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Effect of vasoactive intestinal polypertide on prolactin release in vitro
Pergamon Press
Life Sciences, Vol. 25, pp . 2071-2074 Printed in the D.S .A .
BFBBCT OF {IASOA(.`PIVB I11TS8TINAL POLYPBPTID~B ~ PPOLIICTIN n_or ssç~ ~ VITPO C. J. Shear, J. A. Cleaiens and N . H. Dininger The Lilly Research Laboratories, B11 L111y and Coapany Indianapolis, IN 46206 (Received in final form November 2, 1979) Suasiary Vasoactive intestinal polypeptide (VIP) at concentrations of 1 x 10-811 to 1 x 10 -511 caused release of prolactin fros anterior pituitary tissue in vitro under specific experimental conditions . VIP stiaulatedprolactin release when anterior pituitary tissue was incubated in sodium 199 in glass culture tubes containing the peptide-antibiotic bacitracin or bovine soma albumin . ViP also caused significant release of prolactin when polystyrene instead of glass culture tubes were used as incubation vessels . The results indicate that VIP can cause prolactin reléase by a 8lrect action on anterior pituitary lactotrophes . Addition of bacitracin or bovine seras albumin to the culture medivo oust prevent VIP fros binding to glass . IIse of polystyrene culture tubes elisinates the need for an extraneous protein source to prevent VIP glass binding . Vasoactive intestinal~polypeptide (VIP) was originally isolated and purified fras porcine ®all intestine (1,2) . Imunohistochemical and radioimunological studies desonetrated VIP in neurons of the central and peripheral nervous systea (3-6) . The developsent of a sensitive radiassunoassay for ViP (7) has de~onatrated high levels of VIP insunoreactivity in the hypothalamus (B), and radioimunoassable VIP in hypothalanohypophyaeal portal blood is 19 to as high as 180 tines the concentration of the polypeptlde in systemic arterial blood (9) . In addition to its vasodilatory and hypotensive effects, VIP influences cardiac action, respiration, gastrainteatinal secretion and motility, sugar and lipid metabolise and central nervous eystes activity (10) . VIP alters anterior pituitary (AP) hormone release . Carebrocisternal administration of VIP to rats elevates plasma prolactin (PRL), growth hormone ((~), and luteini:ing hormone (LH) (11) . Synthetic VIP ac~inistered either intraventricularly or intravenously caused dose related elevations in plasma PRL in urethane-anesthetised rats, an effect which was attenuated by simultaneous administration of naloxone, an opiate antagonist, or L-DOPA, a precursor o! dopamine (12) . In vitro experimentation has produced varied results as to the effect of VIP on AP PRL release. Scse researchers reported that while VIP alone had no effect ce PRL release (11, 12) it did attenuate the inhibition of PRL release induced by dopamine in vitro (12) . Others reported VIP (1 x 10 -71I) to be stimuletary to PRL réleâse~n vitro when the peptide-antibiotic, bacitracin, was added to the incubationned~~presumably to prevent ensymatic 8egradation of the peptide (13) . 0024-3205/79/242071-04$02 .00/0 Copyriftht (c) 1979 Perp~amon Press Ltd
2072
VIP and Prolactin Release In Vitro
Vol . 25, Nos . 24 6 25, 1979
It was of interest to us to determine if VIP could cause the release of immunoassayable PRL in paired anterior pituitary halves incubated in vitro . Methods General Procedure Mature male Sprague Dawley rats (Laboratory Supply Co ., Cumberland, IN .) 