bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of membrane trafficking in plasma membrane solute transport
Authors: Bradbury N. A. , Bridges R. J.
Published: December 2017

Cells can rapidly and reversibly alter solute transport rates by changing the kinetics of transport proteins resident within the plasma membrane. Most notably, this can be brought about by reversible phosphorylation of the transporter. An additional mechanism for acute regulation of plasma membrane transport rates is by the regulated exocytic insertion of transport proteins from intracellular vesicles into the plasma membrane and their subsequent regulated endocytic retrieval. Over the past few years, the number of transporters undergoing this regulated trafficking has increased dramatically, such that what was once an interesting translocation of a few transporters has now become a widespread modality for regulating plasma membrane solute permeabilities. The aim of this article is to review the models proposed for the regulated trafficking of transport proteins and what lines of evidence should be obtained to document regulated exocytic insertion and endocytic retrieval of transport proteins. We highlight four transporters, the insulin-responsive glucose transporter, the antidiuretic hormone-responsive water channel, the urinary bladder H(+)-ATPase, and the cystic fibrosis transmembrane conductance regulator Cl- channel, and discuss the various approaches taken to document their regulated trafficking. Finally, we discuss areas of uncertainty that remain to be investigated concerning the molecular mechanisms involved in regulating the trafficking of proteins.
Na+ transport pathways in secretory acinar cells: membrane cross talk mediated by [Cl-]i
Authors: Robertson M. A. , Foskett J. K.
Published: December 2017

Fluid secretion by epithelial cells can be modulated by agents that activate Cl- channels in the apical membrane. To sustain secretion, Cl- influx across the basolateral membrane must also be accelerated. To examine the cellular mechanisms that couple Cl- efflux across the apical membrane to Na(+)-coupled Cl- entry across the basolateral membrane, we employed optical imaging techniques, utilizing single rat salivary acinar cells. Na+ influx was negligible in resting cells but was rapidly increased by carbachol due to activation of a Na(+)-H+ exchanger, a Na(+)-K(+)-2Cl- cotransporter, and, most likely, a nonselective cation channel. Receptor stimulation was not necessary, since elevation of intracellular Ca2+ concentration ([Ca2+]i) by thapsigargin activated the Na+ transporters at equivalent rates. Cell acidification, activation of protein kinase C, cell shrinkage, and other events associated with the rise of [Ca2+]i had little effect on Na+ transport in resting cells. Nevertheless, stimulation of cells in a medium that prevented normal Ca(2+)-induced cell shrinkage prevented activation of all three transport pathways. The block of the activation was not overcome by osmotic shrinkage but was relieved when [Cl-]i was allowed to fall, including conditions in which [Cl-]i fell in the absence of cell shrinkage. Activation of a Na(+)-H+ exchanger, Na(+)-K(+)-2Cl- cotransporter, and nonselective cation channel therefore exhibits a requirement for agonist-induced fall in [Cl-]i. Low [Cl-]i may create a permissive environment for Ca(2+)-dependent activation of multiple Na(+)-transport pathways, providing a mechanism for cross talk that coordinates transport activities of the apical and basolateral membranes in secretory epithelial cells.
31P-MRS measurements of extracellular pH of tumors using 3-aminopropylphosphonate
Authors: Gillies R. J. , Liu Z. , Bhujwalla Z.
Published: December 2017

The extracellular pH (pHex) of tumors is generally acidic. However, it is only recently that noninvasive magnetic resonance spectroscopic (MRS) measurements have determined that the intracellular pH (pHin) of tumor cells in situ is neutral or slightly alkaline compared with that of normal tissues. Thus cells in tumors maintain larger pH gradients than do cells in nontumor tissues. To date, measurements of pHex in tumors have been made using microelectrodes, which preclude measurement of pHex and pHin within the same preparation. In addition, microelectrodes are invasive and have the potential to alter the measured pH values. The present communication describes simultaneous measurement of pHex and pHin in vitro in bioreactor culture and in vivo using 31P-MRS analyses of 3-aminopropylphosphonate (3-APP) and inorganic phosphate. In vitro results indicate that 3-APP is not toxic and that its resonant frequency is sensitive to pH and not significantly affected by temperature or ionic strength. Bioreactor experiments indicate that this compound is neither internalized nor metabolized by cells. Experiments in vivo indicate that 3-APP can be administered intraperitoneally and that RIF-1 tumors maintain a steady-state pHin of 7.25 and a pHex of 6.66. These data have significance to basic tumor cell physiology and to the design of approaches to cancer chemotherapy and hyperthermic therapy, because both of these modalities exhibit pH sensitivity. It is also likely that these techniques will be applicable to localized MRS of other organ systems in vivo.
Mitochondrial biogenesis and development of respiratory chain enzymes in kidney cells: role of glucocorticoids
Authors: Djouadi F. , Bastin J. , Gilbert T. , Rotig A. , Rustin P. , Merlet-Benichou C.
Published: December 2017

We have previously shown that the activity of several mitochondrial oxidative enzymes increased greatly in the medullary thick ascending limb of Henle's loop (MTAL) of developing rat kidney between 16 days after birth and the adult stage. These changes were triggered by the postnatal rise in circulating glucocorticoids. To determine whether these increases are related to a mitochondrial biogenesis we have studied the changes in mitochondrial density of MTAL cells and mitochondrial DNA content in the inner stripe of the outer medulla during the postnatal period. The activities of respiratory chain (RC) complexes II and IV, located in the inner mitochondrial membrane (IMM), were also assayed, and developmental changes in the ultrastructure of the IMM were quantified. Quantitative electron-microscopic data showed a doubling in mitochondrial density from day 16 to the adult, accompanied by twofold increase in mitochondrial DNA, as determined by slot-blot experiments. The surface density of IMM increased by 77%, and there was a concomitant large rise in the activity of both RC enzymes. A possible role of glucocorticoids on the regulation of mitochondrial biogenesis was examined in similar experiments performed in adrenalectomized rat pups. The data demonstrated that glucocorticoids are essential for the rise in RC enzymes and the development of IMM but do not regulate postnatal changes in mitochondrial density and mitochondrial DNA content. Finally, ontogenic changes in oxidative capacities of MTAL cells could be a critical factor in the development of kidney urine concentrating mechanisms in weanling rats.
Adenosine transport in cultured human umbilical vein endothelial cells is reduced in diabetes
Authors: Sobrevia L. , Jarvis S. M. , Yudilevich D. L.
Published: December 2017

