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There is great interest in utilizing butyrate as a chemopreventive agent for colon tumorigenesis because of its ability to promote apoptosis in colon tumor cell lines. Because CD95 (APO-1/Fas) transduces signals resulting in apoptosis, we tested the hypothesis that butyrate-dependent colonocyte apoptosis is mediated by this death receptor. Butyrate (1 mM) exposure for 24 h upregulated expression of Fas and its ligand in young adult mouse colon (YAMC) cells. To delineate the proapoptotic effect of butyrate and to avoid the confounding effects of detachment from the extracellular matrix, adherent cell apoptosis was monitored as loss of plasma membrane asymmetry and dissipation of mitochondrial membrane potential (DeltaPsi(mt)) by laser cytometry. Soluble Fas receptor protein (Fas:Fc chimera) and caspase inhibitors (z-VAD-fmk and z-IETD-fmk) blocked butyrate induction of apoptosis. Treatment with Fas agonistic antibody (clone Jo-2) significantly induced cell death, indicating that Fas in colonocytes is functional. In addition, butyrate promoted apoptosis by inducing loss of DeltaPsi(mt) and phospholipid asymmetry of the plasma membrane after 12 and 24 h of exposure, respectively, before cell detachment. Therefore, Fas receptor-dependent signal transduction is involved in butyrate induction of apoptosis in colonocytes.

Mechanical stimulation of airway epithelial cells generates the Ca2+ mobilization messenger inositol 1,4,5-trisphosphate and the protein kinase (PK) C activator diacylglycerol. Inositol 1,4,5-trisphosphate diffuses through gap junctions to mediate intercellular communication of the mechanical stimulus (a "Ca2+ wave"); the role that diacylglycerol-activated PKC might play in the response is unknown. Using primary cultures of rabbit tracheal cells, we show that 12-O-tetradecanoylphorbol 13-acetate- or 1, 2-dioctanyl-sn-glycerol-induced activation of PKC slows the Ca2+ wave, decreases the amplitude of induced intracellular free Ca2+ concentration ([Ca2+]i) increases, and decreases the number of affected cells. The PKC inhibitors bisindolylmaleimide and Gö 6976 slowed the spread of the wave but did not change the number of affected cells. We show that ATP-induced [Ca2+]i increases and oscillations, responses independent of intercellular communication, were inhibited by PKC activators. Bisindolylmaleimide decreased the amplitude of ATP-induced [Ca2+]i increases and blocked oscillations, suggesting that PKC has an initial positive effect on Ca2+ mobilization and then mediates feedback inhibition. PKC activators also reduced the [Ca2+]i increase that followed thapsigargin treatment, indicating a PKC effect associated with the Ca2+ release mechanism.

Several secretagogues induce mucin secretion in epithelial monolayers, as determined by measuring released granule contents. To assess whether different agonists act on the same granule pool, capacitance changes in intact monolayers of the goblet cell line HT29-Cl.16E were measured by a novel impedance method. Apical ATP (purinergic agonist) and basolateral carbachol (cholinergic agonist) induce rapid exocytosis with maximal capacitance changes within 3 min. The maximal levels of exocytosis that can be induced by optimal concentrations of either agonist are the same and produce a 30-40% increase in total monolayer capacitance. When ATP and carbachol are applied simultaneously, the magnitude of exocytosis is unchanged from the single-secretagogue level. The recovery of capacitance to baseline (endocytosis) is significantly faster after ATP stimulation than after carbachol stimulation. When ATP and carbachol are applied sequentially at doses that give maximal exocytosis, the magnitude of the capacitance increase produced by the second secretagogue is less than or equal to that of the capacitance decrease during the recovery period. Together, these data suggest that purinergic and cholinergic agonists act on the same granule pool.

Calcium transport in the apical-to-basolateral (A-to-B) or B-to-A direction was examined in cells treated with 10 nM 1, 25-dihydroxyvitamin D3 [1,25(OH)2D3, calcitriol] for up to 72 h. Net A-to-B calcium transport was positive at all time points and increased from 0.14 +/- 0.06 to 0.50 +/- 0.01 nmol. well-1. min-1 after 72 h of calcitriol treatment. Neither phenol red transport nor transepithelial electrical resistance was altered by calcitriol treatment, suggesting that the increase in net A-to-B calcium transport was not due to paracellular movement. Neither 25-hydroxyvitamin D3 nor 24,25-dihydroxyvitamin D3 (100 nM, 48 h) alters basal or calcitriol-stimulated A-to-B calcium transport. Treatment with the calmodulin antagonist trifluoperazine (50 microM) reduced calcitriol-stimulated A-to-B Ca transport by 56%. The transcription inhibitor actinomycin D inhibited calcitriol-regulated A-to-B calcium transport as well as calbindin D9k and 24-hydroxylase mRNA accumulation. These data demonstrate that calcitriol-mediated A-to-B calcium transport in Caco-2 cells is a specific, transcellular process that requires transcriptional events normally mediated through the vitamin D receptor.

A novel Na/H exchange activity that requires Cl was recently identified in the apical membrane of crypt cells of the rat distal colon. This study explores the nature of the coupling of Cl and Na/H exchange. A concentration of 100 microM 5-nitro-2-(3-phenylpropylamino)benzoic acid, a Cl channel blocker, inhibited the Cl dependence of both proton gradient-driven 22Na uptake from crypt cell apical membrane vesicles and Na-dependent intracellular pH recovery from an acid load during microperfusion of the crypt lumen. Cl-dependent proton gradient-driven 22Na uptake was inhibited by 94% by 500 microM DIDS but only by 1% by 10 microM DIDS, an anion exchange inhibitor at low concentrations but a Cl channel blocker at high concentrations. In addition, a polyclonal antibody to the cystic fibrosis transmembrane conductance regulator (CFTR) inhibited Cl-dependent proton gradient-driven 22Na uptake by 38%. These results indicate that the Cl dependence of Na/H exchange in the colonic crypt apical membrane involves a Cl channel and not a Cl/anion exchange and permit the speculation that this Cl channel activity represents both CFTR and the outward rectifying Cl conductance.

Colonic luminal short-chain fatty acids (SCFA) stimulate electroneutral sodium absorption via activation of apical Na/H exchange. HT29-C1 cells were used previously to demonstrate that transepithelial SCFA gradients selectively activate polarized Na/H exchangers. Fluorometry and confocal microscopy (with BCECF and carboxy SNARF-1, respectively) are used to measure intracellular pH (pHi) in HT29-C1 cells, to find out which Na/H exchanger isoforms are expressed and if results are due to pHi gradients. Inhibition of Na/H exchange by HOE-694 identified 1) two inhibitory sites [50% inhibitory dose (ID50) = 1.6 and 0.05 microM] in suspended cells and 2) one inhibitory site each in the apical and basolateral membranes of filter-attached cells (apical ID50 = 1.4 microM, basolateral ID50 = 0.3 microM). RT-PCR detected mRNA of Na/H exchanger isoforms NHE1 and NHE2 but not of NHE3. Confocal microscopy of filter-attached cells reported HOE-694-sensitive pHi recovery in response to luminal or serosal 130 mM propionate. Confocal analysis along the apical-to-basal axis revealed that 1) luminal or serosal propionate establishes transcellular pHi gradients and 2) the predominant site of pHi acidification and pHi recovery is the apical portion of cells. Luminal propionate produced a significantly greater acidification of the apical vs. basal portion of the cell (compared with serosal propionate), but no other dependence on the orientation of the SCFA gradient was observed. Results provide direct evidence for a subcellular response that assures robust activation of apical NHE2 and dampening of basolateral NHE1 during pHi regulation.

We performed Ussing chamber experiments on cultured human bronchial epithelial cells to look for the presence of electrogenic dibasic amino acid transport. Apical but not basolateral L-arginine (10-1, 000 microM) increased the short-circuit current. Maximal effect and EC50 were approximately 3.5 microA/cm2 and 80 microM, respectively, in cells from normal subjects and cystic fibrosis patients. The involvement of nitric oxide was ruled out because a nitric oxide synthase inhibitor (NG-nitro-L-arginine methyl ester) did not decrease the arginine-dependent current. Apical L-lysine, L-alanine, and L-proline, but not aspartic acid, were also effective in increasing the short-circuit current, with EC50 values ranging from 26 to 971 microM. Experiments performed with radiolabeled arginine demonstrated the presence of an Na+-dependent concentrative transporter on the apical membrane of bronchial cells. This transporter could be important in vivo to maintain a low amino acid concentration in the fluid covering the airway surface.

