Primary culture of polarized human salivary epithelial cells for use in developing an artificial salivary gland.

Therapeutic irradiation for head and neck cancer, and the autoimmune disease Sjogren's syndrome, lead to loss of salivary parenchyma. They are the two main causes of irreversible salivary gland hypofunction. Such patients cannot produce adequate levels of saliva, leading to considerable morbidity. We are working to develop an artificial salivary gland for such patients. A major problem in this endeavor has been the difficulty in obtaining a suitable autologous cellular component. This article describes a method of culturing and expanding primary salivary cells obtained from human submandibular glands (huSMGs) that is serum free and yields cells that are epithelial in nature. These include morphological (light and transmission electron microscopy [TEM]), protein expression (immunologically positive for ZO-1, claudin-1, and E-cadherin), and functional evidence. Under confocal microscopy, huSMG cells show polarization and appropriately localize tight junction proteins. TEM micrographs show an absence of dense core granules, but confirm the presence of tight and intermediate junctions and desmosomes between the cells. Functional assays showed that huSMG cells have high transepithelial electrical resistance and low rates of paracellular fluid movement. Additionally, huSMG cells show a normal karyotype without any morphological or numerical abnormalities, and most closely resemble striated and excretory duct cells in appearance. We conclude that this culture method for obtaining autologous human salivary cells should be useful in developing an artificial salivary gland.

Increased fluid secretion after adenoviral-mediated transfer of the human aquaporin-1 cDNA to irradiated miniature pig parotid glands.

The treatment of most head and neck cancer patients includes ionizing radiation (IR). Salivary glands in the IR field suffer irreversible damage. Previously, we reported that adenoviral (Ad)-mediated transfer of the human aquaporin-1 (hAQP1) cDNA to rat submandibular glands following IR restored salivary flow to near normal levels. It is unclear if this strategy is useful in larger animals. Herein, we evaluated AdhAQP1-mediated gene transfer after parotid gland IR (20 Gy) in the miniature pig. Sixteen weeks following IR, salivation from the targeted gland was decreased by >80%. AdhAQP1 administration resulted in a dose-dependent increase in parotid salivary flow to approximately 80% of pre-IR levels on day 3. A control Ad vector was without significant effect. The effective AdhAQP1 dose was 2.5 x 10(5) pfu/microl infusate, a dose that leads to comparable transgene expression in murine and minipig salivary glands. Three days after Ad vector administration little change was observed in clinical chemistry and hematology values. These findings demonstrate that localized delivery of AdhAQP1 to IR-damaged salivary glands increases salivary secretion, without significant general adverse events, in a large animal model.

Polarized sorting of aquaporins 5 and 8 in stable MDCK-II transfectants.

Localization of aquaporin (AQP) water channels to either apical or basolateral membranes is important for various epithelial functions. We have established MDCK-II cell transfectants stably expressing AQP5 (RW5 cells) or AQP8 (RW8 cells). The expression of both AQPs was confirmed by the results of immunofluorescence microscopy and immunoblot analysis. When grown on polycarbonate filters, osmotically-obliged transepithelial water flow across RW5 and RW8 monolayers was approximately 3-fold greater than that occurring across a monolayer of the parental cell line. Importantly, results of confocal immunofluorescence microscopy studies showed that AQP5 sorted to the apical membranes of RW5 cells. In contrast, AQP8 sorted to the basolateral membranes of RW8 cells. This is the first report of (i) stable epithelial cell cultures exhibiting a functional, polarized distribution of AQPs 5 and 8, and (ii) a basolateral localization of AQP8 in a polarized epithelial cell.

Cationic liposome-mediated gene transfer to rat salivary epithelial cells in vitro and in vivo.

