Naunyn-Schmiedeberg's
Naunyn-Schmiedeberg's Arch. Pharmacol. 298, 263-266 (1977)
Archivesof Pharmacology 9 by Springer-Verlag1977
.Increased Level of cAMP in the Rat Intestinal Mucosa Caused by Sodium Lauryl Sulphate G. B R I S E I D 1, I. OYE 2, and K. B R I S E I D 1 1Department of Pharmacology, Institute of Pharmacy 2Institute of Pharmacology, University of Oslo, Blindern, Oslo 3, Norway
S u m m a r y . The level of cyclic A M P in the jejunal mucosa from tied loops of anaesthetized rats was found to be significantly increased ( 2 7 - 5 0 %) when sodium lauryl sulphate (SLS) was added to the loop fluid ( 2 - 2 7 raM). Imidazole (25 raM) did not significantly alter the resting level of cyclic AMP, but reduced the increase caused by SLS (17 raM). Theophylline (25 raM) significantly increased the intestinal level of cyclic AMP, and potentiated the increase caused by SLS. Ouabain (2.5 raM) did not alter the level of cyclic A M P in the presence or in the absence of SLS. The results of previous experiments on the increases in intestinal absorption caused by SLS or by dibutyryl cyclic A M P (Briseid et al., 1974, 1976) are discussed in light of the present data. It is concluded that the SLSeffect on absorption can only partly by ascribed to its effect on the intestinal level of cyclic AMP. K e y words: Cyclic A M P -
Imidazole -
Sodium lauryl sulphate Theophylline - Jejunal absorption.
INTRODUCTION It has-previously been shown that the increases in absorption of phenolsulphonphthalein and pralidoxime from jejunal loops of anaesthetized rats caused b y sodium lauryl sulphate (SLS) (Briseid et al., 1974) were potentiated by theophylline a n d reduced by imidazole (Briseid et al., 1976). An increased absorption was also obtained by addition of dibutyryl cyclic A M P to the loop fluid. This indirect evidence of an increased level of cyclic A M P as part of the effect of SLS on the absorption was supported by determinations of the concentration of cyclic A M P in the rat intestinal mucosa in the presence and in the absence of SLS (Briseid et al., 1976). The effect of surfaceSend offprint requests to : Dr. K. Briseid at the above address
active agents on the cyclic A M P level in the rat intestine was also investigated by Binder et al. (1975), who found that taurochenodeoxycholic acid increased the colonic mucosal cyclic A M P level. Donowitz and Binder (1975) obtained similar results using a synthetic detergent, dioctyl sodium sulfosuccinate. In the present work we wanted to verify our preliminary results on the effect of SLS on the level of cyclic A M P in the rat jejunal mucosa (Briseid et al., 1976). We further wanted to evaluate the role of cyclic A M P for the effect of SLS on the absorption of phenolsulphonphthalein and pralidoxime (Briseid et al., 1974, 1976).
METHODS Determination of Cyclic AMP. The concentration of cyclicAMP in
the rat intestinal mucosa was measured under experimental conditions similar to those described for the absorption experiments (Briseid et al., 1974). When the intestinal loop had been washed out, it was opened and sheets of the mucosa were stripped from the submucosal layer. The specimens of fresh tissue were immediately frozen on dry ice and homogenizedin cold 5 % trichloroacetic acid (Gilman, 1970).CyclicAMP was determined in the supernatant after extraction of the trichloroacetic acid with ether (Gilman, 1970) by the protein binding assay described by Christoffersen et al. (1973). It was demonstrated in recoveryexperiments that the amount of SLS which is present in the mucosa extracts under our experimental conditions, does not affect the cAMP assays (Sund and Hillestad, to be pubiished). Test Substances and Surfactant. Imidazole,grade II[ : SigmaChemical Company, St. Louis. Ouabain (g-strophanthin krist., reinst): Merck A.G., Darmstadt. Sodium lauryl sulphate (Texapon KI2), SLS: Henkel and Cie, GmbH., Diisseldorf. Theophylline (theophyllin krist, wasserhaltig): Knoll A.G., Ludwigshafen am Rhein. Test Solutions. Imidazole25 raM, ouabain 2.5 raM, SLS 17 mM and theophylline 25 raM. Tyrode solution (Briseid and Briseid, 1971) was used as test fluid, pH 7.4. Osmotic adjustment of the instilled solutions was carried out by reductions in the amount of sodium chloride. Protein Determination. Protein was determined by the method of
Lowry et al. (1951). Statistical Method. Student's t-test was used. A probability level of
less than 5 % was considered significant.
