0013-7227/91/1293-1355$03.00/0 Endocrinology Copyright© 1991 by The Endocrine Society
Vol. 129, No. 3 Printed in U.S.A.
Signal Transduction Systems in Growth HormoneReleasing Hormone and Somatostatin Release from Perifused Rat Hypothalamic Fragments* CHRISTY D. CUGINI, JR., WILLIAM J. MILLARD, AND JOHN W. LEIDY, JR. Veterans Affairs Medical Center and the Department of Medicine, Marshall University School of Medicine (C.D.C., J. W.L.), Huntingdon, West Virginia 25704 and 25755-9410; and the Department of Pharmacodynamics, J. Hillis Miller Health Center, University of Florida College of Pharmacy (W.J.M.), Gainesville, Florida 32610
ABSTRACT. The role of signal transduction systems was examined in the secretion of GH-releasing hormone (GHRH) and somatostatin (SS) from perifused rat hypothalamic fragments. Forskolin, an adenylate cyclase activator, stimulated the release of GHRH and SS in a concentration-dependent manner (10-100 fiM) with greatest stimulation for GHRH at 100 nM (mean ± SE, 249 ± 14%) and for SS at 30 nM (172 ± 18%). (Bu)2cAMP also augmented GHRH and SS release. The protein kinase-C activator phorbol 12-myristate 13-acetate did not significantly stimulate basal GHRH or SS release at concentrations of 10 nM to 1 nM. The calcium ionophore A23187 enhanced the release of GHRH and SS in a concentration-dependent manner (2-20 /xM), with the greatest responses of 282 ± 50% at 10 »M and 189 ± 24% at 20 nM, respectively. Potentiation by phorbol 12-myristate 13-acetate of forskolin-stimulated GHRH and SS
G
H SECRETION is regulated by the stimulatory and inhibitory hypothalamic releasing hormones, GHreleasing hormone (GHRH) and somatostatin (SS) (1, 2). The neural, hormonal, and metabolic regulation of the secretion and synthesis of these hypothalamic peptides is being investigated currently by in vitro and in vivo methods (3-6). An in vitro experimental approach to examine the regulation of GHRH and SS is measurement of release from hypothalamic fragments in perfusion and static incubation. Experience with hypothalamic perifusion and static incubation systems has established the validity of this technique for physiological investigations (7, 8). Recent studies in the hypothalamus have suggested the importance of signal transduction systems (9-17),
Received December 12,1990. Address all correspondence and requests for reprints to: Dr. John W. Leidy, Jr., Section of Endocrinology, Department of Medicine, Marshall University School of Medicine, Huntington, West Virginia 25755-9410. * Presented in part at the 72nd Annual Meeting of The Endocrine Society, Atlanta, GA, 1990 (Abstract 119). This work supported by the Department of Veterans Affairs and NIH Grant HD-22199 and RR05870.
release was observed. A23187 at 10 fiM did not enhance forskolinstimulated GHRH release, but did potentiate forskolin-stimulated SS release in a more than additive response. We conclude that there is 1) cAMP stimulation of hypothalamic GHRH and SS release, 2) a modulating role of protein kinase-C on cAMP-stimulated release of GHRH and SS, 3) a stimulatory role of the calcium messenger system for GHRH and SS release, 4) interaction of the signal pathways with differences in net GHRH and SS responses, and 5) a modulatory effect of protein kinase-C in perifused hypothalamic fragments which differs from the stimulation of basal GHRH and SS release reported in fetal-derived hypothalamic cell cultures. Our observations suggest an important regulatory role of interacting signal transduction systems in the hypothalamic secretion of GHRH and SS. (Endocrinology 129: 1355-1362,1991)
which are important in the synthesis, release, and action of neurotransmitters, neuropeptides, and hormones. Our investigations were designed to examine the role of signal transduction systems in the secretion of GHRH and SS from the hypothalamus. We have established a perifusion system for rat hypothalamic fragments to measure basal and stimulated release of GHRH and SS. Using this system, the adenylate cyclase-protein kinase-A (cAMP) pathway, protein kinase-C pathway of the phosphoinositide second messenger system, and calcium messenger system were explored. Additionally, the interactions of activation of protein kinase-C or increased intracellular calcium with cAMP-stimulated release of GHRH and SS were investigated. Materials and Methods Hypothalamic fragments Hypothalamic fragments from male Sprague-Dawley rats (150-250 g; 5-8 weeks old; Hilltop Lab Animals, Inc., Scottsdale, PA) were dissected to include selectively the arcuate nucleus and median eminence. Animals were killed by decapitation after CO2 narcosis. Hypothalamic fragments (8-12 mg)
1355
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 21:50 For personal use only. No other uses without permission. . All rights reserved.
