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The effect of prostacyclin on intestinal ion transport in the rat
Pergamon Press
Life Sciences, Vol. 26, pp . 123-131 Printed in the U,S,A,
TSE EFFECT OF PROSTACYCLIN ON INTSSTII~L ION TRANSPORT IN THE RAT J . Hardcastle, P,T. Hardcastle and J.S . Radfern Department of Physiology, The University, Sheffield S10 2TN (Received in final form November 26, 1979) Summary The actions of PGI2 and PGE2 on electrically monitored ton transport in rat jejunum and colon have been determined both in vivo and in vitro. Whilst PCB2 was shown to induce a marked change in ion transport PGI2 was relatively ineffective . The ability of the prostanoids to influence ion transport is related to their capaoity to change mucosal cyclic A1~ levels since in isolated small intestinal anterocytes PGB2 caused a marked stimulation in cyclic AID levels whilst PGI2 had little effect . In colonie mucosal scrapes PGE was more effective than PGI2 in stimulating changes in cyclic A~ levels . It appears doubtful that PGI2 plays a direct role in the regulation of intestinal ia¢~ transport, Proetaglandina have a profound effect on intestinal fluid transport and have bean shown to cause a net secretion into the intestinal lumen (1, 2) . Transport studies reveal that thin secretion is due to a reduction in sodium absorption and a stimulation of chloride secretion (3, 4) . Such changes in intestinal transport probably contribute to the diarrhoea caomonly associated with prostaglaadinrproducing human tumours such as madullary carcinoma of the thyroid (5) . The ahanges in intestinal fluid and electrolyte went brought about by proataglandina are mediated by an increase in mucosal cyclic ALA levels due to a stimulation of adanylate cyclase (2, 6) . Recently a new prostaglandin has been discovered which has been named proatacyclin (PG2 2) . PGI~ is generated by the vascular walls and released into the arterial circulation (7) . It has also been found that PGI~ like activity ie present in homogenates of rat deal mucosa (8), suggestiag the possibility that this prostanoid might influence intestinal transport . Wa have therefore investigated the action of PGI on ion transport and mucosal cyclic ~ levels in the jejunum and colon of ~ rat and have compared its effects with those of PC~2 using electrical techniques to assess net action ion movement . l+lsthods Sxperimente were carried out an male albino rata weighing between 230 and 250q, These were obtained fxnm the Sheffield Field Laboratories and allowed free access to food (diet 86, Oxoid, London) and water .
0024-3205/80/020123-09$02,00/0 Copyright (c) 1980 Pergamon Press Ltd
37oC, the initial amM bathed potential the final transintestinal resistance small uA gas was the an arterial Devices 4made albumin the with dithiothreitol for of across tissue, mM and of modified and intestine intraperitoneal and generated solution 250 was washed dislocation saline small After and cell competitive via jejunum change difference intestine of KC1, and 10 potential contact with tissue 20 proximal were of oxygenated incubated of described one The aerosol The stopped MX2 ylmin pooled min, and and ablood difference the cyclic pellet electrical values incubated traneintestinal intestine incubation cell Krebs The 1containing of electrodes cannula in by cells recorder, administered in to by filled 20 aand intestinal and with stabilization each Weiser mM visual pressure solutions potential resistance give was mM by AMP and suspension colon differences these and was binding bicarbonate this for by colon (DTT) Epithelial phosphate were MgSO cyclic boiling r®moved theophylline injection pair 154 0to The was levels with centrifuged in Hardcastle aresistance (11) réauapended activity 15 the were display the tissues value final 1,5 (14), and washed mM ml were aand ,resistance being was measured sheet AI+IP protein 2oxygenated shaking through and by difference difference mM of solutions potential NaCl left 154 Gut mM was for in from incubated calculated connected means period buffered were measured mounted subtracted concentration was cells aBriefly, ethylenediaminetetra-acetic of saline the three NaH mM of isolated in was can using 3added Ion measurements &at in carotid rats 37oC sodium water min method NaCl using bathing ~ assayed Egqenton recorded acontact intestinal colon PO~~, be was of the obtained Transport were a900 cannula Ag/AgCl was calcium containing times the difference was between (10) dilution to via by calculated wide Prostsnoids killed saline for and peritoneal determined after bath The 1qThe pentobarbitone artery salt from intestinal (12) of measured used isolated same 250 ain medium two for with equilibrated calomel bore of in using Bell filled (9) mM tubes Brown, solution on in at aliquots perapex bridge behave electrodes the were by removal containing and sheets ~ 5successive buffer buffei 5to CaCl of abuffer the epithelial min, asalt 37oC aand tig/ml, magnesium-free Rats Telsec The from calculate was total two were made were blow 1using in with cavity half Howell by Albano, ~ithelial ltuninal jugular qas blood~essure electrodes, , for passing bridges, was in containing vivo pairs results Usainq-type determined the (15 wet of potential 10 were containing then with ohmic resistance every added to 154 cells athe two connected 27 mM pressure intestine discarded 7,5 15 the 5weight by Mistral the acid using Ekins, mM cells mM of vein the anaesthetised cm fluid Tris, min means method glucose, centrifuged channel 958 min, 26, were aresistors minute to supernatant tosodi~ NaCl, cells, head segments salt The current resistance (EDTA) 0phoapate and O2/58 difference the the two in No were to while chambers Since periods 6L 120 The Sqherzi ofwas gauge displayed to and of in bridge the 15 Vibron each after chart bovine 2, pH aThe mM C02 Stern and vivo ml wick the of of the 7,A~ 1980 at
12 4
Vol,
Prostacyclin
.
