Brain Research, 142 (1978) 379-383 © Elsevier/North-Holland Biomedical Press
379
Effects of indomethacin on cholera toxin-induced cerebrospinal fluid production
ARTHUR M. FELDMAN*, THOMAS SMITH, MELVIN H. EPSTEIN and SAUL W. BRUSILOW Department of Pediatrics and Neurosurgery, The Johns Hopkins University School of Medicine, Baltimore Md. 21205 (U.S.A.)
(Accepted September 14th, 1977)
The intraventricular administration of purified cholera toxin in the dog results in an increase in cerebrospinal fluid (CSF) production z. That this secretory response is associated with stimulation of choroid plexus adenylate cyclase and resultant increases in tissue cyclic A M P levels has been shown using an in vitro preparation of rat choroid plexus 4. Furthermore, prostaglandin E~ increases choroid plexus cyclic AMP accumulationS. Recent studies have demonstrated that the non-steroidal anti-inflammatory agents indomethacin or aspirin inhibited cholera toxin-induced intestinal secretion 6,a,1~, but these compounds did not prevent cyclic A M P accumulation in vitro1,10,12,15. The present study was undertaken to investigate the effects of indomethacin on cholera toxin-induced CSF secretion in the cat and to correlate these findings with the in vitro effects of indomethacin on cholera toxin-induced cyclic A M P accumulation in our rat model. In vivo experiments were performed on mongrel cats weighing 2-5 kg. They were anesthetized with pentobarbital, secured in a stereotactic frame, and maintained on positive pressure ventilation. A femoral vein was cannulated for intravenous fluid, anesthesia and drug administration, and a femoral artery was cannulated for monitoring blood pressure. Arterial pressure, heart rate, intracranial pressure, body temperature, pO2, and pCO2 were monitored. CSF production was measured using a modification of the method of Pappenheimer et al. 14 as previously described 2. The cisterna magna was catheterized with an 18-gauge Tygon catheter which was secured with cation-activated glue. Each lateral ventricle was cannulated with an 18-gauge needle through a small drill hole in the skull. Both lateral ventricles were perfused with Elliott's B artificial CSF containing 2/~Ci/100 ml of [14C]carboxyinulin (New England Nuclear) at 0.09 ml/min/ventricle. Purified cholera toxin, 50/~g (Schwarz/Mann), prepared as previously described a, or heat-inactivated cholera toxin in a total volume of 200 #1 of saline, was administered to the animals through both ventricular cannulas. Two hours after cholera toxin challenge, the perfusion was begun and CSF formation * Present address: Department of Otolaryngology, L.S.U. School of Medicine m Shreveport, P.O. Box 33932, Shreveport, La. 71130, U.S.A.
380 5O E
i
tj
2
30 z F-
201
8 ro
Control
i
HCT
i CT
z, I
CT+I
Fig. 1. The effects of cholera toxin (CT), heat-inactivated cholera toxin (HCT), and mdomethacm (1) on cerebrosp:nal fluid production m the cat. Production rates were measured 2-3 h after administration of 50 #g of purified CT to each lateral ventricle. Indomethacin was given i.v. (30 mg/kg) and s.c. (30 mg/kg) at the time of CT challenge, and an i.v. booster dose (30 mg/kg) was given 2.5 h later * represents a significant (P < 0.001) difference from control.
