bluuebunny/crossref_metadata_2025 huggingface dataset:
Slowly activating voltage-dependent K+ conductance is apical pathway for K+ secretion in vestibular dark cells
Authors: Marcus D. C. , Shen Z.
Published: December 2017

Dark cell epithelium secretes K+ into the lumen of the vestibular labyrinth by a previously unidentified apical transport mechanism. Previous single-channel patch-clamp studies demonstrated nonselective cation channels and maxi-K+ channels in the apical membrane, but in too low a density to account for transepithelial K+ transport. In this report, we demonstrated with the cell-attached macro-patch-clamp technique an outward apical membrane current at 0-mV pipette voltage, which was stimulated by elevating bath K+ concentration from 3.6 to 25 mM and inhibited by 10 microM bumetanide, similar to their known effects on transepithelial short-circuit current and K+ secretion. Furthermore, the patch current was activated over several seconds by a sustained depolarization and deactivated over several hundred milliseconds by a hyperpolarization. Current-voltage relationships from tail currents were obtained with either NaCl or KCl in the pipette. Depolarization from -40 to +40 mV led to an increased conductance by a factor of 7.3 +/- 1.7 (n = 7) and 19.2 +/- 7.6 (n = 6) for NaCl and KCl, respectively, and to a reversal voltage near the presumed equilibrium potential for K+. The results demonstrate that dark cell K+ secretion occurs via K(+)-selective channels with characteristics similar to those associated with the IsK protein.
MCP-1-stimulated monocyte attachment to laminin is mediated by beta 2-integrins
Authors: Jiang Y. , Zhu J. F. , Luscinskas F. W. , Graves D. T.
Published: December 2017

Migration of monocytes to sites of inflammation involves a series of attachments and detachments to extracellular matrix proteins. We examined the capacity of a chemokine, monocyte chemoattractant protein-1 (MCP-1), to regulate attachment of human monocytes to laminin, collagen I, collagen IV, or fibronectin. MCP-1 increased monocyte attachment to laminin in a dose- and time-dependent manner and stimulated a lesser increase to the other matrix proteins. Function-blocking monoclonal antibodies (MAbs) to the integrin beta 2-subunit (CD18), including Fab' fragments and alpha M (CD11b) blocked > 70% of attachment, whereas MAbs to the beta 1-integrin subunit reduced attachment by < 30%. This suggests that the CD11b/CD18 integrin is the predominant molecule involved in adhesion of MCP-1-stimulated monocytes to laminin. The association of CD11b with F-actin illustrated by confocal microscopy further supports this concept. In contrast, when monocytes were stimulated with the beta 1-stimulatory MAb TS2/16, monocyte adhesion to laminin occurred through beta 1-integrins. Thus MCP-1 can stimulate monocyte attachment to laminin, and this process is mediated through beta 2-integrins, principally CD11b/CD18.
Different effects of gradual vs. acute adenine nucleotide depletion on ATP cost of muscle contraction
Authors: Foley J. M. , Adams G. R. , Meyer R. A.
Published: December 2017

ATP and phosphocreatine (PCr) levels were chronically reduced in rat fast-twitch muscle by 40 and 87%, respectively, after rats were fed a diet containing 1% beta-guanidinopropionate (beta-GPA) for 9 wk. Myosin heavy chain distribution of superficial gastrocnemius muscle changed from 22:78% type IIA/type IIB in control to 39:61% in GPA-treated muscles. ATP cost of contractions was estimated from the PCr changes measured by gated 31P-nuclear magnetic resonance after brief (< 6 s) bursts of contractions. There was no significant change in the ATP cost of either twitch (0.3 mumol.g-1.twitch-1) or tetanic (2.5 mumol.g-1.100 ms tetanus-1) contractions in gastrocnemius muscle after chronic phosphagen depletion due to beta-GPA feeding. In two other groups of rats, ATP of gastrocnemius muscles was acutely depleted to inosine 5'-monophosphate (IMP) by tetanic stimulation. IMP reanimation was inhibited during a subsequent 75-min recovery period in one group by hadacidin treatment (100 mg/kg), resulting in 46% ATP depletion compared with the similarly stimulated and recovered control group. In contrast to gradual ATP depletion due to beta-GPA feeding, this acute ATP depletion treatment was associated with a 39% decrease in ATP cost of twitch contractions.
Cl(-)-dependent NH4+ transport mechanisms in medullary thick ascending limb cells
Authors: Amlal H. , Paillard M. , Bichara M.
Published: December 2017

To characterize Cl(-)-dependent NH4+ transport mechanisms in renal medullary thick ascending limb (MTAL), intracellular pH (pHi) and membrane potential (PD) were monitored with use of 2',7'-bis(carboxyethyl)- 5(6)-carboxyfluorescein and 3,3'-dipropylthiadicarbocyanine, respectively, in suspensions of rat MTAL tubules in CO2-free media. Exposure of MTAL cells to 4 mM NH4Br caused, after an initial cell alkalinization due to NH3 entry, an NH4(+)-induced fall in pHi that was approximately 67% less pronounced in Cl(-)-free than in Cl(-)-containing media. The following experiments were performed in the presence of 1 microM amiloride to block the MTAL NH4+ conductance. When cells were preincubated in a Cl(-)-free gluconate medium in which K+ and Cl- conductances are greatly reduced, abrupt addition of 100 mN N-methyl-D-glucamine (NMDG)-Cl had no effect on cell PD and pHi in the absence of ammonia, but acutely acidified the cells by approximately 0.2 pH units in the presence of 4 mM NH4Br, which thus indicated nonelectrogenic (NMDG-Cl)-dependent NH4+ influx. The latter also occurred in a Cl(-)-free thiocyanate medium in which the Cl- conductance was blocked by 0.1 mM diphenylamine-2-carboxylate (DPC). An NMDG-Cl- dependent NH4(+)-induced fall in pHi was reduced approximately 33% by 10 mM Ba+, approximately 84% by 0.1 mM bumetanide, and 100% by 1.5 mM furosemide, whereas 1 mM hydrochlorothiazide had no effect; inhibition by Ba+ was observed even in the presence of 0.1 mM verapamil added to block both K+ channels and K+/NH4+ antiport.(ABSTRACT TRUNCATED AT 250 WORDS)
Patterns of tyrosine phosphorylation differ in vascular hypertrophy and hyperplasia
Authors: Ali S. , Dorn G. W.
Published: December 2017