1356
GHRH AND SS SIGNAL TRANSDUCTION
Endo•1991 Vol 129 • No 3
were removed, with the borders of dissection being the posterior margin of the optic chiasm, the anterior edge of the mammillary bodies, and lateral incisions 1.5 mm from the midline at a 45° angle to the midsagittal plane. The wedge-shaped fragment was then bisected with a scalpel in the midline through the third ventricle and median eminence. Previous immunohistochemical studies in our laboratory revealed that most of the immunoreactive GHRH neuronal bodies would be contained in the hypothalamic fragment (18). The GHRH content (determined by RIA) of the hypothalamic fragments (2.54 ± 0.12 ng; n = 32) was 81% that of the entire hypothalamus (3.12 ± 0.18 ng; n = 32), indicating that most of the neuronal cell bodies and axons containing immunoreactive GHRH were included in this preparation.
eluant was 2 ml isopropanol-water (80:20%, with 0.1% TFA). After centrifugal evaporation (Speed-Vac, Savant Instruments, Inc., Hicksville, NY), extracts were reconstituted with 0.01 M acetic acid, pipetted into polystyrene tubes for RIA, and centrifugally evaporated. Extracts of the perifusate medium (KRB plus additions without or with test substances) inhibited binding in the GHRH RIA in a volume-dependent manner, and medium controls of appropriate volume and composition were included in each RIA. The recovery of GHRH from medium was 98 ± 2% (n = 6). SS was measured from unextracted heatinactivated (90 C for 10 min) acidified medium and centrifugally evaporated, using 100 /il (0.1 vol perifusion fraction)/ replicate. The recovery of SS from medium was 86 ± 6% (n = 6).
Perifusion
RIA for GHRH and SS
Sixteen hypothalamic fragments (eight hypothalamic equivalents) per chamber were perifused with Krebs-Ringer bicarbonate buffer (KRB), pH 7.4, with additions of 10 mM glucose, 60 ixM ascorbic acid, 100 nM bacitracin, and 0.05% BSA, and gassed with 95% O2-5% CO2. Hypothalamic fragments were kept in KRB at 4 C until loading into perifusion chambers. Four 1-ml disposable syringes were used as perifusion chambers and submerged in a 37 C water bath, with infusion of perifusate at 100 /il/min by a four-channel peristaltic pump. The perifusion system is similar to that developed for measurement of SS release from hypothalamic fragments by other investigators (19, 20). A fused nylon-polypropylene screen at the end of the column contained the fragments. The total dead volume of the system was 650 n\, which included a chamber size of 500 /il. The lag time of the perifusion system was 5 min. All components of the perifusion apparatus were siliconized. Fractions of 10 min (1.0 ml) were collected in glass testtubes containing 50 n\ 10% trifluoroacetic acid (TFA) and frozen at -20 C until analyses. Forskolin (adenylate cyclase activator), phorbol 12-myristate 13-acetate (PMA; protein kinase-C activator), and A23187 (calcium ionophore) were dissolved in dimethylsulfoxide (DMSO). The final perifusate concentration was 0.5% DMSO for forskolin and A23187 and 0.005% DMSO for PMA. There was no effect on baseline release of GHRH and SS by DMSO with concentrations up to 0.5%. (Bu)2cAMP (dbcAMP) was dissolved directly in KRB. Media were adjusted to pH 7.4. Forskolin, dbcAMP, PMA, and A23187 were obtained from Sigma Chemical Co. (St. Louis, MO). Infusions of test substances were performed for 40 min, followed by 80 min of medium without test substances. For high K+ medium (20-60 mM K+), KC1 was substituted for NaCl isotonically in KRB. High K+ medium was infused for 10 min, followed by normal K+ medium for 50 min.
