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Evidence for TRH-induced influx of extracellular Ca2+ in pituitary GH4C1 cells
Vol. 180, No. 2, 1991 October 31, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 860-866
Evidence for TRH-induced influx of extracellular Ca 2+ in pituitary GH4C 1 cells* Kid T6rnquist Endocrine Research Laboratory, University of Helsinki, Minerva Foundation Institute for Medical Research, Helsinki, Finland Received September 4, 1991
The aim of the study was to investigate the relationship between thyrotropin-releasing hormone (TRH)-induced changes in intracellular free Ca 2+ ([Ca2+]i), and influx of extracellular Ca 2+ in Fura 2 loaded pituitary GH4C1 cells. Stimulating the cells with TRH in a Ca2+-containing buffer induced a biphasic change in [Ca2+] i. First, a transient increase in [Ca2+]i, followed by a sustained phase. In cells stimulated with TRH in a Ca2+-free buffer, the transient increase in [Ca2+]i was decreased (p < 0.05), and the sustained phase was totally abolished. Addition of Ni 2+ prior to TRH blunted the component of the TRH-induced transient increase in [Ca2+]i dependent on influx of Ca 2+. In the presence of extracellular Mn 2+, TRH stimulated quenching of Fura 2 fluorescence. This quenching was blocked by Ni 2+. The results indicate that both the TRH-induced transient increase in [Ca2+]i as well as the sustained phase in [Ca2+]i in GH4C1 cells is dependent on influx of extracellular Ca2+. ®1991 Academic Press, Inc.
The stimulus-secretion coupling induced by binding of thyrotropin-releasing hormone (TRH) to its receptor in pituitary cells is mediated via activation of phospholipase C. This leads to hydrolysis of phosphatidylinositol-4,5bisphosphate (Ptdlns) to inositol-l,4,5-trisphosphate (IP3) and diacylglycerol (DG) (1,2). IP3 stimulates the release of sequestered intracellular Ca 2+ (3), and DG activates protein kinase C (4). Stimulating pituitary cells with TRH induces a biphasic change in intracellular Ca2+ ([Ca2+]i): first a rapid transient increase in [Ca2+]i, followed by a sustained plateau phase (5). The transient increase in [Ca2+]i has generally been considered to be due to the IP3* Supported in part by a grant from the Ella Foundation. o0o6-291x/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
860
and Georg Ehrnrooth
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
induced release of sequestered Ca 2+, while the plateau phase in [Ca2+]i is dependent on influx of extracellular Ca 2+ (5,6). In some cell types, however, agonist-induced release of sequestered Ca 2+ has been linked to a substantial influx of extracellular Ca 2+ (see (7). In the present study in GH4C1 cells we show, that both the TRH-induced transient increase in [Ca2+]i, as well as the plateau phase in [Ca2+]i is dependent on influx of extracellular Ca 2+. A preliminary report of these observations has been presented (8). Materials and M e t h o d s Materials Fura 2-AM was obtained from Molecular Probes (Eugene, OR). TRH was from Sigma (St Louis, MO). Nimodipine was a kind gift from Dr. Seuter (Bayer AG, Germany) All flasks and dishes used for the cell culture were from N U N C Plastics (Kamstrup, Denmark). Cell culture Clonal rat pituitary GH4C1 cells were grown in monolayer culture in Ham's F 10 Nutrient Mixture with 15 % (vol/vol) horse serum and 2.5 % fetal bovine serum in a water-saturated atmosphere of 5 % CO2 and 95 % air at 37 oC, as described previously (9,10). Before an experiment, the cells from a single donor culture were harvested with 0 . 1 % trypsin and subcultured in 100 mm culture dishes for 7-9 days. The cells were fed every 2-3 days, and always the day before an experiment. Measuring [Ca2+li The method for determining [Ca2+]i with Fura 2 in GH4C1 cells has been described (11). In brief, the cells were harvested in Hepes-buffered salt solution (HBSS, containing in millimolar concentrations: NaC1, 118; KCI, 4.6; glucose, 10; CaCI2, 0.4; HEPES, 20.0; pH 7.2) lacking CaC12, and containing 0.02 % EDTA. The cells were then washed twice in HBBS (containing Ca2C1), and incubated for 35 min at 37 °C with 1 IxM Fura 2-AM. Fluorescence was measured in a Hitachi F2000 spectrophotometer (Hitachi, Ltd, Tokyo, Japan), using the excitation wavelengths 340 and 380 nm, and the emission wavelength 510 nm. [Ca2+]i was calculated according to (12) with Kd = 224 nM for Fura-2, using a computer program designed for the fluorometer. Statistics Results are given as the mean + SE, and each experiment was repeated at least five times using at least three different cell preparations. Statistical analyses were made using Student's t-test for comparison b e t w e e n two means. Three or more means were tested using analysis of variance. Results TRH-induced changes in [Ca2+li in GH4C1 cells. Stimulating GH4C1 cells with TRH induced a biphasic change in [Ca2+]i • first a transient increase in [Ca2+]i 861
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
1000 TRH
+
c
Ni 2+ TRH
TRH
500
li,.\ Fig. !. Effect of TRH on [Ca2+]i in GH4C1 cells. The cells were stimulated with 100 nM TRH in: A. Ca2+-containing buffer. B. Nominally Ca2+-free buffer containing 100 I.tM EGTA. C. After addition of 2 mM Ni 2+ to cells in Ca2+ buffer. The horizontal bar in (A) denotes 1 min.