Vascular smooth muscle cells (VSMC) undergo hypertrophy when exposed to thromboxane A2 and hyperplasia when exposed to phorbol 12-myristate 13-acetate (PMA) or platelet-derived growth factor (PDGF). Each of these three agonists stimulate rapid tyrosine phosphorylation of numerous VSMC proteins. The current studies were undertaken to identify proteins that are specifically tyrosine phosphorylated in one or the other growth response. All three agonists increased the phosphotyrosine content of multiple proteins. In Western analysis of phosphotyrosine immunoprecipitates, the hyperplastic agents PDGF and PMA increased tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1), GTPase-activating protein (GAP), and phosphatidylinositol-3-kinase (PI-3-kinase), while the hypertrophic agonist thromboxane failed to tyrosine-phosphorylate either of these three substrates. Tyrosine kinase inhibition with herbimycin A (5 microM) prevented agonist-stimulated tyrosine phosphorylation of PLC-gamma 1, GAP, and PI-3-kinase. In growth studies, herbimycin A inhibited PMA- and PDGF-induced hyperplasia but not thromboxane-stimulated hypertrophy. These results indicate that tyrosine phosphorylation of PLC-gamma 1, GAP, and PI-3-kinase are specific responses for VSMC hyperplasia but not thromboxane-stimulated hypertrophy.
Effects of intracellular acidosis on Ca2+ activation, contraction, and relaxation of frog skeletal muscle
Authors: Baker A. J. , Brandes R. , Weiner M. W.
Published: December 2017

The goal of this study was to determine the effects of intracellular acidosis (pH approximately 6.3) of frog skeletal muscle on force and on intracellular Ca2+ concentration ([Ca2+]i; measured at 20 degrees C using indo 1 fluorescence). Acidosis reduced tetanic force by only 11 +/- 2% (mean +/- SE, n = 8) but increased tetanic [Ca2+]i by 33 +/- 6%, suggesting that acidosis reduced the maximum Ca(2+)-activated force. During relaxation, the [Ca2+]i at half-maximal force was doubled with acidosis, suggesting that acidosis altered the Ca(2+)-force relationship. Acidosis markedly slowed force relaxation and [Ca2+]i decline (time constants fitted to force and [Ca2+]i during relaxation increased by 133 +/- 20 and 68 +/- 13%, respectively, with acidosis), suggesting that slowed force relaxation with acidosis may arise from slowed Ca2+ clearance from the cytosol. Late in relaxation, at approximately 30% of initial force, there was a transient phase of [Ca2+]i increase that was delayed with acidosis in proportion to the slowing of force relaxation. This is consistent with previous suggestions that dissociation of cross-bridges from the thin filament during relaxation promotes Ca2+ release to the cytosol from troponin. This study concludes that in skeletal muscle acidosis has little effect on tetanic force and that the major effects are decreased Ca2+ sensitivity and slower relaxation.
Rectification of whole cell cystic fibrosis transmembrane conductance regulator chloride current
Authors: Overholt J. L. , Saulino A. , Drumm M. L. , Harvey R. D.
Published: December 2017

Whole cell epithelial cystic fibrosis transmembrane conductance regulator (CFTR) Cl- currents exhibited a linear current-voltage (I-V) relationship with high symmetrical transmembrane Cl- concentrations. However, when intracellular Cl- (Cli-) was reduced by replacement with glutamate, I-V relationships were outwardly rectifying. Rectification was not affected by reducing extracellular Cl- to eliminate or reverse the gradient, indicating that rectification is not a function of the Cl- gradient. Rectification was affected by Cli- in a concentration-dependent manner, and it was weaker when Cli- was reduced by replacement with sucrose. These characteristics are identical to those of the cardiac isoform of CFTR, and the experimental data could be simulated by an Eyring rate theory model assuming that permeating anions interact at a single binding site within the channel pore. No evidence was found for multiple binding sites. These results indicate that rectification is a function of the concentration and permeability of the anions inside the cell. It is concluded that rectification of CFTR Cl- current is a property of ion channel permeation that would occur under physiological conditions and that permeation of the epithelial and cardiac isoforms of CFTR is identical.
Cholinergic activation of phospholipase D in lacrimal gland acini is independent of protein kinase C and calcium
Authors: Zoukhri D. , Dartt D. A.
Published: December 2017