monitored at 10-min intervals over a period of I h. Experimental values represented the mean of this collection period. Indomethacin dissolved in Tris buffer, pH 8.0, or a control solution containing the Tris buffer alone was injected intravenously (30 mg/kg) and subcutaneously (30 mg/kg) at the time of cholera toxin administration. At 2.5 h after the initial indomethacin administration, a booster dose (30 mg/kg) was given intravenously. In vitro studies were performed using plexuses isolated from Sprague-Dawley rats weighing 150-250 g. After anesthetizing the animals with pentobarbital (Nembutal, 100 mg/kg) and exposing the cerebral hemispheres, the rats were perfused with saline through a 22-gauge needle placed in the left ventricle in order to clear the choroid plexus of blood. Both choroid plexuses were excised and placed in cold Krebs-Ringer bicarbonate buffer which contained 0.3 M glucose and which was constantly gassed with 95 ~o 02-5 ~ COs. These choroid plexuses were then placed in 1 ml of buffer which was continuously agitated in a Dubnoff shaker both maintained at 38 °C. After a 10-min preincubation, either cholera toxin (10 /~g/ml) or heat-inactivated cholera toxin in a volume of 100 y1 was added to the incubation vessel along with either indomethacin (20 #g/ml) dissolved in 0.1 M Tris buffer, pH 8.0, or a control solution. Sixty minutes after the addition of the cholera toxin the reaction was stopped by the addition of 1 ml of cold 7 ~otrichloroacetic acid, followed immediately by homogenization with a teflon pestle. Theophylline was added to both control and treated reaction mixtures 5 mm to prior the termination of the experiment. After centrifugation at 1800 × g, the supernatant was decanted, ether washed, and heated in a hot water bath at 60 °C for 4 min. Cyclic AMP content was determined using a modification of the radloimmunoassay of Harper and Brooker a. Protein was measured by the method of Lowry et al. 1~. Radioimmunoassay materials were supplied by Schwarz/Mann, Orangeburg, N.Y. Fig. 1. shows that the intraventricular administration of cholera toxin significantly
381 TABLE I Effect of indomethacin on cholera toxin-induced cyclic AMP accumulation (pmoles cyclic AMP/mg protein)
Choroid plexuses were incubated m Krebs-Rmger bicarbonate buffer (pH 7.4) at 37 °C for 60 mm. Theophylline, when present, was added to the incubation medium 5 rain prior to the termination of the experiment. Values represent the mean ± S.E. (n). Experiment 1
Cholera toxin + theophylhne Cholera toxin + indomethacin + theophylhne Inactivated cholera toxin + indomethacm (20/~g/ml) q- theophylline Inactivated cholera toxin + theophylline
121.09 4- 12.1 (4) 82.06 4- 8.6 (4)* 17.45 4- 1.58 (4) 22.09 4- 1.43 (4)
Experiment 2
Cholera toxin Inactivated cholera toxin + indomethacin (20/~g/ml) Inactivated cholera toxin
11.624- 0.63 (4) 11.08 + 3.3 (4) 7.95 4- 0.92 (4)
* P < 0.05.
(P < 0.001) increased CSF production in the cat. The rate of CSF secretion after cholera toxin challenge was 45.4 4- 1.7 (S.E.M.)/d/min, while after administration of bovine serum albumin or heat-inactivated cholera toxin the rates of production were 22.4 4- 1.3 and 25.4 ± 2.6 bd/min respectively. When cholera toxin (CT)-treated animals were pretreated with indomethacin, the rate of production was 37.8 ± 1.8 #l/min. While this value was significantly (P < 0.025) less than the CT-treated animals, it was greater (P < 0.001) than control values and was no different from production rates when indomethacin alone was given to the animals (31.0 4- 2.9 #l/min). The addition of indomethacin and cholera toxin to the in vitro incubation medium resulted in a 33 (P < 0.05) decrease in cyclic A M P accumulation when compared with incubations containing cholera toxin alone (Table I). However, when indomethacin and cholera toxin were present in the incubation medium, cyclic A M P accumulation was still significantly (P < 0.001) greater than in controls. The combination of indomethacin and inactivated cholera toxin had no effect on cyclic A M P levels. When theophylline was not present in the incubation medium, neither cholera toxin nor indomethacin had an effect on cyclic A M P accumulation when compared with the inactivated cholera toxin control. The present studies (Fig. 1) demonstrate a two-fold increase in CSF secretion in cats after intraventricular administration of cholera toxin. While previous studies in our laboratory using dogs demonstrated a similar change in CSF production, the cats provide a better experimental model. When dogs were used there was a larger variation
