bluuebunny/crossref_metadata_2025 huggingface dataset:
Control of intracellular calcium by membrane potential in human melanoma cells
Authors: Nilius B. , Schwarz G. , Droogmans G.
Published: December 2017

The modulation of intracellular calcium ([Ca2+]i) by the membrane potential was investigated in human melanoma cells by combining the nystatin-perforated patch-clamp technique with Ca2+ measurements. Voltage steps to -100 mV induced a rise in [Ca2+]i and a creeping inward current. These effects were absent in Ca(2+)-free solution and could be blocked by Ni2+ or La3+. Voltage ramps revealed a close correlation between [Ca2+]i and voltage, with the strongest voltage dependence around the resting potential. Long-lasting tail currents, closely correlated with the rise in [Ca2+]i and a reversal potential close to the K+ equilibrium potential, occurred if the membrane potential was clamped back to 0 mV. They were absent if intracellular K+ was replaced by Cs+ and blocked by extracellular tetraethylammonium (5 mM), Ba2+ (1 mM), or a membrane-permeable adenosine 3',5'-cyclic monophosphate analogue. These observations are discussed in relation to cell proliferation. The enhanced expression of K+ channels during cell proliferation provides a positive-feedback mechanism resulting in long-term changes in [Ca2+]i required for the G1-S transition in the cell cycle.
Chemotaxis of newt eosinophils: calcium regulation of chemotactic response
Authors: Brundage R. A. , Fogarty K. E. , Tuft R. A. , Fay F. S.
Published: December 2017

Local chemical events underlying chemotaxis were characterized in a new model cell, the newt eosinophil. These cells exhibit a chemotactic response to a trypsin-sensitive component of newt serum. Ca2+ plays a role in this process, since treatments expected to diminish Ca2+ availability from the medium [ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, Co2+, and verapamil], to break down transmembrane Ca2+ gradients (ionomycin), or to interfere with the function of intracellular Ca2+ stores (caffeine and neomycin) inhibited cell polarization and movement. Using imaging techniques we found that cytosolic Ca2+ concentration ([Ca2+]i) increased in response to newt serum. Migrating newt eosinophils exhibited a dynamic heterogeneous distribution of [Ca2+]i. [Ca2+]i was elevated in cells undergoing a change of direction relative to cells migrating persistently in one direction. Migrating cells contained gradients of [Ca2+]i along their long axis, with the front of the cell having consistently lower [Ca2+]i than the rear. When cells were loaded with the cell-permeant form of fura 2, fura 2 acetoxymethyl ester, a caffeine-sensitive membrane-delimited region of elevated [Ca2+]i was seen associated with the microtubule organizing center. A model is proposed relating the distribution of [Ca2+]i and the location of the external stimulus to the generation and interaction of substances within the cell that both simulate and inhibit increases in [Ca2+]i.
Intracellular mechanism of Pb(2+)-induced norepinephrine release from bovine chromaffin cells
Authors: Tomsig J. L. , Suszkiw J. B.
Published: December 2017

The intracellular mechanism of Pb(2+)-induced release of norepinephrine (NE) was investigated in comparison with Ca2+ in bovine chromaffin cells permeabilized with staphylococcal alpha-toxin. Pb2+ activated NE release at considerably lower concentrations [concentration of free metal giving half maximal metal-dependent release (K0.5) 4.6 nM] than Ca2+ (K0.5 2.4 microM). The release of NE was associated with the release of dopamine-beta-hydroxylase but not lactate dehydrogenase. The maximal secretory responses produced by Pb2+ and Ca2+ were similar and nonadditive. Pb(2+)- and Ca(2+)-dependent releases showed a similar requirement for MgATP and were equally enhanced by protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) but not by kinase A activator 8-bromoadenosine 3',5'-cyclic monophosphate free base. The protein kinase C inhibitor staurosporine blocked the TPA-stimulated component of secretion but had no effect on the NE release in the absence of TPA. Calmidazolium, an inhibitor of calmodulin, inhibited the secretion evoked by both metals to similar extent. Agents interacting with microtubules (colchicine and vinblastine) or microfilaments (cytochalasin B and phalloidin) had no effect on secretion induced by either metal cation. These observations indicate that both Pb2+ and Ca2+ act at a common site and activate the exocytotic release of NE by an analogous mechanism.
Ni2+ impairs thrombin-induced signal transduction by acting on the agonist and/or receptor in human platelets
Authors: Azula F. J. , Alonso R. , Marino A. , Trueba M. , Macarulla J. M.
Published: December 2017

We have investigated the effect of NiCl2 on platelet activation induced by thrombin, phorbol 12-myristate 13-acetate, and calcium ionophores. Besides blocking Ca2+ influx, NiCl2 inhibited platelet aggregation, intracellular Ca2+ mobilization, and phospholipase C activation induced by thrombin in a dose-dependent manner. In contrast to ionomycin, NiCl2 completely blocked the platelet aggregation and intracellular Ca2+ mobilization induced by A23187. A23187 was not able to translocate Ni2+ across the plasma membrane. Ni2+ also inhibited phorbol myristate acetate-induced platelet aggregation. The results with staurosporine and low NiCl2 concentrations are in agreement in that increases in intracellular Ca2+ concentration and protein kinase C activation are necessary for full platelet activation mediated by thrombin.
Cl- regulation of a Ca(2+)-activated nonselective cation channel in beta-agonist-treated fetal distal lung epithelium
Authors: Tohda H. , Foskett J. K. , O'Brodovich H. , Marunaka Y.
Published: December 2017

