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Calcium requirement for stimulation of cyclic AMP accumulation in anterior pituitary gland by LH-RH
Molecular and Cellular Endocrinology
2 (1975) 117-124. 0 North-Holland
Publ. Comp.
CALCIUM REQUIREMENT FOR STIMULATION OF CYCLIC AMP ACCUMULATION IN ANTERIOR PITUITARY GLAND BY LH-RH P. BORGEAT*,
P. GARNEAU
and F. LABRIE*
Medical Research Council Group in Molecular Endocrinology, Centre Hospitalier de I’ UniversitL Lava& Quebec, Quebec GI V 4G2, Canada
Received 16 August 1974
Accepted 18 October 1974
Removal of Ca*+ from the incubation medium by addition of 2 mM ethylene glycol bis-(b-aminoethyl ether)-N,N’tetraacetic acid (EGTA) leads to at least 75% inhibition of the luteinizing hormone-releasing hormone (LH-RH)-induced accumulation of adenosine 3’5’monophosphate (cyclic AMP) in rat anterior pituitary gland in vitro. This inhibitory effect of EGTA is reversed by the addition of Ca’ +. A half-maximal effect of Ca* + on LH-RHinduced cyclic AMP accumulation is observed at 2-5 x 1O-5 M free Ca’+. The LH-RHinduced LH and FSH release is completely dependent upon the presence of CaZ+ in the incubation medium, a half-maximal effect being measured at 1-2 x 10e4 M free CaZ+. The basal release of LH is increased upon CaZ+ removal. Keywords: pituitary;
CAMP; releasing hormones;
secretion.
It has been recently demonstrated that synthetic LH-RH (luteinizing hormone-releasing hormone) stimulates cyclic AMP (adenosine 3’5’-monophosphate) accumulation in rat anterior pituitary gland in vitro (Borgeat et al., 1972; Kaneko et al., 1973; Labrie et al., 1973; Makino, 1973; Borgeat et al., 1974). Since calcium (Caz +) was known to be required for the hypothalamic extract-induced release of luteinizing hormone (LH) (Samli and Geschwind, 1968; Wakabayashi et al., 1969), and follicle-stimulating hormone (FSH) (Wakabayashi et al., 1969; Jutisz and Paloma de la Llosa, 1970), it was felt important to use the synthetic neurohormone to study a possible requirement of Ca2+ at a step preceding activation of adenylate cyclase. Such an early site of action in the LH and FSH secretory cells would then be added to the already suspected late site of Ca2 + requirement observed during high K +-induced
* P. B. and F. L. are respectively Fellow and Associate of the Medical Research Council of Canada.
P. Borgeat et al.
118
release of LH and FSH (Samli and Geschwind, 1968; Wakabayashi et al., 1969; Jutisz and Paloma de la Llosa, 1970). The interest of such a study was strengthened by the observations that Ca2 + is required for hormonal activation of adenylate cyclase in many systems. These studies pertain to the stimulatory action of ACTH (adrenocorticotropic hormone) in fat cells (Bar and Hoechter, 1969), adrenal cells (Sayers et al., 1972) and adrenal cell membrane particles (Lefkowitz et al., 1970). Calcium deprivation was also found to decrease cyclic AMP accumulation in response to thyrotropin (TSH) and LH (Kuo, 1970), in isolated adipose cells and to P-lipotropic hormone in fat cells (Lis et al., 1972). The present data show that Ca 2 + is absolutely required for the stimulation of cyclic AMP accumulation and LH and FSH release by LH-RH in rat anterior pituitary gland in vitro.
