bluuebunny/crossref_metadata_2025 huggingface dataset:
CaMKII mediates stimulation of chloride conductance by calcium in T84 cells
Authors: Worrell R. T. , Frizzell R. A.
Published: December 2017

We used the secretory colonic cell line T84 to study the regulatory pathways controlling the Ca-stimulated Cl conductance [GCl(Ca)]. Under whole cell patch clamp, basal (unstimulated) current levels averaged 73 +/- 9 pA/20 pF (n = 93) and increased to 600 +/- 100 pA/20 pF (n = 53; at +100 mV) on exposure to 1-2 microM ionomycin. Bath application of the calmodulin (CaM) antagonists trifluoperazine, calmidazolium, or sphingosine (50 microM) reversibly inhibited GCl(Ca), whereas the protein kinase C antagonists H7 and phloretin (50 microM) were without effect. This suggests that increases in intracellular Ca stimulate GCl(Ca) via a CaM-dependent process rather than activating Cl channels directly. To assess the involvement of protein kinases in the Ca-dependent stimulation of Cl conductance, we employed pseudosubstrate peptide inhibitors of protein kinase C (PKC) and the Ca/CaM-dependent protein kinase II (CaMKII). Cellular concentrations of inhibitors during whole cell recording were estimated to be 4-20 times the inhibitory constant values for kinase inhibition observed in vitro. Pipette solutions containing the PKC peptide inhibitor PKC-(19-36) (7.5 microM) had no effect on GCl(Ca). In contrast, stimulation of GCl(Ca) by ionomycin was abolished when pipette solutions contained 10 microM CaMKII peptide inhibitor CaMKII-(273-302). The truncated peptide CaMKII-(284-302) (20 microM) lacks the CaMKII inhibitory domain and did not affect GCl(Ca). These data suggest that CaM, acting through the multifunctional CaMKII, mediates the Ca-dependent stimulation of Cl conductance in colonic secretory cells.
Humoral factors reduce gap junction sensitivity to cytoplasmic pH. II. In vitro manipulations
Authors: Moreno A. P. , Arellano R. O. , Rivera A. , Ramon F.
Published: December 2017

Our previous studies demonstrated a diurnal rhythm in the response of gap junctions between crayfish giant axons to acidification and that the response was reduced after eyestalk ablation, sinus gland removal, or visual stress. In this paper we describe experiments to test whether compounds in the circulating hemolymph were responsible for modulation of the responsiveness gap junction channels to intracellular pH. In axons from destalked animals in which the hemolymph had been replaced with normal saline, the maximal junctional resistance after acidification (Rjmax) reached control values. In contrast, Rjmax reached only 30% of control after acidification in axons from animals that had been destalked but not perfused. Hemolymph drawn after eyestalk ablation was tested on axons from control animals. Treatment with hemolymph drawn 1 day after destalking resulted in control Rjmax values, while treatment with hemolymph drawn 7 days after destalking resulted in Rjmax values of only 5-40%. Similarly, pretreatment for 1 h with 100 microM ecdysterone resulted in low Rjmax values. These experimental results suggest that a circulating compound, most likely ecdysterone or a related molecule, regulates the physiological properties of gap junctions from crayfish lateral axons.
Effect of exogenous and endogenous nitric oxide on mitochondrial respiration of rat hepatocytes
Authors: Stadler J. , Billiar T. R. , Curran R. D. , Stuehr D. J. , Ochoa J. B. , Simmons R. L.
Published: December 2017

Although nitric oxide (.N = O) biosynthesis is inducible in rat hepatocytes (HC), the physiological significance of .N = O production by these cells is unknown. Short exposure of HC to authentic .N = O led to a concentration-dependent inhibition of mitochondrial aconitase, NADH-ubiquinone oxidoreductase, and succinate-ubiquinone oxidoreductase (complexes I and II of the mitochondrial electron transport chain). Most susceptible to .N = O inhibition was mitochondrial aconitase, in which a reduction in enzyme activity to 20.2 +/- 1.6% of control was observed. In contrast to mitochondrial aconitase, cytosolic aconitase activity was not inhibited by .N = O. After exposure to a maximal inhibitory concentration of .N = O, mitochondrial aconitase activity recovered completely within 6 h. Complex I did not fully recover within this incubation period. Endogenous .N = O biosynthesis was induced in HC by a specific combination of cytokines and lipopolysaccharide. After 18 h of incubation with these stimuli, a significant inhibition of mitochondrial aconitase activity to 70.8 +/- 2.4% of controls was detected. However, this was due only in part to the action of .N = O. A non- .N = O-dependent inhibition of mitochondrial function appeared to be mediated by tumor necrosis factor.
pHi regulation in frog retinal pigment epithelium: two apical membrane mechanisms
Authors: Lin H. , Miller S. S.
Published: December 2017