Nonselective cation (NSC) channels have been identified in the apical membrane of fetal distal lung epithelium (FDLE). However, their physiological role in Na+ transport is uncertain. Because terbutaline, a beta 2-agonist, increases Na+ transport by FDLE, we studied its effect and selected signal transduction mechanisms on NSC channel activity. Using patch-clamp and single-cell imaging techniques, we found that terbutaline activated the NSC channel by 1) increasing its sensitivity to cytosolic Ca2+ concentration ([Ca2+]c) by 100- to 1,000-fold, 2) increasing [Ca2+]c from 35 nM to 3.3 microM, 3) producing a dependency of the NSC channel activity on the cytosolic Cl- concentration ([Cl-]c) at a physiological [Ca2+]c, and 4) inducing a reduction in the [Cl-]c from 45 to 25 mM, which directly activates the beta 2-treated NSC channel. These observations indicate that a beta 2-agonist physiologically activates an amiloride-blockable NSC channel in FDLE through an increase in its sensitivity to [Ca2+]c, resulting in the development of a [Cl-]c dependency at a physiological [Ca2+]c associated with both an increase in [Ca2+]c and a reduction in [Cl-]c. A development of the [Cl-]c dependency and a reduction in [Cl-]c act as a second messenger of the beta-agonist signal transduction pathway in this Na(+)-transporting epithelium.
Permeability properties of rat renal lysosomes
Authors: Piqueras A. I. , Somers M. , Hammond T. G. , Strange K. , Harris H. W. , Gawryl M. , Zeidel M. L.
Published: December 2017

Although lysosomes maintain large pH gradients and may be subjected to significant osmotic gradients in vivo, little is known about their passive permeability properties. In recent studies, vacuolar H(+)-adenosine-triphosphatases (ATPases), such as those found in lysosomes, have been suggested to act as water channels. In addition, the erythrocyte and proximal tubule water channel CHIP28 is present on the plasma membrane of proximal tubule cells and may undergo endocytosis so that it is incorporated in lysosomes. We therefore examined water, proton, and small nonelectrolyte permeabilities in freshly purified lysosomes from rat renal proximal tubule. Lysosomes were purified by differential and Percoll gradient centrifugation. The preparation contained only lysosomes when examined by electron microscopy. Moreover, analysis by flow cytometry showed virtually all particles to be positive for acid phosphatase and cathepsin B activities. Permeabilities were measured on a stopped-flow fluorimeter by monitoring the self-quenching or pH-sensitive quenching of entrapped fluorescein derivatives. Osmotic water permeability (Pf) averaged 0.011 +/- 0.003 cm/s (n = 6), a value similar to that of biological membranes containing water channels. However, Pf was insensitive to the organic mercurial reagent p-chloromercuribenzene-sulfonate and to HgCl2 and exhibited an activation energy of 10.8 +/- 0.8 kcal/mol. These results indicate that water flux in lysosomes occurred via the lipid bilayer, and not via water channels. Addition of ATP led to lysosomal acidification (proton flux = 4.6 +/- 0.8 x 10(-11) mmol H+.s-1.cm-2), which was completely inhibited by 0.1 microM bafilomycin. Pf was insensitive to this agent as was the passive proton permeability (0.36 +/- 0.18 cm/s, n = 4). Permeabilities to small nonelectrolytes varied in proportion to the oil-water partition coefficient, confirming the applicability of Overton's rule to lysosomes. We conclude that proximal tubular lysosomes exhibit high Pf, which occurs via the lipid bilayer and not via vacuolar H(+)-ATPase.
Polyadenylation of Na(+)-K(+)-ATPase beta 1-subunit during early development of Xenopus laevis
Authors: Burgener-Kairuz P. , Corthesy-Theulaz I. , Merillat A. M. , Good P. , Geering K. , Rossier B. C.
Published: December 2017

In fully grown Xenopus oocytes, the synthesis of beta-subunits is limiting for the formation of functional Na(+)-K(+)-adenosinetriphosphatase alpha/beta-complexes (Geering, K. FEBS Lett. 285: 189-193, 1991). In the present study, we show that during oocyte growth (from stage I to stage VI) alpha 1-, but not beta 1- or beta 3-isoform, mRNAs accumulate. In addition, beta-mRNAs are apparently sequestered in an untranslated pool in fully grown oocytes (stage VI). From fertilization to morulation, the total pools of alpha 1-, beta 1-, or beta 3-mRNAs vary little. Whereas polyadenylated [poly(A)+] alpha 1- and beta 3-isoform mRNAs did not change significantly, poly(A)+ beta 1-mRNA abundance increased three- to fourfold at morulation, accompanied by a parallel increase in beta 1-protein synthesis. After midblastula transition (i.e., at early gastrula) and during neurulation, poly(A)+ alpha 1- and beta 3-mRNAs accumulated rapidly, whereas poly(A)+ beta 1-mRNA accumulation was delayed by approximately 2 h, beginning only at early neurula. Our results indicate that 1) the abundance of poly(A)+ beta 1-mRNA is rate limiting during embryonic development for the assembly of alpha 1/beta 1-heterodimers, shown to be involved in the vectorial transport of sodium in kidney cells, and 2) the polyadenylation of beta 1-mRNA is a rate-limiting factor during morulation for the synthesis and assembly of new sodium pumps at the time of blastocoel fluid formation. The 3'-untranslated region of beta 1-mRNA (but not of alpha 1-mRNA) expresses cytoplasmic polyadenylation elements (CPEs) with the consensus sequence AXX-AUUUU(A/U)(A/U)(A/U). A role of CPE in the differential polyadenylation of alpha 1- and beta 1-mRNA is proposed.
Regulatory volume decrease in cultured astrocytes. I. Potassium- and chloride-activated permeability
Authors: Pasantes-Morales H. , Murray R. A. , Lilja L. , Moran J.
Published: December 2017

