Aquaporins (AQPs) are channel proteins that regulate the movement of water through the plasma membrane of secretory and absorptive cells in response to osmotic gradients. In the salivary gland, AQP5 is the major aquaporin expressed on the apical membrane of acinar cells. Previous studies have shown that the volume of saliva secreted by AQP5-deficient mice is decreased, indicating a role for AQP5 in saliva secretion; however, the mechanism by which AQP5 regulates water transport in salivary acinar cells remains to be determined. Here we show that the decreased salivary flow rate and increased tonicity of the saliva secreted by Aqp5 ؊/؊ mice in response to pilocarpine stimulation are not caused by changes in whole body fluid homeostasis, indicated by similar blood gas and electrolyte concentrations in urine and blood in wild-type and AQP5-deficient mice. In contrast, the water permeability in parotid and sublingual acinar cells isolated from Aqp5 ؊/؊ mice is decreased significantly. Water permeability decreased by 65% in parotid and 77% in sublingual acinar cells from Aqp5 ؊/؊ mice in response to hypertonicity-induced cell shrinkage and hypotonicity-induced cell swelling. These data show that AQP5 is the major pathway for regulating the water permeability in acinar cells, a critical property of the plasma membrane which determines the flow rate and ionic composition of secreted saliva.The precise regulation of water and electrolyte transport in the acinar cells of the salivary gland is crucial for proper production of saliva. The fluid component of salivary secretions hydrates the oral cavity, aiding in the mastication and swallowing of food, in the neutralization of acids, and in protection against the invasion of potential pathogens. Clinically, salivary gland hypofunction commonly presents as xerostomia, a symptomatic complaint of dry mouth prevalent in the geriatric population (for review, see Ref. 1) which may result from either systemic or extrinsic causes (for review, see Refs. 1-3).Saliva formation is a two-stage process (4, 5). First, the acinar cells secrete an isotonic plasma-like fluid, and second, ductal cells modify the acinar secretions primarily through the reabsorption of Na ϩ and Cl Ϫ so that the final saliva is hypotonic. This fluid secretion model predicts that saliva formation is primarily caused by transepithelial Cl Ϫ transport and that Cl Ϫ uptake is dependent on an inwardly directed Na ϩ chemical gradient across the basolateral plasma membrane. An increase in intracellular Ca 2ϩ , usually associated with muscarinic receptor stimulation, triggers fluid secretion by simultaneously activating apical Cl Ϫ channels and basolateral K ϩ channels. The efflux of Cl Ϫ and K ϩ across the apical and basolateral membranes, respectively, produces a transepithelial potential difference that is neutralized by paracellular Na ϩ transport across tight junctions. The resulting transepithelial osmotic gradient drives the movement of water, creating a plasma-like primary secretion.In salivary gland acinar cell...

The distribution and function of aquaporins (AQPs) have not previously been defined in sweat glands. In this study, AQP1, AQP3, and AQP5 mRNA were demonstrated in rat paw by reverse transcription (RT)-PCR, but AQP2 and AQP4 were not. AQP1, AQP3, and AQP5 protein were confirmed in these tissues by immunoblotting. AQP1 was identified in capillary endothelial cells by immunohistochemical labeling, but not in sweat glands or epidermis. Abundant AQP3 expression was seen in basal levels of epidermis, but not in sweat glands. AQP2 and AQP4 were not observed in either skin or sweat glands. Immunohistochemical labeling revealed abundant AQP5 in secretory parts of rat and mouse sweat glands, where immunoelectron microscopy demonstrated abundant AQP5 labeling in the apical plasma membrane. AQP5 immunolabeling of human sweat glands yielded a similar pattern. To establish the role of AQP5 in sweat secretion, we tested the response of adult mice to s.c. injection of pilocarpine, as visualized by reaction of secreted amylase with iodine͞starch. The number of active sweat glands was dramatically reduced in AQP5-null (؊͞؊) mice compared with heterozygous (؉͞؊) and wild-type (؉͞؉) mice. We conclude that the presence of AQP5 in plasma membranes of sweat glands is essential for secretion, providing potential insight into mechanisms underlying mammalian thermoregulation, tactile sensitivity, and the pathophysiology of hyperhidrosis.

