Experimental Gerontology, Vol. 27, pp. 211-219, 1992
0531-5565/92 $5.00 + .00 1992 Pergamon Press Ltd.
Printed in the USA. All rights reserved.
AGE-RELATED C H A N G E S IN THE M E C H A N I S M S OF LHRHS T I M U L A T E D LH RELEASE FROM PITUITARY CELLS IN VITRO
ATSUSHI M I Y A M O T O , x TOSHIO M A K I , ~ M A R C R. BLACKMAN 2 and G E O R G E S. R O T H ~ JMolecular Physiology and Genetics Section and 2Endocrinology Section, Gerontology Research Center, N1A, NIH, Francis Scott Key Medical Center, Baltimore, Maryland 21224
- - In vitro release of LH in response to LHRH, phorbol myristate acetate (PMA), the ionophore A23187, and nifedipine was evaluated in primary cultures of anterior pituitary cells from intact mature (6 to 7 month) and old (23 to 24 month) male Wistar rats. LH release from pituitary cells is reduced approximately 30% and 60% after 4 and 48 h of 10-7M LHRH stimulation in cells of old rats, respectively. This impairment may be secondary to a loss of LHRH receptors. LHRH-stimulated LH release from cells of mature rats was inhibited 70% by the voltage-gated calcium channel blocker, nifedipine (10-6M), whereas LHRH-stimulated LH release from cells of old rats was too low to detect the effects of this drug. Age changes can be partially reversed by A23187 and PMA during 4 h, but not 48 hrs of stimulation. It therefore appears that short- and long-term (4 h and 48 h, respectively) stimulation of LH release may proceed through separate mechanisms that are differentially affected by aging. Abstract
Key Words: pituitary cells, LH release, aging
INTRODUCTION REDUCTIONS I N LHRH-stimulated L H release by pituitary cells in vivo and in vitro are well d o c u m e n t e d manifestations o f reproductive senescence in rodents (Tang and Tang, 1981; H u a n g et al., 1985; Chuknyiska et aL, 1987). However, the cellular and molecular mechanisms by which such impairments occur have not yet been well defined. For example, some laboratories have reported loss o f L H R H receptors during aging (Limonta et aL, 1988), while others have observed no change (Sonntag et al., 1984). Such apparent discrepancies may be due to sex/strain or preparation differences. Subsequent to receptor binding, L H R H signal transduction requires calcium mobilization (Conn, 1986, 1989; Stojilkocic et al., 1989; Stutzin et al., 1989). For short-term
Correspondence to: George S. Roth, Molecular Physiology & Genetics Section, Gerontology Research Center NIA, NIH, Francis Scott Key Medical Center, 4940 Eastern Ave., Baltimore, MD 21224. (Received 28 May 1991; Accepted 6 August 1991 ) 211
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responses (i.e., 15 min or less), intracellular calcium stores may be released by generation of inositol 1,4,5-trisphosphate (Kiesel et al., 1987; Mitchell et al., 1988). Longer term responses require entrance of extracellular calcium through voltage-dependent channels (Tasaka et al., 1988). We previously demonstrated that age differences in stimulated LH release could be partially abolished if L H R H receptors were bypassed and intracellular calcium concentrations raised artificially by the ionophore A23187 (Chuknyiska et al., 1987). Thus, selective impairments in calcium mobilization may be at least partially responsible for reduced LH release from pituicytes of aged rats. Most aging studies of the phenomenon have examined L H R H stimulation varying from 4 to 48 h. Although general agreement exists as to the reduced magnitude of LH release during aging, the possibility remained that multiple differential mechanisms might be responsible for response changes at different times following L H R H stimulation. In order to gain further insight into these possible mechanisms, both L H R H receptors and voltagedependent calcium channels were examined. In addition, the effects of calcium-mobilizing and amplifying agents such as A23187 and phorbol myristate acetate (PMA) were studied for periods of 4 or 48 h. Results suggest that multiple differential mechanisms of age changes in L H R H responsiveness may occur at differential times following stimulation. MATERIALS AND METHODS Animals Paired groups of mature (6 to 7 month) and old (23 to 24 month) male Wistar rats were obtained from the