Regulatory volume decrease (RVD) in detached cerebellar astrocytes in culture after acute exposure to hyposmolarity was characterized in this and the accompanying paper [H. Pasantes-Morales, R. A. Murray, R. Sanches-Olea, and J. Moran. Am. J. Physiol. 266 (Cell Physiol. 35): C172-C178, 1994]. RVD was independent of extracellular calcium, was accelerated at pH 8-9 and retarded at pH 6, and was reduced at temperatures < 18 degrees C. The cationic pathway activated by hyposmolarity was specific for K+ and Rb+, since RVD was abolished and secondary swelling occurred when these ions replaced Na+. However, Li+, choline, tris(hydroxymethyl)aminomethane, and glucosamine, all as Cl- salts, did not affect RVD. The anion pathway was unselective, since RVD was inhibited when NaCl was replaced by anion K+ salts with a permeability rank of SCN- = I- > NO3- > Cl- > benzoate > acetate >> SO3- > gluconate. RVD was unaffected by bumetanide (50 microM) and weakly inhibited by furosemide (2 mM). Quinidine but not other K+ channel blockers inhibited RVD, and its effect was reversed by gramicidin. RVD was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and dipyridamole but not by diphenylamine-2-carboxylate or anthracene-9-carboxylate. These results suggest that diffusion possibly via channels rather than cotransporters is involved in the swelling-activated K+ and Cl- fluxes. Gramicidin did not change astrocyte volume in isosmotic conditions, but greatly accelerated RVD, suggesting that low Cl- permeability in isosmotic conditions markedly increases by swelling, thus making K+ permeability the rate-limiting step for RVD.
Epidermis contains platelet-type 12-lipoxygenase that is overexpressed in germinal layer keratinocytes in psoriasis
Authors: Hussain H. , Shornick L. P. , Shannon V. R. , Wilson J. D. , Funk C. D. , Pentland A. P. , Holtzman M. J.
Published: December 2017

Human epidermal cells exhibited none of the cytosolic lipoxygenase activity that is prominent in mucosal epithelial cells, but instead contained a microsomal activity that converted arachidonic acid to 12-hydroxyeicosatetraenoic acid (12-HETE). Identification of the extractable 12-HETE-forming activity as a 12-lipoxygenase (distinct from cytochrome P-450) included (S)-12-stereospecificity of product formation, trapping of 12-hydroperoxyeicosatetraenoic acid as an intermediate reaction product, and lack of NADPH dependence for activity. Epidermal cell poly(A)+ RNA contained high levels of a 2.3-kb mRNA that selectively hybridized with human platelet 12-lipoxygenase cDNA, and partial cDNA sequence of this mRNA indicated identity to platelet 12-lipoxygenase. The epidermal 12-lipoxygenase was not recognized by antibodies against the leukocyte-type 12- and 15-lipoxygenases (found in leukocytes, reticulocytes, and mucosal epithelial cells) but was detected by an antiplatelet 12-lipoxygenase antibody. The epidermal 12-lipoxygenase antigen was selectively expressed in germinal layer keratinocytes in healthy and psoriatic skin, and these layers exhibited hyperplasia and increased immunostaining in inflamed psoriatic skin. Together with previous results, these observations indicate that 1) epidermis generates 12-HETE by either cytochrome P-450 or lipoxygenase-based mechanisms depending on reaction conditions, and 2) 12-lipoxygenases (originally described in hematopoietic cell types) may be expressed in at least two distinct isoforms in epithelial barriers in humans, and in the case of the skin, a microsomal (platelet-type) 12-lipoxygenase is selectively overexpressed in germinal layer keratinocytes during psoriatic inflammation.
PGE2: a mediator of corneal endothelial wound repair in vitro
Authors: Joyce N. C. , Meklir B.
Published: December 2017

Maintenance of the integrity of the corneal endothelial monolayer is essential for corneal clarity. Aging, trauma, inflammation, and diseases, such as diabetes, can compromise monolayer integrity, resulting in corneal edema and loss of visual acuity. In adult humans, repair of the monolayer occurs mainly by two forms of cell movement: "migration," in which individual cells move from the wound edge to repopulate the defect, and "spreading," in which cells enlarge and flatten, causing movement of the monolayer as a sheet to cover the defect. Previous studies from this laboratory have shown that these two forms of movement can be pharmacologically separated. In the current studies, an established tissue culture model was used to determine the effect of prostaglandin E2 (PGE2) and of mediators of the adenosine 3',5'-cyclic monophosphate (cAMP) pathway on individual cell migration. Indomethacin, an inhibitor of prostaglandin synthesis, significantly decreased individual cell migration below levels obtained when wounds were exposed to culture medium alone. PGE2, but not PGF2 alpha, restored this response in a dose-dependent manner. In the presence of indomethacin, forskolin, a direct adenylate cyclase activator, stimulated individual cell migration. 2',5'-Dideoxyadenosine, an adenylate cyclase inhibitor, reversed the stimulatory effects of both forskolin and PGE2. Dibutyryl cAMP (DBcAMP) also stimulated individual cell migration in the presence of indomethacin, whereas, H89, a protein kinase A inhibitor, reversed both the DBcAMP and PGE2-induced effects. These results provide evidence that stimulation of the cAMP pathway enhances individual cell migration and that PGE2, acting via this pathway, may be an endogenous stimulator of this response during corneal endothelial wound repair.
Impaired cardiac function in rats with healed myocardial infarction: cellular vs. myocardial mechanisms
Authors: Cheung J. Y. , Musch T. I. , Misawa H. , Semanchick A. , Elensky M. , Yelamarty R. V. , Moore R. L.
Published: December 2017

