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Inhibition of small intestinal mucosal and smooth muscle cell function by ricinoleic acid and other surfactants
Pergamon Press
Life Sciences Vol . 16, pp . 1595-1606 Printed in the U.S .A .
INHIBITION OF SMALL INTESTINAL MUCOSAL AND SMOOTH MUSCLE CELL FUNCTION BY RICINOLEIC ACID AND OTHER SURFACTANTS Timothy S . Gaginella, John J. Stewart, Gary W . Gullikaon Ward A . Olsen and Paul Bass School of Pharmacy, School of Medicine Center for Health Sciences University of Wisconsin Madison, Wisconsin 53706 (Received in final form May 1, 1975)
Summary Sodium ricinoleate, dioctyl sodium sulfosuccinate, sodium dodecyl (lauryl) sulfate, polysorbate 80, sodium deoxycholate and chenodeoxycholate were found to produce depression of in vitro mucosal and smooth muscle cell function . These actions were assessed by measuring net water and electrolyte transport from everted hamster gut sacs and contractile activity of the electrically stimulated guinea-pig ileum . All compounds were effective depressants of both systems at concentrations which were likely to be below their respective critical micellar concentration . Ricinoleic acid may produce its cathartic effect due to its amphipathic nature, possibly by hydrophobic interaction with membrane lipoproteins . Ricinoleic acid and the other surfactants may be acting through a common mechanism. Ricinoleic acid, the active constituent of castor oil, is an eighteen carbon, hydroxylated, monounsaturated fatty acid (1) . Although castor oil has been used as a safe and effective cathar tic for centuries, its mechanism of action has been only vaguely defined as an "irritant" or "stimulant" to the gastrointestinal tract (2-5) . These descriptions are particularly inappropriate since decreased contractile activity of circular smooth muscle has been reported after oral administration of cathartic doses of castor oil to intact unanesthetized dogs (6) . In addition, ricinoleic acid can inhibit water and electrolyte absorption from the human jejunum (7), dog ileum (8) and human colon (9) . Recently, a new explanation has been proposed to account for castor oil-induced catharsis _in vivo . In this explanation, both inhibition of water and electrolyte absorption and reduced contractile activity of intestinal circular smooth muscle are considered important for the cathartic action of the compound (6) . Long-chain fatty acids such as ricinoleate are amphipathic (hydrophilic-lipophilic) at physiologic pH, and they therefore possess surfactant activity (10) . It has been postulated that ricinoleate may inhibit intestinal fluid absorption as a consequence of its surface activity (8) . As a test of this hypothesis, 1595
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we have compared ricinoleic acid with : two structurally different anionic surfactants, dioctyl sodium sulfosuccinate (DSS) and sodium dodecyl sulfate (SDS) ; a non-ionic surfactant, polysorbate 80 (PS-80) ; and two bile salts, sodium deoxycholate and chenodeoxycholate . All surfactants were evaluated for their relative ability to inhibit net water and electrolyte absorption and intestinal smooth muscle contractile activity _in vitro . Absorption studies were conducted with everted hamster jejunal gut sacs . Electrically (coaxially) stimulated guinea-pig ileal preparations were used in studies comparing the actions of the test agents on contractile activity of intestinal smooth muscle . Materials and Methods Transport Studies . Nonfasted male Golden Syrian hamsters (Mesocricetus auratus weighing 90 to 120 grams were sacrified by cervical dislocation . Approximately a 15 cm segment of jejunum beginning at the ligament of Treitz was quickly removed and placed in mucosal buffer solution (aerated with 959 OZ - 5~ COZ)composed of (m Moles/1) : Na, 150 ; C1, 130 ; K, 5 ; HC03, 25 ; and dextrose 2 . Calcium and magnesium were omitted since these cations precipitated the fatty acid as an insoluble soap . Control experiments indicated that the absence of these cations did not significantly alter absorption . In addition, polyethylene glycol (PEG) 4000 (Ruger Chemical Co ., Irvington, N. J.) was present in a concentration of 5 g/1 . The intestine was reduced to 12 cm by trimming the distal end. Only one segment of jejunum was used from each animal . After stripping off fat and mesentery, gut segments were everted over a stainless steel rod (90 mm X 1 .5 mm) . Each everted segment was tied off at one end and suspended from a narrow-bore glass tube (4 mm OD) inserted through a rubber stopper . The rubber stopper was then placed into a culture tube (16 mm x 150mm) filled with 12 ml of control or test mucosal buffer solution . Teat agents were added to mucosal buffer solution only, except in those experiments were serosal or serosal plus mucosal application of ricinoleate were compared . Mucosal buffer solutions were aerated with 95~ OZ - 5,~ COZ throughout the experiment and kept at 37 to 38~ C by a water bath . The serosal compartment was filled with 1 .0 ml of the same oxygenated buffer but, in addition, contained 0 .02 ~.Ci of 1°C-PEG 4000 (New England Nuclear Corp ., Boston, Mass .) as an indicator of water movement (11) . Oamolarities of all solutions were measured with an Osmette~ Automatic Osmometer (Precision Systems Inc., Waltham, Mass .) and varied between 280 and 290 milliosmolar . All solutions had a pH of approximately 7 .4 after aeration with the oxygen mixture. To assay for water movement, an aliquot (100 ~l) of serosal solution was taken in duplicate at the beginning and end of t~e 30 minute absorptive period and counted in 10 ml of Insta~el (Packard Inst ., Downers Grove, I11 .) scintillation fluid using a Packard 2002 Liquid Scintillation Spectrometer . Total recovery of l 4 C-PEG 4000 from the serosal compartment was 98 .0 ± 1 .2 (n-_6) . Sodium and potassium concentrations were determined from samples (50 W1) of serosal solution using flame photometry (Instrumentation labs Inc ., Lexington, Mass .) . Chloride ion determinations were made from aliquots (100 ~.1) by electrotitration (Buchler Intestinal segments were dried overnight Inst ., Fort Lee, N.J .) .
