bluuebunny/crossref_metadata_2025 huggingface dataset:
Transepithelial acidification by cultures of rabbit proximal tubules grown on filters
Authors: Ford S. M. , Williams P. D. , Grassl S. , Holohan P. D.
Published: December 2017

Transepithelial acidification in the proximal tubule occurs by the simultaneous actions of the Na(+)-H+ exchanger in the brush border and the basolateral Na(+)-HCO3- cotransporter. The presence of these systems has been demonstrated for cultured cells; however, their contributions to the transepithelial movement of acid equivalents has not been confirmed in monolayers. To examine transepithelial acidification by intact cells, tubules were grown on membrane filters. Confluent cultures developed a transepithelial pH gradient within 6 h by decreasing the pH of medium in the apical chamber (6.66 +/- 0.03) while raising the basolateral pH to 7.40 +/- 0.02. Cells maintained on plastic did not acidify the medium during this time. Amiloride (10-100 microM) inhibited development of the gradient only when placed in the top chamber. 4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS; 10-100 microM), which inhibits basolateral Na(+)-HCO3- cotransport, decreased the gradient only when added to the bottom. These results demonstrate that cultured proximal tubule cells can develop a transepithelial pH gradient and that the polarized distribution of the transport systems is maintained in vitro.
Regulation of intracellular pH by cultured opossum kidney cells
Authors: Montrose M. H. , Murer H.
Published: December 2017

Opossum kidney (OK) cells (an epithelial cell line) were examined by flame photometry of cellular Na+ and K+ and by microfluorometric measurements of the intracellular pH (pHi) of single cells loaded with 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). The work concentrates on defining resting pHi values under different experimental conditions and examines factors that contribute to the maintenance of resting pHi. To use nigericin to calibrate the intracellular response of BCECF, cellular K+ levels were measured by a null point analysis, and the stability and magnitude of cellular Na+ and K+ levels were determined vs. time. Resting pHi in medium without added CO2 was high when measured by null point analysis of the population (pHi 7.6) and from measurements of single cells that have recovered from an acid load caused by NH4 prepulse (pHi 7.76 +/- 0.03, n = 20 cells). In single-cell measurements, addition of CO2-HCO3- to the medium results in cellular acidification of the steady-state pHi by 0.35 +/- 0.04 pH units. In medium equilibrated with room air, the resting pHi is shown to be a dynamic steady state composed of net flux due to apical Na(+)-dependent transport (Na(+)H+ exchange) plus acidifying processes. It is concluded that although 5-[N-ethyl-N-isopropyl]amiloride (EIPA) inhibits the forward reaction of Na(+)-H+ exchange, EIPA is either ineffective as an inhibitor of the reverse reaction of Na(+)-H+ exchange or Na(+)-H+ exchange does not reverse measurably in the OK cells.
Effects of substance P on [Ca2+]i and force in intact guinea pig ileal smooth muscle
Authors: Matthijs G. , Himpens B. , Peeters T. L. , Vantrappen G.
Published: December 2017

In intact sheets of the guinea pig ileal longitudinal muscle, loaded with fura-2, both substance P (SP)- and K(+)-induced contractile responses are preceded by cytoplasmic free Ca2+ ([Ca2+]i) variations. In response to SP (10(-10)-10(-7) M), [Ca2+]i and force increased concentration dependently. From 10(-9) M on, the response was biphasic: an initial Ca2+ spike and force transient were followed by a tonic component. The [Ca2+]i and force vs. log [SP] curves were sigmoidal for the initial phasic component, while a homologous receptor desensitization caused a reduced tonic component of the [Ca2+]i and contractile response at higher concentrations of SP. Both intracellular Ca2+ release and Ca2+ influx play a role in the effect of the peptide. In depolarizing solutions (140 mM K+) and in Ca2(+)-free medium (2 mM EGTA), SP induced a transient increase in [Ca2+]i and force. The Ca2+ stores used by SP and acetylcholine (ACh) overlap. [Ca2+]i and force fell to base-line levels when the extracellular Ca2+ was reduced from 1.2 to 0.2 mM during stimulation with SP. Verapamil reduced the tonic response. We also studied the relation between [Ca2+]i and force for the peak and steady-state values after stimulation with increasing concentrations of SP and K+. The phasic force response was linearly related to log [Ca2+]i. During the sustained response to K+, the Ca2+ sensitivity of the contractile/regulatory proteins was decreased, whereas no changes were observed during prolonged stimulation with the peptide.
Apical membrane Cl- channels in airway epithelia: anion selectivity and effect of an inhibitor
Authors: Li M. , McCann J. D. , Welsh M. J.
Published: December 2017

Previous work has investigated the anion selectivity of transepithelial Cl- secretion by airway epithelia and its inhibition by the Cl(-)-channel blocker 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB). Here we report the anion selectivity of the apical membrane and of the outwardly rectifying Cl- channel and the effect of NPPB on the Cl- channel. The anion selectivity sequence of the apical membrane determined with conventional microelectrodes in the native epithelium was SCN- greater than I- greater than Br- greater than NO3- approximately Cl- much greater than SO(4)2- approximately gluconate-. This contrasts with the observation that Cl- and Br- support transepithelial secretion but that I- does not. Thus the anion selectivity of transepithelial transport is determined by the basolateral membrane Cl- entry step. The anion selectivity of the outwardly rectifying Cl- channel studied in excised patches was the same as that of the apical membrane. We also found that NPPB reversibly blocked the outwardly rectifying Cl- channel from both the internal and external surfaces of the patch. NPPB, 10 microM, completely blocked the channel; lower concentrations caused a decrease in the probability of finding the channel in the open state. NPPB also caused the appearance of a subconductance state of the channel, an occurrence which is rarely observed in the absence of NPPB. These data provide further support for the conclusion that the outwardly rectifying Cl- channel is responsible for Cl- exit from the cell across the apical membrane.
Chloride transport across the membrane of parotid secretory granules
Authors: Gasser K. W. , Hopfer U.
Published: December 2017

