bluuebunny/crossref_metadata_2025 huggingface dataset:
High-affinity uptake of 67Cu into a veratridine-releasable pool in brain tissue
Authors: Barnea A. , Hartter D. E. , Cho G.
Published: December 2017

We have previously characterized two saturable, ligand-dependent processes for 67Cu uptake by hypothalamic slices: a high- and low-affinity process (22). In this study, we wished to ascertain if veratridine, a secretagogue that mimics a physiological release process, stimulates the release of newly taken up 67Cu and whether uptake of 67Cu into the releasable pool of copper is dependent on the process of 67Cu uptake. Hypothalamic or caudate slices from male rats were loaded for 30 min with 67Cu complexed to histidine (His) under conditions favoring high- or low-affinity uptake. First, we assessed the stability of the newly taken up 67Cu and found that, regardless of the mode of 67Cu entry into the tissue, greater than or equal to 85% of the 67Cu is retained in tissues incubated for 3 h in 67Cu-free buffer. Moreover, the 67Cu taken up by the high-affinity process was not displaced by 15-fold molar excess of nonradiolabeled Cu2+, histidine, albumin, or Zn2+, and only 20-30% of the 67Cu taken up by the low-affinity process was displaced by 10-fold excess Cu2+ or albumin. Next, we assessed veratridine stimulation of 67Cu release and found that 67Cu release occurred only from tissues loaded with the high- but not with the low-affinity process. This effect of veratridine was calcium dependent and was blocked by Tetrodotoxin, a specific blocker of the voltage-sensitive Na+ channel. In addition, we confirmed our earlier observation that a depolarizing concentration of K+ stimulates 67Cu release.(ABSTRACT TRUNCATED AT 250 WORDS)
Continuous membrane voltage recordings in A10 vascular smooth muscle cells: effect of AVP
Authors: Korbmacher C. , Helbig H. , Stahl F. , Coroneo M. , Haller H. , Lindschau C. , Quass P. , Wiederholt M.
Published: December 2017

Continuous membrane voltage (V) recordings were obtained in A10 vascular smooth muscle cells (rat aorta) using glass microelectrodes. Resting membrane voltage in 262 impalements averaged 54.0 +/- 0.4 (SE) mV. Relative K+ conductance was characterized, and the contribution of electrogenic Na+-K+-ATPase to membrane voltage was investigated. Action potentials could be induced by application of 1 mM barium or 10(-4) M acetylcholine. In a few recordings, spontaneous spike activity occurred, and this could be abolished by 5 mM MgCl2 or by removal of extracellular Ca2+. Barium-induced action potentials were not dependent on the presence of extracellular Na+ and not inhibitable by 10(-6) M tetrodotoxin. Application of 10(-6) M [Arg8] vasopressin (AVP) for 30 s caused a typical biphasic membrane voltage response with an initial transient hyperpolarization of -9.5 +/- 1.1 mV and a more sustained subsequent depolarizing response averaging 28.2 +/- 1.3 mV (mean +/- SE, n = 58). The effect of AVP on membrane voltage was blocked by the V1-antagonist [beta-mercapto-beta,beta-cyclopentamethylenepropionyl1,O-Me- Tyr2,Arg8]vasopressin. The initial hyperpolarizing component of the membrane voltage response to AVP became more prominent when V was predepolarized, for example, by a preceding AVP application. However, when AVP was applied during high K+ depolarization or in the presence of quinidine (1 mM), the initial hyperpolarizing response was practically abolished. The time course of the initial hyperpolarization was shown to be similar to the calcium transient observed in fura-2-loaded A10 cell suspensions after the application of AVP. We conclude that the initial AVP-induced hyperpolarization in A10 cells corresponds to an activation of Ca2+-activated K+ channels.
Propionate induces cell swelling and K+ accumulation in shark rectal gland
Authors: Feldman G. M. , Ziyadeh F. N. , Mills J. W. , Booz G. W. , Kleinzeller A.
Published: December 2017

Small organic anions have been reported to induce cell solute accumulation and swelling. To investigate the mechanism of swelling, we utilized preparations of rectal gland cells from Squalus acanthias incubated in medium containing propionate. Propionate causes cells to swell by diffusing across membranes in its nonionic form, acidifying cell contents, and activating the Na+-H+ antiporter. The Na+-H+ exchange process tends to correct intracellular pH (pHi), and thus it maintains a favorable gradient for propionic acid diffusion and allows propionate to accumulate. Activation of the Na+-H+ antiport also facilitates Na+ entry into the cell and Nai accumulation. At the same time Na+-K+-ATPase activity, unaffected by propionate, replaces Nai with Ki, whereas the K+ leak rate, decreased by propionate, allows Ki to accumulate. As judged by 86Rb+ efflux, the reduction in K+ leak was not due to propionate-induced cell acidification or reduction in Cli concentration. Despite inducing cell swelling, propionate did not disrupt cell structural elements and F actin distribution along cell membranes.
Flow cytometric identification and isolation of hypertrophic type II pneumocytes after partial pneumonectomy
Authors: Uhal B. D. , Rannels S. R. , Rannels D. E.
Published: December 2017