This study demonstrates that the apical membrane of frog retinal pigment epithelium (RPE) contains two intracellular pH (pHi) regulatory mechanisms, an electrogenic Na-HCO3 cotransporter blocked by DIDS and an amiloride-inhibitable Na-H antiporter. pHi was studied using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). In these cells resting pHi equals 7.26 +/- 0.09 (n = 58). After an acid load (NH4Cl prepulse), pHi recovery required apical extracellular Na concentration ([Na]o) in HCO3 or HCO3-free Ringer. In HCO3 Ringer recovery was completely blocked by 1 mM apical DIDS (n = 5) but was not affected by absence of Cl. In HCO3-free Ringer, recovery was completely blocked by 1 mM apical amiloride (n = 3). At resting pHi, the intrinsic pH-buffering capacity of the cell is approximately 7.1 mM/pH and rises monotonically as pHi decreases. In HCO3 Ringer, the initial rate of acidification caused by apical Na removal, 0.39 +/- 0.03 pH/min (n = 26), was 80-90% inhibited by apical DIDS (n = 5) and 16% inhibited by 1 mM apical amiloride (n = 7), but not affected by absence of Cl. In HCO3 Ringer, initial rates of acidification induced by apical DIDS or amiloride were 0.11 +/- 0.06 (n = 5) and 0.03 +/- 0.02 pH/min (n = 7), respectively. These results indicate that the Na-HCO3 cotransporter accounts for 80-90% of the acid extrusion from frog RPE cells. Increasing apical [K]o from 2 to 5 mM approximates the in vivo apical [K]o changes during a light-dark transition and alkalinizes the cells. [K]o-induced alkalinization had an initial rate of 0.11 +/- 0.02 pH/min (n = 16), which was approximately 75% inhibited by apical DIDS (to 0.04 +/- 0.01 pH/min, n = 7) and completely blocked by HCO3/CO2 removal from both bathing solutions. [K]o-induced pHi changes alter RPE transport mechanisms and may affect RPE-photoreceptor interactions.
Rapid increase in inositol phosphate levels in norepinephrine-stimulated vascular smooth muscle
Authors: Gu H. , Martin H. , Barsotti R. J. , LaBelle E. F.
Published: December 2017

We examined the correlation between agonist-stimulated increases in inositol phosphates and force development in vascular smooth muscle. Segments of rat tail artery were preincubated with [3H]inositol and treated with norepinephrine (10(-5) M) for 3-10 s. Tissue levels of inositol monophosphate (IP), inositol bisphosphate (IP2), and inositol trisphosphate (IP3) were measured. IP and IP2 increased significantly after 3 s of norepinephrine treatment. IP3 increased significantly after 5 s of norepinephrine treatment. Analysis of tissue extracts by high-pressure liquid chromatography demonstrated that the only isomer of IP3 present in any tissue extract was the 1,4,5-isomer [Ins(1,4,5)P3]. Contractile response to norepinephrine stimulation showed that the increase in inositol phosphates coincides well with the time course of force development. This is the first report demonstrating such an early increase in Ins(1,4,5)P3 in agonist-stimulated vascular smooth muscle. These results are consistent with the hypothetical role of Ins(1,4,5)P3 as a mediator linking agonist-receptor activation to increased intracellular calcium and force development in norepinephrine-stimulated vascular smooth muscle.
Relaxation, [Ca2+]i, and the latch-bridge hypothesis in swine arterial smooth muscle
Authors: Rembold C. M.
Published: December 2017

During vascular smooth muscle relaxation, myosin light-chain phosphorylation values decrease to resting values more rapidly than do stress values. Because phosphorylation is proportionally low, the latch-bridge hypothesis predicts that stress during relaxation should be predominantly carried by latch bridges. I evaluated the mechanical properties of latch bridges by changing tissue length and measuring myoplasmic Ca2+ concentration ([Ca2+]) with aequorin during relaxation of swine carotid medial tissues. Stress production was predicted with the latch-bridge model of Hai and Murphy, in which the measured aequorin [Ca2+] signal is the only determinant of stress. The aequorin-based latch-bridge model predicted relaxation induced by removal of the histamine stimulation. However, when tissues were relaxed by removal of extracellular Ca2+ or Ca(2+)-channel blockers in the continued presence of histamine, the aequorin-based model modestly underestimated the resulting relaxation. This underestimation was most likely caused by a small increase in the [Ca2+] sensitivity of phosphorylation since a model with an altered [Ca2+] sensitivity of phosphorylation more accurately predicted the resulting relaxation. The time course of relaxation in swine carotid artery was not substantially altered when the tissue was either briefly stretched or shortened and then returned to the original length. Because stretch should detach cross bridges, I modified the aequorin-based latch-bridge model to account for stretch-induced cross-bridge detachment. Because [Ca2+] values were slightly above resting values both before and after the stretch, the model predicted that phosphorylated cross bridges could reattach, be dephosphorylated, and form new latch bridges. The model predicted relaxation except during the first few seconds after stretch. These results suggest that latch-bridge reattachment is not necessary to explain the majority of the response to stretch during relaxation. The rate-limiting step for relaxation appears to be removal of [Ca2+] and not latch-bridge detachment.
Effect of angiotensin II on cytosolic free calcium in neonatal rat cardiomyocytes
Authors: Kem D. C. , Johnson E. I. , Capponi A. M. , Chardonnens D. , Lang U. , Blondel B. , Koshida H. , Vallotton M. B.
Published: December 2017