In Aplysia intestine, stimulation of Na+ absorption with luminal alanine increases apical membrane K+ conductance (GK,a), which presumably regulates enterocyte volume during stimulated Na+ absorption. However, the mechanism responsible for the sustained increase in plasma membrane K+ conductance is not known for any nutrient-absorbing epithelium. In the present study, we have begun to test the hypothesis that the alanine-induced increase in GK,a in Aplysia enterocytes results from exocytic insertion of K+ channels into the apical membrane. We used the fluid-phase marker horseradish peroxidase to assess the effect of alanine on apical membrane exocytosis and conventional microelectrode techniques to assess the effect of alanine on fractional capacitance of the apical membrane (fCa). Luminal alanine significantly increased apical membrane exocytosis from 1.04 +/- 0.30 to 1.39 +/- 0.38 ng. min-1. cm-2. To measure fCa, we modeled the Aplysia enterocyte as a double resistance-capacitance (RC) electric circuit arranged in series. Several criteria were tested to confirm application of the model to the enterocytes, and all satisfied the model. When added to the luminal surface, alanine significantly increased fCa from 0.27 +/- 0. 02 to 0.33 +/- 0.04 (n = 10) after 4 min. There are two possible explanations for our findings: 1) the increase in exocytosis, which adds membrane to the apical plasma membrane, prevents plasma membrane fracture, and 2) the increase in exocytosis delivers K+ channels to the apical membrane by exocytic insertion. After the alanine-induced depolarization of apical membrane potential (Va), there is a strong correlation (r = 0.96) between repolarization of Va, which reflects the increase in GK,a, and increase in fCa. This correlation supports the exocytic insertion hypothesis for activation of GK,a.

We examined the effects of H2O2 on Cl- secretion across human colonic T84 cells grown on permeable supports and mounted in modified Ussing chambers. Forskolin-induced short-circuit current, a measure of Cl- secretion, was inhibited in a concentration-dependent fashion when monolayers were pretreated with H2O2 for 30 min (30-100% inhibition between 500 microM and 5 mM). Moreover, H2O2 inhibited 76% of the Cl- current across monolayers when the basolateral membranes were permeabilized with nystatin (200 micrograms/ml). When the apical membrane was permeabilized with amphotericin B, H2O2 inhibited the Na+ current (a measure of Na+-K+-ATPase activity) by 68% but increased the K+ current more than threefold. In addition to its effects on ion transport pathways, H2O2 also decreased intracellular ATP levels by 43%. We conclude that the principal effect of H2O2 on colonic Cl- secretion is inhibitory. This may be due to a decrease in ATP levels following H2O2 treatment, which subsequently results in an inhibition of the apical membrane Cl- conductance and basolateral membrane Na+-K+-ATPase activity. Alternatively, H2O2 may alter Cl- secretion by direct action on the transporters or alterations in signal transduction pathways.

Dami human leukemia cells express G protein-coupled thrombin receptors that operate through the phospholipase C pathway. When these receptors are activated by alpha-thrombin or by thrombin receptor-activating peptide, an elevation in cytosolic Ca2+ concentration develops that is accompanied by hyperpolarization of the plasma membrane. This transitory phase of hyperpolarization is primarily mediated by inwardly rectifying, Ca2+-activated K+ channels that have an inward conductance of approximately 24 pS. In cell-attached patches the channels open within seconds after superfusion of the cell with thrombin receptor-activating peptide. In inside-out patches, perfusion of submicromolar Ca2+ onto the cytosolic surface of the membrane is sufficient to activate the channels. In outside-out patches, channel opening can be blocked by nanomolar concentrations of charybdotoxin. The function of these intermediate-sized inwardly rectifying, Ca2+-activated K+ channels has not been established; however, by analogy with other cell systems, they may serve to regulate cell volume during cellular activation or to increase the electromotive drive that sustains Na+ and/or Ca2+ influx through ligand-gated cation channels.

Indomethacin and other nonsteroidal anti-inflammatory drugs are commonly used to indirectly deduce the possible role of PGs in a process being studied. The objective of this study was to determine if indomethacin, at concentrations comparable to plasma and tissue levels obtained in humans taking therapeutic doses, predisposes human gastric cells to injury through inhibition of PGs or acts through an alternate mechanism. The role of intracellular Ca2+ in this damaging process was also assessed. Indomethacin pretreatment, although by itself nondamaging, was associated with elevated intracellular Ca2+ concentrations and an increased cellular permeability, an effect that was dependent on extracellular Ca2+. Furthermore, indomethacin pretreatment significantly predisposed AGS cells to injury induced by two dissimilar agents (deoxycholate and A-23187), both of which are associated with intracellular Ca2+ accumulation. The addition of exogenous PGs did not reverse the predisposition to injury induced by indomethacin. The observed effects of indomethacin were dependent on concentration and not on ability to inhibit PG synthesis. Similar effects were not observed with equipotent concentrations of ibuprofen or aspirin. Finally, the exacerbation of deoxycholate-induced injury induced by indomethacin was not observed when extracellular Ca2+ was removed. Indomethacin, by disturbing intracellular Ca2+ homeostasis, predisposes human gastric cells to injury through mechanisms independent of PG synthesis. The current study suggests that data resulting from studies employing only indomethacin as a PG synthesis inhibitor should be interpreted with caution.

In this study, we attempted to investigate the response of polarized cells to inappropriate interaction with the extracellular matrix. Cell lines of epithelial [Madin-Darby canine kidney (MDCK) and LLC-PK1], endothelial [bovine aortic endothelial cells (BAEC)], and mesenchymal (ESK-4 and NIH/3T3) origins were employed. With collagen gel overlay, MDCK cells underwent membrane remodeling and gradually developed lumen formation within 24 h. Apoptosis could also be observed following cell remodeling. The ratio of apoptosis was enhanced from 12.1 +/- 2.4% within 24 h to 58.4 +/- 9.8% at day 3, and finally the monolayer was disintegrated. Collagen gel overlay-induced apoptosis was not a result of physical stress, since agarose gel overlay did not induce any morphological alterations. All epithelial and endothelial cells examined developed apoptosis in response to collagen overlay. In contrast, collagen overlay did not affect growth of fibroblasts at all, although their growth under agarose gel was slightly hindered due to physical stress. Collagen overlay-induced apoptosis seems to be a unique phenomenon for polarized cells and thus is defined as "disoriented cell death." Furthermore, anti-alpha2-integrin antibody could abolish collagen overlay-induced morphological changes and apoptosis in MDCK cells, indicating that signals through alpha2-integrin on the apical membrane are required for disoriented cell death. Finally, Bcl-2 overexpression prolonged survival of MDCK cells in response to collagen overlay, but these cells eventually developed apoptosis due to downregulation of Bcl-2 protein. These findings indicate that inappropriate cell-matrix interaction results in apoptosis, which may account for cell death mechanisms during developmental processes or under pathological conditions.

Decreases in intracellular pH (pHi) potently dilate coronary resistance arteries but constrict small pulmonary arteries. To define the ionic mechanisms of these responses, this study investigated whether acute decreases in pHi differentially regulate K+ currents in single vascular smooth muscle (VSM) cells isolated from rat coronary and pulmonary resistance arteries. In patch-clamp studies, whole cell K+ currents were elicited by 10-mV depolarizing steps between -60 and 0 mV in VSM cells obtained from 50- to 150-micrometers-OD arterial branches, and pHi was lowered by altering the NH4Cl gradient across the cell membrane. Progressively lowering pHi from calculated values of 7.0 to 6.7 and 6.4 increased the peak amplitude of K+ current in coronary VSM cells by 15 +/- 5 and 23 +/- 3% but reduced K+ current in pulmonary VSM cells by 18 +/- 3 and 21 +/- 3%, respectively. These changes were reversed by returning cells to the control pHi of 7.0 and were eliminated by dialyzing cells with pipette solution containing 50 mmol/l HEPES to buffer NH4Cl-induced changes in pHi. Pharmacological block of ATP-sensitive K+ channels and Ca2+-activated K+ channels by 1 micromol/l glibenclamide and 100 nmol/l iberiotoxin, respectively, did not prevent changes in K+ current levels induced by acidotic pHi. However, block of voltage-gated K+ channels by 3 mmol/l 4-aminopyridine abolished acidosis-induced changes in K+ current amplitudes in both VSM cell types. Interestingly, alpha-dendrotoxin (100 nmol/l), which blocks only select subtypes of voltage-gated K+ channels, abolished the acidosis-induced decrease in K+ current in pulmonary VSM cells but did not affect the acidosis-induced increase in K+ current observed in coronary VSM cells. These findings suggest that opposing, tissue-specific effects of pHi on distinct subtypes of voltage-gated K+ channels in coronary and pulmonary VSM membranes may differentially regulate vascular reactivity in these two circulations under conditions of acidotic stress.