The inotropic responsiveness of isolated perfused rat hearts and single left ventricular (LV) myocytes to extracellular Ca2+ ([Ca2+]o) was examined 3 wk after ligation of left main coronary artery. Myocytes isolated from myocardial infarcted (MI) hearts were 10% longer. At [Ca2+]o of 1.1 mM, cell shortening as well as intracellular Ca2+ concentration dynamics were similar between MI and sham LV myocytes. At [Ca2+]o of 4.9 mM, maximal extent of cell shortening was significantly less in MI myocytes (16 +/- 1 vs. 22 +/- 1%), and peak intracellular Ca2+ concentration was also substantially lower. Thus, under conditions of high [Ca2+]o, decreased sarcolemmal Ca2+ influx and Ca2+ release during excitation-contraction may contribute to systolic dysfunction in MI hearts. Perfused working hearts and isovolumic heart preparations with infarcted LV displayed depressed maximal systolic pressure and decreased sensitivity to the inotropic effects of [Ca2+]o. Our data also indicate that, in addition to possible abnormalities in the contractile response of single myocytes, global factors such as loss of functional myocardium, altered chamber geometry, tissue fibrosis, and/or subendocardial ischemia contributed to depressed LV function in post-MI hearts perfused at physiological [Ca2+]o.
Calcium-dependent release of arachidonic acid in response to purinergic receptor activation in airway epithelium
Authors: Lazarowski E. R. , Boucher R. C. , Harden T. K.
Published: December 2017

The effect of purinergic receptor agonists on arachidonic acid release was investigated in [3H]arachidonic acid-prelabeled human airway epithelial cells. Exposure of bronchial epithelial BEAS39 cells to extracellular ATP resulted in a marked release of unesterified [3H]arachidonic acid with maximal effect observed within 60-90 s. [3H]diacylglycerol and [3H]phosphatidic acid accumulated in parallel with [3H]arachidonic acid. ATP-stimulated [3H]arachidonic acid release with a K0.5 of 9 +/- 2 microM and UTP was equipotent; no effect was observed with P2Y- or P2X-purinergic receptor agonists or with adenosine. Similar results were obtained with primary cultures of normal human nasal epithelium, CF/T43 and HBE1 airway epithelial cell lines derived from a cystic fibrosis patient and from a normal donor, respectively, and HT-29 human colon carcinoma cells. ATP stimulated inositol phosphate formation in BEAS39 cells with a concentration dependence identical to that for [3H]arachidonic acid release. The effect of ATP on both [3H]arachidonic acid release and inositol phosphate formation was equally inhibited by pertussis toxin. The Ca2+ ionophore A-23187 mimicked the effects of ATP or UTP on arachidonic acid release, and a marked inhibitory effect was observed with thapsigargin. The protein kinase C inhibitor staurosporine partially inhibited ATP-stimulated [3H]arachidonic acid release. These data are consistent with the hypothesis that phospholipase A2 activation is secondary to P2U-purinergic receptor stimulation of D-myoinositol 1,4,5-trisphosphate production and calcium mobilization from intracellular stores.
Low pH enhances expression of carbonic anhydrase II by cultured rat inner medullary collecting duct cells
Authors: Schwartz G. J. , Brown D. , Mankus R. , Alexander E. A. , Schwartz J. H.
Published: December 2017

Carbonic anhydrase (CA) facilitates the secretion of protons from renal epithelia by catalyzing the buffering of hydroxyl ions by CO2. We have previously found that inner medullary collecting duct (IMCD) cells cultured from rat kidney secrete protons and express CA II. Incubation of IMCD cells in acidic medium for 48 h has been shown to stimulate the secretion of protons by a protein synthesis-dependent process. To establish whether CA II might be involved in this process, IMCD cells were exposed to incubation media supplemented with 10(-7) M deoxycorticosterone acetate, pH 7.0 (acid) or pH 7.7 (control) for 48 h, and CA II mRNA and protein were quantitated. Part of the CA II cDNA was obtained by reverse transcription of total RNA from rat kidney followed by amplification using oligonucleotide primers derived from conserved areas in the coding regions of human, mouse, and chick CA II cDNAs in a polymerase chain reaction. By Northern analysis, steady-state levels of CA II mRNA from acid-incubated cells showed an increase of 80% compared with controls and 70% when expressed relative to a housekeeping mRNA, beta-actin. Western blot analysis using a human antibody to CA II showed an approximate doubling of CA II protein after acid incubation. By immunofluorescence microscopy, the domes of acid-incubated IMCD cells contained considerably more CA II-stained cells than found in control cultures. Thus incubation of IMCD cells in acid medium stimulates the expression of CA II mRNA and protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Effects of insulin and exercise on amino acid transport in rat skeletal muscle
Authors: King P. A.
Published: December 2017

In the present study, the initial rates of amino acid transport by isolated rat skeletal muscle plasma membrane vesicles were investigated. This approach facilitates the study of the transport of naturally occurring amino acids independent of the effects of cellular metabolism. Alanine and glutamine influxes were measured using a rapid filtration technique. Transport was examined in the presence and absence of Na and the properties of membranes from control, insulin-treated, or acutely exercised rats were studied. Both alanine and glutamine were transported by Na-dependent processes. The values for maximum rate of transport (Vmax) for Na-dependent alanine and glutamine transport were 203 and 224 pmol.mg-1.s-1, respectively. The K1/2 values were 2.9 mM alanine and 1.9 mM glutamine. The Vmax for Na-dependent alanine transport was increased by insulin treatment of the animal and by acute exercise. 2-(Methylamino)-isobutyric acid (MeAIB) partially inhibited the control Na-dependent alanine influx and completely inhibited the increase due to insulin or exercise treatment, indicating the importance of both system A and a non-system A, Na-dependent carrier for alanine transport. The Vmax for Na-dependent MeAIB uptake was also increased by insulin or exercise treatments of the rats. Unlike alanine, Na-dependent glutamine transport was not affected by insulin.
Regulation of isotonic shortening velocity by second messengers in tracheal smooth muscle
Authors: Gunst S. J. , al-Hassani M. H. , Adam L. P.
Published: December 2017

