00l3-7227/9l/1291-0011$03.00/0 Endocrinology Copyright (ci 1991 by The Endocrine Society

Vol. 129, No. 1 Printed in U.S.A.

Calcium and Calmodulin Mediation of Growth HormoneReleasing Hormone Release from the Rat Hypothalamus in Vitro J. HONEGGER*, R. D'URSO, G. M. BESSER, AND A. B. GROSSMAN Department of Endocrinology, St. Bartholomew's Hospital, London EC 1A 7BE, United Kingdom

ABSTRACT. A major role for Ca2+ and calmodulin in stimulus-secretion coupling has been suggested for several neuropeptides; however, the cellular mechanisms of GH-releasing hormone (GHRH) release have been little investigated so far. We have used a previously validated acute rat hypothalamic explant system in order to elucidate whether Ca2+ acts as a second messenger in the regulation of GHRH release, and whether calmodulin-dependent pathways are involved. Calcium dependence of somatostatin (SRIH) release was assessed in the same experiments. Calmodulin dependence of SRIH was not investigated in detail, as it has been established previously. The calcium-entry antagonist, verapamil, antagonized K+-stimulated GHRH and SRIH release in a dose-dependent manner, with maximal inhibition shown at 10~4 M. The calmodulin antagonist W7 also blocked K+-evoked GHRH release in a dose-dependent manner, with significant inhibition in the dose range 5 X 10~5 M to 2 x 10""4 M; similarly, a more specific calmodulin inhibitor,

the W7 derivative 5-iodo-C8 (W8), reversed K+-stimulated GHRH release, showing slightly higher potency than W7. W7 also reversed GHRH release in response to the calcium-ionophore A23187, although verapamil had no effect on A23187evoked GHRH or SRIH release. Thapsigargin, which increases the efflux of Ca2+ from calciosomes, did not affect either GHRH or SRIH release at lO"5 M or 10"4 M. The basal release of GHRH was clearly suppressed by W7 and W8 (10~4 M), whereas verapamil had no effect. We conclude that calcium influx is crucial for depolarization-induced GHRH and SRIH release. Calcium entrance in response to A23187 appears to be independent of verapamil-sensitive calcium channels. The lack of effect of thapsigargin suggests that increased intracellular Ca2+ from intracellular stores is not equivalent to an increase in Ca2+ influx. Both basal and depolarization-induced release of GHRH in this system are calmodulin dependent. (Endocrinology 129: 11-16, 1991)

C

ALCIUM (Ca2+) and calmodulin play a crucial role in cellular regulation in the central nervous system. The Ca2+-binding regulatory protein calmodulin, which is able to activate various intracellular enzyme systems and to promote widespread functional and structural changes (1), is found in high concentrations in neural tissue and nerve terminals. It regulates many Ca2+-dependent neural functions and is involved in neurotransmitter release (2, 3). The essential role of Ca2+ and calmodulin in stimulus-secretion coupling for several neuropeptide hormones is now well established; a depolarizing stimulus causes Ca2+ influx through voltagesensitive Ca2+ channels (4), and Ca2+ then binds and activates calcium-dependent intracellular calmodulin (58). However, the intracellular roles of Ca2+ and calmodulin in modulating GH-releasing hormone (GHRH) se-

cretion are not clearly established. Using fetal hypothalamic neurons in culture, one study demonstrated that both cAMP and protein kinase-C pathways could stimulate GHRH release, and that responses to both stimuli could be blocked by verapamil (9). We have previously described and validated an acute hypothalamic explant system in the adult rat for measurement of rat GHRH (10) and somatostatin (SRIH) (11, 12). We have now used this system to investigate the role of Ca2+ as a second messenger in GHRH and SRIH release, and of calmodulin-dependent pathways in GHRH release. The dissection limits used for this study included the dominant locations of GHRH- and SRIH-containing neurons within the hypothalamus and the median eminence, as the major projection and terminal area of their nerve fibers, respectively (13-17).

Received January 21, 1991. Address all correspondence and requests for reprints to: Dr. Ashley Grossman, Department of Endocrinology, St. Bartholomew's Hospital, London EClA 7BE, United Kingdom. * Supported through the Deutscher Akademischer Austauschdienst by Funds of the NATO science fellowship program. Current address: Department of Neurosurgery, University of Erlangen-Nurnberg, 8520Erlangen, Germany.