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at 120° C to determine dry weight . Previously reported formulas (12) were used to determine net water, sodium, chloride and potassium transport . All values of water or ion transport are expressed as units per minute per gram dry weight of intestine . The assumption was made that the tissues equilibrated rapidly with the buffer and that the transport rate was relatively constant over the 30 minute experimental period . Thus, expression of the results as "per minute" implies the above assumption, and the results should be thought of as average rates . Control experiments were done with each day's experimental series since considerable day to day variation was found in the absorption of water . Initial studies indicated that the actively transported sugar, D-3-O-methylglucose, was absorbed by the tissue in a linear manner over a 2 hour period . This was taken as an indication that the tissue remained viable and functionally active throughout at least the 30 minute period utilized in these studies. Data analysis consisted of a standard unpaired t test comparing control with drug treated tissues(13) . Significance was taken at p < 0 .05 . Isolated Intestinal Smooth Muscle Studies . The surfactants were tested for their effects on the coaxially stimulated guinea-pig ileum using a method previously described (14) . Guinea-pigs of either sex weighing 400 to 800 grams were killed by a blow to the head . Six sections of terminal ileum, each 1 cm in length, were removed after discarding the 10 cm segment nearest the ileocecal valve . Each segment was immersed in a 25 ml organ bath filled with a modified Krebs solution of the following composition (mMoles/1) : Na, 140 ; K, 5 .9 ; Ca, 2 .5 ; Mg, 1 .2 ; C1, 122 ; HC03, 25 ; HZPOq, 1 .2 ; SOy, 1 .2 and dextrose, 11 .5 . The baths were gassed with 95~ Oz - 5 ;6 COz and maintained at 37 to 38° C . After placing the tissue over one of the electrodes, one end of the tissue was secured to a fixed clamp and the other end was attached to an isometric strain gage cantilever . Voltage from a stimulator (American Electronics Laboratories, Model 104A, Lansdale, Pa .) was passed through a voltage divider to drive six tissues simultaneously . Stimuli were applied at 8 .5 second intervals, at an intensity of 5 volts, for a duration of 0 .5 milliseconds . Isometric twitch responses of longitudinal smooth muscle were recorded by a Beckman Dynograph Recorder (type 411) . Equal tension was applied to all tissues after which the muscle preparations were allowed to equilibrate for 30 minutes with periodic washing . After stable baseline contractions were obtained, all test agents were added in equal volumes (0 .25 ml) so that each tissue was exposed to a different final bath concentration . All substances were added as their water soluble solutions except PS-80 which was solubilized in propylene glycol (propylene glycol did not affect contractile activity) . To quantify depressant responses, ten twitch contractions immediately preceeding addition of a teat substance were averaged and taken as 100 percent (control) activity . The average height of ten twitch wntractiona after stabilization of the response to the teat substance was then calculated as a percent inhibition of control . Drugs Used . Sodium ricinoleate (NU-Chek Prep, Elysian, Minn .) was pure by TLC using a solvent system of petroleum ether, anhydrous
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diethyl ether and glacial acetic acid (70 :30 :3) ; sodium dodecyl sulfate and dioctyl sodium sulfosuccinate (both ~ 95;8 pure as obtained from Sigma Chemical Co ., St . Louis, Mo .) ; sodium deoxycholate and sodium chenodeoxycholate (Schwartz-Mann, Orangeberg, N.Y . and Calbiochem, Los Angeles, Calif . respectively) . Results Effects on Net Water and Electrolyte Absorption . To determine the surface at which the ricinoleate effect was produced, the drug was added to mucosal and serosal solutions, either separately or to both simultaneously . When the serosal solution contained 2 .0 mM ricinoleate, no effect was produced . Addition of the drug to the mucosal solution produced a significant reduction (p < 0 .001) in net water absorption (Fig . 1) . Sodium and chloride absorption were also inhibited . The effect produced by allowing both surfaces contact with the drug caused an inhibition of net water absorption equal to that resulting from mucosal application alone (Fig . 1) .
FIG . 1 Comparison of the effects of serosal and/or mucosal application of sodium ricinoleate (2 .0 mM) on net water absorption by the hamster jejunum. Means (+ S .E .M .) are shown ; numbers in parentheses are the number of tissues used . P values refer to comparisons with control .
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Reversibility of the ricinoleate effect was examined by incubating the drug-treated tissues for 30 minutes, followed by a second 30 minute period in control buffer . The result was com pared with a 30 minute incubation in control buffer or a 30 minute test with 2 .0 mM ricinoleate (Fig . 2) . No significant difference was found between control ând "reversed" tissues, but a significant (p < 0 .001) effect was induced with the 30 minute application of ricinoleate .
