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Once excess liquid gains access to air spaces of an injured lung, the act of breathing creates and destroys foam and thereby contributes to the wounding of epithelial cells by interfacial stress. Since cells are not elastic continua, but rather complex network structures composed of solid as well as liquid elements, we hypothesize that plasma membrane (PM) wounding is preceded by a phase separation, which results in blebbing. We postulate that interventions such as a hypertonic treatment increase adhesive PM-cytoskeletal (CSK) interactions, thereby preventing blebbing as well as PM wounds. We formed PM tethers in alveolar epithelial cells and fibroblasts and measured their retractive force as readout of PM-CSK adhesive interactions using optical tweezers. A 50-mOsm increase in media osmolarity consistently increased the tether retractive force in epithelial cells but lowered it in fibroblasts. The osmo-response was abolished by pretreatment with latrunculin, cytochalasin D, and calcium chelation. Epithelial cells and fibroblasts were exposed to interfacial stress in a microchannel, and the fraction of wounded cells were measured. Interventions that increased PM-CSK adhesive interactions prevented blebbing and were cytoprotective regardless of cell type. Finally, we exposed ex vivo perfused rat lungs to injurious mechanical ventilation and showed that hypertonic conditioning reduced the number of wounded subpleural alveolus resident cells to baseline levels. Our observations support the hypothesis that PM-CSK adhesive interactions are important determinants of the cellular response to deforming stress and pave the way for preclinical efficacy trials of hypertonic treatment in experimental models of acute lung injury. alveolar epithelial cell; acute lung injury; interfacial stress; osmotic response; cytoprotection doi: 10.1152/ajplung.00217.2010.

Tubular reabsorption of sulfate is achieved by the sodium-dependent sulfate transporter, NaSi-1, located at the apical membrane, and the sulfate-anion exchanger, sat-l, located at the basolateral membrane. To delineate the physiological role of rat sat-l, [[sup.35]S]sulfate and [[sup.14]C]oxalate uptake into sat-l-expressing oocytes was determined under various experimental conditions. Influx of [[sup.35]S]sulfate was inhibited by bicarbonate, thiosulfate, sulfite, and oxalate, but not by sulfamate and sulfide, in a competitive manner with Ki values of 2.7 [+ or -] 1.3 mM, 101.7 [+ or -] 9.7 [micro]M, 53.8 [+ or -] 10.9 [micro]M, and 63.5 [+ or -] 38.7 [micro]M, respectively. Vice versa, [[sup.14]C]oxalate uptake was inhibited by sulfate with a Ki of 85.9 [+ or -] 9.5 [micro]M. The competitive type of inhibition indicates that these compounds are most likely substrates of sat-1. Physiological plasma bicarbonate concentrations (25 mM) reduced sulfate and oxalate uptake by more than 75%. Simultaneous application of sulfate, bicarbonate, and oxalate abolished sulfate as well as oxalate uptake. These data and electrophysiological studies using a two-electrode voltage-clamp device provide evidence that sat-1 preferentially works as an electroneutral sulfate-bicarbonate or oxalatebicarbonate exchanger. In kidney proximal tubule cells, sat-1 likely completes sulfate reabsorption from the ultrafiltrate across the basolateral membrane in exchange for bicarbonate. In hepatocytes, oxalate extrusion is most probably mediated either by an exchange for sulfate or bicarbonate. tubular reabsorption; basolateral membrane

The vast majority (~99%) of all known cases of autosomal dominant polycystic kidney disease (ADPKD) are caused by naturally occurring mutations in two separate, but genetically interacting, loci, pkd1 and pkd2. pkd1 encodes a large multispanning membrane protein (PKD1) of unknown function, while pkd2 encodes a protein (TRPP2, polycystin-2, or PKD2) of the transient receptor potential (TRP) superfamily of ion channels. Biochemical, functional, and genetic studies support a model in which PKD 1 physically interacts with TRPP2 to form an ion channel complex that conveys extracellular stimuli to ionic currents. However, the molecular identity of these extracellular stimuli remains elusive. Functional studies in cell culture show that TRPP2 can be activated in response to mechanical cues (fluid shear stress) and/or receptor tyrosine kinase (RTK) and G protein-coupled receptor (GPCR) activation at the cell surface. Recent genetic studies in Chlamydomonas reinhardtii show that CrPKD2 functions in a pathway linking cell-cell adhesion and [Ca.sup.2+] signaling. The mode of activation depends on protein-protein interactions with other channel subunits and auxiliary proteins. Therefore, understanding the mechanisms underlying the molecular makeup of TRPP2-containing complexes is critical in delineating the mechanisms of TRPP2 activation and, most importantly, the mechanisms by which naturally occurring mutations in pkd1 or pkd2 lead not only to ADPKD, but also to other defects reported in model organisms lacking functional TRPP2. This review focuses on the molecular assembly, function, and regulation of TRPP2 as a cell surface cation channel and discusses its potential role in [Ca.sup.2+] signaling and ADPKD pathophysiology. TRP channels; autosomal dominant polycystic kidney disease; [Ca.sup.2+] signaling

