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Stimulation of luteinising hormone release by luteinising hormone-releasing hormone in the porcine anterior pituitary: The role of cyclic AMP
Molecular and CellularEndocrinology, 10 (1978) 321-341 o Elsevier/North-Holland Scientific Publishers, Ltd.
STIMULATIONOFLUTEINISINGHORMONERELEASE BYLUTEINISING HORMONE-RELEASINGHORMONEINTHEPORCINEANTERIOR PITUITARY:THEROLEOFCYCLICAMP Ameae M. WALKER * and Cohn R. HOPKINS Department of Histology and Cell Biology (Medical), The Medical School, Universityof Liverpool, P.O. Box 147, Liverpool L69 3BX, U.K. Received 25 October 1977; accepted 10 January 1978
Cells dissociated from porcine pituitary glands have been used to examine the relationship between cyclic AMP levels and the stimulation of luteinising hormone (LH) secretion by luteinising hormone*eleasing hormone (LHRH). It has been shown that while dibutyryl cyclic AMP and theophylhne have a modest and, under some conditions, an inhibitory effect on LH release, the phosphodiesterase inhibitors isobutylmethylxanthie and ICI 63197 both induce LH release and enhance LHRH-induced release. The prostaglandins Er and Ea and cholera toxin, which are shown to raise cyclic AMP levels in the dissociated preparation, also induce LH release and increase LHRH-induced LH release. Finally, LHRH is shown to increase the total content of cyclic AMP in the mixed celJ preparation by approximately 30% and it is concluded that, in this system, the nucleotide does play a role in coupling LHRH stimulation to LH release. Keywords: cyclic nucleotides; gonadotropins;
hypophysiotropic
hormones.
The requirement for cyclic AMP in the coupling of LHRH stimulation to LH secretion has been extensively studied using rat pituitary preparations. Nevertheless, although a large body of information on the topic now exists, the question remains unresolved. The evidence in favour of the view that LHRH causes a rise in cyclic AMP which is in turn directly responsible for LH release includes (1) the observation that LHRH can activate adenyl cyclase in both crude homogenates (Deery and Howell, 1973) and plasma membrane fractions (Spona, 1975), (2) that LHRH stimulation causes a rise in the intracellular level of cyclic AMP (Borgeat et al., 1972; Kaneko et al., 1973; Labrie et al., 1973; Makino, 1973; Yanaihara et al., 1973; Naor et al., 1975; Yoshida et al., 1975), (3) that exogenous cyclic AMP will stimulate LH release (Ratner, 1970; Labrie et al., 1973; Makino, 1973) (4) that phosphodiesterase inhibitors induce LH release and potentiate LHRH stimulation
* Present address: Section for Cell Biology, Yale Medical School, New Haven, Corm. 06510, U.S.A. 327
328
A.M. Walker, C.R. Hopkins
(Ratner, 1970; Makino, 1973: Wakabayashi et al., 1973; Tang and Spies, 1976). and (5) that substances shown to increase intracellular levels of cyclic AMP also increase LH release (Ratner, 1970; Makino, 1973). Other studies would not, however, support this view since they have either failed to demonstrate that cyclic AMP (Wakabayashi et al., 1973; Sundberg et al., 1976; Tang and Spies, 1976) and/or phosphodiesterase inhibitors (Wakabayashi et al., 1973; Tang and Spies, 1976) significantly stimulate LH release or they have shown that, while agents such as prostaglandin E, and cholera toxin will increase intracellular levels of cyclic AMP, only LHRH will stimulate LH release (Naor et al., 1975). Recently we have developed a dissociated cell preparation from the porcine anterior pituitary which, in response to LHRH, releases LH in a consistent and predictable manner (Walker and Hopkins, 1977). This preparation is sensitive to LHRH stimulation with a half-maximal response at 4 X lo-‘* M and allows dose-response curves to be derived with less than a 1% variation between replicate aliquots. Moreover, since these preparations can be routinely obtained from abattoir material, quantitative assays for regulatory agents such as cyclic nucleotides are relatively unconstrained by the availability of material. In work reported here we have used this preparation to examine the relationship between cyclic AMP and LHRHstimulated LH release in the porcine pituitary. For conformity we have, as far as possible, used the agents, dose levels and time points used in previously reported studies. We conclude that in this system cyclic AMP does play a role in the LHRHstimulation of LH release.
MATERIALS AND METHODS Materials
The sodium salts of N602 dibutyryl cyclic adenosine monophosphate (dbcAMP), adenosine triphosphate (ATP), adenosine monophosphate (AMP) and cyclic guanosine monophosphate (cGMP) and theophylline were obtained from Sigma (London), Surrey, U.K., and guanosine ~phosphate (GTP) and cyclic adenosine monophosphate (CAMP) (sodium salts) were obtained from Boehringer Corp. (London), Ealing, London, U.K. [8-3H]cAMP was obtained from the Radiochemical Centre, ~er~am, Bucks, U.K. and Norit GSX was kindly supplied by Norit, Clydesdale, Glasgow, UK. Prostaglandins Er and E2 were generously provided by Dr. J.E. Pike, Upjohn, Kalamazoo, Michigan, U.S.A. and ICI 63197 was a gift from Dr. M. Dukes, ICI Pha~aceutic~s, Maccles~eld, U.K. Cholera toxin (lot No. 0972) was kindly supplied by Dr. R.A. Finkelstein under the auspices of the NIAMDD, Bethesda, Md., U.S.A. ~ss~ci~ti~n of pigsty tissue and i~~~~ti~n of diss~c~ted cells The rationale of the method used for dissociation will be published in detail
Stimulation of LH release by LHRH
329
elsewhere (Walker and Hopkins, 1978). In outline the method involves the sequential incubation of chopped tissue blocks in collagenase (0.075% in Krebs-Ringer bicarbonate (KRB) (Krebs, 1950) for 60 min) and trypsin (0.5% in KRB minus Ca’+ and Mg’+) followed by gentle pipetting to disperse the tissue. Cell debris was removed from the suspension by low-speed centrifugation (250 g for 3 min) through 4% bovine serum albumin. Following their resuspension in Dulbecco’s modified Eagle’s minimum essential medium (DMEM) the cells were assessed for viability (normally X5%) and then aliquotted in DMEM + 10% foetal calf serum either into 30~mm petri dishes coated with poly(L-lysine) or into 90.mm petri dishes containing 2 ml 6% gelatin. For the measurement of LH-release dissociated cells attached to poly(L-lysine)coated petri dishes and incubated for 48 h in DMEM/lO% foetal calf serum were used. Each experiment was performed in triplicate. Serum was removed with three S-min rinses in DMEM and then incubated in this serum-free medium for 2 h at 37’C. As an intra- and interexperimental control a dose-response range from lo-” M-10+ M LHRH was normally included in the protocol. LHRH was added (25 1.11to 3 ml DMEM) as a concentrated solution in phosphate-buffered saline. Other additions were made to the medium directly before use, and where the solvent was other than aqueous it was checked for its effect on LH release. (All were without effect). Media were collected, centrifuged to removecell debris at 3000 g and then stored at -20°C until assay. LH was measured by radioimmunoassay using an antiserum raised against LER-7784 (Walker and Hopkins, 1977) which bound 50% iodinated LH (LER786-3) at a final dilution of 1 : 150,000. The cross-reactivity in the range of l-30 ng/ml (RIA standard curve) used in this study was less than 1% for porcine FSH (NIH-P-2) and less than 2% for chromatographically purified porcine TSH *. The sensitivity was better than 0.5 ng/ml, the coefficient of intraassay variation is 11.4% and of interassay variation 17%. Parallelism was demonstrated over the concentration range measured between the LH used as standard (LER-786-3), LH in the medium and LH in cell extracts.