Adenosine transport in cultured human umbilical vein endothelial cells (HUVEC) was characterized and shown to be mediated by a single facilitated diffusion mechanism. Initial rates of adenosine influx at 22 degrees C were saturable [apparent Michaelis constant, 69 +/- 10 microM; maximum velocity (Vmax), 600 +/- 70 pmol.10(6) cells-1.s-1] and inhibited by nitrobenzylthioinosine (NBMPR). Formycin B had an unusually high affinity [inhibitory constant (Ki), 18 +/- 4.3 microM], whereas inosine had a low affinity (Ki, 440 +/- 68 microM) and nucleobases were without effect on adenosine influx. The number of transporters (1.2 x 10(6) sites/cell) was estimated by NBMPR equilibrium binding (apparent dissociation constant, 0.11 +/- 0.01 nM; maximum binding, 2.0 +/- 0.15 pmol/10(6) cells). In addition, we compared these endothelial cells with those obtained from cords from pregnancies complicated by diabetes (HUVEC-D), since embriopathy may occur in these conditions. HUVEC-D exhibited a 2.3-fold reduction in both the Vmax for adenosine influx and the maximum number of NBMPR binding sites (260 +/- 40 pmol.10(6) cells-1.s-1 and 0.86 +/- 0.08 pmol/10(6) cells, respectively). However, the turnover number for each nucleoside transporter in normal and diabetic HUVEC was similar (approximately 300 adenosine molecules/s). Adenosine metabolism at 10 microM in HUVEC-D was modified compared with normal cells. Intracellular phosphorylation (> 90%) was the predominant pathway in normal HUVEC, whereas in HUVEC-D, substantial levels of adenine and adenosine were detected. The present results demonstrate therefore the downregulation of the NBMPR-sensitive nucleoside transporter and changes in adenosine metabolism in HUVEC from diabetic pregnancies.
Tyrosine kinase involvement in IL-1 beta-induced expression of iNOS by beta-cells purified from islets of Langerhans
Authors: Corbett J. A. , Kwon G. , Misko T. P. , Rodi C. P. , McDaniel M. L.
Published: December 2017

Nitric oxide is believed to mediate the inhibitory effects of cytokines on glucose-stimulated insulin secretion by both rat and human islets. The aims of this study were 1) to determine the cellular source of the cytokine-inducible isoform of nitric oxide synthase (iNOS) expressed in islets following cytokine stimulation and 2) to determine whether tyrosine kinase activity participates in cytokine-induced iNOS expression. In this report we demonstrate that the cytokine interleukin-1 beta (IL-1 beta) stimulates the expression of iNOS and the formation of nitric oxide (as determined by nitrite formation, a stable oxidative product of nitric oxide) by isolated intact rat islets and by primary beta-cells purified by fluorescence-activated cell sorting (FACS). Both the expression of iNOS and nitrite formation induced by IL-1 beta were prevented by the mRNA transcriptional inhibitor actinomycin D. IL-1 beta did not induce the expression of iNOS by FACS-purified alpha-cells, the other major endocrine cell type of the islet. The tyrosine kinase inhibitors genistein and herbimycin A prevented IL-1 beta-induced expression of immunoprecipitable iNOS and nitrite release by islets, by insulinoma RINm5F cells, and by FACS-purified beta-cells. Herbimycin A and genistein also prevented IL-1 beta-induced iNOS mRNA accumulation as determined by Northern blot analysis of total RNA isolated from RINm5F cells. These findings indicate tyrosine kinase activation participates in IL-1 beta-induced expression of iNOS by the insulin-secreting beta-cell.(ABSTRACT TRUNCATED AT 250 WORDS)
Effects of tetrodotoxin-induced neural inactivation on single muscle fiber metabolic enzymes
Authors: Michel R. N. , Cowper G. , Chi M. M. , Manchester J. K. , Falter H. , Lowry O. H.
Published: December 2017

Selected enzymes were measured in mixed-fiber bundles and individual fibers from rat plantaris (PL) and soleus (Sol) muscles that had undergone either 2 wk of tetrodotoxin (TTX) inactivation of the sciatic nerve, a sham operation, or were contralateral to the TTX limb. TTX disuse caused severe wasting of PL (46%) and Sol (26%) muscles and of single fibers (50% and 40%, respectively). TTX PL and Sol also had reduced (50%) glycogen content. In TTX, PL, and Sol macro samples and single fibers, the activities (mol.h-1.kg dry wt-1) of hexokinase, glycogen phosphorylase, and lactate dehydrogenase were higher, lower, and unchanged, respectively, compared with controls. Single-fiber data showed that these changes occurred in all fibers. In TTX PL macro samples, activities of glycerol-3-phosphate dehydrogenase (GPDH), pyruvate kinase (PK), malate dehydrogenase (MDH), citrate synthase (CS), beta-hydroxyacyl-CoA dehydrogenase (BOAC), and thiolase were, or tended to be, lower. Single-fiber data showed a disappearance of high-oxidative moderate glycolytic fibers (i.e., usually fast-twitch oxidative in control) and the appearance of more fibers with a metabolic enzyme profile approaching that of control slow-oxidative fibers. In TTX Sol macro samples, GPDH and PK tended to be higher, and thiolase, BOAC, CS, and MDH lower. Single-fiber data corroborated these findings and suggested the appearance of fast fibers with downregulated oxidative enzyme profiles. Our results suggest that neuromuscular activity is a major, but not the sole, determinant of the size and metabolic heterogeneity that exists in muscle cells.
ATP depletion rather than mitochondrial depolarization mediates hepatocyte killing after metabolic inhibition
Authors: Nieminen A. L. , Saylor A. K. , Herman B. , Lemasters J. J.
Published: December 2017