BACKGROUND: Previously we have shown that gene transfer to salivary gland epithelial cells readily occurs via recombinant adenoviruses, although the response is short-lived and results in a potent host immune response. The aim of the present study was to assess the feasibility of using cationic liposomes to mediate gene transfer to rat salivary cells in vitro and in vivo. METHODS: Initially, for transfection in vitro, we used two cationic liposome formulations (GAP-DLRIE/DOPE and DOSPA/DOPE) complexed with plasmid encoding human growth hormone (hGH) as a reporter gene. Thereafter, using GAP-DLRIE/DOPE, plasmids were transferred to rat salivary glands in vivo, and hGH levels measured in saliva, serum and gland extracts. RESULTS: Under optimal conditions, transfection of rat submandibular glands (SMGs) was consistently observed. Approximately 95% of the cells transfected with a plasmid encoding beta-galactosidase were acinar cells. Maximal hGH expression was obtained during the first 48 h post-transfection using a plasmid encoding the hGH cDNA and complexed with GAP-DLRIE/DOPE. hGH was detected in gland extracts and saliva, and occasionally in serum. No systemic or local gland pathology was consistently or significantly observed. CONCLUSIONS: The levels of the reporter gene product, hGH, obtained after GAP-DLRIE/DOPE-mediated gene transfer are considerably lower (<0.5%) than those achieved with adenoviral vectors (10(8) PFU). Nonetheless, cationic liposome-mediated gene transfer to salivary glands may be useful for potential therapeutic applications.

Salivary glands as a model for craniofacial applications of gene transfer.

The potential applications of gene transfer technology to all branches of medicine are increasing. It is quite likely that within the next 10-20 years surgical practice routinely will utilize gene transfer, at least adjunctively. The purpose of this review is to familiarize the oral and maxillofacial surgeon with this technology. Studies performed with salivary glands in animal models are presented as examples of proof of concept.

Construction and function of a recombinant adenovirus encoding a human aquaporin 1-green fluorescent protein fusion product.

Transfer of the human aquaporin 1 (hAQP1) gene provides a novel way to potentially correct the severe salivary hypofunction associated with therapeutic radiation for head and neck cancer. To facilitate the study of individual cells transduced with this gene, we have designed a fusion product of the hAQP1 and jellyfish green fluorescent protein (GFP) cDNAs. An expression plasmid, pACCMVhAQP1GFP, and a recombinant adenovirus, AdhAQP1GFP, encoding this fusion product were constructed. Both the recombinant plasmid and virus directed the expression of the encoded, 55-kDa fusion protein (hAQP1GFP), which was detected in the plasma membranes of several epithelial cell lines (293, SMIE, and A5). hAQP1GFP was functionally active and facilitated fluid movement across a polarized salivary epithelial cell monolayer (approximately 5-fold noninfected controls) in response to an osmotic gradient. In response to a hypotonic challenge, individual epithelial cells expressing the fusion protein exhibited significantly more capacitance (used herein as an indicator of cell swelling) than control cells. Conversely, in response to a hypertonic challenge, individual infected cells shrunk more rapidly (approximately 2- to 3-fold) and to a greater extent than control cells. We conclude that AdhAQP1GFP is a useful experimental tool to identify and study individual cells expressing a water channel transgene.

Trp1, a candidate protein for the store-operated Ca(2+) influx mechanism in salivary gland cells.

The trp gene family has been proposed to encode the store-operated Ca(2+) influx (SOC) channel(s). This study examines the role of Trp1 in the SOC mechanism of salivary gland cells. htrp1, htrp3, and Trp1 were detected in the human submandibular gland cell line (HSG). HSG cells stably transfected with htrp1alpha cDNA displayed (i) a higher level of Trp1, (ii) a 3-5-fold increase in SOC (thapsigargin-stimulated Ca(2+) influx), determined by [Ca(2+)](i) and Ca(2+)-activated K(+) channel current measurements, and (iii) similar basal Ca(2+) permeability, and inhibition of SOC by Gd(3+) but not by Zn(2+), as compared with control cells. Importantly, (i) transfection of HSG cells with antisense trp1alpha cDNA decreased endogenous Trp1 level and significantly attenuated SOC, and (ii) transfection of HSG cells with htrp3 cDNA did not increase SOC. These data demonstrate an association between Trp1 and SOC and strongly suggest that Trp1 is involved in this mechanism in HSG cells. Consistent with this suggestion, Trp1 was detected in the plasma membrane region, the proposed site of SOC, of acinar and ductal cells in intact rat submandibular glands. Based on these aggregate data, we propose Trp1 as a candidate protein for the SOC mechanism in salivary gland cells.

