European Journal of Clinical Investigation (1980) 10,23 1-234

Interaction of laxatives with enzymes of cyclic A M P metabolism from human colonic mucosa

BERND SIMON & HORST KATHER, Medizinische Universitatsklinik Heidelberg, Gastroenterologische Abteilung, Heidelberg, F.R.G.

Received 3 October 1979 and in revised form 8 January 1980

Abstract. The mechanism by which laxatives such as dioctyl sodium sulfosuccinate and ricinoleic acid evoke colonic fluid secretion has been suggested to involve mucosal cyclic AMP. Ricinoleic acid and dioctyl sodium sulfosuccinate were tested for their capacity to modulate the key enzymes of CAMP-metabolisrn- adenylate cyclase and CAMP-phosphodiesterase-in human colonic mucosa.

Both laxatives were ineffective stimuli of human colonic adenylate cyclase. In contrast to ricinoleic acid, dioctyl sodium sulfosuccinate was a competitive in- hibitor of soluble CAMP-phosphodiesterase activity. These experiments suggest that the cathartic properties of dioctyl sodium sulfosuccinate in human colonic mucosa might be mediated by cyclic AMP via inhibi- tion of soluble phosphodiesterase activity.

Key words. Human colonic mucosa, adenylate cyclase activity, phosphodiesterase activity, cyclic AMP, laxa- tives, dihydroxy bile acids, colonic secretion.

Introduction Laxatives such as dioctyl sodium sulfosuccinate and ricinoleic acid are known to modify small and large bowel electrolyte and water transport resulting in a net accumulation of fluid within the lumen of the intestinal tract [ l , 21. The cathartic properties of both com- pounds are believed to be mediated by increased cyclic AMP levels in colonic mucosa [3-51. However, the data concerning the mediator role of this nucleotide in laxa- tive-induced secretion are equivocal [6] . In an attempt to clarify the apparent discrepancies in the proposed cellular mechanism of colonic secretion by laxatives, we have examined the effects of ricinoleic acid and dioctyl sodium sulfosuccinate on the key enzymes of CAMP-metabolism-adenylate cyclase and CAMP- phosphodiesterase-in homogenates of human col- onic mucosa.

Correspondence: Dr k m d Simon, Medizinische Universitiits- klinik Heidelberg, Gastroenterologische Abteilung, Bergheimentr. 58,6900 Heidelberg, F.R.G. 0014-2972/80/06O(rO23 1502.00 0 1980 Blackwell Scientific Publications

Materials and Methods Experiments were performed on histologically normal colonic mucosa obtained from eight patients undergo- ing hemicolectorny for carcinoma or diverticulosis. Three patients were female. Preoperative procedures included an instillation of hypertonic saline solution (5 litres) via a gastric tube. The identity of the mucosa was verified histologically, using conventional haematoxy- lin-eosin stain.

Experimental procedures have been described pre- viously [7]. The mucosa was scraped from the muscle layer and homogenized in a Teflon-glass homogenizer (Colora-Messtechnik, Zell-Homogenisator. Lorch, Wiirttemberg, F.R.G.) in a medium containing 50 mmol/l Tris-HC1 buffer, pH 7.4,3 mmol/l MgClz and 3 mrnol/l rnercaptoethanol.

Adenylate cyclase activity was determined by the method of Salornon er al. [8] at 30°C. The assay mix- ture contained 25 mmol/l Tris-HC1, pH 7.6, 5 mrnol/l MgC12, 20 mmol/l creatinine phosphate, 100 U/ml creatine phosphokinase, 1 mmol/l 3',5'-cyclic adeno- sine monophosphate (CAMP) and d2-P-ATP ( I mmol/l; 40-50 cpm/pmol). The reaction was initiated by addition of 20 p1 of homogenate protein and ter- minated by additions of 0.1 ml of stopping solution composed of2% (w/v) lauryl sulphate, 1 rnmol/l cAMP and 40 mmol/l ATP. cAMP formation was linear up to 25 min using 40-100 pg of protein per assay. Cyclic 32P-cAMP was purified by column chromatography using Dowex AG 50 W-X 4 and neutral alumina [8].

Cyclic AMP phosphodiesterase activity, which was shown to be located mostly in cytosol (unpublished observation), was determined by the method of Poch [9]. This method is based on the use of radioactively 'labelled cyclic 3',5'-AMP as substrate and the quanti- tative removal of the labelled product 5'-AMP.