CAMPwas measured by the method of Gilman using a binding protein prepared from porcine adrenal cortices according to the method of Brown et al. (1971). Samples from the incubation mix containing cells and medium were deproteinized by precipitation with an equal volume of 95% ethanol at -2O’C. Recovery of CAMPas shown by the addition of standard nucleotide before ethanol precipitation was greater than 90%. Since a number of variables including the components of the balanced salt solution were found to alter the nature of the binding of the nucleotide, control media were included in the standard curves at all times. The standard
* Generously provided by Dr. G. Hennen, Institut de Mkdecine, Universit6 de Li&ge, Belgium.
330
A.M. Walker, C.R. Hopkins
curves for the assay showed that 50% of the [3H]cAMP was displaced by 2 pmol [‘HIcAMP and 550 pmol cGMP. 5’-AMP caused minimal displacement of [3H]CAMPat concentrations of less than 1000 pmol. At the levels expected (Hardman, 1971) interference from cGMP and 5’-AMP is therefore negligible. ATP and GTP at the concentrations used are also without significant effect. For experiments in which CAMPwas to be measured the dissociated cells were maintained at high density on a 6% gelatin substrate (Walker and Hopkins, 1978). After 48 h in culture the cells were spun (250g for 5 min) through 2% tic011in DMEM to remove residual gelatin and serum and resuspended with additions as indicated in the appropriate legends. For measuring LHRH and PGE,-induced increases in CAMP the phosphodiesterase inhibitor ICI 63197 was included in the experimental and control media. In the PGEi experiments lo-’ M ATP and 10m4M GTP were also included because although there is no evidence that they were required under the conditions used in the present study, it has been reported (Deery and Howell, 1973) that these nucleotides are required for the activation of adenyl cyclase by PGE in the rat pituitary.
RESULTS LHRH-induced LH release
LHRH induces a highly reproducible dose-related level of LH release from dissociated cells previously incubated 48 h in culture (fig. 1). The curve is always sigmoidal with a half-maximal response at 4 X lo-” M and a maximal response at lo-’ M LHRH. The magnitude of the response does, however, vary from one dissociation to another so that in the data presented below only intraexperimental data are compared. Exogenous CAMP (dbcAiW)
Alone, dbcAMP at 5 X loo3 M induces a significant increase in the release of LH (fig. 2). The increase in release induced by 3 X 10m3M dbcAMP is not statistically significant but always (4 experiments in triplicate) lies between the saline control and 5 X 10m3M and is suggestive of a dose-related response. However, compared to the level of LH release obtained with IO-’ M LHRH with this preparation (20-fold), the maximum level obtained with 5 X 10q3 M dbcAMP is modest (-3-fold). 5 X 10q3 M dbcAMP has an inhibitory effect on a submaximal (low9 M) LHRH stimulation of LH release (fig. 2). Although not significant, the same inhibitory effect was seen when dbcAMP was combined with lo-’ M LHRH. Phosphodiesterase inhibitors i’%eophylline Alone, theophylline at 3 X 10s3 M has a small but significant effect on LH release (fig. 3) while at this dose level it significantly decreases the
~ti~l~~ationof LH release by LHRH
Fig. 1. Dose-response curve for LHRHinduced LH release. Cells were plated at 4 X lo6 cells/ dish incubated 48 h in (DMEM + serum) and then given three S-min rinses in (DMEM - serum) before being incubated for 4 h in (DMEM -serum) (saline control) or (DMEM - serum + LHRH) at the concentrations indicated. Sub~quen~y the incubation media were collected and LH was measured by radio~munoa~y as described in Methods. Basal release was approx. 30 ng LH/dish. Arrow indicates dose level at which the response is half-maximal. Dishes incubated in triplicate, variation bars indicate * SE&. Fig. 2. Effect of dibutyryl cyclic AMP (dbcAMP) on LH release and on LHRH-stimulated LH release. Cells were plated out at 2 X lo6 cells/dish, and incubated for 48 h before use. They were then given three 5-min rinses in (DMEM - serum) and incubated for 2 h in serum-free medium as indicated. Saline = (DMEM - serum). Media were collected and radiohnmunoassayed for LH as described in Methods. Dishes were incubated in triplicate; variation bars * S.E.M.
amount of LH released by a submaximal (1 0e9 M) and a maximal (lo-’ M) dose of LHRH. This effect, though unexpected (see ~scussion), was observed in all of our experiments. ~S~bu~Z~erh~~Qn~hjne fIBMX) This xanthine derivative has been shown to be a potent phosphodiesterase inhibitor (Beavo et al., 1970; Butcher and Sutherland, 1962). Alone, it induces a dose-related increase in LH release and with LHRH it evokes a more than additive response at both submaximal and maximal dose levels of the peptide (fig. 4). ICI 63197 This compound is a triazolopyrimidine which has also been shown to inhibit phosphodiesterase (Somerville et al., 1970). In beef heart and guinea pig
332
Fig. 3. Effect of theophylline fig. 2.
A.M. Walker, C.R. Hopkins
on LH release and on LHRHstimulated
LH release; protocol as in
lung preparations it is, respectively, two and eight times more potent than theophylline in inhibiting CAMP phosphodiesterase (Barrett-Bee and Henderson, 1976). It is also much less effective than theophylline in inhibiting cGMP phosphodiesterase (Barrett-Bee and Henderson, 1976). At lo-’ M it significantly stimulates LH release when used alone and when used in combination with a submaximal (10S9 M) and a maximal (lo-’ M) dose of LHRH it produces a more than additive response (fig. 5). Agents likely to raise cyclic AMP levels in the LH cell Prostaglandins of the E series and cholera toxin have been widely used to elevate intracellular CAMP levels and have been shown to increase CAMP in the rat pituitary (Deery and Howell, 1973; Yanaihara et al., 1973). These compounds were examined in terms of their ability to raise CAMP levels in the porcine pituitary and for their effect on LH release. Prostaglandins El and E2 1O-5-1O4 M PGEr significantly
increases the level of CAMP in dissociated por-
Stimulation of LH release by LHRH
Fig. 4. Effect of isobutylmethylxanthine release; protocol as in fig. 2.