The importance of ATP depletion and mitochondrial depolarization in the toxicity of cyanide, oligomycin, and carbonyl cyanide m-cholorophenylhydrazone (CCCP), an uncoupler, was evaluated in rat hepatocytes. Oligomycin, an inhibitor of the reversible mitochondrial ATP synthase (F1F0-adenosinetriphosphatase), caused dose-dependent cell killing with 0.1 microgram/ml being the minimum concentration causing the maximum cell killing. Oligomycin also caused rapid ATP depletion without causing mitochondrial depolarization. Fructose (20 mM), a potent glycolytic substrate in liver, protected completely against oligomycin toxicity. CCCP (5 microM) also caused rapid killing of hepatocytes. Fructose retarded cell death caused by CCCP but failed to prevent lethal cell injury. Although oligomycin (1.0 microgram/ml) was lethally toxic by itself, in the presence of fructose it protected completely against CCCP-induced cell killing. Cyanide (2.5 mM), an inhibitor of mitochondrial respiration, caused rapid cell killing that was reversed by fructose. CCCP completely blocked fructose protection against cyanide, causing mitochondrial depolarization and rapid ATP depletion. In the presence of fructose and cyanide, oligomycin protected cells against CCCP-induced ATP depletion and cell death but did not prevent mitochondrial depolarization. In every instance, cell killing was associated with ATP depletion, whereas protection against lethal cell injury was associated with preservation of ATP. In conclusion, protection by fructose against toxicity of cyanide, oligomycin, and CCCP was mediated by glycolytic ATP formation rather than by preservation of the mitochondrial membrane potential. These findings support the hypothesis that inhibition of cellular ATP formation is a crucial event in the progression of irreversible cell injury.
Calcium channels in excitable cells: divergent genotypic and phenotypic expression of alpha 1-subunits
Authors: Lievano A. , Bolden A. , Horn R.
Published: December 2017

The Ba2+ currents and mRNA levels of four members of the rat brain family of alpha 1-subunit Ca2+ channel genes were examined and compared in the rat cell lines GH3 and PC-12 and in the mouse lines NIE-115 and AtT-20. The RNA was measured with ribonuclease protection assays using probes derived from rat brain (rb) Ca2+ channel cDNAs (rbA, rbB, rbC, and rbD), and the Ba2+ currents were studied by whole cell patch-clamp recording. L-, N-, P-, and T-type currents were discriminated by the voltage dependence and pharmacological properties of Ba2+ currents. All cell lines expressed all four rat brain Ca2+ channel genes, except GH3 cells, which lacked rbB. The functional diversity of Ba2+ currents, however, was quite different among the cell lines. GH3 cells showed evidence of L- and T-type currents, undifferentiated PC-12 cells of L-type currents, AtT-20 cells of L-, N-, and P-type currents, and undifferentiated NIE-115 cells of a T-type current that was partially blocked by both nifedipine and BAY K 8644. Dimethyl sulfoxide-differentiated NIE-115 cells also had an L-type current. Differentiation of NIE-115 cells caused an increase in the levels of rbB, rbC, and rbD RNAs. Differentiation by nerve growth factor caused an increase in levels of all four genes in PC-12. Our data give further support for the assignment of rbA, rbB, and rbC/rbD gene products as components of P-, N-, and L-type Ca2+ channels, respectively.
Mechanical stretch/relaxation of cultured rat mesangial cells induces protooncogenes and cyclooxygenase
Authors: Akai Y. , Homma T. , Burns K. D. , Yasuda T. , Badr K. F. , Harris R. C.
Published: December 2017

In cultured rat glomerular mesangial cells, continuous cycles of stretching and relaxation (stretch/relaxation) stimulate cell proliferation, protein synthesis, and prostaglandin production. We examined regulation of gene expression that may underlie these alterations in cell functions. Stretch/relaxation caused time-dependent induction of the immediate early genes, c-fos and zif 268/egr-1, with maximal increases occurring between 15 and 30 min. The mitogen-inducible prostaglandin G2/H2 synthase (PGH2S-2) gene was also induced within 30 min of stretch/relaxation, with concomitant increases in the immunoreactive PGH2S-2 protein. These gene inductions were preceded by transient translocation of protein kinase C activity from cytosol to membrane as well as by increases in 45Ca2+ uptake and total cellular calcium content. The stretch/relaxation-induced expression was suppressed by protein kinase C inhibition, whereas less profound inhibition was observed with inhibition of calcium influx in low (100 nM) calcium buffer. These findings indicate that in mesangial cells mechanical stress induces expression of the protooncogenes and the mitogen-inducible cyclooxygenase primarily through protein kinase C-dependent mechanisms.
Voltage-dependent ionic conductances in Chinese hamster ovary cells
Authors: Skryma R. , Prevarskaya N. , Vacher P. , Dufy B.
Published: December 2017