Our objective in this study was to determine the effect of changes in luminal and cytoplasmic pH on cystic fibrosis transmembrane regulator (CFTR) Cl- conductance (GCl). We monitored CFTR GCl in the apical membranes of sweat ducts as reflected by Cl- diffusion potentials (VCl) and transepithelial conductance (GCl). We found that luminal pH (5.0-8.5) had little effect on the cAMP/ATP-activated CFTR GCl, showing that CFTR GCl is maintained over a broad range of extracellular pH in which it functions physiologically. However, we found that phosphorylation activation of CFTR GCl is sensitive to intracellular pH. That is, in the presence of cAMP and ATP [adenosine 5'-O-(3-thiotriphosphate)], CFTR could be phosphorylated at physiological pH (6.8) but not at low pH (approximately 5.5). On the other hand, basic pH prevented endogenous phosphatase(s) from dephosphorylating CFTR. After phosphorylation of CFTR with cAMP and ATP, CFTR GCl is normally deactivated within 1 min after cAMP is removed, even in the presence of 5 mM ATP. This deactivation was due to an increase in endogenous phosphatase activity relative to kinase activity, since it was reversed by the reapplication of ATP and cAMP. However, increasing cytoplasmic pH significantly delayed the deactivation of CFTR GCl in a dose-dependent manner, indicating inhibition of dephosphorylation. We conclude that CFTR GCl may be regulated via shifts in cytoplasmic pH that mediate reciprocal control of endogenous kinase and phosphatase activities. Luminal pH probably has little direct effect on these mechanisms. This regulation of CFTR may be important in shifting electrolyte transport in the duct from conductive to nonconductive modes.

Recent studies indicate that regression of left ventricular hypertrophy (LVH) normalizes the in situ electrophysiological abnormalities of the left ventricle. This study was designed to determine whether regression of LVH also normalizes the abnormalities of individual membrane currents. LVH was induced in rabbits by renal artery banding. Single ventricular myocytes from rabbits with LVH at 3 mo after renal artery banding demonstrated increased cell membrane capacitance, prolonged action potential duration, decreased inward rectifier K+ current density, and increased transient outward K+ current density compared with myocytes from age-matched controls. Additional rabbits were randomized at 3 mo after banding to treatment with either vehicle or captopril for an additional 3 mo. Myocytes from LVH rabbits treated with vehicle showed persistent membrane current abnormalities. However, myocytes isolated from LVH rabbits treated with captopril had normal cell membrane capacitance, action potential duration, and membrane current densities. Captopril had no direct effect on membrane currents of either control or LVH myocytes. These data support the hypothesis that the action potential prolongation and membrane current abnormalities of LVH are reversed by regression. Normalization of membrane currents probably explains the reduced vulnerability to ventricular arrhythmia observed in this LVH model after treatment with captopril.

It has been demonstrated that K+ channel regulation of membrane potential is critical for control of CCK secretion. Because certain K+ channels are pH sensitive, it was postulated that pH affects K+ channel activity in the CCK-secreting cell line STC-1 and may participate in regulating CCK secretion. The present study examines the role of electroneutral Na+/H+ exchange on extracellular acidification and hormone secretion. Treatment of STC-1 cells with the amiloride analog ethylisopropyl amiloride (EIPA) to inhibit Na+/H+ exchange inhibited Na+-dependent H+ efflux and increased basal CCK secretion. Substituting choline for NaCl in the extracellular medium elevated basal intracellular Ca2+ concentration and stimulated CCK release. Stimulatory effects on hormone secretion were blocked by the L-type Ca2+ channel blocker diltiazem, indicating that secretion was dependent on the influx of extracellular Ca2+. To determine whether the effects of EIPA and Na+ depletion were due to membrane depolarization, we tested graded KCl concentrations. The ability of EIPA to increase CCK secretion was inhibited by depolarization induced by 10-50 mM KCl in the bath. Maneuvers to lower intracellular pH (pHi), including reducing extracellular pH (pHo) to 7.0 or treatment with sodium butyrate, significantly increased CCK secretion. To examine whether pH directly affects membrane K+ permeability, we measured outward currents carried by K+, using whole cell patch techniques. K+ current was significantly inhibited by lowering pHo to 7.0. These effects appear to be mediated through changes in pHi, because intracellular dialysis with acidic solutions nearly eliminated current activity. These results suggest that Na+/H+ exchange and membrane potential may be functionally linked, where inhibition of Na+/H+ exchange lowers pHi and depolarizes the membrane, perhaps through inhibition of pH-sensitive K+ channels. In turn, K+ channel closure and membrane depolarization open voltage-dependent Ca2+ channels, leading to an increase in cytosolic Ca2+ and CCK release. The effects of pHi on K+ channels may serve as a potent stimulus for hormone secretion, linking cell metabolism and secretory functions.

Previous studies have shown that the envelope protein of the mouse mammary tumor virus (MMTV) rapidly upregulates prolactin (PRL) receptors by shifting them from internal pools to the cell surface and downregulates epidermal growth factor (EGF) receptors by inducing their internalization and degradation. This study shows that the effect on PRL receptors is mediated by the nitric oxide (NO)/cGMP pathway, since it can be mimicked by an NO donor or 8-bromo-cGMP and can be blocked by an NO synthase inhibitor. In contrast, the effect on EGF receptors is mediated by tyrosine phosphorylation and phosphatidylinositol 3-kinase (PI3K), since it can be blocked by either a tyrosine kinase inhibitor or by a PI3K inhibitor. Both of these pathways can be activated by a calcium ionophore and inhibited by calcium chelation. Therefore, it appears that the mouse mammary tumor virus envelope protein, like other retroviral envelope proteins, initially elevates cytoplasmic calcium, which can then stimulate both the NO/cGMP and the tyrosine phosphorylation/PI3K pathways, leading to PRL receptor upregulation and EGF receptor downregulation, respectively.

We have used fluo 3-loaded mouse pancreatic acinar cells to investigate the relationship between Ca2+ mobilization and intracellular pH (pHi). The Ca2+-mobilizing agonist ACh (500 nM) induced a Ca2+ release in the luminal cell pole followed by spreading of the Ca2+ signal toward the basolateral side with a mean speed of 16.1 +/- 0.3 micron/s. In the presence of an acidic pHi, achieved by blockade of the Na+/H+ exchanger or by incubation of the cells in a Na+-free buffer, a slower spreading of ACh-evoked Ca2+ waves was observed (7.2 +/- 0.6 micron/s and 7.5 +/- 0.3 micron/s, respectively). The effects of cytosolic acidification on the propagation rate of ACh-evoked Ca2+ waves were largely reversible and were not dependent on the presence of extracellular Ca2+. A reduction in the spreading speed of Ca2+ waves could also be observed by inhibition of the vacuolar H+-ATPase with bafilomycin A1 (11.1 +/- 0.6 micron/s), which did not lead to cytosolic acidification. In contrast, inhibition of the endoplasmic reticulum Ca2+-ATPase by 2,5-di-tert-butylhydroquinone led to faster spreading of the ACh-evoked Ca2+ signals (25.6 +/- 1.8 micron/s), which was also reduced by cytosolic acidification or treatment of the cells with bafilomycin A1. Cytosolic alkalinization had no effect on the spreading speed of the Ca2+ signals. The data suggest that the propagation rate of ACh-induced Ca2+ waves is decreased by inhibition of Ca2+ release from intracellular stores due to cytosolic acidification or to Ca2+ pool alkalinization and/or to a decrease in the proton gradient directed from the inositol 1,4, 5-trisphosphate-sensitive Ca2+ pool to the cytosol.

Stimulation of pancreatic acinar cells induces the release of digestive enzymes via the exocytotic fusion of zymogen granules and activates postfusion granule membrane retrieval and receptor cycling. In the present study, changes in membrane surface area of rat single pancreatic acinar cells were monitored by cell membrane capacitance (Cm) measurements and by the membrane fluorescent dye FM1-43. When measured with the Cm method, agonist treatment evoked a graded, transient increase in acinar cell surface area averaging 3. 5%. In contrast, a 13% increase in surface area was estimated using FM1-43, corresponding to the fusion of 48 zymogen granules at a rate of 0.5 s-1. After removal of FM1-43 from the surface-accessible membrane, a residual fluorescence signal was shown by confocal microscopy to be localized in endosome-like structures and confined to the apical regions of acinar cells. The development of an optical method for monitoring the membrane turnover of single acinar cells, in combination with measurements of Cm changes, reveals coincidence of exocytotic and endocytotic activity in acinar cells after hormonal stimulation.