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FIG . 2 Reversibility of the effect of sodium ricinoleate (2 .0 mM) on net water absorption by the hamster jejunum. Means (+ S .E .M .) are shown ; numbers in parentheses are the number of tissues used . The p values compare results of treatments with control . Both PS-SO and SDS significantly inhibited net water absorption . The effect of PS-80 but not SDS was reversible (Table 1) . The bile salts, sodium deoxycholate and chenodeoxycholate, also produced significant decreases in net water absorption which were not reversible (Table 1) . The DSS (2 .0 mM) significantly inhibited net water, sodium and chloride (p < 0 .005) but not potassium absorption (Fig . 3) . The effects of DSS were reversible to within 10 percent of the control values . Effects on Isolated Smooth Muscle Contractile Activity . Sodium ricinoleate, DSS, SDS, deoxycholate and chenodeoxycholate produced
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TABLE 1 Effects of Surfactants on Water Absorption and Reversibility (~1/min/q dry wt) by the Hamster Jejunum In Vitro. Treatmen tb
Control
WATER ABSORPTIONa Test c
Reversibility
PS-80 (1,$ w/v)
176 .3 +_ 10 .7(4)
116 .5 + 11 .9(8)
SDS ( 2 . OtaM)
252 .4 + 20 .1(5)
124 .3 +
6 .7(6)
Deoxycholate (2 .OmM)
279 .3 + 10 .5(5)
104.6 ±
8.4(6)
119.9 + 14 .9(5)
Chenodeoxycholate (2 .OmM)
279 .3 +_ 10 .5(5)
83 .2 _+ 15 .4(6)
131 .8 + 20 .3(5)
199 .9(2) 94 .8 +
8 .6(3)
a+ S .E .M . ; numbers in parentheses are number of tissues used bMucosal solution only cThe unparied t test between control and teat experiments for all treatments were significantly different with a p value at least < 0 .01.
FIG . 3 Effect of dioctyl sodium sulfoauccinate (DSS) on net water and electrolyte absorption by the hamster jejunum. Numbers in parenth ses are the number of tissues used and vertical bars are means (~ .E .M .) . For potassium S .E .M . values were too small to be illustrated .
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a dose dependent depression of the coaxially stimulated guineapig ileum. The dose response relationships for the above compounds were remarkably similar, occurring over the same narrow concentration range. At the lowest concentration (1 .25 X 10 - s M), no response was observed to any of the compounds . At the highest concentration employed (4 X 10' 4 M), the mean percent depression (± S .E .M .) of the twitch for each compound was as follows : sodium ricinoleate (99.4 + 0 .62, n--8) ; DSS (97.1, n~2) ; SDS (92 .6 + 2 .90, n=4) ; deoxycholaté (96.9 + 3.12, n-_6) ; and chenodeoxycholaté (97 .3 ± 1 .77, n=7) . The depressant effects of all agents were completely reversed to control contraction heights within 10 minutes following washing with fresh Krebs buffer . In contrast, PS-80 produced aLnoat 100 percent depression of the twitch contractions over a relatively wide concentration range (0 .025 to 0 .80 w/v) . Even at 0 .025 ;8, for example, the compound produced a 75 .8 + 3 .74 (n=8) percent inhibitory response . The effects of PS-80were also reversible . Figure 4 compares the depressant actions of several surfactants on the twitch contractions .
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FIG " 4 The effects of several test agents on the twitch response of the electrically (coaxially) stimulated guinea-pig ileum. A, sodium ricinoleate (2 X 10 - 4M)P B, DSS (4 X 10 -4M) ; C, PS-80 (0 .4% w/v) ; D, SDS (4 X 20 -4M) and E, sodium deoxycholate (4 X 10 -4M) . Record E shows the reversibility of the depression produced by deoxycholate which was also obtainable with the other test agents . Note different time scale for record E . Discussion In this study, ricinoleic acid depressed both muacosal and smooth muscle cell function in vitro as measured by inhibition of
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net water and electrolyte absorption and smooth muscle contractility . DSS, SDS, PS-80 and two bile salts, all substances with well defined surfactant properties, also depressed both systems . A common (ie ., surfactant) mechanism of action is suspected since all agents produced similar effects, at similar concentrations, even though they differ in chemical structure . The depressant actions of the surfactants on both systems may be either an effect of micellar aggregates or monomeric units . The critical micellar concentration (C .M .C .) for the compounds used in this study have been estimated by various authors (10, 15-18), but because of differences in temperature, counter-ion concentration, pH and the methods of determination, direct extrapolation of these values to our systems cannot be made . It should be noted, however, that with the possible exception of PS-80, literature estimates of C .M .C .'s for these compounds are somewhat higher than the concentrations employed here .* Still, because of the relatively high concentrations of test agents required to produce inhibition of water and electrolyte absorption, the existence of micellar aggregates in mucosal buffer solutions cannot be excluded . The relatively low concentrations required to produce inhibition of smooth muscle contractility makes it more likely that surfactant inhibitory actions on this system are caused by monomeric units . The differences in concentration of surfactants required to depress each system might explain the reversible effects of all compounds on smooth muscle function and the irreversible actions of several agents on mucosal function . The concentrations of teat agents used to inhibit water absorption from the isolated gut sac were approximately five times higher than those which produced complete depression of the twitch response of the electrically stimulated guinea-pig ileum. Thus, the several surfactants producing irreversible effects on mucosal function might have caused irreversible damage to the cells . Other investigations employing different techniques to study water absorption have shown that the inhibitory action of high concentrations of bile salts can be reversed (19-22) . Apparently, reversible inhibitory effects are more difficult to demonstrate in the isolated gut sac preparation . The amphipathic nature of the surfactant compounds is probably responsible for their effects on a number of membrane bound enzyme systems . For example, membrane integrity and continuity are necessary for Na+/K+ ATP'ase activity (23) . Amphipaths such as SDS are known to bind to proteins (24-26) and membrane lipoproteins (27) causing unfolding and reversible denaturation (28, 29) . Both DSS and SDS inhibit active sodium transport y frog skin below 2 .OmM (30) and SDS is known to inhibit Na+/K+ ATP'ase as well as a number of other enzymes below its C .M .C . (31,32) . In addition, DSS is claimed to increase 3' S' cyclic adenosine monophosphate (CAMP) in rat colonic mucosa (33) . A nonspecific change in membrane structure has been proposed as the mechanism by which detergents stimulate adenyl cyclase activity (34) . Like other surfactants, long-chain fatty acids also bind to proteins (35) and lipoproteins (36) . Ricinoleic acid has also been shown to inhibit transport ATP'ase in frog skin and rabbit Potassium ricinoleate, 3 .6mM at 55 °C (10) ; PS-80, 1.4 X 10 -3 mg/ml at 25°C (15) ; DSS, 5 .1mM at 40°C (10) ; SDS, 2 .15 (16) or 8 .4mM (17) at 25 ° C ; deoxycholate and chenodeoxycholate generally 2-6mM (18) .
