Life Sciences, Vol . 23, pp . 2073-2078 Printed in the U .S .A .
Pergamon Press
VARIATIONS IN THE RESPONSE OF ENZYME-DISSOCIATE RAT PITUITARY CELLS TO THYROTROPIN RELEASING HORKflNE C . Michael Moriarty, M. Patricia Leuachea and G.T . Campbell Department of Physiology and Biophysics University of Nebraska College of Medicine Omaha, Nebraska 68105 (Received in final form September 22, 1978) Sumoary While exploring the interaction between thyrotropia releasing hormone (TRH) and normal rat anterior pituitary cells in moaolayer culture we observed that cells dissociated with the use of trypsin did not respond to TRH with an increase is either TSH or prolactin (PRL) release . The dissociated cells were cultured for 3 days, then washed to remove serum proteins a~ exposed to 10"6M TRH for 3 hours. TSH and PRL secretion from ati~lated and unstimulated cultures was determined by radioimmunoaesay and normalized using cell protein. When such tryps1n-dissociated cells were exposed to 0 .5 mM dibutyryl cyclic AMP the release of both TSH a~ PRL doubled indicating that the intracellular aecretory machinery was functional and that the block to TRH was proximal to the formation of cyclic AMP and pres~bly at the level of a TRH surface receptor . Praviwa studies have shown that such trypsin-dissociated celle respond to LHRH and a crude hypothalamic eatract with a dose dependent increase is LH,FSH and ACTH release. This rules out a nonspecific effect of trypsin. When pituitary cells were dissodated with a non-trypain technique, the unstieulated release of both TSH and PRL was comparable to that found with the trypeinHowever, these cultures did respond to diasociated cultures . TRH with an increase in TSH release although again no effect was seen with PRL. The susceptibility of the cells to trypsia suggeata the possibility that a protein moiety may be closely associated with the function of the receptor . The use of isolated cells to study the mechanise of hormone action has Since enzymatic digestion is normally increased markedly in recent years. employed is the isolation procedure it is important to be aware of possible alterations in cell surface receptors and therefore, possible changes in physiological responsiveness of such cells ae a result of the dissociation procedure. For several years we have employed isolated rat pituitary cells is order to investigate the cellular basis of pituitary hormone secretion (1,2) . Such cells were routinely dissociated from the tissue aatria with the use of trypeia and mechanical agitation. We have shown that cells obtained is this manner respond in a dose-dependent fashion to LH-RH with an increase in LH and FSH release (3) a~ to a crude hypothalamic eztract with an increase in ACTH release. We were, therefore, surprised to find that such cells did not 0300-9653/78/1113-2073$02 .00/0 Copyright (c) 1978 Pergamon Press
2074
TRH Receptors
Vol . 23, No . 20, 1978
respond to TRß with a corresponding increase is TSH release. The eaperimente reported below were designed to provide additional information regarding the ability of TRH to stimulate the release of TSH a~/or prolactin from cells dissociated with the use of trypain. Materials and Methods Male rata (200-250 g) of the Sprague-Dawley strain were used throughout this study . They were kept in a room at constant temperature (24° C) and given food and water ad libitum . All rate were acclimatized to a regulated light-dark cycle (lights on at 0600 h, off at 2200 h) for at least one week prior to use . All animals were killed between 0800 and 0900 h. For each ezperiment 12-25 rats were decapitated each within 5-10 sec after removal from their cages . The pituitaries were removed, the n~ro-intermediate lo3es dissected away, and the adenohypophyaes minced into pieces less than 1 mm . Two different procedures were employed in order to dissociate the cells from their tissue matrix . The first was a modification of the procedure of Portaaova _et al . (3) as described previously (1) . Essentially, this procedure employs a combination of trypsis digestion and mechanical agitation. The minced tissue blocks were transferred to a siliconized 50 ml Erlenmeyer flask containing 20 ml of Kreba-Ringer bicarbonate buffer (RRB) plus trypain (2 .5 mg/ml, Worthington Biochmical) . All solutions were passed through a millipore filter (0 .2 U pore size) before use. The tissue suspension was maintained at 37 ° C a~ gassed with 95x 02 :5x C02 while being stirred at 200 rpm with a ailiconized glees paddle . After 20 min the fragments were allowed to settle, the supernatant was decanted with a eiliconized Pasteur pipette and transf erred to a 250 ml Erlenmeyer flask kept oa ice . Fresh trypeiacontaiaing media was added to the original flask and the procedure repeated 3-4 times . The final contests of the 250 ml Erlenmeyer were centrifuged at The supernatant was discarded a~ the cell 475 z g for 20 min at 4° C. pellet resuspended in KRB with amino acids (Grand Island Biological Co .) and washed to remove trypain. The