bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of pH on Ca2+i, Na+i, and pHi of MDCK cells: Na(+)-Ca2+ and Na(+)-H+ antiporter interactions
Authors: Borle A. B. , Bender C.
Published: December 2017

The effects of high and low extracellular pH (8.0 and 6.8) on the intracellular concentration of Ca2+, Na+, and H+ were measured in perfused Madin-Darby canine kidney (MDCK) cells cast in agarose gel threads. Cytosolic free Ca2+ (Ca2+i) was measured with aequorin, while intracellular Na+ (Na+i) and H+ (Hi+ or pHi) were determined with the fluorescent indicators SBFI and BCECF, respectively. In addition, H+ secretion was assayed by the pH-stat method, and Na+ or Ca2+ fluxes were measured with 22Na or 45Ca, respectively. H+ secretion was significantly depressed by several experimental conditions that are known to inhibit the Na(+)-H+ antiporter: H+ secretion decreased 44% in the presence of 10(-5) M ethylisopropylamiloride, 49% in Na+o-free media, 44% in the presence of 10(-4) M ouabain, and 32% in the presence of 10(-4) M 8-bromoadenosine 3',5'-cyclic monophosphate. In addition, pHi decreased by 0.2 pH units in Na+o-free media. Finally, recovery from an intracellular acidosis evoked by 20 mM NH4Cl pulse required the presence of extracellular Na+. When the extracellular pH (pHo) was increased from 7.4 to 8.0, H+ secretion increased 58% from 17.5 to 27.7 nmol.min-1.mg protein-1 and Na+ influx increased 48%. As a result, pHi rose from 7.43 to 7.71 and Na+i increased from 15.6 to 19.7 mM. Finally, Ca2+i rose from 120 to 268 nM. These results suggest that the high pHo stimulated the Na(+)-H+ antiporter, and the subsequent rise in Na+i decreased the Na+ electrochemical potential, thereby activating the reverse mode of Na(+)-Ca2+ exchange (Ca2+ influx vs. Na+ efflux) which led to the rise in Ca2+i.(ABSTRACT TRUNCATED AT 250 WORDS)
Isolation and characterization of rat submandibular intralobular ducts
Authors: Dehaye J. P. , Turner R. J.
Published: December 2017

Intralobular (granular) salivary ducts were purified by isopycnic centrifugation after collagenase/hyaluronidase digestion of the rat submandibular gland. The resulting ductal fraction (density, 1.056 +/- 0.003) was highly enriched in kallikrein (a ductal cell marker) and contained little amylase activity (an acinar cell marker). The resting intracellular calcium level in the ductal preparation was 103 +/- 4 nM. Increased intracellular calcium concentrations (2-3 times resting levels) were observed in response to muscarinic (carbachol) and alpha-adrenergic (epinephrine) agonists, but little response was observed to substance P, suggesting the absence of substance P peptidergic receptors on rat submandibular ducts. Intracellular adenosine 3',5'-cyclic monophosphate levels were increased 35-fold in response to beta-adrenergic stimulation (isoproterenol) and forskolin. The ducts secreted kallikrein in response to epinephrine, carbachol, and isoproterenol but not in response to substance P. Epinephrine was the most potent inducer of kallikrein release with a K0.5 of approximately 3 microM and a maximal secretory rate approximately nine times unstimulated levels. Taken together, these results provide strong evidence for the functional integrity of the ductal preparation. This preparation should prove useful for the further elucidation of the properties of intralobular salivary ducts structures which heretofore have only been studied indirectly.
Cis-inhibition and trans-stimulation of cationic amino acid transport in the perfused rat pancreas
Authors: Sweiry J. H. , Munoz M. , Mann G. E.
Published: December 2017

Transport of cationic amino acids in the isolated perfused rat pancreas was studied using dual-isotope dilution techniques. At 50 microM substrate concentration, unidirectional tracer uptakes for L-arginine (56 +/- 1%), L-lysine (49 +/- 2%), and L-ornithine (44 +/- 3%) were followed by rapid tracer efflux. In the presence of Na+, influx of L-arginine [Michaelis constant (Km) = 1.74 +/- 0.15 mM, maximum velocity (Vmax) = 1.97 +/- 0.07 mumol.min-1.g-1] and L-lysine (Km = 2.48 +/- 0.17 mM, Vmax = 2.42 +/- 0.08 mumol.min-1.g-1) was mediated by a common transport system, sensitive to cis-inhibition by L-ornithine, 2,4-L-diaminobutyric acid, D-lysine, and D-arginine. Substrates for system A [alpha-(methylamino)isobutyric acid] and an anionic carrier (L-aspartate) were poor cis-inhibitors of L-arginine entry. Removal of Na+ resulted in a 40% reduction in cationic amino acid influx. After cell loading (20 min), L-[3H]-lysine cleared predominantly from a slowly exchanging pool with a rate constant of 5.97 +/- 0.67 min. An influx/efflux permeability ratio of 14.5 +/- 1.6 was determined, and efflux of L-lysine was trans-stimulated by vascular challenges with cationic or large neutral amino acids. The specificity, relative Na+ independence, and exchange properties of this saturable cationic amino acid transporter in the pancreatic epithelium resemble those reported for system y+ in cultured fibroblasts and hepatocytes.
Inhibition of phorbol ester-induced contraction by calmodulin antagonists in rat aorta
Authors: Chuprun J. K. , Bazan E. , Chang K. C. , Campbell A. K. , Rapoport R. M.
Published: December 2017