For preliminary experiments with perifusion and static incubation systems, the release of rat GHRH (rGHRH) was measured by a previously reported nonequilibrium double antibody RIA, using antiserum 3138 and [125I] rGHRH purified by reverse phase HPLC (21, 22). The sensitivity with antiserum 3138 was 1.8 pg/tube, with half-maximum displacement at 15 pg/tube. A previously uncharacterized antiserum (WJM 4/5) was used to improve assay sensitivity to 0.79 pg/tube, with half-maximal displacement of 4.8 pg/tube. This antiserum was generated in a New Zealand rabbit using rGHRH coupled to bovine thyroglobulin by carbodiimide (24). The working dilution was 1:100,000 for overnight binding of 30%. Specificity studies revealed less than 1 X 10"4 cross-reactivity with rat secretin, glucagon, gastric inhibitory peptide, vasoactive intestinal peptide, peptide histidine-isoleucine-27, glucagon, rat GH, SS-14, TRH, or GnRH. Cross-reactivity with human GHRH(1-44)NH2 at 1.5 X 10"2 and [His1,Nle27]human GHRH-(132)NH2 at 6.3 x 10' 4 indicated that antiserum WJM 4/5 is Nterminally directed, whereas the cross-reactivity of these peptides with the C-terminally directed antiserum 3138 was less than 10"4. Dilutions of extracts from basal release and 60 mM K+-stimulated release produced parallel RIA displacement curves with GHRH standards. HPLC separation of extracts of pooled media from 60 mM K+-stimulated release demonstrated coelution of immunoreactive GHRH with synthetic GHRH and oxidized GHRH ([Met(O)27]GHRH, oxidized by chloramine-T or H2O2). Duplicate replicates were analyzed and expressed as picograms of GHRH per hypothalamus equivalent (HT)/10 min. The release of SS was measured using a well characterized RIA, as previously described, with an assay sensitivity of 2 pg/ tube (25). Antiserum 693 was generously provided by Dr. S. Reichlin (Tufts-New England Medical Center, Boston, MA). Dilutions of heat-inactivated acidified medium from basal release and 60 mM K+-stimulated release produced displacement curves parallel with those of SS standards. Duplicate replicates were analyzed and expressed as picograms of SS per HT/10 min.
Extractions TFA-acidified media were extracted for measurement of GHRH with octadecyl-silylsilica cartridges (Bond Elut C18, 500 mg sorbent, Analytichem, Harbor City, CA), as previously described (21, 22). Cartridge preconditioning and washing between extractions were modified by the addition of chloroform and methanol washes, which remove substances (lipids) that interfere with GHRH binding and the GHRH RIA (23). The
Statistical analyses Responses to high K+ media, which were infused for 10 min, were integrated over 40 min, which was the duration of the response. Responses to test substances infused for 40 min were
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 21:50 For personal use only. No other uses without permission. . All rights reserved.
GHRH AND SS SIGNAL TRANSDUCTION integrated over 80 min, which was substantially the duration of the response. The response of each channel was normalized by the baseline release of the channel during a 40-min control period between 120-160 min. Normalization was necessary because of the relatively large intrachannel variation of release in some experiments, particularly for SS. The time of the control period was picked by subjective criteria as the first 40 min of relatively stable release for both GHRH and SS release (Figs. 1 and 2). In experimental perifusions, the normalized response to test substances was compared to the normalized release from control perifusions without test substances (n = 8) during the corresponding time periods and expressed as a percentage of control release. Although the release of GHRH and SS was relatively stable, there was sufficient variation during the perifusion to require comparison for accurate measurement of small responses. Responses to test substances were z
2.0
n
Control
2 o
>
1.5-1
1.0
UJ DC
I eg o
0.5
0.0
60
120
180
240
300
360
420
480
TIME, MIN FlG. 1. Time course of GHRH release in control perifusions from hypothalamic fragments. Release is expressed as picograms per HT/10 min. To the left of the vertical dashed line is the unstable initial release. The control period of 120-160 min is indicated by the open bar. The results are plotted as the mean ± SE from two perifusion experiments with four channels each, for a total n = 8.