The effect of angiotensin II (ANG II) on cytosolic free Ca2+ concentration ([Ca2+]i) was studied in cultured neonatal rat ventricular myocytes. [Ca2+]i was estimated in groups of one to three cells by dual-wavelength microfluorometry or in cell populations using conventional fluorometry. ANG II (10(-8) M) produced an acute short-lived increase over the control basal diastolic [Ca2+]i and increased the frequency of the [Ca2+]i transients. The amplitude of the [Ca2+]i transients was decreased to 64.4% of basal values. The effect of ANG II on [Ca2+]i was blocked by the selective AT1 receptor subtype antagonist Du Pont 753 but not by the AT2 antagonist PD 123319. Removal of extracellular Ca2+ or blockade of voltage-gated Ca2+ channels in cells cultured for 5-7 days abolished the [Ca2+]i transients, but only partially diminished the effect of ANG II on [Ca2+]i. Thapsigargin, an inhibitor of sarcoplasmic reticulum Ca(2+)-Mg(2+)-ATPase, reduced or abolished the [Ca2+]i response to ANG II. Phorbol 12-myristate 13-acetate (PMA), 10(-6) and 10(-7) M, also decreased the amplitude of the Ca2+ transients similar to ANG II. Pretreatment with 10(-6) M PMA or 10(-6) M 1-oleoyl-2-acetyl-glycerol (OAG) inhibited the initial rise in [Ca2+]i and the Ca2+ transients. Thus ANG II produces an acute rise in [Ca2+]i which is derived predominantly from sarcoplasmic reticulum intracellular stores. This acute effect is followed by a significant reduction in the amplitude for the Ca2+ transient and may be mediated by activation of protein kinase C.
Differences between OK and LLC-PK1 cells: cystine handling
Authors: States B. , Harris D. , Segal S.
Published: December 2017

Cultured opossum kidney (OK) and porcine kidney (LLC-PK1) cells were compared for biochemical characteristics and cystine transport systems. The cell lines differ in amount of protein per cell, with OK cells having approximately one-half the amount found in LLC-PK1. Both cell lines contain 19 micrograms DNA/10(6) cells. As cells reach confluence, cystine uptake increases in OK and decreases in LLC-PK1 cells. Throughout the growth period, only lysine inhibits cystine uptake in OK, whereas glutamate is the inhibitor in LLC-PK1. The predominant site of cystine transport in OK cells is across the apical membrane, and the basolateral membrane is the corresponding site of transport in LLC-PK1 cells. Although the intracellular reduced glutathione pool is the same, the cysteine pool in OK cells is approximately one-fourth that found in LLC-PK1 cells. The ability of OK cells to reflect the shared cystine-dibasic amino acid transport system and LLC-PK1 to exhibit the cystine-glutamate antiporter system makes available two models for investigation of the development and structure of cystine transport systems.
Chloride conductance pathways in heart
Authors: Hume J. R. , Harvey R. D.
Published: December 2017

Nonelectrogenic movement of Cl- is believed to be responsible for the active accumulation of intracellular Cl- in cardiac muscle. The electro-neutral pathways underlying this nonpassive distribution of Cl- are believed to include Cl(-)-HCO3- exchange, Na(+)-dependent cotransport (operating as Na(+)-Cl- and Na(+)-K(+)-2Cl- cotransport), and K(+)-Cl- cotransport. The electrogenic movement of Cl- in cardiac muscle is particularly interesting from a historical perspective. Until recently, there was some doubt as to whether Cl- carried any current in the heart. Early microelectrode experiments indicated that a Cl- conductance probably played an important role in regulating action potential duration and resting membrane potential. Subsequent voltage-clamp experiments identified a repolarizing, transient outward current that was believed to be conducted by Cl-, yet further investigation suggested that this transient outward current was more likely a K+ current, not a Cl- current. This left some doubt as to whether Cl- played any role in regulating membrane potential in cardiac muscle. More recent studies, however, have identified a highly selective Cl- conductance that is regulated by intracellular adenosine 3',5'-cyclic monophosphate, and it appears that this Cl- current may play an important role in the regulation of action potential duration and resting membrane potential.
Acetylcholine Ca2+ stores refilling directly involves a dihydropyridine-sensitive channel in dog trachea
Authors: Bourreau J. P. , Abela A. P. , Kwan C. Y. , Daniel E. E.
Published: December 2017

Repetitive stimulation of the smooth muscle with acetylcholine (ACh) in the continuous presence of nifedipine resulted in a progressive decrease in the developed tension. This was associated with a decrease in the content of the agonist-sensitive intracellular Ca2+ stores. Agonist-sensitive internal Ca2+ stores appeared to be readily depleted by successive or prolonged agonist stimulation in Ca(2+)-free medium. The refilling of the empty stores when the muscle is at rest required extracellular Ca2+, was decreased by nifedipine, and was increased by BAY K 8644 and by increased external Ca2+ concentration. Refilling of stores during ACh stimulation in Ca(2+)-containing medium was decreased by nifedipine and by cyclopiazonic acid (CPA), an inhibitor of the sarcoplasmic reticulum (SR) Ca2+ pump, and was potentiated by BAY K 8644. BAY K 8644 reversed the inhibitory effect of CPA on stores Ca2+ refilling. Ryanodine in normal Krebs increased muscle resting tension, an effect not observed in Ca(2+)-free medium, blocked by nifedipine and enhanced by BAY K 8644. We propose that the refilling of ACh-sensitive internal Ca2+ stores involves two distinct pathways, one dependent on the uptake of cytosolic Ca2+ via a CPA-sensitive SR Ca(2+)-adenosinetriphosphatase, and the other pathway dependent on extracellular Ca2+ influx via a dihydropyridine-sensitive Ca2+ channel and is CPA insensitive. The refilling pathway between plasmalemma and SR may involve a plasmalemma L-type Ca2+ channel (dihydropyridine sensitive) and the SR Ca2+ release channel (ryanodine sensitive).
Intracellular pH on adrenergic-stimulated cAMP and cGMP production in rat pinealocytes
Authors: Ho A. K. , Girard M. , Young I. , Chik C. L.
Published: December 2017