To determine if rat lacrimal gland acini contain phospholipase D (PLD) activity, we took advantage of PLD's unique ability, in the presence of ethanol, to catalyze a transphosphatidylation reaction to produce phosphatidylethanol (PEth). Lacrimal gland acini were labeled for 3 h with [14C]stearic acid, preincubated for 20 min in the presence of 2% ethanol, and incubated for 20 min with or without agonists. Total cellular lipids were then extracted and analyzed by thin-layer chromatography, and the radioactivity was determined by liquid scintillation counting. Carbachol (1 mM), a cholinergic agonist, stimulated the production of both [14C]PEth and [14C]phosphatidic acid ([14C]PA) twofold. This effect was completely blocked by the muscarinic antagonist atropine (10 microM). [14C]PEth accumulation was also stimulated twofold by the active phorbol esters, 4 beta-phorbol 12-myristate 13-acetate and 4 beta-phorbol 12,13-dibutyrate at 1 microM. Ionomycin (1 microM), a Ca2+ ionophore, also stimulated the production of [14C]PEth twofold. In contrast to carbachol, neither phorbol esters nor ionomycin stimulated [14C]PA production. Neither [14C]PEth nor [14C]PA production was altered by epinephrine (1 mM), a nonselective adrenergic agonist, or phenylephrine (0.1 and 1 mM), a specific alpha 1-adrenergic agonist. We concluded that PLD activity, modulated by muscarinic receptors, protein kinase C, and Ca2+, but not by adrenergic receptors, is present in rat lacrimal gland acini. We also concluded that cholinergic activation of PLD appears to be independent of PKC and Ca2+.
Golgi apparatus is involved in intracellular Ca2+ regulation in epithelial LLC-PK1 cells
Authors: Zha X. , Chandra S. , Ridsdale A. J. , Morrison G. H.
Published: December 2017

Several lines of evidence suggest that the Golgi apparatus is involved in Ca2+ regulation in renal epithelial LLC-PK1 cells. Laser scanning confocal microscopy (LSCM) was employed to establish that a prominent perinuclear region is occupied mainly by the Golgi apparatus in this cell line. LSCM measurements in individual cells with the ionized Ca2+ indicator calcium green revealed that stimulation of LLC-PK1 cells with arginine vasopressin (AVP) resulted in the elevation of ionized Ca2+ levels. However, the vasopressin-induced rise in ionized Ca2+ was attenuated if the Golgi apparatus was disassembled by pretreating the cells with brefeldin A (BFA). Subcellular measurements of total Ca2+ with ion microscopy in cryogenically prepared cells indicated that 1) within 1 min of AVP treatment significant quantities of sequestered Ca2+ were released from the perinuclear Golgi region and 2) the BFA treatment reduced the total Ca2+ stored in the Golgi region. These observations indicate that the Golgi apparatus is sensitive to hormonal stimulation and may play important roles in intracellular Ca2+ regulation in LLC-PK1 cells.
Reconstitution of immunopurified alveolar type II cell Na+ channel protein into planar lipid bilayers
Authors: Senyk O. , Ismailov I. , Bradford A. L. , Baker R. R. , Matalon S. , Benos D. J.
Published: December 2017

Low-amiloride-affinity (L-type) Na+ channels have been functionally and immunologically localized to alveolar type II (ATII) cells. Purified rabbit ATII epithelial cells were isolated by elastase digestion and solubilized with 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate. The solubilized proteins were purified by ion-exchange chromatography, followed by immunoaffinity purification over a column to which rabbit polyclonal antibodies raised against purified bovine renal Na+ channel protein were bound. The proteins eluted from the immunoaffinity column were assayed for specific binding of [3H]Br-benzamil and reconstituted into planar lipid bilayers. Sequential purification steps gave a final enrichment in specific [3H]Br-benzamil binding of > 2,000 compared with the homogenate. Single-channel currents of 25 pS were recorded from the immunopurified rabbit ATII cell protein. Addition of the catalytic subunit of protein kinase A (PKA) plus ATP to the presumed cytoplasmic side of the bilayer resulted in a significant increase in the single-channel open probability (Po), from 0.40 +/- 0.14 to 0.8 +/- 0.12, without altering single-channel conductance. The addition of amiloride or ethylisopropyl amiloride (EIPA) to the side opposite that in which PKA acts reduced Po with no change in single-channel conductance. Rabbit ATII Na+ channels in bilayers had an inhibitory constant for amiloride of 8 microM and 1 microM for EIPA. These data confirm the presence of L-type Na+ channels in adult mammalian ATII cells.
Intracellular H+ inhibits a cloned rat kidney outer medulla K+ channel expressed in Xenopus oocytes
Authors: Tsai T. D. , Shuck M. E. , Thompson D. P. , Bienkowski M. J. , Lee K. S.
Published: December 2017

The pH sensitivity of a cloned rat kidney K+ channel, ROMK1, was examined after expression in Xenopus oocytes. Membrane currents and intracellular pH (pHi) were concomitantly monitored by the two-microelectrode voltage-clamp technique and a pH-sensitive microelectrode. Oocytes injected with ROMK1 cRNA developed a hyperpolarized resting potential of -98.7 +/- 0.98 mV and a slightly inwardly rectifying Ba(2+)-sensitive K+ current. Lowering external pH from 7.4 to 6.7 using membrane-permeable acetate buffer reduced measured pHi from 7.2 to 6.6 and reduced the ROMK1 current by 80%. The H+ blockade of ROMK1 currents was voltage independent. The relationship between ROMK1 slope conductance and pHi fitted to a titration curve suggested binding of four H+ to a site with a pK of 6.79. Extracellular acidification from pH 7.4 to 6.0 using membrane-impermeable biphthalate buffer had no effect on the ROMK1 current. The pH sensitivity of the ROMK1 channel is similar to that reported for a small-conductance native kidney K+ channel.
Decrease in heart peptide initiation during head-down tilt may be modulated by HSP-70
Authors: Menon V. , Yang J. , Ku Z. , Thomason D. B.
Published: December 2017