225-250 g were used as pituitary tissue donors for all incubations . All studies utilized a paired pituitary half incubation system . Following decapitation the AP was rapidly removed, separated fran the posterior pituitary lobe, henisected longitudinally, and each half AP was placed in a 5 ml borosilicate glass (Fisher brand) or polystyrene (Lancer) culture tube containing 2 nl of medium 199 (GIHCO, Grand Island, NY .) . One tube containing half the AP was used to test the effect of VIP, while the tube containing the opposite half of the same AP was used as the control. Six such pairs (12 AP halves) of AP halves were used to test the effect of each dose of VIP on PRL release. The incubations were carried out in a Dubnoff metabolic shaker (60 cycles/min) under oonatant gassing with humidified 95 "02-58002 at 37 + 0 .5° C. The pa regained at 7 .25 + 0.1 throughout the preincubation and incubation . All ~ediuv 199 was képt in a reservoir at 37 + 0.5° C and constantly gassed with 95802-58002 for at least one hour prior to use . Physiological saline (0 .98 NaCl) was used as a control treatment and diluent for the stock solution and all doses of VIP . Synthetic VIP (Peninsula Laboratories, San Carlos, CA . ; Lot No . 00046) was used in all incubations . After a 1 hr preincubation, the median 199 in the tubes was decanted and 2 .0 nl of fresh medium was added to all control tubes . The maiming tube of each pair received 1 .9 ml medium 199 and either 0 .1 ml physiological saline or 0 .1 nl saline containing various amounts of VIP. Osmolarity of the incubation media was measured by freezing point depression and regained at 303 + 17 m0am in all tubes. All incubations lasted for 2.0 hrs . At the end of thé incubation the gedium was decanted, diluted appropriately and frozen at -20° C until assayed for PRL . Pituitary wet weights were recorded and PRL released into the medium was expressed an the basis of tissue weight . Stud ies using bacitaracin and bovine serim albumin in the incubation median In several incübations the peptide-antibiotic, bacitracin (1 x 10 - 1~oc bovine serum albumin (BSA) (1 .4 x 10 -5!!) was added to the median 199 prior to use in the preincabations and incubations . in each incubation a set of control tubes containing medüan 199 without bacitracin or BSA was used to Bake sure that the addition of these polypeptidea had no effect on hormone release . Assay and statistic al procedures Medium 199 aliquots from the incubations were diluted with 18 HSA-phosphate buffered saline and assayed for PRL and GH by radioisaunoassay . Reagents used in the assays were similar to those supplied in NI11L~D kits, and the assays were carried out in accordance with the instructions supplied in the radioimunoassay kite . PRL released into the media was expressed as ng of MI11l~D prolactin RP-1 per gg of AP tissue . 81nce VIP had no effect on GS release from AP tissue in vitro, no data is recorded in subsequent tables . The data were analyzed ûsing Student's " t-test' for paired observations . Results VIP (1 x 10-9!! to 1 x 10-7M) had no significant effect on PRL release when the incubation was conducted in glass culture tubes (Table 1 - 8xperinent 1) .
Vol . 25, Nos. 24 6 25, 1979
VIP and Prolactin Release In Vitro
2073
VPhen the AP tissue halves ++ere incubated in medium 199 containing bacitracin, VIP at concentrations of 1x10 -51l, 1x10 - , 1x10-8M, but not 1x10-91t caused significant PRL release when coapared to control AP halves incubated in medium 199 containing bacitracin alone. Addition of bacitracin to the media had no effect on PRL released from AP tissue (Table 1 Sxperiment 2) . iltsen AP tissue was incubated in glass culture tubes using median 199 containing BSA (1 .4x10 -6M), addition of VIP at concentrations of 1x10-51!, TABLE 1 . The 8ffect of VIP on Anterior Pituitary Prolactin Release In Vitro
Treataent Groupsa
Mean Difference in Prolactin Released bY Treated vs Control Pituitary Halves (ng/g9 AP Tissue)
Sxperiaent No . 1 - Glass Culture Tubes 1. 2. 3. 4.
Control Control Control Control
vs ve vs vs
Saline VIP (10-9M) VIP (10-81!) VIP (10-7M)
+8 .5 +53 .4 +87 .5 +102 .1
+ + + +
50 .3b 59 .2 57 .2 94 .9
Sxperinent No . 2 - Glass Culture Tubes: Bacitracin Present 1. 2. 3. 4. 5.
