GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

86, 359-377 (1992)

Relationship between Cyclic AMP-Stim~latad an Gonadotropin-Releasing Hormone-Stimulated Gonadotropin in the Goldfish JOHN P. CHANG,' Department

of Zoology,

ANDERSON 0. L. WONG,GLEN FREDRICKVANGOOR

University of Alberta, Edmonton, Alberta, University of Guelph, Guelph, Ontario,

VAN DER KRAAM,” AN Canada; Canada

and *Department

of Zoology,

Accepted October 2, 1991 The relationship between drugs elevating intracellular CAMP levels and gonadotropin (GTH)-releasing hormone (GnRH) in the stimulation of GTH secretion in the goldfish was investigated using dispersed goldfish pituitary cells in primary culture. In static incubation experiments, activation of adenylyl cyclase by forskolin and the inhibition of CAMP pbosphodiesterase by 3 isobutyl-I-methylxanthine (IBMX) increased CAMP release and stimulated GTH secretion. The addition of membrane permeant CAMP analogs, Sbromoadenosine 3’:5’-cyclic monophosphate @Br-CAMP), and dibutyryi CAMP also increased GTH release, suggesting that elevation of CAMP levels can induce GTH secretion. In the goldfish, dopamine is a physiological inhibitor of GTH release. Application of the dopamine agonist apomorphine decreased the GTH responses to forskolin, 8Br-CAMP, and salmon GT releasing hormone (sGnRH). The ability of agents that elevate CAMP levels to mimic GnRH action on GTH release suggests that CAMP may mediate GnRH-stimulated GTH secretion in the goldfish; however, this possibility was not substantiated by results from further experiments. In 2-hr static incubation studies, the GTH responses to sGnRH and chicken GnRH-II (cGnRH-II) were enhanced by coincubations with forskolin, IBMX, and 8BrCAMP. The magnitudes of these enhancements were at least additive, if not synergistic. The levels of CAMP released into the media were unaffected by treatment with sGnRH and cGnRH-II, either in the absence or in the presence of IBMX. Replacement of normal testing media with Ca”-deficient media (without Ca” salts and in the presence of 0.1 mM EGTA) decreased sGnRH and cGnRH-II stimulation of GTH release but did not affect forskolin and S&-CAMP actions. These results indicate that sGnRH and cGnRH-II stimulation of short term ( 0.05) are identified by the same underscore.

CAMP AND GnRH-INDUCED TABLE 1 STIMULATION OFCAMP AND GTH RELEASEFROM DEPERSEIPGOLDFISH PITUITARY CELLS BY F~RSK~LINANDIBMX

Treatment For&din 0 E 4 10 40

cAMP Release (pmoliml)

GTH (ng/mU0.25 million cells)“,b

(pMJ

~mJfX (ww 0 I 10 loo 1000

0.60 3.64 20.30 27.12 57.76

f + + t lr

0.06” 0.54b 0.80” 0.95’ 11.74*

0.58 0.63 0.64 I.04 1.05

+k +r I?I

0.04a 0.08” 0.04a 0.04b O.lSb

463 2 711 + 80.5 + 911 f 988 +

19” 26bSC 22”~~ 70d2’ 32’

466 496 488 578 769

21a 29” 22a 29b 75’

+ f k f k

Note. Results (mean 2 SE) are from one of the replicate experiments using pituitary cells from sexually mature fish (April) included in Fig. 1. a CAMP and GTH levels that are similar are identified by the same superscript. ’ GTH data have been included in Fig. 1.

tively. Dispersed pituitary cells obtained from male and female goldfish undergoing gonadal recrudescence as well as from sexually mature (i.e., prespawning) fish were used (February to May). Results were similar regardless of the gonadal conditions of the donor fish. Pooled results are presented in Fig. 2. Exposure of dispersed goldfish pituitary cells to 0.1 to 1 ~.LMand 100 to 2000 ~JLMgBr-CAMP significantly elevated GTH contents in the testing media (Fig. 2, top). Similarly, additions of 0.1 pM and 10 to 2000 FM db-CAMP significantly stimulated GTII release (Fig. 2, bottom). The effects on GTH release of interactions of IBMX with forskolin or 8Br-CAMP were monitored in four replicate experiments, once each with dispersed cells obtained from male and female goldfish undergoing gonadal recrudescence (March) or from sexually mature fish (May), and twice with pituitary cells obtained from fish with regressed gonads (July and August). Similar results were obtained from all four ex-