Chinese hamster ovary (CHO) cells are becoming a widely used biological material. A number of studies report membrane ion conductance changes after transfection of channels and receptors, but there are few data available on the properties of membrane ion conductances of CHO cells before transfection. In this work we studied voltage-dependent ionic conductances in cultures of CHO native (CHO-K1) cells. Three types of voltage-dependent ionic conductances were identified: 1) a K+ conductance showing sensitivity to Ca2+ and a unit conductance of approximately 210 pS in symmetrical 150 mM K+ outside-out patches (this conductance, which did not inactivate during a 160-ms pulse, was inhibited by 30 nM charybdotoxin but not by 30 mM extracellular tetraethylammonium); 2) a rapidly activating and inactivating tetrodotoxin (TTX)-sensitive inward current, peaking at about -10 to 0 mV (this current showed characteristics similar in many respects to Na+ current recorded in neurons); and 3) another voltage-dependent inward current, which had slow inactivation, was TTX insensitive but was blocked by Co2+ (current was also carried by Ba2+, peaked at approximately 0 to +10 mV, was identified as a Ca2+ conductance, and was inhibited by dihydropyridines but not by 10 microM omega-conotoxin). Cell-attached patch recordings of single Ca2+ channel currents demonstrated a unitary conductance of approximately 20 pS.
Relationship between intra- and extracellular pH in primary cultures of rat astrocytes
Authors: Mellergard P. , Ou-Yang Y. , Siesjo B. K.
Published: December 2017

We studied the influence of extracellular pH (pHe) on the mechanisms regulating intracellular pH (pHi) in astrocytes cultured from neonatal rat cortex, using single cell microspectrofluorometry and the pH-sensitive fluorophore 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. When pHe was maintained at control values of 7.35 during acid transients caused by an increased CO2 tension, pHi was rapidly regulated back to normal. However, at pHe 6.9 or below, there was no recovery of pHi. Steady-state pHi was also strongly dependent on pHe (pHi = 1.14 + 0.80 pHe). The pHi recovery after normalization of pHe was very rapid, indicating that a prolonged exposure to a low pH stimulates pH-regulating mechanisms, and was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and amiloride or the removal of Na+. Recovery was also slowed down by Cd2+. The pHe-dependent acidification was not critically influenced by DIDS or amiloride and was not inhibited by tetrodotoxin, tetraethylammonium, Ba2+,2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)-quinoxaline, kynurenic acid, 5-hydroxysaclofen, bicuculline, Cd2+, or albumin. The results emphasize the importance of pHe for pHi regulation and suggest that in astrocytes pHi is not regulated back to normal levels until pHe is normalized.
Tension-voltage relations of single myocytes reflect Ca release triggered by Na/Ca exchange at 35 degrees C but not 23 degrees C
Authors: Vornanen M. , Shepherd N. , Isenberg G.
Published: December 2017

Contractile tension in response to 200-ms voltage-clamp pulses was measured in isolated guinea pig ventricular cells conditioned to constant Ca load. At 23 degrees C, the tension-voltage relation was bell shaped, decaying from a maximum at +20 mV to zero at +100 mV, but at 35 degrees C it was sigmoidal, with similar twitch tensions at +20 and +100 mV. Tension at 35 degrees C and +100 mV was reduced by ryanodine or caffeine and abolished by removal of Ca just before the test pulse. At 35 degrees C and +100 mV, twitch tension increased markedly as the Na concentration in the patch pipette ([Na]p) was varied between 0 and 20 mM. Cd (300 microM) blocked tension at all potentials at 23 degrees C, but tension remained in the presence of Cd at 35 degrees C (29% of control at +2 mV and 100% of control at +100 mV). Cd-resistant tension began to relax during the clamp pulse at all potentials (80 +/- 10 ms at +2 mV and 140 +/- 12 ms at +100 mV). Ni (3.6 mM) both reduced and slowed tension transients at all potentials. The results suggest that fast contractions due to sarcoplasmic reticulum Ca release can be triggered by Ca influx through either Ca current (ICa) or Na/Ca exchange and that those triggered through exchange are much more temperature sensitive than those triggered by ICa.
Dissociation of depolarization-activated and swelling-activated Cl- channels
Authors: Krouse M. E. , Haws C. M. , Xia Y. , Fang R. H. , Wine J. J.
Published: December 2017

In many cells, patch excision and depolarization induce outwardly rectifying Cl- channels (ORDIC channels) whose function and normal mode of regulation are unknown. One possible function is the mediation of swelling-activated Cl- conductance, because in many cells rectifying Cl- currents are activated by cell swelling. However, swelling-activated Cl- channels in some epithelia have larger conductances than ORDIC channels and inactivate more rapidly, although both have similar anion selectivity and are blocked by stilbenes. Thus it has not been possible to determine whether the two types of channel current arise from distinct proteins or alternate states of a single protein. We studied 14 cell lines and found 2 lines, C127 mouse mammary epithelial cells and IEC-6 rat intestinal crypt cells, with very low levels of ORDIC channels. However, despite the near absence of ORDIC channels in these rodent cells, a large swelling-activated Cl-conductance was demonstrated by whole cell, efflux, and single-channel methods. Thus it is likely that ORDIC and swelling-activated channel currents arise from different channel proteins.
Signal transduction in vascular smooth muscle: diacylglycerol second messengers and PKC action
Authors: Lee M. W. , Severson D. L.
Published: December 2017

Agonist-stimulated phospholipid turnover can generate diacylglycerol (DAG), an intracellular second messenger that activates protein kinase C (PKC). DAG can be produced from the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by a phosphoinositide-specific phospholipase C and by the degradation of phosphatidylcholine (PC) by a phospholipase C or the concerted actions of phospholipase D and phosphatidate phosphohydrolase. In vascular smooth muscle, agonist-stimulated DAG accumulation is biphasic; PIP2 hydrolysis produces a transient increase in DAG, which is followed by a sustained phase of DAG accumulation from PC degradation. Metabolism of DAG attenuates PKC activation and thus results in signal termination. The metabolic fates for DAG include 1) ATP-dependent phosphorylation to form phosphatidic acid (DAG kinase), 2) hydrolysis to release fatty acids and glycerol (DAG and monoacylglycerol lipases), 3) synthesis of triacylglycerol (DAG acyltransferase), and 4) synthesis of PC (choline phosphotransferase). Hydrolysis through the lipase pathway is the predominant metabolic fate of DAG in vascular smooth muscle. Activation of PKC in vascular smooth muscle modulates agonist-stimulated phospholipid turnover, produces an increase in contractile force, and regulates cell growth and proliferation. Further research is required to investigate cross talk between signal transduction mechanisms involving lipid second messengers. In addition, spatial considerations such as nuclear PKC activation and the influence of diradylglycerol generation on the duration of PKC activation are important issues.
Shear stress inhibits adhesion of cultured mouse endothelial cells to lymphocytes by downregulating VCAM-1 expression
Authors: Ando J. , Tsuboi H. , Korenaga R. , Takada Y. , Toyama-Sorimachi N. , Miyasaka M. , Kamiya A.
Published: December 2017