This study examines the mechanism of the rapid decrease in cardiac muscle protein synthesis during rodent hindlimb non-weight bearing. Polysomes isolated from rat hearts 8 h after suspension show less RNA in the polysome pool and a shift in polysome size toward fewer ribosomes per mRNA; 18 h after suspension, the size shift persists, but the amount of RNA in the polysome pool returns to control values. These data are consistent with a decrease in the rate of initiation of protein synthesis. At both 8 and 12 h of suspension, the cardiac polysomes show a 78 and 93% increase association with the nascent polypeptide chaperone protein 70-kDa heat-shock cognate/heat-shock protein (HSC/HSP-70), respectively, that persists after 7 days of non-weight bearing. Because the dissociation of HSC/HSP-70 from unfolded protein can be modulated by ATP, we measured the adenosine nucleotide pools and found a 53% decrease in ATP levels after 18 h of suspension. We propose a mechanism in which a shift of HSC/HSP-70 to the nascent polypeptide indirectly inhibits protein synthesis initiation.
Appearance of surfactant-like particles in apical medium of Caco-2 cells may occur via tight junctions
Authors: Engle M. J. , Grove M. L. , Becich M. J. , Mahmood A. , Alpers D. H.
Published: December 2017

Intestinal alkaline phosphatase (AP) is secreted by Caco-2 cells bound to surfactant-like particles (SLP), which can be localized by electron microscopy to the basolateral space and the intestinal lumen, especially over tight junctions. To investigate the hypothesis that SLP are secreted basolaterally and enter the lumen through the tight junction, Caco-2 cells were incubated with agents known to increase permeability at tight junctions. Cytochalasin D and phorbol 12-myristate 13-acetate increased Caco-2 cell monolayer permeability and the appearance of particles in apical medium two- to threefold, as monitored by mannitol movement and AP activity, respectively. Blocking the apical secretory pathway by nocodazole or colchicine had no effect on either parameter. Estimation of SLP content demonstrated an increase in apical media particles similar to that determined by AP activity. Quantitative image analysis established that apical SLP content increased 4-10 times, whereas total cell particle content remained unchanged. These data indicate that SLP may be secreted initially into the basolateral space and then transported to the intestinal lumen through the tight junctions.
Effect of Prozac on whole cell ionic currents in lens and corneal epithelia
Authors: Rae J. L. , Rich A. , Zamudio A. C. , Candia O. A.
Published: December 2017

Prozac (fluoxetine), a compound used therapeutically in humans to combat depression, has substantial effects on ionic conductances in rabbit corneal epithelial cells and in cultured human lens epithelium. In corneal epithelium, it reduces the current due to the large-conductance potassium channels that dominate this preparation. Its effects seem largely to decrease the open probability while leaving the single-channel current amplitude unaltered. In cultured human epithelium, currents from calcium-activated potassium channels and inward rectifiers are unaffected by Prozac. Delayed-rectifier potassium currents are reduced by Prozac in a complicated way that involves both gating and single-channel current amplitude. Fast tetrodotoxin-blockable sodium currents are also decreased by Prozac in this preparation. For all of these ion conductance effects, Prozac concentrations of 10(-5) to 10(-4) M are required. Whereas these levels are 10- to 100-fold higher than the plasma levels achieved in therapeutic use in humans, they are comparable to or less than levels needed for many other blockers of the ionic conductances studied here.
Osmoregulation of GPC:choline phosphodiesterase in MDCK cells: different effects of urea and NaCl
Authors: Kwon E. D. , Zablocki K. , Jung K. Y. , Peters E. M. , Garcia-Perez A. , Burg M. B.
Published: December 2017

The organic osmolyte, glycerophosphocholine (GPC), accumulates in renal cells in response to high concentrations of either NaCl or urea, despite the very different effects of these solutes on cell function and volume. Together, high levels of these solutes increase GPC amount in Madin-Darby canine kidney cells by inhibiting its enzymatic degradation. The present study tests the effects of NaCl and urea, individually, on GPC accumulation and its degradation. A technique was developed to determine the absolute rate of GPC degradation by measuring the initial rate of disappearance of [3H]GPC (pulsed into the cells by hypotonic shock) and the specific activity of GPC in the cells. The mass of GPC in the cells was measured by another newly developed method, a sensitive chemiluminescent assay. We find that exposure to high NaCl or urea decreases the absolute rate of cellular GPC degradation by approximately one-half during the first 20.5 h. Reductions in GPC degradation are accompanied by commensurate decreases in the activity of GPC:choline phosphodiesterase (GPC:PDE; EC 3.1.4.2), an enzyme that catalyzes degradation of GPC. Activity of GPC:PDE falls > 50% in cells exposed for 2 h to high osmolality. Inhibition is sustained for 7 days with high urea alone. In contrast, with high NaCl alone, GPC:PDE activity reverts to control values by 7 days, by which time synthesis of GPC is increased, accounting for sustained GPC accumulation. Collectively, these data suggest that GPC accumulation in response to either high NaCl or urea occurs initially by inhibition of its degradation but that the effect of NaCl on degradation differs, in that it is transient, while that of urea is sustained.
mRNAs of enzymes involved in energy metabolism and mtDNA are increased in endurance-trained athletes
Authors: Puntschart A. , Claassen H. , Jostarndt K. , Hoppeler H. , Billeter R.
Published: December 2017

Improvements in endurance capacity by training are associated with structural and biochemical adaptations of working muscles that affect the mitochondrial compartment. We investigated whether the 1.8-fold higher mitochondrial volume density in a group of endurance-trained athletes compared with untrained subjects was reflected by higher steady-state levels of mRNAs coding for components of the oxidative phosphorylation pathway using a quantitative polymerase chain reaction approach. We found that mitochondrially encoded RNAs (cytochrome-c oxidase subunit I, NADH reductase subunit 6, 16S rRNA), as well as nuclear-encoded RNAs (cytochrome-c oxidase subunit IV, succinate dehydrogenase, fumarase) are all increased coordinately in the athletes (1.54- to 1.94-fold). In addition, mitochondrial (mt) DNA concentration was also 1.55-fold higher in the trained athletes, whereas genomic DNA was not changed. Our findings thus show similar RNA expression of mitochondrially encoded genes in sedentary and endurance-trained subjects, whereas pretranslational control mechanisms account for higher levels of nuclear-encoded RNAs in the athletes.
CFTR mediates electrogenic chloride secretion in mouse inner medullary collecting duct (mIMCD-K2) cells
Authors: Vandorpe D. , Kizer N. , Ciampollilo F. , Moyer B. , Karlson K. , Guggino W. B. , Stanton B. A.
Published: December 2017