final pellet was resuspended is minimum essential medium (lißll) suppleaented with lOx horse serum and 2 .55 fetal calf serum. Penicillin (100/ml), atreptamycin (100 ug/m1) and fungizone (0 .25 ug/ml) were added and the cells seeded into monolayer culture at a density of 40,000 cells/cm2 of growing surface. We did not employ any additional trypain inhibitors as there are sufficient e~ogeaous inhibitors present in the serum to inactivate any residual trypain. The second dissociation procedure employed was that of vale _et al . (4) . In this case the minced blocks were incubated for 45 min at 37 ° C in a HEPES (N 1-2-hydroxyethylpiperazine-Nl-ethaaesulfoaic acid) buffer solution contain ing O .1Z hyaluroaideae, 0 .35x collagenase and O. Lt bovine serum albumin . The procedure was facilitated by gently drawing the tissue fragments through a ailiconized Pasteur pipette every 10 min . This media was then centrifuged at The 475 z g for 10 min and the enayne-containing supernatant discarded . pallet was reauape~ed is 10 ml of the HSPES buffer containing 0.252 Viokase and incubated for 20 min at 37 ° C. The undigested tissue fragments were allowed to settle and the supernatant containing the isolated cells washed 6 times in sterile MEM containing lOx horse serum, 2 .5Z fetal calf serum and antibiotics as indicated above. These cells were seeded into mosolayer culture at the same density as those dissociated with the trypain technique . After medium was serum-free (10-b!f TßH
three days of culture and one medium change the serum-containing removed and each flask washed three times with identical but media. Fresh serum-free media with or without a aecretagogue or 0 .5 mM dibutyryl cyclic AMP) was added and the cells incubated
Vol . 23, No . 20, 1978
TRH Receptors
2075
for three hours at 37 ° C . At the end of the incubation, the medium from each flask was decanted and stored at -70 ° C for subsequent radioimmunoassay of both TSH (2) and prolactin (5) . The cells were scraped off the growing surface with a rubber policeman, lysed and assayed for total protein via the Lowry method (6) . Results Pituitary cells isolated with the use of trypain and mechanical agitation are viable and readily attach and proliferate when seeded into monolayer culture (1,2) . When such cultures are ezposed to appropriate secreta gogues, they respond with a dose-dependent increase of ACTH se well ae LH and FSH . Consequently, ve were surprised to find that cells prepared in this fashion did not respond to TRH with the expected increase in TSH release . Results are shown in Fig . la . Following isolation with the trypaia technique and subsequent monolayer culture for 3 d, there is no increase in TSH release after a 3 h exposure to 10-6M TRH . Similarly, there was ao stimulation of prolactin release under these conditions (Fig . lb) . pf. L
~ROLACTIN
TfH
W H O 6 r L
SO ,~, F
O
~s V
4 7
CONTROL
10'bM TRH
CONTROL
10'6M TRH
FIG . 1 Effect of a 3 h eapoaure to 10- 6M TRH on TSH (Fig, la and prolactia (Fig . lb) release from 3 d old monolayer cultures of rat anterior pituitary cells isolated with the use of trypsia . Values are ~ t SE . N - 5 . The inability of TRH to stimulate TSH release could be due to an alteration at the receptor level and/or to a block in the stimulus-aecretioa coupling pathway at a locus distal to the TRH receptor . To investigate this we tested the ability of 0 .5 mM dibutyryl cyclic AMP to stimulate the release of TSH under the same experimental conditions . Even at this done, which is lover than frequently used for in vitro aecretioa studies, a marked stimulation of TSH release was found (Fig . 2a) . In the same fashion 0 .5 mM dlbutyryl cyclic AMP markedly increased prolactin release (Fig . 2b) . Discussion It is now amply documented that the initial action of the hypophyaiotrophic hormones is a binding to specific receptor sites located primarily in (or on) the plasma membrane of the respective adenohypophysial cells . In the case of TRH there have been numerous studies correlating the extent of membrane binding with subsequent biological activity . Ae a correlate to
2076
TRH Receptors
CONTROL
Vol . 23, No . 20, 1978
O.SmM db CAMS
CONTROL
O.SwM db CAMS
FTG . 2 Effect of a 3 h exposure to 0 .5 mM dibutyryl cyclic AMP on TSH (Fig . 2a) and prolactin (Fig . 2b) release from 3 d old moaolayer cultures of rat anterior pituitary cells isolated with the use of trypain . Values are Z t SE . N ~ 5 . To test the possibility that the TRH receptors were altered during the dissociation procedure, presumably by the proteolytic action of trypsia, we isolated cells by a procedure not employing trypain . Whey cells were iso lated with this procedure (4) and cultured in a monolayer for 3 d, they responded to TRH with a prompt increase in TSH release (Fig . 3a) . As before, however, TRH had ao effect oa prolactin release from such cultures (Fig . 3b) . In each case, the basal or unetimulated release of both TSH and prolactin were similar is cultures prepared by both dissociation techniques .