Measurement The preparation with jejunum transmural electrodes, other electrode . electrometers recorder,
.
Mean connected on
.
.
Prostanoids dose
.2
.
Measurement Sheets and The both current and 100 mucosal resultant of absence
. .
.
Potential an serosal
.
Estimation The cervical (13) filled citrate and buffered 0 .5 at collected centrifuge . NaCl, The serum buffer, proatanoid reaction 2500 using
. min .
t
.
.6
.
.2
.
.18 .
Vol . 26, No . 2, 1980
Prostacyclin and Gut Ion Transport
12 5
and Tampion (15) . Cyclic AMP levels were expressed as p~mole/mq protein ae determined by the method of Lowry, Rosebrough, Farr and Randall (16) using bovine serum albumin as standard . PGI 2 was used as the sodium salt and dissolved in 10 mM Tris HC1, adjusted to pH IO with 1M NaOH . PGß 2 was taken up in ethanol and further diluted with the appropriate buffer . Controls were carried out and in no instance could the obaexved effect be attributed to the vehicles employed . The Student's t-test was used to evaluate the experimental data . A paired t-teat was used for in vivo results ànd an unpaired t-test for those obtained in vitro . Reaulta ßffects of proetanoids in vivo
P
~ 15
~ Epf20ygll4p)
ed.
e
~ iz(zoy9~~
15 12
9
9
6
6
3
3
f~
COLON
0
Tme (mins,)
Fiq . 1 Typical affects of PGE and PQ , administered intravenously an transinteatinal potential differences ~ rat je~unum and colon, together with changea in arterial blood pressure . Both the jejunum and colon spontaneously generate a tranamural potential difference, the aerosol side of the tissue being positive with respect to the mucoeal aide . The magnitude of this potential is 4-7 mV in the jejune and 8-15 mV in the colon . Intravenous administration of PGE 2 (20 yq/kq) caused rapid and traasiant increases in both jejunal and colanic potential differences, while the acme dose of PGI 2 produced a significantly (p < O .ool) ®aller response (Fiq . 1) . The hypotensive actions of PGE 2 and PGI 2 however, ware not significantly different (p > O .1) .
12 6
Proatacyclin and Gut Ion Transport
Vol . 26, No . 2, 1980
6-koto -PGF la , the breakdown product of PG2 2 ,did not significantly affect either the tranaintestinal potential difference (p > 0 .05) or mean arterial blood pressure (p > O .1) . The changes in jejunal and colonic potential differences, caused by PGE 2 and PGI 2 , were dose-dependent over a range of 1 to 40 uq/~ and the dose response relationships were sigmoid (Fig . 2),
Fig . 2 Relationship between the dose of prostanoid administered intravenously and the change in the transintestinal potential difference in rat jejunum and colon . The change in potential difference ( ~ P,d) is plotted against l~q dose prostanoid and each point represents the mean of 6 observations - SEM . O " ~ PGE 2 ~ PGI2 Again these curves illustrate the lack of sensitivity of the intestine to PGI 2 in comparison with PGE2 . On the other hand the two prostanoids produced similar falls in mean arterial blood pressure over the whole dose range Q+Yg 3) Effects - of- prostanoida- in vitro To assess whether the observed electrical responses of the intestine to PGE 2 and PGI 2 reflected changes in net ion transport, the effects of these prostanoida on the current generated by sheets of jejunum and colon were measured (Fig . 4), The addition of 5 uq/ml PGE 2 to the aerosol solution increased the current generated by both regions of the intestine while the same concentration of PGI 2 produced a significantly smaller maximum response both in the jejunum (0 .01 > P > 0 .001) and colon (0 .01 > P > 0 .001) . 6-ketoPGFla caused only a small increase in jejunal and colonié currents .
