- Email: [email protected]
Stereospecific actions of misoprostol on rat colonic electrolyte transport
Prostaglandins 46:221-231, 1993 STEREOSPECIFIC
A C T I O N S OF M I S O P R O S T O L O N R A T C O L O N I C ELECTROLYTE TRANSPORT
S. Won-Kim, J.F. K a c h ~
and T.S. Gaginella 2
G a s t r o i n t e s t i n a l Diseases R e s e a r c h Searle Research & Development Skokle, IL 60077
ABSTRACT Misoprostol (Miso) produces a mild, transient diarrhea in some patients, which is believed to be partly due to intraluminal fluid accumulation. To better understand this diarrheagenic action, we compared the effects of Miso, its 4 stereoisomers (11R16R, 11 R16S, 11S16S, 11 $16R), misoprostol free acid (Miso-FA), and 16,16-dimethyl PGE 2 (dmPGE2) on rat colonic electrolyte transport in vitro. Increases in short-circuit current (Isc) were measured (after serosal addition) in segments of mucosa stripped of muscularis and mounted in Ussing chambers. The rank order of apparent potencies, in terms of threshold, were (uM): 11R, 16S (1.2) = dmPGE 2 (1.0)> Miso-FA (10.0) Miso > > 11R, 16R; 11S, 16R; 11S, 16S (all inactive at 100/~M). The response to dmPGE 2 and Miso was attenuated in the presence of the N a + / K + / C r co-transport inhibitor bumetanide (100 #M). Pretreatment with atropine (0.1 /~M) did not affect the Isc response to Miso, Miso-FA, or dmPGE 2. Tetrodotoxin partially attenuated (39+9% inhibition) the response to Miso-FA, but did not affect Miso or dmPGE 2. In conclusion, Miso increases CI secretion across rat colonic mucosa through a direct action on epithelial cells. The activity resides in the 11R,16S isomer, thus implying a stereospecific interaction at PGE receptors. The effect of Miso to stimulate epithelial CIsecretion might contribute to its diarrheagenic action in vivo.
INTRODUCTION Misoprostol (Miso), a methyl ester analog of PGE~, is currently used clinically for the prevention of gastric ulceration produced by non-steroidal antiinflammatory agents (1). Miso produces diarrhea to the extent of 13.1-39.2% of patients administered 200 /~g four times daily (2,3). Luminal fluid secretion associated with epithelial c r secretion may be a contributing factor in Miso-induced diarrhea.
Send reprint requests to J.F. Kachur, Searle Research and Development, 4901 Searle Parkway, Skokie, IL 60077 2 Present address: Aphton Corporation, Box 1049, Woodland, CA 95776
Copyright © 1993 Butterworth-Heinemann
222
Prostaglandins
Prostaglandins are well known to stimulate intestinal fluid secretion in rat intestinal mucosa (4,5). Rat ileal mucosa mounted in Ussing chambers responded to PGE 1 by secreting chloride ions from the serosal to mucosal medium: an event reflected by an increase in the overall transmucosal short-circuit current (Isc) (6). Prostaglandins are capable of regulating mucosal CI- secretion in the rat by a direct epithelial action or by stimulating enteric nerves (7). Miso is a mixture of two diastereomers and each diastereomer is composed of two equimolar enantiomers (Figure 1). Miso is converted by tissue esterases to its free acid (Miso-FA), in vivo. To better understand the diarrheagenic action of Miso in terms of fluid secretion, its effect on colonic ion transport in the rat was studied. Comparisons were made with its stereoisomers, the free acid and 16,16-dimethyl PGE 2 (dmPGE2).
METHODS
Ion transport studies Male Sprague-Dawley rats (200-300 g, Charles River Laboratories, Wilmington, MA) were maintained on a standard laboratory diet and allowed free access to food and water. After cervical dislocation, the distal colon was excised and placed immediately in oxygenated modified Krebs-Ringer buffer solution of the following composition (millimolar): NaCI 120.2;KCI 5.9; CaCI 2 2.5; MgCI 2 1.2; NaH2PO 4 1.2 and NaHCO 3 25; glucose 11.1. The tissue was stripped of its underlying longitudinal and circular muscle layers by blunt dissection; the resulting preparation consisted of only mucosa and submucosa. Adjacent tissue was mounted as a flat sheet on pins between two lucite half chambers (World Precision Instruments, Inc., New Haven, CT). The area was 0.64 cm 2. Tissues were bathed on both sides by 5 ml of the buffer solution, circulated by gas lift and maintained at 37°C by water jacketed reservoirs. The solution was gassed continuously with 5% CO 2 in 02 and maintained at pH of 7.4. The buffer solution also contained 5.0/~M indomethacin to suppress the endogenous reduction of prostanoids. Electrical measurements were monitored with an automatic voltage clamp (TR 100F, JWT engineering, Overland Park, KS). Direct connecting voltage and current passing electrodes (Worlds Precision Instruments, Inc., New Haven, CT.) were utilized to measure transepithelial potential difference (PD) and short-circuit (Isc). Transepithelial PD was periodically measured and tissue resistance (Rt) was calculated from Ohm's law. Isc was continuously recorded on a Gould model 2800S reorder (Gould Inc., Cleveland, OH). Tissues were equilibrated under short circuit conditions until Isc had stabilized (usually 30-45 minutes). Basal resistance values averaged 96 4- 8 Ohm. cm 2 (n= 18) 30 minutes after mounting. All compounds were added to the serosal bathing medium. Inhibitors (serosal addition) were added 20 minutes prior to misoprostol.