264
Naunyn-Schmiedeberg's Arch. Pharmacol. 298 (1977)
Table 1. Protein/wet weight tissue ratios in the intestinal mucosa from single loop preparations in the rat. Effect of the experimental time period and of the concentration of sodium lauryl sulphate (SLS). Loop fluid Tyrode ofpH 7.4. Each figure represents data from 6 rats. Mg protein/mg wet weight: I without SLS. II SLS 17 raM. III SLS 0 to 27 mM and 71/2min exposure periods. When comparing the results from the longer exposure periods with the results obtained after 2 min (I and II), and also the results obtained in the presence of SLS with the result without SLS (III), no statistically significant differences were seen (P > 0.05) Minutes
I
II
SLS mM
Ill
2 71/2 15 30 60
0.11 0.11 0.12 0.10 0.11
0.11 0.10 0.09 0.09 0.09
0 2 7 12 17 22 27
0.09 0.09 0.10 0.10 0.10 0.10 0.10
Table 2. Effect of sodium lauryl sulphate on the cyclic AMP level in the intestinal mucosa of the rat as a function of concentration. Loop fluid Tyrode of pH 7.4 and an exposure period of 71/2min. Each figure represents data from 6 rats SLS mM
Cyclic AMP pmoles/mg wet wt.
S.E. diff. to SLS 0 mM
Level of significance
0 2 7 12 17 22 27
0.67 0.85 0.91 0.97 0.92 1.08 1.01
0.03 0.05 0.10 0.07 0.04 0.08
0.05 0.02 0.02 0.01
> > > >
P P P P P 0.01 > P
> > > > < >
0.02 0.01 0.01 0.001
0.OOl 0.001
RESULTS
Determination of Protein in Wet Weight Tissue. D e t e r m i n a t i o n s o f the p r o t e i n / w e t weight tissue ratios in the absence a n d in the presence o f SLS 17 m M over the t i m e interval 2 - 6 0 rain did n o t reveal any alterations (Table 1, I,II). A c c o r d i n g to Gafil and H o l z i n g e r (1975) the p r o t e i n d e t e r m i n a t i o n m e t h o d o f L o w r y et al. (1951) as m o d i f i e d by H a r t r e e (1972) m i g h t be slightly influenced by SLS. U n d e r o u r e x p e r i m e n t a l c o n d i t i o n s the p r o t e i n / w e t weight tissue ratios were n o t f o u n d to c h a n g e in e x p e r i m e n t s in w h i c h the SLS c o n c e n t r a t i o n was increased f r o m 0 - 2 7 m M in c o n n e c t i o n with an e x p o s u r e p e r i o d o f 71/2 rain. O n e such e x p e r i m e n t is s h o w n in T a b l e 1, III. This result at the same time p r o v i d e s e v i d e n c e that the s u r f a c t a n t did n o t cause a l t e r a t i o n s in the a m o u n t s o f fluid p r e s e n t in the sheets o f m u c o s a u s e d for the p r o t e i n d e t e r m i n a t i o n s a n d the results o f the assays o f cyclic A M P in the present w o r k are given as p m o l e s / m g wet weight tissue. Effect of Sodium LauryI Sulphate on the Cyclic A M P Level as a Function of Time and as a Function of Concentration. Time-effect e x p e r i m e n t s carried o u t with SLS 17 m M d e m o n s t r a t e d no significant alterations in the cyclic A M P levels o v e r the t i m e interval 2 60 rain. T a b l e 2 shows the results o f an e x p e r i m e n t with c o n c e n t r a t i o n s o f SLS r a n g i n g f r o m 2 - 27 m M . T h e e x p o s u r e p e r i o d was 71/2 rain. A m a x i m u m increase in cyclic A M P c o n c e n t r a t i o n o f a b o u t 50 % was registered for the two highest c o n c e n t r a t i o n s , but also the 27 % increase registered at 2 m M SLS was statistically significant.
Effects of Sodium Lauryl Sulphate, Imidazole, Theophylline and Ouabain on the Cyclic A M P Level in the Jejunal Mucosa of the Rat. T a b l e 3 shows t h a t imid-
Table 3. Effects of sodium laurylsulphate (SLS) 17 mM, imidazole (imidaz.) 25 mM, theophylline (theoph.) 25 mM or ouabain (ouab.) 2.5 mM on the level of cyclic AMP in the rat intestinal mucosa. Loop fluid Tyrode (T) of pH 7.4 and exposure period of 71/2 min. Each figure represents data from 6 rats Experiment
Medium
Cyclic AMP pmoles/mg wet wt.
S.E. diff.
Level of significance
A
I T II T+SLS IIIT+imidaz. IV T+SLS+imidaz.
1.22 1.89 1.03 0.72
I - II:0.22 I - I I I : 0.19 II - I V : 0.33 I I I - I V : 0.31
0.01 0.4 0.01 0.4
B
I T II T+SLS III T+theoph. IV T+SLS+theoph.
0.63 0.81 1.45 2.41
I - II:0.18 I -III:0.17 II - IV: 0.15 I I I - IV: 0.21
0.1 > P > 0.01 > P > P < 0.01 > P >
0.05 0.001 0.001 0.001
C
I T II T+SLS IIIT+ouab. IVT+SLS+ouab.