Evidence suggests that the mechanical behavior of smooth muscle tissues is regulated by Ca(2+)-dependent changes in the phosphorylation of the 20,000-Da light chain of myosin (MLC). However, alternative mechanisms activated by specific kinases may be involved in regulating the shortening velocity in some smooth muscle tissues. To determine how the activation of protein kinases A or C affects the regulation of the shortening velocity in canine tracheal smooth muscle, we evaluated the effects of forskolin (10(-5) M) and phorbol 12,13-dibutyrate (PDBu, 3 x 10(-6) M) on active stress, intracellular Ca2+ ([Ca2+]i), MLC phosphorylation, and isotonic shortening velocity during contractions elicited by 60 mM KCl. Forskolin depressed and PDBu increased active stress, [Ca2+]i, MLC phosphorylation, and shortening velocity; thus the effects of these agents on the shortening velocity may result from changes in Ca(2+)-dependent MLC phosphorylation. In contrast, the decline in velocity that occurred with time during tonic contractions elicited by K+ depolarization was not associated with significant changes in MLC phosphorylation; thus the time-dependent changes in shortening velocity may be regulated by a mechanism other than MLC phosphorylation.
Receptor- and phorbol ester-mediated phospholipase D activation in rat parotid involves two different pathways
Authors: Guillemain I. , Rossignol B.
Published: December 2017

We have investigated phospholipase D (PLD) activation in rat parotid acini prelabeled with [14C]stearic acid. In the presence of 2% ethanol, muscarinic and alpha-adrenergic agonists stimulated the formation of [14C]phosphatidylethanol as a result of a PLD activity. The calcium ionophore, ionomycin, and the phorbol esters, 4 beta-phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu), also stimulated phosphatidylethanol accumulation, but 1-oleyl-2-acetyl-sn-glycerol (OAG), a permeant analogue of diacylglycerol did not. Chelerythrine and staurosporine, two inhibitors of protein kinase C, failed to affect any response. These results suggest that protein kinase C was not involved in the regulation of PLD activity. A difference between PLD regulation by PMA and receptor-mediated agonists was observed with regard to the extracellular calcium requirement. Our results strongly suggest that PLD activation in parotid acini involved different pathways: a calcium-dependent pathway activated by receptor-mediated agonists and a calcium-independent pathway activated by phorbol esters. Moreover, we observed that PLD activation did not result in any change in phosphatidic acid level. We propose that the phosphatidyl transferase activity of PLD reflected a metabolic pathway which may allow a base-exchange reaction in parotid gland.
Analysis of decaying unitary currents in on-cell patches of cells with a high membrane resistance
Authors: Ravesloot J. H. , van Putten M. J. , Jalink K. , Ypey D. L.
Published: December 2017

Under nonideal voltage-clamp conditions, unitary currents in the cell-attached patch (CAP) configuration of the patch-clamp technique are decaying instead of rectangular. From these data, four parameters can be determined when multiple identical channels are present. We analyzed an electrical equivalent circuit of the CAP configuration. This analysis yielded, for single-channel resistance, attached-cell membrane resistance, patch resistance, and attached-cell membrane capacitance, a set of different equations to calculate their values from these four parameters. To choose the most robust equation, those that yielded the same resistor/capacitance were compared on the basis of their sensitivity for inevitable deviations of the parameters from their nominal values. The results of our theoretical study are of methodological interest: 1) because they confirm that, under special conditions, CAP measurements can be used to measure electrical membrane properties of intact cells (this is important inasmuch as the CAP is the only configuration in which the cytoplasm of cells remains undisturbed) and 2) because we identify the level of voltage clamp and the equations with which the electrical properties can be determined with optimal accuracy.
Mechanism of sodium hyperabsorption in cultured cystic fibrosis nasal epithelium: a patch-clamp study
Authors: Chinet T. C. , Fullton J. M. , Yankaskas J. R. , Boucher R. C. , Stutts M. J.
Published: December 2017

Transepithelial Na+ absorption is increased two to three times in cystic fibrosis (CF) compared with normal (NL) airway epithelia. This increase has been associated with a higher Na+ permeability of the apical membrane of airway epithelial cells. Because Na+ absorption is electrogenic and abolished by amiloride, Na+ channels are thought to dominate the apical membrane Na+ permeability. Three Na+ channel-related mechanisms may explain the increase in apical Na+ permeability in CF cells: increased number of channels, increased single-channel conductance, and increased single-channel open probability. We compared the properties of Na(+)-permeable channels in the apical membrane of confluent preparations of human NL and CF nasal epithelial cells cultured on permeable supports. Na(+)-permeable channels were studied using the patch-clamp technique in the excised inside-out and cell-attached configurations. The same types of Na(+)-permeable channels were recorded in CF and NL cells. In excised patches, nonselective (Na+/K+) cation channels were recorded, and no differences between CF and NL were found in the properties, incidence, single-channel conductance, and single-channel open probability. In cell-attached patches, channels with a higher Na+ vs. K+ selectivity dominated. There was no difference between CF and NL cells in the incidence (18.8 vs. 21.4%, respectively) and conductance (17.2 +/- 2.8 vs. 21.4 +/- 1.5 pS, respectively) of Na(+)-permeable channels. However, the open probability was higher in CF cells compared with NL cells (30.0 +/- 3.4%, n = 6, vs. 15.0 +/- 3.9%, n = 13; P < 0.05). We conclude that, in CF nasal epithelial cells, the increase in Na+ permeability of the apical membrane results from an increase in the open probability of Na(+)-permeable channels in the apical membrane.
Protein kinase-independent inhibition of muscarinic K+ channels by staurosporine
Authors: Lo C. F. , Breitwieser G. E.
Published: December 2017