GTH RELEASE

IN GOLDFISH

periments. Pooled results Fig. 3. Incubation with 10 100 pM IBMX, and 1 m creased GTH secretion. In t 100 pM IBMX, the GTW r to 10 pM forskolin and 1 were not significantly differ-en other or from values obse with 10 pM forskolin alone. vations indicate that stimulation o lease by CAMP-dependent mechanis maximized during coincubation wi pM IBMX or 10 l~lM forskolin. The GTH responses to treatment forskolin and 1,9-dideQxy-f~rskoli~~ a active forskolin analog, were co three replicate experiments using cells from male and female goldfis stages of gonadal recrudescence ber). The pooled resul Fig. 4. Additions of 1 significantly increased ever, the applie dideoxy-forsko tion. Inleractions with the dopamine APO. As indicated in the introduct tivation of dopamine D2 GTM secretion. The influ mine agonist APO on s three replicate experiments using cells obtained from sexu and female goldfish (J the pooled results are presen Incubations with 100 nM sG

male and female goldfish and cells from fish

364

CHANG

ET AL.

1

10

8BrcAMP

100

1000

2000

(PM)

180 GJ-H 160 B

0 0.001~0.110 --

1002ooo looo

140 120

80 0

0.001

0.01

0.1 dbcAMP

1

10

100

1000

2000

(@I)

FIG. 2. Dose-dependent stimulation of GTH release by 8Br-CAMP (top) and db-CAMP (bottom). Results presented (mean -+ SE) are pooled from three replicate 8Br-CAMP and five replicate db-CAMP experiments, respectively. Average basal GTH values are 377 k 56 and 309 f 20 rig/ml/0.25 million cells for the 8Br-CAMP and db-CAMP experiments, respectively. In each panel, treatments producing similar GTH responses (ANOVA and Fisher’s LSD test, P > 0.05) are identified by the same underscore.

undergoing gonadal regression (June). In both experiments, sGnRH and cGnRH-II stimulated GTH release, but the amounts of CAMP released into the media were not altered by GnRH treatments (Table 2). Interactions of sGnRH or cGnRH-II with IBMX, forskolin, or 8Br-CAMP. The inter-

actions between sGnRH and IBMX were examined once using dispersed pituitary cells from male and female goldfish during late stages of gonadal recrudescence (March) and an additional three times with pituitary cells from fish undergoing gonadal regression (June to July). Results from these four replicate experiments are similar and the pooled results are presented in Fig. 6A. Additions of sGnRH increased GTH

release in a dose-dependent manner. Coincubation with 100 pM IBMX increased basal GTH release and enhanced the GTH responses to sGnRH. The GTH responses to 1 to 100 ti sGnRH in the presence of IBMX were greater than the added sum of the responses to IBMX and the corresponding doses of sGnRH alone. The interactions between cGnRH-II and IBMX were examined in three replicate experiments using dispersed pituitary cells from male and female goldfish undergoing gonadal regression (June and July). Pooled results are presented in Fig. 6B. cGnRH-II increased GTH release in a dose-dependent manner. In these experiments with cGnRHII, treatment with 100 pM IBMX slightly

cAMP

”

200

2E!

180

#

160

iii

140

s

120

AND

GnRH-INDUCED

GTH

RELEASE

IN

GOLDFISH

100 80 Control

Ford

SBr-CAMP

Control

For&

8Ba-cAMP

FIG. 3. Effects of coincubation with 100 pM IBMX on the GTH responses to 10 FM forskoiin (Forsk) and 1 mM $&-CAMP. Pooled results (mean ? SE) from four replicate experiments are presented. The average basal GTH value in these experiments is 166 f 28 ng/mVQ.25 million cells. Treatment groups are identified by numbers, and treatments producing similar GTH responses (ANOVA and Fisher’s LSD test, P > 0.05) are identified by the same underscore.