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jejunum (37) and increases CAMP in rat colonic mucosa (38) . In addition, ricinoleic acid inhibits the mitochondrial enzymecarrier, adenine nucleotide tranalocase (39), shaving that the compound can also affect enzyme systems present in the membranes of cell organelles . Finally, bile salts reduce ATP production (40) , inhibit Na+/iC } ATP'ase (41,42) and increase CAMP in various tissues (38,43) . Thus, ricinoleic acid and other surfactant compounds appear to have similar effects on cellular and intracellular membrane structures hlhich, in turn, contain systems that regulate fluid absorption and are probably required for contractile activity of smooth muscle. Of undoubted importance to the in vivo actions of castor oil is the fact that ricinoleic acid, present as a triglyceride in the oil, is poorly absorbed (8,44), allowing large quantities to reach the lower small bowel and colon . Assuming a 50 percent absorption of the ricinoleic acid liberated from a 30 gram dose of castor oil [a reasonable assumption from the data of Watson et al., (44)] and a maximum mixing volume of 2 .0 liters present in the distal intestine (45), ricinoleate would be present in the lower gastrointestinal tract at approximately a 20 millimolar concentration . Concentrations of ricinoleate considerably less than this have been found to effectively inhibit water and electrolyte absorption in animals (8) and man (7,9) . The effect of ricinoleic acid on water and electrolyte absorption in vivo is probably a surfactant-induced action of the compound directly on mucosal cells . The depressant effects of ricinoleic acid on the contractile activity of in vitro intestinal smooth muscle probably has little pertinence to the in vivo actions of the compound . Ab sorbed ricinoleic acid is recoverable as monoglyceridea, diglycerides and triglyceridea from the lymph (46) . Apparently, little or no free fatty acid contacts the gastrointestinal smooth muscle . The depressant effects of ricinoleic acid on in vitro intestinal smooth muscle demonstrated in this study, merely serve to show that even then the muscle is bathed in very high concentrations of the compound, the result is not stimulation of smooth muscle contractile activity . In all probability, the inhibitory actions of oral castor oil on the intestinal smooth muscle activity in vivo (6), is an indirect action mediated through a hormonal mechanism (47) . The actions of ricinoleic acid on water and electrolyte absorption and smooth muscle contractility (6-9, 12, 38-39, 48-49), suggests that the pharmacological classification of castor oil as an "irritant" or "stimulant" should be re-evaluated . Acknawledaments The authors wish to thank Dr . Sidney F. Phillips for his comments and suggestions regarding our manuscript . References 1. 2.
H. MEYER, Arch. E~. Path . U. Pharmak . 28, 145-152, (1890) . E . FINGL, ~Pharmac~oc~ical Basof Therapeutics , ed. by L. Goodman and A . Gilman, p. 1020, MacMillan Co., New York (1970) .
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T . SOLLMAN, A Manual of P'harmacology and its Application to Therapeutics and Toxicology , p. 212, W .B . Saundera Co ., Philadelphia, Pa . (1957) . D . BONNYCASTLE, Drill's Pharmacology in Medicine , ed, by J. R. Dipalma, p . 982, McGraw-Hill, New York (1971) . P . C. REYNELL and G . H. SPRAY, GastroenterolocTV 34, 867-873 (1958) . J. J. STEWART, T. S . GAGINELLA, W . A ., OISEN and P. BASS, J. Pharmacol . Eue. Ther . _192, 458-467, 1975 . H . V. AMMON, P. J. THOMAS and S . F . PHILLIPS, J. Clin . Invest . 53, 374-379 (1974) . H . V. AMMON and S . F . PHILLIPS, J. Clin . Invest . 53, 205210 (1974) . H . V. AMMON and S . F . PHILLIPS, Gastroenterology 65, 744749 (1973) . P . MUKERJEE and K . J. MYCSLS, Critical Micelle Concent rations of Aqueous Surfactant Systems , II .S . Department of Commerce, Washington, D.C . (1971) . D . L. MILLER and H . P, SCHEDL, Gastroenterology 58, 40-46 (1970) . P . BRIGHT-ASARE and H. J. BINDER, Gastroenterology 64, 81-88 (1973) . R. G. D. STSS L and J. H. TORRIE, Principles and Procedures of Statistics , p. 67, McGraw-Hill, New York (1960) . P. BASS, J. A . KENNEDY, J. N. WILEY, J. VILLARRF~AL and D . E. BUTLER, J. Pharmacol . Exp . Ther . 186, 183-198 (1973) . L. S . C . WAN and P. F. S . LEE, J. Pharm. SSçi . 63, 136-137 (1974) . J. STEINHP+RDT, N. STOCKER, D. CARROLL and K. S . BIRDI, Biochem. 13, 4461-4468 (1974) . E. D . GODDÂRD and G . C . BENSON, Canad . J. Chem . 35, 986-991 (195 7) . D. M . SMALL, The Bile Acids -Chemistry, Physiology , _and Metabolism (Vol . 1), ed . by P. P. NAIR and D . KRITCHEVSKY, p. 303-308, Plenum Press, New York (1971) . H. S . MEKHJIAN, S . F. PHILLIPS and A . F . HOFMANN, J. Clin . Invest . 50, 1569-1577 (1971) . H. S . MEKHJIAN and S . F. PHILLIPS, Gastroenterology 59, 120-129 (1970) . D . L. WINGATE, S . F. PHILLIPS and A . F . HOFMANN, J. Clin . Invest . 52, 1230-1236 (1973) . M . V. TEEM and S . F. PHILLIPS, Gastroenterology 62, 261-267 267 (1972) . J. C. SKOU, Physiol. _Rev . 45, 596-617 (1965) . J. OAKES, Eur . J. Biochem. 36, 553-558 (1973) . R. W. BONSALL and S . HUNT, Biochem. Biophys , Acta 249, 266280 (1971) . A . K. DUNKER and R. R. RUECKERT, J . Biol . Chew . 244, 50745080 (1969) . A . TZAGOLOFF and H. S . PENEFSKY, Methods in Snzvmologv (Vol . 3IXII) , p . 219, (1971) . C . TANFORD, Advances in Protein Chemistry , Vol. 23, p. 211, Academic Press, New York (1968) . C. TANFORD, Advances in Protein Chemistry , Vol . 24, p. 67, Academic Press, New York (1970) . G . D . WEBB, Acta Physiol . Scand . 63, 377-384 (1965) . s . Acts 135, 45-60 (1967) . P . C . CHAN, Biochem. Bio E . D . WILLS, Biochem . J. 57, 109-120 (1954) .