il~. >.
2 W O W a
iy .
TSH
~~
~ROLACTIN
~ 30 40
.. 1 S r W
O W J W
v 30 e, E
v s ~ 10
w
W F
1
S
20 ~ 10 m d
F
O
a
CONTROL
10"6M TRH
CONTROL
10'6M TRH
FIG . 3 Effect of a 3 h exposure to 10-6M TRH on TSH (Fig . 3a) and prolactin (Fig . 3b) release from 3 d old monolayer cultures of rat anterior pituitary cells isolated with the non-trypain technique . Values are â t SE . N ~ 5.
Vol . 23, No . 20, 1978
TRH Receptors
207 7
these observations, it follows that if the TRH receptor is modified such that TRH binding is reduced, and/or if the transduction of information resulting from such binding ie impaired, the resultant secretory response will be diminished . In the experiments reported here the biological action of TRH waa abWhile we can sent in pituitary cells isolated with the trypain procedure . not with certainty attribute this action to the trypeia her ae since there are some other differences between the two procedures, there are a number of factors which suggest that such may be the case . First, in addition to the proteolytic action of trypain, mechanical agitation was employed . However, similar mechanical agitation was utilized in the non-trypain procedure and In addition, an inthis resulted in cells which were responsive to TRH. creasing amount of evidence indicates that hormone receptors are vulnerable to proteolytic enzymes such as trypain [cf. (7) for references] and isolation of cells using such enzymes might alter the number and/or affinity of receptors on the cell surface . Although there have been numerous studies using enzyme-dissociated anterior pituitary cells, few studies have compared the various dissociating procedures . In one such study, Nakano et al . (8) found that trypaindisaociated cells displayed reduced secretory capabilities when compared to cells diasociated with collagenase and hyaluronidase . They concluded that trypain dissociation resulted in damage to the pituitary cell surface; damage that was not reversed after short term culture in fresh serum-containing medium . Hopkins and Farquhar (9) eaamined in detail the relative cell yields obtained when different dissociation procedures were used in conjunction with anterior pituitaries. They did not however, contrast the responsivenesa to various secretagogues in the various procedures . The action of trypain oa pituitary cells under our experimental conditions appears restricted to thyrotrophs and possibly mammotrophs . Using the same trypain dissociation procedure we have previously found that pituitary cells respond to LH-RH with a dose-dependent increase in LH and FSH release and to a crude hypothalamic extract with a dose-dependent increase in ACTH release . Thus, a protein moiety which is susceptible to proteolytic action may be associated is a functional sense with the TRH receptor in contrast to the LH-RH and possibly the CRH receptors . That the action of trypain is localized at the cell surface is suggested by the ability of dibutyryl cyclic AMP, particularly in the low dose used, to increase the release of both TSH and prolactin . This lndicatea that the intracellular secretory machinery was not impaired by the prior exposure to trypain. Furthermore, the observation that the basal or unstimulated hormone release was the same in cells dissociated with either technique suggests the absence of a non-specific effect of In other tissues significant morphoeither method on membrane permeability . logical damage has been observed in cells isolated with the use of trypain (10) . No intracellular alterations have been observed in pituitary cells diasociated with the u~e of trypain (2,11) reinforcing our opinion that the damage was localized to the surface receptors . Several workers have suggested that the TRH receptors on thyrotrophe~ and mammotrophs are similar if not identical (12,13), While the present experiments are not addressed specifically to this point, it would appear that there may well be a functional difference between the TRH receptors on the two cell types mace TSH release 3s blocked only with the trypain technique . This, however, mây simply be a manifestation of inconsistent response of prolactin to TRH found in pituitary cell cultures particularly in the absence of eatrogens (13,14,15) .