This report demonstrates that Ca2+, Mg2+, and protons alter the ability of polymyxin B (PX, a cationic antibiotic used clinically as a bactericidal agent) to increase the apical membrane conductance of the rabbit urinary bladder. Using electrophysiological methods, we determine that these alterations occur by two mechanisms. First, they blocked the PX-induced conductance in a rapid and reversible manner; second, they competed with PX for a membrane binding site. In addition, Ca2+ (but not Mg2+ or protons) altered the rate at which the induced conductance could be reversed. When solution pH was greater than 8.8, PX was not able to induce a conductance. This ability of high pH to inhibit the action of PX was due to a decrease in the number of positive charges on PX. Further studies demonstrated that for maximal activity, PX required its fatty acid tail. These data were used to develop a model describing the mechanism by which PX can induce a conductance in the apical membrane of the rabbit urinary bladder.

Monolayers of the human colonic epithelial cell line T84 exhibit electrogenic Cl- secretion in response to the Ca2+ agonist thapsigargin and to the cAMP agonist forskolin. To evaluate directly the regulation of apical Cl- conductance by these two agonists, we have utilized amphotericin B to permeabilize selectively the basolateral membranes of T84 cell monolayers. We find that apical anion conductance is stimulated by both forskolin and thapsigargin but that these conductances are differentially sensitive to the anion channel blocker DIDS. DIDS inhibits thapsigargin-stimulated responses completely but forskolin responses only partially. Furthermore, the apical membrane anion conductances elicited by these two agonists differ in anion selectivity (for thapsigargin, I- > Cl-; for forskolin, Cl- > I-). However, the DIDS-sensitive component of the forskolin-induced conductance response exhibits anion selectivity similar to that induced by thapsigargin (I- > Cl-). Thus forskolin-induced apical anion conductance comprises at least two components, one of which has features in common with that elicited by thapsigargin.

We have developed an in vitro model of adaptive cytoprotection induced by deoxycholate (DC) in human gastric cells and have shown that pretreatment with a low concentration of DC (mild irritant, 50 microM) significantly attenuates injury induced by a damaging concentration of DC (250 microM). This study was undertaken to assess the effect of the mild irritant on changes in intracellular Ca2+ and to determine if these perturbations account for its protective action. Protection conferred by the mild irritant was lost when any of its effects on intracellular Ca2+ were prevented: internal Ca2+ store release via phospholipase C and inositol 1,4, 5-trisphosphate sustained Ca2+ influx through store-operated Ca2+ channels or eventual Ca2+ efflux. We also investigated the relationship between Ca2+ accumulation and cellular injury induced by damaging concentrations of DC. In cells exposed to high concentrations of DC, sustained Ca2+ accumulation as a result of extracellular Ca2+ influx, but not transient changes in intracellular Ca2+ content, appeared to precede and induce cellular injury. We propose that the mild irritant disrupts normal Ca2+ homeostasis and that this perturbation elicits a cellular response (involving active Ca2+ efflux) that subsequently provides a protective action by limiting the magnitude of intracellular Ca2+ accumulation.

The nature of the pathway for K+ release activated during regulatory volume decrease (RVD) in A6 epithelia was investigated by measuring cell thickness (Tc) as an index of cell volume and by probing K+ efflux with 86Rb as tracer for K+ (RRb). Cell swelling was induced by sudden reduction of basolateral osmolality (from 260 to 140 mosmol/kgH2O). Experiments were performed in the absence of Na+ transport. Apical RRb was negligible in iso- and hyposmotic conditions. On the other hand, osmotic shock increased basolateral RRb (RblRb) rapidly, reaching a maximum 7 min after the peak in Tc. Quinine (0.5 mM) completely inhibited RVD and RblRb. Also verapamil (0.2 mM) impeded volume recovery considerably; lidocaine (0.2 mM) did not exert a noticeable effect. The K+ channel blocker Ba2+ (30 mM) delayed RVD but could not prevent complete volume recovery. Cs+ inhibited RVD noticeably at concentrations <40 mM. With large Cs+ concentrations (>40 mM), the initial osmometric swelling was followed by a gradual increase of Tc, suggesting activation of Cs+ influx. Chronic exposure of the basolateral surface to 0.5 mM La3+ or Gd3+ completely abolished RVD and RblRb. Acute administration of lanthanides at the time of osmolality decrease did not affect the initial phase of RVD and reduced RblRb only slightly. Apical Gd3+ exerted an inhibitory effect on RVD and RblRb. The effect of Gd3+ should therefore be localized at an intracellular site. The role of Ca2+ entry could be excluded by failure of extracellular Ca2+ removal to inhibit volume recovery. In contrast to lanthanides, chronically and acutely administered Mg2+ (0.5 mM) inhibited RVD and RblRb by approximately 50%. These data suggest that K+ excretion during RVD occurs through a rather poorly selective pathway that does not seem to be directly activated by membrane stretch.

We examined the effect of norepinephrine (NE) on intracellular pH (pHi) and activity of Na+ (aNai) in the isolated perfused kidney proximal tubule of Ambystoma, using single-barreled voltage and ion-selective microelectrodes. In control HCO-3 Ringer, addition of 10(-6) M NE to the bath reversibly depolarized the basolateral membrane potential (V1), the luminal membrane potential (V2), and the transepithelial potential difference (V3) and increased pHi by 0. 14 +/- 0.02. These effects were mimicked by isoproterenol but were abolished after pretreatment with SITS or in the absence of CO2/HCO-3. Removal of bath Na+ depolarized V1 and V2, hyperpolarized V3, and decreased pHi. These effects are largely mediated by the electrogenic Na+-(HCO-3)n cotransporter. In the presence of NE, the effects of Na+ removal on membrane potential differences and the rate of change of pHi were significantly smaller. Reducing bath HCO-3 concentration from 10 to 2 mM at constant CO2 (pH 6.8) depolarized V1 and V2, decreased pHi, and lowered aNai. These changes are also due to Na+-(HCO-3)n. In the presence of NE, reducing bath [HCO-3] caused a smaller depolarizations of V1 and V2, and the rate of pHi decrease was significantly reduced. Our results indicate: 1) NE causes an increase in pHi; 2) the NE-induced alkalinization is mediated by a SITS-sensitive and HCO-3-dependent transporter on the basolateral membrane; and 3) in the presence of NE, the reduced effects caused by basolateral HCO-3 changes or Na+ removal are indicative of an inhibitory effect of NE on Na+-(HCO-3)n cotransport.

We previously demonstrated that nitric oxide (NO) stimulates the basolateral small-conductance K+ channel (SK) via a cGMP-dependent pathway [M. Lu and W. H. Wang. Am. J. Physiol. 270 (Cell Physiol. 39): C1336-C1342, 1996]. Because NO at high concentration has been shown to react with superoxide (O-2) to form peroxynitrite (OONO-) [W. A. Pryor and G. L. Squadrito. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L699-L722, 1995 and M. S. Wolin. Microcirculation 3: 1-17, 1996], we extended our study to examine, using patch-clamp technique, the effect of high concentrations of NO on SK in cortical collecting duct (CCD) of rat kidney. Addition of NO donors [100-200 microM S-nitroso-N-acetyl-penicillamine (SNAP) or sodium nitroprusside (SNP)] reduced channel activity, defined as the product of channel number and open probability, to 15 and 25% of the control value, respectively. The inhibitory effect of NO was completely abolished in the presence of 10 mM Tiron, an intracellular scavenger of O-2. NO donors, 10 microM SNAP or SNP, which stimulate channel activity under control conditions, can also inhibit SK in the presence of an O-2 donor, pyrogallol, or in the presence of an inhibitor of superoxide dismutase, diethyldithiocarbamic acid. The inhibitory effect of NO is still observed in the presence of exogenous cGMP, suggesting that the NO-induced inhibition is not the result of decreased cGMP production. We conclude that the inhibitory effect of NO on channel activity results from an interaction between NO and O-2.

The eosinophil granule protein major basic protein (MBP) is toxic to a wide variety of cell types, by a poorly understood mechanism. To determine whether the action of MBP involves an alteration in membrane permeability, we tested purified MBP on rabbit urinary bladder epithelium using transepithelial voltage-clamp techniques. Addition of nanomolar concentrations of MBP to the mucosal solution caused an increase in apical membrane conductance only when the voltage across the apical membrane was cell interior negative. The magnitude of the MBP-induced conductance was a function of MBP concentration, and the rate of the initial increase in conductance was a function of the transepithelial voltage. The MBP-induced conductance was nonselective for K+ and Cl-. Mucosal Ca2+ reversed the induced conductance, whereas mucosal Mg2+ partially blocked the induced conductance and slowed the rate of the increase in conductance. The induced conductance was partially reversed by changing the voltage gradient across the apical membrane to cell interior positive. Prolonged exposure resulted in an irreversible loss of the barrier function of the urinary bladder epithelium. These results suggest that an increase in cell membrane ion permeability is an initial step in MBP-induced loss of barrier function.