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Life Sciences Vol . 16, pp . 1595-1606 Printed in the U.S .A .
INHIBITION OF SMALL INTESTINAL MUCOSAL AND SMOOTH MUSCLE CELL FUNCTION BY RICINOLEIC ACID AND OTHER SURFACTANTS Timothy S . Gaginella, John J. Stewart, Gary W . Gullikaon Ward A . Olsen and Paul Bass School of Pharmacy, School of Medicine Center for Health Sciences University of Wisconsin Madison, Wisconsin 53706 (Received in final form May 1, 1975)
Summary Sodium ricinoleate, dioctyl sodium sulfosuccinate, sodium dodecyl (lauryl) sulfate, polysorbate 80, sodium deoxycholate and chenodeoxycholate were found to produce depression of in vitro mucosal and smooth muscle cell function . These actions were assessed by measuring net water and electrolyte transport from everted hamster gut sacs and contractile activity of the electrically stimulated guinea-pig ileum . All compounds were effective depressants of both systems at concentrations which were likely to be below their respective critical micellar concentration . Ricinoleic acid may produce its cathartic effect due to its amphipathic nature, possibly by hydrophobic interaction with membrane lipoproteins . Ricinoleic acid and the other surfactants may be acting through a common mechanism. Ricinoleic acid, the active constituent of castor oil, is an eighteen carbon, hydroxylated, monounsaturated fatty acid (1) . Although castor oil has been used as a safe and effective cathar tic for centuries, its mechanism of action has been only vaguely defined as an "irritant" or "stimulant" to the gastrointestinal tract (2-5) . These descriptions are particularly inappropriate since decreased contractile activity of circular smooth muscle has been reported after oral administration of cathartic doses of castor oil to intact unanesthetized dogs (6) . In addition, ricinoleic acid can inhibit water and electrolyte absorption from the human jejunum (7), dog ileum (8) and human colon (9) . Recently, a new explanation has been proposed to account for castor oil-induced catharsis _in vivo . In this explanation, both inhibition of water and electrolyte absorption and reduced contractile activity of intestinal circular smooth muscle are considered important for the cathartic action of the compound (6) . Long-chain fatty acids such as ricinoleate are amphipathic (hydrophilic-lipophilic) at physiologic pH, and they therefore possess surfactant activity (10) . It has been postulated that ricinoleate may inhibit intestinal fluid absorption as a consequence of its surface activity (8) . As a test of this hypothesis, 1595
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we have compared ricinoleic acid with : two structurally different anionic surfactants, dioctyl sodium sulfosuccinate (DSS) and sodium dodecyl sulfate (SDS) ; a non-ionic surfactant, polysorbate 80 (PS-80) ; and two bile salts, sodium deoxycholate and chenodeoxycholate . All surfactants were evaluated for their relative ability to inhibit net water and electrolyte absorption and intestinal smooth muscle contractile activity _in vitro . Absorption studies were conducted with everted hamster jejunal gut sacs . Electrically (coaxially) stimulated guinea-pig ileal preparations were used in studies comparing the actions of the test agents on contractile activity of intestinal smooth muscle . Materials and Methods Transport Studies . Nonfasted male Golden Syrian hamsters (Mesocricetus auratus weighing 90 to 120 grams were sacrified by cervical dislocation . Approximately a 15 cm segment of jejunum beginning at the ligament of Treitz was quickly removed and placed in mucosal buffer solution (aerated with 959 OZ - 5~ COZ)composed of (m Moles/1) : Na, 150 ; C1, 130 ; K, 5 ; HC03, 25 ; and dextrose 2 . Calcium and magnesium were omitted since these cations precipitated the fatty acid as an insoluble soap . Control experiments indicated that the absence of these cations did not significantly alter absorption . In addition, polyethylene glycol (PEG) 4000 (Ruger Chemical Co ., Irvington, N. J.) was present in a concentration of 5 g/1 . The intestine was reduced to 12 cm by trimming the distal end. Only one segment of jejunum was used from each animal . After stripping off fat and mesentery, gut segments were everted over a stainless steel rod (90 mm X 1 .5 mm) . Each everted segment was tied off at one end and suspended from a narrow-bore glass tube (4 mm OD) inserted through a rubber stopper . The rubber stopper was then placed into a culture tube (16 mm x 150mm) filled with 12 ml of control or test mucosal buffer solution . Teat agents were added to mucosal buffer solution only, except in those experiments were serosal or serosal plus mucosal application of ricinoleate were compared . Mucosal buffer solutions were aerated with 95~ OZ - 5,~ COZ throughout the experiment and kept at 37 to 38~ C by a water bath . The serosal compartment was filled with 1 .0 ml of the same oxygenated buffer but, in addition, contained 0 .02 ~.Ci of 1°C-PEG 4000 (New England Nuclear Corp ., Boston, Mass .) as an indicator of water movement (11) . Oamolarities of all