The purpose of the present study was to investigate the relative roles of protein kinase C (PKC) and myosin light chain kinase (MLCK) in phorbol ester-induced contraction of vascular smooth muscle through the use of PKC and calmodulin antagonists. Prior exposure to PKC antagonists staurosporine (0.03 microM) and H-7 (10 microM) had relatively little effect on contractions to phorbol 12-myristate 13-acetate (PMA), while contractions to norepinephrine and KCl were greatly inhibited. Prior exposure to the calmodulin antagonists calmidazolium (3 and 10 microM) and W-7 (10 microM) inhibited contractions to PMA in the presence and absence of extracellular Ca2+, while contractions to norepinephrine and KCl remained relatively unaffected. Calmidazolium and W-7 were relatively weak relaxants when applied during the PMA contraction, and the magnitudes of relaxation were similar to those observed in norepinephrine- and KCl-contracted tissues. Calmidazolium partially inhibited the PMA-induced translocation of PKC. These results suggest that 1) the calmodulin antagonists inhibit the development of PMA-induced contraction, at least in part, through inhibition of PKC translocation; 2) the mechanisms of phorbol ester- and agonist-induced translocation of PKC are distinct; 3) the potencies and inhibitory mechanisms of these agents depend on whether the agents are added before or during the contraction; and 4) the selectivity of these agents, as evaluated in enzyme preparations, may not be consistent with their cellular actions.
Regulation of ACTH secretory pathways in cultured pituitary cells
Authors: Schwartz J. , Gibson S. , White A.
Published: December 2017

Although chloroquine, an agent that disrupts regulated protein secretion, has previously been shown to decrease the adrenocorticotropic hormone (ACTH) secretory response to adenosine 3',5'-cyclic monophosphate or corticotropin-releasing factor (CRF) in AtT-20 and rat anterior pituitary cells, respectively, it has no effect on the response to vasopressin. The present study extended experiments with chloroquine to cultured sheep anterior pituitary cells, which have a greater maximum response to vasopressin. Chloroquine (200 microM) had no effect on basal ACTH secretion or on stimulation by vasopressin. In contrast to the rat, the net response to CRF was tripled by chloroquine in ovine cells. The effect of chloroquine on the response to CRF was more effective by coexposure of cells to CRF and chloroquine than by pretreatment with chloroquine. Monensin or vinblastine did not increase the ACTH response to CRF. The results indicate ACTH release in response to vasopressin is chloroquine insensitive in this way, can be dissociated from the mechanism that responds to CRF, and would be consistent with the CRF response mechanism involving pathways that can alter the secretory pool of ACTH. When chloroquine acts to increase the response to CRF, it is likely not to act by stabilizing the CRF-receptor complex.
Microprobe analysis of maturation-related elemental changes in rat parotid secretory granules
Authors: Wong J. G. , Izutsu K. T. , Robinovitch M. R. , Iversen J. M. , Cantino M. E. , Johnson D. E.
Published: December 2017

Electron probe X-ray microanalysis was use to quantitate the elemental and mass changes that take place during the secretory granule maturation process. A single injection of isoproterenol stimulated the depletion of secretory granules from rat parotid acinar cells. Granules at different stages of maturation were analyzed as they reaccumulated within the cells over time. Dry mass measurements revealed that secretory material becomes concentrated about twofold within maturing granules. Nearly all of the increase in mass concentration could be attributed to a reduction in water space. Data are presented that indicate that Na, K, Cl, and water all efflux from secretory granules during maturation. In contrast, granule S content is positively correlated with maturation. Hence, significant changes in granule elemental and water contents occur during the maturation process.
Two outward K+ currents in bovine pigmented ciliary epithelial cells: IK(Ca) and IK(V)
Authors: Jacob T. J.
Published: December 2017

Pigmented ciliary epithelial cells were studied using the whole cell voltage-clamp technique. Depolarizing steps from a holding potential of -80 mV resulted in a small initial inward current followed by a large outward current. Prolonged depolarizing voltage steps revealed inactivating and noninactivating components of outward current. Outward current was sensitive to the level of Ca2+ in the pipette and was increased by the calcium ionophore A23187; it was blocked by tetraethylammonium (TEA+), quinine, and 4-aminopyridine (4-AP). 4-AP blocked 70% of the outward current with a Ki of 7 x 10(-5) M, and part of the remaining current was abolished by Ni2+. Ni2+ caused a reduction in outward current by blocking IK(Ca) indirectly via decreasing Ca2+ entry through T-type Ca2+ channels. Separating Ni(2+)-sensitive from -insensitive outward conductance gives components that correspond notionally to IK(Ca) and IK(V), respectively. On this basis IK(Ca) represents approximately 28% of K+ outward current. Charybdotoxin blocked 26% of the outward conductance at very depolarized voltage steps as calculated from the slope of the current-voltage curve in this region. It is concluded that there are two major components to the outward current: IK(V), an inactivating voltage-sensitive K+ current, and IK(Ca), which is dependent on the entry of Ca2+ through T-type Ca2+ channels and comprises approximately a quarter of the total K+ outward current under the conditions described. Because of their relative voltage-activation properties, IK(Ca) will be the more important in terms of K+ transport and the secretion of aqueous humor by the ciliary epithelium.
Permanent hexose transport upregulation in a respiration-deficient human fibroblast cell strain
Authors: Andrejchyshyn S. , Continelli L. , Germinario R. J.
Published: December 2017