Evidence that aquaporin-8 is located in the basolateral membrane of rat submandibular gland acinar cells.

It is possible that, during primary saliva formation, aquaporins (AQPs) facilitate transcellular water flow across acinar cells to the lumina of salivary glands. In the rat submandibular gland (rSMG) AQP5 is localized in the apical membranes of acinar cells. The presence of a basolateral AQP in the same cell type has not been reported. We have therefore used immunofluorescence confocal microscopy to determine the subcellular localization of a newly discovered aquaporin, AQP8, in rSMG epithelial cells. The antibodies we used were made against the amino- or carboxyl-terminus (anti-rAQP8NT and anti-rAQP8CT, respectively) of an AQP8 cloned from rat pancreas and liver (rAQP8). Two lines of evidence suggest that both antibodies are suitable for immunolocalization studies. First, results of immunofluorescence confocal microscopy studies show that both antibodies bind to the plasma membranes of 293 cells infected with an adenovirus encoding rAQP8. Second, results of immunoblots of membranes from infected cells suggest that both antibodies bind to glycosylated and non-glycosylated forms of rAQP8. When tested in frozen sections of rSMG, we could not detect the binding of anti-rAQP8NT to any membranes. In contrast, anti-rAQP8CT binds to the basolateral membranes of acinar (but not ductal) epithelia, suggesting that rAQP8 resides in the basolateral membranes of acinar cells. Lack of anti-rAQPNT binding to basolateral membranes suggests that this epitope is not available in the membranes. Our evidence for the basolateral localization of rAQP8 in acinar cells, coupled with previous findings that AQP5 is localized apically in the same cells, raises the possibility that water crosses the acinar epithelium through these channels during primary saliva formation.

Polarized secretion of transgene products from salivary glands in vivo.

Previously (Kagami et al. Hum. Gene Ther. 1996;7:2177-2184) we have shown that salivary glands are able to secrete a transgene-encoded protein into serum as well as saliva. This result and other published data suggest that salivary glands may be a useful target site for vectors encoding therapeutic proteins for systemic delivery. The aim of the present study was to assess in vivo if transgene-encoded secretory proteins follow distinct, polarized sorting pathways as has been shown to occur "classically" in cell biological studies in vitro. Four first-generation, E1-, type 5 recombinant adenoviruses were used to deliver different transgenes to a rat submandibular cell line in vitro or to rat submandibular glands in vivo. Subsequently, the secretory distribution of the encoded proteins was determined. Luciferase, which has no signal peptide, served as a cell-associated, negative control and was used to correct for any nonspecific secretory protein release from cells. The three remaining transgene products tested, human tissue kallikrein (hK1), human growth hormone (hGH), and human alpha1-antitrypsin (halpha1AT), were predominantly secreted (>96%) in vitro. Most importantly, in vivo, after a parasympathomimetic secretory stimulus, both hK1 and hGH were secreted primarily in an exocrine manner into saliva. Conversely, halpha1AT was predominantly secreted into the bloodstream, i.e., in an endocrine manner. The aggregate results are consistent with the recognition of signals encoded within the transgenes that result in specific patterns of polarized protein secretion from rat submandibular gland cells in vivo.

Construction and in vivo efficacy of a replication-deficient recombinant adenovirus encoding murine growth hormone.