Vol . 26, No . 2, 1980
Proatacyclin and Gut Ion Transport
127
120E E a .Q
Y
c c u
Y O
80604020 O at
1 1Ô Prostanoid (Ng 1 Kg)
~1 0
Fiq . 3
Relationship between the dose of prostanoid administered ihtravanously and the change in arterial blood pressure . The decrease in arterial blood pressure is plotted ngain#t log doae~prostanoid and each point represents the mean of 6 O obsarvationa - SEM. " ~ PGEZ ~ PGI2
200 1B0 160
É_ E
120 100
eo 60 40 20 0
Fiq . 4 8ffects of prostanoida on calculated current (I l ) across sheets of rat jejunum and colon . Proataaoide were added to t~ ~erosal solution to give a final oonceat~ation of 5 y~g/ml . Each point rspresents'the mean of 6 O / " ~ PGBZ ~ PGI2 ~ 6-koto-Prwtaglandin Fla observations - SEM . .
12 8
Prostacyclin and Gut Ion Transport
Vol . 26, No . 2, 1980
Effects of prostanotds on cyclic AMP levels Basal cyclic AMP levels averaged 10 pJmole/mg protein/15 min . PGE Z caused a dose~ependent increase in cyclic AMP levels in the isolated small intestinal epithelial cells with a maximal 3-fold increase (Fig . 5) . Neither PGI nor 6-keto-PGF had a significant effect (P > O .1) on cyclic AMP levels ât doses ,up to 10 uggml . At the highest dose used, 100 ug/ml, PGI 2 caused a slight stimulation of cyclic AMP levels, although the specificity of this action is in doubt as 6-keto-PGF~ produced a similar stimulation at the same dose .
5
~05%
01
1
Prostarod Concadrahon yg/ml
10
100
Fig . 5 Effect of varying concentrations of proatanoida on the cyclic AMP levela in isolated cello of rat small intestine . Each poin~ represents the meen of 3 individual experiments carried out in triplicate - SEM . " = PGEZ O a PliI 2 = 6-koto -Prostaglandin F lo An attempt was made to isolate the surface epithelial cells of the colon employing the same technique as used in the small intestine . However, the cell yields were too low to allow for reliable determination of cyclic AMP levels . In order to obtain some assessment of the effects of the proatanoida on colonic cyclic AMP levels, assays were carried out using mucosal scrapes (Fig . 6), even though the colonic surface epithelial cello comprise only a fraction of the total cell mass . PGE 2 caused a dose-dependent increase in cyclic AMP levels, a 15-fold stimulation being achieved at a dose of 20 ug/ml, this response being significantly greater (0 .05 > P > 0 .01) than the atimulat~on with 4 uq/ml PGE 2 . A significant increase in cyclic AMP levels was produced by both 4 ug/ml PGI 2 (0 .01 > P > 0 .001) and 20 ug/ml PGI 2 (0 .05 > P > 0 .01) . Although 4 ug/ml PGI 2 caused a similar stimulation to 4 ug/ml PGE Z (P > O .1), at the higher dose PGI was significantly (P < 0 .001) less active than PGE2 . In both jejunum and coon 20 uq/ml PGI was significantly less effective in stimulating cyclic AMP levels ~ the same concentration of PGE2 fP < 0 .001 in both tissues) . However, PGI 2 was
Dal . 26, No, 2, 1980
Proatacyclin and Gut Ion Transport
129
significantly more active in the colon than 1n the jejunum (d .01 > P > d,d01} .
Fig . 6 Effects of PGE2 and FGIZ an the cyclic AMP levels in mucoaal scrapes of rat proximal salon, The scrapes from 7 rata were pooled and 9 âetsrminatio~a were, made at each proatanoid concentration . Each point represents the mean - 3EM . Diacuseion It .is now well established that prostaglandina cause diarrhoea by inducing a net secretion into the lumen of the intestine (l, 2, 4), an effect mediated by an increase in mucaeal cyclic 11I+ß levels {2, 6} . The newly discovered PG2 2 is a patent stimulator of many prostaglandin induced responses and this suggests that it too may have a role in regulating intestinal transport activity . This suggestion is supported by the reported presence of prostacyclin-like activity in mucoeal extracts of the gastro-intestinal tract {8) and its ability to stimulate human colonic adenylate cyclase activity (17) . The results of the present study show that PGZ 2 is much leas effective in increasing the transintestinal potential difference than PGEZ {Figs . 1 an$ 2}, although both ps~oatanoids cause similar reäuctions in mean arterial blood pressure (Figs . 1 and 3) . The relative effectiveness of PGJ3 2 and PGI Z in producing a vaaodapreasor response is in agreement with the work of Armstrong, Lattimer, esancada and Vane (18) . Thus the lack of responsiveness of the transinteetinal potential difference to PGIZ cannot be attributed to its rapid metabolism and a consequent loss of activity .