(513 + 24
nM; mean + SE; Fig. 1A), followed by a plateau level (135 + 8
nM). If the cells were suspended in a nominally Ca2+-free buffer containing 100 ~tM EGTA, the transient increase in [Ca2+]i was 222 + 33 nM (p < 0.05, compared with control cells), and the plateau phase was totally abolished
(Fig. 1B). Fig. 1C shows, that addition of 2 mM Ni 2+ to GH4C1 cells in suspension reduced basal [Ca2+]i levels from 116 + 8 nM to 77 + 7 nM (p < 0.05), probably by blocking influx of extracellular Ca 2+ via spontaneously active, dihydropyridine sensitive voltage-operated Ca 2+ c h a n n e l s (VOCC). Stimulating the cells with TRH induces a transient increase in [Ca2+]i (330 ± 22 nM), which was significantly smaller (p < 0.05) than that observed in control cells (Table 1). The results in Fig. 2 show that the inhibiting effect of Ni 2+ on the TRH-induced transient in [Ca2+]i was dose-dependent. The
55ot 1
t
250 -~
ol
0.1
1
10
mM Ni2+
Fig. 2. Inhibitory effect of Ni2+ on TRH-induced transient increase in [Ca2+]i in GH4C1 cells. Ni 2+ was added to the cell suspension 1 min prior to stimulating the cells with 100 nM TRH. Each point gives the mean + SE of 5-9 separate experiments. 862
VoI. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
600-
500C
;~ 4-
40O3o0-
2001000
. . . . . . . .
0
i
1
. . . . . . . .
i
10
. . . . . . . .
i
100
. . . . . . . .
1000
nM TRH
Fig. 3. Dose-dependent effect of TRH on the transient increase in [Ca2+]i in GH4C1 cells. The cells were stimulated with the appropriate concentration of TRH in Ca2+ containing buffer ([]), in nominally Ca2+-free buffer containing 100 ~tM EGTA (11), or in Ca2+ buffer containing 2 mM Ni 2+ (O). Each point gives the mean + SE of 5-9 separate experiments. Cells incubated in a nominally Ca2+-free buffer always gave a significantly (p < 0.05) smaller increase in [Ca2+]i compared with cells in Ca2+ buffer. *) p < 0.05 compared with cells stimulated in the presence of Ni 2+. inhibitory effect of Ni 2+ was not the result of blockade of VOCC, as the TRHinduced transient in [Ca2+]i was unaltered in cells incubated with 300 nM the dihydropyridine Ca 2+ channel blocker nimodipine (497 + 56 nM). The plateau phase was, however, significantly decreased (73 + 2 nM, p < 0.05) in the presence of nimodipine. In GH4C1 cells depolarized with 50 mM K + in the presence of nimodipine, the TRH-induced transient increase in [Ca2+]i was not changed (427 + 51 nM), but the plateau phase was significantly decreased (49 + 3 nM, p < 0.05). The transient change in [Ca2+]i in response to increasing concentrations of TRH is shown in Fig. 3. The response obtained in a Ca2+-free buffer was always significantly (p < 0.05) lower compared with that in control cells in Ca2+-containing buffer. In the presence of 2 mM Ni 2+, the T R H - i n d u c e d transient was significantly lower (p < 0.05) than that observed in control cells at the two highest doses of TRH tested. The plateau phase was totally inhibited by Ni 2+ at all the TRH-doses tested (not shown). TRH-stimulated Mn 2+ entry in GH4C1 cells. The results in Fig. 4A show that addition of Mn 2+ to cells in buffer containing 300 nM nimodipine (to prevent influx of Mn2+ via VOCC) produced an immediate drop in fluorescence, suggesting that unstimulated cells are permeable to Mn 2+. Addition of Ni 2+ prior to Mn 2+ had no effect on Mn2+-induced quenching of Fura 2 (Fig 4B). Stimulating the cells with TRH, immediately followed by addition of 1 mM Mn 2+, rapidly decreased the fluorescence, probably due to influx of Mn 2+ and 863
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
B Mn2+
Ni 2+ Mn2+
Dig
Dig
¢1 O =O
O O
8O [D
C
O U,.