increased basal TH values, but no statistical significan could be demonstrated when compared with untreated controls. The GTH responses to cGnRH-II were also increased by coincubation with IBMX. The GTH release responses to 1 to 100 nM cGnRI-I-II in the presence of IBMX were greater than the added sum of the responses to IBMX and the corresponding doses of c II-II alone ~ easurements of CAMP were also performed on sampIes from one of the above experiments testing the effects of IBMX ad-

dition on sGn

are presented in Tab IBMX alone elevated c

urthermore neicompared to controls. ther of these two native tered the amount of CA It may be argued that there is a tendency of treatment wit

160

0

.l Concentration

1 (@VI)

IO

100

FIG. 4. Comparison of the GTH releasing ability of forskolin and its inactive analog, dideoxyforskolin. Pooled results (mean 2 SE) from three replicate experiments are presented. average basal GTH value in these experiments is 140 i 10 ng/m1/0.25 million cells. For forskolin 1,9dideoxyforskolin treatments, concentrations producing similar GTH responses (ANGVA Fisher’s LSD test, P > 0.05) are identified by the same underscore.

P,9The and and

366

CHANG ET AL.

?i 3 a 8 g u

160 140 120 100

control

Forsk8Br-CAMP

control

ForskgBr-CAMP + APO

sCnRH

sGnRH + APO

5. Inhibition of the GTH responses to 10 p,,%fforskolin (Forsk), 1 m&I SBr-CAMP, and 100 ti sGnRH by 1 pM apomorphine (APO). Pooled results (mean 5 SE) from three replicate experiments are presented. The average basal GTH value in these experiments is 84 +4 ng/m1/0.25 million cells. Treatment groups are identified by numbers and treatments producing similar GTH responses (ANOVA and Fisher’s LSD test, P > 0.05) are identified by the same underscore. FIG.

cGnRH-II alone to increase CAMP release (Table 3) but no such trend exists inother cGnRH-II experiments (Table 2). Furthermore, there is even a tendency for cGnRHII to reduce CAMP levels when CAMP degradation has been blocked by IBMX (Table 3), indicating that neither sGnRH nor cGnRH-II increased CAMP release. The effects of coincubation with 10 @‘k! forskolin or 1 mM 8Br-CAMP on sGnRH and cGnRH-II stimulation of GTH release were also examined. Dispersed pituitary cells prepared from male and female goldfish undergoing early stages of gonadal recrudescence were used (December to February). Pooled data from four replicate experiments are presented in Fig. 7. Treatments with 100 nM sGnRH, 100 nM cGnRH-II, 10 PM forskolin, and 1 mM SBrCAMP alone all increased GTH release. The GTH responses to the addition of sGnRH, cGnRH-II, and 8Br-CAMP alone were similar to each other, but all were significantly lower than the forskolinstimulated GTH release. Coincubations of sGnRH or cGnRH-II with forskolin or SBrCAMP resulted in GTH responses that were greater in magnitude than those observed in treatments with these secretagogues alone.

Extracellular Ca 2t-dependence of forskolin, 8Br-CAMP, and GnRH action. The

effects of replacement of normal testing media with Ca2+deficient media on the GTH responses to forskolin, SBr-CAMP, sGnRH, and cGnRH-II were compared in four replicate experiments, once with pituitary cells from fish undergoing gonadal regression (June) and three times with pituitary cells from fish undergoing early stages of gonadal recrudescence (December to February). Results from all replicate experiments are similar regardless of the state of gonadal maturation of the fish. Pooled results from these experiments are presented in Fig. 8. The GTH responses to 100 nM cGnRH-II were similar to those to 10 PM forskolin, and both of these responses were significantly greater than those stimulated by 100 nM sGnRH or 1 mM 8Br-CAMP. Incubation with Ca2+-deficient media reduced the GTH release induced by 100 nM sGnRH and 100 nM cGnRH-II; in contrast, the GTH responses to 10 PM forskolin and 1 mM 8Br-CAMP were not affected. Under e-Ca’+-deficient conditions, 100 nM sGnRH, but not 100 nM cGnRH-II, still significantly elevated GTH secretion above the corresponding controls.