In rat pinealocytes, activation of alpha 1-adrenergic receptors leads to increases in intracellular pH (pHi). In this study, the role of pHi on adrenergic regulation of cyclic nucleotide accumulation was investigated using ammonium chloride, which increased pHi, and sodium propionate, which reduced pHi. Ammonium chloride significantly enhanced the norepinephrine-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) responses, while it selectively potentiated the isoproterenol (ISO)-stimulated cGMP response. Reduction of pHi by sodium propionate reduced the norepinephrine-stimulated cGMP accumulation by 70%, and its effect on the ISO-stimulated cGMP response was stimulatory. Treatment with sodium propionate effectively neutralized the enhancing effects of ammonium chloride on the adrenergic-stimulated cAMP and cGMP responses. These effects of sodium propionate and ammonium chloride on cyclic nucleotides appeared to reflect altered rate of synthesis, and they were also in part secondary to changes in intracellular Ca2+. Our findings indicate that the receptor-mediated changes in pHi may play an integral part in the adrenergic regulation of cAMP and cGMP production in rat pinealocytes.
Muscle-specific regulation of the heme biosynthetic enzyme 5'-aminolevulinate synthase
Authors: Essig D. A. , McNabney L. A.
Published: December 2017

The induction of 5'-aminolevulinate synthase (ALV synthase) activity in adult muscle by overload occurs in the absence of proportional changes in its mRNA content. Complete interpretation of these findings is difficult because little is known of the basal regulation of ALV synthase expression in muscle. In three adult chicken muscle fiber types (n = 5 each), differences in ALV synthase activity were correlated (r greater than or equal to 0.89; P less than 0.05) to the activities of cytochrome oxidase (COX) and citrate synthase (CS) and to levels of the “liver” isoform of ALV synthase mRNA. During posthatch development, ALV synthase activity and mRNA levels (n = 3-6 per time point) also covaried with changes in COX and CS activity. The highest levels of ALV synthase mRNA in muscle are observed early in myogenesis prior to induction of COX activity. The regulation of ALV synthase is also tissue-specific because the higher basal levels of ALV synthase activity in liver mitochondria are associated with disproportionately less oxidative enzyme activity and less of the liver ALV synthase isoform mRNA than in muscle.
Force, length, and Ca(2+)-troponin C affinity in skeletal muscle
Authors: Fuchs F. , Wang Y. P.
Published: December 2017

On the basis of isotopic methods it has been found that force generation promotes increased Ca2+ binding to troponin C in cardiac muscle [P. Hofmann and F. Fuchs. Am. J. Physiol. 253 (Cell Physiol. 22): C541-C546, 1987] but not in skeletal muscle (J. Muscle Res. Cell Motil. 6: 477, 1985). However, studies with skinned rabbit psoas muscle fibers containing substituted fluorescent troponin C analogues indicate that force-generating cross bridges do promote increased Ca2+ binding in skeletal muscle (K. Guth and J. D. Potter. J. Biol. Chem. 262: 13627-13635, 1987). We have reexamined this question using a modified contraction-relaxation protocol in which Ca2+ binding to detergent-treated rabbit psoas fibers was measured either during steady-state force development or after relaxation was induced by one of two myosin ATPase inhibitors, vanadate or 2,3-butanedione monoxime. A standard double-isotope technique was used to measure Ca2+ binding. Another set of experiments was done in which force was reduced by releasing muscle fibers from sarcomere lengths of 2.4-2.6 microns to 1.5-1.7 microns, and bound Ca2+ was determined either before or after the release. No statistically significant effect of force generation or sarcomere length on Ca(2+)-troponin C affinity was observed. Thus the discrepancy remains between results obtained with isotopic and fluorescence methods. It is possible that in skinned fibers emission from fluorescence probes is more closely related to protein-protein interactions than to the amount of Ca2+ bound to troponin C.
Differential expression of Na channels in functional subpopulations of rat lactotropes
Authors: Horta J. , Hiriart M. , Cota G.
Published: December 2017

We have investigated the voltage-dependent Na channel activity of single lactotropes in pituitary cultures from adult male rats by recording whole cell Na+ currents under voltage clamp. Cells were identified by their ability to secrete prolactin in the basal state as measured with the reverse hemolytic plaque assay. We found that cells with elevated secretory rates [large-plaque (LP) lactotropes] present relatively large Na+ currents in response to depolarization, whereas Na+ currents in cells secreting prolactin at low rates [small-plaque (SP) lactotropes] are small or are not detectable. The maximum amplitude of the inward Na+ current, normalized by cell capacitance, is about sixfold larger, on the average, in LP lactotropes than in SP lactotropes. Complete block of the Na channels with external tetrodotoxin inhibits by approximately 72% the amount of prolactin secreted from the entire cell population over a period of 1 h, an effect that depends on a drastic reduction in the proportion of LP lactotropes. The results indicate that Na channel activity promotes basal prolactin secretion in male rat lactotropes and suggest that differences in Na channel expression contribute to explain the functional heterogeneity of these pituitary cells.
Actin filaments regulate epithelial Na+ channel activity
Authors: Cantiello H. F. , Stow J. L. , Prat A. G. , Ausiello D. A.
Published: December 2017