Prostaglandins
223
0
(+_3 OH
HO
Misoprostol (SC-29333) R=CH~ Misoprostol Free Acid (SC-306953 R=H
Stereoisomers 0
O
\
I
cx~ ~ _,,,OH
HO
SC-30249 llR, 16S O
SC-30423 11S, 16R O
Fto
5C-30248 I1R. 16R
Figure 1' Miso.
SC-_042_q"~ 11S. 16S
Chemical structures for Miso, Miso-FA free acid and the stereoisomers of
224
Prostaglandins
Concentration-response curves were constructed by cumulative addition (10 .8- 104 M) of agonist to the serosal side of the tissue. The maximal response was defined as the last incremental concentration of agonist that produced no further increase in Isc. Materials 16,16-dimethyl PGE2, bumetanide, tetrodotoxin and atropine were purchased from Sigma Chemical Company (St. Louis, MO). Misoprostol, misoprostol free acid and isomers were synthesized (8) at Searle (Skokie, IL). The drugs were dissolved in dimethyl sulfoxide (DMSO) and the final concentration of solvent did not exceed 0 2% in the bath. An equivalent amount of DMSO (without drug) added to the serosal and mucosal sides of the tissue had no effect on Isc. Statistics Individual experiments were averaged and the results are given as the mean + SEM. Tissues were paired on the basis of tissue resistance (< 25% difference). Statistical comparisons between a prostaglandin alone and in the presence of TTX or bumetanide were made above the threshold concentration of the agonist, using Student's paired t-test.
RESULTS Application of Miso or Miso-FA to the serosal side of the tissue increased Isc. Although true potencies could not be determined because we were not sure that all the drugs had achieved their maximum, the threshold concentrations required to initiate a response indicated the following order of "apparent potency" (/~V1) of Miso and related agonists were determined (Figure 2) as follows: 11R, 16S (1.2) = dmPGE 2 (1.0) > MisoFA (10.0) ~ Miso (11.1). 11R, 16R; 11S, 16S; and 11S, 16R were inactive at 100/~VI. To examine the ionic basis of its Isc response, Miso was tested in the presence of the Na+/K+/Cr co-transport inhibitor, bumetanide. Pretreatment with bumetanide significantly reduced the Isc response to Miso (Figure 3). The Isc response to dmPGE2, and effect that is known to be due to luminal secretion of CI- (5), was also inhibited by bumetanide. The neural toxin tetrodotoxin (TTX) was used to assess the role of enteric nerves in mediating the response to misoprostol. TTX did not significantly affect the Isc response to Miso, although the maximal response to Miso-FA was reduced by 39% (Figure 4). Pretreatment with atropine did not affect the response to either Miso or MisoFA (Figure 5). The Isc response to dmPGE2 was not reduced by atropine or tetrodotoxin (data not shown).
Prostaglandins
75
225
.... O " "
i i
16,16dmPGE2
mlsoprostol misoprostolfree acid
.... "~'-"
11R 16R
A
11R 16s
:
11,1,,
L~
11s 16R
55
l ,,L ~:~
T
m
~.
35
c
-5
. . . . . . . .
1 0 -8
!
10 -7
•
•
"
" ' ' ' ' 1
"
'
. . . . . .
10 . 6 [AGONIST],
I
10 .5
•
"
"
. . . . .
I
10 .4
. . . . . . . .
10 . 3
M
Figure 2. Effect of increasing concentrations of Miso and related agonists on Isc in distal colon, dmPGE 2 is included here for comparison, n number ranged from 3 to 16 animals. Miso and dmPGE 2 did not significantly affect transmucosal resistance; Miso-FA decreased resistance by 18% (NS). Note that 16,16-dmPGE 2 and the 11R, 16S isomer had a threshold concentration of 1/~M for, whereas Miso-FA and Miso required a 10bold higher concentration before a notable response was seen.
Prostaglandins
226 A. 120 .... -O-----
E
control +bumetanide
100
,< 80
0
o7OOOOOI ,.
r-
o" /
/ m
P 0 e,
'°"1
0 ....... 10 .8
....
~--"
10 .7
10 .6
10 -5
10-4
[MISOPROSTOL], M
B. 100 .... -0"---
E
80
--
control
+ bumetanide
,< 0
60
Q
C
40
CO
..°°'" .°°o°"
20
0 w 10 -8
.......
~ 10 "7
........
i
........
10 -6
| 10 .5
........
, 10 -4
[16,16 dMPGE2], M
Figure 3. Effect of pretreatment with bumetanide (100/~M) on the Isc response to Miso (A) and dmPGE 2 (B). n = 5 and 3 animals for Miso and dmPGE 2 (B), respectively. Bumetanide significantly (P<0.05) inhibited the effect of Miso at and above 10-5M and dmPGE 2 at and above 10-eM. Bumetanide decreased Isc by 6 + 3/~A/cm 2.