Type II pneumocytes were isolated by either Percoll density gradient centrifugation or by immunoglobulin G (IgG) panning from the lungs of normal rats and the right lung of rats subjected to left pneumonectomy. Cells were studied at 7- (pnx-7) and 15- (pnx-15) days postoperative, times during and after, respectively, rapid compensatory growth of the right lung. Acridine orange staining permitted resolution of type II cells from contaminants on the basis of high red fluorescence (greater than 590 nm). Simultaneous measurement of forward-angle light scatter (FALS) suggested a shift of pnx-7 cells toward greater size, which was reversed in pnx-15 cells. By Percoll gradient isolation, approximately 15% of pnx-7 cells analyzed were above the mean FALS of control cells. In contrast, approximately 30% of the pnx-7 cells isolated by IgG panning were above the mean FALS of corresponding control cells. Biochemical analyses of pnx-7 cells separated by cell sorting into "high FALS" and "low FALS" subgroups revealed that high FALS type II cells contained 50% more protein (P less than 0.05) and 140% more RNA (P less than 0.01) than low FALS cells, with no significant change in cellular DNA content. These data are consistent with previous studies of type II cells isolated from the lungs of pneumonectomized animals and confirm the presence of hypertrophic cells in these preparations. They provide a foundation from which to design further flow cytometric studies of the role of hypertrophic type II pneumocytes in compensatory lung growth.
Identification of a 185-kDa band 3-related polypeptide in oxyntic cells
Authors: Thomas H. A. , Machen T. E. , Smolka A. , Baron R. , Kopito R. R.
Published: December 2017

Polyclonal antibodies to the purified mouse erythrocyte anion exchange protein (band 3) and to a conserved COOH-terminal peptide of mouse band 3 (alpha-Ct) recognized a single major 185-kDa polypeptide in immunoblots of a membrane fraction prepared from rabbit gastric glands. Competition studies revealed that the epitopes shared between the rabbit gastric 185-kDa antigen and the approximately 100-kDa mouse erythrocyte band 3 protein are restricted to the COOH-terminal domain of band 3, which is known to contain the catalytic site for anion exchange activity. Immunofluorescence microscopy was used to demonstrate that this band 3-related polypeptide is associated with the plasma membrane in a subpopulation of gastric gland cells composed exclusively of oxyntic cells, as judged by the coincidence of immunofluorescence with alpha-Ct and with a monoclonal antibody to the gastric H+-K+-ATPase. This alpha-Ct-reactive antigen was further localized to the cytoplasmic face of the basolateral membrane of oxyntic cells, which correlates well with the physiologically determined site of anion exchange activity. These data demonstrate the presence in gastric oxyntic cells of a novel member of the family of proteins related to the erythrocyte anion exchanger. The possibility that the 185-kDa polypeptide is an anion exchanger is discussed.
Regulation of intracellular pH in resting and in stimulated parietal cells
Authors: Paradiso A. M. , Townsley M. C. , Wenzl E. , Machen T. E.
Published: December 2017

Microspectrofluorimetry of the pH-sensitive, fluorescent dye 2',7'-biscarboxyethyl-5 (6)-carboxyfluorescein (BCECF) was used to measure intracellular pH (pHi) in single parietal cells (PC) of intact rabbit gastric glands during resting and stimulated conditions. In 61% of PC, histamine plus isobutylmethylxanthine (IBMX) (both 100 microM) caused a small increase in pHi, ranging from 0.04 to 0.21 pH units (average delta pHi = 0.09 +/- 0.04 units over a 6-min period). In the other 39% of PC, pHi remained constant or decreased slightly (maximum decrease was 0.10 unit). The specific inhibitors omeprazole (50 microM, blocks H+- K+-ATPase), 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonic acid (H2DIDS; 100-200 microM, blocks Cl-HCO3 exchange) and amiloride (1 mM, blocks Na-H exchange) were added to both resting and stimulated PC. In stimulated PC, omeprazole caused pHi to decrease by 0.08 unit, but this inhibitor had no effect on pHi of resting PC. H2DIDS caused pHi to increase in stimulated PC five times faster compared with resting PC. Amiloride or Na-free solution (which should reverse the Na-H exchanger and cause cellular acidification) caused pHi to decrease 2.5 or 5 times, respectively, more slowly in stimulated PC compared with resting PC. Also, recovery from NH4-induced acidification (due primarily to Na-H exchange) was 1.8 times faster (measured at pHi 6.7) in resting vs. in stimulated PC. During histamine plus IBMX-induced stimulation, increased H secretion by the H+-K+-ATPase at the apical membrane is accompanied by an increase in activity of the Cl-HCO3 exchanger at the serosal membrane.(ABSTRACT TRUNCATED AT 250 WORDS)
Aldosterone receptors in A6 cells: physicochemical characterization and autoradiographic study
Authors: Claire M. , Machard B. , Lombes M. , Oblin M. E. , Bonvalet J. P. , Farman N.
Published: December 2017

The A6 cell line is derived from the kidney of Xenopus laevis. Aldosterone increases sodium transport across A6 cell epithelia. In the present study, aldosterone binding characteristics were studied in A6 cell cytosol. Both type I (mineralocorticoid) and type II (glucocorticoid) receptors are present in the cytosolic fraction of these cells. Aldosterone and corticosterone had a high affinity for type I sites (Kd = 1.25 and 0.16 nM, respectively) and a lower affinity for type II sites (Kd = 39 and 10 nM, respectively). Testosterone and estradiol did not compete for aldosterone binding. RU 26988, a highly specific glucocorticoid agonist, competed with aldosterone for type II but not for type I sites. Hydrodynamic parameters of both type I and type II corticosterone receptor complexes were identical. Their Stokes radius was approximately 6 nm, as estimated by high-performance size-exclusion chromatography, and their sedimentation coefficient determined by ultracentrifugation on glycerol gradients was approximately 9s. The molecular mass calculated from these parameters was approximately 200 kDa, a value that is very close to the value estimated for nontransformed mineralocorticoid and glucocorticoid receptors of other species. The [3H]aldosterone labeling of intact A6 cells was examined by autohistoradiography. At every concentration tested (2, 20, and 50 nM), all cells were found to be specifically labeled in both cytoplasm and nucleus. At 20 nM, in the presence of an excess of RU 26988, labeling was also detected. At every concentration the labeling data was compatible with a Gaussian distribution, indicating that A6 cells correspond to a homogeneous population with regard to aldosterone binding and that probably both type I and type II sites are present in the same cells.
Localization of Cl- conductance in normal and Cl- impermeability in cystic fibrosis sweat duct epithelium
Authors: Reddy M. M. , Quinton P. M.
Published: December 2017