0.78 0.92 0.75 0.85
I - II:0.07 I - I I I : 0.07 II - I V : 0.10 III--IV:0.10
0.1 0.8 0.7 0,1
0.05 0.7 0.6 0,05
> > > >
> > > >
P>0.001 P > 0.3 P > 0.001 P > 0.3
P> P > P > P>
G. Briseid et al. : Jejunal Absorption- Sodium Lauryl Sulphate and the cAMP System azole significantly reduced the SLS-caused increase in the level of cyclic AMP (Table 3A, I I - I V ) , while imidazole alone did not significantly reduce the resting level of cyclic AMP (Table 3A, I - I I I ) . Table 3 further shows that theophylline significantly increased the intestinal level of cyclic AMP (Table 3 B, I - I I I ) and strongly potentiated the increase caused by SLS (Table 3 B, II - IV). Ouabain did not alter the level of cyclic AMP in the presence (Table 3 C, I I - IV) nor in the absence of SLS (Table 3 C, I - III).
DISCUSSION
Effect of Sodium Lauryl Sulphate on the Level of Cyclic A M P in the Rat Jejunal Mucosa as a Function of Time and as a Function of Concentration. Previous experiments indicated that the increase in the level of intestinal cyclic AMP caused by SLS 17 mM had reached a maximum already after an exposure period of 2 min (Briseid et al., 1976). Experiments carried out in connection with the present work demonstrated that no significant SLS-caused increase in the concentration of cyclic AMP took place over the period 2 - 60 rain, and a standard exposure period of 71/z rain was used throughout. Experiments not shown in this paper demonstrated that the increase in loop fluid volume caused by SLS 17 mM (Briseid et al., 1974, 1976) had taken place already after an exposure period of 5 rain. The lowest concentration of SLS tested (2 raM) caused a significant increase in the level of cyclic AMP (27~) and concentrations of SLS of 1 2 - 2 7 m M caused increases ( 4 5 - 50 ~) which did not differ significantly (Table 2). It should be mentioned that Donowitz and Binder (1975) observed an increase in the level of cyclic AMP of about 60 ~ in the rat colonic mucosa following in vivo incubation with dioctyl sodium sulfosuccinate 6 mM for 3 h. Effects of Sodium LauryI Sulphate, Imidazole, TheophylIine and Ouabain on the Level of Cyclic A M P in the Jejunal Mucosa of the Rat. Under our eXperimental conditions imidazole did not significantly reduce the resting level of cyclic AMP in the intestinal mucosa, but strongly reduced an increase caused by SLS (Table 3 A). Theophylline, on the other hand, both increased the cyclic AMP level and potentiated the increase caused by SLS (Table 3B). It is possible that the additional effect seen when SLS was used simultaneously with theophylline might be partially ascribed to an enhanced rate of diffusion of the last mentioned substance into the mucosa. Ouabain did neither alter the resting level of intestinal cAMP nor the increased level obtained with SLS (Table 3, C). It has previously been pointed out (Briseid et al., 1976) that reports in
265
the literature on the effect of ouabain on components of the adenyl cyclase-cyclic AMP system are few and conflicting, encompassing in vivo experiments that demonstrate stimulation of, and no effect on, and inhibition of adenyl cyclase (0ye and Sutherland, 1966; Ho et al., 1967; Braun et al., 1969; Shimizu et al., 1970; Katz and Tenenhouse, 1973).
Discussion of Previous Data on the Effects of Sodium Lauryl Sulphate, Imidazole, Theophylline and Ouabain on the Jejunal Absorption in the Rat in View of the Present Results on the Effects of the Same Substances on the Level of Jejunal Cyclic AMP. The evidence previously provided for the assumption that an increased level of intestinal cyclic AMP contributed to the absorption-enhancing effect of SLS (Briseid et al., 1976) was extended in the present work. The data obtained demonstrated that SLS will cause a small but significant increase in the mucosal concentration of cyclic AMP, and that this increase can be reduced by imidazole and potentiated by theophylline. Similar results were previously obtained for the increase in absorption caused by SLS under identical experimental conditions (Briseid et al., 1976). The question arises, however, if the whole SLS-effect on absorption can be explained through an increased level of intestinal cyclic AMP. This does not seem very probable. I. Quantitative considerations suggest that an important part of the effect of SLS on absorption is not correlated with an increase in cyclic AMP. In the present work it was found that imidazole completely eliminated the increase in cyclic AMP brought about by SLS (Table 3A), while the increase in absorption caused by SLS were only to a minor extent inhibited by imidazole (Briseid et al., 1976). Likewise, theophylline was found strongly to increase the level of intestinal cyclic AMP and to potentiate the effect of SLS on this level (Table 3 B), but the increases in absorption caused by theophylline were rather small and the effects of SLS only slightly enhanced (Briseid et al., 1976). 2. The increase in the absorption of phenolsulphonphthalein and of pralidoxime caused by SLS were significantly reduced if sodium and chloride or chloride and bicarbonate were replaced by other ions in the loop fluid (Briseid et al., 1974, 1976). These results reflect an ionic dependence of the SLS-caused absorption which points to an increased level of cyclic AMP as the basis for the effect (Field et al., 1968, 1972; Powell et al., 1973; Donowitz and Binder, 1975). The SLScaused absorption of ouabain was, however, not influenced by the mentioned alterations in the ionic composition of the medium (Briseid et al., 1974) and it might be suggested that part of the SLS-caused absorption is not dependent on an increase in the level of cyclic AMP.