solutions were measured with an Osmette~ Automatic Osmometer (Precision Systems Inc., Waltham, Mass .) and varied between 280 and 290 milliosmolar . All solutions had a pH of approximately 7 .4 after aeration with the oxygen mixture. To assay for water movement, an aliquot (100 ~l) of serosal solution was taken in duplicate at the beginning and end of t~e 30 minute absorptive period and counted in 10 ml of Insta~el (Packard Inst ., Downers Grove, I11 .) scintillation fluid using a Packard 2002 Liquid Scintillation Spectrometer . Total recovery of l 4 C-PEG 4000 from the serosal compartment was 98 .0 ± 1 .2 (n-_6) . Sodium and potassium concentrations were determined from samples (50 W1) of serosal solution using flame photometry (Instrumentation labs Inc ., Lexington, Mass .) . Chloride ion determinations were made from aliquots (100 ~.1) by electrotitration (Buchler Intestinal segments were dried overnight Inst ., Fort Lee, N.J .) .
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at 120° C to determine dry weight . Previously reported formulas (12) were used to determine net water, sodium, chloride and potassium transport . All values of water or ion transport are expressed as units per minute per gram dry weight of intestine . The assumption was made that the tissues equilibrated rapidly with the buffer and that the transport rate was relatively constant over the 30 minute experimental period . Thus, expression of the results as "per minute" implies the above assumption, and the results should be thought of as average rates . Control experiments were done with each day's experimental series since considerable day to day variation was found in the absorption of water . Initial studies indicated that the actively transported sugar, D-3-O-methylglucose, was absorbed by the tissue in a linear manner over a 2 hour period . This was taken as an indication that the tissue remained viable and functionally active throughout at least the 30 minute period utilized in these studies. Data analysis consisted of a standard unpaired t test comparing control with drug treated tissues(13) . Significance was taken at p < 0 .05 . Isolated Intestinal Smooth Muscle Studies . The surfactants were tested for their effects on the coaxially stimulated guinea-pig ileum using a method previously described (14) . Guinea-pigs of either sex weighing 400 to 800 grams were killed by a blow to the head . Six sections of terminal ileum, each 1 cm in length, were removed after discarding the 10 cm segment nearest the ileocecal valve . Each segment was immersed in a 25 ml organ bath filled with a modified Krebs solution of the following composition (mMoles/1) : Na, 140 ; K, 5 .9 ; Ca, 2 .5 ; Mg, 1 .2 ; C1, 122 ; HC03, 25 ; HZPOq, 1 .2 ; SOy, 1 .2 and dextrose, 11 .5 . The baths were gassed with 95~ Oz - 5 ;6 COz and maintained at 37 to 38° C . After placing the tissue over one of the electrodes, one end of the tissue was secured to a fixed clamp and the other end was attached to an isometric strain gage cantilever . Voltage from a stimulator (American Electronics Laboratories, Model 104A, Lansdale, Pa .) was passed through a voltage divider to drive six tissues simultaneously . Stimuli were applied at 8 .5 second intervals, at an intensity of 5 volts, for a duration of 0 .5 milliseconds . Isometric twitch responses of longitudinal smooth muscle were recorded by a Beckman Dynograph Recorder (type 411) . Equal tension was applied to all tissues after which the muscle preparations were allowed to equilibrate for 30 minutes with periodic washing . After stable baseline contractions were obtained, all test agents were added in equal volumes (0 .25 ml) so that each tissue was exposed to a different final bath concentration . All substances were added as their water soluble solutions except PS-80 which was solubilized in propylene glycol (propylene glycol did not affect contractile activity) . To quantify depressant responses, ten twitch contractions immediately preceeding addition of a teat substance were averaged and taken as 100 percent (control) activity . The average height of ten twitch wntractiona after stabilization of the response to the teat substance was then calculated as a percent inhibition of control . Drugs Used . Sodium ricinoleate (NU-Chek Prep, Elysian, Minn .) was pure by TLC using a solvent system of petroleum ether, anhydrous
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diethyl ether and glacial acetic acid (70 :30 :3) ; sodium dodecyl sulfate and dioctyl sodium sulfosuccinate (both ~ 95;8 pure as obtained from Sigma Chemical Co ., St . Louis, Mo .) ; sodium deoxycholate and sodium chenodeoxycholate (Schwartz-Mann, Orangeberg, N.Y . and Calbiochem, Los Angeles, Calif . respectively) . Results Effects on Net Water and Electrolyte Absorption . To determine the surface at which the ricinoleate effect was produced, the drug was added to mucosal and serosal solutions, either separately or to both simultaneously . When the serosal solution contained 2 .0 mM ricinoleate, no effect was produced . Addition of the drug to the mucosal solution produced a significant reduction (p < 0 .001) in net water absorption (Fig . 1) . Sodium and chloride absorption were also inhibited . The effect produced by allowing both surfaces contact with the drug caused an inhibition of net water absorption equal to that resulting from mucosal application alone (Fig . 1) .
