bluuebunny/crossref_metadata_2025 huggingface dataset:
Apical membrane potassium conductance in guinea pig gallbladder epithelial cells
Authors: Gunter-Smith P. J.
Published: December 2017

The fractional resistance of the apical membrane (fRa) of guinea pig gallbladder epithelial cells was observed to vary with changes in apical membrane potential (Va). Depolarizing Va from a base-line potential of -60 to -30 mV decreased fRa from 0.79 +/- 0.03 to 0.59 +/- 0.05. A comparable hyperpolarization had no effect on fRa. The potassium channel blocker tetraethylammonium (TEA) inhibited the changes in fRa induced by voltage when added to the mucosal but not when added to the serosal solution. Mucosal addition of Ba2+ and decreased pH also inhibited changes in fRa, whereas quinidine and 4-amino-pyridine did not. These results indicate that an increase in the K+ conductance of the apical membrane is responsible for changes in fRa with membrane depolarization. The current-voltage relation of this TEA-sensitive pathway was determined from differences in transepithelial current in the presence and absence of maximally effective concentrations of TEA and analyzed with respect to the Goldman constant-field equation. Computer-generated, best-fit analysis to the data indicated that they cannot be easily reconciled with K+ movement through a voltage-independent pathway or channel. Taken together, the results suggest that activation of a voltage-dependent K+ conductance in the apical membrane is responsible for changes in fRa with Va. This conductance also appears to be Ca2+-sensitive as ionomycin caused a shift in the relation between Va and fRa.
Interactions between sustained contractile activity and beta-adrenergic receptors in regulation of gene expression in skeletal muscles
Authors: Kraus W. E. , Bernard T. S. , Williams R. S.
Published: December 2017

Continuous electrical stimulation for 10-21 days of the motor nerve innervating the anterior compartment muscles of adult rabbits increased both the density of beta-adrenergic receptors (beta-AR) and tissue concentrations of adenosine 3',5'-cyclic monophosphate (cAMP) by two to threefold. Changes in cAMP and in beta-AR occurred in parallel with stimulation-induced adaptations in the specific activity of mitochondrial enzymes (2- to 6-fold increases) and with changes in steady-state concentrations of mitochondrial RNA, beta-F1ATPase mRNA, and myoglobin mRNA (2- to 11-fold increases). These increases in muscle cAMP, in beta-AR, and in expression of protein and mRNA products of genes encoding proteins of oxidative metabolism occurred even in animals receiving high doses of propranolol during the period of electrical stimulation. In contrast to genes that encode proteins of oxidative metabolism, the direction and the time course of activity-induced changes in expression of the glycolytic enzyme aldolase A appeared to be unrelated to changes in muscle cAMP; suppression of steady-state concentrations of aldolase A mRNA was maximal (20-25% of control) at early time points preceding the maximal rise in cAMP. In addition, administration of propranolol attenuated the suppressive effect of continuous contractile activity on expression of aldolase A, even in the absence of an effect of this drug on cAMP in stimulated muscles. We conclude that activity-induced changes in cAMP, in beta-AR, and in expression of genes that encode proteins important for oxidative metabolism occur as a direct consequence of contractile activity and do not require concomitant stimulation of beta-AR.(ABSTRACT TRUNCATED AT 250 WORDS)
Lack of endothelium-dependent relaxation in coronary resistance arteries of cholesterol-fed rabbits
Authors: Osborne J. A. , Siegman M. J. , Sedar A. W. , Mooers S. U. , Lefer A. M.
Published: December 2017

The endothelium-dependent contractile responses of subepicardial coronary resistance arteries (286 +/- 18 microns ID, n = 22) from rabbits fed either a 0.5 or 2.0% cholesterol-enriched diet or a control diet for 10-12 wk were determined under isometric conditions at the optimum length for active force production (Lo). After the development of tone with 29 mM K+-Krebs, arteries from control rabbits treated with acetylcholine (0.1-10 microM) showed a concentration-dependent relaxation, with a maximum decrease in tone of 63%. In contrast, coronary arteries from animals fed 0.5 and 2.0% cholesterol contracted to acetylcholine (approximately 210% increase in tone). A similar phenomenon was seen with arteries precontracted with 10 nM 9,11-methanoepoxy-prostaglandin H2 (U 46,619), a thromboxane A2 mimetic. The contractile responses to acetylcholine occurred in arteries in which the endothelium was structurally intact and which were devoid of plaque. Arteries from cholesterol-fed animals were poorly responsive to ADP (0.01-10.0 microM), whereas arteries from normal animals relaxed. All arteries relaxed to an equal degree when exposed to acidified nitrite, which produces nitric oxide (NO). The data suggest that as a result of hypercholesterolemia, there may be a dysfunction in the synthesis or release of endothelium-derived relaxing factor (EDRF) by the endothelial cells of coronary resistance arteries, rather than an abnormality of the smooth muscle cells per se.
Na+-H+ exchange and pH regulation in red blood cells: role of uncatalyzed H2CO3 dehydration
Authors: Motais R. , Fievet B. , Garcia-Romeu F. , Thomas S.
Published: December 2017