333
(IBMX) on LH release and LHRH-stimulated
LH
tine pituitary cells (fig. 6). Moreover, when used alone, this agent induces LHrelease in a dose-related manner with a half-maximal dose level at 5 X lo-’ M and a maximum level at lo-’ M (fig. 7). The phosphodiesterase inhibitor ICI 63197 considerably enhances the effect of PGE, even when the prostaglandin is used at a threshold level (10m5 M) (fig. 8). With a submaximum dose of LHRH (10m9 M), the effect of PGE, at 10e4 M is additive (fig. 8). However, the amount of LH released in response to a maximum dose level (1 O-’ M) of LHRH in the presence of a maximum dose level (1 O-’ M) of PCEr is significantly (P = 0.01) more than additive (fig. 8). The results obtained with PGEz were in all respects the same as those obtained with PGE,.An example of the PGE2-induced response is shown in fig. 7.
334
Fig. 5. Effect of ICI 63197 on LH release and on LHRH-stimulated fig. 2.
A.&i. Walker, CR. Hopkim
LH release; protocol as in
Cholera toxin
Cholera toxin at IO fig/ml increased the intracellular level of CAMPin the dissociated cell preparation and this effect increases with time over a 4-h incubation period (fig. 9). Alone, 10 pgjrnl cholera toxin is a potent secretagogue of LH release, equal in its effect over 2 h to that of a maximal dose of LHRH (IO-’ M) (fig. 10). The toxin also slightly increases the effect of a submaxim~ (lo+‘’ M) dose of LHRH and also
Stimulation of LH release by LHRH
COI!TF!OL
335
lo-'N
PGE,
Fig. 6. Effect of prostaglandin Er on the level of CAMP. Cells were incubated in suspension over 6% gelatin in (DMEM + serum) at a concentration of 20 X lo6 cells/ml for 48 h at 25°C. They were then warmed to 37”C, loaded above 2% ficoll and centrifuged at 25Og for 5 min. The pelleted cells were resuspended in KRB and incubated 15 min at 37°C with 10e2 M ATP, lo4 M GTP, lo* M ICI 63197 + PGEr as indicated. At the end of the incubation 95% ethanol at -20°C was added to the preparation and following centrifugation (3000g for 15 mm) the supernatant taken for the assay of CAMP as described in Methods. Each incubation was performed in triplicate, variation bars f S.E.M.
increases the amount of LH released above that obtained with a maximal dose of LHRH (lo-’ M) (fig. 10).
The effect of LHRH on intracellular levels of CAMP Although LH cells comprise only about 15% of the total cell population (Walker
and Hopkins, 1977) it is nevertheless possible to demonstrate an increase in the level of CAMPin response to lo-’ M LHRH (fig. 11). The increase is of the order of 30% and when compared to the levels present in the control it is statistically significant with a P value of <0.05. If it is assumed that CAMP is equally distributed throughout the pituitary cell population, a 30% increase over the total content represents a 200% increase in the LH-cell population.
336
A.M.
Walker, C.R. Hopkins
100 -
80 -
Fig. 7. Effect of PGEl and PGE2 on LH release. Experimental protocol as described for fig. 2.
DISCUSSION The finding that LHRH induces a significant increase in intracellular levels of CAMP within 15 min together with the observation that prostaglandin Er and cholera toxin significantly increase both the level of CAMP and LH release provides strong circumstantial evidence in favour of CAMP playing a direct role in LHRHstimulated LH release. An increase in the level of CAMP induced by LHRH has also been demonstrated in the rat pituitary (Borgeat et al., 1972; Kaneko et al., 1973; Labrie et al., 1973; Makino, 1973; Yanaihara et al., 1973; Naor et al., 1975; Yoshida et al., 1975), and, with the exception of the studies reported by Borgeat et al. (1972), there workers have all shown the increase to occur within the first 15 min of stimulation.
331
Stimulation of LH release by LHRH
EXP.2
EXP.3
Fig. 8. Effect of ICI 63197 on PGElstimulated LH release and the effect of PGEl on LHRHstimulated LH release. Experimental protocol as described for fig. 2.
The results obtained with exogenous cyclic nucleotides and theophylline are at variance with those obtained with IBMX and ICI 63197. Since both the latter two compounds have been shown to be potent and relatively specific inhibitors of CAMP phosphodiesterase (Butcher and Sutherland, 1962; Beavo et al., 1970; Barrett-Bee and Henderson, 1976) and both are effective at a dose level considerably below that at which theophylline exerts an overt effect upon LH-release, it is concluded that, in addition to their anticipated influence on intracellular CAMP levels, both dbcAMP and theophylline exert additional inhibitory effects on gonadotropic cells. In this context it is perhaps worth noting that there is evidence in other systems that at high concentrations both of these agents may be toxic (Sheppard et al., 1971; van Wijk et al., 1972). Amongst the variety of responses reported using dbcAMP and theophylline on the rat pituitary, our observations directly agree with those of Ratner (1970) and Makino (1973) since in both of these studies these agents were both found to produce a small but significant increase in LH release. IBMX and ICI 63197 have not previously been used in this kind of study. However, their effects when used alone and in conjunction with LHRH are entirely consistent with the view that, in acting as phosphodiesterase inhibitors, they increase the level of CAMP and thus enhance LH release. Prostaglandins of the E series have been shown to raise CAMP levels in the rat
338
r
______
----_-_O-----Q_----4
1
1
I
1
2
3
4
TlMt IhRi
Fig. 9. The effect, with time, of cholera toxin on the level of CAMP. Ceils incubated in suspension over 6% gelatin in (DMEM + serum) at a concentration of 5 X lo6 cells/ml for 48 II at 25’C. They were then warmed to 37”C, loaded above 2% ficoll and centrifuged (25Og for 5 min) before being resuspended in KRB and incubated either with or without 10 pg/ml cholera toxin for the times indicated. Following incubation they were deproteinized in 95% ethanol at -20°C and assayed for CAMP. Each incubation in triplicate, variation bars indicate +_S.E.M. Fig. 10. The effect of cholera toxin on LH release and on LHRH-stimulated release. Esperimrntal protocol as described for fig. 2.
pituitary and we have obtained a similar result in the pig for both PGE, and PGE2. Again, in agreement with the observations made by Ratner (1970) and Makino (1973) on the rat we have found that PGEl will also stimulate LH release, and the result indicating that the phosphodiesterase inhibitor ICI 63197 will enhance the effect of PGE on LH release provides additional evidence in favour of the view that, in the porcine system, PGE is acting, at least partially, by raising CAMP levels. It should be noted, however, that Zor and his colleagues (1975) and Sundberg and his co-workers (1976) both failed to stimulate LH release with PGE, from the rat pituitary in vitro. Interestingly, although Makino (1973) has shown that PGE will slightly increase an LHRH-induced rise in CAMP, only in our studies has PGE been
Stirmlation
of LH release by LHRH
KRB
339
KRB 'KRB + lo:'n 10.'MLHRH 4 ICI
63197
lo-"MICI 63197
Fig. 11. The effect of LHRH and ICI 63197 on the level of CAMP. Cells were incubated in suspension over 6% gelatin in (DMEM + serum) at a concentration of 7 X lo6 cells/ml for 48 h at 25’C. They were then warmed to 37”C, loaded above 2% ficoll, centrifuged (25Og for 5 min) before being resuspended in KRB alone, KRB containing lo-’ M ICI 63197 or KRB containing 10” M ICI 63197 + lo-’ M LHRH and incubated for 15 min at 37’C. Following this Incubation the preparations were deproteinized in 95% ethanol at -20°C and assayed for CAMP. Each incubation in triplicate, variation bars indicate + S.E.M.