Monolayers of endothelial cells (EC) cultured from mouse lymph nodes were exposed to controlled levels of shear stress (0-7.1 dyn/cm2) in a parallel plate flow chamber, and binding between the flow-loaded EC and mouse lymph node-derived lymphocytes was assayed. A large number of lymphocytes adhered to the stationary control EC, but in EC exposed to a shear stress of 1.5 dyn/cm2 for 6 h, the adhesion decreased to 68.8 +/- 12.8% (SD; n = 19) of control (n = 29, P < 0.001). The decrease in adhesion induced by flow loading was time and shear stress dependent and reversible. Treatment of stationary EC with a monoclonal antibody (MAb) to vascular cell adhesion molecule-1 (VCAM-1) reduced the adhesion to 70.6 +/- 11.5% (n = 19) of control (P < 0.001), whereas MAb to CD44 and to intercellular adhesion molecule-1 had no effect on it. Flow cytometric analysis revealed that the amount of VCAM-1 expressed on the cell surface was decreased to 48.5 +/- 15.8% (n = 6) of control by flow loading (P < 0.001). Flow loading experiments using two perfusates with different viscosities demonstrated that the decrease in VCAM-1 expression due to flow was shear stress rather than shear rate dependent. The detection of mRNA by reverse transcriptase-polymerase chain reaction showed that VCAM-1 mRNA levels were markedly depressed in EC exposed to flow loading.(ABSTRACT TRUNCATED AT 250 WORDS)
pHi and serum regulate AE2-mediated Cl-/HCO3- exchange in CHOP cells of defined transient transfection status
Authors: Jiang L. , Stuart-Tilley A. , Parkash J. , Alper S. L.
Published: December 2017

Anion exchanger (AE) protein-mediated anion exchange contributes to regulation of intracellular pH (pHi), Cl- concentration, and volume in vertebrate cells. We have extended the functional characterization of recombinant AE2-mediated Cl-/HCO3- exchange in single Chinese hamster ovary cells stably transfected with the polyoma large T antigen (CHOP cells) of defined transient transfection status using a novel surface marker coexpression vector. Marker expression and detection had minimal effect on the low endogenous Cl-/HCO3- exchange activity of CHOP cells, whereas coexpression of marker with AE2 elevated CHOP cell Cl-/HCO3- exchange activity 16-fold. Between pHi of 7.3 and 7.8, AE2-mediated flux of proton equivalents was activated > 11-fold by increasingly alkaline pHi without reaching saturation. This activation may be secondary to allosteric effects of pHi on AE2, in parallel with the obligatory increase in substrate intracellular HCO3- concentration. Nominal removal of CO2/HCO3- reduced AE2 activity by 90%. Addition of 10% calf serum slowly activated AE2 activity severalfold. This activation was slowly reversed after serum removal. Surface marker coexpression vectors improve both the efficiency and reliability of studies of recombinant protein function for a wide range of single cell assays in many cell types.
Large Ca(2+)-activated K+ channels responsive to angiotensin II in cultured human mesangial cells
Authors: Stockand J. D. , Sansom S. C.
Published: December 2017

The patch-clamp method was used to determine the properties and response to angiotensin II (ANG II) of K+ channels in subpassages of human mesangial cell cultures. In cell-attached patches, with 140 mM KCl in the bath and cell potential equal to 40 mV, the open probability (Po) of large K+ channels (MKCa) was 0.8 with 0.5 mM Ca2+ in the bath and < 0.05 if the bath Ca2+ concentration was reduced to 1.0 microM. Open and closed dwell-time histograms of MKCa displayed both fast and slow time constants. Addition of ANG II (100 nM) to the bath solution (Ca2+ = 1.0 microM) increased the Po of MKCa in cyclic bursts by decreasing the time constant of the slow closed state. In excised inside-out patches, the mean single-channel conductance of MKCa was 206 pS in symmetrical 140 mM KCl. The selectivity sequence, established in asymmetrical cationic solutions, was K+ (1.0) > Rb+ (0.54) > NH+4 (0.11) > > Cs+ = Na+ (< 0.05). The Po of MKCa was increased by depolarizing potentials and high bath Ca2+. The Boltzmann distribution was consistent with an effective valence of 1.0, and the Hill coefficient for Ca2+ activation was 0.52. We conclude that MKCa has properties similar to large Ca(2+)-activated K+ channels and may act to repolarize the membrane of mesangial cells in response to an agonist-induced mobilization of intracellular Ca2+.
Intracellular Cl activity: evidence of dual mechanisms of cl absorption in sweat duct
Authors: Reddy M. M. , Quinton P. M.
Published: December 2017