Materials and Methods Animals Male Wistar rats (Banting & Kingham Ltd., Aldbrough, UK), housed four per cage from the time of weaning and kept under constant conditions of room temperature (18 C) and 11

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Ca2+ AND CALMODULIN DEPENDENCE OF GHRH RELEASE

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lighting (0400-1900 h), with free access to food and water, were used for all experiments. Hypothalamic dissection Groups of animals were decapitated between 0900-1000 h, their brains removed, and hypothalamic blocks dissected with the following limits: anterior border of the optic chiasm, anterior border of the mamillary bodies, and the lateral hypothalamic sulci. The depth of dissection was 2 mm. In all experiments the hypothalami were bisected longitudinally through the midsagittal plane. The total dissection time was less than 2 min from decapitation. Incubation system Two hypothalamic halves from the same animal were incubated in polyethylene vials containing 400 ^1 Earle's Balanced Salt Solution (EBSS, GIBCO Biocult, Paisley, Scotland) supplemented with 0.2% human serum albumin, 60 iig/m\ ascorbic acid, and 40 IU/ml aprotinin, in an atmosphere of 95% O2 and 5% CO2. Incubation vials were kept in a shaking water bath at 37 C. Previous experiments have shown that the basal release of GHRH stabilizes from 60 min incubation, and thereafter remains stable for at least 3 h (10); we have therefore chosen an 80-min preincubation period. The incubation medium was aspirated every 20 min and replaced with fresh medium. For investigation of basal release, preincubation was followed by 20 min incubation in medium alone and a subsequent 20-min period with incubation in either medium alone (control) or medium containing the different test substances. For assessment of the effects of different test substances on depolarization-evoked or calcium-ionophore (A23187)-evoked release, we chose a 10-min incubation period with the stimulus alone, and a subsequent 10-min stimulation period either in the absence (control) or presence of the agents to be tested. A 20-min and a 10-min incubation in medium alone was interposed between the two stimulations in order to allow recovery to basal release. For most experiments, we analysed data on between six and eight vials; however, for additional or confirmatory data points, a minimum of four vials were subjected to analysis. The samples were collected and stored at -20 C until assay for GHRH and, in some experiments, also for SRIH, by RIA.

GHRHRIA Rat GHRH was measured according to the method of Tsagarakis et al. (10). Briefly, synthetic rat GHRH(l-43) (Bachem Inc., Torrance, CA) was used as standard and for iodination. For iodination it was labeled with 125I via the chloramine-T method and purified on an octadecylsilica column using a gradient of methanol containing 1% (vol/vol) trifluoroacetic acid. The GHRH antiserum (code RG5BM) was a gift from Dr. B. Rafferty (National Institute of Biological Standards, South Mimms, Herts, UK) and was used at a final dilution of 1:30,000. Antibody-bound [125I]GHRH was separated from free [125I] GHRH by the second antibody-polyethylene glycol assisted method (18). The sensitivity of the assay was 4 pg/tube (100n\ sample), with inter- and intraassay coefficients of variation of 12% and 6%, respectively. Cross-reactivity studies previously have been performed and have shown no significant cross-

Endo'1991 Vol 129 • No 1

reactivity with other hypothalamic peptides (10). Furthermore, Sephadex chromatography and HPLC have previously confirmed that hypothalamic GHRH co-elutes with synthetic rat GHRH(l-43). SRIH RIA RIA of SRIH was performed according to Penman et al. (11), using synthetic cyclic SRIH-14 (Bachem Inc.) as standard and for iodination with I25I by the chloramine-T method. Label was purified on an octadecylsilica column using a gradient of methanol containing 1% (vol/vol) trifluoroacetic acid. The assay procedure, which differs slightly from the previously described method, was as follows: standard curves were constructed using doubling dilutions of SRIH in assay buffer in the range 1-125 pg/ml. Assay buffer contained 0.05 M sodium phosphate buffer pH 7.4 and 0.4% human serum albumin (wt/vol). For assay of SRIH, 200 n\ of standard or 200 ix\ of sample containing 50 /A collected medium and 150 ^1 assay buffer (or 25 /xl medium and 175 /A assay buffer for measuring K+-evoked release) and 100 fA antirat SRIH serum were added and incubated for 24 h at 4 C. The SRIH antiserum (code R9) was a gift from Dr. J. Stewart (Harborview Medical Center, Seattle, WA) and was used at a final dilution of 1:80,000. After this, [125I]uTyr-SRIH tracer was added in 100 ^1 assay buffer. After 24 h incubation, separation of bound from free [125I]"Tyr-SRIH was carried out by double antibody precipitation using a final dilution of donkey antirabbit 7-globulin (IDS, Washington, UK) of 1:96 and a 1:1200 dilution of carrier rabbit serum. The sensitivity of the assay was 4 pg/tube. The intra- and interassay coefficients of variation were 12% and 10%, respectively. Previous studies have shown no significant cross-reactivity with other hypothalamic peptides; chromatography has shown that the peptides measured are SRIH (1-14) and SRIH (1-28), but that the molar ratio of the two peptides remains constant under potassium stimulation (12). Test substances iV-(6-aminohexyl)-5-chloronaphthalene-l-sulphonamide (W7), calcium ionophore A23187, and verapamil were supplied by Sigma (St. Louis, MO). 5-Iodo-C8 (W8) was a gift from Dr. G. M. Blackburn (Department of Chemistry, Sheffield University, Sheffield UK). Thapsigargin was supplied by Scientific Marketing Associates (Barnet, UK). W7, W8, and thapsigargin were dissolved in dimethylsulfoxide (DMSO) and further diluted in EBSS. The final concentrations of the solvent DMSO in medium was 1% or less. These concentrations of DMSO alone had no effect on basal or K+-stimulated GHRH or SRIH release. However, both W7 and W8 cross-reacted in the RIA for SRIH. This was not found for the GHRH assay. KC1, 56 mM, containing medium was prepared by decreasing the concentration of sodium to maintain isotonicity, with the concentration of the remaining salts being the same as in EBSS. Statistical analysis Each vial was incubated in series with incubation medium alone, followed by incubation in the presence of the test substance under investigation. For studying the effects of different agents on basal hormone release, the hormone release during