MATERIALS
AND
METHODS
Hemipituitaries from male Sprague-Dawley rats (225-250 g) were used throughout these studies. Each hemipituitary from the same animal was used respectively as control and experimental. Unless mentioned, three pituitary halves were used in each group. Incubations were performed at 37 “C in Krebs-Ringer bicarbonate buffer containing 11 mM D-glucose (KRBG) as described (Borgeat et al., 1972). When Ca2 +-free KRBG was used, CaCl, was replaced by equivalent amounts of NaCl. The incubation medium was changed at the end of each incubation period. Different concentrations of free Ca2 + in the incubation medium were obtained using 2 mM EGTA (ethylene glycol bis-(P-aminoethyl ether)-N,N’tetraacetic acid) and concentrations of CaCl, calculated according to a dissociation constant of 3.95 x low6 M for the Ca2 +-EGTA interaction (De Meis and Hasselback, 1971). Cyclic AMP was measured by the receptor binding assay of Gilman (1970). LH and FSH release was determined by double-antibody radioimmunoassay (Midgley, 1967; Ode11 et al., 1967) using rat hormones (NIAMD Rat LH-1-3, NIAMD Rat FSH-I-1, NIAMD Rat LH-RP-1 and NIAMD Rat FSH-RP-I) and rabbit antisera (NIAMD Anti-Rat LH Serum 1 and NIAMD Anti-Rat FSH Serum 6) kindly provided by Dr. A. F. Parlow for the National Institute of Arthritis and Metabolic Diseases, Rat Pituitary Hormone Program. Radioimmunoassay data were processed using a desk-top Hewlett-Packard calculator model 9830 essentially as described by Rodbard and Lewald (1970). Results of cyclic AMP determinations are expressed as percent of control (& SD)
Calcium, LH-RH
and cyclic AMP
119
while data of LH and FSH assays are presented determinations.
as mean
& SEM of triplicate
Synthetic LH-RH (AY-24, 031-4) was kindly supplied by Dr. Manfred G&z, Ayerst Research Laboratories, Montreal. [3H] cyclic AMP (20-30 Ci/mmole) and carrier-free l 251 were obtained from New England Nuclear. Goat anti-rabbit y-globulin serum was a product of Endocrinolab, Quebec.
Table 1 Effect of EGTA and Caz + on basal and LH-RH-stimulated levels of cyclic AMP in male rat anterior pituitary gland. Adenohypophyseal tissue was first incubated for 60 min before four further incubations of 1, 1, “I4 and “I4 h carried out in the presence of the indicated compounds. Cyclic AMP content was measured at the end of the last incubation. Additions Control
Experimental
Cyclic AMP % of control k SD
Number of experiments
KRBG
CaZ +-free KRBG + 2 mM EGTA
80 * 10
2
KRBG
Ca’ +-free KRBG
I_ 2 mM EGTA + 7 mM Ca2 +
99 + 21
2
KRBG
KRBG + 2 x 1O-7 M LH-RH
350 h 65
4
KRBG 2 x lo-’ M LH-RH
Ca’+-free KRBG + 2 x 10e7 M LH-RH + 2 mM EGTA
49 i
16
4
KRBG 2 x 1O-7 M LH-RH
CaZ+-free KRBG + 2 x 1O-7 M LH-RH + 2 mM EGTA + 7mMCaZ+
114 *
7
3
_
RESULTS As shown in table 1, removal of Ca ’ + from the incubation medium by the addition of 2 mM EGTA (a chelating agent with a low affinity for MgZ + and a high affinity for Ca2 ‘) leads to an inhibition of the LH-RH-induced accumulation of cyclic AMP in anterior pituitary tissue. This effect of EGTA was completely reversed by the addition of calcium, thus suggesting that the inhibition by EGTA of LH-RH-induced stimulation of cyclic AMP accumulation was related to the removal of Ca z + . Under our experimental conditions,
120
P. Bovgeat
et al.