FIG . 1 Comparison of the effects of serosal and/or mucosal application of sodium ricinoleate (2 .0 mM) on net water absorption by the hamster jejunum. Means (+ S .E .M .) are shown ; numbers in parentheses are the number of tissues used . P values refer to comparisons with control .
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Reversibility of the ricinoleate effect was examined by incubating the drug-treated tissues for 30 minutes, followed by a second 30 minute period in control buffer . The result was com pared with a 30 minute incubation in control buffer or a 30 minute test with 2 .0 mM ricinoleate (Fig . 2) . No significant difference was found between control ând "reversed" tissues, but a significant (p < 0 .001) effect was induced with the 30 minute application of ricinoleate .
(4)
s sa
NS
o
®~ . 2m ®~
mi~
2~
ß 100
p
FIG . 2 Reversibility of the effect of sodium ricinoleate (2 .0 mM) on net water absorption by the hamster jejunum. Means (+ S .E .M .) are shown ; numbers in parentheses are the number of tissues used . The p values compare results of treatments with control . Both PS-SO and SDS significantly inhibited net water absorption . The effect of PS-80 but not SDS was reversible (Table 1) . The bile salts, sodium deoxycholate and chenodeoxycholate, also produced significant decreases in net water absorption which were not reversible (Table 1) . The DSS (2 .0 mM) significantly inhibited net water, sodium and chloride (p < 0 .005) but not potassium absorption (Fig . 3) . The effects of DSS were reversible to within 10 percent of the control values . Effects on Isolated Smooth Muscle Contractile Activity . Sodium ricinoleate, DSS, SDS, deoxycholate and chenodeoxycholate produced
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TABLE 1 Effects of Surfactants on Water Absorption and Reversibility (~1/min/q dry wt) by the Hamster Jejunum In Vitro. Treatmen tb
Control
WATER ABSORPTIONa Test c
Reversibility
PS-80 (1,$ w/v)
176 .3 +_ 10 .7(4)
116 .5 + 11 .9(8)
SDS ( 2 . OtaM)
252 .4 + 20 .1(5)
124 .3 +
6 .7(6)
Deoxycholate (2 .OmM)
279 .3 + 10 .5(5)
104.6 ±
8.4(6)
119.9 + 14 .9(5)
Chenodeoxycholate (2 .OmM)
279 .3 +_ 10 .5(5)
83 .2 _+ 15 .4(6)
131 .8 + 20 .3(5)
199 .9(2) 94 .8 +
8 .6(3)
a+ S .E .M . ; numbers in parentheses are number of tissues used bMucosal solution only cThe unparied t test between control and teat experiments for all treatments were significantly different with a p value at least < 0 .01.
FIG . 3 Effect of dioctyl sodium sulfoauccinate (DSS) on net water and electrolyte absorption by the hamster jejunum. Numbers in parenth ses are the number of tissues used and vertical bars are means (~ .E .M .) . For potassium S .E .M . values were too small to be illustrated .
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a dose dependent depression of the coaxially stimulated guineapig ileum. The dose response relationships for the above compounds were remarkably similar, occurring over the same narrow concentration range. At the lowest concentration (1 .25 X 10 - s M), no response was observed to any of the compounds . At the highest concentration employed (4 X 10' 4 M), the mean percent depression (± S .E .M .) of the twitch for each compound was as follows : sodium ricinoleate (99.4 + 0 .62, n--8) ; DSS (97.1, n~2) ; SDS (92 .6 + 2 .90, n=4) ; deoxycholaté (96.9 + 3.12, n-_6) ; and chenodeoxycholaté (97 .3 ± 1 .77, n=7) . The depressant effects of all agents were completely reversed to control contraction heights within 10 minutes following washing with fresh Krebs buffer . In contrast, PS-80 produced aLnoat 100 percent depression of the twitch contractions over a relatively wide concentration range (0 .025 to 0 .80 w/v) . Even at 0 .025 ;8, for example, the compound produced a 75 .8 + 3 .74 (n=8) percent inhibitory response . The effects of PS-80were also reversible . Figure 4 compares the depressant actions of several surfactants on the twitch contractions .
A
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WW1
`II
I~~II Illll ~
D
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J
w
IIIIIII~----'L~~ I~I
..~ .,L ...~~ ...~ .. . . . . .