found to increase the effect of a submaximal dose of LHRH on LH release. The more than additive increase in LH release observed when PGE is added to a maximal dose (lo-’ M) of LHRH is especially noteworthy. It may indicate that, in this system, prostaglandins play a role in addition to the activation of adenyl cyclase, perhaps by acting as an ionophore at the level of the mitochondrion and thus increasing the intracellular concentration of free calcium (Kirtland and Baum, 1972). In the rat, PGEr and PGE? have both been reported to be without effect on LHRH-stimulated LH release (Somerville et al., 1970; Makino, 1973; Kaneko et al., 1974;Naor et al., 1975;Sundberg et al., 1976). In agreement with the observations made on other tissues (Cuatrecasas et al., 1976) we have found that cholera toxin at a concentration of 10 E.cg/mldramatically increases the intracellular levels of CAMPin pituitary cells. This result agrees with that of Zor et al. (1975) who have shown that the toxin also increases CAMPlevels in the rat pituitary. An important point of difference between our observations and those made on the rat requires emphasis however, since although in the rat it is possible to elevate CAMPlevels with cholera toxin and PGE without affecting LH release, in the porcine preparation both PGEs and cholera toxin are potent secretagogues for LH and both enhance LHRH-induced LH release. The ability of the prostaglandins and cholera toxin to substantially increase LH release above that ob-
340
A.M. ft’alhrr. C.R. Hopkins
tamed with a maximal dose of LHRH is also of interest. Presumably it indicates that the threshold of LHRH-induced release is regulated at a step in the coupling sequence proximal to that at which the cyclic nucleotide is involved. In conclusion, it can be said that with the exception of the results obtained with dbcAMP and theophylline, all of the data obtained in the present study support the view that in the pig pituitary CAMP plays a role in coupling LHRH stimulation to LH release. It has not been possible in this system to dissociate an increase in the level of CAMP from the release of LH. It remains to be shown whether or not CAMP directly controls a rate-limiting step in the LHRH-LH coupling sequence or if, for example, the influence of the nucleotide is exerted indirectly via an intermediary agent such as calcium.
ACKNOWLEDGEMENTS We are most grateful to Dr. Leo Reichert, Jr., Emory University, Atlanta, Ga 30322, U.S.A., for generously supplying the porcine LH used to develop the radioimmunoassay and to the NIAMDD pituitary hormone distribution program for providing the standards used to characterize the assay. This work was carried out during the tenure of an MRC postgraduate studentship and was supported by an MRC project grant (G975/517/C) awarded to Colin R. Hopkins.
REFERENCES Barrett-Bee, K. and Henderson, W. (1976) Biochem. Sot. Trans. 4,699-701. Beavo, J.A., Rogers, N.C., Hardman, J.C., Sutherland, E.W. and Newman, E.V. (1970) Mol. Pharmacol. 6,597-603. Borgeat, P., Chavancy, G., DuPont, A., Labrie, F., Arimura, A. and Schally, A. (1972) Proc. Natl. Acad. Sci. U.SA.69,267-268. Brown, B.L., Albano, J.D.M., Ekins, R.P., Sgherze, A.M. and Tampion, W. (1971) Biochem. J. 121,561~562. Butcher, R.W. and Sutherland, E.W. (1962) J. Biol. Chem. 237, 1244-1250. Cuatrecasas, P., Berret, V., Craig, S., OXeefe, E. and Sahyoun, N. (1976) In: Structural Basis of Membrane Function, Eds.: Y. Hatefi and W. Ohaniance (Academic Press, New York) pp. 275-292. Deery, D.J. and Howell, S.L. (1973) Biochim. Biophys. Acta 329, 17-22. Drouin, J., Ferland, L., Bernard, J. and Labrie, F. (1976) Prostaglandins 11,367-374. Gilman,A.G. (1970) Proc. Natl. Acad. Sci. U.S.A.67,305-312. Hardman, J.G. (1971) In: Cyclic AMP, Eds.: G.A. Robison, R.W. Butcher and E.W. Sutherland (Academic Press, New York) p. 402. Kaneko, T., Oka, H., Munemara, M.J., Susuki, S., Yauida, H. and Oda, T. (1974) Biochem. Biophys. Res. Commun. 61,53-57. Kaneko, T., Saito, S., Oka, H., Oda, T. and Yanaihara, N. (1973) Metabolism 22,77-80. Kirtland, S.J. and Baum, H. (1972) Nature New Biol. 236,47.
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Krebs, H.A. (1950) Biochim. Biophys. Acta 4, 249-256. Labrie, F., Pelletier, G., Borgeat, P., Drouin, J., Savary, M., Cote, J. and Ferland, L. (1973) Gonadotrophins and Gonadal Function. Symp. 33 (Bangalore). Makino, T. (1973) Am. J. Obstet. Gynaecol. 115,606-614. Naor, Z., Koch, Y., Bauminger, S. and Zor, U. (1975) Prostaglandins 9, 211-217. Ratner. A. (1970) Life Sci. 9,1221-1226. Sheppard, J.R. (1971) Proc. Natl. Acad. Sci. U.S.A. 68,1316-1320. Somerville, A.R., Rabouhand, M.L. and Smith, A.A. (1970) Biochem. J. 120,llP. Spona, J. (1975) Endocrinol. Exp. 9. 27-33. Sundberg, D.K., Fawcett, C.P. and McCann, S.M. (1976) Proc. Sot. Exp. Biol. Med. 151, 149156. Tang, L.K.L. and Spies, H.G. (1976) Proc. Sot. Exp. Biol. Med. 151,198-203. Wakabayashi, K., Date, Y. and Tamaoki, B. (1973) Endocrinology 92,698-704. Walker, A.M. and Hopkins, C.R. (1977) Endocrinology (in press). Yanaihara, N., Yanaihara, C., Sakagami, M., Tsuki, K., Hashimoto, T., Kaneko, T., Oka, H. Schally, A., Arimura, A. and Redding,T.W. (1973) J. Med. Chem. 16,373-377. Yoshida, T., Hattori, Y., Hoshiae, H., Hirano, M., Takahashi, K. and Suzuki, M. (1975) Acta Endocrinol. 79,658-659. Van Wijk, R., Wicks, W.D. and Clay, K. (1972) Cancer Res. 32,1905-1911. Zor, U., Kaneko, T., Schneider, H.P.G., McCann, S.M. and Field, J.B. (1970) J. Biol. Chem. 245,2883-2888.