the presence of the chelating agent has no significant effect on basal cyclic AMP levels while excess CaZ + (7 mM) does not influence the basal or LH-RHinduced accumulation of adenohypophyseal cyclic AMP. Fig. 1 illustrates the results of a more detailed study of the effect of free Ca2 + on LH-RH-induced cyclic AMP accumulation. In this experiment, increasing amounts of CaCl, were added to the incubation medium containing in all groups, 2 mM EGTA, 10e7 M LH-RH and 10 mM theophylline. At free Ca2 + concentrations below 5 x lOA M, the stimulatory effect of LH-RH on cyclic AMP accumulation is completely blocked while a progressive increase is observed between 10e5 and lob4 M. A maximal stimulatory effect of LH-RH (2-fold) is found at lo-3 M free Ca2 +. A half-maximal effect of Ca2 + on LH-RH-induced cyclic AMP accumulation is measured at 2 to 5 x 1O-5 M free Ca2 +. As shown in fig. 2A and B, the LH-RH-induced release of LH and FSH is also completely dependent upon the presence of free Ca2 + in the incubation medium; a half-maximal effect of Ca ’ + being observed between 1 to 2 x
L+
I
10-S
I
I
I
10-5 10-4 10-X FREE Co++[ M)
10-Z
Fig. 1. Effect of increasing concentrations of free Ca2+ on LH-RH-induced cyclic AMP accumulation in male rat anterior pituitary gland. Adenohypophyseal tissue was first incubated for 60 min before four successive incubations of 1, 1, “I4 and “I4 h in the presence or absence of the indicated concentrations of free Ca’ + 10m7M LH-RH, lo-’ M theophylline and 2 x 1O-3 M EGTA were present in all groups.
Calcium, LH-RH
B
and cyclic AMP
121
LH-RH + ECTA+Ca++ 7
(b)
____L_
____L_--A
10-4
10~5 FREE
Ca++
m-3
IM)
Fig. 2. Effect of increasing concentrations of Ca2+ on LH-RH-induced release of LH (a) and FSH (b) from male rat anterior pituitary gland. Adenohypophyseal tissue was first incubated for 45 min in 1 ml of normal KRBG before a 35-min incubation in a Ca2+-free medium containing 2 x 10-j M EGTA. Three successive incubations of 3/4, 21/2 and 3/4 h were then carried out in the presence or absence of the indicated concentrations of Caz+. 10e7 M LH-RH, and 2 x 1O-3 M EGTA were present in all groups. The amounts of LH and FSH released during the second incubation period (21/2 h) are presented.
10e4 M and a maximal response being reached at 5 to 8 x 1O-4 M free Ca2 +. Although the magnitude of the LH and FSH responses is different, the LH-RHinduced release of the two pituitary hormones is obtained at the same free Ca’ + concentrations. Since, as observed previously (Borgeat et al., 1972, 1974), the effect of LH-RH is minimal in the first 45 min of incubation, the present data were obtained in the second incubation period ranging from the 45th to 195th min after addition of LH-RH. Qualitatively similar results were obtained in the third incubation period (data not shown). Although the LH-RH-induced release of LH required the presence of Ca* +, an inverse effect of Ca2 + is observed on the basal release of the hormone. As shown in fig. 3, complete removal of Ca2 + by addition of 1 mM EGTA to a Ca2 +-free medium leads to a I- to 2-fold increase of the LH secretion rate. The in vitro stimulation of LH secretion by the absence of Ca2 + is maximal (100 to 200% over control) during the first 2 hours of incubation and it dedreases (50% over control) at later time intervals (fig. 3).
P. Borgeat et al.
122
MINUTES
Fig. 3. release. periods periods
OF
o
EGTA-CA++
0
EGTA
+ CA+’
INCUBATION
Effect of Ca2 + removal from the incubation medium on spontaneous in vitro LH Adenohypophyseal tissue (5 pituitary halves per group) was first incubated for 2 of 30 mia in normal KRBG before incubation for 10 successive 30-min incubation in Caz+-free KRBG medium containing 1 mM EGTA in the presence or absence of 3.5 x 10e3 M CaCI,.