TRH Mn 2+
Dig
TRH
N i2+
ft.
Dig
Mn 2+
H
\ \.
1
Fig. 4. Effect of Mn 2+ on Fura 2 fluorescence in GH4C1 cells. The excitation wavelength was 360 nm, and fluorescence was measured at 510 nm. All experiments contained 300 nM nimodipine. A. Addition of 1 mM Mn 2+ to unstimulated cells, and lysing the cells with 0.05 gg/ml digitonin (Dig). B. Addition of 2 mM Ni 2+ prior to Mn 2÷ and digitonin. C. Stimulating the cells with 100 nM TRH prior to addition of Mn 2+ and lysing the cells with digitonin. D. Addition of Ni 2+ prior to TRH, Mn 2+ and digitonin. The horizontal bar in (A) denotes 1 min.
quenching o f Fura 2 (Fig. 4C). H o w e v e r , if 2 m M Ni 2+ was added to the cell suspension
prior
to stimulation
with
TRH,
and
Mn 2+ was then added, the
quenching o f Fura 2 was reduced (Fig. 4D). Discussion The results in the present report show, that in GH4C1
cells, the T R H - i n d u c e d
transient increase in [Ca2+]i, as well as the T R H - i n d u c e d plateau in [Ca2+]i, is dependent
on
extracellular the
cells
influx
of
Ca 2+.
The
TRH-induced
influx
of
Ca 2+ during the transient in [Ca2+]i is directed to the cytosol of (as
determiner
dihydropyridine-insensitive, agonist-induced several
extracellular
influx
pituitary
corticotrophes,
influx
with
Mn2+-quenching Ni2+-sensitive
of extracellular cell of
types.
In
Ca 2+
has
of
pathway.
Fura The
2
(13)
via
presence
a of
Ca 2+ has been suggested to o c c u r in gonadotrophes, been
864
shown
somatotrophes
to o c c u r in r e s p o n s e
and to
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
stimulation with gonadotrophin-releasing hormone, growth hormonereleasing hormone and corticotropin-releasing hormone, respectively (1416). In these cells, however, the influx of Ca 2+ is mediated primarily via activation of d i h y d r o p y r i d i n e - s e n s i t i v e Ca 2+ channels. The results in the present study showed, that influx of Ca 2+ via dihydropyridine-sensitive Ca 2+ channels is of minor or no importance during the TRH-induced transient influx of Ca 2+ in GH4C1 cells. However, the influx of Ca 2+ during the plateau phase appears to be mediated via activation of both d i h y d r o p y r i d i n e sensitive and -insensitive Ca 2+ channels, in agreement with earlier results
(5,6). Previous studies on TRH-induced influx of Ca 2+ in GH4C1 cells have been contradictory. T R H - i n d u c e d u p t a k e of 4 5 C a 2+ has been detected in enzymatically treated GH4C1 cells (17). However, the uptake of 45Ca2+ was measured after a pretreatment period with TRH, making the results difficult to interpret. We, and others (18), have been unable to show acute TRHinduced uptake of 45Ca2+ in GH-cells. Using the intracellular Ca 2+ indicator Quin 2, it was shown that only a minor part of the TRH-induced transient in [ C a 2 + ] i was due to influx of Ca 2+ (6). Furthermore, part of the transient increase in [Ca2+]i could be blocked by the dihydropyridine Ca 2+ channel blocker nifedipine (6,18). In a recent investigation using both the patchclamp technique and fluorescent measurements of [Ca2+]i, no TRH-induced influx of Ca 2+ could be detected in GH3 cells (19). The reason for the discrepancies in the results is not known. The mechanism for agonist-induced uptake of extracellular Ca 2+ is presently not well understood. In some cells, like neutrophils (20), Ca2+-induced Ca 2+ influx has been suggested to occur. In cardiac cells (21), and chick endothelial cells (22), influx of Ca 2+ has been suggested to occur via activation of a receptor-coupled G protein. Furthermore, both IP3 and IP4 has been proposed to activate influx of Ca 2+ (23,24). TRH-induced influx of Ca 2+ through VOCC is suggested to occur via activation of protein kinase C (6). The m e c h a n i s m for the T R H - i n d u c e d , d i h y d r o p y r i d i n e - i n s e n s i t i v e influx of extracellular Ca 2+ is presently unknown. All the above-mentioned pathways are theoretically possible in GH4C1 cells.