CAMP AND GnRH-INDUCED TABLE 2 cAMP AND GTH RELEASE RESPONSESTO TREATMENTWITH sGnRH AND cGnRH-II

Treatments Experiment Control sGnRH In%4 100 ill%4 cGnRH-II lnlw 100 nh4

CAMP Release (pmoliml)”

GTH (ng/m1/0.25 million cells)b

1 (sexually mature, April) 0.58 ” 0.03 458 + 9a 0.57 k 0.04 8.40 2 0.07

596 t 2Sb 6.51 -+ 30b

0.61 2 0.08 0.63 ? 0.09

6.54 +- 26b 659 k 15b

Experiment 2 (regressing, June) Control 0.72 f 0.02 120 rt sGnRH 0.1 nM 0.66 i 0.05 11.5 +0.1 i-&if Q.71 2 0.03 123 -t 1nM 0.65 z!z0.04 145 rt 10 nM 0.76 f 0.04 146 +100 n&f 0.73 i- 0.04 148 k cGnRH-II 0.01 nM 0.71 2 0.02 148 + 0.1 nM 0.69 t- 0.04 144 2 1 nM 0.70 I 0.03 156 2 10 nM 0.78 t 0.05 152 2 100 nM 0.72 k 0.04 170 +

GTH RELEASE

IN GOLDFISH

367

ration of the GTH responses to initial lenges by 5-min pulses of 100 alone was 3.4 * 0.3 fractions (n = 18); t GTH response was considered ter~~~a~e when the GTH value return than) that observed at the ti cation of the test pulse. The GTH release responses as we1 tion of the response, were calculated an the results are presented in Fig. 10. ations of four 5-min

6a 5” 3” cib 2b 4b 9b 3b 3b 5b.C 6’

~Vote. Results (mean k SE) from two separate experiments using pituitary cells prepared from fish at two different stages of gonadal development are presented. a No significant differences were observed with cAMP values in both experiments. b In each experiment, GTH values that are similar are identified by the same superscript.

Kinetics of the forskolin and IBMX action and their interactions with sGnRH. The acute GTH responses to 10 pM forskolin, BOO pM IBMX, and 100 n&I sGnRH were monitored in cell column perifusion studies. Data from three replicate experiments using dispersed cells obtained from sexually regressed male and female goldfish (Jnly to September) were expressed as a percentage of the pretreatment GTH value and pooled, and the results (mean + SE) are presented in Fig. 9. Basal GTH release decreased by approximately 50% during the experiment in all columns. The average du-

All three parameters

of the GT

test pulse stimulated were not significantly corresponding contra terms of the magnitude of th tal response or the duration

IBMX

did not

100 IEM sGnRH alone to 5-min pulses of sG of previous drug exposures. when compared to controls (Fig. IO). ~~rn~are~ to control columns, ~~rn~~ta~eo~s a~~~~~~~~a~

were longer in durat in magnitude in both

CHANG ET AL.

368

180 -

170 160 = I

150 -

0

.Ol

.I cGnRH-II

1

10

100

(nM)

FIG. 6. Enhancement of the GTH responses to (A) sGnRH and (B) cGnRH-II by coincubation with 100 pJ4 IBMX. Pooled results (mean f SE) from four replicate experiments with sGnRH and three replicate experiments with cGnRH-II are presented. The average basal GTH values are 190 i 32 and 121 k 5 rig/ml/0.25 million cells for the experiments with sGnRH and cGnRH-II, respectively. For incubations in the absence (normal) or the presence of IBMX (+IBMX), sGnRH, or cGnRH-II treatments producing similar GTH responses (ANOVA and Fisher’s LSD test, P > 0.05) are identified by the same underscore.

ulated GTH releases that were significantly greater in total magnitude and duration, but not in the magnitude of the rapid response (Fig. 9, middle and bottom, and Fig. 10). In a preliminary perifusion experiment using dispersed pituitary cells from male and female goldfish during sexual recrudescence (February), simultaneous application of 10 l.~44 forskolin and 100 nJ4 sGnRH similarly enhanced the duration and the magnitudes of both the rapid and the total GTH responses as compared to applications of 100 n44 sGnRH alone (n = 2; data not shown). However, basal GTH release

showed only a slight decrease (615%) in these experiments with pituitary cells from fish during sexual recrudescence, as in previous perifusion studies (Chang et al., 199Oc). DISCUSSION

