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Recently, we demonstrated a novel interaction between large-conductance (maxi-K or [K.sub.Ca]1.1) and intermediate-conductance (IK1 or [K.sub.Ca]3. l) [Ca.sup.2+]-activated K channels: activation of IK1 channels causes the inhibition of maxi-K activity (Thompson J and Begenisich T. J Gen Physiol 127: 159-169, 2006). Here we show that the interaction between these two channels can be regulated by the membrane cholesterol level in parotid acinar cells. Depletion of cholesterol using methyl-[beta]-cyclodextrin weakened, while cholesterol enrichment increased, the ability of IK1 activation to inhibit maxi-K channels. Cholesterol's stereoisomer, epicholesterol, was unable to substitute for cholesterol in the interaction between the two K channels, suggesting a specific cholesterol-protein interaction. This suggestion was strengthened by the results of experiments in which cholesterol was replaced by coprostanol and epicoprostanol. These two sterols have nearly identical effects on membrane physical properties and cholesterol-rich microdomain stability, but had very different effects on the IK1/maxi-K interaction. In addition, the IK1/maxi-K interaction was unaltered in cells lacking caveolin, the protein essential for formation and stability of caveolae. Finally, disruption of the actin cytoskeleton restored the IKl-induced maxi-K inhibition that was lost with cell cholesterol depletion, demonstrating the importance of an intact cytoskeleton for the cholesterol-dependent regulation of the IK1/maxi-K interaction. [K.sub.Ca] 1.1 ; [K.sub.Ca]3.1 ; cholesterol analogs

Transepithelial [Cl.sup.-] transport in salivary gland ducts is a major component of the ion reabsorption process, the final stage of saliva production. It was previously demonstrated that a [Cl.sup.-] current with the biophysical properties of ClC-2 channels dominates the [Cl.sup.-] conductance of unstimulated granular duct cells in the mouse submandibular gland. This inward-rectifying [Cl.sup.-] current is activated by hyperpolarization and elevated intracellular [Cl.sup.-] concentration. Here we show that ClC-2 immunolocalized to the basolateral region of acinar and duct cells in mouse salivary glands, whereas its expression was most robust in granular and striated duct cells. Consistent with this observation, nearly 10-fold larger ClC-2-1ike currents were observed in granular duct cells than the acinar cells obtained from submandibular glands. The loss of inward-rectifying [Cl.sup.-] current in cells from [Clcn2.sup.-/-] mice confirmed the molecular identity of the channel responsible for these currents as ClC-2. Nevertheless, both in vivo and ex vivo fluid secretion assays failed to identify significant changes in the ion composition, osmolality, or salivary flow rate of [Clcn2.sup.-/-] mice. Additionally, neither a compensatory increase in Cftr [Cl.sup.-] channel protein expression nor in Cftr-like [Cl.sup.-] currents were detected in Clcn2 null mice, nor did it appear that ClC-2 was important for blood-organ barrier function. We conclude that ClC-2 is the inwardrectifying [Cl.sup.-] channel in duct cells, but its expression is not apparently required for the ion reabsorption or the barrier function of salivary ductal epithelium. inward-rectifying chloride current; barrier function; NaCl absorption

Atherosclerosis remains a major cause of death in the developed world despite the success of therapies that lower cholesterol and BP. The intermediate-conductance calcium-activated potassium channel KCa3.1 is expressed in multiple cell types implicated in atherogenesis, and pharmacological blockade of this channel inhibits VSMC and lymphocyte activation in rats and mice. We found that coronary vessels from patients with coronary artery disease expressed elevated levels of KCa3.1. In Apoe-/- mice, a genetic model of atherosclerosis, KCa3.1 expression was elevated in the VSMCs, macrophages, and T lymphocytes that infiltrated atherosclerotic lesions. Selective pharmacological blockade and gene silencing of KCa3.1 suppressed proliferation, migration, and oxidative stress of human VSMCs. Furthermore, VSMC proliferation and macrophage activation were reduced in KCa3.1-/- mice. In vivo therapy with 2 KCa3.1 blockers, TRAM-34 and clotrimazole, significantly reduced the development of atherosclerosis in aortas of Apoe-/- mice by suppressing VSMC proliferation and migration into plaques, decreasing infiltration of plaques by macrophages and T lymphocytes, and reducing oxidative stress. Therapeutic concentrations of TRAM-34 in mice caused no discernible toxicity after repeated dosing and did not compromise the immune response to influenza virus. These data suggest that KCa3.1 blockers represent a promising therapeutic strategy for atherosclerosis., Introduction Atherosclerosis is the ultimate pathological consequence of vascular remodeling and is associated with the activation of VSMCs and inflammatory cells (1-3). In atherosclerosis, VSMCs switch from a contractile state [...]