References .
2. 3. 4.
T. M. M. Y.
F. J. Martin, J. Biol. Chem. 258, 14816-14822 (1983). J. Rebecchi, M. C. Gershengorn, Biochem. J. 216, 287-294 (1983). J. Berridge, Proc. R. Soc. Lond. B 234, 359-378 (1988). Nishizuka, Science 233, 305-312 (1986). 865
Vol. 180, No. 2, 1991
5. 6. 7. 8. 9. 10. 1 1. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
P . R . Albert, A. H. Tashjian Jr, J. Biol. Chem. 259, 5827-5832 (1984). P . R . Albert, A. H. Tashjian Jr., J. Biol. Chem. 259, 15350-15363 (1984). J . W . Putney Jr, Cell Calcium 11, 611-624 (1990). K. T6rnquist, Program of the Spring Meeting of the Finnish Pharmacological Society, March 3, Espoo, Finland (1991). A . H . Tashjian Jr., Y. Yasumura, L. Levine, G. H. Sato, M. L. Parker, Endocrinology 82, 342-352 (1968). A . H . Tashjian Jr., Methods Enzymol. 58, 527-535 (1979). K. T6rnquist, A. H. Tashjian Jr., Endocrinology 124, 2765-2766 (1989). G. Gryenkiewicz, M. Poenia, R. Y. Tsien, J. Biol. Chem. 260, 3440-3450 (1985) J . E . Merrit, R. Jacob, T. J. Hallam, J. Biol. Chem. 264, 1522-1527 (1989). J.P. Chang, E. E. McCoy, J. Graeter, K. Tasaka, K. J. Catt, J. Biol. Chem. 261, 9105-9108 (1986). N. Guerineau, J.-B. Corcuff, A. Tabarin, P. Mollard, Endocrinology 129, 409-420 (1991). B . T . Lussier, M. B. French, B. C. Moor, J. Kraicer, Endocrinology 128, 570-582 (1991). K.-N. Tan, A. H. Tashjian Jr, J. Biol. Chem. 256, 8994-9002 (1981). M. C. Gershengorn, C. Thaw, Endocrinology 116, 591-596 (1985). C . D . Benham, J. Physiol. 415, 143-158 (1989). V. von Tscharner, B. Prod'horn, M. Baggiolini, H. Reuter, Nature 324, 369-372 (1986). A. Yatani, et al., Science 248, 1288-1292 (1987). W . W . Chien, R. Mohabir, W. T. Clusin, J. Clin. Invest. 85, 1436-1443 (1990). M. Kuno, P. Gardner, Nature 326, 301-304 (1987). R . F . Irvine, R. M. Moor, Biochem. J. 240, 917-920 (1986).
866
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 860-866
Evidence for TRH-induced influx of extracellular Ca 2+ in pituitary GH4C 1 cells* Kid T6rnquist Endocrine Research Laboratory, University of Helsinki, Minerva Foundation Institute for Medical Research, Helsinki, Finland Received September 4, 1991
The aim of the study was to investigate the relationship between thyrotropin-releasing hormone (TRH)-induced changes in intracellular free Ca 2+ ([Ca2+]i), and influx of extracellular Ca 2+ in Fura 2 loaded pituitary GH4C1 cells. Stimulating the cells with TRH in a Ca2+-containing buffer induced a biphasic change in [Ca2+] i. First, a transient increase in [Ca2+]i, followed by a sustained phase. In cells stimulated with TRH in a Ca2+-free buffer, the transient increase in [Ca2+]i was decreased (p < 0.05), and the sustained phase was totally abolished. Addition of Ni 2+ prior to TRH blunted the component of the TRH-induced transient increase in [Ca2+]i dependent on influx of Ca 2+. In the presence of extracellular Mn 2+, TRH stimulated quenching of Fura 2 fluorescence. This quenching was blocked by Ni 2+. The results indicate that both the TRH-induced transient increase in [Ca2+]i as well as the sustained phase in [Ca2+]i in GH4C1 cells is dependent on influx of extracellular Ca2+. ®1991 Academic Press, Inc.