After the colonic mucosa was prepared as for the adenylate cyclase assay, the homogenate was centri- fuged at 20,000 g for 10 min. 100 pl of supernatant, containing 200400 pg of protein, were added to 200 p1 of incubation mixture. The final composition of the incubation mixture, 300 pl, was 50 mmol/l Tris-HC1 (pH 7.9, 3 mmol/l MgCl2, 1 rnmol/l 5'-AMP and 1 pnol/l cyclic 3',5'-AMP as well as approximately 0.0 1

23 1

232 BERND SIMON & HORST KATHER

pCl of [‘H]cyclic 3’,5’-AMP (specific activity. 70-100 counts/min/pmol). The hydrolysis of cyclic AMP was stopped after 10 min by the addition of 200 pl ZnSOJ (0.17 mol/l). The reaction product 5’-AMP was removed by precipitation using 200 pI of Ba(0H) (0. I5 mol/l).

The protein content of the samples was measured according to Lowry el al. [lo]. The activity of adenylate cyclase is given as pmol cAMP formed per mg protein per 15 min; the activity of phosphodiesterase is expressed as the amount of 3’,5’ cAMP hydrolysedimg protein/min.

Statistical analysis was by the Wilcoxon-test for paired samples [ 1 I ) . Each enzyme determination was performed in triplicate. The coefficient of variation averaged 3-8%.

[d?P]ATP and [3H]cAMP were obtained from the Radiochemical Centre, Amersham, Bucks., U.K. Deoxycholic acid, chenodeoxycholic acid and urso- deoxycholic acid (as their sodium salts) were pur- chased from Calbiochem, Switzerland. Sodium ricino- leate was obtained from K & K Laboratories, Inc. (Planview. N.Y .), dioctyl sodium sulfosuccinate from Ravensberg GmbH (Konstanz, F.R.G.). VIP (highly purified porcine) was purchased from Calbiochem, Switzerland, through Fa. Paesel, FrankfurtiMain, F.R.G. All other chemicals and reagents were of the highest grade commercially available.

Results Basal adenylate cyclase activity in homogenates of human colonic mucosa averaged 250 pmol cAMP/mg protein/ I5 min. Mean basal CAMP-phosphodiesterase activity was 150 k 15 pmoles per mg protein per min.

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Agonist ( - l o g m d / l ) Figure I. E k t of VIP. ricinoleate and dioctyl sodium sulfosuccinate on human large bowel adenylate cyclase. Values are mean SD of six separate experiments. each carried out in triplicate.

Fig. I illustrates the effects of increasing concentra- tions of vasoactive intestinal peptide (VIP), ricinoleic acid and dioctyl sodium sulfosuccinate (DSS) on the human large bowel cyclase. As described previously [7, 151, VIP is a potent activator of the human enzyme. Stimulation by the intact neurotransmitter was detect- able at 1 x lo-’ mol/l and maximal at 1 x lo-’ mol/l. Ricinoleic acid and DSS were tested at concentrations form I x mol/l to 5 x mol/l. No dose-related stimulation of the large bowel adenylate cyclase was observed over this concentration range. Doses of the

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Figure 2. Effect of laxatives (ricinokic acid, DSS) (left part) and dihydroxy bile acids (deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid) (right part) on soluble CAMP-phosphodiesterasc activity in human colonic mucosa. Mean phosphodiester- ase activity is 1502 I 5 pmoles/mg proteinlmin. Phosphodiesterase activity is expressedas’/, inhibition ofcontrol (in the absence of bile acids and laxatives). Values are mean 2 SD of Seven individual experiments carried out in triplicate.

LAXATIVES AND ENZYMES OF CYCLIC AMP METABOLISM 233

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laxatives above 1 x mol/l inhibited basal activity. Average inhibition of about 40-50% occurred at a laxative concentration of 0.5 mmol/l.

Fig. 2 shows the effects of laxatives-ricinoleic acid and DSS (left p a r t e a s well as of dihydroxy bile acids-deoxycholic acid, chenodeoxycholic acid, urso- deoxycholic acid (right part-n soluble CAMP-phos- phodiesterase activity in human colonic mucosa.