Erythrocytes of rainbow trout respond to adrenergic stimulation by activation of a Na+-H+ exchange. When red blood cells are suspended in their own plasma and equilibrated with a convenient gas mixture in a tonometer, the extrusion of H+ induces a fast, very strong acidification of the blood (by 0.5-0.7 pH units), explained as follows. Excretion of H+ into a medium containing HCO3- causes the formation of H2CO3. The uncatalyzed dehydration of H2CO3 is slow so that H+ accumulates above the level that would prevail at equilibrium, promoting a strong acid disequilibrium pH. Then the blood pH progressively returns to a value close to its initial value because of the slow uncatalyzed dehydration of H2CO3 and washout of the CO2 so produced. The period of acid disequilibrium pH, however, is lengthened because part of the CO2 generated by the spontaneous dehydration is not washed out by tonometry but diffuses into the red cells where it is rapidly converted into HCO3- and H+ by carbonic anhydrase and then excreted by Na+-H+ and Cl-HCO3- exchangers. This recycling process "refuels" the ionic reaction, increasing the time needed to reach equilibrium. The anion exchanger does not sense this strong acid disequilibrium pH, since the external HCO3- concentration is practically unchanged at that time. During the extracellular pH (pHe) recovery period, simultaneously extracellular HCO3- content decreases and intracellular Cl- content increases. Thus intracellular pH and pHe appear to be uncoupled. This overall interpretation is confirmed by experiments using carbonic anhydrase and drugs such as propranolol and amiloride.(ABSTRACT TRUNCATED AT 250 WORDS)
Evidence for apical sodium channels in frog lung epithelial cells
Authors: Fischer H. , Van Driessche W. , Clauss W.
Published: December 2017

To reveal the mechanism of Na+ transport across Xenopus lung epithelium, we recorded short-circuit current (Isc), transepithelial resistance (Rt), and current noise spectra while the isolated lung tissues were mounted in an Ussing-type chamber. Mean values of Isc and Rt obtained while the tissue was bilaterally incubated with NaCl-Ringer solution were Isc = 11.57 +/- 1.19 microA.cm-2 and Rt = 0.82 +/- 0.07 k omega.cm2. Amiloride added to the mucosal (apical) side depressed Isc by 61 to 99%. Ouabain abolished Isc totally when added to the basolateral compartment. Adenosine 3',5'-cyclic monophosphate (cAMP), epinephrine, and a variety of other compounds did not alter Isc significantly. Transepithelial depolarization with serosal KCl solution reduced Isc to 6.22 +/- 1.37 microA.cm-2. Amiloride-sensitive current and the kinetics of amiloride interaction were not significantly affected by depolarization. Fluctuation analysis of Isc in the presence of amiloride revealed a Lorentzian component in the power density spectrum indicating apical Na+ channels. Assuming pseudo-first order kinetics, we calculated single channel currents (iNa) and channel density (M): iNa = 0.29 +/- 0.04 pA and M = 0.24 +/- 0.04 micron 2. Our results show that the route for Na+ transport through lung epithelial cells follows the classical Koefoed-Johnson-Ussing model for tight epithelia.
Synthesis of alpha 2-macroglobulin by bovine adrenal cortical cell cultures
Authors: Kirshner N. , Corcoran J. J. , Erickson H. P.
Published: December 2017

Primary cultures of bovine adrenal medullary cells synthesize and secrete a high-molecular-weight protein into the culture medium. The protein was purified from the serum-free medium of cultured cells and was identified as alpha 2-macroglobulin by gel electrophoresis, sedimentation velocity, electron microscopy, immunoprecipitation, immunodiffusion, and autoradiography. Antisera directed against the protein were prepared and used to determine the cell types that synthesize the protein. Immunohistofluorescence studies show that adrenal cortical cells present in the adrenal medullary cell cultures reacted with the antisera to the protein purified from the medium, but adrenal medullary chromaffin cells did not. Cell cultures prepared from bovine adrenal cortex also synthesize and secrete alpha 2-macroglobulin and react with the antisera.
Cellular pathways of potassium transport in renal inner medullary collecting duct
Authors: Kone B. C. , Kikeri D. , Zeidel M. L. , Gullans S. R.
Published: December 2017

The dominant K+ transport pathways in rabbit inner medullary collecting duct (IMCD) cells were identified using an extracellular K+ electrode and fluorometric estimates of membrane potential. Ba2+ (5 mM) caused an initial rate of net K+ influx (61 +/- 6 nmol K+.min-1. mg protein-1) equivalent to the net K+ efflux (59 +/- 5 nmol K+. min-1.mg protein-1) induced by ouabain (0.1 mM). Addition of ouabain to Ba2+ -treated cells caused no net K+ flux. Membrane potential experiments demonstrated a K+ conductance that was inhibited by Ba2+. Thus K+ transport in the IMCD occurs principally via Ba2+ -sensitive K+ conductive pathway(s) and Na+-K+-ATPase. In studies that examine the metabolic determinants of K+ transport in the IMCD, glucose (but not 3-O-methylglucose) augmented oxygen consumption (QO2; + 12%) and cell K+ content (+12%), whereas iodoacetic acid, an inhibitor of glycolysis, promoted a release of cell K+. However, inhibition of mitochondrial oxidative phosphorylation with rotenone demonstrated that glycolysis alone could not maintain cell K+ content. Thus glucose metabolism plays an important role in K+ transport in the IMCD, but both glycolysis and oxidative phosphorylation are required to maintain optimal cellular K+ gradients.
Agonist-specific role for Na+-H+ antiport in prostaglandin release from microvessel endothelium
Authors: Gerritsen M. E. , Perry C. A. , Moatter T. , Cragoe E. J. , Medow M. S.
Published: December 2017