Results from the present study indicate that elevation of CAMP increases GTH release from goldfish pituitary cells and may partly mimic GnRH action, but it is highly unlikely that CAMP mediates sGnRH and cGnRH-II stimulation of GTH release in the goldfish.

cAMP AND GnRH-INDUCED TABLE 3 EFFECTS OF TREATMENT WITH sGnRH AND cGnRH-II ON CAMP RELEASE IN THE ABSENCE OR PRESENCE OF 0.1 mM IBMX GnRH Concentration (n-w

GTH RELEASE

IN GOLDFISH

369

Levavi-Sivan, 1990) and the African catfish (van Asselt, 1989). presence of fun&o teleost pituitary fragment p

CAMP Release (pmol/ml) Normal”

+ IBMX (0.1 nL%l)b

system cannot be determined 0.57 0.50 0.60 0.57 0.63 0.63

+- 0.03a,b t 0.03b r 0.03” I Q.O2”,b +- 0.03” -c-0.03”

0.74 0.62 0.66 0.60 0.69 0.74

2 f * k i i

0.51 0.48 0.55 0.62 cl.62 0.67

+ r i. i r 2

0.71 0.62 0.64 0.65 0.62 0.66

+ 0.04 zk 0.03 t 0.03 + 0.03 I 0.04 rt 0.03

o.04”.b 0.04b 0.05”sb 0.08”.b 0.07”.b 0.08”

0.07 0.05 0.04 0.05 0.04 0.08

iVote. Results presented (mean 2 SE) are from one of the three replicate sGnRH experiments and two of the three replicate cGnRH-II experiments, all performed with pituitary cells obtained from sexually regressed fish (June and July). GTH data from these experiments have been included in Fig. 6. L2For sGnRH or cGnRH-II treatments under normal incubation conditions, CAMP levels that are similar are identified by the same superscript. b No significant differences in CAMP levels were observed for the sGnRH + IBMX or the cGnRH-II + IBMX treatment groups.

Consistent with the hypothesis that elevation of CAMP directly stimulates GTH release, addition of forskolin, a direct stimulator of adenylyl cyclase, and IBMX, an inhibitor of phosphodiesterase, elevated CAMP release and GTH secretion in a dosedependent manner from primary cultures of dispersed goldfish pituitary cells under static incubation conditions. Similarly, the addition of membrane permeant analogs of P, and 8Br-CAMP also caused dose-dependent increases in GTH release. Previously, it has been reported that the addition of CAMP analogs or forskolin stimulated GTH release from pituitary fragments of tilapia (Yaron and

in

CAMP synthesis in shown to increase

+ channels in moll

The EC,, values (l-20 iu. dependent and -independent actions of f5rskolin are similar, and both values are

skolin, an analog of forskolin K+ channels but is inactive ’ adenylyl cyclase (ECSa > 50

H release from She cells of the goldfish. the observation that

370

CHANG ET AL. 240

Control

Conhol s’&RH CcnRH-n

sChRH d&RH-II

+ Forskolin

Conhol SGURH dMH-n + 8Br-cAMP

FIG. 7. Enhancement of the GTH responses to 100 r&f sGnRH and 100 nJ4 cGnRH-II by coincubations with 10 pii forskolin and 1 nu%48Br-CAMP. Pooled results (mean + SE) from four replicate experiments are presented. The average basal GTH value in these experiments is 336 f 26 ngmUO.25 million cells. Treatment groups are identified by numbers and treatments having similar GTH responses (ANOVA and Fisher’s LSD test, P > 0.05) are identified by the same underscore.

role of dopamine D2 receptor-mediated inhibition of basal and GnRH-stimulated GTH release in the regulation of the preovulatory GTH increase in the female goldfish, as well as the role of dopamine in the regulation of brain GnRH activity during spawning behavior in male goldfish, has been documented (Peter et al., 1986, 1990; Yu and Peter, 1990). Like sGnRH in the present study, forskolin treatment caused a rapid increase in GTH release, and the for180

241093857

skolin- and SBr-CAMP-induced elevation in GTH secretion were attenuated by the dopamine agonist APO. These results indicate that CAMP-dependent stimulation of GTH release is physiological, is under the regulation by dopamine, and mimics GnRH action. Since dopamine D2 receptor activation has been linked to a decrease in adenylylcyclase activity in mammals (see introduction), these findings also suggest the possibility that CAMP-dependent path-