Recent studies on the [P2X.sub.7] receptor in 2BH4 cells and peritoneal macrophages have demonstrated that the raise in intracellular [Ca.sup.2+] concentration induces a pore opening similar to [P2X.sub.7] receptor pore. Herein, we have investigated whether the pore activated by the elevation of intracellular [Ca.sup.2+] concentration is associated to [P2X.sub.7] receptor. Using patch clamp in cell attached, whole cell configuration, and dye uptake, we measured the pore opening in cell types that express the [P2X.sub.7] receptor (2BH4 cells and peritoneal macrophages) and in cells that do not express this receptor (HEK-293 and IT45-RI cells). In 2BH4 cells, the stimulation with ionomycin (5-10 [micro]M) increased intracellular free [Ca.sup.2+] concentration and induced pore formation with conductance of 421 [+ or -] 14 pS, half-time (t 1/2) for ethidium bromide uptake of 118 [+ or -] 17 s, and t 1/2 for Lucifer yellow of 122 [+ or -] 11 s. [P2X.sub.7] receptor antagonists did not block these effects. Stimulation of HEK-293 and IT45-RI cells resulted in pore formation with properties similar to those found for 2BH4 cells. Connexin hemichannel inhibitors (carbenoxolone and heptanol) also did not inhibit the pore-induced effect following the increase in intracellular [Ca.sup.2+] concentration. However, 5-(N,N-hexamethylene)-amiloride, a [P2X.sub.7] receptor pore blocker, inhibited the induced pore. Moreover, intracellular signaling modulators, such as calmodulin, phospholipase C, mitogen-activated protein kinase, and cytoskeleton components were important for the pore formation. Additionally, we confirmed the results obtained for electrophysiology by using the flow cytometry, and we discarded the possibility of cellular death induced by raising intracellular [Ca.sup.2+] at the doses used by using lactate dehydrogenase release assay. In conclusion, increased concentration in intracellular [Ca.sup.+2] induces a novel membrane pore pharmacologically different from the [P2X.sub.7] associated pore and hemigap-junction pore. [P2X.sub.7] receptor; pore formation; second messenger

The transport systems involved in intestinal methionine (Met) absorption are described as [Na.sup.+]-dependent and [Na.sup.+]-independent mechanisms. However, since recent studies have suggested the importance of the [H.sup.+] gradient as a driving force for intestinal nutrient absorption, the aim of the present work was to test whether Met transport across the apical membrane of Caco-2 cells is affected by extracellular pH. The results show that Land D-Met uptake was increased by lowering extracellular pH from 7.4 to 5.5, in both the presence and absence of [Na.sup.+]. Cis-inhibition experiments revealed that inhibition of L-Met transport by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) or L-lysine (L-Lys) was higher at a pH of 5.5. Moreover, the BCH-insensitive component was not affected by pH, whereas the L-Lys-insensitive component was increased by lowering extracellular pH, thus suggesting the participation of system L. The contribution of another mechanism, sensitive to both BCH and L-Lys, was also considered. The inhibition obtained with taurine (Tau) was also higher at a pH of 5.5, thus suggesting the involvement of system [B.sup.0,+] on pH-stimulated component. As for D-Met uptake, the results showed higher inhibition with L-Lys and Tau at a pH of 5.5 and no effect on the L-Lys- or Tau-insensitive component. In conclusion, Met transport across the apical membrane of Caco-2 cells is increased by low extracellular pH as the result of the stimulation of two transport systems functionally identified with systems L and [B.sup.0,+] for L-Met and with system [B.sup.0,+] for D-Met. intestine; amino acid transport; [H.sup.+] gradient