We have constructed a recombinant, replication-deficient, first-generation adenovirus-encoding mouse GH (mGH), AdCMVmGH. This virus directed mGH production from an epithelial cell line in vitro in a dose-dependent manner. When injected into the quadriceps muscle or submandibular ducts of mGH-deficient Snell dwarf mice, AdCMVmGH resulted in the production of significantly elevated serum mGH levels. Furthermore, after i.m. injection, dwarf mice increased in weight by 8% over 4 days and close to 100% by 30 days. When AdCMVmGH was administered to 3- to 4-week-old rats by i.v. injection to assess general metabolic responses, serum mGH, insulin-like growth factor 1, triglycerides and cholesterol levels were significantly elevated. AdCMVmGH should be a valuable experimental tool for the controlled, directed expression of mGH in preclinical mouse model studies.

Systemic action of human growth hormone following adenovirus-mediated gene transfer to rat submandibular glands.

We have previously suggested that although salivary glands function in an exocrine manner they might none the less offer a useful way to deliver therapeutic proteins systemically. As a direct functional test of this hypothesis, we constructed a recombinant adenovirus (AdCMVhGH) encoding human growth hormone (hGH) and then studied the biological action of hGH produced following transfer of the hGH gene to rat submandibular glands. At 48 h following infusion of AdCMVhGH into these glands via cannulation of the main excretory duct, serum levels of hGH were approximately 16 ng/ml and rat insulin-like growth factor-1 was elevated approximately 25%. Moreover, serum chemistry profiles of rats subjected to in vivo gene transfer displayed alterations in the BUN:creatinine ratio and triglyceride levels presumably reflecting the anabolic actions of the hGH. These results provide the first demonstration of systemic biological action from a transgene product secreted in an endocrine fashion from the salivary glands.

Relationship between adenovirus-mediated aquaporin 1 expression and fluid movement across epithelial cells.

AdhAQP1, a recombinant adenovirus encoding the human water channel aquaporin 1 (AQP1), has been shown to be useful for gene therapy of salivary glands rendered hypofunctional following irradiation. Here we utilized AdhAQP1 to examine the relationship between AQP1 expression and fluid movement across a polarized salivary epithelial cell monolayer. In response to a 440 to 340 mosm gradient, net fluid movement across cells infected with AdhAQP1 was approximately 10-fold that seen in uninfected cells or cells infected with a control virus. At a multiplicity of infection (MOI) of 5, fluid movement was linear for 15-30 min. Increasing the osmotic gradient resulted in a proportional increase in fluid movement. At low virus MOIs (0.1-1.0), fluid movement was markedly enhanced relative to that seen at higher MOIs (approximately 5.10), where the level of AQP1 expression and number of cells transduced were considerably greater. We conclude that significant, osmotically-obliged fluid movement in a salivary cell monolayer with low basal water permeability does not require high levels of AQP1 expression.

In vitro fusion of endocytic vesicles: effects of reagents that alter endosomal pH.

Ricin, a plant toxin that binds to galactose-terminated glycoproteins and glycolipids on the cell surface, is internalized into endosomes before reaching the cytosol where it exerts its toxic activity. Fusion of early endosomes containing ricin or transferrin was demonstrated by using postnuclear supernatant fractions from K-562 cells. For both ligands, fusion depended on time, temperature, and ATP and was blocked by preincubation with N-ethylmaleimide. Some reagents that increase endosomal pH, the ionophores monensin and nigericin and the weak base chloroquine, stimulated the rate of fusion. However, bafilomycin A1, a specific inhibitor of vacuolar H(+)-ATPases, did not alter the rate of fusion. Moreover, it reduced or eliminated stimulation caused by monensin, nigericin, or chloroquine. Thus, the increased rate of fusion did not correlate with the higher lumenal pH of the endosome. The results suggest instead that fusion was stimulated by reagents that promoted accumulation of cations within the vesicles.

Anti-salivary antibodies in primary Sjögren's syndrome.