Previously we demonstrated that the inner medullary collecting duct cell line mIMCD-K2 secretes Cl- by an electrogenic mechanism [N. L. Kizer, B. Lewis, and B. A. Stanton. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F347-F355, 1995; N. L. Kizer, D. Vandorpe, B. Lewis, B. Bunting, J. Russell, and B. A. Stanton. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F854-F861, 1995]. The goal of the present study was to characterize the Cl- channel responsible for adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated Cl- secretion. To this end, using the patch-clamp technique, we measured Cl- currents. In whole cell patch-clamp experiments, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) activated Cl- currents that were time and voltage independent, inhibited by diphenylamine 2-carboxylate (DPC), and had a linear current-voltage (I-V) relation. In cell-attached patches of the apical membrane, we identified 7-pS Cl- channels that were stimulated by CPT-cAMP. In inside-out patches with Cl- in the pipette and bath solutions, Cl- currents had a linear I-V relation. The halide permeability sequence was PCl = PBr > PI. The Cl- channel inhibitors DPC, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, and glibenclamide blocked the 7-pS Cl- channel, whereas 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid was ineffective. By reverse transcriptase polymerase chain reaction, we isolated a partial cDNA clone encoding the cystic fibrosis transmembrane conductance regulator in mIMCD-K2 cells. We conclude that cAMP stimulates electrogenic Cl- secretion in inner medullary collecting duct cells by activating cystic fibrosis transmembrane conductance regulator Cl- channels.
The mitogenic effect of platelet-derived growth factor in human hepatic stellate cells requires calcium influx
Authors: Failli P. , Ruocco C. , De Franco R. , Caligiuri A. , Gentilini A. , Giotti A. , Gentilini P. , Pinzani M.
Published: December 2017

Platelet-derived growth factor (PDGF) is a key mitogen for hepatic stellate cells (HSC) and has been shown to be implicated in liver tissue repair and fibrogenesis. In this study the relationship between PDGF-induced intracellular Ca2+ concentration ([Ca2+]i) increase and mitogenesis in cultured human HSC was evaluated. In high-density cell cultures (80-90% subconfluence), PDGF induced a significant increase in [Ca2+]i, characterized by a short-lasting peak phase, which was followed by a long-lasting plateau phase. The plateau phase was abolished in the absence of extracellular Ca2+. However, in low-density cell cultures (30-40% subconfluence), the plateau phase was absent or markedly less pronounced. In parallel sets of experiments, PDGF was significantly less effective in inducing mitogenesis in low-density cell cultures than in high-density cell cultures and was totally ineffective in the absence of extracellular Ca2+. These results suggest that 1) spatial and time dynamics of PDGF-induced [Ca2+]i increase are dependent on cell density and 2) PDGF-induced mitogenesis requires extracellular Ca2+ influx.
Micro- and macrovascular endothelial cells in beta-adrenergic regulation of transendothelial permeability
Authors: Zink S. , Rosen P. , Lemoine H.
Published: December 2017

Barrier function of endothelial cells (EC) was modulated using beta-adrenergic agonists, e.g., isoproterenol (ISO) and formoterol (FOR). To get a direct comparison between EC from different vascular sources, we isolated EC from aorta (BAEC) and retina (BREC) of the same calf. For permeability studies, EC were cultured on polycarbonate filters. At confluency, transendothelial exchange of the diffusion marker fluorescein isothiocyanate-dextran was determined. Microvascular retinal EC monolayers are half as permeable as monolayers from macrovascular BAEC. When EC are stimulated with beta-adrenergic receptor (AR) agonists, monolayer permeability decreases, and the amount of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) increases in both cell types. Comparison of the half-maximum concentrations causing change in permeability (pEC50) shows direct coupling between beta-AR and adenylate cyclase. The beta 2-selective agonist FOR stimulates cAMP synthesis in BAEC with a pEC50 value of 9.37 and decreases permeability with a pEC50 value of 9.72. In BREC, the pEC50 values of ISO concerning stimulation of cAMP synthesis and the decrease of permeability are also very similar, 5.32 and 5.34, respectively. BREC are not as sensitive to beta 2-AR agonists as BAEC. The pEC50 value of FOR influence on BREC permeability is 8.77 in comparison with 9.72 for BAEC. These results could be interpreted with different affinities of the beta 2-selective AR agonist in BREC and BAEC.
Regulation of protein synthesis and degradation in adult ventricular cardiomyocytes
Authors: Schluter K. D. , Millar B. C. , McDermott B. J. , Piper H. M.
Published: December 2017

For studies on the regulation of myocardial protein metabolism, isolated adult cardiomyocytes were introduced as an experimental model about a decade ago. When used shortly after isolation, this model represents a tool for studying the properties of normal and diseased myocardium on the cellular level. The influence of various peptide hormones, neurotransmitters, and mechanical stimulation on protein synthesis and degradation in isolated cardiomyocytes has been studied. It has been demonstrated, for example, that alpha 1-adrenoceptor stimulation increases protein synthesis in newly isolated cardiomyocytes, independently of any mechanical effects. Other potential growth stimuli require appropriate conditions to induce cellular responsiveness. Neuropeptide Y, for example, does not stimulate cellular protein synthesis in newly isolated cells, whereas it does so in cells that have been cultured for a week in the presence of serum. Mechanical stretch also represents a growth stimulant. It seems that its signal transduction involves an autocrine loop. Thus different mechanisms, by which exogenous influences can modify cellular protein synthesis and degradation, have been identified on the cellular level, with the use of isolated adult cardiomyocytes.
Activation of adenylyl cyclase downregulates sodium/calcium exchanger of arterial myocytes
Authors: Smith L. , Smith J. B.
Published: December 2017