DISCUSSION As shown in table 1, the addition of 2 mM EGTA to rat hemipituitaries incubated in Ca2 + -free KRBG buffer does not affect the basal intracellular cyclic AMP concentration. However, in order to minimize possible effects of EGTA other than those secondary to removal of calcium ions, the effect of increasing concentrations of Ca2 + was studied by addition of the required amounts of CaCl, to a fixed concentration of EGTA (2 mM) present in control and experimental groups. The amount of CaClz necessary to achieve the desired concentrations of free Ca2 + was calculated using a dissociation constant of 3.95 x 10eh M for the Ca 2 +-EGTA interaction (De Meis and Hasselback, 1971). As noticed for the spontaneous release of LH (Samli and Geschwind, 1968), adrenocorticotropin (Zimmerman and Fleischer, 1970), growth hormone and prolactin (A. Lemay and F. Labrie, unpublished data), the N6, 2’-0-dibutyryl cyclic AMP-induced release of growth hormone and prolactin (A. Lemay and F. Labrie, unpublished data) and the hypothalamic extract-induced release of
Calcium, LH-RH
and cyclic AMP
123
LH (Samli and Geschwind, 1968) and FSH (Jutisz and Paloma de la Llosa, medium has 1970), we have found that removal of Ca 2 + from the incubation no effect on the stimulation of adenohypophyseal cyclic AMP accumulation by LH-RH (data not shown). Since removal of Ca2 + by addition of EGTA is required for the inhibitory effect, it is likely that repeated washings of the tissue leave enough Ca 2 + bound intra- or extracellularly to permit stimulation of cyclic AMP accumulation by LH-RH. LH As shown in fig. 3, not only Ca2 +- is not required for spontaneous release but an appreciably higher (100 to 200 ‘A over control) rate of hormonal release is observed in the absence of Ca 2 +. It should however be mentioned that when 2 mM EGTA is added to Ca ’ +-free KRBG buffer, the stimulatory effect of the absence of Ca2 + on LH release appears to be restricted to this hormone. In fact, no similar effect has been observed on the release of the other pituitary hormones (Samli and Geschwind, 1968; Borgeat and Labrie, unpublished data). Since the gonadotrophs account for only 5 to 8 % of the total cell population in rat anterior pituitary gland, it is possible that complete Ca’ + removal is accompanied by increased levels of cyclic AMP in the cells secreting LH but that the effect is masked by unchanged or decreased cyclic AMP concentrations in the other pituitary cells. It should also be mentioned that the stringent Ca2 + requirements for stimulation of cyclic AMP accumulation by LH-RH are not found with purified growth hormone-releasing hormone (Borgeat and Labrie, unpublished data) or prostaglandin Ez (Cooper et al., 1972; Borgeat and Labrie, unpublished data). In fact, in the absence of Ca 2 +, these two agents lead to an important stimulation of cyclic AMP accumulation while the release of growth hormone is blocked. The present data show that Ca2 +- is required at a step preceding stimulation of cyclic AMP accumulation by LH-RH. The site of Ca2 + requirement could be on the LH-RH receptor, on the adenylate cyclase, on cyclic nucleotide phosphodiesterase, at some intermediate step between binding of LH-RH and activation of adenylate cyclase or at a combination of these sites. As the binding of another releasing hormone, thyrotropin-releasing hormone (Labrie et al., 1972) is not increased by Ca2 f and 1 mM EGTA does not affect fluoridestimulated adenohypophyseal adenylate cyclase activity (Poirier and Labrie, unpublished), it appears more likely that Ca ’ + is required at some step(s) between binding of LH-RH and activation of adenylate cyclase, although an action at the other sites remains possible.
124
P. Borgeat
et al.