Controls va Saline Control vs VIP (10-9M) Control va VIP (10-8M) Control va VIP (10-7M) Control vs VIP (10-51!) Bxperigent po . 3
1 . Control va Saline 2 . Control va VIP (10-81!) 3 . Control va VIP (10-7M)
-35.3 +56 .4 +142 .5 +276 .8 +336 .2
+ + + + +
85 .9 42 .8 35 .2 (p~ .05)d 60 .7 (p< .02) 108.1 (p< .05)
Polystyrene Culture Tubes +1 .4 + 105.7 +248 .4 + 88 .1 (prc .05) +189 .7 + 50 .7 (pc .02)
8xperiaent No . 4 - Glass Culture Tubers BSA in Medium 1. 2. 3. 4. 5.
Controls va Saline Control va VIP (10-91!) Control va VIP (10-ölt) Control va VIP (10-7M) Control va VIP (10-51!)
+24 .3 +49 .6 +164 .8 +306 .4 +402 .7
+ + + + +
36 .2 32 .8 28 .4 (pt .05) 52 .7 (pr .02) 96 .2 (pc .01)
an~6 paired pituitary glands per conditions . bMean difference + S .B . of assn difference . cGroup 1 : aedism 199 containing no bacitracin va 1.9 gl gedium containing containing 10-51t becitracin plus 0.1 gl saline . All regaining control groups contain bacitracin . d8ignificance of wean difference detergined using Student'a 't-test' for paired observations . °Group 1s gedisat 199 containing nso HSA vs 1 .0 gl aedism 199 containing 1 .4 x 10-6M HSA + .1 nl saline . All regaining control groupa contain 88A.
2074
yIP and Prolactin Release In VZtrp
yol . 25, Nos . 24 & 25, 1979
When polystyrene culture tubes were used as incubation vessels, VIP caused significant release of PRL (Table 1 - Experiment 3) . 1x10 -7M, and 1x10 -8M but not 1x10 -9M caused significant release of PRL over that released bY control AP halves incubated in medium 199 containing BSA alone (Table 1 - Experiment 4) . Addition of BSA to the media had no effect on PRL release . Dis cussion Cerebrocisternal administration of VIP to rats produced elevations in plasma PRL, GH, and LH, but in vitro incubation of ng to 10 u g had no effect on hemipituitaries with doses of VIP ranging fron pituitary hormone release (11) . Other workers reported VIP (lx 10-7M) to be stimulatory to PRL release in vitro when bacitracin was present in the incubati~ media (13) . Bacitracin was used presumeably to prevent enzymatic degradation of VIP in vitro . The data in thisreport confirms that VIP releases PRL from AP tissue incubated in medium 199 when bacitracin is present . Further, bacitracin was not necessary when polystyrene (plastic) instead of glass culture tubes were used . When glass culture tubes were used, VIP significantly stimulated AP PRL release in vitro when HSA was substituted for bacitracin in the incubation media . 0uc findings suggest that when bacitracin or BSA was not present as an extraneous protein source, the VIP was inactivated, possibly by being bound to the glass tube walls . The physiological significance of our in vitro findings is not known at this time . However, the presence of highconcentrations of VIP in portal blood and the ability of VIP to stimulate PRL releasé under appropriate conditions suggests a stimulatory role of this ominous polypeptide in the control of PRL release in vivo .