207 8
TRH Receptors
Vol . 23, No . 20, 1978
Finally, Schrey et al . (16) have recently reported that rat pituitary cells isolated with the use of trypein do respond in a dose-dependent fashion to TRH with graded increases is TSH release . This discrepancy might be due to variations in the dissociation procedures used in the two studies . Acknowledgements The technical assistance of Michele Fontaine and Jack Wagoner is gratefully acknowledged . The authors wish to thank Dr . Albert Parlow and the NIAI~D Rat Pituitary Hormone Distribution Program for the TSH and prolactia radioimmunoassay material . The authors also thank Ruth Coaette for secretarial assistance . This work was supported by grants from the National Institutes of Health (AM 18328, AM 19170) and the American Cancer Society (BC 218) . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 .
C .M . MORIARTY,~Mol . Cell . Endocr 6 349-361 (1977) . M .P . LEUSCHEN, R .B . TOBIN and C .M . MORIARTY, Endocrinology 102 509-518 (1978) . R . PORTANOVA, D .R . SMITH and G . SAYERS, Proc . Soc . Exp . Biol . Med . 133 573-576 (1970) . W . VÀLE, G . GRANT, M . AMOSS, R . BLACRWELL and R . GUILLEMIN, Endocrinology 91 562-572 (1972) . J .A . RABAT-~ and G .T . CAMPBELL, Endocrinology 101 890-897 (1977) . T_T. , J . Biol . Chem . O .H . LOWRY, J .N . ROSEBROUGH, A .L . FAIR and R .J . RR 193 265-275 (1951) . C .R . KAHN, J . Cell Biol . _70 261-286 (1976) . H . NARANO, C .P . FAWCETT and S .M . McCANN, Endocrinology 98 278-288 (1976) . C .R . HOPRINS and M .G . FARQUHAR, J . Cell Biol . _59 276-303 (1973) . M . MASSON-PSVET, H .J . JONGSMA and J . de BRULTNE, J . Mol . Cell . Cardiol . _8 747-757 (1976) . 3 . MALAMED, R . PORTANOVA sad G . SAYERS, Proc . Soc . Esp . Biol . Med . 13 8 920-926 (1971) . P .M . HINRLE, E .L . WOROCH sad A .H . TASHJLAN, JR ., J . Biol . Chem . 249 3085-3090 (1974) . W . VALE, R . BT Af`.K4nZf.T , G . GRANT and R . GUILLEMIIQ, Endocrinology 93 26-33 (1973) . C . RIMER sad W . VALE, Endocrinology 95 978-983 (1974) . J .C . GROSHONG, G .E . MILD and W .B . MAIARREY, Life Sci . 20 1821-1828 (1977) . M .P . SCHREY, B .L . BROWN and R .P . ERINS, Mol . Cell . Endocr . _8 271-282 (1977) .