Rabbit coronary microvascular endothelial (RCME) cells synthesize prostaglandin (PG) I2 and PGE2 in response to stimulation with human thrombin, ATP, and the Ca2+ ionophore, A23187. Replacement of extracellular Na+ with choline or N-methylglucamine reduced thrombin-stimulated PG secretion but did not significantly affect either ATP- or A23187-stimulated PG secretion. Pretreatment of RCME cells with Na+ channel or Na+ -Ca2+ exchange blockers did not alter PG release in response to any of these three agonists. Pretreatment of RCME cells with the specific Na+ -H+ antiport blockers 5-(N-ethyl-N-isopropyl)-amiloride (EIPA, 10 microM) and 5-(N,N-hexamethylene)-amiloride (HMA, 0.1 microM) significantly reduced thrombin but not A23187- or ATP-stimulated PG secretion. Studies with the intracellular pH indicator dye 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein demonstrated thrombin activation of Na+ -H+ antiport, an effect blocked by either HMA or EIPA. Since manipulations known to inhibit Na+ -H+ exchange (EIPA, HMA, replacement of Na+ with choline or N-methylglucamine) reduced thrombin-stimulated RCME cell PG release, we conclude that activation of Na+ -H+ exchange is involved in the coupling of thrombin interaction with RCME cells to subsequent phospholipase activation and PG release.
Aldosterone-induced proteins: purification and localization of GP65,70
Authors: Szerlip H. M. , Weisberg L. , Clayman M. , Neilson E. , Wade J. B. , Cox M.
Published: December 2017

Aldosterone stimulates sodium transport in responsive epithelia by inducing "effector" proteins that control or modulate transcellular sodium flux. We have previously identified a group of electrophoretically microheterogeneous (pI 5.8-6.2) and polymorphic (Mr 65 and 70) glycoproteins that are specifically induced by aldosterone in toad urinary bladders (TUBs) and cultured toad kidney cells (A6 cell line). We raised a series of monoclonal antibodies (MAb) to these proteins and, using light and electron immunohistochemistry, localized the higher Mr glycoproteins (GP70) to the apical plasma membrane and subapical granules of the sodium-transporting cell of the TUB epithelium, the granular cell. GP70 appears to be discharged into the bladder lumen; this process is increased by phorbol myristate acetate, an agent known to induce granule exocytosis. These findings are consistent with the possibility that GP70 represent components or modulators of the "high-resistance" renal epithelial sodium channel. MAbs reactive against GP65 did not identify these glycoproteins within TUB epithelial cells; these lower Mr aldosterone-induced proteins may be incompletely processed forms of GP70.
Interaction of signal transduction pathways in mediating acid secretion by rat parietal cells
Authors: Ostrowski J. , Bomsztyk K.
Published: December 2017

To examine the role of protein kinase C (PKC) on the acid secretory activity of isolated rat parietal cells, histamine-and dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP)-stimulated [14C]aminopyrine accumulation was determined in the presence of agents that redistribute PKC activity to plasma membranes. Phorbol 12-myristate 13-acetate (PMA), 1-oleoyl-2-acetylglycerol (OAG), and phospholipase C inhibited, in a dose-dependent fashion, histamine- and DBcAMP-stimulated [14C]aminopyrine accumulation. Because PKC inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) reversed the effect, the results suggest that inhibition of histamine- or DBcAMP-stimulated [14C]aminopyrine accumulation induced by PMA, OAG, or phospholipase C was caused by increased activity of PKC in plasma membrane. To determine where in the cascade of events PKC inhibits acid secretion, histamine-, cholera toxin-, and forskolin-stimulated [14C]aminopyrine accumulation was measured with or without PMA. Because the percent of inhibition by PMA of [14C]aminopyrine accumulation was similar with the three secretagogues, the results suggest that PKC inhibits acid secretion at a point beyond adenosine 3',5'-cyclic monophosphate (cAMP) production. This was supported by the fact that PMA had no effect on histamine-stimulated production of cAMP and by the finding that activation of PKC had the same effect on histamine- or DBcAMP-stimulated [14C]aminopyrine accumulation. Histamine and DBcAMP inhibited PKC activity, suggesting a reciprocal interaction between PKC and histamine-triggered signal transduction pathway.
Multiple calcium-mediated effector mechanisms regulate chloride secretory responses in T84-cells
Authors: Dharmsathaphorn K. , Cohn J. , Beuerlein G.
Published: December 2017

Free cytosolic Ca2+ [( Ca2+]i) has been implicated as a second messenger mediating the ion transport effects of carbachol, histamine, taurodeoxycholate, ionomycin, and 4-bromo-A23187 (4-BrA23187) in T84-cells. In this study, we correlated short-circuit current (Isc, reflective of Cl- secretion) and [Ca2+]i responses in T84-cell monolayers stimulated by these agents to evaluate the role of [Ca2+]i in Cl- secretory responses. Time-course studies showed that the duration of [Ca2+]i and Isc responses did not correlate with one another. Isc responses were more prolonged than [Ca2+]i responses with carbachol and histamine (both derived [Ca2+]i partly from intracellular sources), less prolonged than [Ca2+]i with taurodeoxycholate, and continued to increase after [Ca2+]i stabilized with ionomycin and 4-BrA23187. Isc and [Ca2+]i responses to histamine and carbachol were additive. A comparison of the magnitude of [Ca2+]i and Isc responses in cells stimulated by different agonists showed that the change in [Ca2+]i accompanying equivalent Isc responses varied greatly, suggesting that secretagogues vary in their dependency on [Ca2+]i. These findings suggest the existence of multiple [Ca2+]i-mediated effector mechanisms or the existence of multiple mediators that augment or attenuate the action of [Ca2+]i.
Cytoplasmic pH in pulmonary macrophages: recovery from acid load is Na+ independent and NEM sensitive
Authors: Bidani A. , Brown S. E. , Heming T. A. , Gurich R. , Dubose T. D.
Published: December 2017