Gerontology Research Center (GRC) animal colony at the Francis Scott Key Medical Center in Baltimore. The GRC is fully accredited by the American Association for Accreditation of Laboratory Animal Care. All animals were killed by decapitation. Cell preparation and incubation procedure Dispersed pituitary cells were prepared as described previously (Chuknyiska et al., 1987). In brief, anterior pituitary glands were minced into several pieces and treated for 60 min with 0.2% collagenase, 0.3% hyaluronidase, and 3% bovine serum albumin (BSA) in 0.1 M phosphate-buffered saline (pH 7.4) at 37°C. Then the cells were washed three times with Dulbecco's Modified Eagle's medium (DMEM) containing 10% dextran charcoal-treated calf:rat serum (1:2, v/v). Cells were incubated overnight in DMEM containing the above serum and 0.2 mM glutamine, then washed three times in serum-free DMEM, seeded at ~ 105 cells/well in plastic dishes, and incubated for 48 h at 370C under an atmosphere of 95% 02-5% CO2. The release of LH was measured after (Experiment 1) or during (Experiment 2) 48 h of incubation. In Experiment 1, at the end of 48 h of incubation cells were again washed three times and incubated in serum-free DMEM for 4 h in the absence or presence of LHRH, PMA, and A23187. In Experiment 2, LHRH, PMA A23187, and nifedipine were added at the start of the 48-h incubation, and then every 12 h during the 48-h incubation. The media were collected and stored at - 20"C until assayed. Cell viability was routinely checked by trypan blue exclusion and found to be 85-90% under all experimental conditions.
AGING AND CONTROL OF LH RELEASE
213
Radioimmunoassays LH was measured by double-antibody radioimmunoassays using rat hormones and antisera generously supplied by the National Hormone and Pituitary Agency, as previously described (Chuknyiska et al., 1987). Results are expressed in terms of the RP-2 standards of rat LH.
Membrane preparation Pituitary plasma membranes were prepared according to the method described by Perrin et al. (1983). Anterior pituitaries (n = 5) were homogenized in ice-cold 0.32 M sucrose. The homogenate was centrifuged at 600 X g for 5 min, and the resulting supernatant was centrifuged at 48 000 X g for 20 min at 4°C. The pellet was resuspended in 10 Mm Tris/ HC1 buffer (pH 7.4) containing 1.0 mM dithiothreitol and 0.15% BSA and used immeidately for the radioreceptor assay. Protein was determined by the method of Lowry et al. ( 1951 ) using BSA as standard.
Radioligand preparation A superactive, degradation resistant analogue of LHRH, D-ser(t-Bu)6-des-Gly~° EA (buserelin), used for iodination and as standard was kindly provided by Dr. K. von Richtenberg, Hoechst AG, Frankfurt, Germany. Buserelin was iodinated with chloramine T, and the iodinated peptide was then further purified by HPLC using a C~s column (Perrin et al., 1983). The specific activity of the tracer was ~ 1000 uCi/ug.
L H R H receptor binding assay For saturation analysis, the binding assay contained 25 #1 assay buffer ( 10 Mm Tris/HCl [pH 7.4] containing 1.0 Mm dithiothreitol and 0.15% BSA), 100 ul of the pituitary plasma membrane preparation (0.13-0.14 mg protein) and 100 ul ['25I]buserelin (~8,000350 000 cpm). Nonspecific binding for each sample was monitored by including unlabeled buserelin (100 ng/tube) in the assay buffer. Incubation was carried out on ice for 90 min and terminated by the addition of 3 ml ice-cold assay buffer to each tube, followed by centrifugation at 48 000 × g (20 min at 4°C). The supernatant was aspirated, and the pellets were counted in a gamma spectrometer. Saturation data were analyzed by a computerassisted curve-fitting program (LUNDON-1) equipped with a statistical package (Feldman, 1972).
Statistical analysis Statistical analyses were performed by one-way analysis of variance (ANOVA) followed by Student's t test. RESULTS Figure 1 shows the effects of aging on LH release from rat pituitary cells 4 h after stimulation by maximal concentrations of LHRH (10 -7 M). In agreement with previous reports (Chuknyiska et al., 1987; Tang and Tang, 1981), this response is reduced by 30% when cells from old animals are compared to those of intact mature counterparts (p
0531-5565/92 $5.00 + .00 1992 Pergamon Press Ltd.