Epithelial sodium channels (ENaC) play an essential role in maintaining total body fluid and electrolyte homeostasis. As such, abnormal expression of ENaC at the cell surface is linked to several important human diseases. Although the stability of ENaC subunits has been extensively studied by protein biochemical analysis, the half-life of the functional channel in the apical membrane remains controversial. Because the functional stability of the multisubunit channel may be more physiologically relevant than the stability of individual subunit proteins, we performed studies of functional ENaC channels using A6 epithelial cells, a Xenopus laevis distal nephron cell line. We recorded single-channel activity in over 400 cells with the translation blockers cycloheximide (CHX) or puromycin, as well as the intracellular protein trafficking inhibitors brefeldin A (BFA) or nocodazole. Our cell-attached, single-channel recordings allow us to quantify the channel density in the apical membrane, as well as to determine channel open probability (Po) from control (untreated) cells and from cells at different times of drug treatment. The data suggest that the half-life of ENaC channels is ~3.5 h following puromycin, BFA, and nocodazole treatment. Furthermore, these three drugs had no significant effect on the Po of ENaC for at least 6 h after exposure. A decrease in apical channel number and Po was observed following 2 h of CHX inhibition of protein synthesis, and the apparent channel half-life was closer to 1.5 h following CHX treatment. Treatment of cells with the translation inhibitors does not alter the expression of the protease furin, and therefore changes in protease activity cannot explain changes in ENaC Po. Confocal images show that BFA and nocodazole both disrupt most of the Golgi apparatus after 1-h exposure. In cells with the Golgi totally disrupted by overnight exposure to BFA, 20% of apical ENaC channels remained functional. This result suggests that ENaC is delivered to the apical membrane via a pathway that might bypass the Golgi vesicular trafficking pathway, or that there might be two pools of channels with markedly different half-lives in the apical membrane. cell-attached patch clamp; brefeldin-A; ENaC half-life

The UT-A 1 and UT-A3 urea transporters are expressed in the terminal inner medullary collecting duct (IMCD) and play an important role in the production of concentrated urine. We showed that both hyperosmolarity and vasopressin increase urea permeability in perfused rat terminal IMCDs and that UT-A1 and UT-A3 accumulate in the plasma membrane in response to vasopressin. In this study, we investigated whether hyperosmolarity causes UT-A1 and/or UT-A3 to accumulate in the plasma membrane or represents a complimentary stimulatory pathway. Rat IMCD suspensions were incubated in 450 vs. 900 mosM solutions. We biotinylated the IMCD surface proteins, collected, and analyzed them. Membrane accumulation was assessed by Western blotting of the biotinylated protein pool probed with anti-UT-A1 or anti-UT-A3. We studied the effect of NaCl, urea, and sucrose as osmotic agents. Membrane-associated UT-A1 and UT-A3 increased relative to control levels when either NaCl (UT-A1 increased 37 [+ or -] 6%; UT-A3 increased 46 [+ or -] 13%) or sucrose (UT-A1 increased 81 [+ or -] 13%; UT-A3 increased 60 [+ or -] 8%) was used to increase osmolarity. There was no increase in membrane UT-A1 or UT-A3 when urea was added. Analogously, UT-A1 phosphorylation was increased in NaCl- and sucrose- but not in urea-based hyperosmolar solutions. Hypertonicity also increased UT-A3 phosphorylation. We conclude that the increase in the urea permeability in response to hyperosmolarity reflects both UT-A1 and UT-A3 movement to the plasma membrane and may be a direct response to tonicity. Furthermore, this movement is accompanied by, and may require, increased phosphorylation in response to hypertonicity. renal; osmolality; concentrating mechanism; trafficking