We studied the Cl- permeability properties of apical and basolateral membranes of human reabsorptive sweat duct (RSD) from normal and cystic fibrosis (CF) subjects. In normal ducts, Cl- substitution by impermeant anion gluconate in the lumen increased the voltage divider ratio (VDR) from 4.8 +/- 0.9 to 7.0 +/- 1.1 (n = 8, P less than 0.05), whereas Cl- substitution in the contraluminal bath decreased the VDR from 3.2 +/- 0.7 to 1.9 +/- 0.4 (n = 7, P less than 0.05). These results are consistent with a significant Cl- permeability in both apical and basolateral membranes of normal ducts. Amiloride (10(-4) M) in the lumen of normal ducts resulted in a small increase in VDR from 4.2 +/- 0.6 to 5.0 +/- 0.8 (n = 10, P less than 0.05), whereas the current-induced basolateral membrane voltage deflections (delta Vb) increased from 6.9 +/- 1.3 to 7.7 +/- 1.2 mV, suggesting that inhibition of Na+ permeability decreased basolateral membrane Cl- permeability. In the absence of luminal Cl-, amiloride decreased delta Vb and induced much greater effect on VDR (from 5.2 +/- 1.1 to 10.8 +/- 2.3, n = 9, P less than 0.05) than in the presence of Cl-. Likewise, in the presence of amiloride, Cl- substitution in the lumen had greater effect on VDR (increased from 3.5 +/- 0.5 0.5 to 10.0 +/- 1.5, n = 15, P less than 0.05) than in the absence of amiloride. These results indicate that Na+ conductance in the apical membrane of the normal duct is significantly smaller than Cl- conductance.(ABSTRACT TRUNCATED AT 250 WORDS)
Osmoregulation of glycerophosphorylcholine content of mammalian renal cells
Authors: Nakanishi T. , Burg M. B.
Published: December 2017

Renal medullary cells contain high concentrations of "compatible" organic osmolytes such as glycerophosphorylcholine (GPC), betaine, myo-inositol, and sorbitol. The organic osmolytes occur as an osmoregulatory response to the high and variable interstitial NaCl concentration that is part of the urinary concentrating mechanism. Madin-Darby canine kidney (MDCK) cells in culture were previously shown to accumulate GPC in response to increased osmolality. We demonstrate here that this accumulation occurs in response to elevated extracellular urea concentration as well as to elevated NaCl. GPC does not accumulate unless either choline or GPC is present in the medium. Thus the accumulation results from osmoregulated synthesis of GPC from choline and, possibly, also osmoregulated uptake of extracellular GPC. When the osmolality is decreased from high to normal levels, cell GPC concentration decreases greatly over 24 h, accompanied by efflux of GPC and choline into the medium.
Passively propagated spikes from the soma of cells in pineal gland of guinea pigs
Authors: Parkington H. C. , Coleman H. A. , McCance I.
Published: December 2017

The membrane properties of cells within the pineal gland of guinea pigs were studied using intracellular electrophysiological techniques. The electrotonic responses to intracellular current injection decayed with a single exponential in approximately 60% of cells but was preceded by a quicker component in the remainder. The membrane time constant was 2.8 ms. Depolarization beyond -29 mV activated an outward current that reversed at around the value of the resting potential. Hyperpolarization activated a slow inward current. Spikes occurred in response to activation of alpha-adrenoceptors. They were resistant to tetrodotoxin but were abolished by nifedipine and verapamil, suggesting that calcium carries the current during their upstroke. Spikes could not be evoked by depolarizing current pulses of 1-ms to 2-s duration. The responses to hyperpolarizing current steps or voltage-clamp steps applied during the peak of spikes evoked by nerve stimulation were indistinguishable from the responses to those applied between spikes. During nerve stimulation, fluctuations were observed in the current trace of cells under voltage clamp, indicating that the spikes could not be voltage clamped successfully. It is concluded that the spikes occurring in response to nerve stimulation are generated on the processes of the pinealocytes and are passive in the soma.
Energy-dependent cell volume maintenance in UC-11MG human astrocytomas
Authors: Lomneth R. , Gruenstein E. I.
Published: December 2017

Swelling of astrocytes in the brain is a major cause of the morbidity and mortality associated with stroke and head trauma. Using a human astrocytoma cell line (UC-11MG) as a model system, we studied cell volume changes caused by ATP depletion under conditions mimicking hypoxia. ATP levels were reduced to less than 10% of control using the metabolic inhibitors KCN or antimycin in combination with glucose deprivation. This was sufficient to eliminate ouabain-sensitive 86Rb+ uptake, indicating the Na+-K+-adenosinetriphosphatase was not operating. Furosemide-sensitive 86Rb+ uptake was reduced by approximately 60%, indicating Na+-K+-2Cl- cotransport was also sensitive to ATP loss. ATP depletion resulted in a 30-40% reduction of cell volume within 60 min. ATP depletion also resulted in a net loss of intracellular K+. This loss of K+ could be blocked by Ba2+, indicating the K+ loss was through a conductive channel. When the net K+ loss was blocked by Ba2+, the volume decrease was also prevented. The cells remained viable throughout the time period as judged by exclusion of ethidium bromide by 99% of the cells and recovery of ATP levels to 75% of control within 60 min. We conclude that ATP depletion, following inhibition of glycolysis and oxidative phosphorylation, causes astrocytes to shrink because of a more rapid loss of K+ than uptake of Na+. Thus it appears that ATP depletion alone is not sufficient to account for the rapid phase of astrocytic swelling observed during cerebral ischemia.
Myosin light chain phosphorylation and tension potentiation in mouse skeletal muscle
Authors: Palmer B. M. , Moore R. L.
Published: December 2017