The regulation of hexose transport was studied in a human diploid fibroblast respiration-deficient cell strain (WG750). Transport of 2-deoxy-D-glucose (2-DG) was greater than sixfold higher compared with an in vivo age-matched normal cell strain (MCH55). In addition, 3-O-methylglucose transport and 14CO2 production were elevated in the mutant cell strain. Kinetic analysis revealed that the increased sugar transport in mutant cells was due to an average 5.7-fold increase in the 2-DG maximal transport rate, with no observed differences in the transport Michaelis constant for both normal and mutant cells. Also, the inhibitor constants for D-glucose inhibition of 2-DG transport were nearly identical for both cell types. Glucose deprivation led to a similar time-dependent increase in hexose transport in both cell strains. Serum refeeding of glucose-fed serum-deprived cultures led to a progressive increase in 2-DG transport in normal cells, whereas mutant cells displayed a time-delayed increase in 2-DG transport. Exposure to 67 and 670 nM insulin stimulated 2-DG transport on average 1.99 +/- 0.25- and 2.33 +/- 0.26-fold, respectively, over basal transport in the normal cells, whereas the mutant cells were significantly less sensitive to the stimulatory effects of the hormone. Insulin binding and amino acid transport (i.e., alpha-aminoisobutyric acid uptake) in the normal and mutant cells were not different. Data obtained using Western blot analysis showed that WG750 (mutant) cells expressed an increase (approximately 4-fold) in total cellular HepG2 (erythroid-brain) transporter protein compared with normal cells, thus reflecting the changes seen in hexose transport.(ABSTRACT TRUNCATED AT 250 WORDS)
Thrombin stimulates PDGF production and monocyte adhesion through distinct intracellular pathways in human endothelial cells
Authors: Shankar R. , de la Motte C. A. , DiCorleto P. E.
Published: December 2017

Thrombin stimulates multiple functions in cultured endothelial cells (EC), including an increase in cell surface adhesion sites for monocytes and the production of platelet-derived growth factor (PDGF). We have initiated studies to define the intracellular signaling pathways involved in these two thrombin-induced EC functions by focusing on the possible roles of the Na(+)-H+ antiporter and guanine nucleotide-binding proteins (G proteins). Amiloride suppressed thrombin-stimulated PDGF production by human aortic EC without affecting either basal PDGF production or overall protein synthesis. The steady-state mRNA levels of PDGF-A and PDGF-B chain were not reduced by amiloride. In replicate EC cultures, amiloride had no effect on thrombin-stimulated monocyte adhesion. In addition, thrombin induction of PDGF production, but not monocyte adhesion, was abrogated in the absence of extracellular sodium. Thrombin stimulation of both monocyte adhesion and PDGF production appeared to involve a pertussis toxin-insensitive G protein. Thrombin induced an increase in [35S]guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding to human EC membranes. GTP gamma S, in the presence of a suboptimal concentration of thrombin, caused maximal stimulation of both monocyte adhesion and PDGF production. The effect of GTP gamma S on PDGF production was at the level of transcription. These results indicate that the EC is capable of responding to a pluripotent agonist such as thrombin through multiple signaling pathways, which converge and diverge to achieve differential cellular responses.
Synthesis of cell-impermeable Cl-sensitive fluorescent indicators with improved sensitivity and optical properties
Authors: Biwersi Joachim , Farah Nazih , Wang Yong-Xiong , Ketcham R. , Verkman A. S.
Published: December 2017

Quinolinium compounds have been used as Cl-sensitive fluorescent indicators in cells and cell-free membrane fractions. To improve Cl sensitivity and for conjugation via nucleophilic reaction, the compounds 6-methoxy-N-(n-aminoalkyl)quinolinium bromide hydrochloride (AAQ) with alkyl chain lengths (n) of 2 (AEQ), 3 (APQ), and 4 (ABQ) were synthesized. AAQ was water soluble, fluorescent, and quenched by Cl. The Stern-Volmer constants (KCl) for quenching of protonated AEQ, APQ and ABQ by Cl were 354, 322, and 272 M-1, respectively, higher than KCl for 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ; 118 M-1). To eliminate pH-dependent fluorescence, 6-methoxy-N-(3-trimethylammoniumpropyl)quinolinium dibromide (TMAPQ) was synthesized (KCl, 310 M-1). To red shift fluorescence excitation and emission spectra, 6-phenyl-N-(3-trimethylammoniumpropyl) quinolinium dibromide (phenyl-TMAPQ) (emission 475 nm) and N-(3-trimethylammoniumpropyl)phenanthridinium dibromide (TMAPP) (excitation 380 nm) were synthesized. AEQ and ABQ were conjugated with neutral dextran activated by cyanogen bromide to give indicator-to-dextran mole ratios of 5 to 20. KCl values at pH 7.4 were 132 (AEQ-dextran) and 237 M-1 (ABQ-dextran). To construct a single molecule with Cl-sensitive and insensitive moieties, the bichromophores 6-methoxy-N-(n-dansylsulfonamidoalkyl)quinolinium with alkyl chains of two and four were synthesized. The new Cl-sensitive indicators were used for measurement of intracellular Cl activity and for the labeling of endocytic vesicles in 3T3 fibroblasts and T84 cells. Our results indicate that N-substitution of quinoline with positively charged moieties gives increased Cl sensitivity, and extension of ring conjugation gives indicators with red-shifted fluorescence spectra.