Impairment of the normal spatiotemporal pattern of intracellular [Ca.sup.2+] ([[[Ca.sup.2+]].sub.i]) signaling, and in particular, the transition to an irreversible '[Ca.sup.2+] overload' response, has been implicated in various pathophysiological states. In some diseases, including pancreatitis, oxidative stress has been suggested to mediate this [Ca.sup.2+] overload and the associated cell injury. We have previously demonstrated that oxidative stress with hydrogen peroxide ([H.sub.2][O.sub.2]) evokes a [Ca.sup.2+] overload response and inhibition of plasma membrane [Ca.sup.2+]-ATPase (PMCA) in rat pancreatic acinar cells (Bruce JI and Elliott AC. Am J Physiol Cell Physiol 293: C938-C950, 2007). The aim of the present study was to further examine this oxidant-impaired inhibition of the PMCA, focusing on the role of the mitochondria. Using a [[Ca.sup.2+]]I clearance assay in which mitochondrial [Ca.sup.2+] uptake was blocked with Ru-360, [H.sub.2][O.sub.2] (50[micro]M-1 mM) markedly inhibited the PMCA activity. This [H.sub.2][O.sub.2]-induced inhibition of the PMCA correlated with mitochondrial depolarization (assessed using tetramethylrhodamine methylester fluorescence) but could occur without significant ATP depletion (assessed using Magnesium Green fluorescence). The [H.sub.2][O.sub.2]-induced PMCA inhibition was sensitive to the mitochondrial permeability transition pore (mPTP) inhibitors, cyclosporin-A and bongkrekic acid. These data suggest that oxidant-induced opening of the mPTP and mitochondrial depolarization may lead to an inhibition of the PMCA that is independent of mitochondrial [Ca.sup.2+] handling and ATP depletion, and we speculate that this may involve the release of a mitochondrial factor. Such a phenomenon may be responsible for the [Ca.sup.2+] overload response, and for the transition between apoptotic and necrotic cell death thought to be important in many disease states. calcium overload; oxidative stress; pancreatitis

Stromal interaction molecule 1 (STIM1) and Orail have been identified as crucial elements of the store-operated [Ca.sup.2+] entry (SOCE) pathway, but the mechanism of their functional interaction remains controversial. It is now well established that, upon depletion of the stores, both molecules can accumulate and colocalize in specific areas (puncta) where the endoplasmic reticulum comes in close proximity to the plasma membrane. Some models propose a direct interaction between STIMI and Orail as the most straightforward mechanism for signal transduction from the stores to the plasma membrane. To test some of the predictions of a conformational coupling model, we assessed how tight the relationships are between STIM1 and Orail expression, puncta formation, and SOCE activation. Here we present evidence that STIM1 accumulates in puncta equally well in the presence or absence of Orail expression, that STIM1 accumulation is not sufficient for Orail accumulation in the same areas, and that normal [Ca.sup.2+] release-activated [Ca.sup.2+] current ([I.sub.CRAC]) can be activated in STIMl-deficient cells. These data challenge the idea of direct conformational coupling between STIM 1 and Orai 1 as a viable mechanism of puncta formation and SOCE activation and uncover greater complexity in their relationship, which may require additional intermediate elements. store-operated [Ca.sup.2+] entry

Endothelial nitric oxide (NO) synthase (eNOS) is thought to regulate microvascular permeability via NO production. We tested the hypotheses that the expression of eNOS and eNOS endocytosis by caveolae are fundamental for appropriate signaling mechanisms in inflammatory endothelial permeability to macromolecules. We used bovine coronary postcapillary venular endothelial cells (CVECs) because these cells are derived from the microvascular segment responsible for the transport of macromolecules in inflammation. We stimulated CVECs with platelet-activating factor (PAF) at 100 nM and measured eNOS phosphorylation, NO production, and CVEC monolayer permeability to FITC-dextran 70 KDa (Dx-70). PAF translocated eNOS from plasma membrane to cytosol, induced changes in the phosphorylation state of the enzyme, and increased NO production from 4.3 [+ or -] 3.8 to 467 [+ or -] 22.6 nM. PAF elevated CVEC monolayer permeability to FITC-Dx-70 from 3.4 [+ or -] 0.3 x [10.sup.-6] to 8.5 [+ or -] 0.4 x [10.sup.-6] cm/s. The depletion of endogenous eNOS with small interfering RNA abolished PAF-induced hyperpermeability, demonstrating that the expression of eNOS is required for inflammatory hyperpermeability responses. The inhibition of the caveolar internalization by blocking caveolar scission using transfection of dynamin dominant-negative mutant, dyn2K44A, inhibited PAF-induced hyperpermeability to FITC-Dx-70. We interpret these data as evidence that 1) eNOS is required for hyperpermeability to macromolecules and 2) the internalization of eNOS via caveolae is an important mechanism in the regulation of endothelial permeability. We advance the novel concept that eNOS internalization to cytosol is a signaling mechanism for the onset of microvascular hyperpermeability in inflammation. endothelial nitric oxide; endothelial cells; microvascular permeability; endothelial nitric oxide synthase translocation; acetylcholine; platelet-activating factor