The pulmonary macrophage plays a primary role in the immunological defense of the lung. Although many studies have been devoted to elucidation of its phagocytic and secretory functions, little is known of its membrane transport properties or of how it regulates intracellular pH (pHi). The purpose of this study, therefore, was to determine base-line pHi and the mechanism(s) by which the cell recovers pHi when challenged with an intracellular acid load. Through the use of the pH-sensitive fluorescent dye, 2,7-biscarboxyethyl-5(6)-carboxy-fluorescein (BCECF), base-line pHi was estimated to be 7.24 +/- 0.03. Cells were acidified by two methods, nigericin and weak acids, while recovery (dpHi/dt) was monitored. The rate of recovery was found to be independent of external Na+ and K+ and was insensitive to amiloride. Pretreatment with 4,4'-diiso-thiocyanatostilbene-2,2'-disulfonic acid, an inhibitor of Cl- -HCO3- exchange, was also without effect on recovery from an intracellular acid load in these cells, under nominally HCO3- -free conditions. In contrast, N-ethylmaleimide (NEM) and N,N'-dicyclohexylcarbodiimide, nonspecific inhibitors of proton adenosinetriphosphatases (ATPases), virtually abolished pHi recovery. Efflux of H+ equivalents by pulmonary macrophages was measured by techniques involving both pH stat titration and the effect on fluorescence of extracellular BCECF. Basal H+ extrusion was approximately 2.75 +/- 0.64 nmol H+.min-1.10(6) cells-1 and was enhanced to approximately 26.0 +/- 6.95 nmol H+.min-1.10(6) cells-1 in acid-loaded cell suspensions. The basal rate of H+ extrusion was reduced to approximately 0.84 +/- 0.31 nmol H+.min-1.10(6) cells-1 in the presence of 1 mM NEM. These results suggest that recovery of cytoplasmic pH from an intracellular acid load, as well as regulation of pHi, under the conditions examined, is not mediated by a Na+-H+ exchanger in these cells. Rather, the data are consistent with the presence of an H+-ATPase in the plasma membrane of pulmonary macrophages.
Atrial natriuretic peptide and nitroprusside cause relaxation of cultured rat mesangial cells
Authors: Singhal P. C. , DeCandido S. , Satriano J. A. , Schlondorff D. , Hays R. M.
Published: December 2017

These studies evaluate the effects of atrial natriuretic peptide (ANP) and nitroprusside (NP) on cultured mesangial cells (MC) grown on a flexible silicone rubber surface. The basal tone of the MC produced wrinkles on the silicone rubber surface. A decrease in number or magnitude of wrinkles was considered to represent cell relaxation, whereas an increase represented cell contraction. ANP (10(-9) M) produced a relaxation in greater than 60% of the cells by 10 min. The percentage of cells showing a decrease of wrinkles was significantly higher (P less than 0.05 at 5 min and P less than 0.001 at 10 min) during the ANP-treated period than during the control period. NP (10(-5) M) caused a decrease of wrinkles in greater than 80% of cells (P less than 0.02 at 5 min and P less than 0.01 at 10 min) compared with a 5% decrease in the control period. Dibutyryl guanosine 3',5'-cyclic monophosphate (DBcGMP; 10(-4) M) also produced a decrease of wrinkles in 81% of the cells (P less than 0.02) compared with a 9% decrease in the control period. MC treated with ANP, NP, or DBcGMP and then labeled with 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-phallacidin did not show obvious alteration of morphology of actin filaments compared with untreated cells. ANP could inhibit as well as partially reverse the agonist (angiotensin II)-induced contractile response. ANP (10(-10)-10(-8) M) as well as NP (10(-5) M) increased intracellular cGMP content of MC (P less than 0.005) compared with control cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Two types of chloride channel on duct cells cultured from human fetal pancreas
Authors: Gray M. A. , Harris A. , Coleman L. , Greenwell J. R. , Argent B. E.
Published: December 2017

Using the patch-clamp technique, we have identified two types of chloride channel on duct cells cultured from human fetal pancreas. The channel we observed most frequently exhibited slight outward rectification, had a conductance of 4-7 pS in cell-attached patches, and was present on the apical plasma membrane where it usually occurred in clusters. Its open-state probability was not markedly voltage dependent but was increased up to threefold by exposing duct cells to secretin (10 nM), dibutyryl cyclic AMP (1 mM), or forskolin (1 microM). The other type of chloride channel was only rarely observed. Its current-voltage relationship exhibited marked outward rectification, giving chord conductances of 19 pS for inward currents and 53 pS for outward currents. Although this channel could be activated by sustained depolarization of excised patches, once activated the open state probability was not voltage dependent. While the physiological role of these channels is not firmly established, the small-conductance channel might function in parallel with a Cl- -HCO-3 exchanger to provide a mechanism for electrogenic bicarbonate secretion from the duct cell.
L-glutamate transport in internally dialyzed barnacle muscle fibers
Authors: Horn L. W.
Published: December 2017

The transport of L-glutamate (Glu) by single muscle fibers of Balanus nubilus was studied by means of internal dialysis in an effort to obtain transport data under well-defined intracellular conditions. It is shown that the method effectively controls the sarcoplasmic amino acid composition and Glu metabolism. It was found that the classic neutral amino acid transport systems are either absent or inactive, while Glu is strongly transported by a single system. Nonsaturable Glu "leak" is small relative to mediated transport. Glu influx under 0-trans conditions obeys simple Michaelis-Menten kinetics and shows simple competition with aspartate. Influx is strictly dependent on external Na. Trans-Na reduces influx, whereas trans-Glu has a small stimulatory effect. The preparation may serve as a general model for muscle Glu transport and can be used for a thorough study of the kinetic mechanism.
Electrophysiological effects of mucosal Cl- removal in Necturus gallbladder epithelium
Authors: Stoddard J. S. , Reuss L.
Published: December 2017