266
3. In the present work it was found that ouabain did neither influence significantly the resting level of intestinal cyclic AMP (Table 3, C I - I I I ) nor the increase in the level of cyclic AMP caused by SLS (Table 3, C I I - IV). It has previously been shown that ouabain significantly inhibited the increased absorption of phenolsulphonphthalein and of pralidoxime caused by SLS (Briseid et al., 1974, 1976). Also these data support the assumption that the SLS-effect on absorption only partly can be ascribed to its effect on the intestinal level of cyclic AMP.
REFERENCES Binder, H. J., Filburn, C., Volpe, B. T. : Bile salt alteration of colonic electrolyte transport: Role of cyclic adenosine monophosphate. Gastroenterology 68, 503-- 508 (1975) Braun, T., B~ir, H. P., Soifer, D., Hechter, O. N. : Effect of insulin, prostaglandin El and ouabain on adenyl cyclase of rat fat cell plasma membranes. The Endocrine Society 15th Meeting New York, Abstracts, p. 138 (1969) Briseid, G., Briseid, K. : A comparison of the inhibition by cardiac glycosides of the isolated intestine from the rat and the guinea pig. Acta pharmacol. (Kbh.) 29, 275-283 (1971) Briseid, G., Briseid, K., Bergersen, B.: Studies on the increased absorption of ouabain, phenolsulphonphthalein and pralidoxime caused by sodium lauryl sulphate from single loop preparations in the rat. Naunyn-Schmiedeberg's Arch. Pharmacol. 282, 4 5 57 (1974) Briseid, G., Briseid, K., Kirkevold, K. : Increased intestinal absorption in the rat caused by sodium lauryl sulphate, and its possible relation to the cAMP system. Naunyn-Schmiedeberg's Arch. Pharmacol. 292, 137- 144 (1976) Christoffersen, T., M~brland, J., Osnes, J. B., Oye, I. : Development of cyclic AMP metabolism in rat liver. A correlative study of tissue levels of cyclic AMP, accumulation of cyclic AMP in slices, adenylate cyclase activity, and cyclic nucleotide phosphodiesterase activity. Biochim. biophys. Acta (Amst.) 313, 338 - 349 (1973)
Naunyn-Schmiedeberg's Arch. Pharmacol. 298 (1977) Donowitz, M., Binder, H. J.: Effect of dioctyl sulphosuccinate on colonic fluid and electrolyte movement. Gastroenterology 69, 941 - 950 (1975) Field, M., Fromm, D., AI-Awqati, Q., Greenough, W. B. : Effect of cholea enterotoxin on ion transport across isotated ileal mucosa. J. clin. Invest. 51, 796-804 (1972) Field, M., Plotkin, G. R., Silen, W.: Effects of vasopressin, theophylline and cyclic adenosine monophosphate on short-circuit current across isolated rabbit ileal mucosa. Nature (Lond.) 217, 469-471 (1968) GaA1, D., Holczinger, L. : Effect of detergents and sucrose derivatives on the modified protein determination of Lowry. Acta biochim, biophys. Acad. Sci. (Hung.) 10, 201-206 (1975) Gilman, A. G. : A protein binding assay for adenosine 3': 5'-cyclic monophosphate. Proc. nat. Acad. Sci. (Wash.) 67, 305-312 (1970) Hartree, E. F. : Determination of protein. Modification of the Lowry method that gives a linear photometric response. Analyt. Biochem. 48, 422-427 (1972) Ho, R. J., Jeanrenaud, B., Posternak, T., Renold, A. E. : Insulinlike action of ouabain. II. Primary antilipolytic effect through inhibition of adenyl cyclase. Biochim. biophys. Acta (Amst.) 144, 7 4 - 82 (1967) Katz, S., Tenenhouse, A. : The relation of adenyl cyclase to the activity of other ATP utilizing enzymes and phosphodiesterase in preparations of rat brain; mechanism of stimulation of cyclic AMP accumulation by adrenaline, ouabain and Mn + +. Brit. J. Pharmac. 48, 516- 526 (1973) Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. : Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265-275 (1951) 0ye, I., Sutherland, E. W. : The effect of epinephrine and other agents on adenyl cyclase in the cell membrane of avian erythrocytes. Biochim. biophys. Acta (Amst.) 127, 347-354 (1966) Powell, D. W., Binder, H. J., Curran, P. F. : Active electrolyte secretion stimulated by choleragen in rabbit ileum in vitro. Amer. J. Physiol. 225, 781-787 (1973) Shimizu, H., Creveling, C. R., Daly, J. W. : Cyclic adenosine 3',5'monophosphate formation in brain slices: Stimulation by batrachotoxin, ouabain, veratridine, and potassium ions. Molec. Pharmacol. 6, 184-188 (1970)
Received November 4, 1976~Accepted March 3, 1977
Naunyn-Schmiedeberg's Arch. Pharmacol. 298, 263-266 (1977)
Archivesof Pharmacology 9 by Springer-Verlag1977