Decreasing muscle phosphagen content through dietary administration of the creatine analog [beta]-guanidinopropionic acid ([beta]-GPA) improves skeletal muscle oxidative capacity and resistance to fatigue during aerobic exercise in rodents, similar to that observed with endurance training. Surprisingly, the effect of [beta]-GPA on muscle substrate metabolism has been relatively unexamined, with only a few reports of increased muscle GLUT4 content and insulin-stimulated glucose uptake/clearance in rodent muscle. The effect of chronically decreasing muscle phophagen content on muscle fatty acid (FA) metabolism (transport, oxidation, esterification) is virtually unknown. The purpose of the present study was to examine changes in muscle substrate metabolism in response to 8 wk feeding of [beta]-GPA. Consistent with other reports, [beta]-GPA feeding decreased muscle ATP and total creatine content by ~50 and 90%, respectively. This decline in energy charge was associated with simultaneous increases in both glucose (GLUT4; +33 to 45%, P < 0.01) and FA (FAT/CD36; +28 to 33%, P < 0.05) transporters in the sarcolemma of red and white muscle. Accordingly, we also observed significant increases in insulin-stimulated glucose transport (+47%, P < 0.05) and AICAR-stimulated palmitate oxidation (+77%, P < 0.01) in the soleus muscle of [beta]-GPA-fed animals. Phosphorylation of AMPK (+20%, P < 0.05), but not total protein, was significantly increased in both fiber types in response to muscle phosphagen reduction. Thus the content of sarcolemmal transporters for both of the major energy substrates for muscle increased in response to a reduced energy charge. Increased phosphorylation of AMPK may be one of the triggers for this response. [beta]-guanidinopropionic acid; rat; skeletal muscle; energy charge; substrate metabolism

In osmoregulating teleost fish, urea is a minor nitrogen excretory product, whereas in osmoconforming marine elasmobranchs it serves as the major tissue organic solute and is retained at relatively high concentrations (~400 mmol/1). We tested the hypothesis that urea transport across liver mitochondria is carrier mediated in both teleost and elasmobranch fishes. Intact liver mitochondria in rainbow trout (Oncorhynchus mykiss) demonstrated two components of urea uptake, a linear component at high concentrations and a phloretin-sensitive saturable component [Michaelis constant (Kin) = 0.58 retool/l; maximal velocity ([V.sub.max]) = 0.12 [micro]mol x [h.sup.-1] x mg [protein.sup.-1] at lower urea concentrations ( phloretin; trifluoromethoxyphenylhydrazone; nigericin; valinomycin

[Ca.sup.+] release-activated [Ca.sup.2+] (CRAC) channels are activated when free [Ca.sup.2+] concentration in the intracellular stores is substantially reduced and mediate sustained [Ca.sup.2+] entry. Recent studies have identified Orail as a CRAC channel subunit. Here we demonstrate that passive [Ca.sup.2+] store depletion using the inhibitor of the sarcoendoplasmic reticulum [Ca.sup.2+]-ATPase, thapsigargin (TG), enhances the surface expression of Orail, a process that depends on rises in cytosolic free [Ca.sup.2+] concentration, as demonstrated in cells loaded with dimethyl BAPTA, an intracellular [Ca.sup.2+] chelator that prevented TG-evoked cytosolic free [Ca.sup.2+] concentration elevation. Similar results were observed with a low concentration of carbachol. Cleavage of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor, synaptosomal-assiciated protein-25 (SNAP-25), with botulinum neurotoxin A impaired TG-induced increase in the surface expression of Orail. In addition, SNAP-25 cleaving by botulinum neurotoxin A reduces the maintenance but not the initial stages of store-operated [Ca.sup.2+] entry. In aggregate, these findings demonstrate that store depletion enhances Orail plasma membrane expression in an exocytotic manner that involves SNAP-25, a process that contributes to store-dependent [Ca.sup.2+] entry. Orail; synaptosomal-associated protein-25; [Ca.sup.2+] entry