Chronic elevation of adenosine 3',5'-cyclic monophosphate (cAMP) is known to inhibit the proliferation of cultured vascular smooth muscle cells. The present findings show that the activation of adenylyl cyclase with forskolin decreased Na+/Ca2+ exchanger (NCX) mRNA and activity. Fetal bovine serum restored NCX transcript and activity. The changes in NCX transcript preceded the changes in NCX activity. Incubation of low-passage immortalized myocytes with forskolin plus 3-isobutyl-1-methylxanthine (IBMX), which inhibits cAMP phosphodiesterase, decreased NCX mRNA by 60% in 6 h and 80% in 24 h. After a 6-h lag, forskolin plus IBMX decreased NCX activity almost linearly to 20% of control at 40 h. 1,9-Dideoxyforskolin, which does not activate adenylyl cyclase, had no effect on NCX mRNA or activity. Forskolin plus IBMX decreased the c-Myc transcript, an immediate-early gene whose expression correlates with cell proliferation, but had no effect on plasma membrane Ca(2+)-ATPase transcripts. Removal of forskolin plus IBMX and addition of fetal bovine serum increased NCX and c-Myc transcripts seven- to eightfold in 6 h and restored NCX activity in 24 h. Inhibition of protein or RNA synthesis by cycloheximide or actinomycin D, respectively, prevented the increase in NCX mRNA. In contrast to blocking NCX induction, cycloheximide potentiated c-Myc induction by serum. Transcription factors that regulate myocyte growth may mediate the opposing influences of serum and forskolin on NCX mRNA and activity.
Differential Ca2+ responses of adrenergic and noradrenergic chromaffin cells to various secretagogues
Authors: Nunez L. , De La Fuente M. T. , Garcia A. G. , Garcia-Sancho J.
Published: December 2017

The effects of several physiological agonists on the cytosolic Ca2+ concentration ([Ca2+]i) of immunnocytochemically identified single adrenergic and noradrenergic bovine chromaffin cells were compared. No differences were observed in the responses to stimulation by high-K+ solutions with or without BAY K 8644, suggesting that the density and properties of voltage-dependent Ca2+ channels were similar in both cell types. The increase of [Ca2+]i induced by acetylcholine was greater in adrenergic cells, and this was due to differences in the response mediated through nicotinic receptors. The responses to bradykinin and to ATP were slightly greater in noradrenergic cells. Only a small fraction of the cells (18-28%) was responsive to ATP. The responses to angiotensin II and to histamine were much greater in adrenergic than in noradrenergic cells. Histamine was almost a selective stimulator of adrenergic cells. These differences suggest differential distribution of functional membrane receptors in both cell types and may be relevant to understanding the differential contribution of epinephrine- and norepinephrine-secreting cells during stressful conflicts in physiological or pathophysiological situations.
Cyclocreatine transport and cytotoxicity in rat glioma and human ovarian carcinoma cells: 31P-NMR spectroscopy
Authors: Schiffenbauer Y. S. , Meir G. , Cohn M. , Neeman M.
Published: December 2017

Cyclocreatine (CY), an analogue of creatine, inhibits tumor growth in vivo and proliferation of tumor cells in vitro. The goal of this study was to probe the mechanism of CY transport and cytotoxicity in C6 rat glioma cells and OC238 human ovarian carcinoma cells (creatine kinase activities of 0.16 and 0.016 units/mg protein, respectively). In both cell lines, CY significantly inhibited cell growth with no effect on membrane integrity and on the content of nucleoside triphosphates. An intrinsic 31P-nuclear magnetic resonance (31P-NMR) signal of phosphocreatine, as well as accumulation of phosphocyclocreatine (PCY) after addition of CY, was observed for C6 glioma but not for the OC238 cells. Transport of CY in C6 glioma showed Michaelis-Menten kinetics for an active sodium-dependent component. Transport was reduced more than fivefold in low-glucose medium. The toxicity of CY to C6 glioma cells may be due to PCY accumulation and cellular swelling. Another mechanism must be invoked to explain CY effects on the human ovarian cancer cells in which no PCY accumulation could be detected and no cellular swelling was observed.
A mitochondrial uncoupler increases KCa currents but decreases KV currents in pulmonary artery myocytes
Authors: Yuan X. J. , Sugiyama T. , Goldman W. F. , Rubin L. J. , Blaustein M. P.
Published: December 2017

Intracellular free Ca2+ concentration ([Ca2+]i) and ATP play important roles in the regulation of K- channels in pulmonary artery (PA) myocytes. Previous studies have demonstrated that hypoxia and the metabolic inhibitor, 2-deoxy-D-glucose, decrease voltage-gated K+ (KV) currents [IK(V)] and thereby depolarize PA myocytes; these effects lead to a rise in [Ca2+]i. Here, we used carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP), a protonophore that uncouples mitochondrial respiration from ATP production, to test whether the inhibition of oxidative phosphorylation affects K+ channel activities in rat PA myocytes. Patch-clamp and fluorescent-imaging microscopy techniques were used to measure K+ currents (IK) and [Ca2+]i, respectively. FCCP (3-5 microM) reversibly raised [Ca2-]i in the presence and absence of external Ca2+. This effect was prevented by pretreating the cells with the membrane-permeable Ca2+ chelator, 1,2-bis(2-amino-phenoxy) ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM). This suggests that much of the FCCP-evoked rise in [Ca2-]i was due to Ca2+ release from intracellular stores. Brief exposure to FCCP (approximately 2 min) reversibly enhanced Ik. This augmentation was not influenced by glibenclamide, an ATP-sensitive K channel blocker, but was eliminated by pretreatment with BAPTA-AM. This implies that the FCCP-evoked rise in [Ca2+]i activated Ca(2+)-activated K- (Kca) channels. Furthermore, in BAPTA-treated cells, longer application (> or = 6 min) of FCCP reversibly decreased IK(V) in PA cells bathed in Ca(2+)-free solution. These results demonstrate that FCCP affects KCa and Kv channels by different mechanisms. FCCP increases IK[Ca] by raising [Ca2+]i primarily as a result of Ca2+ release, but decreases IK(V) by a Ca(2+)-independent mechanism, presumably the inhibition of oxidative ATP production.
Permeability characteristics of erythrocyte membrane to okadaic acid and calyculin A
Authors: Namboodiripad A. N. , Jennings M. L.
Published: December 2017