A small, but significant ( P < 0.05) depression of enzyme activity was noted in the presence of 100 pmol/l dihydroxy bile salts (average inhibition about

In contrast to ricinoleic acid which did not influence soluble cAMP phosphodiesterase activity over the concentration range, DSS effectively inhibited the enzyme activity. Inhibitory effects were detected at a DSS-concentration of 1 pmol/l. Complete inhibition occurred at a DSS-concentration of 50 pmol/l.

In order to test whether dioctyl sodium sulfosuc- cinate competes at the catalytic site for the substrate CAMP, the effects of varying cAMP concentrations were examined under conditions which produce linear Lineweaver-Burk plots. As is shown in Fig. 3, dioctyl sodium sulfosuccinate competes with the substrate cAMP in an apparently competitive manner.

30-3573.

Discussion Considerable evidence indicates that alterations in the cyclic nucleotide levels of large intestinal epithelium are responsible for marked changes in cellular trans- port functions [5, 12-14]. Thus, elevations in cyclic AMP by hormonal secretagogues such as vasoactive

intestinal polypeptide (VIP) and prostaglandins cause fluid and electrolyte secretion into the large bowel lumen [12-141. Tissue cAMP is determined by the relative activities of adenylate cyclase and CAMP- phosphodiesterase, the enzyme responsible for degra- dation of this cyclic nucleotide. VIP and prostaglan- dins enhance cAMP via direct interaction with the membrane-bound adenylate cyclase in colonic rnucosa of animals and man [ 12, 15, 161. They have no effect on CAMP-phosphodiesterase activity (unpublished observation).

In addition, cyclic AMP has been supposed to mediate the actions of non-hormonal secretagogues such as laxatives and bile acids in colonic tissue [3, 4, 171. Binder [3] and Donowitz & Binder [4] have found that ricinoleic acid and dioctyl sodium sulfosuccinate significantly increased mucosal cyclic AMP in rat col- onic mucosa. The intracellular enhancement of this nucleotide has been interpreted to be a consequence of adenylate cyclase stimulation rather than phospho- diesterase inhibition.

However, attempts to activate intestinal adenylate cyclase by laxatives and hydroxy fatty acids in vifro have led to conflicting results. Coyne ef al. [ 181 have reported that hydroxy stearic acid increased adenylate cyclase activity in the human colonic mucosa. In this latter study, however, a clear dose-related stimulation of enzyme activity was not given. In contrast, Gaginella ef al. [6] observed a dose-related inhibition of small intestinal cyclase by ricinoleic acid in concen- trations above 0.01 mmol/l.

Direct effects of both laxatives on the enzymes of cyclic AMP metabolism in human colonic mucosa have not yet been reported. Our experiments clearly estab- lish that ricinoleic acid and dioctyl sodium sulfosuc- cinate have no activating effect on the human colonic cyclase. Concentrations of Na+-ricinoleate, effective in cico, are of 2 4 f o l d higher than the highest concentra- tion used in the present in vifro experiments. When applied in this high concentration range both laxatives inhibit adenylate cyclase activity in human colonic mucosa completely (not shown).

In contrast to ricinoleic acid, dioctyl sodium sulfo- succinate is a potent inhibitor of soluble phosphodies- terase activity. At a DSS-concentration of 0.1 mmol/l the enzyme activity is completely inhibited. The degree of potency is similar to that of papaverine; the type of inhibition is apparently competitive with respect to the substrate CAMP. It is now accepted that a reduction of phosphodiesterase activity by more than 60% causes a significant rise in cellular cyclic AMP levels. The inhi- bition cf colonic phosphodiesterase activity by DSS therefore is likely to be of biological significance in contrast to the small decrease of enzyme activity in the presence of dihydroxy bile acids. Our studies suggest that the accumulation of cellular cyclic AMP following administration of dioctyl sodium sulfosuccinate is, at least in part, due to a direct inhibitory action of this laxative on phosphodiesterase activity.

The ricinoleic acid-induced cAMP increase in rat

234 BERND SIMON & HORST KATHER

colonic mucosa, recently confirmed by Racusen & Binder [S], cannot be explained on the basis of our studies. This laxative has no effect on either adenylate cyclase or phosphodiesterase. It is possible that this surfactant laxative may act indirectly by stimulation of prostaglandin biosynthesis, as recently shown for the diphenolic laxatives [19].

Acknowledgment The authors wish to thank Ms Elke Finkensieper for her excellent technical assistance.

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