Earlier studies have described an antibody that recognized salivary ductal epithelium in sera from 15-50% of patients with primary Sjögren's syndrome; however, the specific salivary antigen in those studies was not identified. The present study further investigated this unknown salivary antigen. Twenty-nine of 31 patients (94%) with primary Sjögren's syndrome demonstrated IgG antinuclear antibodies that bound to an epithelial cell line with ductal characteristics derived from a human salivary gland. Seventy-seven percent of these patients had serum antibodies that bound to ductal cells of normal human parotid tissue after formalin fixation. Western blots of cell extracts, immunofluorescence, and adsorption studies indicated that SS-A/Ro and SS-B/La were the antigens recognized in the salivary cell line. The pattern of fluorescence seen when anti-SS-B/La bound to normal parotid tissue was identical to the fluorescence pattern of the anti-salivary ductal antibodies described in earlier literature.

Hypotonically activated chloride current in HSG cells.

Hypotonically induced changes in whole-cell currents and in cell volume were studied in the HSG cloned cell line using the whole-cell, patch clamp and Coulter counter techniques, respectively. Exposures to 10 to 50% hypotonic solutions induced dose-dependent increases in whole-cell conductances when measured using K+ and Cl- containing solutions. An outward current detected at 0 mV, corresponded to a K+ current which was transiently activated, (usually preceding activation of an inward current and had several characteristics in common with a Ca(2+)-activated K+ current we previously described in these cells. The hypotonically induced inward current had characteristics of a Cl- current. This current was inhibited by NPPB (5-nitro-2-(3-phenyl-propylamino)-benzoate) and SITS (4-acetamido-4'-isothiocyanostilbene), and its reversal potentials corresponded to the Cl- equilibrium potentials at high and low external Cl- concentrations. The induced current inactivated at voltages greater than +80 mV, and the I-V curve was outwardly rectifying. The current was unaffected by addition of BAPTA or removal of GTP from the patch pipette, but was inhibited by removal of ATP or by the presence of extracellular arachidonic acid, quinacrine, nordihydroguairetic acid, and cytochalasin D. Moreover, exposure of HSG cells to hypotonic media caused them to swell and then to undergo a regulatory volume decrease (RVD) response. Neither NPPB, SITS or quinine acting alone could inhibit RVD, but NPPB and quinine together totally inhibited RVD. These properties, plus the magnitudes of the induced currents, indicate that the hypotonically induced K+ and Cl- currents may underlie the RVD response. Cytochalasin D also blocked the RVD response, indicating that intact cytoskeletal F-actin may be required for activation of the present currents. Hence, our results indicate that hypotonic stress activates K+ and Cl- conductances in these cells, and that the activation pathway for the K+ conductance apparently involves [Ca2+], while the activation pathway for the Cl- conductance does not involve [Ca2+] nor lipoxygenase metabolism, but does require intact cytoskeletal F-actin.

Characterization of 3'-azido-3'-deoxythymidine inhibition of ricin and Pseudomonas exotoxin A toxicity in CHO and Vero cells.