The rates of transport of the protein phosphatase inhibitors okadaic acid and calyculin A through rabbit erythrocyte membranes have been estimated by measuring protein phosphatase type 2A (PP2A) activity in lysates. High concentrations of okadaic acid (100 nM) cause rapid (t 1/2 approximately 10 min) inhibition of PP2A. However, the t 1/2 for okadaic acid influx is much longer because the concentration is much higher than the concentration inhibiting 50% of the maximal response (IC50). The estimated t 1/2 is over 1 h at 37 degrees C and over 4 h at 25 degrees C. The effect of low extracellular pH indicates that the undissociated acid is the permeant species. It takes hours to reverse the effect of okadaic acid, because the efflux must proceed through several half times before the concentration is below the IC50 for PP2A. The permeation of calyculin A in contrast to okadaic acid is too fast to measure at 25 degrees C. Our results indicate that okadaic acid entry into erythrocytes is slower than is generally believed; it is crucial to consider concentration, temperature, pH, and time of exposure to okadaic acid to interpret the effects of this agent on intact cells.
Interferon-gamma induces persistent depletion of internal Ca2+ stores in a human salivary gland cell line
Authors: Wu A. J. , Chen Z. J. , Baum B. J. , Ambudkar I. S.
Published: December 2017

Interferon-gamma (IFN-gamma), in the presence of tumor necrosis factor-alpha (TNF-alpha), decreases proliferation of a human salivary gland ductal cell line, HSG (Wu, A., R. Kurrasch, J. Katz, P. Fox, B. Baum, and J. Atkinson. J. Cell. Physiol. 161:217-226, 1994). We examined the possible effects of these cytokines (1,000 U/ml IFN-gamma +/- 20 U/ml TNF-alpha for 7 days) on Ca2+ mobilization in HSG cells. In HSG cells, fetal bovine serum (10%) or carbachol (100 microM) stimulated rapid increases in cytosolic Ca2+ concentration ([Ca2+]i), apparently mobilized from different thapsigargin-sensitive intracellular Ca2+ stores. Serum induced a proliferative effect on HSG cells, which was suppressed (> 90%) by treatment with IFN-gamma +/- TNF-alpha, but not with TNF-alpha alone. Serum-, carbachol-, and thapsigargin-stimulated [Ca2+]i elevations were reduced by 90, 60, and > 65%, respectively, in cells treated with IFN-gamma +/- TNF-alpha and 30, 45, and 45%, respectively, in cells treated with TNF-alpha. Removal of the cytokines from the growth medium induced recovery of both cell proliferation and Ca2+ mobilization responses within 7 days. Treatment of HSG cells with thapsigargin (0.02-2 nM) induced a dose-dependent decrease in cell proliferation. Additionally, acute treatment (< 10 min) of cells with IFN-gamma did not affect [Ca2+]i or alter carbachol-, thapsigargin-, or serum-induced changes in [Ca2+]i. These data demonstrate that prolonged treatment of HSG cells with IFN-gamma +/- TNF-alpha leads to a persistent depletion of intracellular Ca2+ stores. We suggest that this may have a role in cell growth.
Protein kinase C modulates natriuretic peptide receptors in astroglial cultures from rat brain
Authors: Paulding W. R. , Sumners C.
Published: December 2017

We determined previously that astroglia cultured from newborn rat brain contain both guanylyl cyclase-coupled and atrial natriuretic peptide (ANP)-C natriuretic peptide receptors. Here, we investigated the effects of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) on these receptor subtypes in cultured astroglia to understand the intracellular processes involved in the modulation of natriuretic peptide receptors in these cells. PMA (10 nM to 1 microM; 15 min to 24 h) treatment elicited a time- and concentration-dependent decrease in the numbers of 125I-labeled ANP specific binding sites, which was inhibited by the PKC antagonist staurosporine (500 nM). Furthermore, PMA (100 nM, 2 or 24 h) treatment elicited a significant decrease in the specific binding of 125I-des-Cys-Cys-ANP, an ANP-C receptor selective ligand. PMA (10 nM to 1 microM; 30 min) treatment also significantly decreased ANP (100 nM)-stimulated guanosine 3', 5'-cyclic monophosphate levels in cultured astroglia, an effect unmodified by phosphodiesterase inhibition. These data indicate that PKC modulates both guanylyl cyclase-coupled and ANP-C natriuretic peptide receptors in cultured astroglia.
Polarity of TRH receptors in transfected MDCK cells is independent of endocytosis signals and G protein coupling
Authors: Yeaman C. , Heinflink M. , Falck-Pedersen E. , Rodriguez-Boulan E. , Gershengorn M. C.
Published: December 2017