The stimulus-secretion coupling induced by binding of thyrotropin-releasing hormone (TRH) to its receptor in pituitary cells is mediated via activation of phospholipase C. This leads to hydrolysis of phosphatidylinositol-4,5bisphosphate (Ptdlns) to inositol-l,4,5-trisphosphate (IP3) and diacylglycerol (DG) (1,2). IP3 stimulates the release of sequestered intracellular Ca 2+ (3), and DG activates protein kinase C (4). Stimulating pituitary cells with TRH induces a biphasic change in intracellular Ca2+ ([Ca2+]i): first a rapid transient increase in [Ca2+]i, followed by a sustained plateau phase (5). The transient increase in [Ca2+]i has generally been considered to be due to the IP3* Supported in part by a grant from the Ella Foundation. o0o6-291x/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
860
and Georg Ehrnrooth
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
induced release of sequestered Ca 2+, while the plateau phase in [Ca2+]i is dependent on influx of extracellular Ca 2+ (5,6). In some cell types, however, agonist-induced release of sequestered Ca 2+ has been linked to a substantial influx of extracellular Ca 2+ (see (7). In the present study in GH4C1 cells we show, that both the TRH-induced transient increase in [Ca2+]i, as well as the plateau phase in [Ca2+]i is dependent on influx of extracellular Ca 2+. A preliminary report of these observations has been presented (8). Materials and M e t h o d s Materials Fura 2-AM was obtained from Molecular Probes (Eugene, OR). TRH was from Sigma (St Louis, MO). Nimodipine was a kind gift from Dr. Seuter (Bayer AG, Germany) All flasks and dishes used for the cell culture were from N U N C Plastics (Kamstrup, Denmark). Cell culture Clonal rat pituitary GH4C1 cells were grown in monolayer culture in Ham's F 10 Nutrient Mixture with 15 % (vol/vol) horse serum and 2.5 % fetal bovine serum in a water-saturated atmosphere of 5 % CO2 and 95 % air at 37 oC, as described previously (9,10). Before an experiment, the cells from a single donor culture were harvested with 0 . 1 % trypsin and subcultured in 100 mm culture dishes for 7-9 days. The cells were fed every 2-3 days, and always the day before an experiment. Measuring [Ca2+li The method for determining [Ca2+]i with Fura 2 in GH4C1 cells has been described (11). In brief, the cells were harvested in Hepes-buffered salt solution (HBSS, containing in millimolar concentrations: NaC1, 118; KCI, 4.6; glucose, 10; CaCI2, 0.4; HEPES, 20.0; pH 7.2) lacking CaC12, and containing 0.02 % EDTA. The cells were then washed twice in HBBS (containing Ca2C1), and incubated for 35 min at 37 °C with 1 IxM Fura 2-AM. Fluorescence was measured in a Hitachi F2000 spectrophotometer (Hitachi, Ltd, Tokyo, Japan), using the excitation wavelengths 340 and 380 nm, and the emission wavelength 510 nm. [Ca2+]i was calculated according to (12) with Kd = 224 nM for Fura-2, using a computer program designed for the fluorometer. Statistics Results are given as the mean + SE, and each experiment was repeated at least five times using at least three different cell preparations. Statistical analyses were made using Student's t-test for comparison b e t w e e n two means. Three or more means were tested using analysis of variance. Results TRH-induced changes in [Ca2+li in GH4C1 cells. Stimulating GH4C1 cells with TRH induced a biphasic change in [Ca2+]i • first a transient increase in [Ca2+]i 861
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
1000 TRH
+
c
Ni 2+ TRH
TRH
500
li,.\ Fig. !. Effect of TRH on [Ca2+]i in GH4C1 cells. The cells were stimulated with 100 nM TRH in: A. Ca2+-containing buffer. B. Nominally Ca2+-free buffer containing 100 I.tM EGTA. C. After addition of 2 mM Ni 2+ to cells in Ca2+ buffer. The horizontal bar in (A) denotes 1 min.