The human sweat duct (SD) reabsorbs NaCl from lumen to blood over a wide range of luminal concentrations. The physiological strategies employed by the SD to cope with such extreme transport loads remain elusive. When we employed intracellular Cl-sensitive microelectrodes, we found that at high (150 mM) luminal NaCl concentrations ([NaCl]) transcellular Cl absorption occurs through passive diffusion that is evidenced by a large Cl conductance (GCl) in both cell membranes and by a favorable electrochemical driving force for Cl (delta psi Cl) across the apical and basolateral membranes. However, lowering the luminal [NaCl] to 15 mM markedly altered the electrochemical gradient for Cl and reversed the direction of delta psi Cl. Under these conditions, passive absorption of Cl was not feasible, so that Cl can only be absorbed by a nonconductive transport carrier. We surmise that, in the face of such changes in delta psi Cl as a function of luminal [NaCl], continuous transcellular Cl transport in SD could only be sustained if both electroconductive and carrier-mediated Cl transport are present in the SD.
Depletion of intracellular Ca2+ stores activates a Ca(2+)-selective channel in vascular endothelium
Authors: Vaca L. , Kunze D. L.
Published: December 2017

The present study was designed to identify the channel responsible for Ca2+ influx after depletion of intracellular Ca2+ stores. Different maneuvers that deplete intracellular Ca2+ stores activated a Ca(2+)-selective channel. Superfusion of single bovine aortic endothelial cells with 50 nmol/l bradykinin, 10 mumol/l ATP, or 10 mumol/l 2,5-di(tert-butyl)-1,4-benzohydroquinone produced activation of channels of the same amplitude in cell-attached patches. Channel activity declined within the first minute after patch excision. The channel showed strong inward rectification and a reversal potential of 0 mV in symmetrical sodium sulfate (Na2SO4) solution. Under these conditions, the conductance was 5 pS in the inward direction. Addition of 10 mmol/l Ca2+ to the extracellular solution shifted the reversal potential to +30 +/- 5 mV, and the conductance for inward current was 11 pS. The reversal potential was used to calculate an ion permeability ratio of Ca2+/Na+ > 10:1.
Acidosis and glucocorticoids concomitantly increase ubiquitin and proteasome subunit mRNAs in rat muscle
Authors: Price S. R. , England B. K. , Bailey J. L. , Van Vreede K. , Mitch W. E.
Published: December 2017

In rat muscle metabolic acidosis increases ATP-dependent protein degradation and levels of mRNAs for ubiquitin (Ub) and proteasome subunits. Because adrenalectomy (ADX) abolishes the proteolytic response to acidosis in muscle, we examined whether glucocorticoids (GCs) are necessary for acidosis-induced changes in Ub and proteasome mRNAs in muscles. Total RNA content of the white fiber extensor digitorum longus or mixed fiber gastrocnemius muscles were lowest in muscles of ADX rats given acid plus GCs. In contrast, the abundance of Ub and C2 and C9 proteasome subunits mRNAs were increased in muscles from this group compared with untreated ADX rats or ADX rats given acid or GCs alone. Because total RNA is reduced, the increase in these mRNAs in muscles of ADX rats receiving acid plus GCs provides evidence for a specific activation of the ATP-dependent-Ub-proteasome pathway. Thus, GCs are required but not sufficient to produce the coordinated increase in mRNAs encoding ubiquitin and proteasome subunits occurring in muscles of acidotic rats.
Coexisting beta-adrenoceptor subtypes: significance for thermogenic process in brown fat cells
Authors: Zhao J. , Unelius L. , Bengtsson T. , Cannon B. , Nedergaard J.
Published: December 2017

The possible significance of the coexisting beta 1-, beta 2-, and beta 3-adrenoceptors in brown adipose tissue for the thermogenic response was investigated. Oxygen consumption of isolated hamster brown fat cells was analyzed as a measure of thermogenesis. Thermogenesis could be evoked not only by the physiological agent norepinephrine but also by BRL-37344 and CGP-12177. No evidence for biphasic inhibition curves was found with either the selective beta 1-antagonist ICI-89406, the beta 2-antagonist ICI-118551, or the beta 1/beta 2-nonselective beta-antagonist propranolol against 1 microM norepinephrine; pI50 (the negative logarithm of the inhibitory constant for an antagonist, as estimated from the dose-response curve for an antagonist vs. a constant agonist concentration) values for ICI-89406 and ICI-118551 were very low (4-5), implying nonselective inhibition; the pI50 for propranolol was approximately 6 (as expected for the beta 3-receptor). Even with suboptimal norepinephrine, no biphasic inhibition was found. CGP-12177 at concentrations where it is primarily an antagonist to the beta 1-receptor did not influence the dose-response curve for either norepinephrine or BRL-37344. BRL-37344- or CGP-12177-induced thermogenesis was inhibited by the beta-antagonists in a manner similar to norepinephrine-induced thermogenesis. Schild plots for propranolol inhibition of norepinephrine-, isoprenaline-, BRL-37344- and CGP-12177-induced thermogenesis yielded similar pA2 (the negative logarithm of the inhibitory constant for an antagonist, as calculated from a series of agonist dose-response curves at different antagonist concentrations) (approximately 5.5), for interaction with either agonist, implying that the same receptor was stimulated by all agonists. Thus, despite the fact that different beta-receptor subtypes coexist in the tissue, we find no evidence for the participation of beta 1- or beta 2-receptors in the thermogenic response. Within the resolution of the experiments, the results therefore imply that it is predominantly or solely the beta 3-receptor that is coupled to thermogenesis, and it is via this beta-adrenergic receptor that not only norepinephrine but also CGP-12177 and BRL-37344 induce thermogenesis.
Redistribution of protein kinase C isoforms in association with vascular hypertrophy of rat aorta
Authors: Liou Y. M. , Morgan K. G.
Published: December 2017