10
References 1 . S . I . SAID and V . MUTT, Science 169 1217-1218 (1970 . 2 . V . MUTT and S . I . SAID, Europ . JBiochem . . _42 581-589 (1974) . 3 . M . G . BRYANT, S . R . BLOOM . J . M . POhA1C, R . H . ALBOQIIBItQUE, I . MODUN, and A . G . E . PEARSE, Lancet _I 991-993 (1976) . 4 . A . GIACHETTI, R . N . POSBCiBERG, and S . I . SAID, Lancet _II 741-742 (1976) . 5 . L .-I . IARSSON, L . EDVINSON, J . FAHRSNRRUG, R . HARANSON, C . OWMAN, O . SCHAFFALITZRY DE MUCICADELL, and F . 3UNDLSR, Brain Research _113 400-404 (1976) . 6 . P . C . AEON, J . FAHRENRRUG, O . B . SCHAFFALITZRY DE MUCRADELL, J . M . JSSSELL, and L . L . IVSRSEN, Brain Research 143 174-178 (1978) . 7 . J . FAHFt~iRRDG and 0 . B . SCHAFFALITSRY D8 MUCRADSLL, J . Lab . Clin . Med _89 1379-1388 (1977) . 8 . L .-I . IAR330N, J . FAHRSNICRUG, 0 . B . SCHAFFALITSRY DE MUCRADELL, F . SUNDLER, R . HARANSON, and J . F . REHFELD, Proc . Natl . Aced . Sci . (OSA) _73 3197-3200 (1976) . 9 . 3 . I . 3AID and J . C . PORTER, Life Sciences _24 227-230 (1979) . 10 . S . I . SAID and G . M . MARHLOUF, in Chey, W . Y . and F . P . Brook (Eds) . Endocrinolo~+ lof the Gut (Thorofare, New Jersey : Charles Slack, Inc . 1974), pp . 63-87 . 11 . E . VIJAYAN, W . R . 3AM30N, S . I . SAID, and S . M . MCCANN, Endocrinology _104 53-57 (1979) . 12 . Y . RATO, Y . IWASARI, J . IWASARI, H . ABS, N . YANAIHARA, and H . IMURA, Endocrinology _103 554-558 (1978) . 13 . M . RUBSRG, W . H . ROR52TEJN, S . ARANCIHIA, J . BSS30N and A . ENJALBERT, Europ . J . Pharm . 51 319-320 (1978) .
Life Sciences, Vol. 25, pp . 2071-2074 Printed in the D.S .A .
BFBBCT OF {IASOA(.`PIVB I11TS8TINAL POLYPBPTID~B ~ PPOLIICTIN n_or ssç~ ~ VITPO C. J. Shear, J. A. Cleaiens and N . H. Dininger The Lilly Research Laboratories, B11 L111y and Coapany Indianapolis, IN 46206 (Received in final form November 2, 1979) Suasiary Vasoactive intestinal polypeptide (VIP) at concentrations of 1 x 10-811 to 1 x 10 -511 caused release of prolactin fros anterior pituitary tissue in vitro under specific experimental conditions . VIP stiaulatedprolactin release when anterior pituitary tissue was incubated in sodium 199 in glass culture tubes containing the peptide-antibiotic bacitracin or bovine soma albumin . ViP also caused significant release of prolactin when polystyrene instead of glass culture tubes were used as incubation vessels . The results indicate that VIP can cause prolactin reléase by a 8lrect action on anterior pituitary lactotrophes . Addition of bacitracin or bovine seras albumin to the culture medivo oust prevent VIP fros binding to glass . IIse of polystyrene culture tubes elisinates the need for an extraneous protein source to prevent VIP glass binding . Vasoactive intestinal~polypeptide (VIP) was originally isolated and purified fras porcine ®all intestine (1,2) . Imunohistochemical and radioimunological studies desonetrated VIP in neurons of the central and peripheral nervous systea (3-6) . The developsent of a sensitive radiassunoassay for ViP (7) has de~onatrated high levels of VIP insunoreactivity in the hypothalamus (B), and radioimunoassable VIP in hypothalanohypophyaeal portal blood is 19 to as high as 180 tines the concentration of the polypeptlde in systemic arterial blood (9) . In addition to its vasodilatory and hypotensive effects, VIP influences cardiac action, respiration, gastrainteatinal secretion and motility, sugar and lipid metabolise and central nervous eystes activity (10) . VIP alters anterior pituitary (AP) hormone release . Carebrocisternal administration of VIP to rats elevates plasma prolactin (PRL), growth hormone ((~), and luteini:ing hormone (LH) (11) . Synthetic VIP ac~inistered either intraventricularly or intravenously caused dose related elevations in plasma PRL in urethane-anesthetised rats, an effect which was attenuated by simultaneous administration of naloxone, an opiate antagonist, or L-DOPA, a precursor o! dopamine (12) . In vitro experimentation has produced varied results as to the effect of VIP on AP PRL release. Scse researchers reported that while VIP alone had no effect ce PRL release (11, 12) it did attenuate the inhibition of PRL release induced by dopamine in vitro (12) . Others reported VIP (1 x 10 -71I) to be stimuletary to PRL réleâse~n vitro when the peptide-antibiotic, bacitracin, was added to the incubationned~~presumably to prevent ensymatic 8egradation of the peptide (13) . 0024-3205/79/242071-04$02 .00/0 Copyriftht (c) 1979 Perp~amon Press Ltd
