Indomethacin and gonadotrophin release from rat pituitaries in vitro P. S. Brown Department ofPharmacology, University ofBristol, Medical School, Bristol BS8 1TD,
U.K.
Sundberg, Fawcett, Illner & McCann (1975) have shown that high concentrations of indomethacin (1 and 2\m=.\7mM) can increase the release of LH and FSH from rat pituitary tissue in vitro. Such concentrations, however, have other actions in addition to the inhibition of prostaglandin (PG) synthetase for which the drug is commonly used experimentally (Flower, 1974). The effects of lower concentra-
tions of indomethacin have therefore been tested. Rat pituitary tissue was incubated in Krebs-Ringer bicarbonate medium containing glucose (KRBG), as described previously (Sharpe, Shahmanesh, Ellwood, Hartog & Brown, 1975). Each flask contained 1 ml medium and three hemi-pituitaries from 60- to 65-day-old male rats. Experi¬ ments were replicated, between three and six times, in blocks usually of 6 or 4 flasks : all glands for one block were provided by a single standardized litter of 10 male rats (Sharpe, Morris, Wyatt & Brown, 1972). Pituitary halves were distributed among a block randomly except that no flask re¬ ceived both halves of the same gland. Incubation was divided into two parts. During an initial control period of 30 min, the tissue was in KRBG without additions. The medium was then replaced by fresh medium, containing various additions, for the experimental incubation lasting 90 min. Indomethacin (Merck, Sharpe & Dohme) and synthetic LH-releasing hormone (LH-RH : Reckitt & Colman) were dissolved in KRBG while PGs were added to the bulk medium in a small volume of ethanol, an equivalent addition of which was made to control flasks. The media from the control and experimental periods were centrifuged to remove any tissue fragments and frozen until the LH and FSH were measured by radioimmuno¬ assay using NIAMDD kits. Hormone release during the experimental period was expressed as pg hormone (NIAMDD RPl)/gland/h and was corrected, after analysis of covariance, for the amount of hormone released in the same flask during the control incubation. This procedure was economic in the amount of tissue required and appeared to reduce variability of response more than methods in which pituitaries are halved and one half used as a simultaneous unstimulated control. The regression coefficient relating release during experimental incubation to that during the control period appeared to be stable from experiment to experiment, and adjustment based on the relation¬ ship reduced error more effectively than simple arithmetic correction for control release. Analysis of variance allowed the removal of variation due to differences among blocks and, when the combined effects of two substances were tested, the significance of their interaction could be assessed. Variance tended to be high when mean hormone release was high (see Table 1), but data were not transformed because Bartlett's test showed no significant heterogeneity of variance within experiments (P > 005). In preliminary experiments, LH release decreased successively with increasing concentrations of indomethacin between 10 µ and 10 pM, but increased again above control at 100 pwc. The release of FSH was unaffected until 100 pM was reached, when it rose significantly. To confirm the apparently biphasic effect on LH release, incubations were performed at a high (200 pM) and a low (5 pM) concen¬ tration of indomethacin (Table 1 ; Exp. 1 ). For LH there was a significant difference among treatment groups (P < 0-01) with a reduced release at the low concentration and increased release at the high concentration, both being significantly different from control (P < 0-05). There were no significant effects on FSH release. Further incubations were performed to confirm the depression of LH release with 5 pM-indomethacin (Table 1 ; Exp. 2). The fall in LH was comparable with that in the previous experiment and was just short of significance (F 4-35; d.f. 1,9). The effect of the two concentrations of indomethacin was tested on pituitary tissue stimulated submaximally by LH-RH (Table 1 ; Exps 3 and 4). The high indomethacin concentration caused a =
=
Table 1. The release of gonadotrophins from rat pituitary tissue incubated with indomethacin Hormone released
Additions to medium
(pg/pituitary/h) LH-RH
Indomethacin
(ng/ml)
(pM)
LH
FSH
-
Exp. 1
0 0 0
0 5 200
1-92 ±0098 1-50* 2-47*
1-19 + 0083 106 116
2
0 0
0 5
1-31 ±0092
0-79 ± 0078 0-71
1-5 1-5
0 5
1-5 1-5
0 200
3 4
l-03f 4-50 ±0-508
2-76 ±0-191 2-82
4-36 3-45 ±0-460 600*
The S.E.M. is shown for one mean only in each
2-24 ± 0-478
3-81f
experiment: it is applicable
to all means in that experiment as it is derived from the pooled error variance. *
0-1) while indomethacin significantly increased both. Although there was a suggestion that the effect of indomethacin on LH release was greater in the presence of PG, there was no significant interaction (P > 0-2) between the effects of the two substances on either hormone. The PGs tested in these experiments showed no inhibitory effects on basal hormone release or any significant effect on the release excited by indomethacin. It seems likely that the effect of high concen¬ trations of this drug may not be due to inhibition of PG synthesis but to some other action such as inhibition of cyclic nucleotide phosphodiesterase (Flower, 1974). Drugs with this action have been tested and will release both LH and FSH in this system. I am grateful to the following for gifts to this laboratory: Dr A. F. Parlow and the Rat Pituitary Hormone Program, NIAMDD, for assay materials; Merck, Sharp & Dohme for indomethacin; Dr J. E. Pike, Upjohn Company, for PGs; and Dr D. Schäfer, Reckitt & Colman, for synthesis and supply of LH-RH. References
Drouin, J. & Labrie, F. (1975) Specificity of the stimu¬ latory effect of prostaglandin on hormone release in rat pituitary cells in culture. Prostaglandins 11, 355365.