In intact mammalian fast-twitch skeletal muscle, a quantitative relationship exists between the phosphate content of myosin P-light chain (PLC) and the extent of isometric twitch tension potentiation. It has been proposed that PLC phosphorylation causes twitch potentiation in intact muscle by rendering the contractile element more sensitive to activation by Ca2+. If this hypothesis is correct, then an obligatory experimental outcome is that the slope of the "PLC phosphate vs. isometric tension potentiation (ITP)" relationship should increase when the amount of Ca2+ available to activate the contractile element is decreased. Intact mouse extensor digitorum longus muscles were studied in the absence and presence of sodium dantrolene, an agent that partially inhibits Ca2+ release from the sarcoplasmic reticulum (SR). Treatment of muscles with dantrolene produced a 73% reduction in isometric twitch tension and an approximately threefold increase in the slope of the PLC phosphate vs. ITP relationship. Under experimental conditions that produced fused, tetanic contractions equal to 0.52, 0.72, 0.94, and 1.0 force production, contraction-induced changes in PLC phosphate content were directly proportional to the extent of contractile element activation, whereas the extent of ITP was inversely proportional to the extent of contractile element activation. These data indicate that the slope of the PLC phosphate vs. ITP relationship varies inversely as a function of the amount of Ca2+ that is released from the SR to activate the contractile element during both twitch and fused, submaximal tetanic contractions. Furthermore, these findings support the hypothesis that ITP in intact skeletal muscle is due to a PLC phosphorylation-induced sensitization of the contractile element to activation by Ca2+.
Intracellular [Ca2+] and inositol phosphates in avian nasal gland cells
Authors: Shuttleworth T. J. , Thompson J. L.
Published: December 2017

Isolated cells from the nasal salt gland of ducklings (Anas platyrhynchos) were evaluated as a model system for the study of the muscarinic activation of exocrine ion secretion. Cells loaded with the fluorescent probe indo-1 were used to study changes in intracellular Ca2+ concentration [( Ca2+]i) after stimulation. Changes in inositol phosphate generation and oxygen consumption were also determined. Loading with the acetomethoxy ester form of indo-1 (indo-1/AM) was rapid, and intracellular cleavage of the ester was essentially complete. Leakage of the dye was negligible over the time course of measurements (up to 20 min). Resting [Ca2+]i was approximately 100 nM. Stimulation with carbachol resulted in progressive increases in the generation of inositol phosphates and rapid four- to fivefold increases in [Ca2+]i. At normal extracellular Ca2+ concentrations, [Ca2+]i remained elevated (approximately 3 times resting levels) for as long as stimulation continued. Experiments showed that the increases in [Ca2+]i were comprised of a combination of release of Ca2+ from intracellular stores and an enhanced entry of Ca2+ from the extracellular medium. It is specifically this latter process that produces the sustained elevations in [Ca2+]i that are the essential signal for secretory activity.
Existence of muscarinic suppression of a K current in PC-12 pheochromocytoma cells
Authors: Nakazawa K. , Fujimori K. , Takanaka A. , Inoue K.
Published: December 2017

Muscarinic influence on membrane currents of PC-12 pheochromocytoma cells were investigated with whole cell voltage-clamp methods. An outward K current was observed when depolarizing voltage steps were applied to the cells. Methacholine (MCh, 300 microM), a selective agonist for muscarinic receptors, partially suppressed the K current, and the suppression was enhanced by removal of external Ca. The effect of MCh was antagonized by a low dose (100 nM) of atropine. Nicotine (10 microM) induced an inward current in these cells but did not affect the K current activated by depolarizing voltage steps. A Ba current flowing through voltage-gated Ca channels was not changed by MCh. The results indicate the existence of a MCh-sensitive K current in PC-12 cells and suggest that the membrane currents of these cells are modulated by cholinergic agents through muscarinic mechanisms in addition to well-known nicotinic mechanisms.
Characteristics of two types of calcium channels in rat pituitary gonadotrophs
Authors: Stutzin A. , Stojilkovic S. S. , Catt K. J. , Rojas E.
Published: December 2017

The properties of Ca2+ channels in cultured rat pituitary gonadotrophs were analyzed by the patch-clamp technique. The inward Ca2+ currents, recorded in the presence of 5.2 mM Ca2+ or Ba2+, included a fast, transient component with activation-inactivation kinetics and a delayed component with slower activation. The midpoint of the activation curve lay at -30 mV for the transient component and at -12 mV for the delayed component. At the midpoint, changes in potential of 9.5 and 13 mV induced an e-fold change in the activation of the transient and delayed components, respectively. The rate of inactivation of the first component was strongly voltage dependent. At -43 mV, a 7.4-mV change in potential induced an e-fold change in the fraction of Ca2+ channels available to conduct Ca2+ current. During long-lasting (100-200 ms) low-frequency depolarizing voltage-clamp pulses, the size of the delayed component of the Ca2+ current remained constant. The differential effects of membrane potential on inactivation and the different time constants for activation of the two components of the Ca2+ conductance indicate the presence of two types of Ca2+ channels in the membrane of the gonadotroph: the rapidly inactivating current appears to be attributable to a T-type channel, and the noninactivating current corresponds to the L-type channel described in many other cell types.
Osmoregulatory fluxes of myo-inositol and betaine in renal cells
Authors: Nakanishi T. , Burg M. B.
Published: December 2017