The functional role of the cytoskeleton in the control of ion channel activity is unknown. In the present study, immunocolocalization of Na+ channels with specific antibodies and fluorescein isothiocyanate-phalloidin to stain the cortical cytoskeleton indicates that actin is always present in close proximity to apical Na+ channels in A6 cells. The patch-clamp technique was used to assess the effect of cortical actin networks on apical Na+ channels in these A6 epithelial cells. The actin filament disrupter, cytochalasin D (5 micrograms/ml), induced Na+ channel activity in cell-attached patches within 5 min of addition. Cytochalasin D also induced and/or increased Na+ channel activity in 90% of excised patches tested within 2 min. Addition of short actin filaments (greater than 5 microM) to excised patches also induced channel activity. This effect was enhanced by addition of ATP and/or cytochalasin D. The effect of actin on Na+ channel activity was reversed by addition of the G actin-binding protein DNase I or completely prevented by treatment of the excised patches with this enzyme. Addition of the actin-binding protein, filamin, reversibly inhibited both spontaneous and actin-induced Na+ channels. Thus actin filament networks, achieved by either depolymerizing endogenous actin filaments by treatment with cytochalasin D, the addition of exogenous short actin filaments plus ATP, or actin plus cytochalasin D, regulate apical Na+ channel activity. This conclusion was supported by the observation that the addition of short actin filaments in the form of actin-gelsolin complexes in molar ratios less than 8:1 was also effective in activating Na+ channels. We have thus demonstrated a functional role for the cortical actin network in the regulation of epithelial Na+ channels that may complement a structural role for membrane protein targetting and assembly.
Distinct voltage-dependent regulation of a heart-delayed IK by protein kinases A and C
Authors: Walsh K. B. , Kass R. S.
Published: December 2017

We have investigated the effects of stimulation of adenosine 3',5'-cyclic monophosphate-dependent protein kinase (protein kinase A) and Ca(2+)-diacylglycerol-dependent protein kinase (protein kinase C) on the delayed rectifier K+ current (IK) in guinea pig ventricular cells using a whole cell arrangement of the patch-clamp procedure. Stimulation of either protein kinase C or A resulted in enhanced IK activity. Augmentation of IK observed during stimulation of protein kinase A occurred in a markedly voltage-dependent manner, with the largest increases occurring at potentials near the threshold for IK activation. Enhancement of IK during stimulation of protein kinase C followed a different pattern, with minimal effects of the enzyme near IK threshold. Neither protein kinase A nor C altered the kinetics of IK activation, although both kinases slightly changed the kinetics of deactivation. Both kinases increased IK maximal conductance, but the effects of each kinase on the voltage-dependence of activation differed. Protein kinase A shifted IK activation toward more negative voltages but did not affect the slope of the activation curve. Protein kinase C, in contrast, changed the slope of the IK activation curve, with only a small effect on the half-maximal voltage of activation. These contrasting effects on the voltage dependence of IK activation are consistent with actions of the kinases at distinct sites on or near the IK channel protein.
Simple harmonic motion of tropomyosin: proposed mechanism for length-dependent regulation of muscle active tension
Authors: Earley J. J.
Published: December 2017

A simple harmonic theory is proposed to describe the regulatory mechanism of tropomyosin in the activation of muscle contraction. The theory proposes that activation-associated displacement of tropomyosin is inherent to tropomyosin, a consequence of the molecule's large-scale vibrational motion (5-10 A root mean square displacement). In association with thin filament the vibrational motion may become less complex, approaching the ideal case of simple harmonic motion. The degree of activation increases as the amplitude of the simple harmonic motion increases, causing tropomyosin to shorten lengthwise, shiftings its position from the periphery of thin filament (OFF) to the actin groove (ON). The amplitude may be regulated in a rectilinear manner by the thick filament electrostatic force, the thin filament hydrophobic force, and the Ca(2+)-dependent force of the troponin complex. The radial and tangenital components of the resultant force may vary as the muscle is stretched, regulating maximum active tension and Ca2+ sensitivity, respectively. This may represent the molecular basis for Starling's law of the heart. The mechanism may be important for describing the regulatory mechanism of tropomyosin in smooth muscle and nonmuscle cells and may facilitate a clinically relevant understanding of the effects of pH, Mg2+ concentration, ionic strength, and ethanol on the regulation of active tension.
ATP-sensitive Na(+)-H+ antiport in type II alveolar epithelial cells
Authors: Brown S. E. , Heming T. A. , Benedict C. R. , Bidani A.
Published: December 2017

Type II alveolar epithelial cells in suspension have been previously shown to possess a Na(+)-H+ antiporter that modulates recovery from an intracellular acid load in the nominal absence of HCO-3 [E. Nord, S. Brown, and E. Crandall. Am. J. Physiol. 252 (Cell Physiol. 21): C490-C498, 1987]. Such a Na(+)-dependent mechanism has also been demonstrated in cultured type II cell monolayers (K. Sano et al. Biochim. Biophys. Acta 939: 449-458, 1988). It has recently been suggested that cultured type II cells possess a H(+)-ATPase that contributes to recovery from an intracellular acid load [R. Lubman, S. Danto, and E. Crandall. Am. J. Physiol. 257 (Lung Cell. Mol. Physiol. 1): L438-L445, 1989]. The present study was undertaken to investigate and characterize the mechanisms by which cultured type II cells recover from an intracellular acid load in the nominal absence of HCO-3. Cultured type II cell monolayers were loaded with the pH-sensitive probe 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein, and the characteristics of recovery from an imposed intracellular acid load were studied. Recovery of intracellular pH (pHi) was found to be strictly Na(+)-dependent and inhibited greater than or equal to 95% by 1 mM amiloride. Initial rate of recovery was highly sensitive to pHi, with recovery rates varying inversely with increasing pHi. An acidic extracellular pH (6.5) abolished pHi recovery. Treatment of type II cells with either the sulfhydryl reagent N-ethylmaleimide, a nonspecific sulfhydryl reagent, or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, a specific vacuolar H(+)-ATPase inhibitor at the concentration tested, resulted in marginal but not statistically significant decrements in pHi recovery. Intracellular ATP depletion, using KCN or replacement of glucose by a nonmetabolizable glucose analogue, reduced pHi recovery by 70-75% relative to control values. Sensitivity to ATP was apparent even under conditions that preserved the transmembrane Na+ gradient. Taken together, these data are most consistent with a single mechanism for pHi recovery in the absence of HCO3-. We interpret this mechanism to be an ATP-sensitive Na(+)-H+ antiporter that acts to reestablish pHi in type II alveolar epithelial cells.
Role of calcium and cAMP messenger systems in intracellular pH regulation of osteoblastic cells
Authors: Green J. , Kleeman C. R.
Published: December 2017