Leptin, the protein product of the ob/ob gene, is thought to have a central site of action, presumably within the hypothalamus, through which it regulates feeding behavior. THe paraventricular nucleus (PVN) is one structure that has been implicated in regulating feeding behavior. Using patch-clamp recording techniques, this study examines the direct membrane effects of leptin on neurons in a coronal PVN slice. Bath application of the physiologically active leptin fragment (amino acids 22-56) elicited dose-related depolarizations in 82% of the type I cells tested (n = 17) and 67% of the type II cells tested (n = 9). By contrast, the physiologically inactive leptin fragment (amino acids 57-92) had no discernible effect on membrane potential (n = 7). The effects of this peptide were unaffected following synaptic isolation of the cells by bath application of the sodium channel blocker tetrodotoxin (n = 5). Voltage clamp recordings in six cells demonstrated that leptin increased a nonspecific cation conductance with a reversal potential near -30 mV. These findings suggest that neurons in PVN may play an important role in the central neuronal circuitry involved in the physiological response to leptin.

We examined the effects of epinephrine (25, 50, and 150 nM) on 1) basal and insulin-stimulated 3-O-methylglucose (3-MG) transport in perfused rat muscles and 2) GLUT-4 in skeletal muscle plasma membranes. Insulin increased glucose transport 330-600% in three types of skeletal muscle [white (WG) and (RG) gastrocnemius and soleus (SOL)]. Glucose transport was also increased by epinephrine (22-48%) in these muscles (P < 0.05). In contrast, the insulin-stimulated 3-MG transport was reduced by epinephrine in all three types of muscles; maximal reductions were observed at 25 nM epinephrine in WG (-25%) and RG (-32.5%). A dose-dependent decrease occurred in SOL (-27% at 25 nM; -55% at 150 nM, P < 0.05). Insulin (20 mU/ml) and epinephrine (150 nM) each translocated GLUT-4 to the plasma membrane, and no differences in translocation were observed between insulin and epinephrine (P > 0.05). In addition, epinephrine did not inhibit insulin-stimulated GLUT-4 translocation, and the combined epinephrine and insulin effects on GLUT-4 translocation were not additive. The increase in surface GLUT-4 was associated with increases in muscle cAMP concentrations, but only when epinephrine alone was present. No relationship was evident between muscle cAMP concentrations and surface GLUT-4 in the combined epinephrine and insulin-stimulated muscles. These studies indicate that epinephrine can translocate GLUT-4 while at the same time increasing glucose transport when insulin is absent, or can inhibit glucose transport when insulin is present.

Activation of phospholipase A2 (PLA2) and accumulation of lysophosphatidylcholine contribute importantly to arrhythmogenesis during acute myocardial ischemia. We examined thrombin stimulation of PLA2 activity in isolated ventricular myocytes. Basal and thrombin-stimulated cardiac myocyte PLA2 activity demonstrated a distinct preference for sn-1 ether-linked phospholipids with arachidonate esterified at the sn-2 position. The majority of PLA2 activity was calcium independent and membrane associated. Thrombin stimulation of membrane-associated PLA2 occurs in a time- and concentration-dependent fashion. An increase in PLA2 activity was also observed using the synthetic peptide SFLLRNPNDKYEPF (the tethered ligand generated by thrombin cleavage of its receptor). Bromoenol lactone, a selective inhibitor of calcium-independent PLA2, completely blocked thrombin-stimulated increases in PLA2 activity and arachidonic acid release. No significant inhibition of thrombin-induced PLA2 was observed following pretreatment with mepacrine or dibucaine. These data confirm the presence of high-affinity thrombin receptors on isolated cardiac myocytes and demonstrate the specific activation of a unique membrane-associated, calcium-independent PLA2 following thrombin receptor ligation.

Neurons are mechanically robust. During prolonged swelling, molluscan neurons can triple their apparent membrane area. They gain surface area and capacitance independent of extracellular Ca concentration ([Ca]e), but it is unknown if an increase in intracellular Ca concentration ([Ca]i) is necessary. If Ca for stimulating exocytosis is unnecessary, it is possible that swelling-induced membrane tension changes directly trigger surface area readjustments. If, however, Ca-mediated but not tension-mediated membrane recruitment is responsible for surface area increases, swelling neurons should sustain elevated levels of [Ca]i. The purpose of this investigation is to determine if the [Ca]i in swelling neurons attains levels high enough to promote exocytosis and if any such increase is required. Lymnaea neurons were loaded with the Ca concentration indicator fura 2. Calibration was performed in situ using 4-bromo-A-23187 and Ca-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), with free Ca concentration ranging from 0 to 5 microM. Swelling perturbations (medium osmolarity reduced to 25% for 5 min) were done at either a standard [Ca]e or very low [Ca]e level (0.9 mM or 0.13 microM, respectively). In neither case did the [Ca]i increase to levels that drive exocytosis. We also monitored osmomechanically driven membrane dynamics [swelling, then formation and reversal of vacuole-like dilations (VLDs)] with the [Ca]i clamped below 40 nM via 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). [Ca]i did not change with swelling, and VLD behavior was unaffected, consistent with tension-driven, [Ca]i-independent surface area adjustments. In addition, neurons with [Ca]i clamped at 0.1 microM via an ionophore could produce VLDs. We conclude that, under mechanical stress, neuronal membranes are compliant by virtue of surface area regulatory adjustments that operate independent of [Ca]i. The findings support the hypothesis that plasma membrane area is regulated in part by membrane tension.

The Cl- secretory response of colonic cells to Ca(2+)-mediated agonists is transient despite a sustained elevation of intracellular Ca2+. We evaluated the effects of second messengers proposed to limit Ca(2+)-mediated Cl- secretion on the basolateral membrane, Ca(2+)-dependent K+ channel (Kca) in colonic secretory cells, T84. Neither protein kinase C (PKC) nor inositol tetrakisphosphate (1,3,4,5 or 3,4,5,6 form) affected Kca in excised inside-out patches. In contrast, arachidonic acid (AA; 3 microM) potently inhibited Kca, reducing NP0, the product of number of channels and channel open probability, by 95%. The apparent inhibition constant for this AA effect was 425 nM. AA inhibited Kca in the presence of both indomethacin and nordihydroguaiaretic acid, blockers of the cyclooxygenase and lipoxygenase pathways. In the presence of albumin, the effect of AA on Kca was reversed. A similar effect of AA was observed on Kca during outside-out recording. We determined also the effect of the cis-unsaturated fatty acid linoleate, the trans-unsaturated fatty acid elaidate, and the saturated fatty acid myristate. At 3 microM, all of these fatty acids inhibited Kca, reducing NP0 by 72-86%. Finally, the effect of the cytosolic phospholipase A2 inhibitor arachidonyltrifluoromethyl ketone (AACOCF3) on the carbachol-induced short-circuit current (Isc) response was determined. In the presence of AACOCF3, the peak carbachol-induced Isc response was increased approximately 2.5-fold. Our results suggest that AA generation induced by Ca(2+)-mediated agonists may contribute to the dissociation observed between the rise in intracellular Ca2+ evoked by these agonists and the associated Cl- secretory response.

We used single-channel recording techniques to identify and characterize a large-conductance, Ca(2+)-independent K+ channel in the colonic secretory cell line T84. In symmetric potassium gluconate, this channel had a linear current-voltage relationship with a single-channel conductance of 161 pS. Channel open probability (Po) was increased at depolarizing potentials. Partial substitution of bath K+ with Na+ indicated a permeability ratio of K+ to Na+ of 25:1. Channel Po was reduced by extracellular Ba2+. Event-duration analysis suggested a linear kinetic model for channel gating having a single open state and three closed states: C3<-->C2<-->C1<-->O. Arachidonic acid (AA) increased the Po of the channel, with an apparent stimulatory constant (Ks) of 1.39 microM. Neither channel open time (O) nor the fast closed time (C1) was affected by AA. In contrast, AA dramatically reduced mean closed time by decreasing both C3 and C2. The cis-unsaturated fatty acid linoleate increased Po also, whereas the saturated fatty acid myristate and the trans-unsaturated fatty acid elaidate did not affect Po. This channel is activated also by negative pressure applied to the pipette during inside-out recording. Thus we determined the effect of the stretch-activated channel blockers amiloride and Gd3+ on the K+ channel after activation by AA. Amiloride (2 mM) on the extracellular side reduced single-channel amplitude in a voltage-dependent manner, whereas Gd3+ (100 microM) had no effect on channel activity. Activation of this K+ channel may be important during stimulation of Cl- secretion by agonists that use AA as a second messenger (e.g., vasoactive intestinal polypeptide, adenosine) or during the volume regulatory response to cell swelling.