Acetylcholine (ACh) binding to atrial muscarinic receptors activates an inwardly rectifying K+ current (IK[ACh]) via a pertussis toxin-sensitive GTP-binding protein (GK). The muscarinic K+ channel (termed GIRK1) has been cloned, and the nucleotide sequence contains nine consensus sites for protein kinase C (PKC) phosphorylation (16). Dephosphorylation of the muscarinic K+ channel has been implicated in rapid IK[ACh] desensitization in the presence of agonist (13). Staurosporine is a widely used membrane-permeant inhibitor of PKC and other protein kinases (7), including G protein-coupled receptor kinases. We investigated the role of phosphorylation in the regulation of IK[ACh] by examining the effect of a variety of protein kinase inhibitors. Staurosporine produced a rapid and reversible dose-dependent decrease in IK[ACh], activated by either GTP or guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). Other PKC inhibitors, including calphostin C and K-252b, were without effect on GTP gamma S-activated IK[ACh]. In excised patches of atrial membrane under nonphosphorylating conditions (0 ATP, 1 mM 5'-adenylylimidodiphosphate), staurosporine reversibly reduced muscarinic K+ channel activity without altering single-channel current amplitude. These results suggest that staurosporine inhibits IK[ACh] by a mechanism independent of intracellular protein kinases.
Mechanisms of heterologous agonist-stimulated phosphorylation of cholecystokinin receptor
Authors: Zhu Z. , Lutz M. , Gates L. K. , Miller L. J.
Published: December 2017

The phosphorylation of one receptor that occurs as a result of the stimulation of a different receptor on a cell is a common mechanism for heterologous regulation or “cross-talk,” which has been implicated in desensitization. In this work, we focus on the mechanisms of phosphorylation of the rat pancreatic acinar cell cholecystokinin (CCK) receptor that occur upon stimulation of this cell by various agonists. Phosphorylation was allowed to occur in dispersed intact acinar cells in response to the experimental manipulation, and the phosphoreceptor was subsequently purified and quantified as an indication of response. Agonists such as vasoactive intestinal polypeptide and secretin, which act via activation of adenylate cyclase, had no effect on CCK receptor phosphorylation, whereas carbamylcholine and bombesin stimulated increased phosphorylation of the CCK receptor. Because these agents would be expected to activate protein kinase C (PKC) as well as a number of calcium-sensitive kinases and phosphatases, these activities were further dissociated by using more direct activators and inhibitors acting intracellularly. Manipulation of calcium independent of PKC by using a calcium ionophore, inhibition of calcium/calmodulin-dependent kinase II, and inhibition of calcium-dependent protein phosphatase type 2B had no effect on the state of CCK receptor phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)
13-HODE increases intracellular calcium in vascular smooth muscle cells
Authors: Stoll L. L. , Morland M. R. , Spector A. A.
Published: December 2017

13-Hydroxyoctadecadienoic acid (HODE) (2 microM) consistently increased porcine aortic and pulmonary artery smooth muscle cell calcium concentrations ([Ca2+]i), whereas 9-HODE and linoleic acid had no significant effect in the aortic cells and a much lesser effect in the pulmonary artery cells. A transient increase in [Ca2+]i occurred with as little as 50 nM 13-HODE. Structural specificity for elevation of [Ca2+]i also was seen with the monohydroxyeicosatetraenoic acids (HETEs), with 12-HETE but not 5- or 15-HETE increasing [Ca2+]i. 13-HODE, but not 9-HODE, increased smooth muscle cell guanosine 3',5'-cyclic monophosphate (cGMP) levels. The [Ca2+]i increase produced by 13-HODE was dependent on extracellular calcium and was inhibited by the calcium channel blockers verapamil and nifedipine and by KT-5823, a cGMP-dependent kinase inhibitor. A similar increase in [Ca2+]i was produced by 8-bromo-cGMP. These results suggest that 13-HODE, a 15-lipoxygenase product formed from linoleic acid, can act as a lipid mediator in vascular smooth muscle. It raises smooth muscle cGMP, causing a secondary increase in [Ca2+]i due to Ca2+ influx through a cGMP kinase-dependent L-type channel.
Interactions between retinoic acid and 1,25(OH)2D in mouse immortalized osteoblastic C1 cells
Authors: Chentoufi J. , Marie P. J.
Published: December 2017

The interactions between 1,25-dihydroxyvitamin D3 [1,25(OH)2D] and retinoic acid (RA) on alkaline phosphatase (ALP) activity and vitamin D receptors (VDR) were determined in the SV40 immortalized osteoblastic cell line C1. Biochemical and cytochemical analysis showed that ALP activity increased with C1 cell density and that 1,25(OH)2D inhibited ALP activity at high density, whereas RA increased ALP activity at low density. The interactions of the two hormones were also cell density dependent, with a predominant stimulatory effect of RA at low density and a predominant inhibitory effect of 1,25(OH)2D at high density. In contrast, the two hormones inhibited C1 cell proliferation independently of cell density. Scatchard analysis and immunocytochemical staining showed that nuclear VDR levels increased with cell density and that RA, 1,25(OH)2D, and their combination upregulated VDR levels mainly at high cell density. Although RA increased VDR levels, RA did not potentiate the effect of 1,25(OH)2D on ALP activity. The results indicate that the effects and interactions of 1,25(OH)2D and RA on ALP activity in osteoblastic C1 cells depend on the state of phenotypic maturation.
Responses of beta-adrenoceptor in rat soleus to phosphorus compound levels and/or unloading
Authors: Ohira Y. , Saito K. , Wakatsuki T. , Yasui W. , Suetsugu T. , Nakamura K. , Tanaka H. , Asakura T.
Published: December 2017