We have recently shown that two mechanisms are involved in the regulation of pHi in the osteoblastic phenotype cell line UMR-106 (Na(+)-H+ antiporter and a Na(+)-independent Cl(-)-HCO 3(-)-OH- exchanger). In the present work, we used the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein as well as isotope fluxes to investigate the influence of second messengers on the activity of these transporters. Elevation in intracellular calcium concentration [( Ca2+]in) in UMR-106 cells (measured by fura-2 fluorescence) is followed by stimulation of the Cl(-)-HCO3- exchanger, leading to cytosolic acidification. Subsequently, cell alkalinization, mediated by the Na(+)-H+ exchanger, restores pHi to its resting value. An acute reduction in [Ca2+]in abruptly stops the activity of the anion exchanger while having no influence on the activity of the Na(+)-H+ exchanger. The stimulatory effect of Ca2+in on the anion exchanger is dose dependent and is abrogated by the calmodulin inhibitors N-(6-aminohexyl)-5-chloro-naphthalenesulfonamide and calmidazolium. An increase in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) brought about by forskolin, 8-bromo-cAMP, or prostaglandin E2 leads to inhibition of activity of both the Na(+)-H+ antiporter and the anion exchanger. The suppressive effect of cAMP on Cl(-)-HCO3- exchange could be overcome by elevating [Ca2+]in. We conclude that 1) Ca2+in and cAMP can influence pHi in osteoblasts by altering the activities of pHi regulatory mechanisms and 2) the effect of Ca2+in is probably mediated by calmodulin.
Corticosteroid receptors in cells derived from rat brain microvessels: mRNA identification and aldosterone binding
Authors: Loffreda N. , Eldin P. , Auzou G. , Frelin C. , Claire M.
Published: December 2017

B7 is a cell clone derived from rat brain microvessels. Expression of an amiloride-sensitive cationic channel has been recently established in these cells. In this study, the polymerase chain reaction (PCR) was used to amplify definite segments of mineralocorticoid and glucocorticoid receptor mRNA in B7 cells. Aldosterone binding was also characterized. Two classes of sites were detected. Aldosterone exhibited a high affinity for type I sites [dissociation constant (Kd) approximately 0.3 nM] and a lower one for type II sites (Kd approximately 20 nM). RU 28362, a highly specific glucocorticoid agonist, did not compete for type I sites. RU 28362 and dexamethasone were better competitors for type II sites than aldosterone. The sedimentation coefficients of aldosterone type I and type II complexes were approximately 9S. These characteristics are close to the one exhibited by aldosterone type I and type II receptors in rat kidney and other target tissues. In intact B7 cells, aldosterone binding expressed as number of acceptor sites per cell was higher (approximately 41,000 for type II and 8,800 for type I) than in the soluble cellular extract (approximately 18,000 for type II and 1,000 for type I).
Transcellular transport of organic cation across monolayers of kidney epithelial cell line LLC-PK
Authors: Saito H. , Yamamoto M. , Inui K. , Hori R.
Published: December 2017

Transcellular transport and the accumulation of [14C]tetraethylammonium, a typical organic cation, by LLC-PK1 cell monolayers grown on microporous membrane filters were studied. Tetraethylammonium was accumulated progressively in the monolayers from the basolateral side and was transported unidirectionally to the apical side. The transcellular transport of tetraethylammonium was saturable, temperature dependent, and sensitive to the pH of the apical side of the monolayers. The apparent Michaelis constant and maximum velocity values for the transport were 67 microM and 222 pmol.mg protein-1.min-1, respectively. Unlabeled tetraethylammonium, amiloride, procainamide, cimetidine, and choline inhibited the basolateral uptake and transcellular transport of [14C]tetraethylammonium. The development of tetraethylammonium transport activity was observed in the differentiating cells. A sulfhydryl reagent inhibited the tetraethylammonium transport at both the basolateral and apical membranes of the LLC-PK1 cells. These findings suggest that these monolayers possess unidirectional transport systems for organic cations, corresponding to the secretion in the renal proximal tubules.
Hyperoxia induces alkalinization and dome formation in MDCK epithelial cells
Authors: Dreher D. , Rochat T.
Published: December 2017

We observed that confluent Madin-Darby canine kidney (MDCK) epithelial cells exposed to 95% O2 showed intensive dome formation, a manifestation of cell differentiation and transepithelial fluid transport, whereas cells exposed to 40% O2 or to normoxia did not. Hyperoxia-induced dome formation (HIDF) was preceded and accompanied by a significant rise in intracellular pH (pHi) on days 2 (7.53 vs. 7.42) and 3 (7.62 vs. 7.45), as compared with controls. Inhibition of the Na(+)-H+ exchanger by 0.1 or 1.0 mM amiloride caused 29 or 69% reduction of HIDF and completely abolished hyperoxia-induced alkalinization of the cells. HIDF was altered by modification of extracellular pH (pHo); there was a decrease by 84% with pHo 6.8, while pHo 7.8 led to earlier and more intensive dome formation (day 2, +472%; day 3, +27%). We also found that adenosine 3',5'-cyclic monophosphate (cAMP) was increased in hyperoxic cells, a change that was independent from the rise of pHi. We conclude that high-level hyperoxia induces dome formation in MDCK epithelial monolayers by a process involving activation of the Na(+)-H+ exchanger, together with increased intracellular cAMP.
Direct evidence for a basolateral membrane Cl- conductance in toad retinal pigment epithelium
Authors: Fujii S. , Gallemore R. P. , Hughes B. A. , Steinberg R. H.
Published: December 2017