The exocrine salivary glands of mammals secrete [K.sup.+] by an unknown pathway that has been associated with HC[O.sup.-.sub.3] efflux. However, the present studies found that [K.sup.+] secretion in the mouse submandibular gland did not require HC[O.sup.-.sub.3], demonstrating that neither [K.sup.+]/HC[O.sup.-.sub.3] cotransport nor [K.sup.+]/[H.sup.+] exchange mechanisms were involved. Because HC[O.sup.-.sub.3] did not appear to participate in this process, we tested whether a K channel is required. Indeed, [K.sup.+] secretion was inhibited >75% in mice with a null mutation in the maxi-K, [Ca.sup.2+]-activated K channel ([K.sub.Ca] 1.1) but was unchanged in mice lacking the intermediate-conductance [IK.sub.Ca] 1 channel ([K.sub.Ca]3.1). Moreover, paxilline, a specific maxi-K channel blocker, dramatically reduced the [K.sup.+] concentration in submandibular saliva. The [K.sup.+] concentration of saliva is well known to be flow rate dependent, the [K.sup.+] concentration increasing as the flow decreases. The flow rate dependence of [K.sup.+] secretion was nearly eliminated in [K.sub.Ca]1.1 null mice, suggesting an important role for [K.sub.Ca] 1.1 channels in this process as well. Importantly, a maxi-K-like current had not been previously detected in duct cells, the theoretical site of [K.sup.+] secretion, but we found that [K.sub.Ca] 1.1 channels localized to the apical membranes of both striated and excretory duct cells, but not granular duct cells, using immunohistochemistry. Consistent with this latter observation, maxi-K currents were not detected in granular duct cells. Taken together, these results demonstrate that the secretion of [K.sup.+] requires and is likely mediated by [K.sub.Ca] 1.1 potassium channels localized to the apical membranes of striated and excretory duct ceils in the mouse submandibular exocrine gland. salivary gland; potassium secretion; calcium-activated K channels; duct cells

Electrolyte transport by airway epithelia regulates the quantity and composition of liquid covering the airways. Previous data indicate that airway epithelia can absorb NaCl. At the apical membrane, cystic fibrosis transmembrane conductance regulator (CFTR) provides a pathway for [Cl.sup.-] absorption. However, the pathways for basolateral [Cl.sup.-] exit are not well understood. Earlier studies, predominantly in cell lines, have reported that the basolateral membrane contains a [Cl.sup.-] conductance. However, the properties have varied substantially in different epithelia. To better understand the basolateral [Cl.sup.-] conductance in airway epithelia, we studied primary cultures of well-differentiated human airway epithelia. The basolateral membrane contained a [Cl.sup.-] current that was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The current-voltage relationship was nearly linear, and the halide selectivity was [Cl.sup.-] > [Br.sup.-] [much greater than] [I.sup.-]. Several signaling pathways increased the current, including elevation of cellular levels of cAMP, activation of protein kinase C (PKC), and reduction of pH. In contrast, increasing cell [Ca.sup.2+] and inducing cell swelling had no effect. The basolateral [Cl.sup.-] current was present in both cystic fibrosis (CF) and non-CF airway epithelia. Likewise, airway epithelia from wild-type mice and mice with disrupted genes for ClC-2 or ClC-3 all showed similar [Cl.sup.-] currents. These data suggest that the basolateral membrane of airway epithelia possesses a [Cl.sup.-] conductance that is not due to CFTR, ClC-2, or ClC-3. Its regulation by cAMP and PKC signaling pathways suggests that coordinated regulation of [Cl.sup.-] conductance in both apical and basolateral membranes may be important in controlling transepithelial CI movement. chloride channel; absorption; adenosine 3',5'-cyclic monophosphate; cystic fibrosis

The strategies available for treating salivary gland hypofunction are limited because relatively little is known about the secretion process in humans. An initial microarray screen detected ion transport proteins generally accepted to be critically involved in salivation. We tested for the activity of some of these proteins, as well as for specific cell properties required to support fluid secretion. The resting membrane potential of human acinar cells was near -51 mV, while the intracellular [[Cl.sup.-]] was ~62 mM, about fourfold higher than expected if Cl ions were passively distributed. Active [Cl.sup.-] uptake mechanisms included a bumetanide-sensitive [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] cotransporter and paired DIDS-sensitive [Cl.sup.-]/HC[O.sup.-.sub.3] and EIPA-sensitive [Na.sup.+]/[H.sup.+] exchangers that correlated with expression of NKCCl, AE2, and NHE1 transcripts, respectively. Intracellular [Ca.sup.2+] stimulated a niflumic acid-sensitive [Cl.sup.-] current with properties similar to the [Ca.sup.2+]-gated Cl channel BEST2. In addition, intracellular [Ca.sup.2+] stimulated a paxilline-sensitive and voltage-dependent, large-conductance K channel and a clotrimazole-sensitive, intermediate-conductance K channel, consistent with the detection of transcripts for KCNMA1 and KCNN4, respectively. Our results demonstrate that the ion transport mechanisms in human parotid glands are equivalent to those in the mouse, confirming that animal models provide valuable systems for testing therapies to prevent salivary gland dysfunction. salivary glands; secretion; fluid; channels; exchangers; cotransporters doi:10.1152/ajpregu.00591.2006