382 among animals. As seen in Fig. 1, cats had small individual differences in CSF secretory rates. Furthermore, while ventricular inflammation was noted in most of the dogs studied, none was seen in the cats after histologic examination. As shown in Fig. l, cholera toxin-induced CSF secretion could be partially inhibited by indomethacin, a finding similar to that described in cholera toxin-induced fluid secretion in the gut 6,9,15. While mdomethacin decreased cholera toxin-induced CSF secretion, indomethacin alone had a stimulatory effect on CSF production (Fig. I ). Similarly, it was recently found that while cholera toxin secretion in the gut was decreased by treatment with indomethacin, indomethacin alone initiated significant absorption of fluid across the gut wall 15. One possible explanation of this paradoxical effect is the presence of multiple secretory pathways all of which are necessary for maximum cerebrospmal production. I f some pathways were stimulated by a prostaglandin while others were inhibited by another prostaglandin, the inhibition of prostaglandln synthesis by indomethacin could result in some secretion via those pathways normally inoperative in the presence of a specific inhibitory prostaglandin. That lndomethacin decreased (29 ~o) but did not block cyclic A M P accumulation in vitro in choroid plexuses isolated from the rat corresponded with the results seen in vivo. However, the present studies differ from previous work in the intestine and the kidney in which lndomethacin had no effect on cyclic A M P accumulation 1,1°,i2. Although indomethacin clearly decreased cholera toxin-induced cyclic A M P accumulation it cannot be assumed that this is the entire mechanism by which indomethacin inhibits cholera-reduced secretion since there is still greater than a three-fold rise in cyclic A M P accumulation. While the biochemical mechanisms of CSF production are unclear, these studies suggest that prostaglandins play a role in CSF secretion. Furthermore, the basic cellular mechanisms involved in CSF production appear to be similar to those seen in other secretory epithelia. The technical assistance of Fallon Maylack and Philip Lyng is gratefully acknowledged. This work was supported in part by N.I.H. Grants AM07145, NS11274, and HD00091, the Kerr Foundation, and the National Foundation March of Dimes.
1 Bourne, H. R., Cholera enterotoxin. Failure of anti-inflammatory agents to prevent cyclic AMP accumulation, Nature (Lond.), 241 (1973) 399. 2 Epstein, M. H., Feldman, A. M., and Brusllow, S. W., Cerebrospmal fluid production: stimulation by cholera toxin, Science, 196 (1977) 1012-1013. 3 Feldman, A. M. and Brusilow, S. W., Effects of cholera toxin on cochlear endolymph production. Model for endolymphatic hydrops, Proc. nat. Acad. Sci. (Wash.), 73 (1976) 1761-1764. 4 Feldman, A. M., Maylack, F., Epstein, M. and Brusilow, S. W., In vitro effects of cholera toxin on rat chorold plexus, Clin. Res., 25 (1977) 430A. 5 Feldman, A. M., Maylack, F., Epstein, M. and Brusllow, S. W., Effects of prostaglandms on rat choroid plexus in vitro, Clin. Res., 25 (1977) 430A. 6 Finch, A. D. and Katz, R. L., Prevention of cholera-reduced intestinal secretion in the cat by aspmn, Nature (Lond.), 238 (1972) 273-274. 7 Frledler, R. M, Kurokawa, K., Coburn, J. W. and Massry, S. G., Renal action of cholera toxin: I. Effects on urinary excretion of electrolyte and cyclic AMP, Kidney Int., 7 (1975) 77-85.
383 8 Harper, J. F. and Brooker, G., Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic GMP after 2'0 acetylation by acetic anhydride in aqueous solution, J. Cyclic Nucleotide Res., 1 (1975) 207-218. 9 Jacoby, H. J. and Marshal, C. H., Antagonism of cholera enterotoxin by anti-inflammatoryagents in the rat, Nature (Lond.), 235 (1972) 163-165. 10 Kimberg, D. V., Field, M. and Johnson, J., Stimulation of intestinal mucosal adenyl cyclase by cholera enterotoxin and prostaglandins, J. clin. Invest., 50 (1971) 1218-1230. 11 Kimberg, D. V., Field M., Gershon, E. and Henderson, A., Effects of prostaglandins and cholera enterotoxm on intestinal mucosal cyclic AMP accumulation. Evidence against an essential role for prostaglandins in the action of toxin, J. clin. Invest., 53 (1974) 941-949. 12 Kurokawa, K., Friedler, R. M. and Massry, S. G., Renal action of cholera toxin: II. Effects on adenylate cyclase-cyclic AMP system, Ktdney Int., 7 (1975) 137-144. 13 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 14 Pappenheimer, J. R., Helsey, S. R., Jordan, E. F. and Downer, J., Perfuslon of the cerebral ventricular system in unanesthetized goats, Amer. J. Physiol., 203 (1962) 763-774. 15 Wald, A., Gotterer, G. S., Rajendra, G. R., Turjman, N. A. and Hendrix, T. R., Effect of indomethacm on cholera-induced fluid movement, unidirectional sodium fluxes, and intestinal cAMP, Gastroenterology, 72 (1977) 106-110
379
Effects of indomethacin on cholera toxin-induced cerebrospinal fluid production
ARTHUR M. FELDMAN*, THOMAS SMITH, MELVIN H. EPSTEIN and SAUL W. BRUSILOW Department of Pediatrics and Neurosurgery, The Johns Hopkins University School of Medicine, Baltimore Md. 21205 (U.S.A.)