13 0
Prostacyclin and Gut Ion Transport
Vol . 26, No, 2, 1980
That the increased tranaintestinal potential differences reflect an alteration in net ion movement is supported by the in vitro studies which show that, both PGE 2 and PGI 2 increase the current generated by the tissue (Fig . 4) . Again PGI caused a smaller response . The increased current generated may be a refection of either an increased cation absorption or an increased anion secretion, or both, but in view of the well established role of the prostaglandins in inducing a net secretion into the lumen it is probable that the increases in potential differences and current generated represent an increase in anion secretion, The secretion induced by prostaglandin has been shown to be mediated by cyclic AMP (2, 6) and this study demonstrated the elevation of cyclic AMP levels following the incubation of intestinal epithelial cells with PGE 2 (Fig . 5), in comparison with the marked elevation in cyclic AMP levels caused by PGE 2 , PGI 2 had no effect in the small intestine except at the highest dose used and even then caused no greater increase than that produced by its metabolite 6-keto -PGF~,,~ In the colonic scrapes PGE 2 was more effective in stimulating cyclic AMP Iévels than PGI2 , although PGI 2 had more effect than in the jejunum . This is an agreement witF~ the findings of Simon and coworkers who found that PGI 2 caused a 2 .5-fold increase in human colonic adenylate cyclase activity (17) whilst PGE 2 caused a 4 .5-fold increase (19), Since cyclic AMP mediates the intestinal secretion induced by proataglandina the fact that in the small intestine PGI 2 is unable to raise the level of this nucleotide in enterocytes is consistent with its relative lack of effect on electrically monitored ion transport . In the colon PGI 2 was relatively ineffective in inducing changes in ion transport and was less effective than PGE 2 in stimulating cyclic AMP levels even though it caused acme stimulation of nucleotide levels . Although same prostaglandina era known to promote intestinal secretion it seems unlikely that PGI 2 has an important effect on this aspect of intestinal function . . Acknowledgements We would like to thank Mr. K, Allen and Miss J . Cookson for skilled technical assistance . Prostaglandina were kindly supplied by Dr. J .E, Pike of the Upjohn Co Kalamazoo, Michigan, U,S,A . J .S,R, is in receipt of a S,R,C, studentship, References 1 . N,F, PIERCE, C .C .J . CARPENTER, H .L, ELLIOTT and W .B, GREENOUGH, Gastroenterology _60 22-32 (1971) . 2, M . TAUB, M,J . COYNE, G .G . HONORRIS, A . CHUNG, B, COYNE and L,J, SCHOENFIELD, Am . J, Gastroenterol, 70 129-135 (1978) . 3, Q, AL-AWQATI and W .B . GREENOUGH, Nature New Biology _238 26-27 (1972), 4 . C . MATUCHANSKY, J . -Y . MARY and J,J . BERNIER, Biol, Gastrcenterol . _5 175-186 (1972) . 5, E .D . WILLIAMS, S .M .M, KARIM and M . SANDLER, Lancet _1 22-23 (1968) . 6, D,V . KIMHERG, M . FIELD, J . JOHNSON, A, HENDERSON and E . GERSHON, J . Clin . Invest, _50 1218-1230 (1971) 7, S . MONCADA, R . KORBUT, S . HUNTING and J,R . VANE, Nature _273 767 (1978), 8, B,J .R, WHITTLE, Br . J . Phaxm, _64 438P (1978), 9, P .T . HARDCASTLE and J, EGGENTON, Biochim, Biophys . Acts _298 95-100 (1973) . 10, H,A, KREBS and K, HENSELEIT, Hoppe-Seyler's Z,Phyaiol, Chem, _210 33-66 (1933) .
Vol . 26, No . 2, 1980
11 . 12, 13 . 14 . 15, 16 . 17, 18, 19 .
Prostacgclin aad Gut Ion Transport
131
T .W . CLARRSOd~I and S .R . TOOLE, Am . J . Physiol . 206 658-668 (1964) . C .J . 8DFlOt~ID6, and J . MARRIOTT, J . Physiol, 194479-494 (1968) . B .R, STERN, Gaetroenterology 51 855-867 (1966) . M .W . WSISER, J . Biol . Chem . _248 2536-2541 (1973) . B,L . BROWN, J,D .M, ALBANO, R,F . ERINS, A .M . SQHERZI and W, TAMPION, Bioahem . J, _121 561-562 (1971) : O .H . hOWRY, N .J . ROSEBROOGH, A .L . FARR and J . RANDALL, J . Biol . Ch~, _193 265-275 (1951) . H . SIMON, H .RATSI:R and B . ICOMMERELL, Z . Gastroenterol . _16 748-751 (1978) . J .M . ARMSTRODiG, N, LATTIMER, S . MONCADA and J,R . VANE, Br . J, Phaxm . _62 125-130 (1978) . B, SIMON, P . CZYGAN, G . SPAAN, J . DITTRICH and H . RATHER Digestion _17 229-233 (1978) .