Information concerning the molecular sorting of G protein-coupled receptors in polarized epithelial cells is limited. Therefore, we have expressed the receptor for thyrotropin-releasing hormone (TRH) in Madin-Darby canine kidney (MDCK) cells by adenovirus-mediated gene transfer to determine its distribution in a model cell system and to begin analyzing the molecular information responsible for its distribution. Equilibrium binding of [methyl-3H]TRH to apical and basolateral surfaces of polarized MDCK cells reveals that TRH receptors are expressed predominantly (>80%) on the basolateral cell surface. Receptors undergo rapid endocytosis following agonist binding; up to 80% are internalized in 15 min. A mutant receptor missing the last 59 residues, C335Stop, is poorly internalized (<10%) but is nevertheless basolaterally expressed (>85%). A second mutant TRH receptor, delta218-263, lacks essentially all of the third intracellular loop and is not coupled to G proteins on binding agonist. This receptor internalizes TRH approximately half as efficiently as wild-type TRH receptors but is nevertheless strongly polarized to the basolateral surface (>90%). These results indicate that molecular sequences responsible for basolateral accumulation of TRH receptors can be segregated from signals for ligand-induced receptor endocytosis and coupling to heterotrimeric G proteins.
Role of nitric oxide and phosphodiesterase isoenzyme II for reduction of endothelial hyperpermeability
Authors: Suttorp N. , Hippenstiel S. , Fuhrmann M. , Krull M. , Podzuweit T.
Published: December 2017

Regulation of endothelial permeability is poorly understood. Previous studies have shown that endothelial cells contain phosphodiesterase (PDE) isoenzymes II-IV and that simultaneous adenylate cyclase activation and/or PDE inhibition blocked endothelial hyperpermeability (J.Clin.Invest. 91: 1421-1428, 1993). We now focused on a possible role for guanosine 3',5'-cyclic monophosphate (cGMP)-dependent mechanisms and studied H2O2-exposed porcine pulmonary artery endothelial cell monolayers. Pretreatment of cells with different nitric oxide (NO) donors or atrial natriuretic peptide (ANP) increased endothelial cGMP-content severalfold and blocked H2O2-related effects on permeability; opposite results were obtained with a NO synthase inhibitor. Determination of cGMP degradation in nitroprusside-exposed endothelial cells identified PDE II as the major cGMP metabolizing pathway, whereas PDE III and IV contributed little or nothing. Inhibition of PDE II reduced H2O2-related endothelial hyperpermeability, an effect that could be enhanced synergistically by simultaneous guanylate cyclase activation. In summary, these studies indicate that cGMP-dependent mechanisms (NO donors, ANP, and dibutyryl-cGMP) blocked H2O2-related increases in endothelial permeability. The major cGMP degrading pathway in endothelial cells was PDE II, thereby substituting the missing PDE V in these cells. Simultaneous guanylate cyclase activation and/or PDE II inhibition may be a valuable approach to treat endothelial hyperpermeability.
Sodium-bicarbonate cotransport in guinea pig ileal crypt cells
Authors: MacLeod R. J. , Redican F. , Lembessis P. , Hamilton J. R. , Field M.
Published: December 2017

Prior studies show that ileal HCO3- secretion is of crypt origin, possibly involving Na+-HCO3- cotransport. To test for the latter, we isolated crypt cells from guinea pig ileum and determined effects of medium HCO3-, Na+, K+, disulfonic stilbenes, and gramicidin on intracellular pH [pHi;2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein fluorescence], cell volume (electronic sizing), and Na+ efflux from 22Na+ -preloaded cells. Ileal crypt cells alkalinized when placed in sodium gluconate-HCO3- medium containing N-5-methyl-5-isobutyl amiloride (1 microM), bumetanide (10 microM) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (250 microM which blocks Cl-/HCO3- exchange but not Na+ dependent HCO3- uptake). Depolarization with either gramicidin (50 microM) or 50 mM K+ caused a further 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS)-inhibitable increase in pHi. Gramicidin also caused SITS-inhibitable cell swelling. Both gramicidin effects were Na+ dependent: at 0 mM Na+, gramicidin acidified and did not alter cell volume; at 25 mM, gramicidin also acidified; at 90 and 140 mM, gramicidin alkalinized and induced cell swelling. HCO3- -dependent SITS-inhibitable Na+ efflux from 22Na+ -preloaded cells was also seen. We conclude that ileal crypt cells engage in electrogenic Na+ -HCO3- symport.
Effects of secretagogues on insulin biosynthesis and secretion in polyamine-depleted pancreatic beta-cells
Authors: Sjoholm A.
Published: December 2017

To extend previous observations on the importance of polyamines for glucose-stimulated insulinogenesis (N. Welsh and A Sjoholm. Polyamines and insulin production in isolated mouse pancreatic islets. Biochem. J. 252: 701-707, 1988), the impact of other secretagogues on insulin secretion of islets partially depleted in polyamines by selective inhibitors of L-ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase was monitored. Glucose-sensitive, but not basal, insulin release was partially abolished in polyamine-deficient islets. Qualitatively similar impairments in insulin secretion were recorded when such islets were stimulated with nonglucidic nutrients (alpha-ketoisocaproic acid + L-glutamine), a cationic amino acid (L-arginine), activators of phospholipase C (carbachol) or protein kinase C (12-O-tetradecanoylphorbol 13-acetate), an adenosine 1', 5'-cyclic monophosphate-raising agent (forskolin), or a hypoglycemic sulfonylurea (glibenclamide). Additionally, glucose-responsive (pro)insulin biosynthesis was preferentially impeded in polyamine-deficient islets. It is concluded that polyamines act as permissive or stimulatory factors in insulin production and release. In addition, they seemingly do not act in an inhibitory manner on phospholipase C, protein kinase C, or Ca2+ flux into these islets, in contrast to reports in which insulinoma and other cells were used.