The Cl- transport pathways in secretory granules isolated from the parotid glands of rats were characterized by the technique of ionophore-induced lysis in defined salt solutions. The granules were shown to possess a Cl- conductance that exhibited a distinct anion selectivity with a sequence I- greater than Br- greater than Cl- greater than F- greater than SO4(2-) much greater than gluconate-. This conductance could be reduced approximately 40% by the stilbene 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) from the cytoplasmic side; the half-maximal concentration for inhibition was 50 microM. Furthermore, the apparent Cl- conductance was reduced by outwardly directed granule H+ gradients and stimulated by inwardly directed gradients. An outwardly directed H+ gradient mimics the in vivo environment and may serve in a regulatory capacity, providing for a tonic inhibition of transport until the granule fuses with the luminal membrane. The granules also possessed a Cl(-)-HCO3- exchange based on electroneutrality of Cl- uptake and stimulation of this uptake by HCO3-. This pathway displayed a different anion selectivity, I- greater than Br- greater than F- greater than Cl- much greater than SO4(2-) much greater than gluconate-, and was not inhibited by SITS on the cytoplasmic side. The presence of these electrolyte transport pathways in the granule membrane is consistent with the production of primary fluid by parotid acinar cells after fusion of granules with the luminal plasma membrane.
A factor in serum lowers resistance and opens tight junctions of MDCK cells
Authors: Conyers G. , Milks L. , Conklyn M. , Showell H. , Cramer E.
Published: December 2017

During an inflammatory reaction, factors in blood affect the permeability of endothelium and possibly organ epithelium. In this study we partially characterized a factor in human and canine blood that lowered the transepithelial electrical resistance (TER) of canine kidney epithelial cells (MDCK) and examined whether vascular permeability factors [complement component C3a and C5a and platelet-activating factor (PAF)] were responsible for this reaction. C3a and C5a caused a small (10-13%) dose-related decrease in the TER (alpha = 0.05), whereas PAF had no effect. In contrast, the factor found in both serum and plasma caused a large (60-83%) dose-dependent decrease (saturated at 30%) in the TER that was reversible within 60 min. The blood factor, which does not appear to be albumin, was heat stable and has an apparent molecular mass of 67 kDa. It preferentially decreased the TER of the epithelium when it came in contact with its basolateral surface and significantly lowered the resistance within 60 min by opening the zonula occludentes. These findings suggest that C3a, C5a, and a factor in blood can directly modulate the permeability of renal epithelium.
PT pretreatment inhibits 48/80-induced activation of Ca(+)-permeable channels in rat peritoneal mast cells
Authors: Kuno M. , Kawaguchi J. , Mukai M. , Nakamura F.
Published: December 2017

We recently reported that the secretagogue, compound 48/80, activated Ca2(+)-permeable channels of mast cells possibly via a second messenger [Kuno, Okada, and Shibata. Am. J. Physiol. 256 (Cell Physiol. 25): C560-C568, 1989]. The effects of pretreatment with pertussis toxin (PT) on compound 48/80 (48/80)-induced activation of the Ca2(+)-permeable channel have now been investigated by measuring Ca2+ signals of cell suspensions using the Ca2+ indicator fura-2 and by recording Ba2+ currents through the channel using the patch-clamp technique. In the presence of extracellular Ca2+, the fluorescence change was biphasic, with an immediate rise and a delayed peak at room temperature. The delayed peak, mainly due to Ca2+ entry through plasma membranes, was greatly reduced by pretreatment with PT. The quenching of the fluorescence by 48/80-induced Mn2+ influx was also decreased by PT, whereas the Ca2+ transients due to Ca2+ release from the intracellular stores apparently did not change. In patch-clamp recordings from cell-attached patches with pipettes containing isotonic Ba2+, the 48/80-induced Ba2+ currents were either suppressed or delayed in the PT-treated cells, under conditions where degranulation was absent. These results suggest that PT-sensitive GTP-binding protein is involved in activating the Ca2(+)-permeable channel in mast cells during stimulus-secretion coupling.
Erythrocyte cation permeability induced by mechanical stress: a model for sickle cell cation loss
Authors: Johnson R. M. , Gannon S. A.
Published: December 2017

Human red blood cells were subjected to mechanical shearing in a Couette viscometer at 37 degrees C, using polyvinylpyrrolidone to increase the medium viscosity. At stresses greater than 300 dyn/cm2, movement of both Na and K down their concentration gradients was observed. The net rate of both monovalent cation fluxes appeared to be linear with applied stress in the range of 300-910 dyn/cm2. The applied shear forces caused no fragmentation of the cells. Observed hemolysis was slight. The observed cation fluxes are not a result of hemolysis because the amount of K released by the hemolyzed cells is quantitatively inadequate to account for the net K efflux, and there is a net uptake of Na by the stressed erythrocytes, which cannot be a consequence of hemolysis. The rates of net Na uptake and K efflux were nearly equal (ratio = 0.93 +/- 0.40, n = 6). The stress-induced permeabilities were reversible when shearing was halted. This work demonstrates the existence of cation permeability inducible in the red cell membrane by mechanical deformation, which may be a model for the sickling-induced monovalent cation exchange observed in deoxygenated sickle cells.
Role of polyamines in mitogenic and secretory responses of pancreatic beta-cells to growth factors
Authors: Sjoholm A. , Welsh N. , Sandler S. , Hellerstrom C.
Published: December 2017