Life Sciences, Vol. 26, pp . 123-131 Printed in the U,S,A,
TSE EFFECT OF PROSTACYCLIN ON INTSSTII~L ION TRANSPORT IN THE RAT J . Hardcastle, P,T. Hardcastle and J.S . Radfern Department of Physiology, The University, Sheffield S10 2TN (Received in final form November 26, 1979) Summary The actions of PGI2 and PGE2 on electrically monitored ton transport in rat jejunum and colon have been determined both in vivo and in vitro. Whilst PCB2 was shown to induce a marked change in ion transport PGI2 was relatively ineffective . The ability of the prostanoids to influence ion transport is related to their capaoity to change mucosal cyclic A1~ levels since in isolated small intestinal anterocytes PGB2 caused a marked stimulation in cyclic AID levels whilst PGI2 had little effect . In colonie mucosal scrapes PGE was more effective than PGI2 in stimulating changes in cyclic A~ levels . It appears doubtful that PGI2 plays a direct role in the regulation of intestinal ia¢~ transport, Proetaglandina have a profound effect on intestinal fluid transport and have bean shown to cause a net secretion into the intestinal lumen (1, 2) . Transport studies reveal that thin secretion is due to a reduction in sodium absorption and a stimulation of chloride secretion (3, 4) . Such changes in intestinal transport probably contribute to the diarrhoea caomonly associated with prostaglaadinrproducing human tumours such as madullary carcinoma of the thyroid (5) . The ahanges in intestinal fluid and electrolyte went brought about by proataglandina are mediated by an increase in mucosal cyclic ALA levels due to a stimulation of adanylate cyclase (2, 6) . Recently a new prostaglandin has been discovered which has been named proatacyclin (PG2 2) . PGI~ is generated by the vascular walls and released into the arterial circulation (7) . It has also been found that PGI~ like activity ie present in homogenates of rat deal mucosa (8), suggestiag the possibility that this prostanoid might influence intestinal transport . Wa have therefore investigated the action of PGI on ion transport and mucosal cyclic ~ levels in the jejunum and colon of ~ rat and have compared its effects with those of PC~2 using electrical techniques to assess net action ion movement . l+lsthods Sxperimente were carried out an male albino rata weighing between 230 and 250q, These were obtained fxnm the Sheffield Field Laboratories and allowed free access to food (diet 86, Oxoid, London) and water .
0024-3205/80/020123-09$02,00/0 Copyright (c) 1980 Pergamon Press Ltd
37oC, the initial amM bathed potential the final transintestinal resistance small uA gas was the an arterial Devices 4made albumin the with dithiothreitol for of across tissue, mM and of modified and intestine intraperitoneal and generated solution 250 was washed dislocation saline small After and cell competitive via jejunum change difference intestine of KC1, and 10 potential contact with tissue 20 proximal were of oxygenated incubated of described one The aerosol The stopped MX2 ylmin pooled min, and and ablood difference the cyclic pellet electrical values incubated traneintestinal intestine incubation cell Krebs The 1containing of electrodes cannula in by cells recorder, administered in to by filled 20 aand intestinal and with stabilization each Weiser mM visual pressure solutions potential resistance give was mM by AMP and suspension colon differences these and was binding bicarbonate this for by colon (DTT) Epithelial phosphate were MgSO cyclic boiling r®moved theophylline injection pair 154 0to The was levels with centrifuged in Hardcastle aresistance (11) réauapended activity 15 the were display the tissues value final 1,5 (14), and washed mM ml were aand ,resistance being was measured sheet AI+IP protein 2oxygenated shaking through and by difference difference mM of solutions potential NaCl left 154 Gut mM was for in from incubated calculated connected means period buffered were measured mounted subtracted concentration was cells aBriefly, ethylenediaminetetra-acetic of saline the three NaH mM of isolated in was can using 3added Ion measurements &at in carotid rats 37oC sodium water min method NaCl using bathing ~ assayed Egqenton recorded acontact intestinal colon PO~~, be was of the obtained Transport were a900 cannula Ag/AgCl was calcium containing times the difference was between (10) dilution to via by calculated wide Prostsnoids killed saline for and peritoneal determined after bath The 1qThe pentobarbitone artery salt from intestinal (12) of measured used isolated same 250 ain medium two for with equilibrated calomel bore of in using Bell filled (9) mM tubes Brown, solution on in at aliquots perapex bridge behave electrodes the were by removal containing and sheets ~ 5successive buffer buffei 5to CaCl of abuffer the epithelial min, asalt 37oC aand tig/ml, magnesium-free Rats Telsec The from calculate was total two were made were blow 1using in with cavity half Howell by Albano, ~ithelial ltuninal jugular qas blood~essure electrodes, , for passing bridges, was in containing vivo pairs results Usainq-type determined the (15 wet of potential 10 were containing then with ohmic resistance every added to 154 cells athe two connected 27 mM pressure intestine discarded 7,5 15 the 5weight by Mistral the acid using Ekins, mM cells mM of vein the anaesthetised cm fluid Tris, min means method glucose, centrifuged channel 958 min, 26, were aresistors minute to supernatant tosodi~ NaCl, cells, head segments salt The current resistance (EDTA) 0phoapate and O2/58 difference the the two in No were to while chambers Since periods 6L 120 The Sqherzi ofwas gauge displayed to and of in bridge the 15 Vibron each after chart bovine 2, pH aThe mM C02 Stern and vivo ml wick the of of the 7,A~ 1980 at
12 4
Vol,
Prostacyclin
.