6-methoxy-N-(3-sulfopropyl)quinolinium; dextran; fibroblast

Submitted on December 20, 1990

Accepted on June 10, 1991
Regulation of annexin I in adipogenesis: cAMP-independent action of methylisobutylxanthine
Authors: Wong W. T. , Nick H. S. , Frost S. C.
Published: December 2017

The differentiation of 3T3-L1 fibroblasts to adipocytes can be accelerated by the addition of 1-methyl-3-isobutylxanthine (MIX), insulin, and dexamethasone to the culture medium. During differentiation, we have demonstrated that the level of both annexin I mRNA and protein decreases. The half-times for this reduction were 2 h and 10 h for annexin I mRNA and protein, respectively. Of the added agents in the differentiation medium, only MIX caused a decline in annexin I expression in 3T3-L1 fibroblasts. The MIX effect in fibroblasts was reversible and required de novo transcription but not protein synthesis. Although MIX could be replaced by high levels of theophylline, neither agonists of the beta-adrenergic receptor nor intracellular second messengers, cAMP and cGMP, were able to reduce annexin I. The potential role of annexin I in cellular differentiation is discussed.
ADH-induced depolymerization of F-actin in the toad bladder granular cell: a confocal microscope study
Authors: Holmgren K. , Magnusson K. E. , Franki N. , Hays R. M.
Published: December 2017

Antidiuretic hormone (ADH) induces the fusion of cytoplasmic vesicles containing water channels with the apical membrane of the toad bladder granular cell. Fusion is accompanied by a 30% depolymerization of F-actin. We have used confocal microscopy to determine the region in the cell that undergoes depolymerization. Bladders were mounted in a split chamber, and control halves and halves stimulated by ADH for 15 min were fixed and then stained with rhodamine phalloidin. Vertical sections through the cells were obtained by confocal microscopy, and the fluorescence intensity of the apical and side regions of the cells was determined. To normalize the data, the apex-side intensity was determined for each cell, and these ratios measured for control and ADH-treated halves. In six paired experiments, the ratio for control halves was 3.69 +/- 0.50 and for ADH-treated halves was 2.61 +/- 0.33; the decrease was significant and in good agreement with earlier studies. Thus actin depolymerization takes place in a hormone-sensitive apical pool where vesicle fusion occurs and supports the view that actin depolymerization may be required for fusion.
Human intestinal glucose transporter expression and localization of GLUT5
Authors: Davidson N. O. , Hausman A. M. , Ifkovits C. A. , Buse J. B. , Gould G. W. , Burant C. F. , Bell G. I.
Published: December 2017

We have studied the developmental and regional expression of mRNAs encoding sodium-dependent and facilitative glucose transporter proteins in human fetal and adult small intestine. The abundance of mRNAs encoding the Na(+)-glucose cotransporter isoform SGLT1 and the facilitative glucose transporter isoforms GLUT2 and GLUT5 is developmentally modulated with highest levels in adult small intestine. By contrast, the levels of GLUT1 mRNA are higher in fetal than adult small intestine. Immunohistochemical analysis of adult small intestine localized GLUT5 to the luminal surface of mature enterocytes, a finding confirmed by Western blot analysis of purified human jejunal brush-border membranes. By contrast, in the fetal small intestine, GLUT5 was localized along the intercellular junctions of the developing villus, indicating that both its expression and localization are developmentally regulated. The localization of GLUT5 to the luminal surface of mature absorptive epithelial cells implies that this protein participates in the uptake of dietary sugars.
Protein kinase C affects microfilaments, bone resorption, and [Ca2+]o sensing in cultured osteoclasts
Authors: Teti A. , Colucci S. , Grano M. , Argentino L. , Zambonin Zallone A.
Published: December 2017

The effects of protein kinase C (PKC) in the control of osteoclast activity are still unknown. We investigated the role of the enzyme in the control of microfilament organization, podosome assembly, bone resorption, and extracellular Ca2+ sensing in chicken and rabbit osteoclasts treated with agents known to affect PKC activity. Cells were treated for 20 min with a PKC activator [phorbol 12-myristate 13-acetate (PMA)], a PKC inhibitor (staurosporine), a protein kinase A (PKA) inhibitor (H-9), a guanosine 3',5'-cyclic monophosphate-dependent protein kinase-PKA-PKC inhibitor (H-7), or with the inactive phorbol, 4 alpha-phorbol, to examine microfilaments by decoration with rhodamine-phalloidin. In PMA-treated osteoclasts, the number of microfilament-containing adhesion structures (podosomes) per cell decreased. However, enlarged microfilamentous cores in podosomes and stress fiber-like filaments, otherwise absent in controls, appeared. Whereas H-7 induced increase of the number of podosomes, staurosporine, H-9, and 4 alpha-phorbol failed to change microfilament organization. Chicken osteoclasts received also long-term treatment with the agents in the presence of [3H]proline-prelabeled chicken or rat bone particles to measure bone resorption. PMA, as well as staurosporine and H-7, stimulated the resorbing activity, whereas cells were insensitive to H-9 and 4 alpha-phorbol. Measurement of cytosolic free calcium concentration in PMA-treated fura-2-loaded single osteoclasts demonstrated a synergistic effect of PKC activation on the inhibitory extracellular calcium concentration-sensing mechanism, which was, by contrast, blocked by H-7, staurosporine, and H-9 and was insensitive to 4 alpha-phorbol. These results indicate that PKC regulates osteoclast activity inducing both morphological and functional modifications.
Regulation of oxygen consumption in fast- and slow-twitch muscle
Authors: Kushmerick M. J. , Meyer R. A. , Brown T. R.
Published: December 2017

Phosphorus nuclear magnetic resonance spectra and steady-state O2 consumption rates were obtained from ex vivo arterially perfused cat biceps brachii (fast twitch) and soleus (slow twitch) muscles during and after periods of isometric twitch stimulation at 30 degrees C. In the biceps muscles, steady-state O2 consumption increased and phosphocreatine (PCr) concentration decreased progressively with stimulation. PCr recovery after these stimulation periods followed first-order kinetics with a half time of 10 min. The results in the biceps could be explained by a feedback control of cellular respiration by ADP concentration. In the soleus, steady-state O2 consumption also increased and PCr concentration decreased as stimulation rates increased. The half time for PCr recovery in the soleus was approximately 5 min, but, in contrast to the pattern in the biceps, the kinetics was not first order. There was an overshoot during the recovery period in the PCr content of soleus and a corresponding undershoot of Pi compared with resting values. Mitochondrial regulation by ADP is not sufficient to account for respiratory control in slow-twitch soleus. The respiration rate in neither muscle was dependent on the Pi content. Thus we conclude that the mechanism of control of cellular respiration is both quantitatively and qualitatively different in fast- and slow-twitch skeletal muscle.
Evidence for the involvement of a K+ channel in isosmotic cell shrinking in vestibular dark cells
Authors: Wangemann P. , Shiga N. , Welch C. , Marcus D. C.
Published: December 2017