--

160 E

80 COlltd

sGnRH

CGIIRH-II

Forskolin

8Br-CAMP

8. Effects of the replacement of normal testing media with Ca 2+-deficient media (without Ca2’ salts and with 0.1 m&4 EGTA) on the GTH responses to 100 nM sGnRH, 100 n&f cGnRH-II, 10 @4 forskolin, and 1 mM 8Br-CAMP. Pooled results (mean 2 SE) from four replicate experiments are presented. The average basal GTH value in these experiments is 269 +- 11 ng/ml/O.25 million cells. Treatment groups are identified by numbers, and those having GTH values (ANOVA and Fisher’s LSD test, P > 0.05) are identified by the same underscore. FIG.

cAMP AND GnRM-INDUCED

10

GTH RELEASE

20

30

371

IN GGLDFIS

40

so

60

40

58

60

Fraction Number

10

20

30

Fraction Number

401

. 0

I

10

.

1

.

20

1

30

.

,

40

I

#

50

.

*

QQ

Fraction Number 9. CTH responses to S-min pulses of 100 n&4 sGnRH, 10 I& forskolin, and 100 p+M IB cell. column perifusion studies. Results presented (mean + SE; controls, top; forskolin treatment, middle; IBMX treatment, bottom; n = 6 columns per panel) are pooled from three replicate experiments using dispersed goldfish pituitary cells from fish with regressed gonads. GTH values are expressed as a percentage of pretreatment. Applications of 5-min pulses of the different secretagogues are identified by the arrows. FIG.

ways may be involved in the actions of the two native GnRH peptides on goldfish gonadotropes. However, results from other experiments in the present study (discussed below) strongly indicate that CAMP does

not mediate sGnRH and cG lation of GTH release in the Several observations in cate that CAMP does not p GTH release response to the two native

372

CHANG ET AL. A) Rapid Response 200

P 4 v& k

180 160 140 120 100 so 60 40 20 0

B) Total Response Column j-J conuol

n L

E z1

50 0

Forskolin

EI’BMX

Pulse1

Pulse 2

Pulse 3

Pulse 4

C) Duration 14 12 10 8 6 4 2 0

Pulse 1 Pulse 2 10. Summary of the GTH responses to 5-min pulses of 100 nM sGnRH, 10 pM forskolin, and 100 pit4 IBMX in cell column perifusion studies. Results presented (mean 2 SE) are the estimated (A) rapid and (B) total response, and (C) duration of the responses calculated from data presented in Fig. 9. For each set of columns, pulses 1 to 4 refer to the corresponding sequential test treatment pulses as illustrated in Fig. 9. (A, P < 0.05, within pulse comparisons, ANOVA and Fisher’s LSD test). FIG.

GnRHs in the goldfish. Coincubation with IBMX significantly elevated the GTH responses to sGnRH and cGnRH-II; the enhancement of the GTH release was more than additive even at maximal sGnRH and cGnRH-II doses of 100 nJ4. Cotreatment

with 1 rn44 8Br-CAMP or 10 pJ4 forskolin increased the GTH responses to both 100 nM sGnRH and 100 nA4 cGnRH-II under static incubation conditions. Applications of 10 $W forskolin together with 100 n44 sGnRH also produced a greater GTH re-