Blebbistatin is a myosin II-specific inhibitor. However, the mechanism and tissue specificity of the drug are not well understood. Blebbistatin blocked the chemotaxis of vascular smooth muscle cells (VSMCs) toward sphingosylphosphorylcholine (I[C.sub.50] = 26.1 [+ or -] 0.2 and 27.5 [+ or -] 0.5 [micro]M for GbaSM-4 and A7r5 cells, respectively) and platelet-derived growth factor BB (I[C.sub.50] = 32.3 [+ or -] 0.9 and 31.6 [+ or -] 1.3 [micro]M for GbaSM-4 and A7r5 cells, respectively) at similar concentrations. Immunofluorescence and fluorescent resonance energy transfer analysis indicated a blebbistatin-induced disruption of the actin-myosin interaction in VSMCs. Subsequent experiments indicated that blebbistatin inhibited the [Mg.sup.2+]-ATPase activity of the unphosphorylated (I[C.sub.50] = 12.6 [+ or -] 1.6 and 4.3 [+ or -] 0.5 [micro]M for gizzard and bovine stomach, respectively) and phosphorylated (I[C.sub.50] = 15.0 [+ or -] 0.6 [micro]M for gizzard) forms of purified smooth muscle myosin II, suggesting a direct effect on myosin II motor activity. It was further observed that the [Mg.sup.2+]-ATPase activities of gizzard myosin II fragments, heavy meromyosin (I[C.sub.50] = 14.4 [+ or -] 1.6 [micro]M) and subfragment 1 (I[C.sub.50] = 5.5 [+ or -] 0.4 [micro]M), were also inhibited by blebbistatin. Assay by in vitro motility indicated that the inhibitory effect of blebbistatin was reversible. Electron-microscopic evaluation showed that blebbistatin induced a distinct conformational change (i.e., swelling) of the myosin II head. The results suggest that the site of blebbistatin action is within the S1 portion of smooth muscle myosin II. Boyden chamber; fluorescence resonance energy transfer; ATPase; in vitro motility assay; electron microscopy

Impedance measurements in whole lenses showed that lens fiber cells possess different permeability properties to the epithelial cells from which they differentiate. To confirm these observations at the cellular level, we analyzed the membrane properties of fiber cells isolated in the presence of the nonselective cation channel inhibitor [Gd.sup.3+]. Isolated fiber cells were viable in physiological [[Ca.sup.2+]] and exhibited a range of lengths that reflected their stage of differentiation. Analysis of a large population of fiber cells revealed a subgroup of cells whose conductivity matched values measured in the whole lens (1). In this group of cells, membrane resistance, conductivity, and reversal potential all varied with cell length, suggesting that the process of differentiation is associated with a change in the membrane properties of fiber cells. Using pharmacology and ion substitution experiments, we showed that newly differentiated fiber cells (< 150 [micro] m) contained variable combinations of [Ba.sup.2+]-and tetraethylammonium-sensitive [K.sup.+] currents. Longer fiber cells (150-650 [micro] m) were dominated by a lyotropic anion conductance, which also appears to plays a role in the intact lens. Longer cells also exhibited a low-level, nonselective conductance that was eliminated by the replacement of extracellular [Na.sup.+] with N-methyl-D-glucamine, indicating that the lens contains both [Gd3.sup.+]-sensitive and -insensitive nonselective cation conductances. Fiber cell differentiation is therefore associated with a shift in membrane permeability from a dominant [K.sup.+] conductance(s) toward larger contributions from anion and nonselective cation conductances as fiber cells elongate. electrophysiology; potassium channel; anion channel; nonselective cation channel

Proton-coupled folate transporter/heine carrier protein 1 (PCFT/HCP1) has recently been identified as a transporter that mediates the translocation of folates across the cellular membrane by a proton-coupled mechanism and suggested to be the possible molecular entity of the carrier-mediated intestinal folate transport system. To further clarify its role in intestinal folate transport, we examined the functional characteristics of rat PCFT/ HCP1 (rPCFT/HCP1) expressed in Xenopus laevis oocytes and compared with those of the carrier-mediated folate transport system in the rat small intestine evaluated by using the everted tissue sacs. rPCFT/ HCP1 was demonstrated to transport folate and methotrexate more efficiently at lower acidic pH and, as evaluated at pH 5.5, with smaller Michaelis constant ([K.sub.m]) for the former (2.4 [micro]M) than for the latter (5.7 [micro]M), indicating its characteristic as a proton-coupled folate transporter that favors folate than methotrexate as substrate, rPCFT/ HCP1-mediated folate transport was found to be inhibited by several but limited anionic compounds, such as sulfobromophthalein and sulfasalazine. All these characteristics of rPCFT/HCP1 were in agreement with those of carrier-mediated intestinal folate transport system, of which the [K.sub.m] values were 1.2 and 5.8 [micro]M for folate and methotrexate, respectively, in the rat small intestine. Furthermore, the distribution profile of the folate transport system activity along the intestinal tract was in agreement with that of rPCFT/HCP1 mRNA. This study is the first to clone rPCFT/HCP1, and we successfully provided several lines of evidence that indicate its role as the molecular entity of the intestinal folate transport system. methotrexate; proton-coupled folate transporter; heme carrier protein; everted sac; oocyte expression system