Control
60
120
180
240
300
360
420
480
TIME, MIN FlG. 2. Time course of SS release in control perifusions from hypothalamic fragments. To the left of the vertical dashed line is the unstable initial release. Values for fractions 1-3 are off the scale and are 192 ± 13,103 ± 5, and 55.1 ± 1.6 pg/HT-10 min. The control period of 120160 min is indicated by the open bar. The results are plotted as the mean ± SE from two perifusion experiments with four channels each, for a total n = 8.
1357
measured in two or more experiments. Comparisons between control and stimulated release were performed by paired t test, one-way analysis of variance, or two-way analysis of variance with Dunnett's or Student-Newman-Keuls multiple range test where appropriate (26). The significance level was set at P < 0.05. Results are expressed or plotted as the mean ± SE.
Results After an initial unstable period of 120 min, relatively stable release rates of GHRH and SS for 360 min were observed (Figs. 1 and 2). The patterns of release were reproducible, with GHRH release declining until 270290 min, then slowly rising, and SS release declining until 350-360 min, then slowly rising. The rate of release for the control period defined from 120-160 min was 0.61 ± 0.05 pg/HT-10 min for GHRH and 11.44 ± 1.39 pg/ HT-10 min for SS (n = 18 perifusions, 4 channels per perifusion). The minimum detectable release for GHRH was 0.27 ± 0.02 pg/HT-10 min, and the ratio of basal release to minimum detectable release was 2.3-fold. For SS, the minimum detectable release was 3.11 ± 0.34 pg/ HT • 10 min, and the ratio of basal release to minimum detectable release was 3.7. During the 360 min of stable release, the mean percent control release varied from 67134% for GHRH and from 71-111% for SS. The SD of mean percent control release was 17% for GHRH and 12% for SS. After 420 min of perifusion, instability of GHRH and SS release from a channel occurred infrequently (
Vol. 129, No. 3 Printed in U.S.A.
Signal Transduction Systems in Growth HormoneReleasing Hormone and Somatostatin Release from Perifused Rat Hypothalamic Fragments* CHRISTY D. CUGINI, JR., WILLIAM J. MILLARD, AND JOHN W. LEIDY, JR. Veterans Affairs Medical Center and the Department of Medicine, Marshall University School of Medicine (C.D.C., J. W.L.), Huntingdon, West Virginia 25704 and 25755-9410; and the Department of Pharmacodynamics, J. Hillis Miller Health Center, University of Florida College of Pharmacy (W.J.M.), Gainesville, Florida 32610
ABSTRACT. The role of signal transduction systems was examined in the secretion of GH-releasing hormone (GHRH) and somatostatin (SS) from perifused rat hypothalamic fragments. Forskolin, an adenylate cyclase activator, stimulated the release of GHRH and SS in a concentration-dependent manner (10-100 fiM) with greatest stimulation for GHRH at 100 nM (mean ± SE, 249 ± 14%) and for SS at 30 nM (172 ± 18%). (Bu)2cAMP also augmented GHRH and SS release. The protein kinase-C activator phorbol 12-myristate 13-acetate did not significantly stimulate basal GHRH or SS release at concentrations of 10 nM to 1 nM. The calcium ionophore A23187 enhanced the release of GHRH and SS in a concentration-dependent manner (2-20 /xM), with the greatest responses of 282 ± 50% at 10 »M and 189 ± 24% at 20 nM, respectively. Potentiation by phorbol 12-myristate 13-acetate of forskolin-stimulated GHRH and SS
G
H SECRETION is regulated by the stimulatory and inhibitory hypothalamic releasing hormones, GHreleasing hormone (GHRH) and somatostatin (SS) (1, 2). The neural, hormonal, and metabolic regulation of the secretion and synthesis of these hypothalamic peptides is being investigated currently by in vitro and in vivo methods (3-6). An in vitro experimental approach to examine the regulation of GHRH and SS is measurement of release from hypothalamic fragments in perfusion and static incubation. Experience with hypothalamic perifusion and static incubation systems has established the validity of this technique for physiological investigations (7, 8). Recent studies in the hypothalamus have suggested the importance of signal transduction systems (9-17),
Received December 12,1990. Address all correspondence and requests for reprints to: Dr. John W. Leidy, Jr., Section of Endocrinology, Department of Medicine, Marshall University School of Medicine, Huntington, West Virginia 25755-9410. * Presented in part at the 72nd Annual Meeting of The Endocrine Society, Atlanta, GA, 1990 (Abstract 119). This work supported by the Department of Veterans Affairs and NIH Grant HD-22199 and RR05870.