Freshly enzymatically isolated cells from the aorta of a rat model of vascular hypertrophy were used to investigate the role of protein kinase C (PKC) isoforms during a physiologically relevant growth response. With the combination of immunofluorescence with digital imaging microscopy, PKC isoforms alpha, delta, and zeta were found to be present in single cells from control and hypertrophied rat aortas. The alpha- and zeta-isoforms were distributed in the cytoplasm of control cells; however, in hypertrophied cells, sustained translocations of alpha-PKC to the surface membrane and zeta-PKC to the intranuclear area were seen. delta-PKC was concentrated in the perinuclear area in control cells but appeared to translocate to a more diffuse localization in the cytosol of hypertrophied cells. Staining of mitochondria with rhodamine 123 indicated some similarity in the spatial distribution compared with that of delta-PKC. In control cells, translocation of isoforms alpha and delta was activated by phenylephrine or 12-deoxyphorbol 13-isobutyrate 20-acetate. Agonist stimulation produced translocation of no isoforms in the hypertrophied cells. These results indicate that isoform-specific spatial distribution and translocation of PKC occur in association with the growth response of vascular hypertrophy.
Inhibition of smooth muscle cell growth by nitric oxide and activation of cAMP-dependent protein kinase by cGMP
Authors: Cornwell T. L. , Arnold E. , Boerth N. J. , Lincoln T. M.
Published: December 2017

Recent studies indicate that nitric oxide (NO) and guanosine 3',5'-cyclic monophosphate (cGMP) may inhibit the proliferation of vascular smooth muscle cells (SMC) in vitro. The purpose of this study was to investigate the mechanism of NO- and cGMP-dependent inhibition of cultured rat aortic SMC. The cytokine interleukin-1 beta (IL-1 beta) inhibited serum- and platelet-derived growth factor-stimulated [3H]thymidine incorporation into DNA in subcultured rat aortic SMC. Incubation with IL-1 beta for 24 h markedly increased cGMP levels but not adenosine 3',5'-cyclic monophosphate (cAMP) levels. However, the IL-1 beta-induced increase in cGMP was correlated with an activation of the cAMP-dependent protein kinase (cAMP kinase) activity ratio. The activation of the cAMP kinase was prevented by treatments that blocked NO and cGMP production. The NO-generating vasodilator, S-nitroso-N-acetylpenicillamine (SNAP) also inhibited DNA synthesis and elevated cGMP levels. The inhibition of DNA synthesis by both IL-1 beta and SNAP was observed only when cGMP levels were elevated to high levels (10-fold or more). As was the case for IL-1 beta, SNAP increased the activity ratio of cAMP kinase. Selective inhibition of cAMP kinase using (R)-p-bromoadenosine 3',5'-cyclic monophosphorothioate prevented the inhibition of proliferation by IL-1 beta. By contrast, the inhibitor of the cGMP-dependent protein kinase, (R)-p-bromoguanosine 3',5'-cyclic monophosphorothioate, had no effect on IL-1 beta-induced inhibition of cellular proliferation. These studies suggest that cGMP-dependent activation of the cAMP kinase may be responsible in part at least for the NO-dependent inhibition of proliferation of subcultured rat aortic SMC.
Aminopyridine inhibition and voltage dependence of K+ currents in smooth muscle cells from cerebral arteries
Authors: Robertson B. E. , Nelson M. T.
Published: December 2017

Voltage-dependent K+ currents were characterized using the patch-clamp technique in smooth muscle cells isolated from rabbit cerebral (basilar) arteries. This study focused on the voltage dependence and the pharmacology of these K+ currents, since this information will be useful for the investigation of the role of the voltage-dependent K+ channels in arterial function. Currents through Ca(2+)-activated K+ (KCa) channels were minimized by buffering intracellular Ca2+ to low levels and by blockers (tetraethylammonium and iberiotoxin) of these channels. Membrane depolarization increased K+ currents, independent of changes in the driving force for K+ movement. With 140 mM internal and external K+, activation of K+ currents by membrane depolarization was half maximal at about -10 mV and increased as much as e-fold per 11 mV. Inactivation also depended on voltage, with a midpoint at -44 mV. 3,4-Diaminopyridine (3,4-DAP),4-aminopyridine(4-AP),3-amino-pyridine(3-AP), and 2-aminopyridine (2-AP) inhibited voltage-dependent K+ currents. At 0 mV, 3,4-DAP, 4-AP, 3-AP, and 2-AP (5 mM) inhibited the K+ currents by 84, 66, 36, and 8%, respectively. Phencyclidine (100 microM) inhibited the current by 53% at 0 mV. Steady-state whole cell currents through these channels were measured at physiological membrane potentials. At -40 mV, 4-AP (5 mM) reduced the steady-state outward current by 2.5 pA. These results are consistent with the idea that voltage-dependent K+ channels are involved in the regulation of the membrane potential of arterial smooth muscle.
Potassium cotransport at the rat choroid plexus
Authors: Keep R. F. , Xiang J. , Betz A. L.
Published: December 2017

The choroid plexuses are involved in cerebrospinal fluid (CSF) secretion and CSF K homeostasis. We examined K transport mechanisms present in the isolated rat choroid plexus that may be involved in these functions, predominantly using 86Rb as a marker for K. The study demonstrates that there are two primary uptake mechanisms. Ouabain-sensitive Na-K-adenosinetriphosphatase and bumetanide-sensitive cotransport, probably of the Na-K-2Cl form, account for 48 and 46% of uptake, respectively. Efflux studies demonstrate that the primary K efflux mechanism is also bumetanide-sensitive cotransport with the other major component probably being by K channels as it is inhibitable by barium or quinidine. Efflux via the cotransporter was not inhibited by R(+)-butylindazone, a KCl cotransport inhibitor, but it was enhanced in the presence of ouabain (P < 0.001) or increased extracellular Na concentration (P < 0.01). Furthermore, Na efflux was bumetanide sensitive (P < 0.05). In all, these data suggest that the efflux cotransporter is also of the Na-K-Cl form and that it is the same transporter as the influx mechanism operating in both directions. The evidence presented leads us to hypothesize that this cotransporter is on the apical membrane of the choroid plexus and that it may have a central role in CSF secretion and perhaps CSF K homeostasis.
K(+)-induced alkalinization in mouse cerebral astrocytes mediated by reversal of electrogenic Na(+)-HCO3- cotransport
Authors: Brookes N. , Turner R. J.
Published: December 2017