REFERENCES Bar, H. P. and Hechter, 0. (1969) Biochem. Biophys. Res. Commun. 35, 681. Borgeat, P., Chavancy, G., DuPont, A., Labrie, F., Arimura, A. and Schally, A. V. (1972) Proc. Natl. Acad. Sci. U.S. 69, 2677. Borgeat, P., Labrie, F., Cot& J., Ruel, F., Schally, A. V., Coy,D. H., Coy, E. .I. and Yanaihara, N. (1974) J. Mol. Cell. Endocrinol. 1, 7. Cooper, R. H., McPherson, M. and Schofield, .I. G. (1972) Biochem. J. 127, 143. De Meis, L. and Hasselback, W. (1971) J. Biol. Chem. 246, 4759. Gilman, A. G. (1970) Proc. Nat]. Acad. Sci. U.S. 67, 305. Jutisz, M. and Paloma de la Llosa, M. (1970) Endocrinology 86, 761. Kaneko, T., Saito, S., Oka, H., Oka, T. and Yanaihara, N. (1973) Metabolism 22, 77. Kuo, J. F. (1970) Biochim. Biophys. Acta 208, 509. Labrie, F., Barden, N., Poirier, G. and De Lean, A. (1972) Proc. Natl. Acad. Sci. U.S. 69, 283. Labrie, F., Pelletier, G., Lemay, A., Borgeat, P., Barden, N., DuPont, A., Savary, M., C&e, J. and Boucher, R. (1973) In: 6th Karolinska Symposium on Res. Meth. Reproductive Endocrinol., Ed.: E. Diczfalusy, p. 301. Lefkowitz, R. J., Roth, J. and Pastan, I. (1970) Nature 228, 864. Lis, M., Gilardeau, C. and Chretien, M. (1972) Proc. Sot. Exptl. Biol. Med. 139, 680. Makino, T. (1973) Amer. J. Obst. Gynec. 115, 606. Midgley, A. R., Jr. (1967) J. Clin. Endocrinol. Metab. 27, 295. Odell, W. D., Rayford, P. L. and Ross, G. T. (1967) J. Lab. Clin. Med. 70, 973. Rodbard, D. and Lewald, J. E. (1970) In: 2nd Karolinska Symposium on Res. Meth. Reproductive Endocrinol., Ed.: E. Diczfalusy, p. 79. Samli, M. H. and Geschwind, I. 1. (1968) Endocrinology 82, 225. Sayers, G., Beall, R. J. and Seeling, S. (1972) Science 175, 1113. Wakabayashi, K., Kamberi, I. A. and McCann, S. M. (1969) Endocrinology 85, 1046. Zimmerman, G. and Fleischer, N. (1970) Endocrinology 87, 426.
2 (1975) 117-124. 0 North-Holland
Publ. Comp.
CALCIUM REQUIREMENT FOR STIMULATION OF CYCLIC AMP ACCUMULATION IN ANTERIOR PITUITARY GLAND BY LH-RH P. BORGEAT*,
P. GARNEAU
and F. LABRIE*
Medical Research Council Group in Molecular Endocrinology, Centre Hospitalier de I’ UniversitL Lava& Quebec, Quebec GI V 4G2, Canada
Received 16 August 1974
Accepted 18 October 1974
Removal of Ca*+ from the incubation medium by addition of 2 mM ethylene glycol bis-(b-aminoethyl ether)-N,N’tetraacetic acid (EGTA) leads to at least 75% inhibition of the luteinizing hormone-releasing hormone (LH-RH)-induced accumulation of adenosine 3’5’monophosphate (cyclic AMP) in rat anterior pituitary gland in vitro. This inhibitory effect of EGTA is reversed by the addition of Ca’ +. A half-maximal effect of Ca* + on LH-RHinduced cyclic AMP accumulation is observed at 2-5 x 1O-5 M free Ca’+. The LH-RHinduced LH and FSH release is completely dependent upon the presence of CaZ+ in the incubation medium, a half-maximal effect being measured at 1-2 x 10e4 M free CaZ+. The basal release of LH is increased upon CaZ+ removal. Keywords: pituitary;
CAMP; releasing hormones;
secretion.
It has been recently demonstrated that synthetic LH-RH (luteinizing hormone-releasing hormone) stimulates cyclic AMP (adenosine 3’5’-monophosphate) accumulation in rat anterior pituitary gland in vitro (Borgeat et al., 1972; Kaneko et al., 1973; Labrie et al., 1973; Makino, 1973; Borgeat et al., 1974). Since calcium (Caz +) was known to be required for the hypothalamic extract-induced release of luteinizing hormone (LH) (Samli and Geschwind, 1968; Wakabayashi et al., 1969), and follicle-stimulating hormone (FSH) (Wakabayashi et al., 1969; Jutisz and Paloma de la Llosa, 1970), it was felt important to use the synthetic neurohormone to study a possible requirement of Ca2+ at a step preceding activation of adenylate cyclase. Such an early site of action in the LH and FSH secretory cells would then be added to the already suspected late site of Ca2 + requirement observed during high K +-induced
* P. B. and F. L. are respectively Fellow and Associate of the Medical Research Council of Canada.