STIMULATIONOFLUTEINISINGHORMONERELEASE BYLUTEINISING HORMONE-RELEASINGHORMONEINTHEPORCINEANTERIOR PITUITARY:THEROLEOFCYCLICAMP Ameae M. WALKER * and Cohn R. HOPKINS Department of Histology and Cell Biology (Medical), The Medical School, Universityof Liverpool, P.O. Box 147, Liverpool L69 3BX, U.K. Received 25 October 1977; accepted 10 January 1978
Cells dissociated from porcine pituitary glands have been used to examine the relationship between cyclic AMP levels and the stimulation of luteinising hormone (LH) secretion by luteinising hormone*eleasing hormone (LHRH). It has been shown that while dibutyryl cyclic AMP and theophylhne have a modest and, under some conditions, an inhibitory effect on LH release, the phosphodiesterase inhibitors isobutylmethylxanthie and ICI 63197 both induce LH release and enhance LHRH-induced release. The prostaglandins Er and Ea and cholera toxin, which are shown to raise cyclic AMP levels in the dissociated preparation, also induce LH release and increase LHRH-induced LH release. Finally, LHRH is shown to increase the total content of cyclic AMP in the mixed celJ preparation by approximately 30% and it is concluded that, in this system, the nucleotide does play a role in coupling LHRH stimulation to LH release. Keywords: cyclic nucleotides; gonadotropins;
hypophysiotropic
hormones.
The requirement for cyclic AMP in the coupling of LHRH stimulation to LH secretion has been extensively studied using rat pituitary preparations. Nevertheless, although a large body of information on the topic now exists, the question remains unresolved. The evidence in favour of the view that LHRH causes a rise in cyclic AMP which is in turn directly responsible for LH release includes (1) the observation that LHRH can activate adenyl cyclase in both crude homogenates (Deery and Howell, 1973) and plasma membrane fractions (Spona, 1975), (2) that LHRH stimulation causes a rise in the intracellular level of cyclic AMP (Borgeat et al., 1972; Kaneko et al., 1973; Labrie et al., 1973; Makino, 1973; Yanaihara et al., 1973; Naor et al., 1975; Yoshida et al., 1975), (3) that exogenous cyclic AMP will stimulate LH release (Ratner, 1970; Labrie et al., 1973; Makino, 1973) (4) that phosphodiesterase inhibitors induce LH release and potentiate LHRH stimulation
* Present address: Section for Cell Biology, Yale Medical School, New Haven, Corm. 06510, U.S.A. 327
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A.M. Walker, C.R. Hopkins
(Ratner, 1970; Makino, 1973: Wakabayashi et al., 1973; Tang and Spies, 1976). and (5) that substances shown to increase intracellular levels of cyclic AMP also increase LH release (Ratner, 1970; Makino, 1973). Other studies would not, however, support this view since they have either failed to demonstrate that cyclic AMP (Wakabayashi et al., 1973; Sundberg et al., 1976; Tang and Spies, 1976) and/or phosphodiesterase inhibitors (Wakabayashi et al., 1973; Tang and Spies, 1976) significantly stimulate LH release or they have shown that, while agents such as prostaglandin E, and cholera toxin will increase intracellular levels of cyclic AMP, only LHRH will stimulate LH release (Naor et al., 1975). Recently we have developed a dissociated cell preparation from the porcine anterior pituitary which, in response to LHRH, releases LH in a consistent and predictable manner (Walker and Hopkins, 1977). This preparation is sensitive to LHRH stimulation with a half-maximal response at 4 X lo-‘* M and allows dose-response curves to be derived with less than a 1% variation between replicate aliquots. Moreover, since these preparations can be routinely obtained from abattoir material, quantitative assays for regulatory agents such as cyclic nucleotides are relatively unconstrained by the availability of material. In work reported here we have used this preparation to examine the relationship between cyclic AMP and LHRHstimulated LH release in the porcine pituitary. For conformity we have, as far as possible, used the agents, dose levels and time points used in previously reported studies. We conclude that in this system cyclic AMP does play a role in the LHRHstimulation of LH release.
MATERIALS AND METHODS Materials
The sodium salts of N602 dibutyryl cyclic adenosine monophosphate (dbcAMP), adenosine triphosphate (ATP), adenosine monophosphate (AMP) and cyclic guanosine monophosphate (cGMP) and theophylline were obtained from Sigma (London), Surrey, U.K., and guanosine ~phosphate (GTP) and cyclic adenosine monophosphate (CAMP) (sodium salts) were obtained from Boehringer Corp. (London), Ealing, London, U.K. [8-3H]cAMP was obtained from the Radiochemical Centre, ~er~am, Bucks, U.K. and Norit GSX was kindly supplied by Norit, Clydesdale, Glasgow, UK. Prostaglandins Er and E2 were generously provided by Dr. J.E. Pike, Upjohn, Kalamazoo, Michigan, U.S.A. and ICI 63197 was a gift from Dr. M. Dukes, ICI Pha~aceutic~s, Maccles~eld, U.K. Cholera toxin (lot No. 0972) was kindly supplied by Dr. R.A. Finkelstein under the auspices of the NIAMDD, Bethesda, Md., U.S.A. ~ss~ci~ti~n of pigsty tissue and i~~~~ti~n of diss~c~ted cells The rationale of the method used for dissociation will be published in detail
Stimulation of LH release by LHRH
329
elsewhere (Walker and Hopkins, 1978). In outline the method involves the sequential incubation of chopped tissue blocks in collagenase (0.075% in Krebs-Ringer bicarbonate (KRB) (Krebs, 1950) for 60 min) and trypsin (0.5% in KRB minus Ca’+ and Mg’+) followed by gentle pipetting to disperse the tissue. Cell debris was removed from the suspension by low-speed centrifugation (250 g for 3 min) through 4% bovine serum albumin. Following their resuspension in Dulbecco’s modified Eagle’s minimum essential medium (DMEM) the cells were assessed for viability (normally X5%) and then aliquotted in DMEM + 10% foetal calf serum either into 30~mm petri dishes coated with poly(L-lysine) or into 90.mm petri dishes containing 2 ml 6% gelatin. For the measurement of LH-release dissociated cells attached to poly(L-lysine)coated petri dishes and incubated for 48 h in DMEM/lO% foetal calf serum were used. Each experiment was performed in triplicate. Serum was removed with three S-min rinses in DMEM and then incubated in this serum-free medium for 2 h at 37’C. As an intra- and interexperimental control a dose-response range from lo-” M-10+ M LHRH was normally included in the protocol. LHRH was added (25 1.11to 3 ml DMEM) as a concentrated solution in phosphate-buffered saline. Other additions were made to the medium directly before use, and where the solvent was other than aqueous it was checked for its effect on LH release. (All were without effect). Media were collected, centrifuged to removecell debris at 3000 g and then stored at -20°C until assay. LH was measured by radioimmunoassay using an antiserum raised against LER-7784 (Walker and Hopkins, 1977) which bound 50% iodinated LH (LER786-3) at a final dilution of 1 : 150,000. The cross-reactivity in the range of l-30 ng/ml (RIA standard curve) used in this study was less than 1% for porcine FSH (NIH-P-2) and less than 2% for chromatographically purified porcine TSH *. The sensitivity was better than 0.5 ng/ml, the coefficient of intraassay variation is 11.4% and of interassay variation 17%. Parallelism was demonstrated over the concentration range measured between the LH used as standard (LER-786-3), LH in the medium and LH in cell extracts.