We have investigated the effects of glucose and the polypeptide growth factor growth hormone (GH), platelet-derived growth factor (PDGF), insulin-like growth factor I (IGF-I), epidermal growth factor (EGF), and transforming growth factor alpha (TGF alpha) on the polyamine content, in relation to proliferation and insulin secretion and content, of pancreatic beta-cells. Fetal rat pancreatic islets containing a high proportion of beta-cells were cultured for 3 days with growth factors. beta-cell replication was significantly increased by glucose, GH, and PDGF plus IGF-I in parallel with increased islet polyamine contents. In contrast, neither EGF nor TGF alpha influenced the islet DNA synthesis rate, polyamine content, insulin content, or insulin accumulation in culture medium. When the increased polyamine content evoked by growth-promoting agents was prevented by inhibitors of polyamine synthesis, elevated DNA synthesis rates persisted or were even augmented. However, subcellular fractionation analysis of islet homogenates revealed that the nuclear polyamine content was not affected by the inhibitors. On the other hand, islet insulin content and glucose-regulated insulin release were decreased by polyamine synthesis inhibitors. Glucose oxidation rates remained unchanged, suggesting that inhibitors were not toxic to islet cells. We conclude that prevention of increases in total cellular content of polyamines in response to glucose, GH, or PDGF plus IGF-I does not prevent mitogenicity of these growth factors. However, when their synthesis is inhibited normal levels of polyamines seem to be maintained in the cell nucleus, an event that may be sufficient to permit a mitotic signal to be translated into a proliferative response.
Acute changes in channel density of amphibian diluting segment
Authors: Hurst A. M. , Hunter M.
Published: December 2017

Intracellular pH is a well-established modulator of the apical membrane potassium conductance of the amphibian diluting segment (early distal tubule). We investigated the modulation of this apical potassium conductance at the single-channel level in everted early distal tubules of the frog. Alkalinization of the bath fluid increased mean channel open probability (NPo) both in the presence and absence of the potassium-hydrogen ionophore nigericin. Reciprocal changes were seen with acidification. Because these effects were observed in cell-attached patches, where the composition of the fluid in the pipette is assumed to remain constant, the observed changes in channel activity were attributed to changes in intracellular pH. Further analysis of the data revealed that the changes in channel activity were produced exclusively by changes in the functional number of channels within the patch (N). We were unable to detect any significant changes in the single-channel open probability (Po). This suggests that the density of channels within a membrane may be far more dynamic than previously assumed.
VIP and forskolin enhance carbachol-induced K+ efflux from rat salivary gland fragments by a Ca2(+)-sensitive mechanism
Authors: Larsson O. , Detsch T. , Fredholm B. B.
Published: December 2017

The effect of vasoactive intestinal peptide (VIP) and forskolin on carbachol-induced K+ release from superfused rat submandibular and parotid gland fragments was examined using a K(+)-sensitive electrode. Carbachol (0.1, 1, and 10 microM) superfused over the glandular fragments for 15 min caused a concentration-dependent, transient elevation of K+ efflux, with a peak value after approximately 5 min. The carbachol-induced release of K+ could be divided into two distinct components, one transient peak lasting 5-8 min independent of extracellular Ca2+ and a second component of K+ release dependent on Ca2+ in the perfusion medium. VIP (1 microM) lacked effect on K+ efflux on its own but increased the carbachol (1 microM)-evoked K+ release. The VIP effects on K+ efflux were mimicked by forskolin (10 microM). Omission of Ca2+ from the medium totally abolished the augmenting effect of VIP and forskolin on carbachol-evoked K+ efflux. The Ca2+ ionophore A23187 (1 or 10 microM) induced a prolonged low-rate efflux of K+, which was dependent on Ca2+ in the medium. This effect of A23187 on K+ secretion was potentiated by forskolin (10 microM). The Na(+)-K(+)-ATPase blocker ouabain did not affect K+ release on its own, a lack of effect which remained following pretreatment with forskolin. It is concluded that VIP, by increasing the intracellular levels of cAMP in the glandular cell, potentiates carbachol-evoked Ca2(+)-dependent K+ efflux. These results may help to explain the synergistic effects of the coexisting transmitters VIP and acetylcholine.
Swelling-activated KCl cotransport in rabbit red cells: flux is determined mainly by cell volume rather than shape
Authors: Jennings M. L. , Schulz R. K.
Published: December 2017

The effect of cell shape on ouabain-insensitive 86Rb+ fluxes was examined in rabbit red blood cells. The purpose of the study was to assess the role of mechanical deformations of the membrane in the activation of KCl cotransport by cell swelling. Conversion of cells to echinocytes with low concentrations of amphiphilic agents (anionic and cationic detergents and dipyridamole) in an isotonic medium activates KCl cotransport only very slightly. Hypotonic swelling of echinocytes causes a large increase in KCl cotransport flux just as in swollen discocytes; both the rate and the extent of activation are unaffected by the shape change. Stomatocyte (cup cell) formation with 20 microM chlorpromazine in isotonic medium causes slight activation of KCl cotransport. The KCl cotransport flux induced by cell swelling is approximately 20% higher in swollen stomatocytes than in swollen discocytes. It is concluded that major changes in cell shape have only minor effects on the swelling sensor, signal transduction apparatus, and KCl cotransport protein. We interpret these findings as evidence against the idea that the cell detects its volume by way of a membrane-associated mechanical sensor. As an alternative to a mechanical volume sensor, a hypothetical mechanism for swelling activation of transport is presented in which dilution of the cytoplasm, by mass action alone, can have very large effects on KCl cotransport.
Relationship between vasopressin-sensitive water transport and plasma membrane fluidity in kidney collecting tubule
Authors: Fushimi K. , Verkman A. S.
Published: December 2017