Prostaglandins
227 A.
80 ---O--
70 ¸
control
60500 C
40302010-
oJ
1 0 "9
10 -8
10-7
10-6
10-5
[MISOPROSTOL],
10-4
M
B.
80.... -0---
.. E
o
control
+ "l-rx
60'
;o;
O m
40'
r"
°.oj~T~ *'**~**
O
20' O C
o-; .... ; o ~ .... ; - o ; "
lO-'
,o-'
,o"
[MISOPROSTOL FREE ACID], M
Figure 4. Effect of pretreatment with TFX (0.1/~M) on the Isc response to Miso (A) and Miso-FA (B). n = 3 and 8 animals for Miso and Miso-FA respectively. Tetrodotoxin significantly (P<0.05) inhibited only the effect of Miso-FA, at and above 105M. Tetrodotoxin decreased Isc by 10 + 3/~A/cm 2.
Prostaglandins
228 A. 7060' E ,~ ::I.
so.
e,"
30"
control
.... - O - - ~
+ atropine
C 10"
O-- . . . . . . ~= ...... 10-9
10-8
10-7
.
10-6
[MISOPROSTOL],
10-5
10-4
M
B. 80' .... - O " ' " m
A
E
control
60.
,,,.,,. (.1
--
40"
C
4) OI rJ C
2o
0-9
10-8
10-7
[MISOPROSTOL
10-6
10-5
FREE ACID],
10-4
M
Figure 5. Effect of atropine (0.1 /~M) on the Isc response to Miso (A) and Miso-FA (B). n = 3 and 8 for misoprostol and misoprostol free acid, respectively. Atropine did not affect Isc.
Prostaglandins
229
DISCUSSION
The results of this study demonstrate that the secretory action of Miso on rat colonic mucosal ion transport resembles that of other diarrheagenic prostaglandins (e.g. dmPGE2). Moreover, the electrical response to Miso-FA was similar to that of Miso. Inhibition of the Miso-induced increase in Isc was bumetanide sensitive, suggesting that electrogenic c r secretion is a major contributor to the colonic Isc response. Bumetanide is reported to inhibit PGEl-stimulated c r secretion in the rabbit colon (9). Miso was previously reported to reduce active Na ÷ absorption in the rat proximal colon (10). It seems likely that both stimulation of electrogenic c r secretion, as well as inhibition of active electrolyte absorption contribute to the overall secretory response to Miso in vivo. In the absence of data on unidirectional fluxes of 22Na+ and 36Cl we cannot absolutely identify the ion responsible for the change in Isc. 11R, 16S was the only active isomer of Miso. Its apparent potency was approximately equal to that of dmPGE 2. Miso, which contains equal amounts of all 4 isomers, was approximately 4x less active than 11R, 16S. These observations reveal the stereospecific nature of prostaglandin receptors that mediate ion transport in the colon. The stereospecific response of Miso extends to the small intestine as well, because the same rank order of potency for the isomers was observed in the ileum as in the colon (data not shown). Other studies in which misoprostol was compared with its isomers, relative to inhibition of gastric secretion and stimulation of gerbil colon contractility (11), yielded a rank order essentially the same as that which we observed. It is reported that prostaglandin receptors exist on intestinal epithelial cells (12). The receptor activated changes in epithelial function that lead to c r secretion are probably linked to adenylate cyclase (5,12,13). In the guinea pig, PGE 2 and Miso may differ in their abilities to stimulate epithelial cell adenylate cyclase (14), suggesting either varying abilities to interact with one type of prostanoid receptor or an action on different receptor sub-types. At least three prostaglandin receptor sub types have been classified (15). The colonic mucosa is innervated by neurons that can release neurotransmitters or bioactive peptides, which stimulate epithelial cells to secrete CI. Prostaglandins may evoke intestinal fluid secretion by activating local secretory reflexes in the enteric nervous system (16). Diener et al (7) observed that the prostacyclin derivative iloprost induces CI secretion solely by activating submucosal neurons, whereas PGE 2 appears to act on both the neurons and the epithelium. Their results also suggested that the neuronal effect of prostaglandins was mediated, in part, by the release of acetylcholine. Our results with TTX indicate that M~so and Miso-FA act primarily on the epithelium, although a small contribution of the Miso-FA Isc response was TTX sensitive. The failure of atropine to block the Isc response to Miso-FA or Miso, however, rules out a role for the cholinergic nervous system in mediating their secretory action.