We developed a two-dimensional model of transport-dependent intracellular calcium signaling in endothelial cells (ECs). Our purpose was to evaluate the effects of spatial colocalization of endothelial nitric oxide synthase (eNOS) and capacitative calcium entry (CCE) channels in caveolae on eNOS activation in response to ATP. Caveolae are specialized microdomains of the plasma membrane that contain a variety of signaling molecules to optimize their interactions and regulate their activity. In ECs, these molecules include CCE channels and eNOS. To achieve a quantitative understanding of the mechanisms of microdomain calcium signaling and the preferential sensitivity of eNOS to calcium entering the cell through CCE channels, we constructed a mathematical model incorporating the cell morphology and cellular physiological processes. The model predicts that the spatial segregation of calcium channels in ECs can create transport-dependent sharp gradients in calcium concentration within the cell. The calcium concentration gradient is affected by channel density and cell geometry. This transport-dependent calcium signaling specificity effect is enhanced in ECs by increasing the spatial segregation of the caveolar signaling domains. Our simulation significantly advances the understanding of how [Ca.sup.2+], despite its many potential actions, can mediate selective activation of signaling pathways. We show that diffusion-limited calcium transport allows functional compartmentalization of signaling pathways based on the spatial arrangements of [Ca.sup.2+] sources and targets. capacitative calcium entry; caveolae; endothelial cells: endothelial nitric oxide synthase; mathematical model

Five secretory carrier membrane proteins (SCAMP-1, -2, -3, -4, and -5) have been characterized in mammalian cells. Previously, SCAMP-1 and -2 have been implicated to function in exocytosis. RNA inhibitor-mediated deficiency of one or both of these SCAMPs interferes with dense core vesicle (DCV) exocytosis in neuroendocrine PC12 cells as detected by amperometry. Knockdowns of these SCAMPs each decreased the number and frequency of depolarization-induced exocytotic events. SCAMP-2 but not SCAMP-1 depletion also delayed the onset of exocytosis. Both knockdowns, however, altered fusion pore dynamics, increasing rapid pore closure and decreasing pore dilation. In contrast, knockdowns of SCAMP-3 and -5 only interfered with the frequency of fusion pore opening and did not affect the dynamics of newly opened pores. None of the knockdowns noticeably affected upstream events, including the distribution of DCVs near the plasma membrane and calcium signaling kinetics, although norepinephrine uptake/storage was moderately decreased by deficiency of SCAMP-1 and -5. Thus, SCAMP-1 and -2 are most closely linked to the final events of exocytosis. Other SCAMPs collaborate in regulating fusion sites, but the roles of individual isoforms appear at least partially distinct. neuroendocrine secretion; membrane fusion; amperometry

In the epididymis, low luminal bicarbonate and acidic pH maintain sperm quiescent during maturation and storage. The vacuolar [H.sup.+]-ATPase (V-ATPase) in epididymal clear cells plays a major role in luminal acidification. We have shown previously that cAMP, luminal alkaline pH, and activation of the bicarbonate-regulated soluble adenylyl cyclase (sAC) induce V-ATPase apical accumulation in these cells, thereby stimulating proton secretion into the epididymal lumen. Here we examined whether protein kinase A (PKA) is involved in this response. Confocal immunofluorescence labeling on rat epididymis perfused in vivo showed that at luminal acidic pH (6.5), V-ATPase was distributed between short apical microvilli and subapical endosomes. The specific PKA activator [N.sup.6]-monobutyryl-3'-5'-cyclic monophosphate (6-MB-cAMP, 1 mM) induced elongation of apical microvilli and accumulation of V-ATPase in these structures. The PKA inhibitor myristoylated-PKI (mPKI, 10 [micro]M) inhibited the apical accumulation of V-ATPase induced by 6-MB-cAMP. Perfusion at pH 6.5 with 8-(4-chlorophenylthio)-2-0-methyl-cAMP (8CPT-2-0-MecAMP; 10 p,M), an activator of the exchange protein activated by cAMP (Epac), did not induce V-ATPase apical accumulation. When applied at a higher concentration (100 [micro]M), 8CPT-2-0-Me-cAMP induced V-ATPase apical accumulation, but this effect was completely inhibited by mPKI, suggesting crossover effects on the PKA pathway with this compound at high concentrations. Importantly, the physiologically relevant alkaline pH-induced apical V-ATPase accumulation was completely inhibited by pretreatment with mPKI. We conclude that direct stimulation of PKA activity by cAMP is necessary and sufficient for the alkaline pH-indueed accumulation of V-ATPase in clear cell apical microvilli. vas deferens; Epac; protein kinase inhibitor