Flower, R.J. (1974) Drugs which inhibit prostaglandin biosynthesis, Pharm. Rev. 26, 33-67.
Sharpe, R.M., Morris, A. Wyatt, A.C. & Brown, P.S. (1972) Variability of response in cross-fostered rats. Lab. Anim. 6, 225-234. Sharpe, R.M., Shahmanesh, M., Ellwood, M.G., Hartog, M. & Brown, P.S. (1975) Discrepancy between radioimmunoassay and radio-ligand re-
ceptor assay of luteinizing hormone released in vitro by pituitary tissue from male rats of different ages. /. Endocr. 65, 265-273. Sundberg, D.K., Fawcett, C.P., Illner, P. & McCann, S.M. (1975) The effect of various prosta¬
glandins and on
a
rat anterior
prostaglandin synthetase inhibitor pituitary cyclic AMP levels and
hormone release in vitro. Proc. Soc. exp. Biol. Med. 148, 54-59.
Zor, U., Taneko, T., Schneider, H.P.G., McCann, S.M. & Field, J.B. (1970) Further studies of stimu¬ lation of anterior pituitary cyclic adenosine 3'5'monophosphate formation by hypothalamic extract and prostaglandins. /. biol. Chem. 245, 2883-2888. Received 1 December 1976
U.K.
Sundberg, Fawcett, Illner & McCann (1975) have shown that high concentrations of indomethacin (1 and 2\m=.\7mM) can increase the release of LH and FSH from rat pituitary tissue in vitro. Such concentrations, however, have other actions in addition to the inhibition of prostaglandin (PG) synthetase for which the drug is commonly used experimentally (Flower, 1974). The effects of lower concentra-
tions of indomethacin have therefore been tested. Rat pituitary tissue was incubated in Krebs-Ringer bicarbonate medium containing glucose (KRBG), as described previously (Sharpe, Shahmanesh, Ellwood, Hartog & Brown, 1975). Each flask contained 1 ml medium and three hemi-pituitaries from 60- to 65-day-old male rats. Experi¬ ments were replicated, between three and six times, in blocks usually of 6 or 4 flasks : all glands for one block were provided by a single standardized litter of 10 male rats (Sharpe, Morris, Wyatt & Brown, 1972). Pituitary halves were distributed among a block randomly except that no flask re¬ ceived both halves of the same gland. Incubation was divided into two parts. During an initial control period of 30 min, the tissue was in KRBG without additions. The medium was then replaced by fresh medium, containing various additions, for the experimental incubation lasting 90 min. Indomethacin (Merck, Sharpe & Dohme) and synthetic LH-releasing hormone (LH-RH : Reckitt & Colman) were dissolved in KRBG while PGs were added to the bulk medium in a small volume of ethanol, an equivalent addition of which was made to control flasks. The media from the control and experimental periods were centrifuged to remove any tissue fragments and frozen until the LH and FSH were measured by radioimmuno¬ assay using NIAMDD kits. Hormone release during the experimental period was expressed as pg hormone (NIAMDD RPl)/gland/h and was corrected, after analysis of covariance, for the amount of hormone released in the same flask during the control incubation. This procedure was economic in the amount of tissue required and appeared to reduce variability of response more than methods in which pituitaries are halved and one half used as a simultaneous unstimulated control. The regression coefficient relating release during experimental incubation to that during the control period appeared to be stable from experiment to experiment, and adjustment based on the relation¬ ship reduced error more effectively than simple arithmetic correction for control release. Analysis of variance allowed the removal of variation due to differences among blocks and, when the combined effects of two substances were tested, the significance of their interaction could be assessed. Variance tended to be high when mean hormone release was high (see Table 1), but data were not transformed because Bartlett's test showed no significant heterogeneity of variance within experiments (P > 005). In