(Accepted September 14th, 1977)
The intraventricular administration of purified cholera toxin in the dog results in an increase in cerebrospinal fluid (CSF) production z. That this secretory response is associated with stimulation of choroid plexus adenylate cyclase and resultant increases in tissue cyclic A M P levels has been shown using an in vitro preparation of rat choroid plexus 4. Furthermore, prostaglandin E~ increases choroid plexus cyclic AMP accumulationS. Recent studies have demonstrated that the non-steroidal anti-inflammatory agents indomethacin or aspirin inhibited cholera toxin-induced intestinal secretion 6,a,1~, but these compounds did not prevent cyclic A M P accumulation in vitro1,10,12,15. The present study was undertaken to investigate the effects of indomethacin on cholera toxin-induced CSF secretion in the cat and to correlate these findings with the in vitro effects of indomethacin on cholera toxin-induced cyclic A M P accumulation in our rat model. In vivo experiments were performed on mongrel cats weighing 2-5 kg. They were anesthetized with pentobarbital, secured in a stereotactic frame, and maintained on positive pressure ventilation. A femoral vein was cannulated for intravenous fluid, anesthesia and drug administration, and a femoral artery was cannulated for monitoring blood pressure. Arterial pressure, heart rate, intracranial pressure, body temperature, pO2, and pCO2 were monitored. CSF production was measured using a modification of the method of Pappenheimer et al. 14 as previously described 2. The cisterna magna was catheterized with an 18-gauge Tygon catheter which was secured with cation-activated glue. Each lateral ventricle was cannulated with an 18-gauge needle through a small drill hole in the skull. Both lateral ventricles were perfused with Elliott's B artificial CSF containing 2/~Ci/100 ml of [14C]carboxyinulin (New England Nuclear) at 0.09 ml/min/ventricle. Purified cholera toxin, 50/~g (Schwarz/Mann), prepared as previously described a, or heat-inactivated cholera toxin in a total volume of 200 #1 of saline, was administered to the animals through both ventricular cannulas. Two hours after cholera toxin challenge, the perfusion was begun and CSF formation * Present address: Department of Otolaryngology, L.S.U. School of Medicine m Shreveport, P.O. Box 33932, Shreveport, La. 71130, U.S.A.
380 5O E
i
tj
2
30 z F-
201
8 ro
Control
i
HCT
i CT
z, I
CT+I
Fig. 1. The effects of cholera toxin (CT), heat-inactivated cholera toxin (HCT), and mdomethacm (1) on cerebrosp:nal fluid production m the cat. Production rates were measured 2-3 h after administration of 50 #g of purified CT to each lateral ventricle. Indomethacin was given i.v. (30 mg/kg) and s.c. (30 mg/kg) at the time of CT challenge, and an i.v. booster dose (30 mg/kg) was given 2.5 h later * represents a significant (P < 0.001) difference from control.