The factors responsible for the cell membrane hyperpolarization elicited in Necturus gallbladder epithelium on Cl- removal from the mucosal bathing solution were evaluated with conventional and ion-sensitive microelectrode techniques. Cl- removal causes reversal of apical Cl- -HCO3- exchange, resulting in a fall in intracellular Cl- activity (aiCl) and an increase in intracellular pH (pHi). Concomitantly, the cell membranes hyperpolarize to values close to the K+ equilibrium potential (EK), aiNa falls, and aiK rises. The observed changes in membrane voltage are not attributable to a pHi-dependent increase in cell membrane K+ permeability (PK), because 1) the cell membrane resistances increased and 2) elevating solution partial pressure of CO2 (PCO2) to counterbalance the cellular alkalinization on mucosal Cl- removal caused a further hyperpolarization of the cell membranes to values greater than EK. This additional hyperpolarization was related to the activity of the Na+ pump, inasmuch as it was accompanied by an increase in aiNa and was ouabain sensitive. These results are consistent with, but do not prove, pump electrogenicity. During the period of Cl- removal from the mucosal bathing solution, the cell membrane depolarization caused by raising serosal K+ concentration was increased, whereas the depolarization caused by lowering serosal Cl- concentration was decreased, compared with substitutions under control conditions. These results indicate that mucosal Cl- removal causes a decrease in basolateral PCl, which we speculate could be due to a decrease in cell volume. We conclude that the hyperpolarization of the cell membranes on mucosal Cl- removal is primarily due to the combined effects of the fall in basolateral PCl and the increase in basolateral ECl.
Insulin and IGF I receptor-mediated Na+ transport in toad urinary bladders
Authors: Blazer-Yost B. L. , Cox M. , Furlanetto R.
Published: December 2017

We compared the concentration dependence of insulin- and insulin-like growth factor I (IGF I)-stimulated Na+ transport with ligand-receptor affinities in the urinary bladder of the toad Bufo marinus. Threshold, half-maximal, and maximal natriferic concentrations of both peptides were approximately 0.1, 1, and 10 nM, respectively. Amiloride, but not ethyl isopropyl amiloride, (10(-5) M), abolished Na+ transport. Maximal responses to either peptide rendered the tissue insensitive to challenge with the other. Separate insulin and IGF I receptors were identified by equilibrium binding and polyacrylamide gel electrophoresis of cross-linked ligand-receptor complexes. For both peptides, half-maximal binding occurred at 3-10 nM; crossover binding to the other receptor occurred with 10- and 100-fold lower affinity. Thus, in this model "high-resistance" renal epithelium, 1) ligand binding to specific insulin and IGF I receptors stimulates transcellular Na+ flux, 2) the natriferic effects of insulin and IGF I apparently depend on activation of apical Na+ channels rather than Na+-H+ antiporters, and 3) the natriferic pathways activated by insulin and IGF I appear to converge subsequent to ligand-receptor binding but before the final transport ("effector") step(s).
Membrane effects of chloride substitutes in guinea pig gallbladder epithelial cells
Authors: Wehner F. , Sigrist G. , Petersen K. U.
Published: December 2017

Differences in the responses of guinea pig gallbladder epithelial cells to replacement of luminal Cl- with either isethionate (I), gluconate (G), sulfate (S), or cyclamate (C) were investigated in vitro using intracellular microelectrode techniques. In prostaglandin E1 (PGE1)-treated tissues (10(-6) M, serosal side), where electrodiffusive apical membrane Cl- permeability (PCla) is high, replacement of luminal Cl- caused transient membrane depolarizations of similar magnitudes but different times to peak (C greater than G = S greater than I). The subsequent shifts in membrane voltages were, at steady state, straight correlated with the concomitant increases in apparent ratio of apical to basolateral membrane resistances (Ra/Rb). Increases followed the rank order I greater than G = S greater than C, which was also found to be the case in the peak membrane hyperpolarizations on restoring luminal Cl-. Under control conditions (no PGE1, low PCla), three of the substitutes caused a slow hyperpolarization, C greater than G = S, whereas an I-for-Cl- substitution evoked a transient depolarization and a drop in Ra/Rb. Under both control and PGE1 conditions, a transient depolarization followed luminal I-for-C substitution. Our results are best explained by a stimulatory effect of I (and, less marked, G and S) on PCla. Intrinsic effects of cyclamate are not ruled out; however, among the substitutes examined, it is the most inert.
Effects of the halothane-sensitivity gene on sarcoplasmic reticulum function
Authors: Mickelson J. R. , Gallant E. M. , Rempel W. E. , Johnson K. M. , Litterer L. A. , Jacobson B. A. , Louis C. F.
Published: December 2017

Pigs heterozygous for the halothane-sensitivity gene exhibit a distinct phenotype with regard to both in vivo and in vitro muscle responses to halothane (E. M. Gallant, J. R. Mickelson, B. D. Roggow, S. K. Donaldson, C. F. Louis, and W. E. Rempel. Am. J. Physiol. 257 (Cell Physiol. 26): C781-C786, 1989). In this paper heavy sarcoplasmic reticulum (SR) preparations were isolated from the muscles of pigs of all three genotypes. The rate of calcium release from SR of pigs homozygous for the halothane-sensitivity gene was approximately twice that of SR from pigs homozygous for the normal allele. Furthermore, in the presence of 6 microM Ca2+, the binding of [3H]ryanodine to SR isolated from the homozygous halothane-sensitive pigs was of a higher affinity than was the binding to SR isolated from the homozygous normal pigs (Kd = 70-90 vs. 265 nM, respectively). The SR from pigs heterozygous for the halothane-sensitivity gene, however, demonstrated intermediate values for the rate of calcium release and the affinity for [3H]ryanodine (Kd = 192 nM). Thus the alterations in heavy SR calcium release and [3H]ryanodine binding in the pigs containing one copy of the halothane-sensitivity gene demonstrate a distinct heterozygote phenotype. These data also suggest that the protein product of this gene is closely associated with, and perhaps identical to, the SR calcium release channel-ryanodine receptor protein.
Cardiolipin-sensitive phospholipase C in subcellular fractions of rabbit myocardium
Authors: Wolf R. A.
Published: December 2017