release was observed. A23187 at 10 fiM did not enhance forskolinstimulated GHRH release, but did potentiate forskolin-stimulated SS release in a more than additive response. We conclude that there is 1) cAMP stimulation of hypothalamic GHRH and SS release, 2) a modulating role of protein kinase-C on cAMP-stimulated release of GHRH and SS, 3) a stimulatory role of the calcium messenger system for GHRH and SS release, 4) interaction of the signal pathways with differences in net GHRH and SS responses, and 5) a modulatory effect of protein kinase-C in perifused hypothalamic fragments which differs from the stimulation of basal GHRH and SS release reported in fetal-derived hypothalamic cell cultures. Our observations suggest an important regulatory role of interacting signal transduction systems in the hypothalamic secretion of GHRH and SS. (Endocrinology 129: 1355-1362,1991)
which are important in the synthesis, release, and action of neurotransmitters, neuropeptides, and hormones. Our investigations were designed to examine the role of signal transduction systems in the secretion of GHRH and SS from the hypothalamus. We have established a perifusion system for rat hypothalamic fragments to measure basal and stimulated release of GHRH and SS. Using this system, the adenylate cyclase-protein kinase-A (cAMP) pathway, protein kinase-C pathway of the phosphoinositide second messenger system, and calcium messenger system were explored. Additionally, the interactions of activation of protein kinase-C or increased intracellular calcium with cAMP-stimulated release of GHRH and SS were investigated. Materials and Methods Hypothalamic fragments Hypothalamic fragments from male Sprague-Dawley rats (150-250 g; 5-8 weeks old; Hilltop Lab Animals, Inc., Scottsdale, PA) were dissected to include selectively the arcuate nucleus and median eminence. Animals were killed by decapitation after CO2 narcosis. Hypothalamic fragments (8-12 mg)
1355
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 21:50 For personal use only. No other uses without permission. . All rights reserved.
1356
GHRH AND SS SIGNAL TRANSDUCTION
Endo•1991 Vol 129 • No 3
were removed, with the borders of dissection being the posterior margin of the optic chiasm, the anterior edge of the mammillary bodies, and lateral incisions 1.5 mm from the midline at a 45° angle to the midsagittal plane. The wedge-shaped fragment was then bisected with a scalpel in the midline through the third ventricle and median eminence. Previous immunohistochemical studies in our laboratory revealed that most of the immunoreactive GHRH neuronal bodies would be contained in the hypothalamic fragment (18). The GHRH content (determined by RIA) of the hypothalamic fragments (2.54 ± 0.12 ng; n = 32) was 81% that of the entire hypothalamus (3.12 ± 0.18 ng; n = 32), indicating that most of the neuronal cell bodies and axons containing immunoreactive GHRH were included in this preparation.
eluant was 2 ml isopropanol-water (80:20%, with 0.1% TFA). After centrifugal evaporation (Speed-Vac, Savant Instruments, Inc., Hicksville, NY), extracts were reconstituted with 0.01 M acetic acid, pipetted into polystyrene tubes for RIA, and centrifugally evaporated. Extracts of the perifusate medium (KRB plus additions without or with test substances) inhibited binding in the GHRH RIA in a volume-dependent manner, and medium controls of appropriate volume and composition were included in each RIA. The recovery of GHRH from medium was 98 ± 2% (n = 6). SS was measured from unextracted heatinactivated (90 C for 10 min) acidified medium and centrifugally evaporated, using 100 /il (0.1 vol perifusion fraction)/ replicate. The recovery of SS from medium was 86 ± 6% (n = 6).
