythrocytes and possibly corresponds to Gi3, was shown to reconstitute the stimulation of K+ channels via muscarinic receptors in isolated membrane patches of pertussis toxin-treated GH3 cells (4,14). No data exist concerning the identity of endogenous Gproteins activated by muscarinic receptors in GH3 cells. The aim of the present study was 1) to elucidate the mechanism by which carbachol lowers cytosolic Ca2+,
INTRODUCTION The rat pituitary cell line GH3 provides an informative model for studies on the molecular mechanisms of 0888-8809/91/0995-1002$03.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society
995
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 05:47 For personal use only. No other uses without permission. . All rights reserved.
Vol 5 No. 7
MOL ENDO-1991 996
B 30 -
CO
X
20 -
O
¥
10-
I Carb Con
Carb
Carb + Atr
Carb +PT
Fig. 1. Effect of Carbachol on Ca2+ Channel Currents in GH3 Cells Whole-cell Ca2+ currents were recorded under voltage-clamp conditions by depolarizing pulses from - 8 0 to 0 mV. A, Representative current traces. Con, Control currents obtained before application of agents; Carb, currents recorded during superfusion of cells with 10 HM carbachol; W, currents recorded after removal of carbachol; Carb + Atr, current obtained during superfusion of cells with 10 HM carbachol plus 50 HM atropine; PT, currents recorded after treatment of cells with pertussis toxin (25 ng/ml for 24 h). Dashed lines represent zero current. B, Reduction of Ca2+ currents, as a percentage of control peak currents. lCa, Ca2+ current. Columns represent the mean ± SEM (n = 6-16).
and 2) to examine which endogenous G-proteins are activated by muscarinic receptors in membranes of GH3 cells. Part of the data has been published in abstract form (15).
RESULTS
c
9 8 7 6 carbachol (M,-log)
Fig. 2. Effect of Carbachol on High Affinity GTPase in Membranes of GH3 Cells Membranes (5 Mg protein) of control cells (O) and pertussis toxin-treated cells (•) were employed, c, Control (without hormone). Shown are mean values of triplicates (SD < 5% of values).
Voltage-dependent Ca2+ currents were measured in the whole-cell configuration of the patch-clamp technique (16). Ba2+ was used as charge carrier, and Cs+ was employed to block K+ outward currents. GH3 cells were kept at a holding potential of - 8 0 mV. Ca2+ currents were activated by 200-msec pulses to 0 mV. The Ca2+ channel blocker (-t-)isradipine (1 ^M) reversibly inhibited, and the Ca2+ channel agonist Bay K 8644 (1 HM) reversibly stimulated Ca2+ currents by 93 ± 3% (n = 7) and 62 ± 9% (n = 8), respectively (means ± SEM). Ca2+ currents were not sensitive to the N-type Ca2+ channel blocker w-conotoxin (10 HM; n = 3). Within 1020 sec after the application of carbachol (10 HM) to the extracellular solution, Ca2+ currents were reduced by 27.4 ± 2.6% (mean ± SEM; n = 16; Fig. 1). After removal of carbachol, currents increased immediately, and within 20-30 sec, they reached an amplitude close to that of control currents. The inhibitory effect of car-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 05:47 For personal use only. No other uses without permission. . All rights reserved.
Carbachol-lnduced Inhibition of Ca2+ Currents in GH3 Cells
997
9467-
-94 -67
67
•
43-
-43
43
•
30DF-
-30 — DF
CXj2 CXjC ( X c CX0C&02
> >1 o H
~0
>
1
CX0c(
o H
/
untreated
PT-treated
Fig. 3. G-Protein a-Subunits in Membranes of GH3 Cells Membrane proteins of untreated and pertussis toxin (PT)treated cells were photolabeled with [a-32P]GTP azidoanilide, separated electrophoretically, and transferred onto nitrocellulose filters. The filters were autoradiographed (AA-GTP) and subsequently incubated with the indicated antibodies («c, acommon-peptide antibody; aic, aicommon-peptide antibody; aa, ai2peptide antibody; ax, aocOmmon-peptide antibody; ao2, «02-peptide antibody). Bound antibodies were visualized by a color reaction. Values on the left and right margins indicate molecular masses of marker proteins (kilodaltons). DF, Dye front. In contrast to other experiments (see Figs. 4-8), photolabeling was performed in the absence of GDP. Under this condition, the 40-kDa proteins were predominantly labeled.
bachol (10 MM) was not observed in the presence of the muscarinic receptor antagonist atropine (50 ^ M ; n = 6) or after treatment of cells with pertussis toxin (n = 10). The data indicate that muscarinic receptors inhibit dihydropyridine-sensitive Ca2+ currents via pertussis toxinsensitive G-proteins. In membranes of GH3 cells, carbachol dose-dependently stimulated a high-affinity GTPase, the enzymatic activity of G-proteins (Fig. 2). The stimulation was halfmaximal and maximal at carbachol concentrations of about 0.3 and 1 HM, respectively. In membranes prepared from pertussis toxin-treated cells, the basal GTPase activity was decreased, and the effect of carbachol was abolished. The data show that the membrane preparation is suitable to study the interaction of muscarinic receptors and G-proteins. To identify the pertussis toxin-sensitive G-proteins activated by muscarinic receptors, we probed membranes of GH3 cells with the photoreactive GTP analog [a-32P]GTP azidoanilide and with antibodies directed against different G-protein a-subunits. For identication
— 67
c -8 -7 -6 -5 -4 carbachol (log M) Fig. 4. Influence of Carbachol on Photolabeling of Proteins in Membranes of GH3 Cells Membrane proteins were photolabeled at increasing concentrations of carbachol (abscissa). Shown is an autoradiogram of a SDS-gel. Values on the left and right margins indicate the molecular masses of marker proteins (kilodaltons). c, Control (without carbachol); DF, dye front.
of photolabeled proteins, membranes of GH3 cells were incubated with [a-32P]GTP azidoanilide, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto nitrocellulose filters. The filters were first autoradiographed and then incubated with antibodies; filter-bound antibodies were visualized by a color reaction. In membranes of control cells (Fig. 3, left panels), the aCOmmon-peptide antibody, which recognizes a-subunits of Gi and Go, reacted with proteins of 39, 40, and 41 kDa. The aOCommon-peptide antibody detected Go a-subunits with molecular masses of 39 and 40 kDa; by the use of a subtype-specific antibody (13), the former was identified as the recently described G02 «subunit (17, 18). Since only two a-subunits of Go proteins are known, i.e. Goi and Go2 a-subunits (19), the 40-kDa species is likely to represent the Goi a-subunit. In addition, two G, a-subunits were detected by the aicommon-peptide antibody, of which the 40-kDa form was identified as the Gi2 a-subunit. The 41-kDa Gi a-subunit may represent the a-subunit of one of the other Gi subtpyes, Gn or Gi3. The identity of immunoreactive proteins with molecular masses greater than 41 kDa is not known. In particular, the ai2- and ao2-peptide antibodies, which, in contrast to the other employed antibodies, were not affinity-purified (see Materials and Methods), reacted with a number of unknown high molecular mass proteins. [a-32P]GTP azidoanilide was incorporated mainly into proteins of 40 and 39 kDa, comigrating with the 40-kDa a-subunits of Gi and Go and the 39-kDa a-subunit of G02, respectively. Photo-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 05:47 For personal use only. No other uses without permission. . All rights reserved.
Vol 5 No. 7
MOL ENDO-1991 998
o o> TJ-
CO
T T
O 0>