~ ! IDI
FIG " 4 The effects of several test agents on the twitch response of the electrically (coaxially) stimulated guinea-pig ileum. A, sodium ricinoleate (2 X 10 - 4M)P B, DSS (4 X 10 -4M) ; C, PS-80 (0 .4% w/v) ; D, SDS (4 X 20 -4M) and E, sodium deoxycholate (4 X 10 -4M) . Record E shows the reversibility of the depression produced by deoxycholate which was also obtainable with the other test agents . Note different time scale for record E . Discussion In this study, ricinoleic acid depressed both muacosal and smooth muscle cell function in vitro as measured by inhibition of
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net water and electrolyte absorption and smooth muscle contractility . DSS, SDS, PS-80 and two bile salts, all substances with well defined surfactant properties, also depressed both systems . A common (ie ., surfactant) mechanism of action is suspected since all agents produced similar effects, at similar concentrations, even though they differ in chemical structure . The depressant actions of the surfactants on both systems may be either an effect of micellar aggregates or monomeric units . The critical micellar concentration (C .M .C .) for the compounds used in this study have been estimated by various authors (10, 15-18), but because of differences in temperature, counter-ion concentration, pH and the methods of determination, direct extrapolation of these values to our systems cannot be made . It should be noted, however, that with the possible exception of PS-80, literature estimates of C .M .C .'s for these compounds are somewhat higher than the concentrations employed here .* Still, because of the relatively high concentrations of test agents required to produce inhibition of water and electrolyte absorption, the existence of micellar aggregates in mucosal buffer solutions cannot be excluded . The relatively low concentrations required to produce inhibition of smooth muscle contractility makes it more likely that surfactant inhibitory actions on this system are caused by monomeric units . The differences in concentration of surfactants required to depress each system might explain the reversible effects of all compounds on smooth muscle function and the irreversible actions of several agents on mucosal function . The concentrations of teat agents used to inhibit water absorption from the isolated gut sac were approximately five times higher than those which produced complete depression of the twitch response of the electrically stimulated guinea-pig ileum. Thus, the several surfactants producing irreversible effects on mucosal function might have caused irreversible damage to the cells . Other investigations employing different techniques to study water absorption have shown that the inhibitory action of high concentrations of bile salts can be reversed (19-22) . Apparently, reversible inhibitory effects are more difficult to demonstrate in the isolated gut sac preparation . The amphipathic nature of the surfactant compounds is probably responsible for their effects on a number of membrane bound enzyme systems . For example, membrane integrity and continuity are necessary for Na+/K+ ATP'ase activity (23) . Amphipaths such as SDS are known to bind to proteins (24-26) and membrane lipoproteins (27) causing unfolding and reversible denaturation (28, 29) . Both DSS and SDS inhibit active sodium transport y frog skin below 2 .OmM (30) and SDS is known to inhibit Na+/K+ ATP'ase as well as a number of other enzymes below its C .M .C . (31,32) . In addition, DSS is claimed to increase 3' S' cyclic adenosine monophosphate (CAMP) in rat colonic mucosa (33) . A nonspecific change in membrane structure has been proposed as the mechanism by which detergents stimulate adenyl cyclase activity (34) . Like other surfactants, long-chain fatty acids also bind to proteins (35) and lipoproteins (36) . Ricinoleic acid has also been shown to inhibit transport ATP'ase in frog skin and rabbit Potassium ricinoleate, 3 .6mM at 55 °C (10) ; PS-80, 1.4 X 10 -3 mg/ml at 25°C (15) ; DSS, 5 .1mM at 40°C (10) ; SDS, 2 .15 (16) or 8 .4mM (17) at 25 ° C ; deoxycholate and chenodeoxycholate generally 2-6mM (18) .
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jejunum (37) and increases CAMP in rat colonic mucosa (38) . In addition, ricinoleic acid inhibits the mitochondrial enzymecarrier, adenine nucleotide tranalocase (39), shaving that the compound can also affect enzyme systems present in the membranes of cell organelles . Finally, bile salts reduce ATP production (40) , inhibit Na+/iC } ATP'ase (41,42) and increase CAMP in various tissues (38,43) . Thus, ricinoleic acid and other surfactant compounds appear to have similar effects on cellular and intracellular membrane structures hlhich, in turn, contain systems that regulate fluid absorption and are probably required for contractile activity of smooth muscle. Of undoubted importance to the in vivo actions of castor oil is the fact that ricinoleic acid, present as a triglyceride in the oil, is poorly absorbed (8,44), allowing large quantities to reach the lower small bowel and colon . Assuming a 50 percent absorption of the ricinoleic acid liberated from a 30 gram dose of castor oil [a reasonable assumption from the data of Watson et al., (44)] and a maximum mixing volume of 2 .0 liters present in the distal intestine (45), ricinoleate would be present in the lower gastrointestinal tract at approximately a 20 millimolar concentration . Concentrations of ricinoleate considerably less than this have been found to effectively inhibit water and electrolyte absorption in animals (8) and man (7,9) . The effect of ricinoleic acid on water and electrolyte absorption in vivo is probably a surfactant-induced action of the compound directly on mucosal cells . The depressant effects of ricinoleic acid on the contractile activity of in vitro intestinal smooth muscle probably has little pertinence to the in vivo actions of the compound . Ab sorbed ricinoleic acid is recoverable as monoglyceridea, diglycerides and triglyceridea from the lymph (46) . Apparently, little or no free fatty acid contacts the gastrointestinal smooth muscle . The depressant effects of ricinoleic acid on in vitro intestinal smooth muscle demonstrated in this study, merely serve to show that even then the muscle is bathed in very high concentrations of the compound, the result is not stimulation of smooth muscle contractile activity . In all probability, the inhibitory actions of oral castor oil on the intestinal smooth muscle activity in vivo (6), is an indirect action mediated through a hormonal mechanism (47) . The actions of ricinoleic acid on water and electrolyte absorption and smooth muscle contractility (6-9, 12, 38-39, 48-49), suggests that the pharmacological classification of castor oil as an "irritant" or "stimulant" should be re-evaluated . Acknawledaments The authors wish to thank Dr . Sidney F. Phillips for his comments and suggestions regarding our manuscript . References 1. 2.
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