The role of plasma membrane fluidity in the regulation of kidney tubule water permeability has been uncertain. We have used new methods to image the fluorescence anisotropy of fluidity-sensitive fluorophores (Fushimi, Dix, and Verkman. Biophys. J. 57: 241-254, 1990) to quantitate membrane fluidity in cells of the vasopressin-sensitive cortical collecting tubule (CCT) and water-impermeable cortical thick ascending limb (CTAL). Isolated tubule segments from rabbit kidney were perfused in vitro, and apical or basolateral plasma membranes were stained with trimethylammonium diphenylhexatriene (TMA-DPH). TMA-DPH anisotropy (r) was imaged quantitatively by an epifluorescence microscope equipped with rotatable polarizers; TMA-DPH nanosecond lifetime (tau) was measured by flash-lamp excitation and gated photomultiplier detection. In CCT, apical membrane r (0.254 +/- 0.003) was similar to basolateral r (0.252 +/- 0.005). Serosal vasopressin at a dose that increased water permeability greater than 10-fold (250 microU/ml) did not affect apical membrane r (delta r = 0.002 +/- 0.003; 7 tubules). A 0.002 change in r was less than that produced by a 2 degrees C temperature variation. In CTAL, apical membrane r was 0.249 +/- 0.002, similar to r from basolateral membrane of proximal tubule (0.24), but much less than that of proximal tubule apical membrane (0.29). These results establish methodology to quantitate fluidity in intact kidney tubule segments and provide the first measurements of plasma membrane fluidity in CTAL and CCT. Our results suggest that regulation of bulk membrane fluidity in CCT apical membrane is not a component of the hydrosmotic action of vasopressin and that low apical membrane fluidity is not responsible for the low water and NH3 permeabilities in CTAL.
Actions of mercurials on cell volume regulation of dissociated MDCK cells
Authors: Rothstein A. , Mack E.
Published: December 2017

The mercurial, p-chloromercuribenzoylsulfonate (PCMBS), blocks volume recovery of dissociated, osmotically swollen, Madin-Darby canine kidney cells (MDCK) and, at higher concentrations, induces substantial swelling. In the absence of Na+ the rate of volume recovery is, in contrast, substantially increased. PCMBS does not inhibit the "normal" volume-regulating pathways, K+ and Cl- conductances. Rather, its blocking action is due to substantial activation of Na+ and K+ permeabilities, especially the former. Consequently, the normal reshrinking mechanism, loss of KCl, is counterbalanced by PCMBS-induced gains of NaCl. In isotonic cells, PCMBS, at higher concentrations, induces cell swelling, indicating that Cl- permeability is also increased, a conclusion confirmed by direct measurement of 36Cl- fluxes. HgCl2 produces similar effects except that it is more potent and more rapid in its action. Activation of conductive ion permeabilities to Na+, K+, and Cl- are associated with appropriate changes in membrane potential. A small bumetanide-sensitive swelling component (Na(+)-Cl- cotransport) is activated by HgCl2 but not by PCMBS. Another effect is elevation of cytoplasmic Ca2+, apparently by mobilization from internal stores. Some of the functional sites (Na+ and K+) appear to be located externally, rapidly accessible to both HgCl2 and PCMBS, whereas others (Cl- and Ca2+) appear to be internal, rapidly accessible to the permeant HgCl2 but slowly to relatively impermeant PCMBS. In conclusion, the disturbances of volume regulation are largely due to the increases in conductive ion fluxes.
NaF-induced amylase release from rat parotid cells is mediated by PI breakdown leading to Ca2+ mobilization
Authors: Tojyo Y. , Tanimura A. , Matsui S. , Matsumoto Y. , Sugiya H. , Furuyama S.
Published: December 2017

We have examined the effects of sodium fluoride (NaF) on amylase release, cellular adenosine 3',5'-cyclic monophosphate (cAMP) level, inositol phosphate formation, and cytosolic free Ca2+ concentration ([Ca2+]i) in dispersed rat parotid acini or cells. At concentrations greater than 1 mM, NaF significantly increased amylase release. The maximum response was observed at 10 mM NaF and was comparable to that of the muscarinic-cholinergic agonist carbachol. Removal of extracellular Ca2+ with EGTA markedly suppressed the NaF-induced secretory response. At concentrations up to 10 mM, NaF did not increase the cellular level of cAMP, indicating that the NaF-induced amylase release is not mediated by cAMP. NaF (1-20 mM) caused a slow increase in [Ca2+]i in a concentration-dependent manner, as monitored with the fluorescent Ca2+ indicator fura-2, and the increased [Ca2+]i did not decline for at least 10 min after addition of NaF. In the absence of extracellular Ca2+, NaF evoked only a small and transient increase in [Ca2+]i. The addition of 10 mM NaF produced a significant accumulation of inositol monophosphate, inositol bisphosphate, and inositol trisphosphate. These results suggest that the NaF-induced amylase release is mediated by a breakdown of phosphoinositides leading to Ca2+ mobilization. The effects of fluoride may be through the action of F- on the GTP-binding protein(s) coupled to phospholipase C.
Dual effect of glycine on isolated rat suprachiasmatic neurons
Authors: Ito C. , Wakamori M. , Akaike N.
Published: December 2017