230
Prostaglandins
Tissue desensitization has been noted to the effects of prostaglandins on Isc (16). It is conceivable that in our study we did not reach a maximum with several of the prostanoids because of this phenomenon. Nevertheless, we believe our results are valid in terms of the relative non-maximal (threshold) responses. Each agonist conceivably could (produce different degrees of desensitization) but this seems unlikely because of cross-desensitization among prostanoids (17). Our results confirm that Miso alters ion transport in the rat in such a manner that in vivo there would be accumulation of fluid in the gut lumen. If this occurs to an extent greater than the ability of the colon to reabsorb the fluid then diarrhea will result. We cannot conclude that the secretion of fluid induced by prostaglandins is the principal or only means by which they provoke diarrhea. In other studies (unpublished observations) we noted agonist effects of Miso on ileal and colonic muscle contractility in vitro. It seems likely that the diarrhea observed with Miso and other prostaglandins is probably due to combined effects on motor and mucosal function of the small and large bowel.
ACKNOWLEDGEMENTS The authors wish to thank Jeanette Fraser, Sherri Gallagher and Isabelle Murti for preparation of the manuscript. The technical expertise of visiting scientist Y. Xie (National Institutes of Pharmaceutical R&D, Beijing, P.R.C.) was much appreciated. We also thank Dr. Paul Collins for his helpful comments. REFERENCES 1.
Jones, J.B. and R.T. Bailey Jr. Misoprostol: a prostaglandin E analog with antisecretory and cytoprotective properties. DICP, the Annals of Pharmacology, 2_.33:276-282. 1989.
2.
Graham, D.Y., N.M. Agrawal, and S.H. Roth. Prevention of NSAID induced gastric ulcer with misoprostol: muticentre, double-blind, placebo-controlled trial. Lancet, 2:1277-1280. 1988.
3.
Pappo, R., P.W. Collins, M.S. Bruhn, A.F. Gasiecki, C.J. Jung, H.W. Sause and J.A. Schulz. Recent developments in the synthesis of antisecretory prostaglandins. In: Chemistry, Biochemistry and Pharmacological Activity of Prostanoids. (S. M. Roberts and F. Scheinmann, eds.) Pergamon Press, New York, 1979, p. 17-26.
4.
Robert, A., J.E. Nezamis, C. Lancaster, A.J. Hanchard, and M.S. Klepper. Enteropooling assay: a test for diarrhea produced by prostaglandins. Prostaglandins, 1_~1:809-828. 1976.
5.
Gaginella, T.S. Eicosanoid-mediated intestinal secretion. In: Textbook of Secretory Diarrhea. (E. Lebenthal and M. Duffey, eds.) Raven Press Ltd, New York, 1990, p 15-30.
Prostaglandins
231
6.
AI-Awqati, Q. and W.B. Greenough Ill. Prostaglandins inhibit intestinal sodium transport. Nature (new Biol) 238:26-27. 1972.
7.
Diener, M., R.J. Bridges, S.F. Knobloch, W. Rummel. Neuronally mediated and direct effects of prostaglandins on ion transport in rat colon descendents. NaunynSchmiedebergs Arch Pharmaco1337:74-78. 1988.
8.
Hefting, R.L. and G.A. Clay. Overview of clinical safety with misoprostol. Digestive Diseases and Sciences 3__0:1855-1935. 1985.
9.
Scheffler, A., K. Heintze and K. Mussier. Bumetanide as an inhibitor of PGE 1- and theophylline-stimulated electrogenic chloride secretion in rabbit colon in vitro. In: Intestinal Absorption and Secretion. (E. Skadhauge and K. Heintze, eds.) MPT Press, New York, 1983.
10. Sernka, T.J., R.P. Rood, M.Y. Mah and C.H. Tseng. Antiabsorptive effects of 16,16 dimethyl prostaglandin E2 in isolated rat colon. Prostaglandins 2;3:411-426. 1982. 11. Bauer, R.F. Misoprostol preclinical pharmacology. Sciences. 3(3118S-125S. 1985.
Digestive Diseases and
12. Wu-Wang, C.Y., S.L. Wang, B. Stevens and J. Neu. Localization and characterization of prostaglandin E receptor in rat small intestine. Prostaglandins, Leukotrienes, and Essential Fatty Acids, 3._66:129-134. 1989. 13. Smith, G., G. Warhurst, M. Lees, L. Turnberg. Evidence that PGE2 stimulates intestinal epithelial cell adenylate cyclase by a receptor-mediated mechanism. Digestive Diseases and Sciences, 3___22:71-75. 1987. 14. Pawlotsky, J-M., P. Ruszniewski, F. ReyI-Desmars, M. Bourgeois and M.J.M. Levin. Effects of PGE 2, Misoprostol, and Enprostil on guinea pig enterocyte adenylate cyclase. Digestive Diseases and Sciences, 3._88:316-320. 1993. 15. Gardiner, P.J. Classification of Prostanoid Receptors. Advances in Prostaglandin, Thromboxane, and Leukotriene Research. (B. Samuelsson, editor), Raven Press, 1990, p.110-118. 16. Brunsson, I., A. Sjoqvist, M. Jodal, and O. Lundgren. Mechanisms underlying small intestinal secretion caused by arachidonic acid, prostaglandin E1 and prostaglandin E2 in the rat in vivo. Acta Physiol Scand 130:633-642. 1987. 17. Musch, M.W., M. Field, R.J. Miller, J.S. Stoff. Homologous desensitization to prostaglandins in rabbit ileum. Am J Physio1252:G120-G127. 1981. Edltor
: A.