Intratracheal infection of mice with adenovirus is associated with subsequent pulmonary inflammation and edema. Water movement through the air space-capillary barrier in the distal lung is facilitated by aquaporins (AQPs). To investigate the possibility that distal lung AQPs undergo altered regulation under conditions of aberrant fluid handling in the lung, we analyzed messenger RNA (mRNA) and protein expression of AQPs 1 and 5 in the lungs of mice 7 and 14 d after infection with adenovirus. Here, we demonstrate that AQP1 and AQP5 show decreased expression following adenoviral infection. Northern blot analysis showed significantly decreased mRNA levels of AQP1, which is expressed in the capillary endothelium, and AQP5, which is expressed in alveolar epithelium, in the lungs of mice both 7 and 14 d after infection. Immunoblotting studies demonstrated significantly reduced levels of AQP1 and AQP5 protein after infection as well. In addition, mRNA expression of the alpha subunit of the epithelial sodium channel was reduced in the lungs of mice 7 and 14 d after adenoviral infection. In contrast, mRNA expression of the alpha1 subunit of the Na,K-adenosine triphosphatase in the lung was unaltered. Immunohistochemical analysis demonstrated that the decreases in AQP1 and AQP5 expression were not localized to regions of overt inflammation but were found throughout the lung. Thus, this study provides the first report of AQP gene regulation in an in vivo model of pulmonary inflammation and edema. Decreased AQP1 and AQP5 levels during adenoviral infection suggest a role for AQP1 and AQP5 in the abnormal fluid fluxes detected during pulmonary inflammation.

Physiological and pharmacological studies have demonstrated that extracellular ATP, acting through P2Y(2) purinoceptor, modulates water permeability of renal medullary collecting duct cells and the secretion of ions, mucin, and surfactant phospholipids by respiratory epithelia. Here we provide direct molecular evidence for the expression of P2Y(2) purinoceptor in these cells. RT-PCR confirmed P2Y(2) purinoceptor mRNA expression in rat lung and kidney and demonstrated expression in renal collecting ducts. Northern analysis showed that both lung and kidney express one 3.6-kb P2Y(2) purinoceptor mRNA transcript. Immunoblots using peptide-derived polyclonal antibody to P2Y(2) purinoceptor showed that inner medullary collecting ducts (IMCD) express two distinct and specific products (47 and 105 kDa) and account for the majority of the receptor expression in inner medulla, whereas the 105-kDa form is predominant in lung. Immunoperoxidase labeling on cryosections showed localization of receptor protein in the apical and basolateral domains of IMCD principal cells and in the secretory cells (Clara cells and goblet cells) of the terminal respiratory bronchioles.

Cope's gray tree frog Hyla chrysoscelis accumulates glycerol during cold acclimation. We hypothesized that, during this process, gray tree frogs adjust renal filtration and/or reabsorption rates to retain accumulated glycerol. During cold acclimation, plasma concentrations of glycerol rose >200-fold, to 51 mmol/l. Although fractional water reabsorption decreased, glomerular filtration rate (GFR) and, consequently, urine flow were <5% of warm levels, and fractional glycerol reabsorption increased. In contrast, dehydrated frogs increased fractional water reabsorption, decreased GFR, and did not accumulate glycerol. We hypothesized that expression of proteins from the aquaporin (AQP)/glyceroporin (GLP) family was associated with changing patterns of water and glycerol movement. We cloned the cDNA for three such proteins, quantified mRNA expression in nine tissues using real-time quantitative PCR, and functionally characterized them using a Xenopus oocyte expression system. HC-1, an AQP1-like water channel conferring low glycerol permeability, is expressed ubiquitously in warm- and cold-acclimated tissues. HC-2, a water channel most similar to AQP2, is primarily expressed in organs of osmoregulation. HC-3, which is most similar to AQP3, is functionally characterized as a GLP, with low permeability to water but high permeability to glycerol. Aspects of expression levels and functional characteristics varied between cold and warm conditions for each of the three AQPs, suggesting a complex pattern of involvement in osmoregulation related to thermal acclimation.