Pergamon Press
VARIATIONS IN THE RESPONSE OF ENZYME-DISSOCIATE RAT PITUITARY CELLS TO THYROTROPIN RELEASING HORKflNE C . Michael Moriarty, M. Patricia Leuachea and G.T . Campbell Department of Physiology and Biophysics University of Nebraska College of Medicine Omaha, Nebraska 68105 (Received in final form September 22, 1978) Sumoary While exploring the interaction between thyrotropia releasing hormone (TRH) and normal rat anterior pituitary cells in moaolayer culture we observed that cells dissociated with the use of trypsin did not respond to TRH with an increase is either TSH or prolactin (PRL) release . The dissociated cells were cultured for 3 days, then washed to remove serum proteins a~ exposed to 10"6M TRH for 3 hours. TSH and PRL secretion from ati~lated and unstimulated cultures was determined by radioimmunoaesay and normalized using cell protein. When such tryps1n-dissociated cells were exposed to 0 .5 mM dibutyryl cyclic AMP the release of both TSH a~ PRL doubled indicating that the intracellular aecretory machinery was functional and that the block to TRH was proximal to the formation of cyclic AMP and pres~bly at the level of a TRH surface receptor . Praviwa studies have shown that such trypsin-dissociated celle respond to LHRH and a crude hypothalamic eatract with a dose dependent increase is LH,FSH and ACTH release. This rules out a nonspecific effect of trypsin. When pituitary cells were dissodated with a non-trypain technique, the unstieulated release of both TSH and PRL was comparable to that found with the trypeinHowever, these cultures did respond to diasociated cultures . TRH with an increase in TSH release although again no effect was seen with PRL. The susceptibility of the cells to trypsia suggeata the possibility that a protein moiety may be closely associated with the function of the receptor . The use of isolated cells to study the mechanise of hormone action has Since enzymatic digestion is normally increased markedly in recent years. employed is the isolation procedure it is important to be aware of possible alterations in cell surface receptors and therefore, possible changes in physiological responsiveness of such cells ae a result of the dissociation procedure. For several years we have employed isolated rat pituitary cells is order to investigate the cellular basis of pituitary hormone secretion (1,2) . Such cells were routinely dissociated from the tissue aatria with the use of trypeia and mechanical agitation. We have shown that cells obtained is this manner respond in a dose-dependent fashion to LH-RH with an increase in LH and FSH release (3) a~ to a crude hypothalamic eztract with an increase in ACTH release. We were, therefore, surprised to find that such cells did not 0300-9653/78/1113-2073$02 .00/0 Copyright (c) 1978 Pergamon Press
2074
TRH Receptors
Vol . 23, No . 20, 1978
respond to TRß with a corresponding increase is TSH release. The eaperimente reported below were designed to provide additional information regarding the ability of TRH to stimulate the release of TSH a~/or prolactin from cells dissociated with the use of trypain. Materials and Methods Male rata (200-250 g) of the Sprague-Dawley strain were used throughout this study . They were kept in a room at constant temperature (24° C) and given food and water ad libitum . All rate were acclimatized to a regulated light-dark cycle (lights on at 0600 h, off at 2200 h) for at least one week prior to use . All animals were killed between 0800 and 0900 h. For each ezperiment 12-25 rats were decapitated each within 5-10 sec after removal from their cages . The pituitaries were removed, the n~ro-intermediate lo3es dissected away, and the adenohypophyaes minced into pieces less than 1 mm . Two different procedures were employed in order to dissociate the cells from their tissue matrix . The first was a modification of the procedure of Portaaova _et al . (3) as described previously (1) . Essentially, this procedure employs a combination of trypsis digestion and mechanical agitation. The minced tissue blocks were transferred to a siliconized 50 ml Erlenmeyer flask containing 20 ml of Kreba-Ringer bicarbonate buffer (RRB) plus trypain (2 .5 mg/ml, Worthington Biochmical) . All solutions were passed through a millipore filter (0 .2 U pore size) before use. The tissue suspension was maintained at 37 ° C a~ gassed with 95x 02 :5x C02 while being stirred at 200 rpm with a ailiconized glees paddle . After 20 min the fragments were allowed to settle, the supernatant was decanted with a eiliconized Pasteur pipette and transf erred to a 250 ml Erlenmeyer flask kept oa ice . Fresh trypeiacontaiaing media was added to the original flask and the procedure repeated 3-4 times . The final contests of the 250 ml Erlenmeyer were centrifuged at The supernatant was discarded a~ the cell 475 z g for 20 min at 4° C. pellet resuspended in KRB with amino acids (Grand Island Biological Co .) and washed to remove trypain. The final pellet was resuspended is minimum essential