Phosphatidylinositol-specific phospholipase C was characterized in the soluble phase and in membrane fractions prepared from rabbit myocardium. Four subforms of soluble phospholipase C were identified and characterized. Activity of one subform was inhibited 80% when cardiolipin was present in substrate vesicles, whereas three subforms were stimulated 2- to 10-fold by cardiolipin. A cationic subform, molecular mass 67 kDa, was stimulated threefold when cardiolipin comprised 2% of the total phospholipid and fivefold when it comprised 12%. The major mechanism for the cardiolipin effect was a decrease in the apparent Michaelis constant (Km) of this subform for substrate. Competition experiments were consistent with binding of this subform to cardiolipin. Phospholipase C activity was present in mitochondrial, microsomal, and sarcolemmal membrane fractions that were essentially free of contamination by cytosol. Detection of membrane-associated phospholipase C was facilitated by cardiolipin. Thus rabbit myocardium contains multiple subforms of soluble phospholipase C that differ substantially in surface charge, molecular mass, and sensitivity to cardiolipin. Anionic phospholipids may be important determinants of intracellular distribution of phospholipase C in myocardial tissue.
Hyperthyroid adult rat cardiomyocytes. II. Single cell electrophysiology and free calcium transients
Authors: Li Q. , Guan Z. , Biagi B. A. , Stokes B. T. , Altschuld R. A.
Published: December 2017

The effects of hyperthyroidism on electrophysiological properties and intracellular free calcium transients in single adult rat cardiomyocytes were studied using conventional microelectrodes and time-resolved single cell fura-2 fluorescence microscopy. Under control conditions, resting membrane potentials and triggered action potentials were not different in euthyroid and hyperthyroid myocytes. Calcium transients produced by electrical stimulation, however, were markedly abbreviated in hyperthyroid myocytes. During a train of stimuli, the duration of the calcium transients at half peak amplitude (half time) was 124 +/- 14 ms at the fifth beat in hyperthyroid cells vs. 287 +/- 35 ms in euthyroid cells. Isoproterenol (1 microM) prolonged time to 50% repolarization (APD50) of the action potentials and increased the peak calcium transients in both euthyroid and hyperthyroid myocytes. It also shortened the half time of the calcium transients in euthyroid myocytes but had little effect on the half time in hyperthyroid cells. These data are consistent with the electrophysiology and mechanical performance in intact euthyroid and hyperthyroid cardiac tissues, and the intrinsic changes in hyperthyroid tissues can therefore be illustrated in single ventricular myocytes. Furthermore, the results suggest that alterations in intracellular calcium handling by sarcoplasmic reticulum may account for contractile changes of the heart induced by hyperthyroidism.
Aequorin luminescence, myosin phosphorylation, and active stress in tracheal smooth muscle
Authors: Gerthoffer W. T. , Murphey K. A. , Gunst S. J.
Published: December 2017

During muscarinic activation of canine tracheal smooth muscle with carbachol, myosin phosphorylation is significantly more sensitive than stress to the external Ca2+ concentration ([Ca2+]o) [W. T. Gerthoffer. Am. J. Physiol. 250 (Cell Physiol. 19): C597-C604, 1986]. To determine whether the intracellular Ca2+ concentration ([Ca2+]i) correlated more closely with changes in phosphorylation or force, we measured isometric force and light emitted by the luminescent intracellular Ca2+ indicator aequorin as [Ca2+]o was increased in the presence of 1 microM carbachol or 60 mM K+. Myosin phosphorylation was measured using an immunoblot assay in a second set of muscle strips treated identically. Stimulation with carbachol increased aequorin luminescence slightly in strips incubated in Ca2+-free solution. Active stress and aequorin luminescence subsequently increased in parallel as [Ca2+]o was increased. Myosin phosphorylation at 0.05 mM [Ca2+]o (0.30 +/- 0.04 mol Pi/mol light chain) was significantly higher than phosphorylation in Ca2+-free solution with no carbachol (0.12 +/- 0.048 mol Pi/mol light chain) and increased to a maximum of 0.56 +/- 0.03 mol Pi/mol light chain at 1.6 mM [Ca2+]o. In contrast, active stress and aequorin luminescence remained low at low [Ca2+]o and reached a maximum at 2.4 mM [Ca2+]o. Stimulation with carbachol produced greater increases in myosin phosphorylation and active stress for a given change in aequorin luminescence than did K+ depolarization. Stimulation with carbachol also produced a different phosphorylation-stress relationship than did K+ depolarization. These observations are consistent with the possibility that carbachol induces increases in the Ca2+ sensitivity of contractile proteins in tracheal smooth muscle.
Impaired control of respiration in iron-deficient muscle mitochondria
Authors: Willis W. T. , Dallman P. R.
Published: December 2017