The time course of the onset of nephrogenic diabetes insipidus and its relationship to aquaporin-2 (AQP2) expression in K(+) deprivation (KD) remains unknown. Rats were fed a K(+)-free diet and killed after 12 h, 1, 2, 3, 6, or 21 days. Serum K(+) concentration was decreased only after, but not before, 3 days of a K(+)-free diet. Urine osmolality, however, decreased as early as 12 h of KD (1,061 +/- 26 vs. 1,487 +/- 102 mosmol/kgH(2)O in control, P < 0.01). It decreased further at 24 h (to 858 +/- 162 mosmol/kgH(2)O in KD, P < 0.004) and remained low at 21 days of KD (436 +/- 58 mosmol/kgH(2)O, P < 0.0001 compared with baseline). Water intake decreased at 12 h (P < 0.002) but increased at 24 h (P < 0.05) and remained elevated at 21 days of KD. Urine volume increased at 24 h of KD (8 +/- 2 to 15 +/- 2 ml/24 h, P < 0.05) and remained elevated at 21 days. Immunoblot analysis demonstrated that AQP2 protein abundance in the outer medulla remained unchanged at 12 h (P > 0.05), decreased at 24 h ( approximately 44%, P < 0.001), and remained suppressed ( approximately 52%, P < 0.03) at 21 days of KD. In the inner medulla the AQP2 protein abundance remained unchanged at both 12 and 24 h of KD. AQP2 protein abundance in the cortex, however, decreased at 12 h ( approximately 47%, P < 0.01) and remained suppressed at 24 h ( approximately 77%, P < 0.001) of KD. Northern blot analysis showed that AQP2 mRNA decreased as early as 12 h of KD in both cortex (P < 0.02) and outer medulla (P < 0.01) and remained suppressed afterward. In conclusion, the urinary concentrating defect in KD is an early event and precedes the onset of hypokalemia. These studies further suggest that the very early urinary concentrating defect in KD (after 12 but before 24 h) results primarily from the suppression of cortical AQP2, whereas the later onset of a urinary concentrating defect (after 24 h) also involves a downregulation of medullary AQP2.

Aquaporin 5 (AQP5), the major water channel expressed in alveolar, tracheal, and upper bronchial epithelium, is significantly down-regulated during pulmonary inflammation and edema. The mechanisms that underlie this decrease in AQP5 levels are therefore of considerable interest. Here we show that AQP5 expression in cultured lung epithelial cells is decreased 2-fold at the mRNA level and 10-fold at the protein level by the proinflammatory cytokine tumor necrosis factor ␣ (TNF-␣). Treatment of murine lung epithelial cells (MLE-12) with TNF-␣ results in a concentration-and time-dependent decrease in AQP5 mRNA and protein expression. Activation of the p55 TNF-␣ receptor (TNFR1) with an agonist antibody is sufficient to cause decreased AQP5 expression, demonstrating that the TNF-␣ effect is mediated through TNFR1. Inhibition of nuclear factor B (NF-B) translocation to the nucleus blocks the effect of TNF-␣ on AQP5 expression, indicating that activation of NF-B is required, whereas inhibition of extracellular signal-regulated or p38 mitogenactivated protein kinases showed no effect. These data show that TNF-␣ decreases AQP5 mRNA and protein expression and that the molecular pathway for this effect involves TNFR1 and activated NF-B. The ability of inflammatory cytokines to decrease aquaporin expression may help explain the connection between inflammation and edema. Aquaporins (AQPs)1 are water channel proteins that function to increase plasma membrane water permeability in secretory and absorptive cells in response to osmotic gradients (1). Aquaporins are found in tissues where the rapid and regulated transport of fluid is necessary such as in the kidney, salivary glands, and lung. Deficiency of AQPs results in human diseases such as nephrogenic diabetes insipidus due to mutations in AQP2 and cataract formation from AQP0 mutations (2, 3). AQP5 is a mammalian water channel expressed on alveolar type I and II cells, tracheal and bronchial epithelium in the lung, in salivary and lacrimal gland epithelia, and in corneal epithelium (4, 5).2 Mice that are deficient in AQP5 have decreased production of saliva as well as altered saliva composition (6).3 In addition, AQP5 knockout mice have a 90% decrease in airspace-capillary water permeability (7). These studies demonstrate the importance of AQP5 under normal conditions in both the salivary gland and lung.Several AQPs have recently been demonstrated to undergo complex regulation; for instance, AQP2 is regulated in response to vasopressin both at the transcriptional and post-translational levels as well as through shuttling of the protein to the membrane (8). In addition, multiple AQPs are regulated under pathophysiological conditions such as altered expression of AQP1, AQP2, AQP3, and AQP4 in the kidney in a number of water balance disorders (9). Recently, through intratracheal infection of mice with adenovirus (10, 11), Towne et al. (12) showed that AQP5 mRNA and protein expression are decreased in a mouse model of pulmonary inflammation and edema. The decreased expression...

CP-induced polyuria in rats is associated with a significant decrease in the expression of collecting duct (AQP2 and AQP3) and proximal nephron and microvascular (AQP1) water channels in the inner medulla.