Measurement The preparation with jejunum transmural electrodes, other electrode . electrometers recorder,
.
Mean connected on
.
.
Prostanoids dose
.2
.
Measurement Sheets and The both current and 100 mucosal resultant of absence
. .
.
Potential an serosal
.
Estimation The cervical (13) filled citrate and buffered 0 .5 at collected centrifuge . NaCl, The serum buffer, proatanoid reaction 2500 using
. min .
t
.
.6
.
.2
.
.18 .
Vol . 26, No . 2, 1980
Prostacyclin and Gut Ion Transport
12 5
and Tampion (15) . Cyclic AMP levels were expressed as p~mole/mq protein ae determined by the method of Lowry, Rosebrough, Farr and Randall (16) using bovine serum albumin as standard . PGI 2 was used as the sodium salt and dissolved in 10 mM Tris HC1, adjusted to pH IO with 1M NaOH . PGß 2 was taken up in ethanol and further diluted with the appropriate buffer . Controls were carried out and in no instance could the obaexved effect be attributed to the vehicles employed . The Student's t-test was used to evaluate the experimental data . A paired t-teat was used for in vivo results ànd an unpaired t-test for those obtained in vitro . Reaulta ßffects of proetanoids in vivo
P
~ 15
~ Epf20ygll4p)
ed.
e
~ iz(zoy9~~
15 12
9
9
6
6
3
3
f~
COLON
0
Tme (mins,)
Fiq . 1 Typical affects of PGE and PQ , administered intravenously an transinteatinal potential differences ~ rat je~unum and colon, together with changea in arterial blood pressure . Both the jejunum and colon spontaneously generate a tranamural potential difference, the aerosol side of the tissue being positive with respect to the mucoeal aide . The magnitude of this potential is 4-7 mV in the jejune and 8-15 mV in the colon . Intravenous administration of PGE 2 (20 yq/kq) caused rapid and traasiant increases in both jejunal and colanic potential differences, while the acme dose of PGI 2 produced a significantly (p < O .ool) ®aller response (Fiq . 1) . The hypotensive actions of PGE 2 and PGI 2 however, ware not significantly different (p > O .1) .
12 6
Proatacyclin and Gut Ion Transport
Vol . 26, No . 2, 1980
6-koto -PGF la , the breakdown product of PG2 2 ,did not significantly affect either the tranaintestinal potential difference (p > 0 .05) or mean arterial blood pressure (p > O .1) . The changes in jejunal and colonic potential differences, caused by PGE 2 and PGI 2 , were dose-dependent over a range of 1 to 40 uq/~ and the dose response relationships were sigmoid (Fig . 2),
Fig . 2 Relationship between the dose of prostanoid administered intravenously and the change in the transintestinal potential difference in rat jejunum and colon . The change in potential difference ( ~ P,d) is plotted against l~q dose prostanoid and each point represents the mean of 6 observations - SEM . O " ~ PGE 2 ~ PGI2 Again these curves illustrate the lack of sensitivity of the intestine to PGI 2 in comparison with PGE2 . On the other hand the two prostanoids produced similar falls in mean arterial blood pressure over the whole dose range Q+Yg 3) Effects - of- prostanoida- in vitro To assess whether the observed electrical responses of the intestine to PGE 2 and PGI 2 reflected changes in net ion transport, the effects of these prostanoida on the current generated by sheets of jejunum and colon were measured (Fig . 4), The addition of 5 uq/ml PGE 2 to the aerosol solution increased the current generated by both regions of the intestine while the same concentration of PGI 2 produced a significantly smaller maximum response both in the jejunum (0 .01 > P > 0 .001) and colon (0 .01 > P > 0 .001) . 6-ketoPGFla caused only a small increase in jejunal and colonié currents .