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Ca->+ AND CALMODULIN DEPENDENCE OF GHRH RELEASE

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(8)

1

18)

1.0

KCI-evoked GHRH release S2:S1 ratio

(8)

(4)

T

T

KCI-evoked GHRH release S2:S1 ratio

(8) T (4)

0.5

Control

10-6

10 s

10 4

1.0

oL

(8)

Control KCI-evoked SRIH release S2:S1 ratio

10"6

10"5

5x10"5

1CH

2x10' 4

W7 concentration (M)

FIG. 2. Effect of W7 on KC1 (56 mM)-evoked GHRH release. Bars represent means ± SEM of the S2:Sl ratio (see Materials and Methods). Number in parenthesis, number of incubation vials, each containing 1 hypothalamus. *, P < 0.05; **, P < 0.01, compared to control.

0.5

(8) (8) 0L Control

10"6

10"5

1.0

Verapamil concentration (M)

FIG. 1. Effect of verapamil on KC1 (56 mM)-evoked GHRH {upper panel) and SRIH (lower panel) release. Bars represent means ± SEM of the S2:Sl ratio (see Materials and Methods). Number in parenthesis, number of incubation vials, each containing 1 hypothalamus. *, P < 0.05; **, P < 0.01, compared to control.

KCI-evoked GHRH release S2:S1 ratio 0.5

the second (B2) and the first (Bl) incubations were measured for each vial, and the ratio of the two (B2/B1) computed. The ratios derived from hypothalami which were incubated in the presence of test substances during the second incubation were compared with parallel control incubations carried out within each experiment. Similarly, for investigation of stimulated release, the ratio between GHRH or SRIH release during the second stimulation period (S2) and first stimulation (Si) was calculated for each vial. Ratios of data incubated during the S2 period in the presence of different agents were compared to data from parallel incubations without such agents. The effects of individual doses of the test substances were then compared with parallel controls by means of Student's t test, with Bonferroni's adjustment for multiple comparisons where appropriate. Data are expressed as mean values ± SE; significance is set at P < 0.05.

Results KC1, 56 mM, caused a 2.5-fold increase in GHRH release (from 25 ± 5 to 62 ± 12 pg/hypothalamus • 10 min; mean ± SD; n = 8) and a 7.6-fold increase of SRIH (from 103 ± 36 to 784 ± 274 pg/hypothalamus • 10 min)

oL Control

10-6

10' 5

W8 concentration (M)

FlG. 3. Effect of W8 on KC1 (56 mM)-evoked GHRH release. Bars represent means ± SEM of the S2:Sl ratio (see Materials and Methods). Number in parenthesis, number of incubation vials, each containing 1 hypothalamus. **, P < 0.01, compared to control.

compared with the preceding incubation period without KC1 supplementation. Verapamil, in the dose range 10"610~4 M, produced a dose-dependent inhibition of K+evoked GHRH release (Fig. 1). Similarly, verapamil antagonized K+-evoked SRIH release (Fig. 1). W7 caused pronounced dose-dependent suppression of K+-induced GHRH release from 5 x 10"5 M to 2 x 10"4 M, whereas lower concentrations (10~6 M and 10~5 M) had no effect (Fig. 2). K+-stimulated release of GHRH was also

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Ca2+ AND CALMODULIN DEPENDENCE OF GHRH RELEASE

14

(4)

(10)

I

1 0

A23187 (1O"SM) evoked GHRH release S2:S1 ratio

Basal GHRH release B2:B1 ratio

(6)

(4)

(4)

(4)

Endo'1991 Voll29«Nol

• (4)

A23187 (lO^M) evoked SRIH release S2:S1 ratio

(4)

1

0.5

Control

2x1