P. Borgeat et al.
118
release of LH and FSH (Samli and Geschwind, 1968; Wakabayashi et al., 1969; Jutisz and Paloma de la Llosa, 1970). The interest of such a study was strengthened by the observations that Ca2 + is required for hormonal activation of adenylate cyclase in many systems. These studies pertain to the stimulatory action of ACTH (adrenocorticotropic hormone) in fat cells (Bar and Hoechter, 1969), adrenal cells (Sayers et al., 1972) and adrenal cell membrane particles (Lefkowitz et al., 1970). Calcium deprivation was also found to decrease cyclic AMP accumulation in response to thyrotropin (TSH) and LH (Kuo, 1970), in isolated adipose cells and to P-lipotropic hormone in fat cells (Lis et al., 1972). The present data show that Ca 2 + is absolutely required for the stimulation of cyclic AMP accumulation and LH and FSH release by LH-RH in rat anterior pituitary gland in vitro.
MATERIALS
AND
METHODS
Hemipituitaries from male Sprague-Dawley rats (225-250 g) were used throughout these studies. Each hemipituitary from the same animal was used respectively as control and experimental. Unless mentioned, three pituitary halves were used in each group. Incubations were performed at 37 “C in Krebs-Ringer bicarbonate buffer containing 11 mM D-glucose (KRBG) as described (Borgeat et al., 1972). When Ca2 +-free KRBG was used, CaCl, was replaced by equivalent amounts of NaCl. The incubation medium was changed at the end of each incubation period. Different concentrations of free Ca2 + in the incubation medium were obtained using 2 mM EGTA (ethylene glycol bis-(P-aminoethyl ether)-N,N’tetraacetic acid) and concentrations of CaCl, calculated according to a dissociation constant of 3.95 x low6 M for the Ca2 +-EGTA interaction (De Meis and Hasselback, 1971). Cyclic AMP was measured by the receptor binding assay of Gilman (1970). LH and FSH release was determined by double-antibody radioimmunoassay (Midgley, 1967; Ode11 et al., 1967) using rat hormones (NIAMD Rat LH-1-3, NIAMD Rat FSH-I-1, NIAMD Rat LH-RP-1 and NIAMD Rat FSH-RP-I) and rabbit antisera (NIAMD Anti-Rat LH Serum 1 and NIAMD Anti-Rat FSH Serum 6) kindly provided by Dr. A. F. Parlow for the National Institute of Arthritis and Metabolic Diseases, Rat Pituitary Hormone Program. Radioimmunoassay data were processed using a desk-top Hewlett-Packard calculator model 9830 essentially as described by Rodbard and Lewald (1970). Results of cyclic AMP determinations are expressed as percent of control (& SD)
Calcium, LH-RH
and cyclic AMP
119
while data of LH and FSH assays are presented determinations.
as mean
& SEM of triplicate
Synthetic LH-RH (AY-24, 031-4) was kindly supplied by Dr. Manfred G&z, Ayerst Research Laboratories, Montreal. [3H] cyclic AMP (20-30 Ci/mmole) and carrier-free l 251 were obtained from New England Nuclear. Goat anti-rabbit y-globulin serum was a product of Endocrinolab, Quebec.
Table 1 Effect of EGTA and Caz + on basal and LH-RH-stimulated levels of cyclic AMP in male rat anterior pituitary gland. Adenohypophyseal tissue was first incubated for 60 min before four further incubations of 1, 1, “I4 and “I4 h carried out in the presence of the indicated compounds. Cyclic AMP content was measured at the end of the last incubation. Additions Control
Experimental
Cyclic AMP % of control k SD
Number of experiments
KRBG
CaZ +-free KRBG + 2 mM EGTA
80 * 10
2
KRBG
Ca’ +-free KRBG
I_ 2 mM EGTA + 7 mM Ca2 +
99 + 21
2
KRBG
KRBG + 2 x 1O-7 M LH-RH
350 h 65
4
KRBG 2 x lo-’ M LH-RH
Ca’+-free KRBG + 2 x 10e7 M LH-RH + 2 mM EGTA
49 i
16
4
KRBG 2 x 1O-7 M LH-RH
CaZ+-free KRBG + 2 x 1O-7 M LH-RH + 2 mM EGTA + 7mMCaZ+
114 *
7
3
_
RESULTS As shown in table 1, removal of Ca ’ + from the incubation medium by the addition of 2 mM EGTA (a chelating agent with a low affinity for MgZ + and a high affinity for Ca2 ‘) leads to an inhibition of the LH-RH-induced accumulation of cyclic AMP in anterior pituitary tissue. This effect of EGTA was completely reversed by the addition of calcium, thus suggesting that the inhibition by EGTA of LH-RH-induced stimulation of cyclic AMP accumulation was related to the removal of Ca z + . Under our experimental conditions,
120
P. Bovgeat
et al.