Pharmacological properties of strychnine-sensitive and -insensitive glycine receptors have been investigated in rat suprachiasmatic nucleus (SCN) neurons. Because the SCN neurons were too small for stable intracellular recordings by the glass-microelectrode technique, a conventional whole cell mode patch-clamp technique was employed on the acutely dissociated SCN neurons. Dissociated SCN neurons were morphologically heterogeneous and could be distinguished into several types. All cells responded to glycine in a concentration-dependent manner. The glycine-induced current was primarily Cl- sensitive and competitively blocked by strychnine. The SCN neurons also responded to excitatory amino acids: glutamate, quisqualate, kainate, and N-methyl-D-aspartate (NMDA). Responses to glutamate and aspartate, which are endogenous neurotransmitter candidates, were enhanced by adding glycine. Glycine especially augmented the maximum response to NMDA in a full concentration range. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) did not suppress the strychnine-sensitive glycine response but did suppress the strychnine-insensitive NMDA response in a competitive manner for glycine. The results suggest that glycine influences neural activity in the SCN as a classical inhibitory neurotransmitter and an excitatory neuromodulator.
Mitochondrial biogenesis in striated muscles: rapid induction of citrate synthase mRNA by nerve stimulation
Authors: Annex B. H. , Kraus W. E. , Dohm G. L. , Williams R. S.
Published: December 2017

Tonic contractile activity induces mitochondrial biogenesis in mammalian skeletal muscles, necessitating regulation of both nuclear and mitochondrial genes encoding mitochondrial proteins. In this study we compared the time course of induction of citrate synthase (CS) mRNA, a nuclear gene product, to that of genes encoded by mitochondrial DNA during the adaptive response to indirect nerve stimulation in tibialis anterior muscles of adult rabbits. A CS cDNA probe was prepared from a rabbit heart cDNA library by the polymerase chain reaction using synthetic oligonucleotide primers based on the published sequence of the porcine gene. This cDNA probe hybridized to a single band on Northern blots of total or polyadenylated RNA from adult rabbit tissues. Nerve stimulation for 3 days increased the abundance of CS mRNA relative to total cellular RNA by 2.3 +/- 0.2-fold (mean +/- SE, n = 8; P less than 0.01). In contrast, CS enzyme activity and mitochondrial RNA transcripts were not significantly increased at this time point. However, when nerve stimulation was continued for 21 days, the increases in CS mRNA and mitochondrial RNAs were similar. These results support the hypothesis that genetic signaling mechanisms triggered by neural input are sensed initially within the nucleus and that expression of mitochondrial genes is regulated as a secondary event.
Swelling-activated K-Cl cotransport: metabolic dependence and inhibition by vanadate and fluoride
Authors: O'Neill W. C.
Published: December 2017

Activation of K-Cl cotransport by cell swelling was studied by measuring K influx in isotonic and hypotonic media in human red blood cells after depletion of cellular ATP and after exposure to vanadate or fluoride. Preincubation of red blood cells with 2-deoxyglucose resulted in an inhibition of swelling-activated K-Cl cotransport that paralleled the decline in cellular ATP. Subsequent repletion of ATP by incubation in glucose, phosphate, and guanosine partially restored swelling-activated K-Cl cotransport. Swelling-activated K-Cl cotransport was also inhibited by 200 microM vanadate. This inhibition was partially blocked by DIDS, indicating an intracellular action, and required a 40-min preincubation, suggesting that inhibition was due to vanadyl rather than vanadate. Activation of K-Cl cotransport in swollen cells was also blocked by 16 mM fluoride, an effect that was immediate and independent of Cl concentration. Incubation of cells with 1 mM adenosine 3',5'-cyclic monophosphate (cAMP) to raise intracellular cAMP levels did not inhibit swelling-activated K-Cl cotransport, indicating that fluoride was not acting through adenyl cyclase. No inhibition of Cl-dependent or bumetanide-sensitive K influx in isotonic medium (Na-K-2Cl cotransport) was observed with ATP depletion, vanadate, fluoride, or cAMP. Activation of K-Cl cotransport by N-ethylmaleimide (NEM) was inhibited by ATP depletion but only partially inhibited by fluoride and not inhibited by vanadate. Fluoride inhibited K-Cl cotransport only when added before NEM treatment. These results suggest that activation of K-Cl cotransport by cell swelling requires ATP and involves a phosphohydrolase or phosphotransferase reaction that is inhibited by vanadyl and fluoride.(ABSTRACT TRUNCATED AT 250 WORDS)
Localization of alpha-isoforms of Na(+)-K(+)-ATPase in rat kidney by in situ hybridization
Authors: Farman N. , Corthesy-Theulaz I. , Bonvalet J. P. , Rossier B. C.
Published: December 2017

The expression of the three alpha-isoforms of Na(+)-K(+)-adenosine triphosphatase (ATPase) was examined in rat brain and rat kidney by Northern blot analysis. All three isoforms were detected in brain tissue while alpha 1-isoform was observed in whole kidney, suggesting that either this isoform was solely expressed in this organ or that alpha 2- and/or alpha 3-isoforms were not detected only because of their restricted distribution among a minority of specialized tubular cells. To distinguish between these two possibilities, in situ hybridization with rat alpha 1-, alpha 2-, and alpha 3-ATPase cRNA was performed on rat kidney sections. Results show that alpha 1-isoform expression largely predominates in the loop of Henle, distal tubule, and cortical collecting tubule. The labeling was drastically reduced by preincubation of sections with RNase. A sense cRNA probe, used as control, did not hybridize. With alpha 2- and alpha 3-probes, the labeling was low and uniformly distributed. In contrast, these two isoforms were clearly expressed in the brain, together with alpha 1. We conclude that only alpha 1-isoform of the Na(+)-K(+)-ATPase is detectable along the rat nephron. Its expression predominates in the tubular segments known to have a high Na(+)-K(+)-ATPase activity.
cAMP-activated C1 conductance is expressed in Xenopus oocytes by injection of shark rectal gland mRNA
Authors: Sullivan S. K. , Swamy K. , Field M.
Published: December 2017