Dubols
:
Recelved:
7-31-92
Accepted:
7-28-93
A C T I O N S OF M I S O P R O S T O L O N R A T C O L O N I C ELECTROLYTE TRANSPORT
S. Won-Kim, J.F. K a c h ~
and T.S. Gaginella 2
G a s t r o i n t e s t i n a l Diseases R e s e a r c h Searle Research & Development Skokle, IL 60077
ABSTRACT Misoprostol (Miso) produces a mild, transient diarrhea in some patients, which is believed to be partly due to intraluminal fluid accumulation. To better understand this diarrheagenic action, we compared the effects of Miso, its 4 stereoisomers (11R16R, 11 R16S, 11S16S, 11 $16R), misoprostol free acid (Miso-FA), and 16,16-dimethyl PGE 2 (dmPGE2) on rat colonic electrolyte transport in vitro. Increases in short-circuit current (Isc) were measured (after serosal addition) in segments of mucosa stripped of muscularis and mounted in Ussing chambers. The rank order of apparent potencies, in terms of threshold, were (uM): 11R, 16S (1.2) = dmPGE 2 (1.0)> Miso-FA (10.0) Miso > > 11R, 16R; 11S, 16R; 11S, 16S (all inactive at 100/~M). The response to dmPGE 2 and Miso was attenuated in the presence of the N a + / K + / C r co-transport inhibitor bumetanide (100 #M). Pretreatment with atropine (0.1 /~M) did not affect the Isc response to Miso, Miso-FA, or dmPGE 2. Tetrodotoxin partially attenuated (39+9% inhibition) the response to Miso-FA, but did not affect Miso or dmPGE 2. In conclusion, Miso increases CI secretion across rat colonic mucosa through a direct action on epithelial cells. The activity resides in the 11R,16S isomer, thus implying a stereospecific interaction at PGE receptors. The effect of Miso to stimulate epithelial CIsecretion might contribute to its diarrheagenic action in vivo.
INTRODUCTION Misoprostol (Miso), a methyl ester analog of PGE~, is currently used clinically for the prevention of gastric ulceration produced by non-steroidal antiinflammatory agents (1). Miso produces diarrhea to the extent of 13.1-39.2% of patients administered 200 /~g four times daily (2,3). Luminal fluid secretion associated with epithelial c r secretion may be a contributing factor in Miso-induced diarrhea.
Send reprint requests to J.F. Kachur, Searle Research and Development, 4901 Searle Parkway, Skokie, IL 60077 2 Present address: Aphton Corporation, Box 1049, Woodland, CA 95776
Copyright © 1993 Butterworth-Heinemann
222
Prostaglandins
Prostaglandins are well known to stimulate intestinal fluid secretion in rat intestinal mucosa (4,5). Rat ileal mucosa mounted in Ussing chambers responded to PGE 1 by secreting chloride ions from the serosal to mucosal medium: an event reflected by an increase in the overall transmucosal short-circuit current (Isc) (6). Prostaglandins are capable of regulating mucosal CI- secretion in the rat by a direct epithelial action or by stimulating enteric nerves (7). Miso is a mixture of two diastereomers and each diastereomer is composed of two equimolar enantiomers (Figure 1). Miso is converted by tissue esterases to its free acid (Miso-FA), in vivo. To better understand the diarrheagenic action of Miso in terms of fluid secretion, its effect on colonic ion transport in the rat was studied. Comparisons were made with its stereoisomers, the free acid and 16,16-dimethyl PGE 2 (dmPGE2).
METHODS
Ion transport studies Male Sprague-Dawley rats (200-300 g, Charles River Laboratories, Wilmington, MA) were maintained on a standard laboratory diet and allowed free access to food and water. After cervical dislocation, the distal colon was excised and placed immediately in oxygenated modified Krebs-Ringer buffer solution of the following composition (millimolar): NaCI 120.2;KCI 5.9; CaCI 2 2.5; MgCI 2 1.2; NaH2PO 4 1.2 and NaHCO 3 25; glucose 11.1. The tissue was stripped of its underlying longitudinal and circular muscle layers by blunt dissection; the resulting preparation consisted of only mucosa and submucosa. Adjacent tissue was mounted as a flat sheet on pins between two lucite half chambers (World Precision Instruments, Inc., New Haven, CT). The area was 0.64 cm 2. Tissues were bathed on both sides by 5 ml of the buffer solution, circulated by gas lift and maintained at 37°C by water jacketed reservoirs. The solution was gassed continuously with 5% CO 2 in 02 and maintained at pH of 7.4. The buffer solution also contained 5.0/~M indomethacin to suppress the endogenous reduction of prostanoids. Electrical measurements were monitored with an automatic voltage clamp (TR 100F, JWT engineering, Overland Park, KS). Direct connecting voltage and current passing electrodes (Worlds Precision Instruments, Inc., New Haven, CT.) were utilized to measure transepithelial potential difference (PD) and short-circuit (Isc). Transepithelial PD was periodically measured and tissue resistance (Rt) was calculated from Ohm's law. Isc was continuously recorded on a Gould model 2800S reorder (Gould Inc., Cleveland, OH). Tissues were equilibrated under short circuit conditions until Isc had stabilized (usually 30-45 minutes). Basal resistance values averaged 96 4- 8 Ohm. cm 2 (n= 18) 30 minutes after mounting. All compounds were added to the serosal bathing medium. Inhibitors (serosal addition) were added 20 minutes prior to misoprostol.