Renal medullary cells contain high concentrations of "compatible" organic osmolytes, such as myo-inositol, betaine, sorbitol, and glycero-phosphorylcholine. These organic osmolytes accumulate as an osmoregulatory response to the high and variable interstitial NaCl concentration that is part of the urinary concentrating mechanism. Madin-Darby canine kidney (MDCK) cells in culture were previously shown to accumulate myo-inositol and betaine in response to increased NaCl. These organic osmolytes are taken up by sodium-dependent active transport into the cells from the medium. The maximum concentration is not reached until 2-4 days after an increase in medium osmolality. The purpose of this study was to characterize the response to a decrease in medium osmolality of cells that had been grown at a high osmolality. The initial response to decreased osmolality was a rapid, transient efflux of both myo-inositol and betaine from the cells. Efflux was greatest during the first 15 min and resulted in a reduction of cell myo-inositol and betaine by almost 13 and 22%, respectively, after 3 h. Active myo-inositol and betaine influx fell more slowly, reaching a lower limit after approximately 1-2 days. The reduced influx was followed by progressive decrease in cell myo-inositol and betaine to approximately 30% of the initial value after 6 days. Thus, after a decrease in medium osmolality, MDCK cell myo-inositol and betaine fell because of a rapid, transient increase in efflux and a slow, sustained decrease in active influx.
An in vivo examination of rat brain during sepsis with 31P-NMR spectroscopy
Authors: Hotchkiss R. S. , Long R. C. , Hall J. R. , Shires G. T. , Brouillard R. G. , Millikan W. J. , Jones D. P.
Published: December 2017

Neurological symptoms including lethargy, obtundation, and confusion are early and common findings in patients with sepsis. The etiology of the mental status changes that occur during severe infection is not known. We investigated the effects of sepsis on the levels of high-energy phosphates to determine whether decreased energy metabolism was a factor in the depressed neurological state. The time course of changes in brain pH and brain high-energy phosphate metabolites during an Escherichia coli infusion was determined from sequential phosphorus-31 nuclear magnetic resonance (31P-NMR) spectra of ketamine-xylazine-anesthetized rats. A second group of rats received 0.9% saline infusion and served as a control group. Despite severe obtundation and near loss of righting reflex, the rats in the septic group had no significant differences in the brain pH, the ratio of phosphocreatine (PCr) to beta-adenosine 5'-triphosphate (beta-ATP), or in the ratio of PCr to Pi. The only significant decrease in brain high-energy phosphates or pH occurred terminally in the septic rat group and corresponded with a rapidly falling arterial blood pressure. We conclude that the severe neurological depression that is characteristic of sepsis is not due to decreased levels of brain high-energy phosphates or brain acidosis.
Delayed K+ current and external K+ in single cardiac Purkinje cells
Authors: Scamps F. , Carmeliet E.
Published: December 2017

The effect of external K+ on the delayed K+ current was investigated in rabbit single Purkinje cells. Whole cell voltage clamp and intracellular dialysis were used. At K+ concentrations less than 1 mM the kinetics of the delayed K+ current were not changed, but the conductance was markedly reduced. This effect was due to a direct change at an extracellular site and not due to secondary changes in intracellular Na+ or Ca2+ concentrations. A rise in intracellular Na+ or Ca2+ rather increased the delayed K+ current. The decrease in the delayed K+ current in low external K+ was absent when the experiments were done in Na+-free solution. It is concluded that external Na+ exerts an inhibitory effect on the conductance of the delayed K+ current.
Proximal tubule volume regulation in hyperosmotic media: intracellular K+, Na+, and Cl-
Authors: Rome L. , Grantham J. , Savin V. , Lohr J. , Lechene C.
Published: December 2017

Nonperfused proximal S2 segments from rabbit kidney cortex have been shown to keep cell volume constant as medium osmolality is slowly raised but to shrink and not exhibit regulatory volume increase (RVI) if medium osmolality is abruptly elevated (J. Lohr and J. Grantham. J. Clin. Invest. 78: 1165-1172, 1986). In the current study, 0.5 mM butyrate in the medium 1) extended the range from 361 to 450 mosmol/kgH2O over which cells maintained volume constant as osmolality was gradually raised and 2) restored RVI after cell shrinkage when osmolality was rapidly raised from 295 to 400 mosmol/kgH2O. Volume regulation was associated with net increases in intracellular Na+ and Cl- but no change in K+ (measured by electron probe). The increments in Na+ and Cl- were insufficient to account for the total addition of osmolytes required for volume maintenance or restoration. The fraction of the expected increase in intracellular osmoles accounted for by the increase in [(K+)i + (Na+)i + (Cl-)i] was 52 and 21% for gradual and rapid osmotic changes, respectively. We conclude that butyrate enhances the capacity of S2 segments to regulate volume in hyperosmotic medium by promoting addition of Na+ and Cl- and by other undermined factors.
Ca2(+)-induced Ca2+ release as examined by photolysis of caged Ca2+ in single ventricular myocytes
Authors: Nabauer M. , Morad M.
Published: December 2017