Development of reliable expression systems for use in identification and functional characterization of proteins required for secretory Cl channel activity is key to understanding the molecular basis of cystic fibrosis (CF). Until now, heterologous expression of epithelial Cl channels had not been accomplished. We show here that Xenopus oocytes express an adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl conductance after injection of mRNA from shark rectal gland. Current through this conductance was rapidly activated by intracellular application of cAMP, reversed near the chloride equilibrium potential (ECl), blocked by the Cl channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoate, and was not affected by preincubation with the intracellular calcium buffer bis-(2-amino-5-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester, a condition that prohibits activation of the endogenous Ca-activated Cl conductance.
Bombesin-like peptides induce Ca2(+)-activated K+ conductance increases in mouse fibroblasts
Authors: Kusano K. , Gainer H.
Published: December 2017

Peptide receptor-activated membrane currents were studied in two mouse fibroblast cell lines, Swiss and Balb/c 3T3 cells, using a patch-electrode voltage-clamp technique. About 50% of the Swiss 3T3 cells examined responded to bombesin (Bn; 10(-9) to 10(-6) M), either by inducing outward current flow or inward current flow at the membrane holding potential (Vh) of -60 mV. The outward current type was more common (approximately 70%) than the inward current type (30%). The Bn-induced outward current (IBn) was reversed as the Vh was held to more negative than -90 mV (avg reversal potential, Erev = -82 mV). This Erev was closer to the equilibrium potential for K+ and shifted by altering the extracellular-to-intracellular K+ concentration ratio, in a Nernst-like relationship. The chance of recording this type of IBn was greatly reduced when K+ conductance blockers were present in the bathing solution (i.e., tetraethylammonium, Ba2+) or in the pipette solution (i.e., Cs). It was also reduced by recording with the pipette containing 5-10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Application of Ca2+ ionophore A23187 (5 microM) induced a similar membrane current with conductance increase. Thus the outward IBn in Swiss 3T3 cells appears to be induced by the intracellular Ca2(+)-dependent K+ conductance increase. Applications of bradykinin (Bk), arginine vasopressin (AVP), neuromedin B (NmB), and gastrin releasing peptide (GRP) to Swiss 3T3 cells also induced receptor-activated currents similar to IBn. Balb/c 3T3 cells rarely generated outward currents in response to Bn, GRP, and NmB but did not respond to both AVP and Bk with outward current flows.
Thy-1 expresses two signals for apical localization in epithelial cells
Authors: Powell S. K. , Lisanti M. P. , Rodriguez-Boulan E. J.
Published: December 2017

Recent work has shown that anchoring via glycosyl phosphatidylinositol (GPI) results in apical targeting for a variety of endogenous and transfected plasma membrane proteins expressed in epithelial cells. To further determine the correlation between GPI anchoring and apical localization, we expressed GPI-anchored and secretory forms of Thy-1 in Madin-Darby canine kidney cells by transfection. Native GPI-anchored Thy-1, normally expressed in thymocytes and neurons, was localized to the apical surface. A truncated form of Thy-1, lacking 22 out of 31 hydrophobic amino acids at the COOH-terminus, was also constructed; this deletion blocked attachment of the GPI anchor and resulted in apical secretion of Thy-1. In combination with previous results, our observations indicate that Thy-1 contains apical targeting information in its protein sequence as well as in the GPI anchor.
Calcium transport, Ca2(+)-ATPase, and lipid order in rabbit ocular lens membranes
Authors: Delamere N. A. , Paterson C. A. , Borchman D. , King K. L. , Cawood S. A.
Published: December 2017

Calcium transport was monitored by measuring ATP-dependent 45Ca uptake into membrane vesicles prepared from rabbit lens cortex. Calcium-stimulated adenosinetriphosphatase (Ca2(+)-ATPase) activity was also measured in the same membrane preparation. Both uptake and Ca2(+)-ATPase activity were inhibited by vanadate. Calcium activation of the uptake process was similar to that of the Ca2(+)-ATPase. Calcium uptake was prevented by calcium ionophore A23187, suggesting that the calcium transported into the vesicles remains diffusible. The ATP-dependent calcium uptake probably represents the transport of calcium into “inside-out” membrane vesicles by the Ca2(+)-ATPase mechanism that normally shifts calcium outward from the lens cytoplasm. The temperature dependence of the Ca2(+)-ATPase and the calcium uptake process was determined. Because lipid order can influence Ca2(+)-ATPase function, we attempted to correlate calcium transport with the physical state of the membrane lipids. Infrared spectroscopy was used to determine the temperature dependence of the CH2 symmetric stretching frequency (an order parameter) in the lipids. A similarity was noted between the temperature-dependence curves for lipid order, Ca2(+)-ATPase, and calcium uptake rate. Entropy, enthalpy, and transition temperature calculated for the Ca2(+)-ATPase and calcium uptake process were in the same range as those parameters calculated for the lipid-phase transition.
[3H]bumetanide binding to mouse kidney membranes: identification of corresponding membrane proteins
Authors: Haas M. , Dunham P. B. , Forbush B.
Published: December 2017