Dietary iron deficiency (ID) decreases iron-containing proteins and hence respiratory capacity of skeletal muscle mitochondria (SMM), but noniron components are much less affected. Using a hexokinase plus glucose ATP-utilizing system, we studied control of respiration in isolated SMM from rats of variable iron status: ID, ID 3 days after intraperitoneal treatment with iron dextran, and control. We found that sensitivity of respiratory control (e.g., ATP/ADP at a given oxygen consumption) was positively related to state 3 respiratory capacity. Titration studies with carboxyatractyloside, a noncompetitive inhibitor of adenine nucleotide translocase (AdNT), revealed that AdNT concentration was unaffected by iron status. However, the turnover number of AdNT was markedly reduced by ID and improved with iron treatment. We conclude that in ID SMM, decreased maximal respiratory capacity is paralleled by impaired sensitivity to putative controllers of oxidative phosphorylation at any respiratory rate, despite normal levels of AdNT. A second study was designed to determine possible consequences of impaired sensitivity of respiratory control on motor unit recruitment during exercise. ID and normal rats were subjected to a program of walking treadmill exercise. Although exercise failed to induce any changes in oxidative enzyme levels in control rat, ID animals and exhibited substantial mitochondrial enzyme adaptation in hindlimb skeletal muscle. Furthermore, the most consistent enzymatic changes were observed to occur in fast glycolytic muscle fibers. These results suggest marked alterations in the pattern of muscle fiber recruitment during mild exercise in ID rodents and support the hypothesis that sensitivity of respiratory control in SMM is an important determinant of motor unit recruitment during aerobic exercise.
Isoproterenol activates a chloride current, not the transient outward current, in rabbit ventricular myocytes
Authors: Harvey R. D. , Hume J. R.
Published: December 2017

The effects of beta-adrenergic stimulation on the Ca2(+)-insensitive transient outward current (Ito) in rabbit ventricular myocytes were examined. Exposure to isoproterenol (ISO; 1 microM) activated a time-dependent current at positive membrane potentials. To determine whether this ISO-induced current was associated with Ito, sensitivity to the K+ channel antagonist, 3,4-diaminopyridine (DAP; 200 microM) was compared before and after exposure to ISO. The DAP-sensitive current was not enhanced by ISO, suggesting that the ISO-induced current was not a component of Ito. Ito and the ISO-induced current could also be dissociated by changing the membrane holding potential. Positive holding potentials, which produced significant inactivation of Ito, had little effect on the ISO-induced membrane current. Furthermore, the ISO-induced current could be observed when K+ was replaced by Cs+. The reversal potential of the ISO-induced current agreed with the predicted Cl- equilibrium potential, and exposure to Cl(-)-free extracellular solutions eliminated the response to ISO. Therefore, we conclude that ISO does not directly activate Ito in rabbit ventricular myocytes, but instead, activates a time-independent chloride current (ICl) similar to that recently described in guinea pig ventricular myocytes and shown to be regulated by adenylate cyclase (R. D. Harvey and J. R. Hume. Science Wash. DC 244: 983-985, 1989).
Effects of EDCF and endothelin on phosphatidylinositol hydrolysis and contraction in rat aorta
Authors: Rapoport R. M. , Stauderman K. A. , Highsmith R. F.
Published: December 2017

Endothelium-derived constricting factor (EDCF) and endothelin are peptidergic substances produced and released from endothelial cells that induce contraction of vascular smooth muscle. The purpose of the present study was to investigate possible mechanisms by which EDCF and endothelin elicit contraction. Exposure of rat aorta to EDCF or synthetic endothelin resulted in time- and concentration-dependent increases in tension and levels of inositol monophosphate, a breakdown product of the phosphatidylinositides. A 10-s exposure to endothelin elevated levels of inositol 1,4,5-trisphosphate. Trypsinization or heating of EDCF prevented the contraction and inositol monophosphate formation. To assess whether EDCF and endothelin may act as endogenous agonists of the dihydropyridine-sensitive Ca2+ channel, we evaluated the ability of the dihydropyridine Ca2+ channel agonist (+)-S202-791 to increase the formation of the inositol phosphates. (+)-S202-791 increased inositol monophosphate formation. However, in contrast to that elicited by EDCF and endothelin, the increase in inositol monophosphate because of (+)-S202-791 was abolished by pretreatment with the cyclooxygenase inhibitor indomethacin (10 microM). These results suggest that contractions induced by EDCFs may be mediated through activation of phospholipase C and subsequent production of second messengers.
An ultracompliant glass microelectrode for intracellular recording
Authors: Fedida D. , Sethi S. , Mulder B. J. , ter Keurs H. E.
Published: December 2017

We describe the simple fabrication of a glass microelectrode that was designed for the stable impalement of contracting isolated cardiac preparations. The electrodes can be made with the aid of a standard pipette puller. The design uses a long-shanked glass electrode that is stepped below the shoulder at 70 degrees to the shaft and again near the tip. The electrodes are of marginally higher resistance than equivalent length straight electrodes (34.9 vs. 26.0 M omega). The stepped design imparts a low stiffness to the electrode in the vertical and horizontal planes, and the mechanical basis for this performance is described and discussed with regard to measured behavior. The duration of impalements with stepped electrodes is significantly longer than for straight electrodes and can usually be maintained for indefinite periods with minimal damage to small, highly contractile muscle preparations.
Vasoactive intestinal peptide stimulates active K+ transport and Na(+)-K(+)-Cl- cotransport in HT-29 cells
Authors: Turner J. T. , Franklin C. C. , Bollinger D. W. , Kim H. D.
Published: December 2017