Ricin (RIC), modeccin (MOD), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) are protein toxins that enter cells by receptor-mediated endocytosis. After intracellular transport and membrane translocation to the cytosol, these toxins inhibit protein synthesis by enzymatically removing a specific adenine residue from ribosomal RNA (RIC, MOD), or by ADP-ribosylation of elongation factor-2 (PE, DT). Recently, Thompson and Pace (1992) reported that AZT (3'-azido-3'-deoxythymidine) inhibited RIC toxicity in Vero cells, and this inhibition was not due to a block of RIC enzymatic activity. This paper extends these findings and examines the effects of AZT treatment on the toxicities of other protein toxins in Chinese hamster ovary (CHO) and Vero cell lines. AZT treatment did not significantly alter the toxicity of DT or MOD in either cell line, but it markedly reduced RIC and PE toxicity in both cell lines. The ID50 values (concentration of toxin required to inhibit protein synthesis by 50%) for RIC and PE in CHO cells increased approximately 6.5- and 12.5-fold, respectively; while in Vero cells the ID50 values increased ca. 8.5- and 4.5-fold, respectively. Results of further studies revealed differences in the mechanisms by which AZT inhibited RIC and PE toxicity. Results of cell-free translation indicated that, unlike its effects on RIC, AZT blocked the ability of PE to perform its enzymatic activity. As AZT did not block RIC enzymatic activity, we examined the effects of AZT on earlier steps in the RIC intoxication process. AZT treatment did not inhibit cell-surface binding or internalization of [125I]-RIC. Results of kinetic studies showed that when AZT was incubated with cells at the time of RIC exposure, it caused no major change in the lag phase, during which RIC reaches the site of translocation. However, it clearly reduced the subsequent first-order reduction in the rate of protein synthesis, suggesting an effect on translocation. Monensin (an ionophore that perturbs intracellular trafficking and increases the toxicities of RIC and PE) reduced AZT protection against both toxins. Nocodazole and colchicine (agents that disrupt microtubules and some routes of intracellular trafficking) reduced the ability of AZT to inhibit RIC, but not PE, toxicity. In summary, our results suggest that (1) AZT acts within the cytosol to inhibit (directly or indirectly) the enzymatic action of PE, and (2) the AZT inhibition of RIC cytotoxicity does not involve perturbations of RIC cell-surface binding, internalization, or enzymatic activity but might result from an alteration in RIC translocation.

Characteristics and regulation of a muscarinically activated K current in HSG-PA cells.

Whole cell currents were measured in HSG-PA cells (a proposed model for salivary gland duct cells) after muscarinic receptor activation or exposure to known signaling agents. Exposure to carbachol or oxotremorine M produced large and often oscillatory increases in outward current whose reversal potentials indicated a K current. The current was sensitive to extracellular atropine, charybdotoxin, and quinine, but not apamin, and to 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in the pipette. The response was prolonged or increased by guanosine 5'-O-(3-thiotriphosphate) and mimicked by D-myo-inositol 1,4,5-trisphosphate (IP3) or heparin in the pipette and by extracellular Ca ionophores. Tetraethylammonium indirectly inhibited the response via the muscarinic receptor. Fura 2 in cell suspensions showed that muscarinic agonists increased cytosolic Ca ion concentration ([Ca2+]i) five- to sevenfold, and measurements with indo 1 in individual cells showed that the oscillatory changes in outward current were tightly correlated with parallel changes in [Ca2+]i. The results indicate that muscarinic receptor stimulation of HSG-PA cells activates Ca(2+)-activated K channels through a signaling pathway involving a G protein, IP3 production, and increased [Ca2+]i levels. These findings are similar to those in salivary gland acinar cells.

The role of calcium ions for the expression of ricin toxicity in cultured macrophages.

Ricin toxin, which consists of two distinct polypeptide moieties, A and B chains, is cytotoxic to the cultured macrophage cell line, J774A.1. Ricin is a protein synthesis inhibitor, and incubating macrophages for 4 hours with ricin (1 pM to 10 nM) in standard medium containing calcium and magnesium inhibited 3H-leucine incorporation into protein (97%, at 1 nM ricin). However, in Ca(2+)-free medium, protein synthesis was inhibited only 19%. EGTA pretreatment (to deplete intracellular calcium) also partly protected cells from protein synthesis inhibition, in spite of added calcium (2 mM) in the incubation medium. Decreased toxicity in the absence of extracellular calcium resulted from decreased toxin binding. Adding or deleting Mg2+ did not affect protein synthesis or binding of 125I-ricin in cultured macrophages. We conclude that calcium is required for ricin to exert its inhibitory effect on protein synthesis in cultured macrophages.

Responsiveness of a human parotid epithelial cell line (HSY) to autonomic stimulation: muscarinic control of K+ transport.