Responses of beta-adrenoceptor (beta-AR) in rat soleus to gravitational unloading and/or changes in the levels of phosphorus compounds by feeding either creatine or its analogue beta-guanidinopropionic acid (beta-GPA) were studied. A decrease in the density of beta-AR (about -35%) was induced by 10 days of hindlimb suspension, but the affinity of the receptor was unaffected. Suspension unloading tended to increase the levels of adenosine triphosphate and phosphocreatine and decrease inorganic phosphate. Even without unloading, the beta-AR density decreased after an oral creatine supplementation (about -20%), which also tended to elevate the high-energy phosphate levels in muscle. However, an elevation of beta-AR density was induced (about +36%) after chronic depletion of high-energy phosphates by feeding beta-GPA (about +125%). Data suggest that the density of beta-AR in muscle is elevated if the high-energy phosphate contents are chronically decreased and vice versa. However, it may not be directly related to the degree of muscle contractile activity.
Development of a H(+)-selective conductance during granulocytic differentiation of HL-60 cells
Authors: Qu A. Y. , Nanda A. , Curnutte J. T. , Grinstein S.
Published: December 2017

The NADPH oxidase is one of the main microbicidal systems of granulocytes. Stimulation of the oxidase during infection leads to a burst of metabolic acid generation. Potentially deleterious cytosolic acidification is prevented by the simultaneous activation of homeostatic H+ extrusion mechanisms, including a recently described H+ conductance. Studies in granulocytes from chronic granulomatous disease patients have suggested a relationship between the oxidase and the H+ conductive pathway. In this report we compared the expression of the H+ conductance and the NADPH oxidase during granulocytic differentiation of dimethyl sulfoxide-induced HL-60 cells. Patch-clamp determinations demonstrated that the H(+)-selective current detectable in differentiated HL-60 cells is virtually absent in uninduced cells. The H+ conductance was also estimated fluorimetrically, measuring changes in the cytosolic pH of suspended cells. Imposition of an inward protonmotive force failed to induce significant cytosolic acidification. In contrast, a sizable conductive H+ extrusion was detected in acid-loaded differentiated cells, consistent with the rectifying properties of the current measured electrophysiologically. By the spectroscopic method, the H+ conductance was not detectable in uninduced cells, developing gradually during granulocytic differentiation. Development of the conductive pathway was found to parallel the biochemical and functional appearance of the NADPH oxidase. These findings suggest that the H+ extrusion mechanisms required for the maintenance of the intracellular pH during granulocyte activation develop pari passu with the acid generating systems and suggest a functional and possibly structural association between the H+ conductance and the NADPH oxidase.
NMR-sensitive fluorinated and fluorescent intracellular calcium ion indicators with high dissociation constants
Authors: London R. E. , Rhee C. K. , Murphy E. , Gabel S. , Levy L. A.
Published: December 2017

A new series of high-dissociation constant (KD) Ca2+ indicators has been developed to reduce perturbations due to buffering of transients, to carry out measurements in cells and organelles with high basal Ca2+ concentrations, and to measure cytosolic Ca2+ levels in the presence of perturbations that may significantly increase these levels. A tetrafluorinated derivative of the chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 1,2-bis(2-amino-5,6-difluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (TF-BAPTA), has a KD of 65 microM and exhibits two fluorine nuclear magnetic resonances, one of which is insensitive to Ca2+ chelation and the second of which shifts by approximately 10 ppm upon Ca2+ binding. TF-BAPTA has pK values of approximately 5.0 and Mg2+ dissociation constants > 50 mM. At a field of 8.5 T, the Ca(2+)-sensitive resonance is in fast-intermediate exchange. Correction factors for the effects of intermediate exchange and for the effect of protonation (pK approximately 5.0) and Mg2+ complexation are discussed. An analogous approach has been used to synthesize 2-[2-(5-carboxyoxazole)]-5-[2-(2-bis(carboxymethyl) amino-5,6-difluorophenoxy)]ethoxy-6-bis(carboxymethyl)aminobenz ofuran (fura F), a structural analogue of fura 2, which exhibits fluorescence characteristics similar to those of fura 2, but has a KD of 20 microM.
ESR spectral transition by arteriovenous cycle in nitric oxide hemoglobin of cytokine-treated rats
Authors: Kosaka H. , Sawai Y. , Sakaguchi H. , Kumura E. , Harada N. , Watanabe M. , Shiga T.
Published: December 2017

Nitric oxide (NO) generation was induced in rats by Escherichia coli lipopolysaccharide (LPS) as detected by electron spin resonance (ESR) signals of NO hemoglobin (HbNO). However, there were inconsistencies in ESR spectral shape among them. We have therefore carried out a systematic study to clarify the in vivo spectral changes. First, the spectra of the alpha-NO heme species had the distinct three-line hyperfine structure in venous blood but not in arterial blood in all rats treated with tumor necrosis factor, interleukin-1, and/or LPS, and methemoglobin was not detected at the g = 6 (high-spin methemoglobin) region. Second, when the treated rats died, the three-line hyperfine structure was very distinct even in arterial blood. Third, even if HbNO was formed by injection of nitrite to rats, the three-line hyperfine structure of HbNO in venous blood was more marked than that in arterial blood, independent of the appearance of the methemoglobin signal. Fourth, an ex vivo study using whole blood demonstrated that the three-line hyperfine structure intensified lineally when O2 saturation of hemoglobin decreased but disappeared on reoxygenation of hemoglobin. These results directly demonstrate in vivo quaternary structural transition of the hemoglobin tetramer from the high-affinity state in the arterial cycle to the low-affinity state in the venous cycle. The transition makes the diverse ESR spectra of HbNO in vivo.
Differential modulation of large-conductance KCa channels by PKA in pregnant and nonpregnant myometrium
Authors: Perez G. , Toro L.
Published: December 2017