We examined the functional role of large-conductance Ca(2+)-activated K+ (KCa) channels in the hamster cremasteric microcirculation by intravital videomicroscopy and characterized the single-channel properties of these channels in inside-out patches of membrane from enzymatically isolated cremasteric arteriolar muscle cells. In second-order (39 +/- 1 microns, n = 8) and third-order (19 +/- 2 microns, n = 8) cremasteric arterioles with substantial resting tone, superfusion with the KCa channel antagonists tetraethylammonium (TEA, 1 mM) or iberiotoxin (IBTX, 100 nM) had no significant effect on resting diameters (P > 0.05). However, TEA potentiated O2-induced arteriolar constriction in vivo, and IBTX enhanced norepinephrine-induced contraction of cremasteric arteriolar muscle cells in vitro. Patch-clamp studies revealed unitary K(+)-selective and IBTX-sensitive currents with a single-channel conductance of 240 +/- 2 pS between -60 and 60 mV (n = 7 patches) in a symmetrical 140 mM K+ gradient. The free Ca2+ concentration ([Ca2+]) for half-maximal channel activation was 44 +/- 3, 20 +/- 1, 6 +/- 0.4, and 3 +/- 0.5 microM at membrane potentials of -60, -30, +30, and +60 mV, respectively (n = 5), with a Hill coefficient of 1.9 +/- 0.2. Channel activity increased e-fold for a 16 +/- 1 mV (n = 6) depolarization. The plot of log[Ca2+] vs. voltage for half-maximal activation (V1/2) was linear (r2 = 0.9843, n = 6); the change in V1/2 for a 10-fold change in [Ca2+] was 84 +/- 5 mV, and the [Ca2+] for half-maximal activation at 0 mV (Ca0; the Ca2+ set point) was 9 microM. Thus, in vivo, KCa channels are silent in cremasteric arterioles at rest but can be recruited during vasoconstriction. We propose that the high Ca0 is responsible for the apparent lack of activity of these channels in resting cremasteric arterioles, and we suggest that this may result from expression of unique KCa channels in the microcirculation.

It has been proposed that capillaries can detect changes in tissue metabolites and generate signals that are communicated upstream to resistance vessels. The mechanism for this communication may involve changes in capillary endothelial cell membrane potentials which are then conducted to upstream arterioles. We have tested the capacity of capillary endothelial cells in vivo to respond to pharmacological stimuli. In a hamster cheek pouch preparation, capillary endothelial cells were labeled with the voltage-sensitive dye di-8-ANEPPS. Fluorescence from capillary segments (75-150 microns long) was excited at 475 nm and recorded at 560 and 620 nm with a dual-wavelength photomultiplier system. KCl was applied using pressure injection, and acetylcholine (ACh) and phenylephrine (PE) were applied iontophoretically to these capillaries. Changes in the ratio of the fluorescence emission at two emission wavelengths were used to estimate changes in the capillary endothelial membrane potential. Application of KCl resulted in depolarization, whereas application of the vehicle did not. Application of ACh and PE resulted in hyperpolarization and depolarization, respectively. The capillary responses could be blocked by including a receptor antagonist (atropine or prazosin, respectively) in the superfusate. We conclude that the capillary membrane potential is capable of responding to pharmacological stimuli. We hypothesize that capillaries can respond to changes in the milieu of surrounding tissue via changes in endothelial membrane potential.

We asked whether inclusion of the FLAG epitope in the fourth extracellular loop of the cystic fibrosis transmembrane conductance regulator (M2-901/CFTR), which permits detection of cell surface expression, affected CFTR's biophysical properties or channel regulation by kinases, phosphatases, and nucleotides. Channel activity of M2-901/CFTR was evaluated in numerous cell types and expression systems to characterize its gating and regulation. Our results show that M2-901/CFTR required adenosine 3',5'-cyclic monophosphate-dependent protein kinase phosphorylation to initiate channel activity. Subsequently, ATP alone was sufficient to support channel gating, and ADP inhibited channel opening. Current fluctuation analysis indicated that the nucleotide-dependent gating rates were indistinguishable from those of wild-type (wt) cystic fibrosis transmembrane conductance regulator (CFTR). Channel conductance in symmetric Cl- (11.2 pS), anion permeability ratio (1.66), and block by gluconate indicate that the anion conduction pathway is indistinguishable from wtCFTR. Sulfonylureas (glibenclamide and LY-295501) inhibited M2-901/ CFTR channel activity by an identical mechanism to that described for wtCFTR. Finally, CFTR-dependent insertion and retrieval of cell membrane was unaffected by the presence of the FLAG epitope. These results indicate that this structural alteration does not affect the control mechanisms for channel gating and suggest that the fourth extracellular loop of CFTR does not contribute to the ion pore. Detection of M2-901/CFTR by a commercially available monoclonal antibody (M2), together with presentation of normal functional properties, makes M2-901/CFTR a valuable tool to evaluate CFTR protein expression and cellular location.

Transport of phosphatidylcholine (PC) molecules across canalicular plasma membranes of the liver is essential for their secretion into bile. To test for evidence of protein-mediated translocation of natural long-chain PCs, we investigated whether hepatocyte membrane subfractions reconstituted into proteoliposomes promoted transmembrane translocation of radiolabeled PCs. Translocation of PC molecules in proteoliposomes was measured by an assay that employed multilamellar acceptor vesicles and the specific PC transfer protein purified from liver. As inferred from the percentage of radiolabel removed from proteoliposomes, facilitated PC translocation occurred in microsomes and canalicular and basolateral plasma membranes from rat liver but not in erythrocyte ghosts, microsomes, homogenates of COS and H35 cells, or Xenopus laevis oocytes. Heat denaturation in the presence of 2-mercaptoethanol and Pronase digestion of solubilized membrane proteins inhibited translocation. In contrast to the mdr2 gene product (Mdr2), which promotes ATP-dependent, verapamil-inhibitable PC translocation, ATP did not enhance and verapamil failed to block PC translocation. These data support the possibility that an ATP-independent PC translocator, possibly distinct from Mdr2, may be present in hepatocyte canalicular plasma membranes.

The role of P1 purinoceptor subtypes, the adenylyl cyclase (AC) pathway, and ATP-sensitive K+ (KATP) channels in adenosine (Ado)-induced membrane hyperpolarization was investigated in isolated segments of the guinea pig coronary artery using conventional microelectrode techniques. Ado (1-100 microM) elicited concentration-dependent hyperpolarization (half-maximal effective concentration 7.5 +/- 0.5 microM) that averaged 28.6 +/- 2.9 mV at 100 microM Ado. The A1 selective agonist N6-cyclopentyladenosine (CPA), the A1/A2 agonist 2-chloroadenosine, and the A2a/A2b agonist 5-(N-ethylcarboxamido)adenosine (NECA) each induced glibenclamide (3 microM)-sensitive hyperpolarization at 10 microM. However, the selective A2a-receptor agonists CGS-21680 and N6-[2-(3,5-dimethoxyphenyl)-2-(2-methoxyphenyl]ethyladenosine (10 microM each) were without effect. Responses to CPA and NECA were significantly reduced by the AC inhibitor SQ-22,536 (100 microM). Activation of the AC-adenosine 3',5'-cyclic monophosphate (cAMP)-protein kinase A (PKA) pathway by four additional methods, i.e., 1) forskolin (0.3-1 microM), 2) isoproterenol (0.1-1 microM), 3) combined milrinone (0.4 microM) and rolipram (30 microM), and 4) combined N6-phenyladenosine 3',5'-monophosphate, 8-(6-aminohexyl)aminoadenosine 3',5'-cyclic monophosphate, and the Sp-isomer of 5,6-dichloro-1-D-ribofuranosylbenzimidazole-3', 5'-cyclic monophosphothioate (100 microM each), also gave rise to glibenclamide-sensitive hyperpolarization. These results suggest that stimulation of A2b receptors coupled to AC represents the predominant mechanism by which Ado elicits hyperpolarization in this vessel. The ensuing increase in cAMP activates PKA, which then increases the activity of KATP channels. Our results further suggest that KATP channels are an important target for numerous pathways that raise cAMP levels in the coronary artery.