There is now evidence that a Cl- conductance on the basal membrane of the retinal pigment epithelium (RPE) is involved in the generation of both the fast oscillation and the light peak of the direct-current electroretinogram as well as being critical for transepithelial fluid and salt movement. In the present study, we characterized the basolateral membrane Cl- conductance of an in vitro preparation of toad RPE-choroid using conventional and Cl(-)-selective microelectrodes. Under control conditions, the potential across the apical (Vap) and basal (Vba) membranes averaged -60 +/- 2 and -45 +/- 2 mV, respectively (n = 40). Intracellular Cl- activity (aiCl = 20 +/- 1 mM) was distributed above equilibrium across both membranes, consistent with active accumulation of Cl-. A sixfold decrease in Cl- in the basal bath depolarized Vba by 12 +/- 1 mV (n = 17) and increased the apparent basal membrane resistance. By sequential measurement of aiCl and subepithelial Cl- activity during a step decrease in basal Cl-, we constructed the change in Cl- equilibrium potential (ECl) across the basal membrane. Estimation of the change in basal membrane electromotive force during the change in ECl gave an average value for the Cl- transference number (TCl) of 0.45. Further evidence for a Cl- conductance was obtained by measuring changes in aiCl induced by transepithelial current. Depolarizing Vba elevated aiCl, whereas hyperpolarizing Vba had the opposite effect, consistent with conductive Cl- movement across the basal membrane. Both the amplitude of the Cl- diffusion potential and the current-induced changes in aiCl were reduced by basal perfusion with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (250-500 microM), a blocker of Cl- channels in some epithelia.
Distribution of actin filament lengths measured by fluorescence microscopy
Authors: Burlacu S. , Janmey P. A. , Borejdo J.
Published: December 2017

We analyzed the distribution of actin filament lengths by optical microscopy (OM). OM avoids possible alterations in the size or structure of actin filaments occurring during sample preparation for electron microscopy (EM). Images of F-actin labeled with tetramethylrhodamine isothiocyanate (TRITC)-phalloidin were analyzed for both size distribution and flexibility. In the standard buffer [25 mM potassium acetate, 4 mM MgSO4, 25 mM tris(hydroxymethyl)aminomethane acetate, pH 7.5, 20 mM beta-mercaptoethanol] filaments did not aggregate into bundles and remained stable at nanomolar concentrations for at least 1 h. At the same concentration, actin labeled directly with rhodamine (no phalloidin) formed unstable filaments whose average length decreased with time. The number average length of TRITC-phalloidin labeled filaments (Ln) was 4.90 microns, the ratio (rho) of the weight average length to the number average length was 2.06, and the correlation length (1/lambda) was 8.33 microns. These parameters were in good agreement with the values determined by EM for filaments shorter than 8 microns. Passing G-actin through a Sephadex G-150 column before polymerization did not have a significant effect on the distribution of lengths but made filaments more stiff (1/lambda = 12.5 microns). Millimolar concentration of ATP increased the correlation length, and gelsolin had the expected fragmenting effect on filaments. These results show that OM can be used as a fast and reliable method to analyze the distribution and flexibility of actin filaments and suggest that, in spite of extensive manipulation of actin filaments during sample preparation, EM is a valid tool for determination of size parameters of actin filaments.
Sarcolemmal Na(+)-K(+)-ATPase activity in congestive heart failure due to myocardial infarction
Authors: Dixon I. M. , Hata T. , Dhalla N. S.
Published: December 2017

Because the Na+ pump is considered to modulate the contractile force development by the cardiac muscle and depressed cardiac pump function is the hallmark of congestive heart failure, we characterized the sarcolemmal Na(+)-K(+)-ATPase activity in failing rat hearts after myocardial infarction. For this purpose, the left ventricular coronary artery was ligated, and hearts were examined 4, 8, and 16 wk later; sham-operated animals served as controls. Hemodynamic assessment revealed the presence of abnormal cardiac function at 4, 8, and 16 wk. Although accumulation of ascites in the abdominal cavity was present in experimental animals at 4 wk, other clinical signs of congestive heart failure in experimental rats including lung congestion and cardiac dilatation were evident 8 and 16 wk after induction of myocardial infarction. The depression in Na(+)-K(+)-ATPase activity in purified sarcolemmal membrane from the uninfarcted experimental left ventricle at 8 wk was associated with depressed Vmax without any changes in the affinities for Mg-ATP, Na+, and K+ or the pH optimum for the enzyme. The Kd values of both the high- and low-affinity binding sites for [3H]ouabain, which is believed to interact with Na(+)-K(+)-ATPase, were increased; however, no change in the density of either class of ouabain binding site was evident. The depression of Na(+)-K(+)-ATPase activity in failing hearts at 16 wk of myocardial infarction was not different from that observed at 8 wk but the enzyme activity was not altered at 4 wk of coronary occlusion. These data support the view that depression of Na(+)-K(+)-ATPase activity may serve as an adaptive mechanism during the development of congestive heart failure.
Tetrodotoxin-insensitive sodium channels in a cardiac cell line from a transgenic mouse
Authors: Sculptoreanu A. , Morton M. , Gartside C. L. , Hauschka S. D. , Catterall W. A. , Scheuer T.
Published: December 2017