the presence of the chelating agent has no significant effect on basal cyclic AMP levels while excess CaZ + (7 mM) does not influence the basal or LH-RHinduced accumulation of adenohypophyseal cyclic AMP. Fig. 1 illustrates the results of a more detailed study of the effect of free Ca2 + on LH-RH-induced cyclic AMP accumulation. In this experiment, increasing amounts of CaCl, were added to the incubation medium containing in all groups, 2 mM EGTA, 10e7 M LH-RH and 10 mM theophylline. At free Ca2 + concentrations below 5 x lOA M, the stimulatory effect of LH-RH on cyclic AMP accumulation is completely blocked while a progressive increase is observed between 10e5 and lob4 M. A maximal stimulatory effect of LH-RH (2-fold) is found at lo-3 M free Ca2 +. A half-maximal effect of Ca2 + on LH-RH-induced cyclic AMP accumulation is measured at 2 to 5 x 1O-5 M free Ca2 +. As shown in fig. 2A and B, the LH-RH-induced release of LH and FSH is also completely dependent upon the presence of free Ca2 + in the incubation medium; a half-maximal effect of Ca ’ + being observed between 1 to 2 x
L+
I
10-S
I
I
I
10-5 10-4 10-X FREE Co++[ M)
10-Z
Fig. 1. Effect of increasing concentrations of free Ca2+ on LH-RH-induced cyclic AMP accumulation in male rat anterior pituitary gland. Adenohypophyseal tissue was first incubated for 60 min before four successive incubations of 1, 1, “I4 and “I4 h in the presence or absence of the indicated concentrations of free Ca’ + 10m7M LH-RH, lo-’ M theophylline and 2 x 1O-3 M EGTA were present in all groups.
Calcium, LH-RH
B
and cyclic AMP
121
LH-RH + ECTA+Ca++ 7
(b)
____L_
____L_--A
10-4
10~5 FREE
Ca++
m-3
IM)
Fig. 2. Effect of increasing concentrations of Ca2+ on LH-RH-induced release of LH (a) and FSH (b) from male rat anterior pituitary gland. Adenohypophyseal tissue was first incubated for 45 min in 1 ml of normal KRBG before a 35-min incubation in a Ca2+-free medium containing 2 x 10-j M EGTA. Three successive incubations of 3/4, 21/2 and 3/4 h were then carried out in the presence or absence of the indicated concentrations of Caz+. 10e7 M LH-RH, and 2 x 1O-3 M EGTA were present in all groups. The amounts of LH and FSH released during the second incubation period (21/2 h) are presented.
10e4 M and a maximal response being reached at 5 to 8 x 1O-4 M free Ca2 +. Although the magnitude of the LH and FSH responses is different, the LH-RHinduced release of the two pituitary hormones is obtained at the same free Ca’ + concentrations. Since, as observed previously (Borgeat et al., 1972, 1974), the effect of LH-RH is minimal in the first 45 min of incubation, the present data were obtained in the second incubation period ranging from the 45th to 195th min after addition of LH-RH. Qualitatively similar results were obtained in the third incubation period (data not shown). Although the LH-RH-induced release of LH required the presence of Ca* +, an inverse effect of Ca2 + is observed on the basal release of the hormone. As shown in fig. 3, complete removal of Ca2 + by addition of 1 mM EGTA to a Ca2 +-free medium leads to a I- to 2-fold increase of the LH secretion rate. The in vitro stimulation of LH secretion by the absence of Ca2 + is maximal (100 to 200% over control) during the first 2 hours of incubation and it dedreases (50% over control) at later time intervals (fig. 3).