2072
VIP and Prolactin Release In Vitro
Vol . 25, Nos . 24 6 25, 1979
It was of interest to us to determine if VIP could cause the release of immunoassayable PRL in paired anterior pituitary halves incubated in vitro . Methods General Procedure Mature male Sprague Dawley rats (Laboratory Supply Co ., Cumberland, IN .) 225-250 g were used as pituitary tissue donors for all incubations . All studies utilized a paired pituitary half incubation system . Following decapitation the AP was rapidly removed, separated fran the posterior pituitary lobe, henisected longitudinally, and each half AP was placed in a 5 ml borosilicate glass (Fisher brand) or polystyrene (Lancer) culture tube containing 2 nl of medium 199 (GIHCO, Grand Island, NY .) . One tube containing half the AP was used to test the effect of VIP, while the tube containing the opposite half of the same AP was used as the control. Six such pairs (12 AP halves) of AP halves were used to test the effect of each dose of VIP on PRL release. The incubations were carried out in a Dubnoff metabolic shaker (60 cycles/min) under oonatant gassing with humidified 95 "02-58002 at 37 + 0 .5° C. The pa regained at 7 .25 + 0.1 throughout the preincubation and incubation . All ~ediuv 199 was képt in a reservoir at 37 + 0.5° C and constantly gassed with 95802-58002 for at least one hour prior to use . Physiological saline (0 .98 NaCl) was used as a control treatment and diluent for the stock solution and all doses of VIP . Synthetic VIP (Peninsula Laboratories, San Carlos, CA . ; Lot No . 00046) was used in all incubations . After a 1 hr preincubation, the median 199 in the tubes was decanted and 2 .0 nl of fresh medium was added to all control tubes . The maiming tube of each pair received 1 .9 ml medium 199 and either 0 .1 ml physiological saline or 0 .1 nl saline containing various amounts of VIP. Osmolarity of the incubation media was measured by freezing point depression and regained at 303 + 17 m0am in all tubes. All incubations lasted for 2.0 hrs . At the end of thé incubation the gedium was decanted, diluted appropriately and frozen at -20° C until assayed for PRL . Pituitary wet weights were recorded and PRL released into the medium was expressed an the basis of tissue weight . Stud ies using bacitaracin and bovine serim albumin in the incubation median In several incübations the peptide-antibiotic, bacitracin (1 x 10 - 1~oc bovine serum albumin (BSA) (1 .4 x 10 -5!!) was added to the median 199 prior to use in the preincabations and incubations . in each incubation a set of control tubes containing medüan 199 without bacitracin or BSA was used to Bake sure that the addition of these polypeptidea had no effect on hormone release . Assay and statistic al procedures Medium 199 aliquots from the incubations were diluted with 18 HSA-phosphate buffered saline and assayed for PRL and GH by radioisaunoassay . Reagents used in the assays were similar to those supplied in NI11L~D kits, and the assays were carried out in accordance with the instructions supplied in the radioimunoassay kite . PRL released into the media was expressed as ng of MI11l~D prolactin RP-1 per gg of AP tissue . 81nce VIP had no effect on GS release from AP tissue in vitro, no data is recorded in subsequent tables . The data were analyzed ûsing Student's " t-test' for paired observations . Results VIP (1 x 10-9!! to 1 x 10-7M) had no significant effect on PRL release when the incubation was conducted in glass culture tubes (Table 1 - 8xperinent 1) .