In cardiac muscle, entry of Ca2+ through the voltage-gated Ca2+ channel and its interaction with an intracellular site are thought to trigger the release of the intracellular Ca2+ pools and to activate contraction. The availability of a novel "caged calcium" compound, and its effective use in neuronal and heart cells to modulate Ca2+ channel and contraction, made it possible to examine directly the Ca2(+)-induced Ca2+ release hypothesis in intact mammalian cardiac myocytes. We used the caged Ca2+ compound DM-nitrophen, which on photolysis, rapidly (less than 200 microseconds) changes its Ca2(+)-binding affinity from 3 X 10(-9) to 2 X 10(-3) M at pH 7.0. In isolated whole cell clamped guinea pig ventricular myocytes dialyzed with unphotolyzed DM-nitrophen (Ca2+ buffered to values less than 10(-7) M), we found that a 160-microseconds light pulse photoreleased sufficient Ca2+ to activate contraction. Photorelease of Ca2+ failed to activate significant contraction in myocytes pretreated with caffeine, supporting the idea that the release of Ca2+ from intracellular pools was necessary to generate tension. However, photorelease of Ca2+ after the depolarization-induced Ca2+ release failed to suppress contraction, as predicted from the Ca2(+)-induced inactivation hypothesis. The failure to suppress contraction was not sufficient to definitively reject the Ca2(+)-induced inactivation hypothesis, since the intracellular Ca2+ concentration may not have risen sufficiently to inactivate the release channel.
Intracellular pH and membrane potassium conductance in rabbit distal colon
Authors: Duffey M. E. , Devor D. C.
Published: December 2017

Intracellular pH (pHc) was measured in the short-circuited epithelium of rabbit distal colon using H(+)-selective microelectrodes. pHc was 6.91 +/- 0.02 (SE) when the bath pH was 7.4. Intracellular HCO3- activity (acHCO3-) was estimated from these measurements to be 8 +/- 0 mM. When we replaced all Cl- in the tissue bathing solutions with the impermeant anion gluconate, pHc rose to 7.44 +/- 0.08 and acHCO3- increased to 30 +/- 6 mM. These results demonstrate that this tissue contains a Cl(-)-HCO3- exchange mechanism. During the Cl- replacement the apical membrane electrical potential difference hyperpolarized from -55 +/- 1 to -74 +/- 3 mV, suggesting that membrane ionic conductance had changed. Elevation of either the apical or basolateral membrane bathing solution K+ concentration produced a greater depolarization of membrane potential during Cl- replacement than when tissues were bathed in normal electrolyte solutions. In additional experiments, pHc was raised by lowering the bath CO2 concentration while the bath Cl- concentration was kept normal. Under these conditions, membrane potential hyperpolarized and was more sensitive to the elevation of bath K+ concentration than when pHc was normal. These results suggest that membrane K+ conductance in this tissue is increased by intracellular alkalinization.
Taurine transport by microvillous membrane vesicles and the perfused cotyledon of the human placenta
Authors: Karl P. I. , Fisher S. E.
Published: December 2017

Human placental uptake and maternal-to-fetal (M-to-F) net transfer of taurine were evaluated in purified microvillous membrane vesicles (MMV) and the isolated perfused cotyledon. Taurine uptake by MMV was specifically stimulated by an inward Na+ gradient [maximum velocity (Vmax), 24.5 +/- 0.6 pmol.mg-1.30 s-1; Michaelis constant (Km), 6.2 +/- 0.7 microM], with uptake stoichiometry showing approximately 2 Na+/taurine molecule. In the presence or absence of Na+, Cl- did not stimulate uptake. Na(+)-stimulated uptake was enhanced by an outward K+ gradient. beta-Alanine and hypotaurine competitively inhibited uptake of taurine in MMV. Two-way uptake inhibition studies showed no interaction between taurine and non-beta-amino acids. When MMV were preloaded with taurine there was enhancement of Na(+)-stimulated uptake. In the perfused placentas, saturable M-to-F net transfer of taurine was not observed, despite saturation of tissue uptake from the maternal circulation. During 3 h of perfusion, no fetal-to-maternal (F-to-M) gradient formed for taurine; yet, a 2:1 gradient simultaneously occurred for histidine. This study demonstrates that taurine uptake by the microvillous membrane of the human placenta is highly specific, of high affinity, and Na+ coupled.
Mechanics of K(+)-induced isotonic and isometric contractions in isolated canine coronary microarteries
Authors: Boels P. J. , Claes V. A. , Brutsaert D. L.
Published: December 2017

The effects of shortening in isotonic contractions on the mechanics of microvascular smooth muscle were investigated. Intramyocardial canine coronary microarteries (in situ diameter 60 +/- 3 microns) were mounted as rings, connected to a newly developed photoelectromagnetic force-length transducer, and activated with 125 mM K+. Shortening during isotonic contractions depressed the length-force relation (shortening deactivation) compared with the length-force relation obtained from isometric contractions; the effect was present at the earliest moments after activation, suggesting that a fundamental mechanism associated with the actual sliding of contractile filaments delayed onset of contractile activity in isotonic contractions compared with isometric contractions. Force-velocity relations were obtained by isotonic quick releases from isotonic and isometric contractions at various times. Isotonic shortening before the quick releases reduced the constants of the apparent hyperbolic force-velocity relations and maximal velocity of shortening (Vmax) compared with isometric contractions released at the same time. Increasing contraction duration reduced Vmax but more so in isotonic than in isometric contractions. Vmax also decreased with decreasing instantaneous length. A possible effect of force development on Vmax before the isotonic quick release was also described. Quick increments of load during isotonic contractions were sustained during active shortening in the phasic part, but during the tonic part loading resulted in a pronounced transient relaxation. Thus, in microvascular preparations, active isotonic shortening altered the length-force, force-velocity, and velocity-time relations and uncovered a time-dependent sensitivity to loading conditions. These experiments suggested that the mechanics of smooth muscle contraction may contribute significantly to the mechanisms of the physiological control of coronary microvascular diameter.
Comparison of leukotriene B4-induced neutrophil migration through different cellular barriers
Authors: Casale T. B. , Abbas M. K.
Published: December 2017