Large volumes of saliva are generated by transepithelial [Cl.sup.-] movement during parasympathetic muscarinic receptor stimulation. To gain further insight into a major [Cl.sup.-] uptake mechanism involved in this process, we have characterized the anion exchanger (AE) activity in mouse serous parotid and mucous sublingual salivary gland acinar cells. The AE activity in acinar cells was [Na.sup.+] independent, electroneutral, and sensitive to the anion exchange inhibitor DIDS, properties consistent with the AE members of the SLC4A gene family. Localization studies using a specific antibody to the ubiquitously expressed AE2 isoform labeled acini in both parotid and sublingual glands. Western blot analysis detected an ~170-kDa protein that was more highly expressed in the plasma membranes of sublingual than in parotid glands. Correspondingly, the DIDS-sensitive [Cl.sup.-]/HC[O.sup.-.sub.3] exchanger activity was significantly greater in sublingual acinar cells. The carbonic anhydrase antagonist acetazolamide markedly inhibited, whereas muscarinic receptor stimulation enhanced, the [Cl.sup.-]/HC[O.sup.-.sub.3] exchanger activity in acinar cells from both glands. Intracellular [Ca.sup.2+] chelation prevented muscarinic receptorinduced upregulation of the AE, whereas raising the intracellular [Ca.sup.2+] concentration with the [Ca.sup.2+]-ATPase inhibitor thapsigargin mimicked the effects of muscarinic receptor stimulation. In summary, carbonic anhydrase activity was essential for regulating [Cl.sup.-]/HC[O.sup.-.sub.3] exchange in salivary gland acinar cells. Moreover, muscarinic receptor stimulation enhanced AE activity through a [Ca.sup.2+]-dependent mechanism. Such forms of regulation may play important roles in modulating fluid and electrolyte secretion by salivary gland acinar cells. secretion; carbonic anhydrase; [Cl.sup.-]/HC[O.sup.-.sub.3] exchanger; calcium regulation

Brown, David A., James E. Melvin, and David I. Yule. Critical role for NHE1 in intracellular pH regulation in pancreatic acinar cells. Am J Physiol Gastrointest Liver Physiol 285: G804-G812, 2003. First published July 3, 2003; 10.1152/ajpgi.00150.2003.--The primary function of pancreatic acinar cells is to secrete digestive enzymes together with a NaCl-rich primary fluid which is later greatly supplemented and modified by the pancreatic duct. A [Na.sup.+]/[H.sup.+] exchanger(s) [NHE(s)] is proposed to be integral in the process of fluid secretion both in terms of the transcellular flux of [Na.sup.+] and intracellular pH (p[H.sub.i]) regulation. Multiple NHE isoforms have been identified in pancreatic tissue, but little is known about their individual functions in acinar cells. The [Na.sup.+]/[H.sup.+] exchange inhibitor 5-(N-ethyl-N-isopropyl) amiloride completely blocked p[H.sub.1] recovery after an N[H.sub.4]Cl-induced acid challenge, confirming a general role for NHE in pHi regulation. The targeted disruption of the Nhe1 gene also completely abolished pHi recovery from an acid load in pancreatic acini in both HC[O.sup.-.sub.3]-containing and HC[O.sup.-.sub.3]-free solutions. In contrast, the disruption of either Nhe2 or Nhe3 had no effect on pHi recovery. In addition, NHE1 activity was upregulated in response to muscarinic stimulation in wild-type mice but not in NHEl-deficient mice. Fluctuations in pHi could potentially have major effects on [Ca.sup.2.sup.+] signaling following secretagogue stimulation; however, the targeted disruption of Nhel was found to have no significant effect on intracellular [Ca.sup.2.sup.+] homeostasis. These data demonstrate that NHE1 is the major regulator of pHi in both resting and muscarinic agonist-stimulated pancreatic acinar cells. fluid secretion; exocrine glands; intracellular pH regulation; intracellular calcium

Experimental evidence shows that Na(super +)/H(super +) exchanger (NHE) genes play important functions in parotid acinar and ductal cells in rats. This observation was confirmed in an experiment that investigated the molecular and pharmacological properties of NHE in rat parotid salivary gland cells. Results showed that NHE1 is the dominant NHE isoform in acinar cells while NHE3 is a significant contributor to pH regulation in ductal cells. In addition, NHE activity was suppressed by low concentrations of the amiloride derivative ethylisopropyl amiloride in the case of acinar cells superfused with a physiological salt solution.