.Increased Level of cAMP in the Rat Intestinal Mucosa Caused by Sodium Lauryl Sulphate G. B R I S E I D 1, I. OYE 2, and K. B R I S E I D 1 1Department of Pharmacology, Institute of Pharmacy 2Institute of Pharmacology, University of Oslo, Blindern, Oslo 3, Norway
S u m m a r y . The level of cyclic A M P in the jejunal mucosa from tied loops of anaesthetized rats was found to be significantly increased ( 2 7 - 5 0 %) when sodium lauryl sulphate (SLS) was added to the loop fluid ( 2 - 2 7 raM). Imidazole (25 raM) did not significantly alter the resting level of cyclic AMP, but reduced the increase caused by SLS (17 raM). Theophylline (25 raM) significantly increased the intestinal level of cyclic AMP, and potentiated the increase caused by SLS. Ouabain (2.5 raM) did not alter the level of cyclic A M P in the presence or in the absence of SLS. The results of previous experiments on the increases in intestinal absorption caused by SLS or by dibutyryl cyclic A M P (Briseid et al., 1974, 1976) are discussed in light of the present data. It is concluded that the SLSeffect on absorption can only partly by ascribed to its effect on the intestinal level of cyclic AMP. K e y words: Cyclic A M P -
Imidazole -
Sodium lauryl sulphate Theophylline - Jejunal absorption.
INTRODUCTION It has-previously been shown that the increases in absorption of phenolsulphonphthalein and pralidoxime from jejunal loops of anaesthetized rats caused b y sodium lauryl sulphate (SLS) (Briseid et al., 1974) were potentiated by theophylline a n d reduced by imidazole (Briseid et al., 1976). An increased absorption was also obtained by addition of dibutyryl cyclic A M P to the loop fluid. This indirect evidence of an increased level of cyclic A M P as part of the effect of SLS on the absorption was supported by determinations of the concentration of cyclic A M P in the rat intestinal mucosa in the presence and in the absence of SLS (Briseid et al., 1976). The effect of surfaceSend offprint requests to : Dr. K. Briseid at the above address
active agents on the cyclic A M P level in the rat intestine was also investigated by Binder et al. (1975), who found that taurochenodeoxycholic acid increased the colonic mucosal cyclic A M P level. Donowitz and Binder (1975) obtained similar results using a synthetic detergent, dioctyl sodium sulfosuccinate. In the present work we wanted to verify our preliminary results on the effect of SLS on the level of cyclic A M P in the rat jejunal mucosa (Briseid et al., 1976). We further wanted to evaluate the role of cyclic A M P for the effect of SLS on the absorption of phenolsulphonphthalein and pralidoxime (Briseid et al., 1974, 1976).
METHODS Determination of Cyclic AMP. The concentration of cyclicAMP in
the rat intestinal mucosa was measured under experimental conditions similar to those described for the absorption experiments (Briseid et al., 1974). When the intestinal loop had been washed out, it was opened and sheets of the mucosa were stripped from the submucosal layer. The specimens of fresh tissue were immediately frozen on dry ice and homogenizedin cold 5 % trichloroacetic acid (Gilman, 1970).CyclicAMP was determined in the supernatant after extraction of the trichloroacetic acid with ether (Gilman, 1970) by the protein binding assay described by Christoffersen et al. (1973). It was demonstrated in recoveryexperiments that the amount of SLS which is present in the mucosa extracts under our experimental conditions, does not affect the cAMP assays (Sund and Hillestad, to be pubiished). Test Substances and Surfactant. Imidazole,grade II[ : SigmaChemical Company, St. Louis. Ouabain (g-strophanthin krist., reinst): Merck A.G., Darmstadt. Sodium lauryl sulphate (Texapon KI2), SLS: Henkel and Cie, GmbH., Diisseldorf. Theophylline (theophyllin krist, wasserhaltig): Knoll A.G., Ludwigshafen am Rhein. Test Solutions. Imidazole25 raM, ouabain 2.5 raM, SLS 17 mM and theophylline 25 raM. Tyrode solution (Briseid and Briseid, 1971) was used as test fluid, pH 7.4. Osmotic adjustment of the instilled solutions was carried out by reductions in the amount of sodium chloride. Protein Determination. Protein was determined by the method of
Lowry et al. (1951). Statistical Method. Student's t-test was used. A probability level of
less than 5 % was considered significant.