medium (lißll) suppleaented with lOx horse serum and 2 .55 fetal calf serum. Penicillin (100/ml), atreptamycin (100 ug/m1) and fungizone (0 .25 ug/ml) were added and the cells seeded into monolayer culture at a density of 40,000 cells/cm2 of growing surface. We did not employ any additional trypain inhibitors as there are sufficient e~ogeaous inhibitors present in the serum to inactivate any residual trypain. The second dissociation procedure employed was that of vale _et al . (4) . In this case the minced blocks were incubated for 45 min at 37 ° C in a HEPES (N 1-2-hydroxyethylpiperazine-Nl-ethaaesulfoaic acid) buffer solution contain ing O .1Z hyaluroaideae, 0 .35x collagenase and O. Lt bovine serum albumin . The procedure was facilitated by gently drawing the tissue fragments through a ailiconized Pasteur pipette every 10 min . This media was then centrifuged at The 475 z g for 10 min and the enayne-containing supernatant discarded . pallet was reauape~ed is 10 ml of the HSPES buffer containing 0.252 Viokase and incubated for 20 min at 37 ° C. The undigested tissue fragments were allowed to settle and the supernatant containing the isolated cells washed 6 times in sterile MEM containing lOx horse serum, 2 .5Z fetal calf serum and antibiotics as indicated above. These cells were seeded into mosolayer culture at the same density as those dissociated with the trypain technique . After medium was serum-free (10-b!f TßH
three days of culture and one medium change the serum-containing removed and each flask washed three times with identical but media. Fresh serum-free media with or without a aecretagogue or 0 .5 mM dibutyryl cyclic AMP) was added and the cells incubated
Vol . 23, No . 20, 1978
TRH Receptors
2075
for three hours at 37 ° C . At the end of the incubation, the medium from each flask was decanted and stored at -70 ° C for subsequent radioimmunoassay of both TSH (2) and prolactin (5) . The cells were scraped off the growing surface with a rubber policeman, lysed and assayed for total protein via the Lowry method (6) . Results Pituitary cells isolated with the use of trypain and mechanical agitation are viable and readily attach and proliferate when seeded into monolayer culture (1,2) . When such cultures are ezposed to appropriate secreta gogues, they respond with a dose-dependent increase of ACTH se well ae LH and FSH . Consequently, ve were surprised to find that cells prepared in this fashion did not respond to TRH with the expected increase in TSH release . Results are shown in Fig . la . Following isolation with the trypaia technique and subsequent monolayer culture for 3 d, there is no increase in TSH release after a 3 h exposure to 10-6M TRH . Similarly, there was ao stimulation of prolactin release under these conditions (Fig . lb) . pf. L
~ROLACTIN
TfH
W H O 6 r L
SO ,~, F
O
~s V
4 7
CONTROL
10'bM TRH
CONTROL
10'6M TRH
FIG . 1 Effect of a 3 h eapoaure to 10- 6M TRH on TSH (Fig, la and prolactia (Fig . lb) release from 3 d old monolayer cultures of rat anterior pituitary cells isolated with the use of trypsia . Values are ~ t SE . N - 5 . The inability of TRH to stimulate TSH release could be due to an alteration at the receptor level and/or to a block in the stimulus-aecretioa coupling pathway at a locus distal to the TRH receptor . To investigate this we tested the ability of 0 .5 mM dibutyryl cyclic AMP to stimulate the release of TSH under the same experimental conditions . Even at this done, which is lover than frequently used for in vitro aecretioa studies, a marked stimulation of TSH release was found (Fig . 2a) . In the same fashion 0 .5 mM dlbutyryl cyclic AMP markedly increased prolactin release (Fig . 2b) . Discussion It is now amply documented that the initial action of the hypophyaiotrophic hormones is a binding to specific receptor sites located primarily in (or on) the plasma membrane of the respective adenohypophysial cells . In the case of TRH there have been numerous studies correlating the extent of membrane binding with subsequent biological activity . Ae a correlate to
2076
TRH Receptors
CONTROL
Vol . 23, No . 20, 1978
O.SmM db CAMS
CONTROL
O.SwM db CAMS
FTG . 2 Effect of a 3 h exposure to 0 .5 mM dibutyryl cyclic AMP on TSH (Fig . 2a) and prolactin (Fig . 2b) release from 3 d old moaolayer cultures of rat anterior pituitary cells isolated with the use of trypain . Values are Z t SE . N ~ 5 . To test the possibility that the TRH receptors were altered during the dissociation procedure, presumably by the proteolytic action of trypsia, we isolated cells by a procedure not employing trypain . Whey cells were iso lated with this procedure (4) and cultured in a monolayer for 3 d, they responded to TRH with a prompt increase in TSH release (Fig . 3a) . As before, however, TRH had ao effect oa prolactin release from such cultures (Fig . 3b) . In each case, the basal or unetimulated release of both TSH and prolactin were similar is cultures prepared by both dissociation techniques .
il~. >.
2 W O W a
iy .