Vol . 25, Nos. 24 6 25, 1979
VIP and Prolactin Release In Vitro
2073
VPhen the AP tissue halves ++ere incubated in medium 199 containing bacitracin, VIP at concentrations of 1x10 -51l, 1x10 - , 1x10-8M, but not 1x10-91t caused significant PRL release when coapared to control AP halves incubated in medium 199 containing bacitracin alone. Addition of bacitracin to the media had no effect on PRL released from AP tissue (Table 1 Sxperiment 2) . iltsen AP tissue was incubated in glass culture tubes using median 199 containing BSA (1 .4x10 -6M), addition of VIP at concentrations of 1x10-51!, TABLE 1 . The 8ffect of VIP on Anterior Pituitary Prolactin Release In Vitro
Treataent Groupsa
Mean Difference in Prolactin Released bY Treated vs Control Pituitary Halves (ng/g9 AP Tissue)
Sxperiaent No . 1 - Glass Culture Tubes 1. 2. 3. 4.
Control Control Control Control
vs ve vs vs
Saline VIP (10-9M) VIP (10-81!) VIP (10-7M)
+8 .5 +53 .4 +87 .5 +102 .1
+ + + +
50 .3b 59 .2 57 .2 94 .9
Sxperinent No . 2 - Glass Culture Tubes: Bacitracin Present 1. 2. 3. 4. 5.
Controls va Saline Control vs VIP (10-9M) Control va VIP (10-8M) Control va VIP (10-7M) Control vs VIP (10-51!) Bxperigent po . 3
1 . Control va Saline 2 . Control va VIP (10-81!) 3 . Control va VIP (10-7M)
-35.3 +56 .4 +142 .5 +276 .8 +336 .2
+ + + + +
85 .9 42 .8 35 .2 (p~ .05)d 60 .7 (p< .02) 108.1 (p< .05)
Polystyrene Culture Tubes +1 .4 + 105.7 +248 .4 + 88 .1 (prc .05) +189 .7 + 50 .7 (pc .02)
8xperiaent No . 4 - Glass Culture Tubers BSA in Medium 1. 2. 3. 4. 5.
Controls va Saline Control va VIP (10-91!) Control va VIP (10-ölt) Control va VIP (10-7M) Control va VIP (10-51!)
+24 .3 +49 .6 +164 .8 +306 .4 +402 .7
+ + + + +
36 .2 32 .8 28 .4 (pt .05) 52 .7 (pr .02) 96 .2 (pc .01)
an~6 paired pituitary glands per conditions . bMean difference + S .B . of assn difference . cGroup 1 : aedism 199 containing no bacitracin va 1.9 gl gedium containing containing 10-51t becitracin plus 0.1 gl saline . All regaining control groups contain bacitracin . d8ignificance of wean difference detergined using Student'a 't-test' for paired observations . °Group 1s gedisat 199 containing nso HSA vs 1 .0 gl aedism 199 containing 1 .4 x 10-6M HSA + .1 nl saline . All regaining control groupa contain 88A.