preliminary experiments, LH release decreased successively with increasing concentrations of indomethacin between 10 µ and 10 pM, but increased again above control at 100 pwc. The release of FSH was unaffected until 100 pM was reached, when it rose significantly. To confirm the apparently biphasic effect on LH release, incubations were performed at a high (200 pM) and a low (5 pM) concen¬ tration of indomethacin (Table 1 ; Exp. 1 ). For LH there was a significant difference among treatment groups (P < 0-01) with a reduced release at the low concentration and increased release at the high concentration, both being significantly different from control (P < 0-05). There were no significant effects on FSH release. Further incubations were performed to confirm the depression of LH release with 5 pM-indomethacin (Table 1 ; Exp. 2). The fall in LH was comparable with that in the previous experiment and was just short of significance (F 4-35; d.f. 1,9). The effect of the two concentrations of indomethacin was tested on pituitary tissue stimulated submaximally by LH-RH (Table 1 ; Exps 3 and 4). The high indomethacin concentration caused a =
=
Table 1. The release of gonadotrophins from rat pituitary tissue incubated with indomethacin Hormone released
Additions to medium
(pg/pituitary/h) LH-RH
Indomethacin
(ng/ml)
(pM)
LH
FSH
-
Exp. 1
0 0 0
0 5 200
1-92 ±0098 1-50* 2-47*
1-19 + 0083 106 116
2
0 0
0 5
1-31 ±0092
0-79 ± 0078 0-71
1-5 1-5
0 5
1-5 1-5
0 200
3 4
l-03f 4-50 ±0-508
2-76 ±0-191 2-82
4-36 3-45 ±0-460 600*
The S.E.M. is shown for one mean only in each
2-24 ± 0-478
3-81f
experiment: it is applicable
to all means in that experiment as it is derived from the pooled error variance. *
0-1) while indomethacin significantly increased both. Although there was a suggestion that the effect of indomethacin on LH release was greater in the presence of PG, there was no significant interaction (P > 0-2) between the effects of the two substances on either hormone. The PGs tested in these experiments showed no inhibitory effects on basal hormone release or any significant effect on the release excited by indomethacin. It seems likely that the effect of high concen¬ trations of this drug may not be due to inhibition of PG synthesis but to some other action such as inhibition of cyclic nucleotide phosphodiesterase (Flower, 1974). Drugs with this action have been tested and will release both LH and FSH in this system. I am grateful to the following for gifts to this laboratory: Dr A. F. Parlow and the Rat Pituitary Hormone Program, NIAMDD, for assay materials; Merck, Sharp & Dohme for indomethacin; Dr J. E. Pike, Upjohn Company, for PGs; and Dr D. Schäfer, Reckitt & Colman, for synthesis and supply of LH-RH. References
Drouin, J. & Labrie, F. (1975) Specificity of the stimu¬ latory effect of prostaglandin on hormone release in rat pituitary cells in culture. Prostaglandins 11, 355365.
Flower, R.J. (1974) Drugs which inhibit prostaglandin biosynthesis, Pharm. Rev. 26, 33-67.
Sharpe, R.M., Morris, A. Wyatt, A.C. & Brown, P.S. (1972) Variability of response in cross-fostered rats. Lab. Anim. 6, 225-234. Sharpe, R.M., Shahmanesh, M., Ellwood, M.G., Hartog, M. & Brown, P.S. (1975) Discrepancy between radioimmunoassay and radio-ligand re-
ceptor assay of luteinizing hormone released in vitro by pituitary tissue from male rats of different ages. /. Endocr. 65, 265-273. Sundberg, D.K., Fawcett, C.P., Illner, P. & McCann, S.M. (1975) The effect of various prosta¬
glandins and on
a
rat anterior
prostaglandin synthetase inhibitor pituitary cyclic AMP levels and
hormone release in vitro. Proc. Soc. exp. Biol. Med. 148, 54-59.
Zor, U., Taneko, T., Schneider, H.P.G., McCann, S.M. & Field, J.B. (1970) Further studies of stimu¬ lation of anterior pituitary cyclic adenosine 3'5'monophosphate formation by hypothalamic extract and prostaglandins. /. biol. Chem. 245, 2883-2888. Received 1 December 1976