The canonical transient receptor potential channels TRPC3 and TRPC6 are abundantly expressed along with the water channel aquaporin-2 (AQP2) in principal cells of the cortical and medullary collecting duct. Although TRPC3 is selectively localized to the apical membrane and TRPC6 is found in both the apical and basolateral domains, immunofluorescence is often observed in the cytoplasm, suggesting that TRPC3 and TRPC6 may exist in intracellular vesicles and may shuttle to and from the membrane in response to receptor stimulation. To test this hypothesis, the effect of arginine-vasopressin (AVP) on the subcellular distribution of TRPC3, TRPC6, and AQP2 was examined in the rat kidney and in cultured cell lines from the cortical (M1) and inner medullary (IMCD-3) collecting duct. Immunofluorescence analysis revealed that TRPC3, but not TRPC6, colocalized with AQP2 in intracellular vesicles. AVP caused the insertion and accumulation of TRPC3 and AQP2 in the apical membrane but had no effect on the subcellular distribution of TRPC6. TRPC3, but not TRPC6, coimmunoprecipitared with AQP2 from both medulla and M1 and IMCD-3 cell lysates. Apical-to-basolateral transepithelia1 [sup.45][Ca.sup.2+] flux in polarized IMCD-3 cell monolayers was stimulated by diacylglycerol analogs or by the purinergic receptor agonist ATP but not by thapsigargin. Stimulated [sup.45][Ca.sup.2+] flux was increased by overexpression of TRPC3 and attenuated by a dominant-negative TRPC3 construct. Furthermore, [sup.45][Ca.sup.2+] flux was greatly reduced by the pyrazole-derivative BTP2, a known inhibitor of TRPC3 channels. These results demonstrate that 1) TRPC3 and TRPC6 exist in different vesicle populations, 2) TRPC3 physically associates with APQ2 and shuttles to the apical membrane in response to AVP, and 3) TRPC3 is responsible for transepithelial [Ca.sup.2+] flux in principal cells of the renal collecting duct. ion channels; renal nephron; immunoprecipitation; immunofluorescence; subcellular localization; [Ca.sup.2+] flux

Caffeine-activated, large-conductance, nonselective cation channels (LCCs) have been found in the plasma membrane of isolated cardiac myocytes in several species. However, little is known about the effects of opening these channels. To examine such effects and to further understand the caffeine-activation mechanism, we carried out studies using wholecell patch-clamp techniques with freshly isolated cardiac myocytes from rats and mice. Unlike previous studies, thapsigargin was used so that both the effect of opening LCCs and the action of caffeine were independent of [Ca.sup.2+] release from intracellular stores. These [Ca.sup.2+]-permeable LCCs were found in a majority of the cells from atria and ventricles, with a conductance of ~370 pS in rat atria. Caffeine and all its direct metabolic products (theophylline, theobromine, and paraxanthine) activated the channel, while isocaffeine did not. Although they share some similarities with ryanodine receptors (RyRs, the openings of which give rise to [Ca.sup.2+] sparks), LCCs also showed some different characteristics. With simultaneous [Ca.sup.2+] imaging and current recording, the localized fluorescence increase due to [Ca.sup.2+] entry through a single opening of an LCC (SCCaFT) was detected. When membrane potential, instead of current, was recorded, SCCaFT-like fluorescence transients (indicating single LCC openings) were found to accompany membrane depolarizations. To our knowledge, this is the first report directly linking membrane potential changes to a single opening of an ion channel. Moreover, these events in cardiac cells suggest a possible additional mechanism by which caffeine and theophylline contribute to the generation of cardiac arrhythmias. ryanodine receptor; methylxanthine; single-channel calcium fluorescence transients; calcium sparks; cardiac arrhythmia