Prostaglandins
223
0
(+_3 OH
HO
Misoprostol (SC-29333) R=CH~ Misoprostol Free Acid (SC-306953 R=H
Stereoisomers 0
O
\
I
cx~ ~ _,,,OH
HO
SC-30249 llR, 16S O
SC-30423 11S, 16R O
Fto
5C-30248 I1R. 16R
Figure 1' Miso.
SC-_042_q"~ 11S. 16S
Chemical structures for Miso, Miso-FA free acid and the stereoisomers of
224
Prostaglandins
Concentration-response curves were constructed by cumulative addition (10 .8- 104 M) of agonist to the serosal side of the tissue. The maximal response was defined as the last incremental concentration of agonist that produced no further increase in Isc. Materials 16,16-dimethyl PGE2, bumetanide, tetrodotoxin and atropine were purchased from Sigma Chemical Company (St. Louis, MO). Misoprostol, misoprostol free acid and isomers were synthesized (8) at Searle (Skokie, IL). The drugs were dissolved in dimethyl sulfoxide (DMSO) and the final concentration of solvent did not exceed 0 2% in the bath. An equivalent amount of DMSO (without drug) added to the serosal and mucosal sides of the tissue had no effect on Isc. Statistics Individual experiments were averaged and the results are given as the mean + SEM. Tissues were paired on the basis of tissue resistance (< 25% difference). Statistical comparisons between a prostaglandin alone and in the presence of TTX or bumetanide were made above the threshold concentration of the agonist, using Student's paired t-test.
RESULTS Application of Miso or Miso-FA to the serosal side of the tissue increased Isc. Although true potencies could not be determined because we were not sure that all the drugs had achieved their maximum, the threshold concentrations required to initiate a response indicated the following order of "apparent potency" (/~V1) of Miso and related agonists were determined (Figure 2) as follows: 11R, 16S (1.2) = dmPGE 2 (1.0) > MisoFA (10.0) ~ Miso (11.1). 11R, 16R; 11S, 16S; and 11S, 16R were inactive at 100/~VI. To examine the ionic basis of its Isc response, Miso was tested in the presence of the Na+/K+/Cr co-transport inhibitor, bumetanide. Pretreatment with bumetanide significantly reduced the Isc response to Miso (Figure 3). The Isc response to dmPGE2, and effect that is known to be due to luminal secretion of CI- (5), was also inhibited by bumetanide. The neural toxin tetrodotoxin (TTX) was used to assess the role of enteric nerves in mediating the response to misoprostol. TTX did not significantly affect the Isc response to Miso, although the maximal response to Miso-FA was reduced by 39% (Figure 4). Pretreatment with atropine did not affect the response to either Miso or MisoFA (Figure 5). The Isc response to dmPGE2 was not reduced by atropine or tetrodotoxin (data not shown).
Prostaglandins
75
225
.... O " "
i i
16,16dmPGE2
mlsoprostol misoprostolfree acid
.... "~'-"
11R 16R
A
11R 16s
:
11,1,,
L~
11s 16R
55
l ,,L ~:~
T
m
~.
35
c
-5
. . . . . . . .
1 0 -8
!
10 -7
•
•
"
" ' ' ' ' 1
"
'
. . . . . .
10 . 6 [AGONIST],
I
10 .5
•
"
"
. . . . .
I
10 .4
. . . . . . . .
10 . 3
M
Figure 2. Effect of increasing concentrations of Miso and related agonists on Isc in distal colon, dmPGE 2 is included here for comparison, n number ranged from 3 to 16 animals. Miso and dmPGE 2 did not significantly affect transmucosal resistance; Miso-FA decreased resistance by 18% (NS). Note that 16,16-dmPGE 2 and the 11R, 16S isomer had a threshold concentration of 1/~M for, whereas Miso-FA and Miso required a 10bold higher concentration before a notable response was seen.
Prostaglandins
226 A. 120 .... -O-----
E
control +bumetanide
100
,< 80
0
o7OOOOOI ,.
r-
o" /
/ m
P 0 e,
'°"1
0 ....... 10 .8
....
~--"
10 .7
10 .6
10 -5
10-4
[MISOPROSTOL], M
B. 100 .... -0"---
E
80
--
control
+ bumetanide
,< 0
60
Q
C
40
CO
..°°'" .°°o°"
20
0 w 10 -8
.......
~ 10 "7
........
i
........
10 -6
| 10 .5
........
, 10 -4
[16,16 dMPGE2], M
Figure 3. Effect of pretreatment with bumetanide (100/~M) on the Isc response to Miso (A) and dmPGE 2 (B). n = 5 and 3 animals for Miso and dmPGE 2 (B), respectively. Bumetanide significantly (P<0.05) inhibited the effect of Miso at and above 10-5M and dmPGE 2 at and above 10-eM. Bumetanide decreased Isc by 6 + 3/~A/cm 2.