CAMPwas measured by the method of Gilman using a binding protein prepared from porcine adrenal cortices according to the method of Brown et al. (1971). Samples from the incubation mix containing cells and medium were deproteinized by precipitation with an equal volume of 95% ethanol at -2O’C. Recovery of CAMPas shown by the addition of standard nucleotide before ethanol precipitation was greater than 90%. Since a number of variables including the components of the balanced salt solution were found to alter the nature of the binding of the nucleotide, control media were included in the standard curves at all times. The standard
* Generously provided by Dr. G. Hennen, Institut de Mkdecine, Universit6 de Li&ge, Belgium.
330
A.M. Walker, C.R. Hopkins
curves for the assay showed that 50% of the [3H]cAMP was displaced by 2 pmol [‘HIcAMP and 550 pmol cGMP. 5’-AMP caused minimal displacement of [3H]CAMPat concentrations of less than 1000 pmol. At the levels expected (Hardman, 1971) interference from cGMP and 5’-AMP is therefore negligible. ATP and GTP at the concentrations used are also without significant effect. For experiments in which CAMPwas to be measured the dissociated cells were maintained at high density on a 6% gelatin substrate (Walker and Hopkins, 1978). After 48 h in culture the cells were spun (250g for 5 min) through 2% tic011in DMEM to remove residual gelatin and serum and resuspended with additions as indicated in the appropriate legends. For measuring LHRH and PGE,-induced increases in CAMP the phosphodiesterase inhibitor ICI 63197 was included in the experimental and control media. In the PGEi experiments lo-’ M ATP and 10m4M GTP were also included because although there is no evidence that they were required under the conditions used in the present study, it has been reported (Deery and Howell, 1973) that these nucleotides are required for the activation of adenyl cyclase by PGE in the rat pituitary.
RESULTS LHRH-induced LH release
LHRH induces a highly reproducible dose-related level of LH release from dissociated cells previously incubated 48 h in culture (fig. 1). The curve is always sigmoidal with a half-maximal response at 4 X lo-” M and a maximal response at lo-’ M LHRH. The magnitude of the response does, however, vary from one dissociation to another so that in the data presented below only intraexperimental data are compared. Exogenous CAMP (dbcAiW)
Alone, dbcAMP at 5 X loo3 M induces a significant increase in the release of LH (fig. 2). The increase in release induced by 3 X 10m3M dbcAMP is not statistically significant but always (4 experiments in triplicate) lies between the saline control and 5 X 10m3M and is suggestive of a dose-related response. However, compared to the level of LH release obtained with IO-’ M LHRH with this preparation (20-fold), the maximum level obtained with 5 X 10q3 M dbcAMP is modest (-3-fold). 5 X 10q3 M dbcAMP has an inhibitory effect on a submaximal (low9 M) LHRH stimulation of LH release (fig. 2). Although not significant, the same inhibitory effect was seen when dbcAMP was combined with lo-’ M LHRH. Phosphodiesterase inhibitors i’%eophylline Alone, theophylline at 3 X 10s3 M has a small but significant effect on LH release (fig. 3) while at this dose level it significantly decreases the
~ti~l~~ationof LH release by LHRH
Fig. 1. Dose-response curve for LHRHinduced LH release. Cells were plated at 4 X lo6 cells/ dish incubated 48 h in (DMEM + serum) and then given three S-min rinses in (DMEM - serum) before being incubated for 4 h in (DMEM -serum) (saline control) or (DMEM - serum + LHRH) at the concentrations indicated. Sub~quen~y the incubation media were collected and LH was measured by radio~munoa~y as described in Methods. Basal release was approx. 30 ng LH/dish. Arrow indicates dose level at which the response is half-maximal. Dishes incubated in triplicate, variation bars indicate * SE&. Fig. 2. Effect of dibutyryl cyclic AMP (dbcAMP) on LH release and on LHRH-stimulated LH release. Cells were plated out at 2 X lo6 cells/dish, and incubated for 48 h before use. They were then given three 5-min rinses in (DMEM - serum) and incubated for 2 h in serum-free medium as indicated. Saline = (DMEM - serum). Media were collected and radiohnmunoassayed for LH as described in Methods. Dishes were incubated in triplicate; variation bars * S.E.M.
amount of LH released by a submaximal (1 0e9 M) and a maximal (lo-’ M) dose of LHRH. This effect, though unexpected (see ~scussion), was observed in all of our experiments. ~S~bu~Z~erh~~Qn~hjne fIBMX) This xanthine derivative has been shown to be a potent phosphodiesterase inhibitor (Beavo et al., 1970; Butcher and Sutherland, 1962). Alone, it induces a dose-related increase in LH release and with LHRH it evokes a more than additive response at both submaximal and maximal dose levels of the peptide (fig. 4). ICI 63197 This compound is a triazolopyrimidine which has also been shown to inhibit phosphodiesterase (Somerville et al., 1970). In beef heart and guinea pig
332
Fig. 3. Effect of theophylline fig. 2.
A.M. Walker, C.R. Hopkins
on LH release and on LHRHstimulated
LH release; protocol as in
lung preparations it is, respectively, two and eight times more potent than theophylline in inhibiting CAMP phosphodiesterase (Barrett-Bee and Henderson, 1976). It is also much less effective than theophylline in inhibiting cGMP phosphodiesterase (Barrett-Bee and Henderson, 1976). At lo-’ M it significantly stimulates LH release when used alone and when used in combination with a submaximal (10S9 M) and a maximal (lo-’ M) dose of LHRH it produces a more than additive response (fig. 5). Agents likely to raise cyclic AMP levels in the LH cell Prostaglandins of the E series and cholera toxin have been widely used to elevate intracellular CAMP levels and have been shown to increase CAMP in the rat pituitary (Deery and Howell, 1973; Yanaihara et al., 1973). These compounds were examined in terms of their ability to raise CAMP levels in the porcine pituitary and for their effect on LH release. Prostaglandins El and E2 1O-5-1O4 M PGEr significantly
increases the level of CAMP in dissociated por-
Stimulation of LH release by LHRH
Fig. 4. Effect of isobutylmethylxanthine release; protocol as in fig. 2.