Cell volume changes were measured in dark cells. Isosmotic addition of 21.4 mM K+, Rb+, Cs+, or NH4+ to a control solution containing 3.6 mM K+ caused piretanide-sensitive cell swelling (initial rate for K+, 0.100 +/- 0.005 microns/s; n = 119), suggesting dependence on the Na(+)-Cl(-)-K+ cotransporter. Subsequent isosmotic removal of 21.4 mM K+ caused piretanide-insensitive cell shrinking (initial rate, -0.104 +/- 0.005 microns/s; n = 119), which was inhibited by barium, lidocaine, quinidine, quinine, verapamil, and 4-aminopyridine but not tetraethylammonium (TEA) or glibenclamide, suggesting the involvement of K+ channel(s). Barium, lidocaine, quinine, quinidine, and 4-aminopyridine caused cell swelling in control solution (initial rate for barium, 0.011 +/- 0.004 microns/s; n = 6), suggesting that the K+ channel is also involved in efflux under control conditions. Cell shrinking was slowed by 21.4 mM extracellular K+, Rb+, or Cs+ but unaffected by Na+, Li+, TEA+, or NH4+ (all in the presence of piretanide and compared with N-methyl-D-glucamine), supporting the notion that the efflux mechanism is permeable to and/or inhibited by K+, Rb+, and Cs+. Cell shrinking was slowed by the presumed replacement of intracellular K+ by Cs+ but not by Rb+. Circumstantial evidence suggests that this putative K+ channel is present in the basolateral membrane. The physiological relevance of such a K+ channel might encompass regulatory volume decrease during K+ secretion.
Evidence for an InsP3-gated channel protein in isolated rat olfactory cilia
Authors: Restrepo D. , Teeter J. H. , Honda E. , Boyle A. G. , Marecek J. F. , Prestwich G. D. , Kalinoski D. L.
Published: December 2017

Stimulation of rat olfactory cilia (ROC) with odorants leads to a transient elevation in the levels of either cAMP or inositol trisphosphate (InsP3). We have characterized the binding of [3H]InsP3 to isolated ROC. Unlabeled InsP3 displaced [3H]InsP3 binding in a dose-dependent manner (dissociation constant = 3.9 +/- 0.65 microM). Binding was stereospecific and dependent on the number of phosphates in the inositol ring. A ciliary protein of 120 kDa molecular mass was labeled specifically upon exposure of cilia membranes to ultraviolet light in the presence of the 125I-labeled InsP3 analogue 1-O-[N-(4-azidosaliciloxy)-3-aminopropyl-1-phospho]-myo-inositol 4,5-bisphosphate. Labeling of this protein displayed the same stereospecificity as binding of [3H]InsP3 to ROC. In addition, ROC membranes incorporated into a phospholipid bilayer at the tip of a patch pipette displayed an increase in conductance upon exposure to micromolar D-myoinositol 1,4,5-trisphosphate in 45% of the trials (n = 88). The InsP3-gated conductance is relatively nonspecific for cations and is distinct from the cAMP-gated conductance. The conductance displayed stereospecificity consistent with the InsP3 binding experiments. The results suggest that the site of action for odorant-stimulated elevations in InsP3 concentration in rat olfactory cilia is at a ciliary InsP3-gated channel.
Lysophosphatidic acid induces a pertussis toxin-sensitive Ca(2+)-activated Cl- current in Xenopus laevis oocytes
Authors: Durieux M. E. , Salafranca M. N. , Lynch K. R. , Moorman J. R.
Published: December 2017

Lysophosphatidic acid (LPA) induces a Ca(2+)-activated Cl- current in defolliculated Xenopus laevis oocytes. The response appears mediated by a specific membrane receptor, because no current is induced when related compounds [phosphatidic acid (PA), lysophosphatidylcholine (LPC), and lysophosphatidylserine (LPS)] are applied extracellularly or when LPA is injected intracellularly. Incubation in pertussis toxin prevents the response. The response is mediated by a Ca(2+)-activated Cl- current because 1) it is abolished by intracellular ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA; 5 mM) but not affected by changes in extracellular Ca2+ concentration and 2) the reversal potential becomes more positive at lower Cl- concentrations. Suramin (2 mM) blocks the LPA-induced current, but PA, LPS, LPC, and the platelet-activating factor antagonist WEB-2086 do not. The response is dose dependent for LPA concentrations from 10(-8) to 10(-3) M. Incubation of oocytes in LPA does not induce germinal vesicle breakdown. These findings suggest that this novel oocyte response to LPA is mediated by a specific membrane receptor linked to a pertussis toxin-sensitive G protein.
Rat kidney Na-K pumps incorporated into low-K+ sheep red blood cell membranes are stimulated by anti-Lp antibody
Authors: Xu Z. C. , Dunham P. B. , Munzer J. S. , Silvius J. R. , Blostein R.
Published: December 2017

