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To investigate potential physiological interactions between the transcellular and paracellular pathways of water transport, we asked whether targeted deletion of Aquaporin 5 (AQP5), the major transcellular water transporter in salivary acinar cells, affected paracellular transport of 4-kDa FITC-labeled dextran (FITC-D), which is transported through the paracellular but not the transcellular route. After i.v. injection of FITC-D into either AQP5 wild-type or AQP5-/- mice and saliva collection for fixed time intervals, we show that the relative amount of FITC-D transported in the saliva of AQP5-/- mice is half that in matched AQP5+/+ mice, indicating a 2-fold decrease in permeability of the paracellular barrier in mice lacking AQP5. We also found a significant difference in the proportion of transcellular vs. paracellular transport between male and female mice. Freeze-fracture electron microscopy revealed an increase in the number of tight junction strands of both AQP5+/+ and AQP5-/- male mice after pilocarpine stimulation but no change in strand number in female mice. Average acinar cell volume was increased by [approximately equal to] 1.4-fold in glands from AQP5-/- mice, suggesting an alteration in the volumesensing machinery of the cell. Western blots revealed that expression of Claudin-7, Claudin-3, and Occludin, critical proteins that regulate the permeability of the tight junction barrier, were significantly decreased in AQP5-/- compared with AQP5+/+ salivary glands. These findings reveal the existence of a genderinfluenced molecular mechanism involving AQP5 that allows transcellular and paracellular routes of water transport to act in conjunction. epithelium | fluid absorption and secretion

Activity of the epithelial Na+ channel (ENaC) is limiting for [Na.sup.+] reabsorption at the distal nephron. Gain-of-function mutations in ENaC cause Liddle's syndrome: a severe form of inheritable hypertension. Several polymorphisms in [alpha]-hENaC possibly associated with abnormal Na+ handling by the kidney and the salt-sensitive hypertension prevalent in black populations have been reported. The functional effects of [alpha]-hENaC polymorphisms on channel activity, however, remain controversial and have not been directly tested in a mammalian background. We ask here whether polymorphisms at positions 334, 618, and 663 in [alpha]-hENaC influence channel activity. Activity of wild-type (A334, C618, A663) and polymorphic ENaC expressed in Chinese hamster ovary cells was assessed with patch-clamp electrophysiology. While the A334T polymorphism had little effect on macroscopic ENaC currents, the C618F and A663T polymorphisms significantly increased ENaC activity >3.3- and 1.6-fold, respectively. Similarly, polymorphic ENaC had greater activity compared with wild-type channels in excised patches with activity of C618F and A663T channels increased 3.8- and 2.6-fold, respectively. Unitary channel conductances and reversal potentials were not different for polymorphic and wild-type ENaC. Increases in activity resulted primarily from increases in the apparent number of active (polymorphic) channels in the plasma membrane. Moreover, addition of a reducing agent to the cytosol significantly increased activity of wild-type ENaC equal to that of C618F polymorphic channels but had no effect on these latter channels. These results are consistent with the C618F and A663T polymorphisms leading to elevated ENaC activity with the possibility that they facilitate altered [Na.sup.+] handling by the kidney. hypertension; Liddle's syndrome; sequence variations

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 iodine/starch. 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.

Although aquaporin 5 (AQP5) is the major water channel expressed in alveolar type I cells in the lung, its actual role in the lung is a matter of considerable speculation. By using immunohistochemical staining, we show that AQP5 expression in mouse lung is not restricted to type I cells, but is also detected in alveolar type II cells, and in tracheal and bronchial epithelium. Aqp5 knockout (Aqp[5.sup.-/-]) mice were used to analyze AQP5 function in pulmonary physiology. Compared with Aqp[5.sup.+/+] mice, Aqp[5.sup.-/-] mice show a significantly increased concentration-dependent bronchoconstriction to intravenously administered Ach, as shown by an increase in total lung resistance and a decrease in dynamic lung compliance (P < 0.05). Likewise, Penh, a measure of bronchoconstriction, was significantly enhanced in Aqp[5.sup.-/-] mice challenged with aerosolized methacholine (P < 0.05). The hyperreactivity to bronchoconstriction observed in the Aqp[5.sup.-/-] mice was not due to differences in tracheal smooth muscle contractility in isolated preparations or to altered levels of surfactant protein B. These data suggest a novel pathway by which AQP5 influences bronchoconstriction. This observation is of special interest because studies to identify genetic loci involved in airway hyperresponsiveness associated with asthma bracket genetic intervals on human chromosome 12q and mouse chromosome 15, which contain the Aqp5 gene.

Evidence that lymphangiomyomatosis (LAM) is caused by TSC2 (ital) mutations has been found but genetic transmission of LAM has not been reported. On chromosome 16p13 loss of heterozygosity has been found in angiomyolipomas and lymph nodes of women with LAM, a rare disease which involves diffuse proliferation of smooth muscle cells and can only be treated by transplanting lungs. LAM may or may not be associated with tuberous sclerosis complex (TSC), an autosomal dominant tumor suppressor gene syndrome. Loss of heterozygosity is found in the chromosomal region for the TSC2 (ital) gene in 60% of tuberous sclerosis complex (TSC)-associated angiomyolipomas. It seems pathogenesis of sporadic LAM may be related to TSC2 (ital). Women with LAM may have low-penetrance germ-line TSC2 (ital) mutations or may be mosaic, having TSC2 (ital) mutations in the kidney and lungs and not in other organs.

We previously reported the presence of a novel variant ([Beta]-T594M) of the amiloride-sensitive [Na.sup.+] channel (ASSC) in which the threonine residue at position 594 in the [Beta]-subunit has been replaced by a methionine residue. Electrophysiological studies of the ASSC on Epstein-Barr virus (EBV)-transformed lymphocytes carrying this variant showed that the 8-(4-chlorophenyithio) adenosine 3[prime]:5[prime]-cyclic monophosphate (8cpt-cAMP)-induced responses were enhanced when compared to wild-type EBV-transformed lymphocytes. Furthermore, in wild-type EBV-transformed cells, the 8cpt-cAMP-induced response was totally blocked by the phorbol ester, phorbol 12-myristate 13-acetate (PMA). This inhibitory effect of PMA was blocked by a protein kinase C inhibitor, chelerythrine. We now have identified individuals who are homozygous for this variant, and showed that PMA had no effect on the 8cpt-cAMP-induced responses in the EBV-transformed lymphocytes from such individuals. Cells heterozygous for this variant showed mixed responses to PMA, with the majority of cells partially inhibited by PMA. Our results demonstrate that an alteration in a single amino acid residue in the [Beta]-subunit of the ASSC can lead to a total loss of inhibition to PMA, and establish the [Beta]-subunit as having an important role in conferring a regulatory effect on the ASSC of lymphocytes.

The identification of a candidate gene for neurofibromatosis 2 (NF2), a dominantly inherited disorder characterized by the occurrence of bilateral schwannomas and other central nervous system tumors such as multiple meningiomas, is reported. Previous studies have suggested the inactivation of a tumor suppressor gene in choromosome 22q12 as a potential cause of the disorder. The candidate NF2 gene encodes a 587-amino acid protein which is related to the membrane-organizing extension spike protein-ezrin-radixin family of proteins that link cytoskeletal proteins with proteins in the cell membrane.

Physical Mapping of a Translocation Breakpoint in Neurofibromatosis VON RECKLINGHAUSEN NEUROFI-bromatosis (NF1) is an autosomaldominant human genetic disease, characterized by cafe-au-lait spots, multiple neurofibromas that increase in size and number [...]