Prostaglandins
227 A.
80 ---O--
70 ¸
control
60500 C
40302010-
oJ
1 0 "9
10 -8
10-7
10-6
10-5
[MISOPROSTOL],
10-4
M
B.
80.... -0---
.. E
o
control
+ "l-rx
60'
;o;
O m
40'
r"
°.oj~T~ *'**~**
O
20' O C
o-; .... ; o ~ .... ; - o ; "
lO-'
,o-'
,o"
[MISOPROSTOL FREE ACID], M
Figure 4. Effect of pretreatment with TFX (0.1/~M) on the Isc response to Miso (A) and Miso-FA (B). n = 3 and 8 animals for Miso and Miso-FA respectively. Tetrodotoxin significantly (P<0.05) inhibited only the effect of Miso-FA, at and above 105M. Tetrodotoxin decreased Isc by 10 + 3/~A/cm 2.
Prostaglandins
228 A. 7060' E ,~ ::I.
so.
e,"
30"
control
.... - O - - ~
+ atropine
C 10"
O-- . . . . . . ~= ...... 10-9
10-8
10-7
.
10-6
[MISOPROSTOL],
10-5
10-4
M
B. 80' .... - O " ' " m
A
E
control
60.
,,,.,,. (.1
--
40"
C
4) OI rJ C
2o
0-9
10-8
10-7
[MISOPROSTOL
10-6
10-5
FREE ACID],
10-4
M
Figure 5. Effect of atropine (0.1 /~M) on the Isc response to Miso (A) and Miso-FA (B). n = 3 and 8 for misoprostol and misoprostol free acid, respectively. Atropine did not affect Isc.
Prostaglandins
229
DISCUSSION
The results of this study demonstrate that the secretory action of Miso on rat colonic mucosal ion transport resembles that of other diarrheagenic prostaglandins (e.g. dmPGE2). Moreover, the electrical response to Miso-FA was similar to that of Miso. Inhibition of the Miso-induced increase in Isc was bumetanide sensitive, suggesting that electrogenic c r secretion is a major contributor to the colonic Isc response. Bumetanide is reported to inhibit PGEl-stimulated c r secretion in the rabbit colon (9). Miso was previously reported to reduce active Na ÷ absorption in the rat proximal colon (10). It seems likely that both stimulation of electrogenic c r secretion, as well as inhibition of active electrolyte absorption contribute to the overall secretory response to Miso in vivo. In the absence of data on unidirectional fluxes of 22Na+ and 36Cl we cannot absolutely identify the ion responsible for the change in Isc. 11R, 16S was the only active isomer of Miso. Its apparent potency was approximately equal to that of dmPGE 2. Miso, which contains equal amounts of all 4 isomers, was approximately 4x less active than 11R, 16S. These observations reveal the stereospecific nature of prostaglandin receptors that mediate ion transport in the colon. The stereospecific response of Miso extends to the small intestine as well, because the same rank order of potency for the isomers was observed in the ileum as in the colon (data not shown). Other studies in which misoprostol was compared with its isomers, relative to inhibition of gastric secretion and stimulation of gerbil colon contractility (11), yielded a rank order essentially the same as that which we observed. It is reported that prostaglandin receptors exist on intestinal epithelial cells (12). The receptor activated changes in epithelial function that lead to c r secretion are probably linked to adenylate cyclase (5,12,13). In the guinea pig, PGE 2 and Miso may differ in their abilities to stimulate epithelial cell adenylate cyclase (14), suggesting either varying abilities to interact with one type of prostanoid receptor or an action on different receptor sub-types. At least three prostaglandin receptor sub types have been classified (15). The colonic mucosa is innervated by neurons that can release neurotransmitters or bioactive peptides, which stimulate epithelial cells to secrete CI. Prostaglandins may evoke intestinal fluid secretion by activating local secretory reflexes in the enteric nervous system (16). Diener et al (7) observed that the prostacyclin derivative iloprost induces CI secretion solely by activating submucosal neurons, whereas PGE 2 appears to act on both the neurons and the epithelium. Their results also suggested that the neuronal effect of prostaglandins was mediated, in part, by the release of acetylcholine. Our results with TTX indicate that M~so and Miso-FA act primarily on the epithelium, although a small contribution of the Miso-FA Isc response was TTX sensitive. The failure of atropine to block the Isc response to Miso-FA or Miso, however, rules out a role for the cholinergic nervous system in mediating their secretory action.
230
Prostaglandins
Tissue desensitization has been noted to the effects of prostaglandins on Isc (16). It is conceivable that in our study we did not reach a maximum with several of the prostanoids because of this phenomenon. Nevertheless, we believe our results are valid in terms of the relative non-maximal (threshold) responses. Each agonist conceivably could (produce different degrees of desensitization) but this seems unlikely because of cross-desensitization among prostanoids (17). Our results confirm that Miso alters ion transport in the rat in such a manner that in vivo there would be accumulation of fluid in the gut lumen. If this occurs to an extent greater than the ability of the colon to reabsorb the fluid then diarrhea will result. We cannot conclude that the secretion of fluid induced by prostaglandins is the principal or only means by which they provoke diarrhea. In other studies (unpublished observations) we noted agonist effects of Miso on ileal and colonic muscle contractility in vitro. It seems likely that the diarrhea observed with Miso and other prostaglandins is probably due to combined effects on motor and mucosal function of the small and large bowel.