264
Naunyn-Schmiedeberg's Arch. Pharmacol. 298 (1977)
Table 1. Protein/wet weight tissue ratios in the intestinal mucosa from single loop preparations in the rat. Effect of the experimental time period and of the concentration of sodium lauryl sulphate (SLS). Loop fluid Tyrode ofpH 7.4. Each figure represents data from 6 rats. Mg protein/mg wet weight: I without SLS. II SLS 17 raM. III SLS 0 to 27 mM and 71/2min exposure periods. When comparing the results from the longer exposure periods with the results obtained after 2 min (I and II), and also the results obtained in the presence of SLS with the result without SLS (III), no statistically significant differences were seen (P > 0.05) Minutes
I
II
SLS mM
Ill
2 71/2 15 30 60
0.11 0.11 0.12 0.10 0.11
0.11 0.10 0.09 0.09 0.09
0 2 7 12 17 22 27
0.09 0.09 0.10 0.10 0.10 0.10 0.10
Table 2. Effect of sodium lauryl sulphate on the cyclic AMP level in the intestinal mucosa of the rat as a function of concentration. Loop fluid Tyrode of pH 7.4 and an exposure period of 71/2min. Each figure represents data from 6 rats SLS mM
Cyclic AMP pmoles/mg wet wt.
S.E. diff. to SLS 0 mM
Level of significance
0 2 7 12 17 22 27
0.67 0.85 0.91 0.97 0.92 1.08 1.01
0.03 0.05 0.10 0.07 0.04 0.08
0.05 0.02 0.02 0.01
> > > >
P P P P P 0.01 > P
> > > > < >
0.02 0.01 0.01 0.001
0.OOl 0.001
RESULTS
Determination of Protein in Wet Weight Tissue. D e t e r m i n a t i o n s o f the p r o t e i n / w e t weight tissue ratios in the absence a n d in the presence o f SLS 17 m M over the t i m e interval 2 - 6 0 rain did n o t reveal any alterations (Table 1, I,II). A c c o r d i n g to Gafil and H o l z i n g e r (1975) the p r o t e i n d e t e r m i n a t i o n m e t h o d o f L o w r y et al. (1951) as m o d i f i e d by H a r t r e e (1972) m i g h t be slightly influenced by SLS. U n d e r o u r e x p e r i m e n t a l c o n d i t i o n s the p r o t e i n / w e t weight tissue ratios were n o t f o u n d to c h a n g e in e x p e r i m e n t s in w h i c h the SLS c o n c e n t r a t i o n was increased f r o m 0 - 2 7 m M in c o n n e c t i o n with an e x p o s u r e p e r i o d o f 71/2 rain. O n e such e x p e r i m e n t is s h o w n in T a b l e 1, III. This result at the same time p r o v i d e s e v i d e n c e that the s u r f a c t a n t did n o t cause a l t e r a t i o n s in the a m o u n t s o f fluid p r e s e n t in the sheets o f m u c o s a u s e d for the p r o t e i n d e t e r m i n a t i o n s a n d the results o f the assays o f cyclic A M P in the present w o r k are given as p m o l e s / m g wet weight tissue. Effect of Sodium LauryI Sulphate on the Cyclic A M P Level as a Function of Time and as a Function of Concentration. Time-effect e x p e r i m e n t s carried o u t with SLS 17 m M d e m o n s t r a t e d no significant alterations in the cyclic A M P levels o v e r the t i m e interval 2 60 rain. T a b l e 2 shows the results o f an e x p e r i m e n t with c o n c e n t r a t i o n s o f SLS r a n g i n g f r o m 2 - 27 m M . T h e e x p o s u r e p e r i o d was 71/2 rain. A m a x i m u m increase in cyclic A M P c o n c e n t r a t i o n o f a b o u t 50 % was registered for the two highest c o n c e n t r a t i o n s , but also the 27 % increase registered at 2 m M SLS was statistically significant.
Effects of Sodium Lauryl Sulphate, Imidazole, Theophylline and Ouabain on the Cyclic A M P Level in the Jejunal Mucosa of the Rat. T a b l e 3 shows t h a t imid-
Table 3. Effects of sodium laurylsulphate (SLS) 17 mM, imidazole (imidaz.) 25 mM, theophylline (theoph.) 25 mM or ouabain (ouab.) 2.5 mM on the level of cyclic AMP in the rat intestinal mucosa. Loop fluid Tyrode (T) of pH 7.4 and exposure period of 71/2 min. Each figure represents data from 6 rats Experiment
Medium
Cyclic AMP pmoles/mg wet wt.
S.E. diff.
Level of significance
A
I T II T+SLS IIIT+imidaz. IV T+SLS+imidaz.
1.22 1.89 1.03 0.72
I - II:0.22 I - I I I : 0.19 II - I V : 0.33 I I I - I V : 0.31
0.01 0.4 0.01 0.4
B
I T II T+SLS III T+theoph. IV T+SLS+theoph.
0.63 0.81 1.45 2.41
I - II:0.18 I -III:0.17 II - IV: 0.15 I I I - IV: 0.21
0.1 > P > 0.01 > P > P < 0.01 > P >
0.05 0.001 0.001 0.001
C
I T II T+SLS IIIT+ouab. IVT+SLS+ouab.