Regulation of active K+ influx and Na(+)-K(+)-Cl- cotransport activity in HT-29 cells by vasoactive intestinal peptide (VIP) was investigated. Both active K+ influx, defined as the ouabain-sensitive component, and Na(+)-K(+)-Cl- cotransport, defined as the ouabain-resistant bumetanide-sensitive component, of total K+ uptake were increased by VIP. VIP increased the maximum velocity (Vmax) values for both components with no change in apparent Michaelis constant (Km) values. Three lines of evidence support the role of adenosine 3',5'-cyclic monophosphate (cAMP) as a mediator of the VIP effects. 1) The rank order potencies of VIP and peptide histidineisoleucineamide (PHI) in binding and cAMP production (J. T. Turner, S. B. Jones, and D. B. Bylund, Peptides Fayetteville 7: 849, 1986) and K+ uptake were consistent; 2) alpha 2-adrenergic agonists inhibited both VIP-stimulated cAMP production (J. T. Turner, C. Ray-Prenger, and D. B. Bylund, Mol. Pharmacol. 28: 422, 1985) and K+ uptake; and 3) forskolin, but not dideoxyforskolin, mimicked the effects of VIP on K+ uptake. Because amiloride blocked the VIP-stimulated active K+ component, the VIP effects on active K+ influx may be secondary to a Na(+)-H+ antiporter-mediated increase in cellular Na+ content. Additional experiments indicated that pretreatment of cells with a protein kinase C activator, previously shown to decrease basal Na(+)-K(+)-Cl- cotransport activity and the apparent number of cotransporters in HT-29 cells (C. C. Franklin, J. T. Turner, and H. D. Kim, J. Biol. Chem. 264: 6667, 1989), did not change the magnitude of response of the remaining cotransporters after adenylate cyclase activation.(ABSTRACT TRUNCATED AT 250 WORDS)
Temperature dependence of myofilament Ca sensitivity of rat, guinea pig, and frog ventricular muscle
Authors: Harrison S. M. , Bers D. M.
Published: December 2017

Cooling the superfusate of intact ventricular muscle, from 30 degrees C to below 4 degrees C in less than 2 s, leads to contractures thought to reflect the amount of Ca available for release from the sarcoplasmic reticulum (SR). On rewarming, tension transiently increases in guinea pig and rat ventricular muscle. It has been proposed that this rewarming tension spike reflects changes in myofilament Ca sensitivity and maximum Ca-activated force (Cmax) associated with rewarming. There are differences in intracellular Ca regulation among cardiac muscle preparations. Some characteristics of rapid-cooling contractures (e.g., the magnitude of the rewarming spike) also differ between species. Therefore, the Ca sensitivity of skinned ventricular muscle from the rat, guinea pig, and frog was determined at 29 (22 degrees C for frog ventricular preparations), 8, and 1 degrees C. The results show that cooling rat and guinea pig ventricular muscle from 29 to 1 degrees C shifts the pCa vs. tension relationship toward higher [Ca2+] by 0.65 and 0.55 pCa units, respectively. Cooling to 1 degrees C also reduced Cmax to 3.3 and 7.8% of that at 29 degrees C in rat and guinea pig ventricular muscle, respectively. Similar results were found for frog ventricular muscle, in which cooling from 22 to 1 degrees C reduced Ca sensitivity by 0.6 pCa units and Cmax to 45.7% of its value at 22 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
Glycogen phosphorylase in fish muscle: demonstration of three interconvertible forms
Authors: Schmidt H. , Wegener G.
Published: December 2017

White skeletal muscle of crucian carp contains a single isoenzyme of glycogen phosphorylase, which was purified approximately 300-fold to a specific activity of approximately 13 mumol.min-1.mg protein-1 (assayed in the direction of glycogen breakdown at 25 degrees C). Tissue extracts of crucian muscle produced three distinct peaks of phosphorylase activity when separated on DEAE-Sephacel. Peaks 1 and 3 were identified, in terms of kinetic properties and by interconversion experiments, as phosphorylase b and a, respectively. Peak 2 was shown to be a phospho-dephospho hybrid. The three interconvertible forms of phosphorylase were purified and shown to be dimeric molecules at 20 degrees C. At 5 degrees C, a and the hybrid tended to form tetramers. The Mr of the subunit was estimated to be 96,400 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hybrid is kinetically homogeneous, and its kinetic properties are intermediate between those of b and a forms. The b, hybrid, and a forms of phosphorylase can be isolated from rapidly frozen muscle of crucian but in different proportions, depending on whether fish were anesthetized or forced to muscular activity for 20 s. Muscle of anesthetized crucian had 36, 36, and 28% of phosphorylase b, hybrid, and a forms, respectively, whereas the corresponding values for exercised fish were 12, 37, and 51%. Results suggest that three interconvertible forms of phosphorylase exist simultaneously in crucian muscle and that hybrid phosphorylase is active in contracting muscle in vivo.
Insulin activation of brain Na(+)-K(+)-ATPase is mediated by alpha 2-form of enzyme
Authors: Brodsky J. L.
Published: December 2017

The sensitivity of the synaptosomal Na(+)-K(+)-ATPase to insulin was examined and found to be stimulated by the hormone when physiological intracellular sodium concentrations were present. Activation was not mediated by a sodium influx into the vesicles, as shown using sodium uptake experiments and by the fact that tetrodotoxin did not inhibit insulin action. Because the brain Na(+)-K(+)-ATPase catalytic subunit exists as two forms with different affinities for the inhibitory cardiac glycoside ouabain, the sensitivity of each form for insulin was examined. As previously observed in adipocytes, only the high-affinity component, alpha 2, was insulin sensitive. A dose-response curve of insulin activation of the Na(+)-K(+)-ATPase demonstrated a maximal insulin effect at relatively high hormone concentrations. It is unknown, therefore, whether stimulation of the brain Na(+)-K(+)-ATPase occurs in vivo.