monitored at 10-min intervals over a period of I h. Experimental values represented the mean of this collection period. Indomethacin dissolved in Tris buffer, pH 8.0, or a control solution containing the Tris buffer alone was injected intravenously (30 mg/kg) and subcutaneously (30 mg/kg) at the time of cholera toxin administration. At 2.5 h after the initial indomethacin administration, a booster dose (30 mg/kg) was given intravenously. In vitro studies were performed using plexuses isolated from Sprague-Dawley rats weighing 150-250 g. After anesthetizing the animals with pentobarbital (Nembutal, 100 mg/kg) and exposing the cerebral hemispheres, the rats were perfused with saline through a 22-gauge needle placed in the left ventricle in order to clear the choroid plexus of blood. Both choroid plexuses were excised and placed in cold Krebs-Ringer bicarbonate buffer which contained 0.3 M glucose and which was constantly gassed with 95 ~o 02-5 ~ COs. These choroid plexuses were then placed in 1 ml of buffer which was continuously agitated in a Dubnoff shaker both maintained at 38 °C. After a 10-min preincubation, either cholera toxin (10 /~g/ml) or heat-inactivated cholera toxin in a volume of 100 y1 was added to the incubation vessel along with either indomethacin (20 #g/ml) dissolved in 0.1 M Tris buffer, pH 8.0, or a control solution. Sixty minutes after the addition of the cholera toxin the reaction was stopped by the addition of 1 ml of cold 7 ~otrichloroacetic acid, followed immediately by homogenization with a teflon pestle. Theophylline was added to both control and treated reaction mixtures 5 mm to prior the termination of the experiment. After centrifugation at 1800 × g, the supernatant was decanted, ether washed, and heated in a hot water bath at 60 °C for 4 min. Cyclic AMP content was determined using a modification of the radloimmunoassay of Harper and Brooker a. Protein was measured by the method of Lowry et al. 1~. Radioimmunoassay materials were supplied by Schwarz/Mann, Orangeburg, N.Y. Fig. 1. shows that the intraventricular administration of cholera toxin significantly
381 TABLE I Effect of indomethacin on cholera toxin-induced cyclic AMP accumulation (pmoles cyclic AMP/mg protein)
Choroid plexuses were incubated m Krebs-Rmger bicarbonate buffer (pH 7.4) at 37 °C for 60 mm. Theophylline, when present, was added to the incubation medium 5 rain prior to the termination of the experiment. Values represent the mean ± S.E. (n). Experiment 1
Cholera toxin + theophylhne Cholera toxin + indomethacin + theophylhne Inactivated cholera toxin + indomethacm (20/~g/ml) q- theophylline Inactivated cholera toxin + theophylline
121.09 4- 12.1 (4) 82.06 4- 8.6 (4)* 17.45 4- 1.58 (4) 22.09 4- 1.43 (4)
Experiment 2
Cholera toxin Inactivated cholera toxin + indomethacin (20/~g/ml) Inactivated cholera toxin
11.624- 0.63 (4) 11.08 + 3.3 (4) 7.95 4- 0.92 (4)
* P < 0.05.
(P < 0.001) increased CSF production in the cat. The rate of CSF secretion after cholera toxin challenge was 45.4 4- 1.7 (S.E.M.)/d/min, while after administration of bovine serum albumin or heat-inactivated cholera toxin the rates of production were 22.4 4- 1.3 and 25.4 ± 2.6 bd/min respectively. When cholera toxin (CT)-treated animals were pretreated with indomethacin, the rate of production was 37.8 ± 1.8 #l/min. While this value was significantly (P < 0.025) less than the CT-treated animals, it was greater (P < 0.001) than control values and was no different from production rates when indomethacin alone was given to the animals (31.0 4- 2.9 #l/min). The addition of indomethacin and cholera toxin to the in vitro incubation medium resulted in a 33 (P < 0.05) decrease in cyclic A M P accumulation when compared with incubations containing cholera toxin alone (Table I). However, when indomethacin and cholera toxin were present in the incubation medium, cyclic A M P accumulation was still significantly (P < 0.001) greater than in controls. The combination of indomethacin and inactivated cholera toxin had no effect on cyclic A M P levels. When theophylline was not present in the incubation medium, neither cholera toxin nor indomethacin had an effect on cyclic A M P accumulation when compared with the inactivated cholera toxin control. The present studies (Fig. 1) demonstrate a two-fold increase in CSF secretion in cats after intraventricular administration of cholera toxin. While previous studies in our laboratory using dogs demonstrated a similar change in CSF production, the cats provide a better experimental model. When dogs were used there was a larger variation
382 among animals. As seen in Fig. 1, cats had small individual differences in CSF secretory rates. Furthermore, while ventricular inflammation was noted in most of the dogs studied, none was seen in the cats after histologic examination. As shown in Fig. l, cholera toxin-induced CSF secretion could be partially inhibited by indomethacin, a finding similar to that described in cholera toxin-induced fluid secretion in the gut 6,9,15. While mdomethacin decreased cholera toxin-induced CSF secretion, indomethacin alone had a stimulatory effect on CSF production (Fig. I ). Similarly, it was recently found that while cholera toxin secretion in the gut was decreased by treatment with indomethacin, indomethacin alone initiated significant absorption of fluid across the gut wall 15. One possible explanation of this paradoxical effect is the presence of multiple secretory pathways all of which are necessary for maximum cerebrospmal production. I f some pathways were stimulated by a prostaglandin while others were inhibited by another prostaglandin, the inhibition of prostaglandln synthesis by indomethacin could result in some secretion via those pathways normally inoperative in the presence of a specific inhibitory prostaglandin. That lndomethacin decreased (29 ~o) but did not block cyclic A M P accumulation in vitro in choroid plexuses isolated from the rat corresponded with the results seen in vivo. However, the present studies differ from previous work in the intestine and the kidney in which lndomethacin had no effect on cyclic A M P accumulation 1,1°,i2. Although indomethacin clearly decreased cholera toxin-induced cyclic A M P accumulation it cannot be assumed that this is the entire mechanism by which indomethacin inhibits cholera-reduced secretion since there is still greater than a three-fold rise in cyclic A M P accumulation. While the biochemical mechanisms of CSF production are unclear, these studies suggest that prostaglandins play a role in CSF secretion. Furthermore, the basic cellular mechanisms involved in CSF production appear to be similar to those seen in other secretory epithelia. The technical assistance of Fallon Maylack and Philip Lyng is gratefully acknowledged. This work was supported in part by N.I.H. Grants AM07145, NS11274, and HD00091, the Kerr Foundation, and the National Foundation March of Dimes.