P. Borgeat et al.
122
MINUTES
Fig. 3. release. periods periods
OF
o
EGTA-CA++
0
EGTA
+ CA+’
INCUBATION
Effect of Ca2 + removal from the incubation medium on spontaneous in vitro LH Adenohypophyseal tissue (5 pituitary halves per group) was first incubated for 2 of 30 mia in normal KRBG before incubation for 10 successive 30-min incubation in Caz+-free KRBG medium containing 1 mM EGTA in the presence or absence of 3.5 x 10e3 M CaCI,.
DISCUSSION As shown in table 1, the addition of 2 mM EGTA to rat hemipituitaries incubated in Ca2 + -free KRBG buffer does not affect the basal intracellular cyclic AMP concentration. However, in order to minimize possible effects of EGTA other than those secondary to removal of calcium ions, the effect of increasing concentrations of Ca2 + was studied by addition of the required amounts of CaCl, to a fixed concentration of EGTA (2 mM) present in control and experimental groups. The amount of CaClz necessary to achieve the desired concentrations of free Ca2 + was calculated using a dissociation constant of 3.95 x 10eh M for the Ca 2 +-EGTA interaction (De Meis and Hasselback, 1971). As noticed for the spontaneous release of LH (Samli and Geschwind, 1968), adrenocorticotropin (Zimmerman and Fleischer, 1970), growth hormone and prolactin (A. Lemay and F. Labrie, unpublished data), the N6, 2’-0-dibutyryl cyclic AMP-induced release of growth hormone and prolactin (A. Lemay and F. Labrie, unpublished data) and the hypothalamic extract-induced release of
Calcium, LH-RH
and cyclic AMP
123
LH (Samli and Geschwind, 1968) and FSH (Jutisz and Paloma de la Llosa, medium has 1970), we have found that removal of Ca 2 + from the incubation no effect on the stimulation of adenohypophyseal cyclic AMP accumulation by LH-RH (data not shown). Since removal of Ca2 + by addition of EGTA is required for the inhibitory effect, it is likely that repeated washings of the tissue leave enough Ca 2 + bound intra- or extracellularly to permit stimulation of cyclic AMP accumulation by LH-RH. LH As shown in fig. 3, not only Ca2 +- is not required for spontaneous release but an appreciably higher (100 to 200 ‘A over control) rate of hormonal release is observed in the absence of Ca 2 +. It should however be mentioned that when 2 mM EGTA is added to Ca ’ +-free KRBG buffer, the stimulatory effect of the absence of Ca2 + on LH release appears to be restricted to this hormone. In fact, no similar effect has been observed on the release of the other pituitary hormones (Samli and Geschwind, 1968; Borgeat and Labrie, unpublished data). Since the gonadotrophs account for only 5 to 8 % of the total cell population in rat anterior pituitary gland, it is possible that complete Ca’ + removal is accompanied by increased levels of cyclic AMP in the cells secreting LH but that the effect is masked by unchanged or decreased cyclic AMP concentrations in the other pituitary cells. It should also be mentioned that the stringent Ca2 + requirements for stimulation of cyclic AMP accumulation by LH-RH are not found with purified growth hormone-releasing hormone (Borgeat and Labrie, unpublished data) or prostaglandin Ez (Cooper et al., 1972; Borgeat and Labrie, unpublished data). In fact, in the absence of Ca 2 +, these two agents lead to an important stimulation of cyclic AMP accumulation while the release of growth hormone is blocked. The present data show that Ca2 +- is required at a step preceding stimulation of cyclic AMP accumulation by LH-RH. The site of Ca2 + requirement could be on the LH-RH receptor, on the adenylate cyclase, on cyclic nucleotide phosphodiesterase, at some intermediate step between binding of LH-RH and activation of adenylate cyclase or at a combination of these sites. As the binding of another releasing hormone, thyrotropin-releasing hormone (Labrie et al., 1972) is not increased by Ca2 f and 1 mM EGTA does not affect fluoridestimulated adenohypophyseal adenylate cyclase activity (Poirier and Labrie, unpublished), it appears more likely that Ca ’ + is required at some step(s) between binding of LH-RH and activation of adenylate cyclase, although an action at the other sites remains possible.
124
P. Borgeat
et al.
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