Raising extracellular K+ concentration ([K+]o) induces an alkaline shift of intracellular pH (pHi) in astrocytes. The mechanism of this effect was examined using the fluorescent pHi indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein in primary cultures of mouse cerebral astrocytes. Raising [K+]o from 3 to 12 mM increased pHi by 0.28 pH units in 26 mM HCO(3-)-buffered solution. In nominally HCO(3-)-free solution (containing approximately 95 microM HCO3-), the alkalinization fell to 0.21 pH units and further to 0.08 pH units on removal of atmospheric CO2, suggesting a process with high affinity for HCO3-. This effect was Na+ dependent, Cl- independent, and inhibited by 0.5 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, indicating the involvement of Na(+)-HCO3- cotransport. The relationship between pHi and log[K+]o was found to be linear and to predict a stoichiometry of at least two HCO3- transported with each Na+. After removal of exogenous CO2/HCO3-, the direction of changes in pHi elicited by adding 1 mM HCO3- showed that net flux of HCO3- via the Na(+)-HCO3- cotransporter was outward at rest and was reversed by depolarization.
Type IIx myosin heavy chain transcripts are expressed in type IIb fibers of human skeletal muscle
Authors: Smerdu V. , Karsch-Mizrachi I. , Campione M. , Leinwand L. , Schiaffino S.
Published: December 2017

Several members of the sarcomeric myosin heavy chain (MHC) gene family have been mapped in the human genome but many of them have not yet been identified. In this study we report the identification of two human skeletal MHC genes as fast IIa and IIx MHC based on pattern of expression and sequence homology with the corresponding rat genes in the 3'-translated and untranslated regions. The distribution of these two gene products as well as that of the beta/slow MHC gene was analyzed in human skeletal muscles by in situ hybridization. The distribution of beta/slow, IIa, and IIx MHC transcripts defines three major muscle fiber types expressing a single MHC mRNA, i.e., either beta/slow, IIa, or IIx MHC mRNA, and two populations of hybrid fibers coexpressing beta/slow with IIa or IIa with IIx MHC mRNA. Fiber typing by ATPase histochemistry shows that IIa MHC transcripts are more abundant in histochemical type IIa fibers, whereas IIx MHC transcripts are more abundant in histochemical type IIb fibers.
Interaction of cA-kinase and cG-kinase in mediating relaxation of dispersed smooth muscle cells
Authors: Murthy K. S. , Makhlouf G. M.
Published: December 2017

The signaling pathways mediating relaxation by vasoactive intestinal peptide (VIP), peptide histidine-isoleucine amide (PHI), isoproterenol (ISO), and sodium nitroprusside (SNP) were examined in dispersed rabbit and guinea pig gastric muscle cells. In rabbit muscle cells, SNP stimulated only guanosine 3',5'-cyclic monophosphate (cGMP) and cGMP-dependent protein kinase (cG-kinase) activity; VIP stimulated adenosine 3',5'-cyclic monophosphate (cAMP) and cGMP, and both cG-kinase and cAMP-dependent protein kinase (cA-kinase) activities; PHI and ISO stimulated only cAMP and cA-kinase activity, and at higher concentrations, cross-activated cG-kinase. All four agents elicited concentration-dependent relaxation. N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89; 1 microM) selectively inhibited cA-kinase activity and abolished relaxation when only cA-kinase was elevated. 8R,9S, 11S-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy- 1H,8H,11H-2,7b,11a-trizadibenzo-(a,g)-cy-cloocta-(c,d,e)- trinden-1-one (KT-5823; 1 microM) selectively inhibited cG-kinase activity and abolished relaxation when only cG-kinase was elevated. When both kinases were elevated, H-89 and KT-5823 partially inhibited relaxation and abolished relaxation in combination. In permeabilized guinea pig and rabbit muscle cells, all agents elicited relaxation and inhibited inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release. Both functions were inhibited in parallel fashion by protein kinase inhibitor PKI(6-22) and by KT-5823. We conclude that cA-kinase and cG-kinase act separately and in concert to inhibit IP3-dependent Ca2+ release and induce relaxation.
Hypertonic activation of AE2 anion exchanger in Xenopus oocytes via NHE-mediated intracellular alkalinization
Authors: Humphreys B. D. , Jiang L. , Chernova M. N. , Alper S. L.
Published: December 2017

Xenopus oocytes express endogenous Na+/H+ exchange activity but lack significant endogenous Cl-/HCO3- exchange activity. Coupled operation of Na+/H+ exchange and Cl-/HCO3- exchange contributes in many cell types to the cellular response to hypertonic stress. We therefore examined in Xenopus oocytes the osmotic regulation of chloride transport mediated by recombinant anion exchanger proteins AE2 and AE1. Hypotonicity was without effect on either anion transporter. Hypertonicity activated AE2-associated 36Cl- influx and efflux in a time- and osmolarity-dependent manner, whether incremental osmoles were charged or uncharged, but had no measurable effect on AE1 function. Hypertonic stimulation of AE2 was completely inhibited by Na+ removal or by addition of amiloride. In contrast, neither maneuver altered isotonic activity of AE2. Hypertonicity also induced amiloride-sensitive elevation of oocyte intracellular pH (pHi), and shifted the sigmoidal relationship of extracellular pH vs. AE2 activity > or = 0.5 units to the acid. Injection of pH 7.4 buffer into oocytes attenuated both hypertonic alkalinization and activation of AE2-associated 36Cl- influx, without inhibition of isotonic AE2 function. These data demonstrate that recombinant AE2 expressed in Xenopus oocytes is activated by increased pHi and that hypertonic activation of AE2 is secondary to hypertonic activation of Na+/H+ exchange.