Crude plasma membranes from whole mouse kidneys have two classes of [3H]bumetanide binding sites. High-affinity sites (K1/2 approximately equal to 0.04 microM; Bmax = 1-2 pmol/mg protein) are similar to those identified on dog kidney membranes (B. Forbush and H.C. Palfrey. J. Biol. Chem. 258: 11787-11792, 1983) both with respect to affinity and in that Na, K, and Cl are required for [3H]bumetanide binding. Low-affinity sites (K1/2 approximately equal to 1 microM; Bmax = 7-14 pmol/mg) are unaffected by removal of these ions; such sites are not seen with dog kidney. When mouse kidney membranes are photolabeled with 4-[3H]benzoyl-5-sulfamoyl-3-(3-thenyloxy)benzoic acid [( 3H]BSTBA), a photoreactive bumetanide analogue, specific incorporation of the label is seen in two regions. As with dog kidney [M. Haas and B. Forbush. Am. J. Physiol. 253 (Cell Physiol. 22): C243-C252, 1987], an approximately 150-kDa protein is labeled with high affinity (K1/2 approximately equal to 0.05 microM). This labeling also requires Na, K, and Cl and appears to correspond to the high-affinity [3H]bumetanide binding sites and to the Na-K-Cl cotransport system. A second peak of [3H]BSTBA photolabeling, centered at approximately 75 kDa, incorporates the label with lower affinity (K1/2 = 2-3 microM). The photolabeling at approximately 75 kDa is unaffected by Na, K, and Cl concentrations and thus may correspond, at least in part, to the low-affinity [3H]bumetanide binding sites. Western blot analysis of [3H]BSTBA-labeled mouse kidney membranes was performed using an antiserum raised to proteins of approximately 82 and approximately 39 kDa isolated from mouse Ehrlich ascites tumor cells using a bumetanide affinity gel (P. B. Dunham, F. Jessen, and E. K. Hoffmann. Proc. Natl. Acad. Sci. USA 87: 6828-6832, 1990). This antiserum cross-reacts with a approximately 150-kDa mouse kidney protein, the staining profile of which on Western blot corresponds very closely to the peak of specific [3H]BSTBA incorporation in this region. The antiserum also reacts with proteins in the range of 65-85 kDa, overlapping the low-affinity peak of [3H]BSTBA incorporation.
Hypercapnic acidosis and increased H2PO4- concentration do not decrease force in cat skeletal muscle
Authors: Adams G. R. , Fisher M. J. , Meyer R. A.
Published: December 2017

Peak tetanic tension was measured during acidosis resulting from either hypercapnia or repetitive tetanic stimulation in isolated, arterially perfused cat biceps brachii (predominantly fast twitch) or soleus (slow twitch) muscles. Phosphocreatine (PCr), Pi, intracellular pH (pHi), and extracellular pH (pHo) were monitored by 31P-nuclear magnetic resonance spectroscopy. During repetitive stimulation under normocapnic conditions (5% CO2, pHo 7.4) Pi increased, pHi decreased from 7.1 to 6.3, and there were significant correlations between both pHi and calculated [H2PO4-] vs. peak tetanic force in both muscle types. However, hypercapnic perfusion (70% CO2, pHo, 6.7, pHi 6.4-6.5) had no effect on peak tetanic force, and there was no significant correlation between pHi or [H2PO4-] during hypercapnia in either muscle. The results indicate that decreased peak tetanic force during repetitive stimulation is not directly due to changes in pHi or diprotonated phosphate.
Tyrosine phosphorylation and oxygen consumption induced by G proteins in neutrophils
Authors: Grinstein S. , Furuya W.
Published: December 2017

In neutrophils, receptor-mediated activation of the respiratory burst requires ATP, possibly for phosphotransferase reactions. The oxidative response is only partially inhibited by blockers of protein kinase C, suggesting the involvement of other kinases. Recent evidence has demonstrated activation of tyrosine phosphorylation in chemoattractant-stimulated cells. This effect is likely mediated by G proteins because it is obliterated by pretreatment with pertussis toxin. In this report we have attempted to correlate the respiratory burst and phosphotyrosine accumulation induced by activation of G proteins, accomplished by treatment of electroporated cells with nonhydrolyzable analogues of GTP. In cells stimulated with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) both responses displayed similar time course and concentration dependence. The guanine nucleotide selectivity sequence and the divalent cation requirements were also similar for both responses. These similarities suggest a relationship between tyrosine phosphorylation and the activation of the NADPH oxidase. GTP gamma S-induced phosphotyrosine accumulation was found to be inhibited by pretreatment of the cells with phorbol esters, underlining the existence of regulatory interactions between different signal transduction pathways in neutrophils.
Apical and basolateral membrane conductances in the TBM cell line
Authors: Horisberger J. D.
Published: December 2017

Cultured cell lines present several advantages over whole organ or ex vivo isolated epithelium for the physiological and biochemical study of epithelial transport. We have developed a new technique allowing for simultaneous intracellular and transepithelial electrophysiological measurements in the epithelium formed by a cultured cell line grown on thin collagen membranes. This technique was applied to the TBM 18/23 (toad bladder origin) cell line. The transepithelial and basolateral membrane potentials were -30 +/- 11 and -72 +/- 8 (SD) mV (n = 36), respectively. With the use of the effect of amiloride, which partially blocked the apical membrane conductance, and circuit analysis, the apical and basolateral membrane conductances were estimated to 0.7 +/- 0.1 and 2.8 +/- 0.4 mS/cm2, respectively. A sodium-selective conductive pathway was demonstrated in the apical membrane, and a barium-sensitive K(+)-selective conductance was shown to be present in the basolateral membrane. The basolateral membrane conductance was not modified by sudden inhibition of sodium transport by amiloride, but it was significantly reduced after a long-term decrease of Na+ transport. The cultured TBM cell line appears to be a convenient model to investigate the regulation of membrane ionic conductances in tight epithelia.
Phorbol ester inhibition of chicken intestinal brush-border sodium-proton exchange
Authors: Chang E. B. , Musch M. W. , Drabik-Arvans D. , Rao M. C.
Published: December 2017