TSH
~~
~ROLACTIN
~ 30 40
.. 1 S r W
O W J W
v 30 e, E
v s ~ 10
w
W F
1
S
20 ~ 10 m d
F
O
a
CONTROL
10"6M TRH
CONTROL
10'6M TRH
FIG . 3 Effect of a 3 h exposure to 10-6M TRH on TSH (Fig . 3a) and prolactin (Fig . 3b) release from 3 d old monolayer cultures of rat anterior pituitary cells isolated with the non-trypain technique . Values are â t SE . N ~ 5.
Vol . 23, No . 20, 1978
TRH Receptors
207 7
these observations, it follows that if the TRH receptor is modified such that TRH binding is reduced, and/or if the transduction of information resulting from such binding ie impaired, the resultant secretory response will be diminished . In the experiments reported here the biological action of TRH waa abWhile we can sent in pituitary cells isolated with the trypain procedure . not with certainty attribute this action to the trypeia her ae since there are some other differences between the two procedures, there are a number of factors which suggest that such may be the case . First, in addition to the proteolytic action of trypain, mechanical agitation was employed . However, similar mechanical agitation was utilized in the non-trypain procedure and In addition, an inthis resulted in cells which were responsive to TRH. creasing amount of evidence indicates that hormone receptors are vulnerable to proteolytic enzymes such as trypain [cf. (7) for references] and isolation of cells using such enzymes might alter the number and/or affinity of receptors on the cell surface . Although there have been numerous studies using enzyme-dissociated anterior pituitary cells, few studies have compared the various dissociating procedures . In one such study, Nakano et al . (8) found that trypaindisaociated cells displayed reduced secretory capabilities when compared to cells diasociated with collagenase and hyaluronidase . They concluded that trypain dissociation resulted in damage to the pituitary cell surface; damage that was not reversed after short term culture in fresh serum-containing medium . Hopkins and Farquhar (9) eaamined in detail the relative cell yields obtained when different dissociation procedures were used in conjunction with anterior pituitaries. They did not however, contrast the responsivenesa to various secretagogues in the various procedures . The action of trypain oa pituitary cells under our experimental conditions appears restricted to thyrotrophs and possibly mammotrophs . Using the same trypain dissociation procedure we have previously found that pituitary cells respond to LH-RH with a dose-dependent increase in LH and FSH release and to a crude hypothalamic extract with a dose-dependent increase in ACTH release . Thus, a protein moiety which is susceptible to proteolytic action may be associated is a functional sense with the TRH receptor in contrast to the LH-RH and possibly the CRH receptors . That the action of trypain is localized at the cell surface is suggested by the ability of dibutyryl cyclic AMP, particularly in the low dose used, to increase the release of both TSH and prolactin . This lndicatea that the intracellular secretory machinery was not impaired by the prior exposure to trypain. Furthermore, the observation that the basal or unstimulated hormone release was the same in cells dissociated with either technique suggests the absence of a non-specific effect of In other tissues significant morphoeither method on membrane permeability . logical damage has been observed in cells isolated with the use of trypain (10) . No intracellular alterations have been observed in pituitary cells diasociated with the u~e of trypain (2,11) reinforcing our opinion that the damage was localized to the surface receptors . Several workers have suggested that the TRH receptors on thyrotrophe~ and mammotrophs are similar if not identical (12,13), While the present experiments are not addressed specifically to this point, it would appear that there may well be a functional difference between the TRH receptors on the two cell types mace TSH release 3s blocked only with the trypain technique . This, however, mây simply be a manifestation of inconsistent response of prolactin to TRH found in pituitary cell cultures particularly in the absence of eatrogens (13,14,15) .
207 8
TRH Receptors
Vol . 23, No . 20, 1978
Finally, Schrey et al . (16) have recently reported that rat pituitary cells isolated with the use of trypein do respond in a dose-dependent fashion to TRH with graded increases is TSH release . This discrepancy might be due to variations in the dissociation procedures used in the two studies . Acknowledgements The technical assistance of Michele Fontaine and Jack Wagoner is gratefully acknowledged . The authors wish to thank Dr . Albert Parlow and the NIAI~D Rat Pituitary Hormone Distribution Program for the TSH and prolactia radioimmunoassay material . The authors also thank Ruth Coaette for secretarial assistance . This work was supported by grants from the National Institutes of Health (AM 18328, AM 19170) and the American Cancer Society (BC 218) . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 .
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