A genetic dimorphism of sheep red blood cells characterized by differences in the intracellular K+ concentration of mature red blood cells (low-K+ or high-K+ cells) reflects differences in their Na-K pumps and is known to be linked to the ML blood group system. We investigated the relationship of Na-K pumps in red blood cells from sheep of the low-K+ phenotype with an antigen, Lp, that is restricted to low-K+ cells. Anti-Lp antibody stimulates the Na-K pumps in these cells presumably by relieving inhibition of the pumps by Lp. The questions addressed were as follows: is Lp a molecular entity distinct from pumps and, if so, can it interact with pumps of exogenous origin? Rat kidney Na-K pumps were incorporated by fusion of microsomes into either low-K+ or high-K+ sheep red blood cells. The activity of the exogenous kidney pumps was distinguished from that of the endogenous red blood cell pumps by the low sensitivity of rodent pumps to ouabain. Anti-Lp stimulated by > 50% rat kidney pumps incorporated into immature low-K+ sheep cells. This indicates that Lp is a distinct molecular entity free to dissociate from endogenous pumps and inhibit exogenous pumps. Anti-Lp did not stimulate kidney pumps incorporated into mature low-K+ cells but did stimulate kidney pumps following in vitro maturation of microsome fused reticulocytes, probably reflecting restriction of lateral movement of pumps and antigens by the cytoskeleton in mature cells.
Endothelin-1 stimulates DNA synthesis and proliferation of pulmonary artery smooth muscle cells
Authors: Janakidevi K. , Fisher M. A. , Del Vecchio P. J. , Tiruppathi C. , Figge J. , Malik A. B.
Published: December 2017

Endothelin-1 (ET-1), a 21-amino acid peptide released from the endothelium, elicits a variety of biological effects that include vascular smooth muscle cell (VSMC) contraction, release of secondary mediators, and cell proliferation. The present study was undertaken to examine the proliferative potential of ET-1 toward pulmonary artery VSMC in culture. In the presence of low serum and epidermal growth factor (EGF), ET-1 stimulated marked DNA synthesis and proliferation of VSMC. The contributing factor from serum appeared to be platelet-derived growth factor (PDGF) because the antibody to PDGF eliminated the stimulatory activity. The antibody to EGF also prevented the stimulation, suggesting that both PDGF and EGF are required for the full expression of the VSMC growth-promoting activity of ET-1. A paradoxical aspect of ET-1 effect on VSMC was the ability of ET-1 to inhibit the EGF-stimulated DNA synthesis when the two factors were added together to a high baseline DNA synthetic activity. The inhibition was prevented if ET-1 was added 12-18 h after the addition of EGF or if ET-1 and EGF were added to a protein kinase C-depleted VSMC. The inhibition by ET-1 may be mediated by protein kinase C activation followed by inhibition of EGF binding to its receptor. The results indicate that ET-1 under appropriate conditions can modulate the growth of pulmonary artery VSMC in both positive and negative directions.
Receptor-activated Ca influx in human airway smooth muscle: use of Ca imaging and perforated patch-clamp techniques
Authors: Murray R. K. , Fleischmann B. K. , Kotlikoff M. I.
Published: December 2017

Previous studies have demonstrated a dihydropyridine-insensitive, receptor-activated calcium influx pathway in cultured human airway smooth muscle (ASM) cells. To further define the biophysical characteristics of this pathway, the relationship between membrane potential and cytosolic free calcium ([Ca2+]i) was studied with the combined methods of the patch-clamp technique and single cell calcium imaging. The nystatin perforated-patch method was used to maintain normal intracellular calcium buffering and receptor-activated signal transduction processes in voltage-clamped cells. Single voltage-clamped human ASM cells responded to exposure to histamine (200 microM) with an initial transient rise in [Ca2+]i followed by a secondary sustained elevation that was dependent on extracellular calcium. Before agonist activation, step changes in holding potential produced only slight changes in [Ca2+]i, whereas, after activation, cells developed a sustained rise in [Ca2+]i that showed a large variation as a function of membrane potential. Depolarization from -80 to 0 mV caused a fall in the steady-state [Ca2+]i to basal levels or slightly below. Repolarization to -80 mV caused the redevelopment of the sustained phase of the calcium response. When calcium was removed from the extracellular fluid by the addition of a stoichiometric excess of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), the voltage dependence of the sustained phase was abolished. In a series of experiments, agonist addition evoked a 54-fold increase in the voltage dependence of calcium.(ABSTRACT TRUNCATED AT 250 WORDS)
Transport of lactate and other monocarboxylates across mammalian plasma membranes
Authors: Poole R. C. , Halestrap A. P.
Published: December 2017

Transport of L-lactate across the plasma membrane is of considerable importance to almost all mammalian cells. In most cells a specific H(+)-monocarboxylate cotransporter is largely responsible for this process; the capacity of this carrier is usually very high, to support the high rates of production or utilization of L-lactate. The best characterized H(+)-monocarboxylate transporter is that of the erythrocyte membrane, which transports L-lactate and a wide range of other aliphatic monocarboxylates, including pyruvate and the ketone bodies acetoacetate and beta-hydroxybutyrate. This carrier is inhibited by alpha-cyanocinnamate derivatives and some stilbene disulfonates and has been identified as a protein of 35-50 kDa on the basis of purification and specific labeling experiments. Other cells possess similar alpha-cyanocinnamate-sensitive H(+)-linked monocarboxylate transporters, but in some cases there are significant differences in the properties of these systems, sufficient to suggest the existence of a family of such carriers. In particular, cardiac muscle and tumor cells have transporters that differ in their Km values for certain substrates (including stereoselectivity for L- over D-lactate) and in their sensitivity to inhibitors. Mitochondria, bacteria, and yeast also possess H(+)-monocarboxylate transporters that share some properties in common with those in the mammalian plasma membrane but are adapted to their specific roles. However, there are distinct Na(+)-monocarboxylate cotransporters on the luminal surface of intestinal and kidney epithelia, which enable active uptake of lactate, pyruvate, and ketone bodies in these tissues. This article reviews the properties of these transport systems and their role in mammalian metabolism.
TNF-alpha induces endothelial cell F-actin depolymerization, new actin synthesis, and barrier dysfunction
Authors: Goldblum S. E. , Ding X. , Campbell-Washington J.
Published: December 2017