Skin from larval bullfrogs was mounted in an Ussing-type chamber in which the apical surface was bathed with a Ringer solution containing 115 mM K+ and the basolateral surface was bathed with a Ringer solution containing 115 mM Na+. Ion transport was measured as the short-circuit current (Isc) with a low-noise voltage clamp, and skin resistance (Rm) was measured by applying a direct current voltage pulse. Membrane impedance was calculated by applying a voltage signal consisting of 53 sine waves to the command stage of the voltage clamp. From the ratio of the Fourier-transformed voltage and current signals, it was possible to calculate the resistance and capacitance of the apical and basolateral membranes of the epithelium (Ra and Rb, Ca and Cb, respectively). With SO4(2-) as the anion, Rm decreased rapidly within 5 min following the addition of 150 U/ml nystatin to the apical solution, whereas Isc increased from 0.66 to 52.03 microA/cm2 over a 60-min period. These results indicate that nystatin becomes rapidly incorporated into the apical membrane and that the increase in basolateral K+ permeability requires a more prolonged time course. Intermediate levels of Isc were obtained by adding 50, 100, and 150 U/ml nystatin to the apical solution. This produced a progressive decrease in Ra and Rb while Ca and Cb remained constant. With Cl- as the anion, Isc values increased from 2.03 to 89.57 microA/cm2 following treatment with 150 U/ml nystatin, whereas with gluconate as the anion Isc was only increased from 0.63 to 11.64 microA/cm2. This suggests that the increase in basolateral K+ permeability produced by nystatin treatment, in the presence of more permeable anions, is due to swelling of the epithelial cells of the tissue rather than the gradient for apical K+ entry. Finally, Cb was not different among skins exposed to Cl-, SO4(2-), or gluconate, despite the large differences in Isc, nor did inhibition of Isc by treatment with hyperosmotic dextrose cause significant changes in Cb. These results support the hypothesis that increases in cell volume activate K+ channels that are already present in the basolateral membrane of epithelial cells.

Verotoxin-producing Escherichia coli (VTEC) are pathogenic bacteria associated with diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. Verotoxins (VTs) elaborated by these organisms produce cytopathic effects on a restricted number of cell types, including endothelial cells lining the microvasculature of the bowel and the kidney. Because human intestinal epithelial cells lack the globotriaosylceramide receptor for VT binding, it is unclear how the toxin moves across the intestinal mucosa to the systemic circulation. The aims of this study were to determine the effects of VT-1 on intestinal epithelial cell function and to characterize VT-1 translocation across monolayers of T84 cells, an intestinal epithelial cell line. VT-1 at concentrations up to 1 microgram/ml had no effect on the barrier function of T84 monolayers as assessed by measuring transmonolayer electrical resistance (102 +/- 8% of control monolayers). In contrast, both VT-positive and VT-negative VTEC bacterial strains lowered T84 transmonolayer resistance (45 +/- 7 and 38 +/- 6% of controls, respectively). Comparable amounts of toxin moved across monolayers of T84 cells, exhibiting high-resistance values, as monolayers with VTEC-induced decreases in barrier function, suggesting a transcellular mode of transport. Translocation of VT-1 across T84 monolayers paralleled the movement of a comparably sized protein, horseradish peroxidase. Immunoelectron microscopy confirmed transcellular transport of VT-1, since the toxin was observed within endosomes and associated with specific intracellular targets, including the Golgi network and endoplasmic reticulum. These data present a mode of VT-1 uptake by toxin-insensitive cells and suggest a general mechanism by which bacterial toxins lacking specific intestinal receptors can penetrate the intestinal epithelial barrier.

A major shortcoming of renal proximal tubular cells (RPTC) in culture is the gradual modification of their energy metabolism from the oxidative type to the glycolytic type. To test the possible reduction of glycolysis by naturally occurring long-chain fatty acids, RPTC were cultured in a two-chamber system, with albumin-bound palmitate (0.4 mM) added to the basolateral chamber after confluency. Twenty-four hours of contact with palmitate decreased glycolysis by 38% provided that carnitine was present; lactate production was decreased by 38%, and the decrease in glycolysis resulted from a similar decrease of basolateral and apical net uptake of glucose. In contrast to the previously described effect of the nonphysiological oxidative substrate heptanoate, palmitate promoted a long-term decrease in lactate production and sustained excellent cellular growth. After 4 days of contact, decreased glycolysis was maintained even in the absence of carnitine and resulted from a decrease of basolateral uptake only, suggestive of long-term regulation different from the earlier effects. Thus, although cultured RPTC lost their oxidative phenotype, they exhibited a type of regulation (Randle effect) that is found in the oxidative-type but not in the glycolytic-type tissues, therefore unmasking a regulative capacity barely detectable in fresh RPTC. Low PO2 (50 mmHg in the apical chamber) could be a major cause of elevated glycolysis and could hinder the effects of palmitate.

In mammals and amphibians, increases in extracellular Ca2+ can activate bicarbonate secretion and other protective functions of gastric mucosa. We hypothesized that the recently cloned extracellular Ca(2+)-sensing receptor (CaR) is functioning in the gastric mucosa. In Necturus maculosus gastric mucosa, reverse transcription-polymerase chain reaction using primers based on previously cloned CaR sequences amplified a 326-bp DNA fragment that had 84% nucleotide sequence identity with the rat kidney CaR. Immunohistochemical localization of the CaR using specific anti-CaR antiserum revealed its presence on the basal aspect of gastric epithelial cells. In microelectrode studies of Necturus antral mucosa, exposure to elevated Ca2+ (4.8 mM) and the CaR agonists NPS-467 and neomycin sulfate resulted in significant hyperpolarizations of basal membrane electrical potentials and increases in apical-to-basal membrane resistance ratios. Circuit analysis revealed that these changes reflected specific decreases in basolateral membrane resistance. Inhibition of prostaglandin synthesis using indomethacin significantly attenuated these effects. We conclude that the CaR is present and functioning in Necturus gastric antrum.

Luminal addition of 20 mM NH4+ produced a rapid acidification of rabbit macula densa (MD) cells from 7.50 +/- 0.06 to 6.91 +/- 0.05 at an initial rate of 0.071 +/- 0.008 pH unit/s. In the luminal presence of 5 microM bumetanide, 5 mM Ba2+ or both, the acidification rate was reduced by 57%, 35% and 93% of control levels. In contrast, intracellular pH (pHi) recovery after removing luminal NH4+ was unaffected by bumetanide and Ba2+ but was sensitive to 1 mM luminal amiloride (71% inhibition). The bumetanide-sensitive acidification rate represents most certainly the NH4+ flux mediated by the apical Na+:K+ (NH4+):2Cl- cotransporter, but the Ba(2+)-sensitive portion does not seem to be associated with the apical K+ channels previously characterized by us. The effects of NH4+ entry across the apical membrane were simulated using a simple model involving five adjustable parameters: apical and basolateral permeabilities for NH4+ and NH3 and a parameter describing a pH-regulating mechanism. The model shows that the apical membrane of MD cells is much more permeable to NH3 than it is to NH4+ and, under control conditions, the apical NH4+ flux appears surprisingly high (11-20 mM/s) and challenges the notion that MD cells present a low intensity of ionic transport.

During a survey of dipeptides that might be transported by the renal PEPT2 transporter in proximal tubule cells, we discovered that acidic dipeptides could stimulate transient secretory anion current and conductance increases in intact cell monolayers. The stimulatory effect of acidic dipeptides was observed in several proximal tubule cell lines that have been recently developed by immortalization of early proximal tubule primary cultures from the Wistar-Kyoto and spontaneously hypertensive rat strains and humans, suggesting that this phenomenon is a characteristic of proximal tubule cells. The electrical current induced in intact monolayers by Ala-Asp, a representative of these acidic dipeptides, must represent Cl- secretion rather than Na+ or H+ absorption, because 1) it was Na+ independent, 2) it showed a pH dependence different from that of the PEPT2 cotransporter, and 3) it correlated with an Ala-Asp-induced increase in Cl- conductance of the apical membrane in basolaterally amphotericin B-permeabilized monolayers. The secretory current could be inhibited by stilbene disulfonates, but not diphenylamine-2-carboxylates, suggesting a non-cystic fibrosis transmembrane conductance regulator type of Cl- conductance. The effect of Ala-Asp was dose dependent, with an apparent 50% effective concentration of approximately 1 mM. Ala-Asp also produced intracellular acidification, suggesting that acidic dipeptides are also substrates for an H(+)-peptide cotransporter.