Salivary electrolyte secretion is under the control of the autonomic nervous system. In this paper we report that HSY, an epithelial cell line derived from the acinar-intercalated duct region of the human parotid gland, responds to muscarinic-cholinergic (generation of Ca2+ signal) and beta-adrenergic (generation of cAMP signal), but not to alpha-adrenergic (lack of Ca2+ signal), receptor stimulation. The muscarinic response was studied in detail. Carbachol (10(-4) M, muscarinic agonist) or A23187 (5 microM, calcium ionophore) stimulation of HSY cells increases both 86Rb (K+) influx and efflux, resulting in no change in net equilibrium 86Rb content. Atropine (10(-5) M, muscarinic antagonist) blocks both the carbachol-generated Ca2+ signal and carbachol-stimulated 86Rb fluxes, but has no effect on either the A23187-generated Ca2+ signal or A23187-stimulated 86Rb fluxes. Carbachol- and A23187-stimulated 86Rb fluxes are substantially inhibited by two K+ channel blockers, quinine (0.3 mM) and scorpion venom containing charybdotoxin (33 micrograms/ml). The inhibition of these stimulated fluxes by another K+ channel blocker, tetraethylammonium chloride (5 mM), is less pronounced. Protein kinase C (PKC) seems to be involved in the regulation of the 86Rb fluxes as 10(-7) M PMA (phorbol ester, phorbol-12-myristate-13-acetate) substantially inhibits the muscarinic-stimulated 86Rb efflux and influx. Because this concentration of PMA totally inhibits the carbachol-generated Ca2+ signal and only 80% of the muscarinic-stimulated 86Rb influx, it seems that a portion of the carbachol-stimulated 86Rb flux (i.e. that portion not inhibited by PMA) might occur independently of the Ca2+ signal. PMA fails to inhibit the A23187-stimulated 86Rb fluxes, however, suggesting that PKC regulates Ca(2+)-sensitive K+ channel activity by regulating the Ca2+ signal, and not steps distal to this event. 4-alpha-Phorbol-12,13-didecanoate, a phorbol ester which fails to activate PKC, fails to inhibit either the carbachol-stimulated increase in intracellular free Ca2+, or carbachol-stimulated 86Rb fluxes.

N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) stimulation of K+ transport in a human salivary epithelial cell line.

Treatment of a human salivary epithelial cell line, HSG-PA, with the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7; 20-70 microM) increased 86Rb (K+) influx and efflux in a manner similar to that resulting from muscarinic (carbachol; Cch) or calcium ionophore (A23187) stimulation. Unlike the Cch or A23187 responses, the W7 responses were not blocked by 0.1 mM atropine (muscarinic antagonist) or phorbol-12-myristate-13-acetate (0.1 microM). Like Cch- or A23187-stimulated 86Rb fluxes, W7-stimulated 86Rb fluxes were substantially blocked by the K+ channel inhibitors quinine (0.25 mM) and scorpion venom-containing charybdotoxin (33 micrograms/mL), while 5 mM tetraethylammonium chloride (K+ channel blocker), furosemide (0.1 mM; Na+,K+,2Cl- co-transport inhibitor) and ouabain (10 microM; Na+,K(+)-ATPase inhibitor) were ineffective. Purified charybdotoxin (10 nM) also blocked W7-stimulated 86Rb influx, as well as 86Rb influx stimulated by Cch or A23187. Although Quin 2 fluorescence measurements indicated that W7 increased free intracellular Ca2+ concentration ([Ca2+]i), the magnitude of the increase appeared to be insufficient to solely account for the W7-stimulated increases in 86Rb fluxes (i.e. K+ channel activity). Ca2+ was involved in the W7 response, however, as lack of Ca2+ in the incubation medium reduced the W7-stimulated increases in 86Rb influx and efflux. Taken together, our results suggest that W7 increased K+ fluxes in HSG-PA cells by interacting, directly or indirectly, with the K+ transport machinery (K+ channels) in a manner different from that observed during muscarinic stimulation, and also in a manner not accounted for solely by the formation of a typical muscarinic- or calcium ionophore-generated calcium signal.