Tumor necrosis factor-alpha (TNF-alpha) influences pulmonary vascular endothelial barrier function in vitro. We studied whether recombinant TNF-alpha (rTNF-alpha) regulates endothelial barrier function through actin reorganization. Postconfluent bovine pulmonary artery endothelial cell monolayers were exposed to human rTNF-alpha (1,000 U/ml) and evaluated for 1) transendothelial [14C]albumin flux, 2) F-actin organization with fluorescence microscopy, 3) F-actin quantitation by spectrofluorometry, and 4) monomeric G-actin levels by the deoxyribonuclease I inhibition assay. rTNF-alpha induced increments in [14C]albumin flux (P < 0.04) and intercellular gap formation at > or = 2-6 h. During this same time, the endothelial F-actin pool decreased (P = 0.0064), with reciprocal increases in the G-actin pool (P < 0.0001). Prior F-actin stabilization with phallicidin protected against the rTNF-alpha-induced increments in G-actin (P < 0.002) as well as changes in barrier function (P < 0.01). Prior protein synthesis inhibition enhanced the rTNF-alpha-induced decrement in F-actin (P < 0.0001), blunted the G-actin increment (P < 0.002), and increased rTNF-alpha-induced changes in endothelial barrier function (P < 0.003). Therefore, rTNF-alpha induces pulmonary vascular endothelial F-actin depolymerization, intercellular gap formation, and barrier dysfunction. rTNF-alpha also increased total actin (P < 0.02) and new actin synthesis (P < 0.002), which may be a compensatory endothelial cell response to rTNF-alpha-induced F-actin depolymerization.
Protective effects of adenosine in the perfused rat heart: changes in metabolism and intracellular ion homeostasis
Authors: Fralix T. A. , Murphy E. , London R. E. , Steenbergen C.
Published: December 2017

Increased concentrations of intracellular H+, Na+, and Ca2+ have been observed during ischemia, and these ionic alterations have been correlated with several indexes of cell injury in a number of studies. Recently, adenosine was proposed to play a role in ischemic preconditioning, since adenosine antagonists block the protective effects of these brief intermittent periods of ischemia and reflow. In this study we evaluated the protective effects of adenosine (20 microM) on high-energy phosphate metabolism, H+ and Ca2+ accumulation, and glycolytic rate during 30 min of no-flow ischemia. Adenosine was observed to slow the onset of contracture (7.0 +/- 0.9 min) and to improve left ventricular developed pressure (62 +/- 7% of initial) during reperfusion compared with untreated hearts (5.0 +/- 0.6 min and 18 +/- 5%, respectively). Intracellular Ca accumulation at the end of 30 min of ischemia was higher in the untreated (2,835 +/- 465 nM) than in the adenosine-treated (2,064 +/- 533 nM) hearts, while intracellular pH fell more in the untreated (5.85 +/- 0.17) than in the adenosine-treated hearts (6.27 +/- 0.16). Glycolytic rate and the rate of ATP decline were significantly attenuated in the adenosine-treated hearts during ischemia. Thus adenosine treatment slowed the rate of metabolism and delayed the accumulation of H+ and Ca2+ during ischemia, resulting in better recovery of function upon reflow.
Altered expression and localization of the tight junction protein ZO-1 after common bile duct ligation
Authors: Fallon M. B. , Mennone A. , Anderson J. M.
Published: December 2017

Hepatocyte tight junctions form the intercellular barrier between bile and blood. Cholestasis due to common bile duct ligation (CBDL) results in structural changes in the tight junction (TJ) and an overt paracellular leak, although the molecular basis for these alterations is undefined. Using the epithelial isoform of the TJ protein ZO-1 (ZO-1 alpha +) as a marker for molecular changes in hepatocyte TJs, we investigated the effects of CBDL on ZO-1 alpha + immunofluorescence (IF) localization and on ZO-1 alpha + mRNA and protein expression over 2 wk of CBDL. ZO-1 alpha + IF staining was altered after 2 days of CBDL and appeared to accumulate in pericanalicular regions after 7 and 9 days. Quantitative immunoblotting and ribonuclease protection revealed a marked increase in hepatic ZO-1 alpha + protein expression and ZO-1 alpha + mRNA levels, respectively. In contrast to changes in ZO-1 alpha + IF, which occurred throughout the lobule, and changes in mRNA and protein expression, which were maximal after 9 days of ligation, the maximal hepatocyte proliferation occurred within 2 days after CBDL and was confined to periportal regions. CBDL results in altered hepatic localization and increased expression of the TJ protein ZO-1 alpha + and appears to represent a specific response by hepatocytes to pathological junction injury independent of cell proliferation.
Expression of hepatocyte growth factor in growing and regenerating rat skeletal muscle
Authors: Jennische E. , Ekberg S. , Matejka G. L.
Published: December 2017