1 Bourne, H. R., Cholera enterotoxin. Failure of anti-inflammatory agents to prevent cyclic AMP accumulation, Nature (Lond.), 241 (1973) 399. 2 Epstein, M. H., Feldman, A. M., and Brusllow, S. W., Cerebrospmal fluid production: stimulation by cholera toxin, Science, 196 (1977) 1012-1013. 3 Feldman, A. M. and Brusilow, S. W., Effects of cholera toxin on cochlear endolymph production. Model for endolymphatic hydrops, Proc. nat. Acad. Sci. (Wash.), 73 (1976) 1761-1764. 4 Feldman, A. M., Maylack, F., Epstein, M. and Brusilow, S. W., In vitro effects of cholera toxin on rat chorold plexus, Clin. Res., 25 (1977) 430A. 5 Feldman, A. M., Maylack, F., Epstein, M. and Brusllow, S. W., Effects of prostaglandms on rat choroid plexus in vitro, Clin. Res., 25 (1977) 430A. 6 Finch, A. D. and Katz, R. L., Prevention of cholera-reduced intestinal secretion in the cat by aspmn, Nature (Lond.), 238 (1972) 273-274. 7 Frledler, R. M, Kurokawa, K., Coburn, J. W. and Massry, S. G., Renal action of cholera toxin: I. Effects on urinary excretion of electrolyte and cyclic AMP, Kidney Int., 7 (1975) 77-85.
383 8 Harper, J. F. and Brooker, G., Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic GMP after 2'0 acetylation by acetic anhydride in aqueous solution, J. Cyclic Nucleotide Res., 1 (1975) 207-218. 9 Jacoby, H. J. and Marshal, C. H., Antagonism of cholera enterotoxin by anti-inflammatoryagents in the rat, Nature (Lond.), 235 (1972) 163-165. 10 Kimberg, D. V., Field, M. and Johnson, J., Stimulation of intestinal mucosal adenyl cyclase by cholera enterotoxin and prostaglandins, J. clin. Invest., 50 (1971) 1218-1230. 11 Kimberg, D. V., Field M., Gershon, E. and Henderson, A., Effects of prostaglandins and cholera enterotoxm on intestinal mucosal cyclic AMP accumulation. Evidence against an essential role for prostaglandins in the action of toxin, J. clin. Invest., 53 (1974) 941-949. 12 Kurokawa, K., Friedler, R. M. and Massry, S. G., Renal action of cholera toxin: II. Effects on adenylate cyclase-cyclic AMP system, Ktdney Int., 7 (1975) 137-144. 13 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 14 Pappenheimer, J. R., Helsey, S. R., Jordan, E. F. and Downer, J., Perfuslon of the cerebral ventricular system in unanesthetized goats, Amer. J. Physiol., 203 (1962) 763-774. 15 Wald, A., Gotterer, G. S., Rajendra, G. R., Turjman, N. A. and Hendrix, T. R., Effect of indomethacm on cholera-induced fluid movement, unidirectional sodium fluxes, and intestinal cAMP, Gastroenterology, 72 (1977) 106-110