The electrophysiological properties of a cardiac cell line (MCM1) originating from a transgenic mouse were characterized. The dominant current in these cells is a sodium current that is insensitive to concentrations of tetrodotoxin (TTX) up to 100 microM. It activates and inactivates rapidly with half-maximal activation at -40 mV and half-maximal inactivation at -79 mV. This sodium current is reduced by agents that increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP) and activate cAMP-dependent protein kinase including isoproterenol, 8-bromo-cAMP, and isobutylmethylxanthine. The phenylalkylamine desmethoxyverapamil blocks the TTX-insensitive sodium current in MCM1 cells in both tonic and use-dependent fashion. Membrane depolarization enhances this block. It is proposed that the TTX-insensitive sodium current in these cells may be similar in origin to the embryonic type of TTX-insensitive sodium current described in other cardiac and skeletal muscle preparations.
Cloning, sequencing, and expression of Na(+)-H+ antiporter cDNAs from human tissues
Authors: Takaichi K. , Wang D. , Balkovetz D. F. , Warnock D. G.
Published: December 2017

Two types of Na(+)-H+ antiporter with different sensitivities to amiloride analogues have been identified in mammalian plasma membranes. A human Na(+)-H+ antiporter cDNA was obtained by Sardet and co-workers (C. Sardet, L. Counillon, A. Franchi, and J. Pouyssegur. Cell 56: 271-280, 1989) using mutant mouse fibroblasts lacking Na(+)-H+ antiporter transformed with human genomic DNA. However, the amiloride sensitivity of this cloned Na(+)-H+ antiporter was not precisely determined. Furthermore, the reported cDNA sequence may be a chimera of human and mouse genes. Hence we isolated a Na(+)-H+ antiporter cDNA actually expressed in human tissues and characterized its amiloride sensitivity. Our 4 kb cDNA obtained from human kidney cortex contained the identical open reading frame to that previously reported and the entire 3' terminus, which was quite different from that reported. This discrepancy was not due to differences in tissue-specific expression because cDNAs from different human tissues were identical, and single bands were observed under high stringency on Northern blots of various human tissues. Na(+)-H+ antiporter activity of mutant mouse fibroblasts deficient in Na(+)-H+ antiporter activity transfected with the cloned cDNA was very sensitive to amiloride and 5-N substituted analogues of amiloride. Thus the cloned cDNA represents the NHE-1 isoform of the Na(+)-H+ antiporter.
Direct role for potassium channel inhibition in hypoxic pulmonary vasoconstriction
Authors: Post J. M. , Hume J. R. , Archer S. L. , Weir E. K.
Published: December 2017

Cellular mechanisms responsible for hypoxic pulmonary vasoconstriction were investigated in pulmonary arterial cells, isolated perfused lung, and pulmonary artery rings. Three K+ channel antagonists, Leiurus quinquestriatus venom, tetraethylammonium, and 4-aminopyridine, mimicked the effects of hypoxia in isolated lung and arterial rings by increasing pulmonary artery pressure and tension and also inhibited whole cell K+ currents in isolated pulmonary arterial cells. Reduction of oxygen tension from normoxic to hypoxic levels directly inhibited K+ currents and caused membrane depolarization in isolated canine pulmonary arterial smooth muscle cells but not in canine renal arterial smooth muscle cells. Nisoldipine or high buffering of intracellular Ca2+ concentration with [1,2-bis(2)aminophenoxy] ethane-N,N,N',N'-tetraacetic acid prevented hypoxic inhibition of K+ current, suggesting that a Ca(2+)-sensitive K+ channel may be responsible for the hypoxic response. These results indicate that K+ channel inhibition may be a key event that links hypoxia to pulmonary vasoconstriction by causing membrane depolarization and subsequent Ca2+ entry.
Regulation of agonist-evoked [Ca2+]i oscillation by intracellular Ca2+ and Ba2+ in AR42J cells
Authors: Zhang B. X. , Zhao H. , Loessberg P. A. , Muallem S.
Published: December 2017

Measurements of intracellular Ca2+ ([Ca2+]i) and intracellular Ba2+ ([Ba2+]i) in single AR42J cells were used to evaluate the effect of [Ca2+]i and [Ba2+]i on agonist-evoked [Ca2+]i oscillations. Variations in [Ca2+]i and [Ba2+]i were imposed by gradual activation of entry through voltage-activated Ca2+ channels (VACC) present in the plasma membrane of these cells. Activation of high K+ was followed by partial inactivation of the channels and stabilization of [Ca2+]i at a new steady-state level depending on the extent of depolarization. Activation by BAY K 8644 was followed by complete inactivation and return of [Ca2+]i to resting levels. Ba2+ activated the channels and entered the cells but could not be removed from the cytosol by cellular Ca2+ pumps. The use of channel blockers and the ability to increase [Ca2+]i and [Ba2+]i by channel activation during [Ca2+]i oscillations showed that VACC do not contribute to or are activated during agonist-stimulated Ca2+ oscillation in this cell type. Graded activation of VACC showed that an increase in [Ca2+]i between the spikes to below 200 nM increased the frequency of the oscillation. Further increase in [Ca2+]i caused gradual reduction in the frequency. At [Ca2+]i above 500 nM, [Ca2+]i oscillations were inhibited. The inhibitory but not the stimulatory effects of [Ca2+]i on the oscillations can be mimicked by [Ba2+]i. These observations suggest that [Ca2+]i levels between the spikes play an important role in regulating the oscillations.