The expression of hepatocyte growth factor (HGF) was studied in rat skeletal muscle during postnatal growth and during regeneration after ischemic injury. By Northern blot analysis two RNA transcripts with estimated sizes of 6.0 and 3.1 kb, respectively, could be detected in immature skeletal muscle at 2, 4, and 10 days after birth, whereas no signal could be detected in adult skeletal muscle. In regenerating muscle, HGF mRNA was reexpressed, and a distinct signal was evident during the first days after the injury. One week after the injury only a weak signal was obtained. By in situ hybridization staining, HGF mRNA could be demonstrated in the immature muscle tissue of newborn rats but not in adult skeletal muscle. In regenerating muscle, the staining for HGF mRNA could be demonstrated in the regenerating muscle cells during the early phase of the regeneration. One week after the injury, only faint staining for HGF mRNA persisted in the regenerated fibers. It is concluded that HGF expression is developmentally regulated in skeletal muscle and that HGF is reexpressed in a transient manner during postischemic muscle regeneration.
Improved in vitro rheological system for studying the effect of fluid shear stress on cultured cells
Authors: Schnittler H. J. , Franke R. P. , Akbay U. , Mrowietz C. , Drenckhahn D.
Published: December 2017

A rheological in vitro system has been developed to study and quantify cellular adhesion under precisely defined external shear forces. The system is similar to a cone-and-plate viscosimeter. A rotating transparent cone produces both steady and pulsatile flow profiles on cultured cells. Direct visualization of cells by phase-contrast or fluorescence optics and connection of the optical system to a computer-controlled x/y-linear stage allows automatic recording of any point of the cell cultures. With the use of up to 12 individual rheological units, this setup allows the quantitative analysis of cell substrate adhesion by determination of cell detachment kinetics. Two examples of application of this rheological system have been studied. First, we show that the extracellular matrix protein laminin strongly increases endothelial cell adhesion under fluid shear stress. In a second approach, we obtained further support for the concept that shear stress-induced formation of actin filament stress fibers is important for endothelial cells to resist the fluid shear stress; inhibition of stress fiber formation by doxorubicin resulted in significant detachment of endothelial cells exposed to medium levels of fluid shear stress (5 dyn/cm2). No detachment was seen under resting conditions.
Endothelin increases [Ca2+]i in M-1 mouse cortical collecting duct cells by a dual mechanism
Authors: Korbmacher C. , Boulpaep E. L. , Giebisch G. , Geibel J.
Published: December 2017

We tested the effects of endothelin-1 (ET-1) on intracellular calcium concentration ([Ca2+]i) of cultured M-1 mouse cortical collecting duct cells. [Ca2+]i was measured using fura 2 and a fluorescent imaging system. At a concentration of extracellular calcium ([Ca2+]o) of 1 mM, ET-1 (10(-12) to 10(-7) M) increased [Ca2+]i. A second application of ET-1 had no effect on Ca2+. In contrast, application of arginine vasopressin after an initial exposure to ET-1 induced a second Ca2+ response. In the absence of extracellular Ca2+ (1 mM EGTA) ET-1 also elicited a Ca2+ peak, indicating participation of Ca2+ release from intracellular stores in the initial Ca2+ peak. At [Ca2+]o of 10 mM, ET-1 also induced an intracellular Ca2+ peak but [Ca2+]i remained significantly elevated. The Ca2+ plateau phase was abolished by nickel (10 or 100 microM) and nifedipine (0.1 or 1 microM). We conclude that ET-1 mediates an increase in [Ca2+]i by Ca2+ release from intracellular stores and activation of a nickel- and nifedipine-sensitive Ca2+ entry mechanism.
Computer modeling of the oxygen supply and demand of cells of the avian growth cartilage
Authors: Haselgrove J. C. , Shapiro I. M. , Silverton S. F.
Published: December 2017

We have used computer modeling studies to investigate the oxygen supply to the prehypertrophic and hypertrophic regions of avian growth plate. We measured experimentally the characteristics of the oxygen consumption of chondrocytes at different oxygen tensions. The oxygen consumption decreases at low oxygen tensions. This relation between oxygen tension and oxygen consumption serves as a protective mechanism that prevents the cells in the prehypertrophic zone from becoming anoxic in the regions farthest from the blood vessels. The results of the calculations, when combined with redox measurements of the cells in the growth plate, indicate that the metabolism of the chondrocytes is not controlled simply by the available oxygen supply.
Calcium-mediated agonists activate an inwardly rectified K+ channel in colonic secretory cells
Authors: Devor D. C. , Frizzell R. A.
Published: December 2017

Single-channel recording techniques were used to identify and characterize the K+ channel activated by Ca(2+)-mediated secretory agonists in T84 cells. Carbachol (CCh; 100 microM) and taurodeoxycholate (TDC; 0.75 mM) stimulated oscillatory outward K+ currents. With K gluconate in bath and pipette, cell-attached single-channel K+ currents stimulated by CCh and ionomycin (2 microM) were inwardly rectified and reversed at 0 mV. The single-channel chord conductance was 32 pS at -90 mV and 14 pS at +90 mV. Similar properties were observed in excised inside-out patches in symmetric K+, permitting further characterization of channel properties. Partial substitution of bath or pipette K+ with Na+ gave a K(+)-to-Na+ selectivity ratio of 5.5:1. Channel activity increased with increasing bath Ca2+ concentration in the physiological range of 50-800 nM. Maximal channel activity occurred at intracellular pH 7.2 and decreased at more acidic or alkaline pH values. Extracellular charybdotoxin (CTX; 50 nM) blocked inward but not outward currents. Extracellular tetraethylammonium (TEA; 10 mM) reduced single-channel amplitude at all voltages. No apparent block of the channel was observed with extracellular Ba2+ (1 mM), apamin (1 microM), 4-aminopyridine (4-AP; 4 mM), quinine (500 microM), or glyburide (10 microM). Cytosolic quinine and 4-AP blocked both inward and outward currents, whereas Ba2+ blocked only outward currents. Apamin, CTX, TEA, and glyburide did not affect channel activity. The agonist activation and pharmacological profile of this inwardly rectified K+ channel indicate that it is responsible for the increase in basolateral K+ conductance stimulated by Ca(2+)-mediated agonists in T84 cells.