0.78 0.92 0.75 0.85
I - II:0.07 I - I I I : 0.07 II - I V : 0.10 III--IV:0.10
0.1 0.8 0.7 0,1
0.05 0.7 0.6 0,05
> > > >
> > > >
P>0.001 P > 0.3 P > 0.001 P > 0.3
P> P > P > P>
G. Briseid et al. : Jejunal Absorption- Sodium Lauryl Sulphate and the cAMP System azole significantly reduced the SLS-caused increase in the level of cyclic AMP (Table 3A, I I - I V ) , while imidazole alone did not significantly reduce the resting level of cyclic AMP (Table 3A, I - I I I ) . Table 3 further shows that theophylline significantly increased the intestinal level of cyclic AMP (Table 3 B, I - I I I ) and strongly potentiated the increase caused by SLS (Table 3 B, II - IV). Ouabain did not alter the level of cyclic AMP in the presence (Table 3 C, I I - IV) nor in the absence of SLS (Table 3 C, I - III).
DISCUSSION
Effect of Sodium Lauryl Sulphate on the Level of Cyclic A M P in the Rat Jejunal Mucosa as a Function of Time and as a Function of Concentration. Previous experiments indicated that the increase in the level of intestinal cyclic AMP caused by SLS 17 mM had reached a maximum already after an exposure period of 2 min (Briseid et al., 1976). Experiments carried out in connection with the present work demonstrated that no significant SLS-caused increase in the concentration of cyclic AMP took place over the period 2 - 60 rain, and a standard exposure period of 71/z rain was used throughout. Experiments not shown in this paper demonstrated that the increase in loop fluid volume caused by SLS 17 mM (Briseid et al., 1974, 1976) had taken place already after an exposure period of 5 rain. The lowest concentration of SLS tested (2 raM) caused a significant increase in the level of cyclic AMP (27~) and concentrations of SLS of 1 2 - 2 7 m M caused increases ( 4 5 - 50 ~) which did not differ significantly (Table 2). It should be mentioned that Donowitz and Binder (1975) observed an increase in the level of cyclic AMP of about 60 ~ in the rat colonic mucosa following in vivo incubation with dioctyl sodium sulfosuccinate 6 mM for 3 h. Effects of Sodium LauryI Sulphate, Imidazole, TheophylIine and Ouabain on the Level of Cyclic A M P in the Jejunal Mucosa of the Rat. Under our eXperimental conditions imidazole did not significantly reduce the resting level of cyclic AMP in the intestinal mucosa, but strongly reduced an increase caused by SLS (Table 3 A). Theophylline, on the other hand, both increased the cyclic AMP level and potentiated the increase caused by SLS (Table 3B). It is possible that the additional effect seen when SLS was used simultaneously with theophylline might be partially ascribed to an enhanced rate of diffusion of the last mentioned substance into the mucosa. Ouabain did neither alter the resting level of intestinal cAMP nor the increased level obtained with SLS (Table 3, C). It has previously been pointed out (Briseid et al., 1976) that reports in
265
the literature on the effect of ouabain on components of the adenyl cyclase-cyclic AMP system are few and conflicting, encompassing in vivo experiments that demonstrate stimulation of, and no effect on, and inhibition of adenyl cyclase (0ye and Sutherland, 1966; Ho et al., 1967; Braun et al., 1969; Shimizu et al., 1970; Katz and Tenenhouse, 1973).
Discussion of Previous Data on the Effects of Sodium Lauryl Sulphate, Imidazole, Theophylline and Ouabain on the Jejunal Absorption in the Rat in View of the Present Results on the Effects of the Same Substances on the Level of Jejunal Cyclic AMP. The evidence previously provided for the assumption that an increased level of intestinal cyclic AMP contributed to the absorption-enhancing effect of SLS (Briseid et al., 1976) was extended in the present work. The data obtained demonstrated that SLS will cause a small but significant increase in the mucosal concentration of cyclic AMP, and that this increase can be reduced by imidazole and potentiated by theophylline. Similar results were previously obtained for the increase in absorption caused by SLS under identical experimental conditions (Briseid et al., 1976). The question arises, however, if the whole SLS-effect on absorption can be explained through an increased level of intestinal cyclic AMP. This does not seem very probable. I. Quantitative considerations suggest that an important part of the effect of SLS on absorption is not correlated with an increase in cyclic AMP. In the present work it was found that imidazole completely eliminated the increase in cyclic AMP brought about by SLS (Table 3A), while the increase in absorption caused by SLS were only to a minor extent inhibited by imidazole (Briseid et al., 1976). Likewise, theophylline was found strongly to increase the level of intestinal cyclic AMP and to potentiate the effect of SLS on this level (Table 3 B), but the increases in absorption caused by theophylline were rather small and the effects of SLS only slightly enhanced (Briseid et al., 1976). 2. The increase in the absorption of phenolsulphonphthalein and of pralidoxime caused by SLS were significantly reduced if sodium and chloride or chloride and bicarbonate were replaced by other ions in the loop fluid (Briseid et al., 1974, 1976). These results reflect an ionic dependence of the SLS-caused absorption which points to an increased level of cyclic AMP as the basis for the effect (Field et al., 1968, 1972; Powell et al., 1973; Donowitz and Binder, 1975). The SLScaused absorption of ouabain was, however, not influenced by the mentioned alterations in the ionic composition of the medium (Briseid et al., 1974) and it might be suggested that part of the SLS-caused absorption is not dependent on an increase in the level of cyclic AMP.