Vol . 26, No . 2, 1980
Proatacyclin and Gut Ion Transport
127
120E E a .Q
Y
c c u
Y O
80604020 O at
1 1Ô Prostanoid (Ng 1 Kg)
~1 0
Fiq . 3
Relationship between the dose of prostanoid administered ihtravanously and the change in arterial blood pressure . The decrease in arterial blood pressure is plotted ngain#t log doae~prostanoid and each point represents the mean of 6 O obsarvationa - SEM. " ~ PGEZ ~ PGI2
200 1B0 160
É_ E
120 100
eo 60 40 20 0
Fiq . 4 8ffects of prostanoida on calculated current (I l ) across sheets of rat jejunum and colon . Proataaoide were added to t~ ~erosal solution to give a final oonceat~ation of 5 y~g/ml . Each point rspresents'the mean of 6 O / " ~ PGBZ ~ PGI2 ~ 6-koto-Prwtaglandin Fla observations - SEM . .
12 8
Prostacyclin and Gut Ion Transport
Vol . 26, No . 2, 1980
Effects of prostanotds on cyclic AMP levels Basal cyclic AMP levels averaged 10 pJmole/mg protein/15 min . PGE Z caused a dose~ependent increase in cyclic AMP levels in the isolated small intestinal epithelial cells with a maximal 3-fold increase (Fig . 5) . Neither PGI nor 6-keto-PGF had a significant effect (P > O .1) on cyclic AMP levels ât doses ,up to 10 uggml . At the highest dose used, 100 ug/ml, PGI 2 caused a slight stimulation of cyclic AMP levels, although the specificity of this action is in doubt as 6-keto-PGF~ produced a similar stimulation at the same dose .
5
~05%
01
1
Prostarod Concadrahon yg/ml
10
100
Fig . 5 Effect of varying concentrations of proatanoida on the cyclic AMP levela in isolated cello of rat small intestine . Each poin~ represents the meen of 3 individual experiments carried out in triplicate - SEM . " = PGEZ O a PliI 2 = 6-koto -Prostaglandin F lo An attempt was made to isolate the surface epithelial cells of the colon employing the same technique as used in the small intestine . However, the cell yields were too low to allow for reliable determination of cyclic AMP levels . In order to obtain some assessment of the effects of the proatanoida on colonic cyclic AMP levels, assays were carried out using mucosal scrapes (Fig . 6), even though the colonic surface epithelial cello comprise only a fraction of the total cell mass . PGE 2 caused a dose-dependent increase in cyclic AMP levels, a 15-fold stimulation being achieved at a dose of 20 ug/ml, this response being significantly greater (0 .05 > P > 0 .01) than the atimulat~on with 4 uq/ml PGE 2 . A significant increase in cyclic AMP levels was produced by both 4 ug/ml PGI 2 (0 .01 > P > 0 .001) and 20 ug/ml PGI 2 (0 .05 > P > 0 .01) . Although 4 ug/ml PGI 2 caused a similar stimulation to 4 ug/ml PGE Z (P > O .1), at the higher dose PGI was significantly (P < 0 .001) less active than PGE2 . In both jejunum and coon 20 uq/ml PGI was significantly less effective in stimulating cyclic AMP levels ~ the same concentration of PGE2 fP < 0 .001 in both tissues) . However, PGI 2 was
Dal . 26, No, 2, 1980
Proatacyclin and Gut Ion Transport
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significantly more active in the colon than 1n the jejunum (d .01 > P > d,d01} .
Fig . 6 Effects of PGE2 and FGIZ an the cyclic AMP levels in mucoaal scrapes of rat proximal salon, The scrapes from 7 rata were pooled and 9 âetsrminatio~a were, made at each proatanoid concentration . Each point represents the mean - 3EM . Diacuseion It .is now well established that prostaglandina cause diarrhoea by inducing a net secretion into the lumen of the intestine (l, 2, 4), an effect mediated by an increase in mucaeal cyclic 11I+ß levels {2, 6} . The newly discovered PG2 2 is a patent stimulator of many prostaglandin induced responses and this suggests that it too may have a role in regulating intestinal transport activity . This suggestion is supported by the reported presence of prostacyclin-like activity in mucoeal extracts of the gastro-intestinal tract {8) and its ability to stimulate human colonic adenylate cyclase activity (17) . The results of the present study show that PGZ 2 is much leas effective in increasing the transintestinal potential difference than PGEZ {Figs . 1 an$ 2}, although both ps~oatanoids cause similar reäuctions in mean arterial blood pressure (Figs . 1 and 3) . The relative effectiveness of PGJ3 2 and PGI Z in producing a vaaodapreasor response is in agreement with the work of Armstrong, Lattimer, esancada and Vane (18) . Thus the lack of responsiveness of the transinteetinal potential difference to PGIZ cannot be attributed to its rapid metabolism and a consequent loss of activity .