Migration of neutrophils across epithelial or endothelial barriers in response to chemotactic stimuli occurs in inflammation and host defense. Leukotriene B4 (LTB4) may be synthesized by and certainly induces chemotaxis of neutrophils. To better understand the interaction between LTB4, neutrophils, and endothelium and epithelium, we compared the effects of LTB4 on human peripheral blood neutrophil migration through filters alone and on human umbilical vein endothelial (HUVE) cells and three different epithelial cell types, Madin-Darby canine kidney (MDCK) cells, human colon carcinoma (T84) cells, and rat type II alveolar cells, cultured on these filters. Significant LTB4-stimulated neutrophil migration occurred at the lowest (1 nM) dose and in the shortest period of time (15 min) across endothelial cells vs. all three epithelial cell types, and interestingly, vs. filters alone. Dose-response experiments (1-100 nM) indicated that at equimolar LTB4 concentrations neutrophil migration across endothelium was two- to threefold greater than that observed across filters alone and the three epithelial barriers. At higher LTB4 concentrations (100 nM), the degree of neutrophil migration through the three epithelial barriers was equivalent to that observed for filters alone. Overall, the data indicate that the various cellular barriers play an active role in inflammatory processes by regulating the transmigration of neutrophils in response to certain inflammatory chemotactic stimuli.
Effects of phenylarsine oxide on stimulation of glucose transport in rat skeletal muscle
Authors: Henriksen E. J. , Holloszy J. O.
Published: December 2017

The trivalent arsenical phenylarsine oxide (PAO) inhibits insulin-stimulated glucose transport in adipocytes and skeletal muscle through direct interactions with vicinal sulfhydryls. In muscle, glucose transport is also activated by contractile activity and hypoxia. It was therefore the purpose of the present study to investigate whether vicinal sulfhydryls are involved in the stimulation of glucose transport activity in the isolated rat epitrochlearis muscle by hypoxia or contractions. PAO (greater than 5 microM) caused a twofold increase in rate of transport of the nonmetabolizable glucose analogue 3-O-methylglucose (3-MG) that was completely prevented by cytochalasin B, the vicinal dithiol dimercaptopropanol, dantrolene, or 9-aminoacridine, both inhibitors of sarcoplasmic reticulum Ca2+ release, or omission of extracellular Ca2+. Although PAO treatment (greater than or equal to 20 microM) prevented approximately 80% of the increase in 3-MG transport caused by insulin, it resulted in only a approximately 50% inhibition of the stimulation of 3-MG transport by either hypoxia or contractile activity. PAO treatment (40 microM) of muscles already maximally stimulated by insulin, contractile activity, or hypoxia did not reverse the enhanced rate of 3-MG transport. These data suggest that vicinal sulfhydryls play a greater role in the activation of glucose transport by insulin than by muscle contractions or hypoxia. The finding that PAO inhibits the stimulation of glucose transport, but does not affect glucose transport after it has been stimulated, provides evidence that vicinal sulfhydryls are involved in the pathways for glucose transport activation in muscle, but not in the glucose transport mechanism itself.
Combined force and voltage measurement in rapidly superfused guinea pig heart cells
Authors: Shepherd N. , Fisher V. J.
Published: December 2017

We describe the construction and use of a setup that allows the rapid exchange of the solution surrounding an isolated guinea pig heart cell while simultaneously measuring the isometric force and membrane potential (Em). Cells were stably attached, by means of poly-L-lysine, to a force transducer which was adapted from one previously used for a study of frog atrial cells [N. Shepherd and F. Kavaler.Am. J. Physiol. 251 (Cell Physiol. 20): C653-C661, 1986]. The modified transducer is simple to construct and use and can be readily added to existing patch-clamp setups. The strength of attachment of a cell to the transducer exceeded the strength of the gigaseal in all of the experiments. The membrane potential was measured by means of patch electrodes and a high-impedance voltage follower. Rapidly changing extracellular K concentration [( K]o) from 5.4 to 10.8 mM caused a positive change of Em by 16.5 +/- 1.4 mV with a half-time (t1/2) of 27 +/- 4 ms. Replacing calcium in the perfusate by magnesium instantly abolished the contraction and shortened the action potential. Twitch tension returned stepwise to the control value on return of calcium to the perfusate. Our initial observations show that the patch electrode can be used successfully in conjunction with the isometric force transducer and rapid extracellular solution changes for studies of excitation and contraction coupling in isolated mammalian heart cells.
Alterations in adipocyte response to lipolytic hormones during cold acclimation
Authors: Rochon L. , Bukowiecki L. J.
Published: December 2017

The effects of cold exposure (7 days, 5 degrees C) and cold acclimation (21 days, 5 degrees C) on the regulation of lipolysis were investigated in adipocytes isolated from epididymal fat pads of rats. Catecholamines stimulated lipolysis in an affinity sequence typical of the beta 1-adrenoceptor subtype: one-half maximum velocity (1/2 Vmax) isoproterenol (35 nM) much greater than 1/2 Vmax norepinephrine (150 nM) approximately 1/2 Vmax epinephrine (200 nM). Cold exposure markedly decreased the sensitivity (1/2 Vmax) and the responsiveness (Vmax) of the adipocytes to the lipolytic action of catecholamines. Addition of adenosine deaminase to fat cells isolated from cold-exposed rats did not normalize the lipolytic activity, suggesting that extracellular adenosine was not responsible for the obtunded lipolysis. This effect of cold exposure was transient as the lipolytic response to catecholamines was normal in fully cold-acclimated animals. Remarkably, the responsiveness of adipocytes to the lipolytic action of glucagon (200 nM) and adrenocorticotropic hormone (ACTH, 1 microM) progressively increased during cold acclimation. Adipocyte lipolytic response to dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) and theophylline was normal in cold-exposed rats, indicating that the lipolytic defect resides at an early step in the lipolytic cascade (pre-cAMP). On the other hand, the antilipolytic effect of insulin on norepinephrine-induced lipolysis significantly decreased during cold acclimation, particularly at physiological levels of insulin (nanomolar level). These results demonstrate that the transient decrease in the lipolytic action of catecholamines observed during cold acclimation is compensated by 1) an increased responsiveness of adipocytes to glucagon and ACTH and 2) by a decreased effectiveness of insulin to induce antilipolysis.(ABSTRACT TRUNCATED AT 250 WORDS)
Intracellular Ca transients in rat cardiac myocytes: role of Na-Ca exchange in excitation-contraction coupling
Authors: Bers D. M. , Lederer W. J. , Berlin J. R.
Published: December 2017