Uterine excitability depends on ion channel activity, the expression of which is regulated by sexual hormones. We show now that the action of protein kinase A (PKA) on large-conductance calcium-activated K+ (KCa) channel activity also depends on the hormonal status. PKA-dependent phosphorylation of reconstituted KCa channels from midpregnant rats usually stimulated channel activity; in contrast, KCa channels from nonpregnant rat and human myometrium were primarily inhibited by this mechanism. Both effects were reversible by phosphatase treatment. These results suggest that one important factor modulating uterine contractility during pregnancy or the regular cycle may be the differential response of KCa channels toward PKA-induced phosphorylation.
In situ determination of [Ca2+]i threshold for translocation of the alpha-protein kinase C isoform
Authors: Khalil R. A. , Lajoie C. , Morgan K. G.
Published: December 2017

Because of inherent difficulties in maintaining physiological conditions in biochemical assays, the intracellular free Ca2+ concentration ([Ca2+]i) required for activation of protein kinase C (PKC) in intact cells remains unclear. In the present study, [Ca2+]i was measured in freshly isolated vascular smooth muscle cells loaded with fura 2 while, in parallel, the distribution of the Ca(2+)-dependent alpha-PKC isoform was monitored using digital imaging microscopy. The [Ca2+]i alpha-PKC translocation threshold was determined by changing extracellular free Ca2+ concentration in steps while monitoring [Ca2+]i. In the absence of agonists, increasing [Ca2+]i caused < 25% of maximal translocation. In the presence of phenylephrine, maximum translocation occurred at [Ca2+]i > or = 198 nM. Phenylephrine augmented translocation of alpha-PKC primarily by increasing the slope of the [Ca2+]i-PKC translocation relationship. These results indicate that the [Ca2+]i threshold of alpha-PKC translocation in situ is less than that reported in most in vitro assays and are consistent with an effect of agonist-induced generation of other second messengers that cause cooperative interactions leading to translocation.
Radiotracer studies of cystic fibrosis transmembrane conductance regulator expressed in Xenopus oocytes
Authors: Ohrui T. , Skach W. , Thompson M. , Matsumoto-Pon J. , Calayag C. , Widdicombe J. H.
Published: December 2017

We measured fluxes of radiotracers in Xenopus oocytes expressing the cystic fibrosis transmembrane conductance regulator (CFTR). Addition of adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents [forskolin and 3-isobutyl-1-methylxanthine (I/F)] led to large increases in uptake of 36Cl, 125I, and 82Br into oocytes expressing wild-type CFTR or delta F508 CFTR but not sham-injected oocytes. I/F also stimulated halide efflux from CFTR and delta F508 oocytes in the sequence Cl > Br > I. cAMP-induced increases in 36Cl efflux from delta F508 oocytes were approximately 20% of those in CFTR oocytes. Increases in halide efflux were blocked by diphenylamine-2-carboxylic acid but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The phorbol ester, phorbol 12-myristate 13-acetate, also stimulated 36Cl efflux from CFTR oocytes. ATP uptakes into CFTR and sham oocytes were similar, and both were reduced by I/F. However, ATP uptake into I/F-treated CFTR oocytes was slightly greater (approximately 40%) than into I/F-treated sham oocytes. Urea uptake into CFTR and sham oocytes was similar and in both cases was increased by I/F. However, the I/F-induced increase in urea uptake into CFTR oocytes was significantly greater than for sham oocytes. I/F stimulated formate uptake into CFTR oocytes but not into sham oocytes. Fluxes of 22Na, 86Rb, 35SO4, 32PO4, and mannitol were unaltered by expression and activation of CFTR.
Regulation of cell volume and [Ca2+]i in attached human fibroblasts responding to anisosmotic buffers
Authors: Bibby K. J. , McCulloch C. A.
Published: December 2017

Alteration of the cell volume of attached fibroblasts with anisosmotic buffers was used to examine the relationship between cell membrane perturbation and intracellular Ca2+ concentration ([Ca2+]i) and to study pathways that may be involved in transducing this response. Human periodontal ligament gingival fibroblasts grown on cover slips were loaded with fura 2-acetoxymethyl ester. The relative cell volume change of single fibroblasts was estimated by measurement of fluorescence intensity at the isosbestic wavelength (356 nm), and [Ca2+]i was calculated from ratiometric fura 2 emission with excitation at 345 and 380 nm. Isotonic buffer (300 mosmol/kgH2O) was substituted with either hypertonic (600 mosmol/kgH2O) or hypotonic (150 mosmol/kgH2O) buffer after baseline recordings. Attached cells exhibited a rapid decrease in cell volume and [Ca2+]i after hypertonic buffer treatment, which was associated with an increase in filamentous actin staining. In contrast, cells treated with hypotonic buffers demonstrated an increase in cell volume (mean approximately 10%), a significant decrease in filamentous actin staining, and a rapid transient elevation in [Ca2+]i (mean approximately 280 nM). This [Ca2+]i rise was significantly inhibited by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, gadolinium ions (P < 0.05), and inhibitors of actin assembly. These results indicate that [Ca2+]i fluxes in response to hypotonic cell swelling in attached fibroblasts are mediated by stretch-activated ion channels and are dependent on actin filaments.