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That the increased tranaintestinal potential differences reflect an alteration in net ion movement is supported by the in vitro studies which show that, both PGE 2 and PGI 2 increase the current generated by the tissue (Fig . 4) . Again PGI caused a smaller response . The increased current generated may be a refection of either an increased cation absorption or an increased anion secretion, or both, but in view of the well established role of the prostaglandins in inducing a net secretion into the lumen it is probable that the increases in potential differences and current generated represent an increase in anion secretion, The secretion induced by prostaglandin has been shown to be mediated by cyclic AMP (2, 6) and this study demonstrated the elevation of cyclic AMP levels following the incubation of intestinal epithelial cells with PGE 2 (Fig . 5), in comparison with the marked elevation in cyclic AMP levels caused by PGE 2 , PGI 2 had no effect in the small intestine except at the highest dose used and even then caused no greater increase than that produced by its metabolite 6-keto -PGF~,,~ In the colonic scrapes PGE 2 was more effective in stimulating cyclic AMP Iévels than PGI2 , although PGI 2 had more effect than in the jejunum . This is an agreement witF~ the findings of Simon and coworkers who found that PGI 2 caused a 2 .5-fold increase in human colonic adenylate cyclase activity (17) whilst PGE 2 caused a 4 .5-fold increase (19), Since cyclic AMP mediates the intestinal secretion induced by proataglandina the fact that in the small intestine PGI 2 is unable to raise the level of this nucleotide in enterocytes is consistent with its relative lack of effect on electrically monitored ion transport . In the colon PGI 2 was relatively ineffective in inducing changes in ion transport and was less effective than PGE 2 in stimulating cyclic AMP levels even though it caused acme stimulation of nucleotide levels . Although same prostaglandina era known to promote intestinal secretion it seems unlikely that PGI 2 has an important effect on this aspect of intestinal function . . Acknowledgements We would like to thank Mr. K, Allen and Miss J . Cookson for skilled technical assistance . Prostaglandina were kindly supplied by Dr. J .E, Pike of the Upjohn Co Kalamazoo, Michigan, U,S,A . J .S,R, is in receipt of a S,R,C, studentship, References 1 . N,F, PIERCE, C .C .J . CARPENTER, H .L, ELLIOTT and W .B, GREENOUGH, Gastroenterology _60 22-32 (1971) . 2, M . TAUB, M,J . COYNE, G .G . HONORRIS, A . CHUNG, B, COYNE and L,J, SCHOENFIELD, Am . J, Gastroenterol, 70 129-135 (1978) . 3, Q, AL-AWQATI and W .B . GREENOUGH, Nature New Biology _238 26-27 (1972), 4 . C . MATUCHANSKY, J . -Y . MARY and J,J . BERNIER, Biol, Gastrcenterol . _5 175-186 (1972) . 5, E .D . WILLIAMS, S .M .M, KARIM and M . SANDLER, Lancet _1 22-23 (1968) . 6, D,V . KIMHERG, M . FIELD, J . JOHNSON, A, HENDERSON and E . GERSHON, J . Clin . Invest, _50 1218-1230 (1971) 7, S . MONCADA, R . KORBUT, S . HUNTING and J,R . VANE, Nature _273 767 (1978), 8, B,J .R, WHITTLE, Br . J . Phaxm, _64 438P (1978), 9, P .T . HARDCASTLE and J, EGGENTON, Biochim, Biophys . Acts _298 95-100 (1973) . 10, H,A, KREBS and K, HENSELEIT, Hoppe-Seyler's Z,Phyaiol, Chem, _210 33-66 (1933) .
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11 . 12, 13 . 14 . 15, 16 . 17, 18, 19 .
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T .W . CLARRSOd~I and S .R . TOOLE, Am . J . Physiol . 206 658-668 (1964) . C .J . 8DFlOt~ID6, and J . MARRIOTT, J . Physiol, 194479-494 (1968) . B .R, STERN, Gaetroenterology 51 855-867 (1966) . M .W . WSISER, J . Biol . Chem . _248 2536-2541 (1973) . B,L . BROWN, J,D .M, ALBANO, R,F . ERINS, A .M . SQHERZI and W, TAMPION, Bioahem . J, _121 561-562 (1971) : O .H . hOWRY, N .J . ROSEBROOGH, A .L . FARR and J . RANDALL, J . Biol . Ch~, _193 265-275 (1951) . H . SIMON, H .RATSI:R and B . ICOMMERELL, Z . Gastroenterol . _16 748-751 (1978) . J .M . ARMSTRODiG, N, LATTIMER, S . MONCADA and J,R . VANE, Br . J, Phaxm . _62 125-130 (1978) . B, SIMON, P . CZYGAN, G . SPAAN, J . DITTRICH and H . RATHER Digestion _17 229-233 (1978) .