266
3. In the present work it was found that ouabain did neither influence significantly the resting level of intestinal cyclic AMP (Table 3, C I - I I I ) nor the increase in the level of cyclic AMP caused by SLS (Table 3, C I I - IV). It has previously been shown that ouabain significantly inhibited the increased absorption of phenolsulphonphthalein and of pralidoxime caused by SLS (Briseid et al., 1974, 1976). Also these data support the assumption that the SLS-effect on absorption only partly can be ascribed to its effect on the intestinal level of cyclic AMP.
REFERENCES Binder, H. J., Filburn, C., Volpe, B. T. : Bile salt alteration of colonic electrolyte transport: Role of cyclic adenosine monophosphate. Gastroenterology 68, 503-- 508 (1975) Braun, T., B~ir, H. P., Soifer, D., Hechter, O. N. : Effect of insulin, prostaglandin El and ouabain on adenyl cyclase of rat fat cell plasma membranes. The Endocrine Society 15th Meeting New York, Abstracts, p. 138 (1969) Briseid, G., Briseid, K. : A comparison of the inhibition by cardiac glycosides of the isolated intestine from the rat and the guinea pig. Acta pharmacol. (Kbh.) 29, 275-283 (1971) Briseid, G., Briseid, K., Bergersen, B.: Studies on the increased absorption of ouabain, phenolsulphonphthalein and pralidoxime caused by sodium lauryl sulphate from single loop preparations in the rat. Naunyn-Schmiedeberg's Arch. Pharmacol. 282, 4 5 57 (1974) Briseid, G., Briseid, K., Kirkevold, K. : Increased intestinal absorption in the rat caused by sodium lauryl sulphate, and its possible relation to the cAMP system. Naunyn-Schmiedeberg's Arch. Pharmacol. 292, 137- 144 (1976) Christoffersen, T., M~brland, J., Osnes, J. B., Oye, I. : Development of cyclic AMP metabolism in rat liver. A correlative study of tissue levels of cyclic AMP, accumulation of cyclic AMP in slices, adenylate cyclase activity, and cyclic nucleotide phosphodiesterase activity. Biochim. biophys. Acta (Amst.) 313, 338 - 349 (1973)
Naunyn-Schmiedeberg's Arch. Pharmacol. 298 (1977) Donowitz, M., Binder, H. J.: Effect of dioctyl sulphosuccinate on colonic fluid and electrolyte movement. Gastroenterology 69, 941 - 950 (1975) Field, M., Fromm, D., AI-Awqati, Q., Greenough, W. B. : Effect of cholea enterotoxin on ion transport across isotated ileal mucosa. J. clin. Invest. 51, 796-804 (1972) Field, M., Plotkin, G. R., Silen, W.: Effects of vasopressin, theophylline and cyclic adenosine monophosphate on short-circuit current across isolated rabbit ileal mucosa. Nature (Lond.) 217, 469-471 (1968) GaA1, D., Holczinger, L. : Effect of detergents and sucrose derivatives on the modified protein determination of Lowry. Acta biochim, biophys. Acad. Sci. (Hung.) 10, 201-206 (1975) Gilman, A. G. : A protein binding assay for adenosine 3': 5'-cyclic monophosphate. Proc. nat. Acad. Sci. (Wash.) 67, 305-312 (1970) Hartree, E. F. : Determination of protein. Modification of the Lowry method that gives a linear photometric response. Analyt. Biochem. 48, 422-427 (1972) Ho, R. J., Jeanrenaud, B., Posternak, T., Renold, A. E. : Insulinlike action of ouabain. II. Primary antilipolytic effect through inhibition of adenyl cyclase. Biochim. biophys. Acta (Amst.) 144, 7 4 - 82 (1967) Katz, S., Tenenhouse, A. : The relation of adenyl cyclase to the activity of other ATP utilizing enzymes and phosphodiesterase in preparations of rat brain; mechanism of stimulation of cyclic AMP accumulation by adrenaline, ouabain and Mn + +. Brit. J. Pharmac. 48, 516- 526 (1973) Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. : Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265-275 (1951) 0ye, I., Sutherland, E. W. : The effect of epinephrine and other agents on adenyl cyclase in the cell membrane of avian erythrocytes. Biochim. biophys. Acta (Amst.) 127, 347-354 (1966) Powell, D. W., Binder, H. J., Curran, P. F. : Active electrolyte secretion stimulated by choleragen in rabbit ileum in vitro. Amer. J. Physiol. 225, 781-787 (1973) Shimizu, H., Creveling, C. R., Daly, J. W. : Cyclic adenosine 3',5'monophosphate formation in brain slices: Stimulation by batrachotoxin, ouabain, veratridine, and potassium ions. Molec. Pharmacol. 6, 184-188 (1970)
Received November 4, 1976~Accepted March 3, 1977