CAMP

AND

GnRH-INDUCED

sponse than that induced by sGnRH or forskolin alone in cell column perifusion studies. Furthermore, sGnRH and cGnRH-II addition did not increase CAMP release in either the presence or the absence of IBMX; also, no correlation was found between CAMP levels and the GTH release response during GnRH treatments with or without IBMX (results not shown). In addition, sGnRH and cGnRH-II actions on GTH release are dependent on e-Ca*‘, as in previous studies (Chang et al., 1990a,b,c, 1991a; Jobin and @hang, 1990), whereas the responses to forskolin and 8Br-CAMP were not affected by incubation with Ca*+deficient media. Moreover, in a preliminary experiment, 1 p& H8, a partially selective inhibitor of cyclic nucleotide-dependent protein kinases, reduced the GTH response 10 t.& forskolin, but not the responses to nILa sGnRH or 10 nM tetradecanoyl orbol-13-acetate (TPA), a PKC activator (GTH responses to control, forskolin, sGnRH, and TPA treatments were 272 % 6, 333 .YL14,325 rt 11 and 656 2 60 nglmV0.25 million cells in the absence of HS and 271 + 16,264 & 10, 344 ? 11 and 712 & 22 rig/ml/ 0.25 million cells in the presence of H8, respectively). It should be noted that IBMX is also a known blocker of adenosine Al receptor. nosine modulates the hormone actions secretory processes in mammalian crine and neuroendocrine tissues (Preston et ai., 1987; Gross et al., 1987; Nikodijevic and Klein, 1989), and the stimulation of Al receptor inhibits sGnRHstimulated GTH release in dispersed goldfish pituitary cells (J. P. Chang and H. R. Habibi, unpublished results). Therefore, sent potentiating effects of IBMX on stimulated GTH release can also be ed by the release from adenosine inhibition. Nevertheless, om the current study support the hypothesis that CAMP-dependent chanisms do not mediate sGnRH and r&H-II stimulation of GTH release in

GTH

RELEASE

EN GOLDFHS

373

the goldfish; a similar lack of c participation has also been proposed for s term GnRH stimulation of L mammals (see introduction). In spite of the lack of evidence for the involvement of CAMP-dependent met nisms in sGnRH and cGnRH-II action on short-term (~2 release, results from the present investigation ~o~s~ste~t~y demonstrated the ability of stimulators of CAMP-dependent processes to enhance GnRH-stimulated GTH release. malian pituitary cell ~reparat~~~s 1 els mediates

Gn

mRNA transcription, an (for a review, see Couni Under static ~~c~batio~ conditions in the present study, i.t is possible that some of these actions of CAMP-dependent processes mediate the effects of forskolin,

ceptor upregulati~m by c the transcriptional, trans translational responses usua lag of at least 30 min to actions of forskolin and experiments in the present study were almost instantaneous ( hancement of the s GTH release by forskolin and I fected the magnitude o phase (first 1.5 min) as well as the duration of the entire response, The possibility that G lize camp-de~ende~t

374

CHANG

CAMP release response following 2-hr static incubations with sGnRH and cGnRH-II in the presence of IBMX does not support a role for CAMP in mediating long-term GnRH actions. The absence of a CAMP release response following 2-hr sGnRH and cGnRH-II treatment in both the presence and the absence of IBMX is at variance with other studies. GnRHstimulated CAMP production in rat anterior pituitary cells following a 2-hr incubation with or without the addition of IBMX (Sen and Menon, 1979). Perifusion of pituitary fragments of the African catfish by a mammalian GnRH analog, Buserelin, has been reported to increase CAMP release (van Asselt, 1989). Species and tissue-preparation differences may account for these divergent observations. However, it should be noted that the dispersed pituitary cells used in the present study are a mixed population of all pituitary cell types, and that GnRH peptides do not act on all these cell types. Although it is unlikely, small CAMP responses may have been undetected. Primary cultures of dispersed goldfish pituitary cells retain some of the seasonal properties, as in intact pituitaries in vivo, but the CAMP-dependent stimulation of basal GTH release and enhancement of GnRH-stimulated GTH release do seem to have obvious seasonal differences. In the present investigation, the average basal GTH release from static incubations of dispersed goldfish pituitary cells varied from between 100 and 150 ng/m1/0.25 million cells in pituitary cell cultures obtained from sexually regressed fish, to between 150 and 300 ng during gonadal recrudescence, to between 300 and 450 ng when the fish were sexually mature (prespawning). These seasonal changes in in vitro basal GTH release parallel the reported seasonal variation in basal serum GTH levels and the sensitivity to GnRH challenges in viva (Habibi et al., 1989). Seasonal differences in the stimulatory ability of neuropeptide Y (NPY) on GTH release have also been demonstrated

ET AL.