333
(IBMX) on LH release and LHRH-stimulated
LH
tine pituitary cells (fig. 6). Moreover, when used alone, this agent induces LHrelease in a dose-related manner with a half-maximal dose level at 5 X lo-’ M and a maximum level at lo-’ M (fig. 7). The phosphodiesterase inhibitor ICI 63197 considerably enhances the effect of PGE, even when the prostaglandin is used at a threshold level (10m5 M) (fig. 8). With a submaximum dose of LHRH (10m9 M), the effect of PGE, at 10e4 M is additive (fig. 8). However, the amount of LH released in response to a maximum dose level (1 O-’ M) of LHRH in the presence of a maximum dose level (1 O-’ M) of PCEr is significantly (P = 0.01) more than additive (fig. 8). The results obtained with PGEz were in all respects the same as those obtained with PGE,.An example of the PGE2-induced response is shown in fig. 7.
334
Fig. 5. Effect of ICI 63197 on LH release and on LHRH-stimulated fig. 2.
A.&i. Walker, CR. Hopkim
LH release; protocol as in
Cholera toxin
Cholera toxin at IO fig/ml increased the intracellular level of CAMPin the dissociated cell preparation and this effect increases with time over a 4-h incubation period (fig. 9). Alone, 10 pgjrnl cholera toxin is a potent secretagogue of LH release, equal in its effect over 2 h to that of a maximal dose of LHRH (IO-’ M) (fig. 10). The toxin also slightly increases the effect of a submaxim~ (lo+‘’ M) dose of LHRH and also
Stimulation of LH release by LHRH
COI!TF!OL
335
lo-'N
PGE,
Fig. 6. Effect of prostaglandin Er on the level of CAMP. Cells were incubated in suspension over 6% gelatin in (DMEM + serum) at a concentration of 20 X lo6 cells/ml for 48 h at 25°C. They were then warmed to 37”C, loaded above 2% ficoll and centrifuged at 25Og for 5 min. The pelleted cells were resuspended in KRB and incubated 15 min at 37°C with 10e2 M ATP, lo4 M GTP, lo* M ICI 63197 + PGEr as indicated. At the end of the incubation 95% ethanol at -20°C was added to the preparation and following centrifugation (3000g for 15 mm) the supernatant taken for the assay of CAMP as described in Methods. Each incubation was performed in triplicate, variation bars f S.E.M.
increases the amount of LH released above that obtained with a maximal dose of LHRH (lo-’ M) (fig. 10).
The effect of LHRH on intracellular levels of CAMP Although LH cells comprise only about 15% of the total cell population (Walker
and Hopkins, 1977) it is nevertheless possible to demonstrate an increase in the level of CAMPin response to lo-’ M LHRH (fig. 11). The increase is of the order of 30% and when compared to the levels present in the control it is statistically significant with a P value of <0.05. If it is assumed that CAMP is equally distributed throughout the pituitary cell population, a 30% increase over the total content represents a 200% increase in the LH-cell population.
336
A.M.
Walker, C.R. Hopkins
100 -
80 -
Fig. 7. Effect of PGEl and PGE2 on LH release. Experimental protocol as described for fig. 2.
DISCUSSION The finding that LHRH induces a significant increase in intracellular levels of CAMP within 15 min together with the observation that prostaglandin Er and cholera toxin significantly increase both the level of CAMP and LH release provides strong circumstantial evidence in favour of CAMP playing a direct role in LHRHstimulated LH release. An increase in the level of CAMP induced by LHRH has also been demonstrated in the rat pituitary (Borgeat et al., 1972; Kaneko et al., 1973; Labrie et al., 1973; Makino, 1973; Yanaihara et al., 1973; Naor et al., 1975; Yoshida et al., 1975), and, with the exception of the studies reported by Borgeat et al. (1972), there workers have all shown the increase to occur within the first 15 min of stimulation.
331
Stimulation of LH release by LHRH
EXP.2
EXP.3
Fig. 8. Effect of ICI 63197 on PGElstimulated LH release and the effect of PGEl on LHRHstimulated LH release. Experimental protocol as described for fig. 2.
The results obtained with exogenous cyclic nucleotides and theophylline are at variance with those obtained with IBMX and ICI 63197. Since both the latter two compounds have been shown to be potent and relatively specific inhibitors of CAMP phosphodiesterase (Butcher and Sutherland, 1962; Beavo et al., 1970; Barrett-Bee and Henderson, 1976) and both are effective at a dose level considerably below that at which theophylline exerts an overt effect upon LH-release, it is concluded that, in addition to their anticipated influence on intracellular CAMP levels, both dbcAMP and theophylline exert additional inhibitory effects on gonadotropic cells. In this context it is perhaps worth noting that there is evidence in other systems that at high concentrations both of these agents may be toxic (Sheppard et al., 1971; van Wijk et al., 1972). Amongst the variety of responses reported using dbcAMP and theophylline on the rat pituitary, our observations directly agree with those of Ratner (1970) and Makino (1973) since in both of these studies these agents were both found to produce a small but significant increase in LH release. IBMX and ICI 63197 have not previously been used in this kind of study. However, their effects when used alone and in conjunction with LHRH are entirely consistent with the view that, in acting as phosphodiesterase inhibitors, they increase the level of CAMP and thus enhance LH release. Prostaglandins of the E series have been shown to raise CAMP levels in the rat
338
r
______
----_-_O-----Q_----4
1
1
I
1
2
3
4
TlMt IhRi
Fig. 9. The effect, with time, of cholera toxin on the level of CAMP. Ceils incubated in suspension over 6% gelatin in (DMEM + serum) at a concentration of 5 X lo6 cells/ml for 48 II at 25’C. They were then warmed to 37”C, loaded above 2% ficoll and centrifuged (25Og for 5 min) before being resuspended in KRB and incubated either with or without 10 pg/ml cholera toxin for the times indicated. Following incubation they were deproteinized in 95% ethanol at -20°C and assayed for CAMP. Each incubation in triplicate, variation bars indicate +_S.E.M. Fig. 10. The effect of cholera toxin on LH release and on LHRH-stimulated release. Esperimrntal protocol as described for fig. 2.
pituitary and we have obtained a similar result in the pig for both PGE, and PGE2. Again, in agreement with the observations made by Ratner (1970) and Makino (1973) on the rat we have found that PGEl will also stimulate LH release, and the result indicating that the phosphodiesterase inhibitor ICI 63197 will enhance the effect of PGE on LH release provides additional evidence in favour of the view that, in the porcine system, PGE is acting, at least partially, by raising CAMP levels. It should be noted, however, that Zor and his colleagues (1975) and Sundberg and his co-workers (1976) both failed to stimulate LH release with PGE, from the rat pituitary in vitro. Interestingly, although Makino (1973) has shown that PGE will slightly increase an LHRH-induced rise in CAMP, only in our studies has PGE been
Stirmlation
of LH release by LHRH
KRB
339
KRB 'KRB + lo:'n 10.'MLHRH 4 ICI
63197
lo-"MICI 63197
Fig. 11. The effect of LHRH and ICI 63197 on the level of CAMP. Cells were incubated in suspension over 6% gelatin in (DMEM + serum) at a concentration of 7 X lo6 cells/ml for 48 h at 25’C. They were then warmed to 37”C, loaded above 2% ficoll, centrifuged (25Og for 5 min) before being resuspended in KRB alone, KRB containing lo-’ M ICI 63197 or KRB containing 10” M ICI 63197 + lo-’ M LHRH and incubated for 15 min at 37’C. Following this Incubation the preparations were deproteinized in 95% ethanol at -20°C and assayed for CAMP. Each incubation in triplicate, variation bars indicate + S.E.M.