2074
yIP and Prolactin Release In VZtrp
yol . 25, Nos . 24 & 25, 1979
When polystyrene culture tubes were used as incubation vessels, VIP caused significant release of PRL (Table 1 - Experiment 3) . 1x10 -7M, and 1x10 -8M but not 1x10 -9M caused significant release of PRL over that released bY control AP halves incubated in medium 199 containing BSA alone (Table 1 - Experiment 4) . Addition of BSA to the media had no effect on PRL release . Dis cussion Cerebrocisternal administration of VIP to rats produced elevations in plasma PRL, GH, and LH, but in vitro incubation of ng to 10 u g had no effect on hemipituitaries with doses of VIP ranging fron pituitary hormone release (11) . Other workers reported VIP (lx 10-7M) to be stimulatory to PRL release in vitro when bacitracin was present in the incubati~ media (13) . Bacitracin was used presumeably to prevent enzymatic degradation of VIP in vitro . The data in thisreport confirms that VIP releases PRL from AP tissue incubated in medium 199 when bacitracin is present . Further, bacitracin was not necessary when polystyrene (plastic) instead of glass culture tubes were used . When glass culture tubes were used, VIP significantly stimulated AP PRL release in vitro when HSA was substituted for bacitracin in the incubation media . 0uc findings suggest that when bacitracin or BSA was not present as an extraneous protein source, the VIP was inactivated, possibly by being bound to the glass tube walls . The physiological significance of our in vitro findings is not known at this time . However, the presence of highconcentrations of VIP in portal blood and the ability of VIP to stimulate PRL releasé under appropriate conditions suggests a stimulatory role of this ominous polypeptide in the control of PRL release in vivo .
10
References 1 . S . I . SAID and V . MUTT, Science 169 1217-1218 (1970 . 2 . V . MUTT and S . I . SAID, Europ . JBiochem . . _42 581-589 (1974) . 3 . M . G . BRYANT, S . R . BLOOM . J . M . POhA1C, R . H . ALBOQIIBItQUE, I . MODUN, and A . G . E . PEARSE, Lancet _I 991-993 (1976) . 4 . A . GIACHETTI, R . N . POSBCiBERG, and S . I . SAID, Lancet _II 741-742 (1976) . 5 . L .-I . IARSSON, L . EDVINSON, J . FAHRSNRRUG, R . HARANSON, C . OWMAN, O . SCHAFFALITZRY DE MUCICADELL, and F . 3UNDLSR, Brain Research _113 400-404 (1976) . 6 . P . C . AEON, J . FAHRENRRUG, O . B . SCHAFFALITZRY DE MUCRADELL, J . M . JSSSELL, and L . L . IVSRSEN, Brain Research 143 174-178 (1978) . 7 . J . FAHFt~iRRDG and 0 . B . SCHAFFALITSRY D8 MUCRADSLL, J . Lab . Clin . Med _89 1379-1388 (1977) . 8 . L .-I . IAR330N, J . FAHRSNICRUG, 0 . B . SCHAFFALITSRY DE MUCRADELL, F . SUNDLER, R . HARANSON, and J . F . REHFELD, Proc . Natl . Aced . Sci . (OSA) _73 3197-3200 (1976) . 9 . 3 . I . 3AID and J . C . PORTER, Life Sciences _24 227-230 (1979) . 10 . S . I . SAID and G . M . MARHLOUF, in Chey, W . Y . and F . P . Brook (Eds) . Endocrinolo~+ lof the Gut (Thorofare, New Jersey : Charles Slack, Inc . 1974), pp . 63-87 . 11 . E . VIJAYAN, W . R . 3AM30N, S . I . SAID, and S . M . MCCANN, Endocrinology _104 53-57 (1979) . 12 . Y . RATO, Y . IWASARI, J . IWASARI, H . ABS, N . YANAIHARA, and H . IMURA, Endocrinology _103 554-558 (1978) . 13 . M . RUBSRG, W . H . ROR52TEJN, S . ARANCIHIA, J . BSS30N and A . ENJALBERT, Europ . J . Pharm . 51 319-320 (1978) .