(513 + 24
nM; mean + SE; Fig. 1A), followed by a plateau level (135 + 8
nM). If the cells were suspended in a nominally Ca2+-free buffer containing 100 ~tM EGTA, the transient increase in [Ca2+]i was 222 + 33 nM (p < 0.05, compared with control cells), and the plateau phase was totally abolished
(Fig. 1B). Fig. 1C shows, that addition of 2 mM Ni 2+ to GH4C1 cells in suspension reduced basal [Ca2+]i levels from 116 + 8 nM to 77 + 7 nM (p < 0.05), probably by blocking influx of extracellular Ca 2+ via spontaneously active, dihydropyridine sensitive voltage-operated Ca 2+ c h a n n e l s (VOCC). Stimulating the cells with TRH induces a transient increase in [Ca2+]i (330 ± 22 nM), which was significantly smaller (p < 0.05) than that observed in control cells (Table 1). The results in Fig. 2 show that the inhibiting effect of Ni 2+ on the TRH-induced transient in [Ca2+]i was dose-dependent. The
55ot 1
t
250 -~
ol
0.1
1
10
mM Ni2+
Fig. 2. Inhibitory effect of Ni2+ on TRH-induced transient increase in [Ca2+]i in GH4C1 cells. Ni 2+ was added to the cell suspension 1 min prior to stimulating the cells with 100 nM TRH. Each point gives the mean + SE of 5-9 separate experiments. 862
VoI. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
600-
500C
;~ 4-
40O3o0-
2001000
. . . . . . . .
0
i
1
. . . . . . . .
i
10
. . . . . . . .
i
100
. . . . . . . .
1000
nM TRH
Fig. 3. Dose-dependent effect of TRH on the transient increase in [Ca2+]i in GH4C1 cells. The cells were stimulated with the appropriate concentration of TRH in Ca2+ containing buffer ([]), in nominally Ca2+-free buffer containing 100 ~tM EGTA (11), or in Ca2+ buffer containing 2 mM Ni 2+ (O). Each point gives the mean + SE of 5-9 separate experiments. Cells incubated in a nominally Ca2+-free buffer always gave a significantly (p < 0.05) smaller increase in [Ca2+]i compared with cells in Ca2+ buffer. *) p < 0.05 compared with cells stimulated in the presence of Ni 2+. inhibitory effect of Ni 2+ was not the result of blockade of VOCC, as the TRHinduced transient in [Ca2+]i was unaltered in cells incubated with 300 nM the dihydropyridine Ca 2+ channel blocker nimodipine (497 + 56 nM). The plateau phase was, however, significantly decreased (73 + 2 nM, p < 0.05) in the presence of nimodipine. In GH4C1 cells depolarized with 50 mM K + in the presence of nimodipine, the TRH-induced transient increase in [Ca2+]i was not changed (427 + 51 nM), but the plateau phase was significantly decreased (49 + 3 nM, p < 0.05). The transient change in [Ca2+]i in response to increasing concentrations of TRH is shown in Fig. 3. The response obtained in a Ca2+-free buffer was always significantly (p < 0.05) lower compared with that in control cells in Ca2+-containing buffer. In the presence of 2 mM Ni 2+, the T R H - i n d u c e d transient was significantly lower (p < 0.05) than that observed in control cells at the two highest doses of TRH tested. The plateau phase was totally inhibited by Ni 2+ at all the TRH-doses tested (not shown). TRH-stimulated Mn 2+ entry in GH4C1 cells. The results in Fig. 4A show that addition of Mn 2+ to cells in buffer containing 300 nM nimodipine (to prevent influx of Mn2+ via VOCC) produced an immediate drop in fluorescence, suggesting that unstimulated cells are permeable to Mn 2+. Addition of Ni 2+ prior to Mn 2+ had no effect on Mn2+-induced quenching of Fura 2 (Fig 4B). Stimulating the cells with TRH, immediately followed by addition of 1 mM Mn 2+, rapidly decreased the fluorescence, probably due to influx of Mn 2+ and 863
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
B Mn2+
Ni 2+ Mn2+
Dig
Dig
¢1 O =O
O O
8O [D
C
O U,.