The subcellular spatial and temporal organization of agonist-induced Ca2+ signals was investigated in single cultured vascular endothelial cells. Extracellular application of ATP initiated a rapid increase of intracellular Ca2+ concentration ([Ca2+]i) in peripheral cytoplasmic processes from where activation propagated as a [Ca2+]i wave toward the central regions of the cell. The average propagation velocity of the [Ca2+]i wave in the peripheral processes was 20-60 microns/s, whereas in the central region the wave propagated at < 10 microns/s. The time course of the recovery of [Ca2+]i depended on the cell geometry. In the peripheral processes (i.e., regions with a high surface-to-volume ratio) [Ca2+]i declined monotonically, whereas in the central region [Ca2+]i decreased in an oscillatory fashion. Propagating [Ca2+]i waves were preceded by small, highly localized [Ca2+]i transients originating from 1- to 3-micron-wide regions. The average amplitude of these elementary events of Ca2+ release was 23 nM, and the underlying flux of Ca2+ amounted to approximately 1-2 x 10(-18) mol/s or approximately 0.3 pA, consistent with a Ca2+ flux through a single or small number of endoplasmic reticulum Ca(2+)-release channels.

The BeWo cell line (b30 clone) has been examined as a potential in vitro system to study transplacental transport. At the light and electron microscope level, the cells were observed to form confluent monolayers on polycarbonate filters in approximately 5 days and morphologically resembled the typical human trophoblast. BeWo monolayers developed a modest transepithelial electrical resistance and a molecular size-dependent permeability to hydrophilic passive diffusion markers, fluorescein, and selected fluorescein-labeled dextrans. Linoleic acid permeation across BeWo monolayers was asymmetric, saturable, and inhibited by low temperature and excess competing fatty acid. Forskolin and 8-bromoadenosine 3',5'-cyclic monophosphate treatments stimulated morphological changes in BeWo cultures and enhanced the asymmetric passage of linoleic acid across the BeWo monolayers while having minimal effects on passive permeability, affirming that the differentiation state of the cells can influence membrane transporters and transmonolayer permeability. The basic permeability properties of the BeWo monolayers suggest that the cells grown on permeable supports may be examined as a convenient in vitro system to evaluate some transplacental transport mechanisms.

Calcium entry in nonexcitable cells occurs through Ca(2+)-selective channels activated secondarily to store depletion and/or through receptor- or second messenger-operated channels. In amphibian liver, hormones that stimulate the production of adenosine 3',5'-cyclic monophosphate (cAMP) also regulate the opening of an ion gate in the plasma membrane, which allows a noncapacitative inflow of Ca2+. To characterize this Ca2+ channel, we studied the effects of inhibitors of voltage-dependent Ca2+ channels and of nonselective cation channels on 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP)-dependent Ca2+ entry in single axolotl hepatocytes. Ca2+ entry provoked by 8-BrcAMP in the presence of physiological Ca2+ followed first-order kinetics (apparent Michaelis constant = 43 microM at the cell surface). Maximal values of cytosolic Ca2+ (increment approximately 300%) were reached within 15 s, and the effect was transient (half time of 56 s). We report a strong inhibition of cAMP-dependent Ca2+ entry by nifedipine [half-maximal inhibitory concentration (IC50) = 0.8 microM], by verapamil (IC50 = 22 microM), and by SK&F-96365 (IC50 = 1.8 microM). Depolarizing concentrations of K+ were without effect. Gadolinium and the anti-inflammatory compound niflumate, both inhibitors of nonselective cation channels, suppressed Ca2+ influx. This "profile" indicates a novel mechanism of Ca2+ entry in nonexcitable cells.

PC-12 cells depolarize during hypoxia and release dopamine. The hypoxia-induced depolarization is due to inhibition of an O2-sensitive K+ current. The role of dopamine released during hypoxia is uncertain, but it could act as an autocrine to modulate membrane conductance during hypoxia. The current study was undertaken to investigate this possibility. Reverse transcription-polymerase chain reaction and sequence analysis revealed that the D2 isoform of the dopamine receptor is expressed in rat PC-12 cells. Exogenously applied dopamine and the D2 agonist quinpirole elicited inhibition of a voltage-dependent K+ current (I(K)) that was prevented by sulpiride, a D2 receptor antagonist. Dopamine and quinpirole applied during hypoxia potentiated the inhibitory effect of hypoxia on I(K). We also found that quinpirole caused reversible inhibition of a voltage-dependent Ca2+ current (I(Ca)) and attenuation of the increase in intracellular free Ca2+ during hypoxia. Our results indicate that dopamine released from PC-12 cells during hypoxia acts via a D2 receptor to "autoregulate" I(K) and I(Ca).

The role of water and electrolyte influxes in anoxia-induced plasma membrane disruption was investigated using rabbit proximal tubule suspension. The results indicated that normal proximal tubule (PT) cells have a great capacity for expanding cell volume in response to water influx, whereas anoxia increases the susceptibility to water influx-induced disruption, and this was attenuated by glycine. However, resistance of anoxic plasma membranes to water influx-induced stress is not lost, although their mechanical strength was diminished, compared with normoxic membranes. Anoxic membranes did not disrupt under an intra-to-extracellular osmotic difference as great as 150 mosM. Potentiating or attenuating water influx by incubating PT cells in hypotonic or hypertonic medium, respectively, during anoxia, did not affect anoxia-induced membrane disruption. After the transmembrane electrolyte concentration gradient was eliminated by a "intracellular" buffer or by permeabilizing the plasma membrane to molecules <4 kDa using alpha-toxin, anoxia still caused further membrane disruption that was prevented by glycine or low pH. These results demonstrate that 1) water or net electrolyte influxes are probably not a primary cause for anoxia-induced membrane disruption and 2) glycine could prevent the plasma membrane disruption during anoxia independently from its effect on transmembrane electrolyte or water influxes. The present data support a biochemical rather than a mechanical alteration of the plasma membrane as the underlying cause of membrane disruption during anoxia.

The patch-clamp technique was used to study the effects of carbachol (CCh) on HT-29 cells. During CCh exposure, the cells (n = 23) depolarized close to the equilibrium potential for Cl- (E(Cl-); -48 mV) and the membrane potential then started to oscillate (16/23 cells). In voltage-clamp experiments, similar oscillations in whole cell currents could be demonstrated. The whole cell conductance increased from 225 +/- 25 pS in control solution to 6,728 +/- 1,165 pS (means +/- SE, n = 17). In substitution experiments (22 mM Cl- in bath solution, E(Cl-) = 0 mV), the reversal potential changed from -41.6 +/- 2.2 mV (means +/- SE, n = 9) to -3.2 +/- 2.0 mV (means +/- SE, n = 7). When the cells were loaded with the calcium-sensitive fluorescent dye, fluo 3, and simultaneously patch clamped, CCh caused a synchronous oscillating pattern of fluorescence and membrane potential. In cell-attached patches, the CCh-activated currents reversed at a relative membrane potential of 1.9 +/- 3.7 mV (means +/- SE, n = 11) with control solution in the pipette and at 46.2 +/- 5.3 mV (means +/- SE, n = 10) with a 15 mM Cl- solution in the pipette. High K+ (144 mM) did not change the reversal potential significantly (P < or = 0.05, n = 8). In inside-out patches, calcium-dependent Cl- channels could be demonstrated with a conductance of 19 pS (n = 7). It is concluded that CCh causes oscillations in membrane potential that involve calcium-dependent Cl- channels and a K+ permeability.

Exchange of physiological salt solution with Na(+)-free solution caused an increase in intracellular Ca2+ concentration ([Ca2+]i) in 86.3% of cultured airway epithelial cells within 75 s. [Ca2+]i returned to near baseline levels within 45 s and frequently showed oscillatory increases thereafter. When extracellular Na+ concentration ([Na+]o) was reduced to 10 and 60 mM, 59.0 and 8.0% of the cells increased [Ca2+]i, respectively. Low [Na+]o-induced increase in [Ca2+]i was not blocked by amiloride, benzamil, La3+, or the absence of extracellular Ca2+. Low [Na+]o-induced [Ca2+]i increase did not occur after thapsigargin treatment. These results indicated that low [Na+]o-induced [Ca2+]i increase is due to release of Ca2+ from intracellular stores. Because mechanical stimulation of a single cell causes a Ca2+ increase among many cells (Sanderson, M. J., A. C. Charles, and E. R. Dirksen. Mechanical stimulation and intercellular communication increases intracellular Ca2+ in epithelial cells. Cell Regul. 1: 585-596, 1990.) we assayed the effect of low [Na+]o on this mechanically induced response. In low [Na+]o, mechanically induced [Ca2+]i increase in the stimulated cell was reduced; however, [Ca2+]i increase in adjacent cells was normal. We suggest that a mechanically induced Na+ conductance in the stimulated cell contributes to [Ca2+]i changes. These signaling pathways may be involved in the maintenance of periciliary ion concentrations.