Perifusion
RIA for GHRH and SS
Sixteen hypothalamic fragments (eight hypothalamic equivalents) per chamber were perifused with Krebs-Ringer bicarbonate buffer (KRB), pH 7.4, with additions of 10 mM glucose, 60 ixM ascorbic acid, 100 nM bacitracin, and 0.05% BSA, and gassed with 95% O2-5% CO2. Hypothalamic fragments were kept in KRB at 4 C until loading into perifusion chambers. Four 1-ml disposable syringes were used as perifusion chambers and submerged in a 37 C water bath, with infusion of perifusate at 100 /il/min by a four-channel peristaltic pump. The perifusion system is similar to that developed for measurement of SS release from hypothalamic fragments by other investigators (19, 20). A fused nylon-polypropylene screen at the end of the column contained the fragments. The total dead volume of the system was 650 n\, which included a chamber size of 500 /il. The lag time of the perifusion system was 5 min. All components of the perifusion apparatus were siliconized. Fractions of 10 min (1.0 ml) were collected in glass testtubes containing 50 n\ 10% trifluoroacetic acid (TFA) and frozen at -20 C until analyses. Forskolin (adenylate cyclase activator), phorbol 12-myristate 13-acetate (PMA; protein kinase-C activator), and A23187 (calcium ionophore) were dissolved in dimethylsulfoxide (DMSO). The final perifusate concentration was 0.5% DMSO for forskolin and A23187 and 0.005% DMSO for PMA. There was no effect on baseline release of GHRH and SS by DMSO with concentrations up to 0.5%. (Bu)2cAMP (dbcAMP) was dissolved directly in KRB. Media were adjusted to pH 7.4. Forskolin, dbcAMP, PMA, and A23187 were obtained from Sigma Chemical Co. (St. Louis, MO). Infusions of test substances were performed for 40 min, followed by 80 min of medium without test substances. For high K+ medium (20-60 mM K+), KC1 was substituted for NaCl isotonically in KRB. High K+ medium was infused for 10 min, followed by normal K+ medium for 50 min.
For preliminary experiments with perifusion and static incubation systems, the release of rat GHRH (rGHRH) was measured by a previously reported nonequilibrium double antibody RIA, using antiserum 3138 and [125I] rGHRH purified by reverse phase HPLC (21, 22). The sensitivity with antiserum 3138 was 1.8 pg/tube, with half-maximum displacement at 15 pg/tube. A previously uncharacterized antiserum (WJM 4/5) was used to improve assay sensitivity to 0.79 pg/tube, with half-maximal displacement of 4.8 pg/tube. This antiserum was generated in a New Zealand rabbit using rGHRH coupled to bovine thyroglobulin by carbodiimide (24). The working dilution was 1:100,000 for overnight binding of 30%. Specificity studies revealed less than 1 X 10"4 cross-reactivity with rat secretin, glucagon, gastric inhibitory peptide, vasoactive intestinal peptide, peptide histidine-isoleucine-27, glucagon, rat GH, SS-14, TRH, or GnRH. Cross-reactivity with human GHRH(1-44)NH2 at 1.5 X 10"2 and [His1,Nle27]human GHRH-(132)NH2 at 6.3 x 10' 4 indicated that antiserum WJM 4/5 is Nterminally directed, whereas the cross-reactivity of these peptides with the C-terminally directed antiserum 3138 was less than 10"4. Dilutions of extracts from basal release and 60 mM K+-stimulated release produced parallel RIA displacement curves with GHRH standards. HPLC separation of extracts of pooled media from 60 mM K+-stimulated release demonstrated coelution of immunoreactive GHRH with synthetic GHRH and oxidized GHRH ([Met(O)27]GHRH, oxidized by chloramine-T or H2O2). Duplicate replicates were analyzed and expressed as picograms of GHRH per hypothalamus equivalent (HT)/10 min. The release of SS was measured using a well characterized RIA, as previously described, with an assay sensitivity of 2 pg/ tube (25). Antiserum 693 was generously provided by Dr. S. Reichlin (Tufts-New England Medical Center, Boston, MA). Dilutions of heat-inactivated acidified medium from basal release and 60 mM K+-stimulated release produced displacement curves parallel with those of SS standards. Duplicate replicates were analyzed and expressed as picograms of SS per HT/10 min.