Printed in the USA. All rights reserved.
AGE-RELATED C H A N G E S IN THE M E C H A N I S M S OF LHRHS T I M U L A T E D LH RELEASE FROM PITUITARY CELLS IN VITRO
ATSUSHI M I Y A M O T O , x TOSHIO M A K I , ~ M A R C R. BLACKMAN 2 and G E O R G E S. R O T H ~ JMolecular Physiology and Genetics Section and 2Endocrinology Section, Gerontology Research Center, N1A, NIH, Francis Scott Key Medical Center, Baltimore, Maryland 21224
- - In vitro release of LH in response to LHRH, phorbol myristate acetate (PMA), the ionophore A23187, and nifedipine was evaluated in primary cultures of anterior pituitary cells from intact mature (6 to 7 month) and old (23 to 24 month) male Wistar rats. LH release from pituitary cells is reduced approximately 30% and 60% after 4 and 48 h of 10-7M LHRH stimulation in cells of old rats, respectively. This impairment may be secondary to a loss of LHRH receptors. LHRH-stimulated LH release from cells of mature rats was inhibited 70% by the voltage-gated calcium channel blocker, nifedipine (10-6M), whereas LHRH-stimulated LH release from cells of old rats was too low to detect the effects of this drug. Age changes can be partially reversed by A23187 and PMA during 4 h, but not 48 hrs of stimulation. It therefore appears that short- and long-term (4 h and 48 h, respectively) stimulation of LH release may proceed through separate mechanisms that are differentially affected by aging. Abstract
Key Words: pituitary cells, LH release, aging
INTRODUCTION REDUCTIONS I N LHRH-stimulated L H release by pituitary cells in vivo and in vitro are well d o c u m e n t e d manifestations o f reproductive senescence in rodents (Tang and Tang, 1981; H u a n g et al., 1985; Chuknyiska et aL, 1987). However, the cellular and molecular mechanisms by which such impairments occur have not yet been well defined. For example, some laboratories have reported loss o f L H R H receptors during aging (Limonta et aL, 1988), while others have observed no change (Sonntag et al., 1984). Such apparent discrepancies may be due to sex/strain or preparation differences. Subsequent to receptor binding, L H R H signal transduction requires calcium mobilization (Conn, 1986, 1989; Stojilkocic et al., 1989; Stutzin et al., 1989). For short-term
Correspondence to: George S. Roth, Molecular Physiology & Genetics Section, Gerontology Research Center NIA, NIH, Francis Scott Key Medical Center, 4940 Eastern Ave., Baltimore, MD 21224. (Received 28 May 1991; Accepted 6 August 1991 ) 211
212
A. MIYAMOTO et al.
responses (i.e., 15 min or less), intracellular calcium stores may be released by generation of inositol 1,4,5-trisphosphate (Kiesel et al., 1987; Mitchell et al., 1988). Longer term responses require entrance of extracellular calcium through voltage-dependent channels (Tasaka et al., 1988). We previously demonstrated that age differences in stimulated LH release could be partially abolished if L H R H receptors were bypassed and intracellular calcium concentrations raised artificially by the ionophore A23187 (Chuknyiska et al., 1987). Thus, selective impairments in calcium mobilization may be at least partially responsible for reduced LH release from pituicytes of aged rats. Most aging studies of the phenomenon have examined L H R H stimulation varying from 4 to 48 h. Although general agreement exists as to the reduced magnitude of LH release during aging, the possibility remained that multiple differential mechanisms might be responsible for response changes at different times following L H R H stimulation. In order to gain further insight into these possible mechanisms, both L H R H receptors and voltagedependent calcium channels were examined. In addition, the effects of calcium-mobilizing and amplifying agents such as A23187 and phorbol myristate acetate (PMA) were studied for periods of 4 or 48 h. Results suggest that multiple differential mechanisms of age changes in L H R H responsiveness may occur at differential times following stimulation. MATERIALS AND METHODS Animals Paired groups of mature (6 to 7 month) and old (23 to 24 month) male Wistar rats were obtained from the Gerontology Research Center (GRC) animal