Membrane current and intracellular Ca concentration ([Ca]i) transients were recorded from isolated rat ventricular myocytes under voltage-clamp control. The cells were dialyzed by the patch pipette solution, which contained the fluorescent Ca indicator indo-1 and 0.5 mM Na. Under these experimental conditions, Ca entry via Na-Ca exchange did not appear to be appreciable even in the absence of extracellular Na. Increasing the duration of voltage-clamp pulses from 5 to 80 ms produced [Ca]i transients of increasing amplitude, while the peak Ca current was not changed. This duration dependence of the [Ca]i transient was most demonstrable at more negative test potentials (e.g., -20 to -30 mV) and was not qualitatively modified by Na-free solutions. This latter result indicates that Ca extrusion by Na-Ca exchange is not responsible for the smaller [Ca]i transients observed when the membrane is repolarized after very brief depolarizations. Although the peak Ca current was not changed by increasing pulse duration, the integrated Ca current was increased. These observations are consistent with a Ca-release mechanism in cardiac excitation-contraction coupling in which 1) the Ca-release process can be modulated by membrane potential or 2) the Ca entering the cell via Ca channels has a preferential access [compared with Ca from the sarcoplasmic reticulum (SR)] to the site(s) that control SR Ca release. The role of Na-Ca exchange in the decline of [Ca]i during relaxation was also explored. Removal of extracellular Na (Nao) resulted in 20% slowing of the decline in [Ca]i during relaxation. From this, we conclude that the Na-Ca exchange competes with SR to remove Ca from the cytoplasm and that under our control conditions the exchanger may account for 20% of this decline. The Nao dependence of relaxation was reduced at more positive membrane potentials and increased by SR Ca loading.
Effect of compartmentalized Ca2+ ions on electrical bursting activity of pancreatic beta-cells
Authors: Chay T. R.
Published: December 2017

Patch-clamp single-channel and whole cell recordings have revealed new insights into the ionic channel properties in the pancreatic beta-cells. I have modeled the electrical events during the burst activity based on the observations that 1) the whole cell Ca2+ current has two functionally distinct components (fast and slow), 2) a fast component is inhibited by intracellular Ca2+, 3) a slow component is inactivated by depolarization, and 4) a significant fraction of the outward current is carried by the Ca2(+)-sensitive, voltage-gated K+ channels [K(Ca, V) channels]. The model contains a feature that the Ca2+ concentration in the submembrane compartment ([Ca2+]s) is higher than that in the cellular phase. At the plateau phase, [Ca2+]s is high enough to activate the K(Ca, V) channels. In addition to the K(Ca, V) channels, the model contains a voltage-activated Ca2+ channel that is quickly blocked by Ca2+ and slowly inhibited by voltage. Because the Ca2+ channel has an intracellular Ca2(+)-dependent inactivation gate, the increase in [Ca2+]s can inactivate the Ca2+ channels. According to this model, the spikes during the plateau phase are caused by a rapid movement of Ca2+ into and out of the compartment. Because of a rapid change in [Ca2+]s, the two competing currents, ICa and IK(Ca, V), fluctuate rapidly; the fluctuation leads to an emergence of spikes. The slow underlying wave is due to a voltage-dependent inactivation gate of the Ca2+ channels, which slowly closes as a result of depolarization. This model differs radically from my previous models, which featured a slowly varying intracellular Ca2+ concentration that was responsible for the underlying slow wave. Although the previous models give plateau fractions (the ratio between the plateau duration and cyclic time) to be far less than unity, the present model is the first of its kind that allows plateau fractions to be in the near-unity range.
Aging does not affect contractile properties of type IIb FDL muscle in Fischer 344 rats
Authors: Walters T. J. , Sweeney H. L. , Farrar R. P.
Published: December 2017

The purpose of this study was to examine the influence of aging on the contractile, metabolic, and histochemical properties of the predominantly fast-twitch flexor digitorum longus muscle (FDL) in the Fischer 344 (F344) rat strain. Contractile measurements were made in situ by stimulating the motoneuron while the animals were under pentobarbital sodium anesthesia. Fiber-type composition, capillarity, aerobic capacity, and myosin light chain composition were also examined. The results of this investigation were striking in that there was no alteration in any parameter measured. Collectively, these results suggest that aging does not induce a functional alteration in the neuromuscular relationship in the predominantly fast-twitch FDL of F344 rats, and consequently, there is no loss of functional capacity of the muscle. This study demonstrates that alteration in predominantly fast-twitch muscle is not a universal phenomenon and that care must be taken when generalizing about the influence of aging on skeletal muscle. Some of the conflicting results regarding the influence of aging on predominantly fast-twitch muscle in rodents may be either strain or muscle specific.