Fluid transport is a major function of the gastrointestinal (GI) tract with more than 9 litres of fluid being absorbed or secreted across epithelia in human salivary gland, stomach, the hepatobiliary tract, pancreas, small intestine and colon. This review evaluates the evidence that aquaporin-type water channels are involved in GI fluid transport. The aquaporins are a family of small (•30 kDa) integral membrane proteins that function as water channels. At least seven aquaporins are expressed in various tissues in the GI tract: AQP1 in intrahepatic cholangiocytes, AQP4 in gastric parietal cells, AQP3 and AQP4 in colonic surface epithelium, AQP5 in salivary gland, AQP7 in small intestine, AQP8 in liver, pancreas and colon, and AQP9 in liver. There are functional data suggesting that some GI cell types expressing aquaporins have high or regulated water permeability; however, there has been no direct evidence for a role of aquaporins in GI physiology. Recently, transgenic mice have been generated with selective deletions of various aquaporins. Preliminary evaluation of GI function suggests a role for AQP1 in dietary fat processing and AQP4 in colonic fluid absorption. Further study of aquaporin function in the GI tract should provide new insights into normal GI physiology and disease mechanisms, and may yield novel therapies to regulate fluid movement in GI diseases.

Secretory diarrhea is the leading cause of infant death in developing countries and a major cause of morbidity in adults. The cystic fibrosis transmembrane conductance regulator (CFTR) protein is required for fluid secretion in the intestine and airways and, when defective, causes the lethal genetic disease cystic fibrosis. We screened 50,000 chemically diverse compounds for inhibition of cAMP/flavone-stimulated Cl-transport in epithelial cells expressing CFTR. Six CFTR inhibitors of the 2-thioxo-4-thiazolidinone chemical class were identified. The most potent compound discovered by screening of structural analogs, CFTRinh-172, reversibly inhibited CFTR short-circuit current in less than 2 minutes in a voltage-independent manner with KI approximately 300nM. CFTRinh-172 was nontoxic at high concentrations in cell culture and mouse models. At concentrations fully inhibiting CFTR, CFTRinh-172 did not prevent elevation of cellular cAMP or inhibit non-CFTR Cl-channels, multidrug resistance protein-1 (MDR-1), ATP-sensitive K+ channels, or a series of other transporters. A single intraperitoneal injection of CFTRinh-172 (250

Water channel aquaporin-1 (AQP1) is strongly expressed in kidney in proximal tubule and descending limb of Henle epithelia and in vasa recta endothelia. The grossly normal phenotype in human subjects deficient in AQP1 (Colton null blood group) and in AQP4 knockout mice has suggested that aquaporins (other than the vasopressin-regulated water channel AQP2) may not be important in mammalian physiology. We have generated transgenic mice lacking detectable AQP1 by targeted gene disruption. In kidney proximal tubule membrane vesicles from knockout mice, osmotic water permeability was reduced 8-fold compared with vesicles from wild-type mice. Although the knockout mice were grossly normal in terms of survival, physical appearance, and organ morphology, they became severely dehydrated and lethargic after water deprivation for 36h. Body weight decreased by 35 6 2%, serum osmolality increased to>500mOsm, and urinary osmolality (657 659 mOsm) did not change from that before water deprivation. In contrast, wild-type and heterozygous mice remained active after water deprivation, body weight decreased by 20-22%, serum osmolality remained normal (310-330mOsm), and urine osmolality rose to>2500mOsm. Urine [Na1] in water-deprived knockout mice was<10mM, and urine osmolality was not increased by the V2 agonist DDAVP. The results suggest that AQP1 knockout mice are unable to create a hypertonic medullary interstitium by countercurrent multiplication. AQP1 is thus required for the formation of a concentrated urine by the kidney.

Aquaporin-3 (AQP3) is a water channel expressed at the basolateral plasma membrane of kidney collecting-duct epithelial cells. The mouse AQP3 cDNA was isolated and encodes a 292-amino acid wateryglycerol-transporting glycoprotein expressed in kidney, large airways, eye, urinary bladder, skin, and gastrointestinal tract. The mouse AQP3 gene was analyzed, and AQP3 null mice were generated by targeted gene disruption. The growth and phenotype of AQP3 null mice were grossly normal except for polyuria. AQP3 deletion had little effect on AQP1 or AQP4 protein expression but decreased AQP2 protein expression particularly in renal cortex. Fluid consumption in AQP3 null mice was more than 10-fold greater than that in wild-type litter mates, and urine osmolality (<275 milliosmol) was much lower than in wild-type mice (>1,200 milliosmol). After 1-desamino-8-D-arginine-vasopressin administration or water deprivation, the AQP3 null mice were able to concentrate their urine partially to 30% of that in wild-type mice. Osmotic water permeability of cortical collecting-duct basolateral membrane, measured by a spatial filtering optics method, was >3-fold reduced by AQP3 deletion. To test the hypothesis that the residual concentrating ability of AQP3 null mice was due to the inner medullary collecting-duct water channel AQP4, AQP3yAQP4 double-knockout mice were generated. The double-knockout mice had greater impairment of urinary-concentrating ability than did the AQP3 single-knockout mice. Our findings establish a form of nephrogenic diabetes insipidus produced by impaired water permeability in collecting-duct basolateral membrane. Basolateral membrane aquaporins may thus provide blood-accessible targets for drug discovery of aquaretic inhibitors.

Deletion of Phe-508 (F508) is the most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) causing cystic fibrosis. △F508-CFTR has defects in both channel gating and endoplasmic reticulum-to-plasma membrane processing. We identified six novel classes of high affinity potentiators of defective F508-CFTR Cl-channel gating by screening 100,000 diverse small molecules. Compounds were added 15min prior to assay of iodide uptake in epithelial cells co-expressing F508-CFTR and a high sensitivity halide indicator (YFP-H148Q/I152L) in which F508-CFTR was targeted to the plasma membrane by culture at 27℃ for 24h. Thirty-two compounds with submicromolar activating potency were identified; most had tetrahydrobenzothiophene, benzofuran, pyramidinetrione, dihydropyridine, and anthraquinone core structures (360-480 daltons). Further screening of>1000 structural analogs revealed tetrahydrobenzothiophenes that activated F508-CFTR Cl- conductance reversibly with Kd<100nM. Single-cell voltage clamp analysis showed characteristic CFTR currents after F508-CFTR activation. Activation required low concentrations of a cAMP agonist, thus mimicking the normal physiological response. A Bayesian computational model was developed using tetrahydrobenzothiophene structure-activity data, yielding insight into the physical character and structural features of active and inactive potentiators and successfully predicting the activity of structural analogs. Efficient potentiation of defective F508-CFTR gating was also demonstrated in human bronchial epithelial cells from a F508 cystic fibrosis subject after 27℃ temperature rescue. In conjunction with correctors of defective F508-CFTR processing, small molecule potentiators of defective F508-CFTR gating may be useful for therapy of cystic fibrosis caused by the F508 mutation.