found to increase the effect of a submaximal dose of LHRH on LH release. The more than additive increase in LH release observed when PGE is added to a maximal dose (lo-’ M) of LHRH is especially noteworthy. It may indicate that, in this system, prostaglandins play a role in addition to the activation of adenyl cyclase, perhaps by acting as an ionophore at the level of the mitochondrion and thus increasing the intracellular concentration of free calcium (Kirtland and Baum, 1972). In the rat, PGEr and PGE? have both been reported to be without effect on LHRH-stimulated LH release (Somerville et al., 1970; Makino, 1973; Kaneko et al., 1974;Naor et al., 1975;Sundberg et al., 1976). In agreement with the observations made on other tissues (Cuatrecasas et al., 1976) we have found that cholera toxin at a concentration of 10 E.cg/mldramatically increases the intracellular levels of CAMPin pituitary cells. This result agrees with that of Zor et al. (1975) who have shown that the toxin also increases CAMPlevels in the rat pituitary. An important point of difference between our observations and those made on the rat requires emphasis however, since although in the rat it is possible to elevate CAMPlevels with cholera toxin and PGE without affecting LH release, in the porcine preparation both PGEs and cholera toxin are potent secretagogues for LH and both enhance LHRH-induced LH release. The ability of the prostaglandins and cholera toxin to substantially increase LH release above that ob-
340
A.M. ft’alhrr. C.R. Hopkins
tamed with a maximal dose of LHRH is also of interest. Presumably it indicates that the threshold of LHRH-induced release is regulated at a step in the coupling sequence proximal to that at which the cyclic nucleotide is involved. In conclusion, it can be said that with the exception of the results obtained with dbcAMP and theophylline, all of the data obtained in the present study support the view that in the pig pituitary CAMP plays a role in coupling LHRH stimulation to LH release. It has not been possible in this system to dissociate an increase in the level of CAMP from the release of LH. It remains to be shown whether or not CAMP directly controls a rate-limiting step in the LHRH-LH coupling sequence or if, for example, the influence of the nucleotide is exerted indirectly via an intermediary agent such as calcium.
ACKNOWLEDGEMENTS We are most grateful to Dr. Leo Reichert, Jr., Emory University, Atlanta, Ga 30322, U.S.A., for generously supplying the porcine LH used to develop the radioimmunoassay and to the NIAMDD pituitary hormone distribution program for providing the standards used to characterize the assay. This work was carried out during the tenure of an MRC postgraduate studentship and was supported by an MRC project grant (G975/517/C) awarded to Colin R. Hopkins.
REFERENCES Barrett-Bee, K. and Henderson, W. (1976) Biochem. Sot. Trans. 4,699-701. Beavo, J.A., Rogers, N.C., Hardman, J.C., Sutherland, E.W. and Newman, E.V. (1970) Mol. Pharmacol. 6,597-603. Borgeat, P., Chavancy, G., DuPont, A., Labrie, F., Arimura, A. and Schally, A. (1972) Proc. Natl. Acad. Sci. U.SA.69,267-268. Brown, B.L., Albano, J.D.M., Ekins, R.P., Sgherze, A.M. and Tampion, W. (1971) Biochem. J. 121,561~562. Butcher, R.W. and Sutherland, E.W. (1962) J. Biol. Chem. 237, 1244-1250. Cuatrecasas, P., Berret, V., Craig, S., OXeefe, E. and Sahyoun, N. (1976) In: Structural Basis of Membrane Function, Eds.: Y. Hatefi and W. Ohaniance (Academic Press, New York) pp. 275-292. Deery, D.J. and Howell, S.L. (1973) Biochim. Biophys. Acta 329, 17-22. Drouin, J., Ferland, L., Bernard, J. and Labrie, F. (1976) Prostaglandins 11,367-374. Gilman,A.G. (1970) Proc. Natl. Acad. Sci. U.S.A.67,305-312. Hardman, J.G. (1971) In: Cyclic AMP, Eds.: G.A. Robison, R.W. Butcher and E.W. Sutherland (Academic Press, New York) p. 402. Kaneko, T., Oka, H., Munemara, M.J., Susuki, S., Yauida, H. and Oda, T. (1974) Biochem. Biophys. Res. Commun. 61,53-57. Kaneko, T., Saito, S., Oka, H., Oda, T. and Yanaihara, N. (1973) Metabolism 22,77-80. Kirtland, S.J. and Baum, H. (1972) Nature New Biol. 236,47.
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Krebs, H.A. (1950) Biochim. Biophys. Acta 4, 249-256. Labrie, F., Pelletier, G., Borgeat, P., Drouin, J., Savary, M., Cote, J. and Ferland, L. (1973) Gonadotrophins and Gonadal Function. Symp. 33 (Bangalore). Makino, T. (1973) Am. J. Obstet. Gynaecol. 115,606-614. Naor, Z., Koch, Y., Bauminger, S. and Zor, U. (1975) Prostaglandins 9, 211-217. Ratner. A. (1970) Life Sci. 9,1221-1226. Sheppard, J.R. (1971) Proc. Natl. Acad. Sci. U.S.A. 68,1316-1320. Somerville, A.R., Rabouhand, M.L. and Smith, A.A. (1970) Biochem. J. 120,llP. Spona, J. (1975) Endocrinol. Exp. 9. 27-33. Sundberg, D.K., Fawcett, C.P. and McCann, S.M. (1976) Proc. Sot. Exp. Biol. Med. 151, 149156. Tang, L.K.L. and Spies, H.G. (1976) Proc. Sot. Exp. Biol. Med. 151,198-203. Wakabayashi, K., Date, Y. and Tamaoki, B. (1973) Endocrinology 92,698-704. Walker, A.M. and Hopkins, C.R. (1977) Endocrinology (in press). Yanaihara, N., Yanaihara, C., Sakagami, M., Tsuki, K., Hashimoto, T., Kaneko, T., Oka, H. Schally, A., Arimura, A. and Redding,T.W. (1973) J. Med. Chem. 16,373-377. Yoshida, T., Hattori, Y., Hoshiae, H., Hirano, M., Takahashi, K. and Suzuki, M. (1975) Acta Endocrinol. 79,658-659. Van Wijk, R., Wicks, W.D. and Clay, K. (1972) Cancer Res. 32,1905-1911. Zor, U., Kaneko, T., Schneider, H.P.G., McCann, S.M. and Field, J.B. (1970) J. Biol. Chem. 245,2883-2888.