Extractions TFA-acidified media were extracted for measurement of GHRH with octadecyl-silylsilica cartridges (Bond Elut C18, 500 mg sorbent, Analytichem, Harbor City, CA), as previously described (21, 22). Cartridge preconditioning and washing between extractions were modified by the addition of chloroform and methanol washes, which remove substances (lipids) that interfere with GHRH binding and the GHRH RIA (23). The
Statistical analyses Responses to high K+ media, which were infused for 10 min, were integrated over 40 min, which was the duration of the response. Responses to test substances infused for 40 min were
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 21:50 For personal use only. No other uses without permission. . All rights reserved.
GHRH AND SS SIGNAL TRANSDUCTION integrated over 80 min, which was substantially the duration of the response. The response of each channel was normalized by the baseline release of the channel during a 40-min control period between 120-160 min. Normalization was necessary because of the relatively large intrachannel variation of release in some experiments, particularly for SS. The time of the control period was picked by subjective criteria as the first 40 min of relatively stable release for both GHRH and SS release (Figs. 1 and 2). In experimental perifusions, the normalized response to test substances was compared to the normalized release from control perifusions without test substances (n = 8) during the corresponding time periods and expressed as a percentage of control release. Although the release of GHRH and SS was relatively stable, there was sufficient variation during the perifusion to require comparison for accurate measurement of small responses. Responses to test substances were z
2.0
n
Control
2 o
>
1.5-1
1.0
UJ DC
I eg o
0.5
0.0
60
120
180
240
300
360
420
480
TIME, MIN FlG. 1. Time course of GHRH release in control perifusions from hypothalamic fragments. Release is expressed as picograms per HT/10 min. To the left of the vertical dashed line is the unstable initial release. The control period of 120-160 min is indicated by the open bar. The results are plotted as the mean ± SE from two perifusion experiments with four channels each, for a total n = 8.
Control
60
120
180
240
300
360
420
480
TIME, MIN FlG. 2. Time course of SS release in control perifusions from hypothalamic fragments. To the left of the vertical dashed line is the unstable initial release. Values for fractions 1-3 are off the scale and are 192 ± 13,103 ± 5, and 55.1 ± 1.6 pg/HT-10 min. The control period of 120160 min is indicated by the open bar. The results are plotted as the mean ± SE from two perifusion experiments with four channels each, for a total n = 8.
1357
measured in two or more experiments. Comparisons between control and stimulated release were performed by paired t test, one-way analysis of variance, or two-way analysis of variance with Dunnett's or Student-Newman-Keuls multiple range test where appropriate (26). The significance level was set at P < 0.05. Results are expressed or plotted as the mean ± SE.
Results After an initial unstable period of 120 min, relatively stable release rates of GHRH and SS for 360 min were observed (Figs. 1 and 2). The patterns of release were reproducible, with GHRH release declining until 270290 min, then slowly rising, and SS release declining until 350-360 min, then slowly rising. The rate of release for the control period defined from 120-160 min was 0.61 ± 0.05 pg/HT-10 min for GHRH and 11.44 ± 1.39 pg/ HT-10 min for SS (n = 18 perifusions, 4 channels per perifusion). The minimum detectable release for GHRH was 0.27 ± 0.02 pg/HT-10 min, and the ratio of basal release to minimum detectable release was 2.3-fold. For SS, the minimum detectable release was 3.11 ± 0.34 pg/ HT • 10 min, and the ratio of basal release to minimum detectable release was 3.7. During the 360 min of stable release, the mean percent control release varied from 67134% for GHRH and from 71-111% for SS. The SD of mean percent control release was 17% for GHRH and 12% for SS. After 420 min of perifusion, instability of GHRH and SS release from a channel occurred infrequently (