Phorbol esters, specific activators of protein kinase C, inhibit amiloride-sensitive Na uptake from the mucosal medium in intact intestinal mucosa as well as in isolated chicken villus enterocytes. In isolated cells, maximal inhibition is observed at 60 s, and influx returns to control values within 15 min. This effect can be measured either as initial 22Na influx rates or by following changes in intracellular pH using the pH-sensitive fluorescent dye 5,6-carboxyfluorescein. The effects of amiloride and phorbol esters were not additive, suggesting inhibition of a common transport system, i.e., Na-H exchange. In brush-border membrane vesicles (BBMV) made from villus enterocytes, amiloride-sensitive Na-H exchange activity was significantly inhibited in phorbol ester-treated cells. The degree of inhibition of 22Na uptake by BBMV had the same time course and dose-effect relationship as phorbol ester-inhibited cellular Na uptake. Similarly, the time course of protein kinase C translocation from cytosol to particulate or brush-border membrane fractions correlated with Na uptake measurements made in whole cells and BBMV. These results suggest that protein kinase C activation in chicken villus enterocytes inhibits brush-border membrane Na-H exchange activity.
Agonist-induced frequency modulation of Ca2+ oscillations in salt gland secretory cells
Authors: Crawford K. M. , Stuenkel E. L. , Ernst S. A.
Published: December 2017

Oscillations in intracellular calcium concentration ([Ca2+]i) induced by the acetylcholine analogue carbachol (CCh) were characterized by microspectrofluorimetry of fura-2 in single secretory cells from the avian salt gland. The frequency of oscillations increased in graded fashion with [CCh] between 25 nM (2.7 +/- 0.6 min-1) and 250 nM (11.8 +/- 1.4 min-1), whereas the amplitude of the spikes was independent of [CCh]. An interperiod return to prestimulatory [Ca2+]i was generally seen only at very low (25 nM) CCh. Between 50 and 250 nM CCh, oscillations were associated with sustained elevated [Ca2+]i levels. The amplitude of the oscillatory spikes was found not to exceed that of initial spikes arising from prestimulatory [Ca2+]i, despite the dose-dependent [effective concentration at 50% (EC50) = 200 nM CCh] sustained rise in [Ca2+]i. At 1 microM CCh, oscillations gave way to a maximal sustained increase in [Ca2+]i. Reduction of [Ca2+]o to 1.5 microM during an oscillatory train or blockage of Ca2+ influx with Ni+ resulted in a reduction in sustained Ca2+i levels and in frequency, but not amplitude, of oscillations. A relationship between the sustained partial rise in [Ca2+]i derived from Ca2+ influx and the oscillatory frequency at a given [CCh] was further indicated by the lower frequency (P less than 0.01) of the early spikes in a train when interspike [Ca2+]i initially returned to near-basal levels. In some cells, oscillations were slow enough (less than 2 min-1) to resolve an interperiod of elevated baseline [Ca2+]i, showing that the latter can occur independent of the repetitive Ca2+ spikes. (ABSTRACT TRUNCATED AT 250 WORDS)
Inhibition of electrical slow waves and Ca2+ currents of gastric and colonic smooth muscle by phosphatase inhibitors
Authors: Ward S. M. , Vogalis F. , Blondfield D. P. , Ozaki H. , Fusetani N. , Uemura D. , Publicover N. G. , Sanders K. M.
Published: December 2017

The effects of calyculin A, a phosphatase inhibitor isolated from the marine sponge Discodermia calyx, on the electrical activity of colonic and gastric muscles were studied. Calyculin A reduced the amplitude and duration of slow waves, primarily by inhibiting the plateau component. Okadaic acid, another phosphatase inhibitor, also reduced the amplitude and duration of gastric slow waves. The mechanism of action of calyculin A was investigated by studying its effects on inward currents of isolated gastric and colonic myocytes. Calyculin A reduced the amplitude of the peak and the sustained components of the inward current. Okadaic acid had similar effects. These data suggest that phosphorylation of Ca2+ channels of gastrointestinal smooth muscles may inhibit Ca2+ currents. This mechanism may provide an important means of regulating the currents responsible for excitation-contraction coupling in these muscles.
Inositol phosphates and [Ca2+]i signals in a differentiating exocrine cell
Authors: Hildebrandt J. P. , Shuttleworth T. J.
Published: December 2017

Generation of inositol phosphates and changes in intracellular Ca2+ concentration [( Ca2+]i) upon muscarinic receptor activation were studied in isolated cells from the nasal salt gland of Anas platyrhynchos, comparing responses in the poorly differentiated cells from ducks drinking only tap water (naive cells) with the more fully differentiated actively secreting cells from ducks drinking 1% NaCl solution for 48 h before the experiment (stressed cells). On stimulation, naive cells showed a rapid five- to sevenfold increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], accompanied by similar changes in Ins(1,3,4,5)P4, whereas both values increased only twofold in stressed cells. [3H]quinuclidinyl benzilate binding experiments revealed that these differences in inositol phosphate production were correlated with differences in the numbers of muscarinic acetylcholine receptors. Continuous recordings of [Ca2+]i revealed that Ca2+ release from intracellular stores upon stimulation was similar in both cell types, but the sustained [Ca2+]i signal (dependent on Ca2+ entry) was three times more pronounced in stressed cells. The results suggest that the adaptive differentiation of salt gland cells is associated with the increased expression of the receptor-mediated Ca2+ entry mechanism. In addition, the high rate of phosphoinositide hydrolysis in naive cells upon receptor activation may have a significance in cell growth, proliferation, and differentiation, which are elements of the development of the salt transport capabilities in these cells.