TRH Mn 2+
Dig
TRH
N i2+
ft.
Dig
Mn 2+
H
\ \.
1
Fig. 4. Effect of Mn 2+ on Fura 2 fluorescence in GH4C1 cells. The excitation wavelength was 360 nm, and fluorescence was measured at 510 nm. All experiments contained 300 nM nimodipine. A. Addition of 1 mM Mn 2+ to unstimulated cells, and lysing the cells with 0.05 gg/ml digitonin (Dig). B. Addition of 2 mM Ni 2+ prior to Mn 2÷ and digitonin. C. Stimulating the cells with 100 nM TRH prior to addition of Mn 2+ and lysing the cells with digitonin. D. Addition of Ni 2+ prior to TRH, Mn 2+ and digitonin. The horizontal bar in (A) denotes 1 min.
quenching o f Fura 2 (Fig. 4C). H o w e v e r , if 2 m M Ni 2+ was added to the cell suspension
prior
to stimulation
with
TRH,
and
Mn 2+ was then added, the
quenching o f Fura 2 was reduced (Fig. 4D). Discussion The results in the present report show, that in GH4C1
cells, the T R H - i n d u c e d
transient increase in [Ca2+]i, as well as the T R H - i n d u c e d plateau in [Ca2+]i, is dependent
on
extracellular the
cells
influx
of
Ca 2+.
The
TRH-induced
influx
of
Ca 2+ during the transient in [Ca2+]i is directed to the cytosol of (as
determiner
dihydropyridine-insensitive, agonist-induced several
extracellular
influx
pituitary
corticotrophes,
influx
with
Mn2+-quenching Ni2+-sensitive
of extracellular cell of
types.
In
Ca 2+
has
of
pathway.
Fura The
2
(13)
via
presence
a of
Ca 2+ has been suggested to o c c u r in gonadotrophes, been
864
shown
somatotrophes
to o c c u r in r e s p o n s e
and to
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
stimulation with gonadotrophin-releasing hormone, growth hormonereleasing hormone and corticotropin-releasing hormone, respectively (1416). In these cells, however, the influx of Ca 2+ is mediated primarily via activation of d i h y d r o p y r i d i n e - s e n s i t i v e Ca 2+ channels. The results in the present study showed, that influx of Ca 2+ via dihydropyridine-sensitive Ca 2+ channels is of minor or no importance during the TRH-induced transient influx of Ca 2+ in GH4C1 cells. However, the influx of Ca 2+ during the plateau phase appears to be mediated via activation of both d i h y d r o p y r i d i n e sensitive and -insensitive Ca 2+ channels, in agreement with earlier results
(5,6). Previous studies on TRH-induced influx of Ca 2+ in GH4C1 cells have been contradictory. T R H - i n d u c e d u p t a k e of 4 5 C a 2+ has been detected in enzymatically treated GH4C1 cells (17). However, the uptake of 45Ca2+ was measured after a pretreatment period with TRH, making the results difficult to interpret. We, and others (18), have been unable to show acute TRHinduced uptake of 45Ca2+ in GH-cells. Using the intracellular Ca 2+ indicator Quin 2, it was shown that only a minor part of the TRH-induced transient in [ C a 2 + ] i was due to influx of Ca 2+ (6). Furthermore, part of the transient increase in [Ca2+]i could be blocked by the dihydropyridine Ca 2+ channel blocker nifedipine (6,18). In a recent investigation using both the patchclamp technique and fluorescent measurements of [Ca2+]i, no TRH-induced influx of Ca 2+ could be detected in GH3 cells (19). The reason for the discrepancies in the results is not known. The mechanism for agonist-induced uptake of extracellular Ca 2+ is presently not well understood. In some cells, like neutrophils (20), Ca2+-induced Ca 2+ influx has been suggested to occur. In cardiac cells (21), and chick endothelial cells (22), influx of Ca 2+ has been suggested to occur via activation of a receptor-coupled G protein. Furthermore, both IP3 and IP4 has been proposed to activate influx of Ca 2+ (23,24). TRH-induced influx of Ca 2+ through VOCC is suggested to occur via activation of protein kinase C (6). The m e c h a n i s m for the T R H - i n d u c e d , d i h y d r o p y r i d i n e - i n s e n s i t i v e influx of extracellular Ca 2+ is presently unknown. All the above-mentioned pathways are theoretically possible in GH4C1 cells.
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