Research was conducted to study the regulation of Ca2+-activated chloride channels in cells from the human colonic cell line T84 and acinar cells from rat parotid glands. Total RNA was isolated from parotid glands using TRIzol reagent while DNA was synthesized according to standard methods. Results showed that Ca2+-dependent chloride channels were influenced by calmodulin-dependent protein kinase II and that channels in parotid acinar cells were regulated by a different Ca2+-dependent mechanism.

The intercellular calcium (Ca2+)(sub i) level and membrane potential affect the activation of rat parotid acinar cells' Ca2+ -dependent chloride (Cl-) channels. The Cl- current increases with an increase in (Ca2+)(sub i) at all membrane potentials. The current is outwardly rectifying and linear, respectively, at low and high (Ca2+)(sub i) levels. The observed properties of the current are explained by a model which constrains the binding of Ca2+ to two binding sites within the membrane electric field. This produces a conformation change necessary for Cl- conductance.

Baculovirus was used to express a rat anion exchanger (AE2) in Spodoptera frugiperda (Sf9) insect cells. The tests did not show any Cl--HCO3- exchange activity in wild-type or sham-infected Sf9 cells. Anion exchange functional expression in AE2 recombinant baculovirus-infected cells were observed with in the first day after transfection and was sustained for three days. The results of the study demonstrate that the AE2 rat protein is suitable for studies of AE2 function.

The role of Na+ - K+ - 2Cl- cotransport and Na+ - H+ exchange in Na+ influx was investigated. The study protocol involved the use of dual-wavelength microfluorometry of the fluorescent Na+ indicator sodium-binding benzofuran isophthalate to determine the mechanisms of muscarinic acid-stimulated uptake of Na+ in rat sublingual mucous acini. The results showed that the muscarinic acid-induced increase in intracellular Na+ was almost exclusively mediated by Na+ - K+ -2Cl- cotransport and Na+ - H+ exchange mechanisms.

The widely expressed Anoctamin 6 (Ano6) supports different Ca2+‐dependent functions, but little is known about its role in salivary glands. Mouse submandibular gland (SMG) acinar cells exhibited a robust regulatory volume decrease (RVD) following cell swelling that was reduced approximately 70% in Ano6–/– mice. Ca2+‐free conditions nearly eliminated the RVD response suggesting that Ano6 is an obligatory component of the cell volume‐activated, Ca2+‐dependent RVD pathway in salivary gland acinar cells. Ex vivo agonist‐stimulated secretion of water and ions was unaffected by Ano6 disruption under both isotonic and hypotonic conditions suggesting that Ano6 does not play a major role in fluid and electrolyte secretion. In contrast, the total amount of β‐adrenergic‐dependent protein secretion by the SMG was significantly reduced in Ano6–/– mice. Closer inspection of these latter results revealed that protein secretion was affected only in the female SMG by Ano6 disruption. These results indicate that Ano6 modulates the RVD response and protein secretion by salivary gland acinar cells. [ABSTRACT FROM AUTHOR]

The nonselective anion exchanger Slc26a6, also known as putative anion transporter 1 and chloride/formate exchanger, is thought to play a major role in HCO3- transport in exocrine glands. In this study, Slc26a6 null mice were used to explore the function of Slc26a6 in the exocrine pancreas. Slc26a6 primarily localized to the apical membrane of pancreatic exocrine acinar cells. The volume of stimulated juice secretion by the ex vivo pancreas was significantly reduced ~35% in Slc26a6-/- mice, but no changes occurred in the gross structure or gland weights of Slc26a6 null mice. The secretion of pancreatic juice by Slc26a6+/+ mice was dependent on HCO3- while, in contrast, fluid secretion by Slc26a6-/- mice was independent of HCO3-, suggesting that Slc26a6 mediates the HCO3--dependent component of fluid secretion. Consistent with these observations, disruption of Slc26a6 also significantly reduced HCO3- secretion by the pancreas ~35%. Taken together, these results demonstrate that the apical Slc26a6 anion exchanger in acinar cells is involved in HCO3--dependent fluid secretion but that another major HCO3--independent pathway is the primary driver of the fluid secretion process in the mouse pancreas. [ABSTRACT FROM AUTHOR]

The physiological roles of individual proteins are demonstrated through the subsequent characterization of mice with null mutations in many of the genes. The recent developments in determining the molecular identifications of the proteins that regulate the fluid secretion process are focused.