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, an apical plasma membrane (APM) water channel in salivary glands, lacrimal glands, and airway epithelium, has an important role in fluid secretion. M 3 muscarinic acetylcholine receptor (mAChR)-induced changes in AQP5 localization in rat parotid glands were investigated with immunofluorescence or immunoelectron microscopy, detergent solubility, and gradient density floatation assays. Confocal microscopy revealed AQP5 localization in intracellular vesicles of interlobular duct cells in rat parotid glands and AQP5 trafficking to the APM 10 min after injection of the mAChR agonist cevimeline. Conversely, 60 min after injection, there was a diffuse pattern of AQP5 staining in the cell cytoplasm. The calcium ionophore A-23187 mimicked the effects of cevimeline. Immunoelectron microscopic studies confirmed that cevimeline induced AQP5 trafficking from intracellular structures to APMs in the interlobular duct cells of rat parotid glands. Lipid raft markers flotillin-2 and GM1 colocalized with AQP5 and moved with AQP5 in response to cevimeline. Under control conditions, the majority of AQP5 localized in the Triton X-100-insoluble fraction and floated to the light-density fraction on discontinuous density gradients. After 10-min incubation of parotid tissue slices with cevimeline or A-23187, AQP5 levels decreased in the Triton X-100-insoluble fraction and increased in the Triton X-100-soluble fraction. Thus AQP5 localizes in the intracellular lipid rafts, and M 3 mAChR activation induces AQP5 trafficking to the APM with lipid rafts via intracellular Ca 2ϩ signaling and induces AQP5 dissociation from lipid rafts to nonrafts on the APM in the interlobular duct cells of rat parotid glands.translocation; aquaporin-5 AQUAPORINS (AQPs) form water channels that selectively transport water across the plasma membrane (19). Thirteen mammalian AQPs, AQP0 -AQP12, have been identified (1, 27). AQP5, initially cloned from rat submandibular glands (32), is an apical membrane water channel that is distributed to epithelial cells in several secretory glands, such as salivary glands (10). Salivary fluid secretion is defective in transgenic mice lacking AQP5, indicating that AQP5 has an important role in fluid secretion (24).The parotid glands are innervated by both sympathetic and parasympathetic nerves (2). The activation of M 3 muscarinic acetylcholine receptors (mAChRs) and ␣ 1 -adrenoceptors increases intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) and induces salivary fluid secretion (2). In vitro experiments using rat parotid slices demonstrated that ACh and epinephrine acting at M 3 mAChRs and ␣ 1 -adrenoceptors, respectively, induce a rapid increase in the AQP5 levels in the apical plasma membrane (APM) by increasing [Ca 2ϩ ] i (14, 15). We previously investigated (16) the possible role of Ca 2ϩ -mediated intracellular signal transduction in the M 3 mAChR agonist-induced increase in AQP5 levels in the APM and demonstrated that activation of endogenous nitric oxide synthase and protein kinase G in the cells is coupled with t...

It has been demonstrated that gene transfer by in vivo electroporation of mouse muscle increases the level of gene expression by more than 100-fold over simple plasmid DNA injection. We tested continuous rat erythropoietin (Epo) delivery by this method in normal rats, using plasmid DNA expressing rat Epo (pCAGGS-Epo) as the vector. A pair of electrodes was inserted into the thigh muscles of rat hind limbs and 100 microg of pCAGGS-Epo was injected between the electrodes. Eight 100-V, 50-msec electric pulses were delivered through the electrodes. Each rat was injected with a total of 400 microg of pCAGGS-Epo, which was delivered to the medial and lateral sides of each thigh. The presence of vector-derived Epo mRNA at the DNA injection site was confirmed by RT-PCR. The serum Epo levels peaked at 122.2 +/- 33.0 mU/ml on day 7 and gradually decreased to 35.9 +/- 18.2 mU/ml on day 32. The hematocrit levels increased continuously, from the preinjection level of 49.5 +/- 1.1 to 67.8 +/- 2.2% on day 32 (p < 0.001). In pCAGGS-Epo treated rats, endogenous Epo secretion was downregulated on day 32. In a control experiment, intramuscular injection of pCAGGS-Epo without subsequent electroporation did not significantly enhance the serum Epo levels. These results demonstrate that muscle-targeted pCAGGS-Epo transfer by in vivo electroporation is a useful procedure for the continuous delivery of Epo.

Transmission electron microscopy (TEM) and x-ray microanalysis (XMA) were used for the study of the ultrastructure of the lumens of dentinal tubules in superficial layers of dentin specimens obtained by use of a new biopsy technique from both hypersensitive and naturally desensitized areas of exposed root surfaces, in vivo. The TEM images showed clearly that the lumens of most of the tubules were occluded with mineral crystals in naturally desensitized areas, but such lumens were empty and surrounded with peritubular and intertubular dentin in hypersensitive areas. Moreover, electron-dense structures that lined peritubular dentin were observed in the empty lumens of dentinal tubules.