ACKNOWLEDGEMENTS The authors wish to thank Jeanette Fraser, Sherri Gallagher and Isabelle Murti for preparation of the manuscript. The technical expertise of visiting scientist Y. Xie (National Institutes of Pharmaceutical R&D, Beijing, P.R.C.) was much appreciated. We also thank Dr. Paul Collins for his helpful comments. REFERENCES 1.
Jones, J.B. and R.T. Bailey Jr. Misoprostol: a prostaglandin E analog with antisecretory and cytoprotective properties. DICP, the Annals of Pharmacology, 2_.33:276-282. 1989.
2.
Graham, D.Y., N.M. Agrawal, and S.H. Roth. Prevention of NSAID induced gastric ulcer with misoprostol: muticentre, double-blind, placebo-controlled trial. Lancet, 2:1277-1280. 1988.
3.
Pappo, R., P.W. Collins, M.S. Bruhn, A.F. Gasiecki, C.J. Jung, H.W. Sause and J.A. Schulz. Recent developments in the synthesis of antisecretory prostaglandins. In: Chemistry, Biochemistry and Pharmacological Activity of Prostanoids. (S. M. Roberts and F. Scheinmann, eds.) Pergamon Press, New York, 1979, p. 17-26.
4.
Robert, A., J.E. Nezamis, C. Lancaster, A.J. Hanchard, and M.S. Klepper. Enteropooling assay: a test for diarrhea produced by prostaglandins. Prostaglandins, 1_~1:809-828. 1976.
5.
Gaginella, T.S. Eicosanoid-mediated intestinal secretion. In: Textbook of Secretory Diarrhea. (E. Lebenthal and M. Duffey, eds.) Raven Press Ltd, New York, 1990, p 15-30.
Prostaglandins
231
6.
AI-Awqati, Q. and W.B. Greenough Ill. Prostaglandins inhibit intestinal sodium transport. Nature (new Biol) 238:26-27. 1972.
7.
Diener, M., R.J. Bridges, S.F. Knobloch, W. Rummel. Neuronally mediated and direct effects of prostaglandins on ion transport in rat colon descendents. NaunynSchmiedebergs Arch Pharmaco1337:74-78. 1988.
8.
Hefting, R.L. and G.A. Clay. Overview of clinical safety with misoprostol. Digestive Diseases and Sciences 3__0:1855-1935. 1985.
9.
Scheffler, A., K. Heintze and K. Mussier. Bumetanide as an inhibitor of PGE 1- and theophylline-stimulated electrogenic chloride secretion in rabbit colon in vitro. In: Intestinal Absorption and Secretion. (E. Skadhauge and K. Heintze, eds.) MPT Press, New York, 1983.
10. Sernka, T.J., R.P. Rood, M.Y. Mah and C.H. Tseng. Antiabsorptive effects of 16,16 dimethyl prostaglandin E2 in isolated rat colon. Prostaglandins 2;3:411-426. 1982. 11. Bauer, R.F. Misoprostol preclinical pharmacology. Sciences. 3(3118S-125S. 1985.
Digestive Diseases and
12. Wu-Wang, C.Y., S.L. Wang, B. Stevens and J. Neu. Localization and characterization of prostaglandin E receptor in rat small intestine. Prostaglandins, Leukotrienes, and Essential Fatty Acids, 3._66:129-134. 1989. 13. Smith, G., G. Warhurst, M. Lees, L. Turnberg. Evidence that PGE2 stimulates intestinal epithelial cell adenylate cyclase by a receptor-mediated mechanism. Digestive Diseases and Sciences, 3___22:71-75. 1987. 14. Pawlotsky, J-M., P. Ruszniewski, F. ReyI-Desmars, M. Bourgeois and M.J.M. Levin. Effects of PGE 2, Misoprostol, and Enprostil on guinea pig enterocyte adenylate cyclase. Digestive Diseases and Sciences, 3._88:316-320. 1993. 15. Gardiner, P.J. Classification of Prostanoid Receptors. Advances in Prostaglandin, Thromboxane, and Leukotriene Research. (B. Samuelsson, editor), Raven Press, 1990, p.110-118. 16. Brunsson, I., A. Sjoqvist, M. Jodal, and O. Lundgren. Mechanisms underlying small intestinal secretion caused by arachidonic acid, prostaglandin E1 and prostaglandin E2 in the rat in vivo. Acta Physiol Scand 130:633-642. 1987. 17. Musch, M.W., M. Field, R.J. Miller, J.S. Stoff. Homologous desensitization to prostaglandins in rabbit ileum. Am J Physio1252:G120-G127. 1981. Edltor
: A.
Dubols
:
Recelved:
7-31-92
Accepted:
7-28-93