colony at the Francis Scott Key Medical Center in Baltimore. The GRC is fully accredited by the American Association for Accreditation of Laboratory Animal Care. All animals were killed by decapitation. Cell preparation and incubation procedure Dispersed pituitary cells were prepared as described previously (Chuknyiska et al., 1987). In brief, anterior pituitary glands were minced into several pieces and treated for 60 min with 0.2% collagenase, 0.3% hyaluronidase, and 3% bovine serum albumin (BSA) in 0.1 M phosphate-buffered saline (pH 7.4) at 37°C. Then the cells were washed three times with Dulbecco's Modified Eagle's medium (DMEM) containing 10% dextran charcoal-treated calf:rat serum (1:2, v/v). Cells were incubated overnight in DMEM containing the above serum and 0.2 mM glutamine, then washed three times in serum-free DMEM, seeded at ~ 105 cells/well in plastic dishes, and incubated for 48 h at 370C under an atmosphere of 95% 02-5% CO2. The release of LH was measured after (Experiment 1) or during (Experiment 2) 48 h of incubation. In Experiment 1, at the end of 48 h of incubation cells were again washed three times and incubated in serum-free DMEM for 4 h in the absence or presence of LHRH, PMA, and A23187. In Experiment 2, LHRH, PMA A23187, and nifedipine were added at the start of the 48-h incubation, and then every 12 h during the 48-h incubation. The media were collected and stored at - 20"C until assayed. Cell viability was routinely checked by trypan blue exclusion and found to be 85-90% under all experimental conditions.
AGING AND CONTROL OF LH RELEASE
213
Radioimmunoassays LH was measured by double-antibody radioimmunoassays using rat hormones and antisera generously supplied by the National Hormone and Pituitary Agency, as previously described (Chuknyiska et al., 1987). Results are expressed in terms of the RP-2 standards of rat LH.
Membrane preparation Pituitary plasma membranes were prepared according to the method described by Perrin et al. (1983). Anterior pituitaries (n = 5) were homogenized in ice-cold 0.32 M sucrose. The homogenate was centrifuged at 600 X g for 5 min, and the resulting supernatant was centrifuged at 48 000 X g for 20 min at 4°C. The pellet was resuspended in 10 Mm Tris/ HC1 buffer (pH 7.4) containing 1.0 mM dithiothreitol and 0.15% BSA and used immeidately for the radioreceptor assay. Protein was determined by the method of Lowry et al. ( 1951 ) using BSA as standard.
Radioligand preparation A superactive, degradation resistant analogue of LHRH, D-ser(t-Bu)6-des-Gly~° EA (buserelin), used for iodination and as standard was kindly provided by Dr. K. von Richtenberg, Hoechst AG, Frankfurt, Germany. Buserelin was iodinated with chloramine T, and the iodinated peptide was then further purified by HPLC using a C~s column (Perrin et al., 1983). The specific activity of the tracer was ~ 1000 uCi/ug.
L H R H receptor binding assay For saturation analysis, the binding assay contained 25 #1 assay buffer ( 10 Mm Tris/HCl [pH 7.4] containing 1.0 Mm dithiothreitol and 0.15% BSA), 100 ul of the pituitary plasma membrane preparation (0.13-0.14 mg protein) and 100 ul ['25I]buserelin (~8,000350 000 cpm). Nonspecific binding for each sample was monitored by including unlabeled buserelin (100 ng/tube) in the assay buffer. Incubation was carried out on ice for 90 min and terminated by the addition of 3 ml ice-cold assay buffer to each tube, followed by centrifugation at 48 000 × g (20 min at 4°C). The supernatant was aspirated, and the pellets were counted in a gamma spectrometer. Saturation data were analyzed by a computerassisted curve-fitting program (LUNDON-1) equipped with a statistical package (Feldman, 1972).
Statistical analysis Statistical analyses were performed by one-way analysis of variance (ANOVA) followed by Student's t test. RESULTS Figure 1 shows the effects of aging on LH release from rat pituitary cells 4 h after stimulation by maximal concentrations of LHRH (10 -7 M). In agreement with previous reports (Chuknyiska et al., 1987; Tang and Tang, 1981), this response is reduced by 30% when cells from old animals are compared to those of intact mature counterparts (p