(Peng et al., 1990). In contrast, forskolin and 8Br-CAMP elevated GTH secretion in cultures of dispersed pituitary cells obtained~from goldfish with gonads that were sexually mature (i.e., prespawning), regressed, or undergoing gonadal recrudescence, without consistent seasonal differences in their effectiveness. Similarly, forskolin treatment in static incubation and cell perifusion experiments, and IBMX addition in static incubation studies, enhanced the GnRH-stimulated GTH release from dispersed goldfish pituitary cells regardless of the state of gonadal maturation of the donor fish. Previously, we have hypothesized that sGnRH and cGnRH-II stimulation of GTH release in the goldfish may be mediated by dissimilar mechanisms. Compared to sGnRH, cGnRH-II action on GTH release from goldfish pituitary cells has been shown to be more effective, more dependent on e-Ca2+ and e-Ca2+ entry through VSCC, more sensitive to inhibition by APO and other dopamine D2 agonists, and more dependent on PKC, and in contrast to sGnRH, cGnRH-II action does not involve an arachidonic acid metabolism component (Chang et al., 1990a,b,c, 1991a,c; Jobin and Chang, 1991). Similar to these reports, 100 niW cGnRH is more potent than 100 nM sGnRH in stimulating GTH release from the same cell preparation in most of the experiments included in this paper (Table 2; Figs, 6-S). The difference in dose-response characteristics (Fig. 6), as well as the difference in the e-Ca2+-dependence of the GTH response to sGnRH and cGnRH-II, is also confirmed in the present study (Fig. 8). These data lend further support to the hypothesis that the GTH responses to sGnRH and cGnRH-II are mediated differently. Despite these differences, the GTH response to both of these GnRH peptides is enhanced by the activation of CAMP production and CAMP-dependent signaling components. Based on the present results, it is possi-

CAMP AND GnRH-INDUCED

ble that another neuroendocrine regulator directly regulates GTH release and synthesis, and potentiates GnRH action in the goldfish via cAMP-dependent mechanisms, pathways that are independent of those utilized by the GnRH peptides to induce GTH release. In rats, corticotropin release is stimulated by the synergistic actions of corticotropin-releasing factor, and angiotensin II or vasopressin, via the activations of the CAMP- and the Ca2’- and phospholipiddependent PKC systems, respectively (Abou-Samra et al., 1986). Whether a similar synergistic interaction exists between P- and Ca2+fPKC-dependent mechain goldfish gonadotropes to regulate rapid GTH release has not been directly investigated. However, as described in the introduction, GnRH actions in fish have been shown to be dependent on e-Ca2+ and PKC activation. The identity of the possible neuroendocrine regulator that may stimulate CAMPdependent mechanisms to directly elicit GTH release and to potentiate GnRH actions in the goldfish is not known, but several identified stimulatory regulators of GTH release in the goldfish can be eliminated as potential candidates. These include norepinephrine (NE), serotonin ), y-aminobutyric acid (GABA), and . The receptor subtypes that have shown to mediate their stimulatory actions on goldfish GTH release are not traditionally coupled to increases in adenylyl cyclase activity (NE acts through al receptors, Chang et al., 1991b; 5HT acts through 5HT2 receptors, Somoza et al., 19881, OF these regulators do not act directly on pitucells (GABA, Kah et al., 1992; Chang, unpublished results), or tions with GnRH are only additive at best (NE, Chang et al., 1991b; C. Peng, J. P. Chang, F. Van Goor, OE. Peter, unpublished results). As for dopamine, activation of Dl receptors has been linked to activation of adenylyl cyclase in mammals; however, in tbe gold-

GTH RELEASE

fish pituitary

IN GOLDFISH

375

cell preparations,

tion results in the i~bibition of GT (Chang et al., 1990~; Peter et a In summary, elevations of CA and the activation of

independent manner, but CAMP appear to be involved in medi GTH-releasing actions of sGn cGnRH-II. CAMP analogs or agents evate intracellular cAMP levels gre hance the GTH release response to the na-

GTH release response will be further investigated.

This study was funded by NSERC Grants U-0552 and U-0554 to J.P.C. and G.V.D.K., respectively. A.O.L.W. was supported by a graduate teaching assistantship from the Dept. of Zoology, Univ. of berta, and by funds from NSERC Grant A-6371 to R. E. Peter. We are grateful to Dr. K. J. Catt of the Endocrinology and Reproduction Research Branch, NICHD, NIH for the gift of the antiserum B4-Sept-74 used in the cAMP radioimmunoassays.

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