bluuebunny/crossref_metadata_2025 huggingface dataset:
Electrophysiology and noise analysis of K+-depolarized epithelia of frog skin
Authors: Tang J. , Abramcheck F. J. , Van Driessche W. , Helman S. I.
Published: December 2017

Epithelia of frog skin bathed either symmetrically with a sulfate-Ringer solution or bathed asymmetrically and depolarized with a 112 mM K+ basolateral solution (Kb+) were studied with intracellular microelectrode techniques. Kb+ depolarization caused an initial decrease of the short-circuit current (Isc) with a subsequent return of the Isc toward control values in 60-90 min. Whereas basolateral membrane resistance (Rb) and voltage were decreased markedly by high [Kb+], apical membrane electrical resistance (Ra) was decreased also. After 60 min, intracellular voltage averaged -27.3 mV, transcellular fractional resistance (fRa) was 86.8%, and Ra and Rb were decreased to 36.1 and 13.0%, of their control values, respectively. Amiloride-induced noise analysis of the apical membrane Na+ channels revealed that Na+ channel density was increased approximately 72% while single-channel Na+ current was decreased to 39.9% of control, roughly proportional to the decrease of apical membrane voltage (34.0% of control). In control and Kb+-depolarized epithelia, the Na+ channel density exhibited a phenomenon of autoregulation. Inhibition of Na+ entry (by amiloride) caused large increases of Na+ channel density toward saturating values of approximately 520 X 10(6) channels/cm2 in Kb+-depolarized tissues.
Delay of desensitization onset by potassium ion in voltage-clamped frog muscle fibers
Authors: Manthey A. A.
Published: December 2017

Increase in extracellular K+ concentration causes delay in desensitization onset during prolonged application of carbamylcholine to the postjunctional membrane in muscle. This could be due to a direct action of K+ on acetylcholine receptors or to some change in the receptors related to K+-induced effects on transmembrane potential. The question of direct vs. voltage-dependent action of K+ was investigated in frog muscle (Rana pipiens) using a point-source voltage clamp. In conductance measurements first without voltage control, desensitization rate in bath media containing 33 mM K+ was -0.198 s-1 among fibers showing an average potential of -30 mV and -0.104 s-1 in 165 mM K+ where the average potential was -2 mV, a decrease of 47%. By comparison, in voltage-clamp tests at a nominal holding potential of +20 mV, increasing extracellular K+ from 33 to 165 mM caused a decrease of 61% in desensitization rate from -0.151 to -0.059 s-1. Another series in 165 mM K+ at a holding level of +10 mV showed a decrease of 67% to a rate of 0.047 s-1. It is concluded that increases in extracellular K+ can delay desensitization onset independently of effects on transmembrane potential. It is suggested that this could result from a direct interaction of K+ with sites on the outer receptor moiety or within channels, but probably not at the inner membrane face, if the latter are considered in equilibrium with bulk intracellular K+.
Insulin stimulates macromolecular synthesis in cultured glial cells from rat brain
Authors: Clarke D. W. , Boyd F. T. , Kappy M. S. , Raizada M. K.
Published: December 2017

The effect of insulin on macromolecular synthesis in glial cells cultured from brains of 1-day-old rats was studied to investigate the role of insulin in brain growth. Insulin caused a dose-dependent stimulation of protein synthesis (measured by [3H]valine incorporation into protein) that became significant by 7 nM insulin. Maximal stimulation of protein synthesis of 145% of control occurred with 18 nM insulin. Long-term protein synthesis was also stimulated to 136% of control by insulin in a dose-dependent manner after 6 days of insulin incubation. Insulin also stimulated net RNA and DNA synthesis (measured by [3H]uridine and [3H]thymidine incorporation into RNA or DNA, respectively) with significant stimulation by 2 nM insulin. Net RNA synthesis stimulation was maximal at 120% of control by 18 nM insulin. Plateau stimulation of DNA synthesis of 175% of control was reached by 200 nM insulin. The effects of insulin on glial protein and RNA synthesis appear to be mediated completely by the insulin receptor. Insulin, in physiological concentrations, stimulated glial DNA synthesis via its interaction with the insulin receptor (46% of total response). At supraphysiological concentrations insulin may have stimulated DNA synthesis via its cross-reactivity with the insulinlike growth factor I receptor (54% of total response). Thus insulin, at concentrations known to exist in the brain, stimulates the processes necessary for growth in the glial cell and is an important growth factor in the developing rat brain.
Stimulation rate, potentiators, and sarcomere length-tension relationship of muscle
Authors: Rome L. C. , Morgan D. L. , Julian F. J.
Published: December 2017

Isometric tetani of single muscle fibers of Rana temporaria were studied as a function of stimulation rate, sarcomere length (1.7-2.3 micron), twitch-to-tetanus ratio, and exposure to twitch potentiators (Zn2+ and NO3-) at 20 degrees C. As the stimulation rate was decreased below a maximal level, tension generation decreased. This depression in tension generation was more pronounced at shorter sarcomere lengths. Therefore the magnitude and shape of the sarcomere length curve was dependent on stimulation rate. Although the depression in tension generation was always accompanied by a noticeable ripple in the tension record in fibers with large twitch-to-tetanus ratios, it could be observed even during well-fused tetani in fibers with low twitch-to-tetanus ratios. In all fibers, however, high stimulation rates or exposure to potentiators resulted in maximum tension generation at each length, and the sarcomere length-tension curve followed that found by Gordon, Huxley, and Julian. This indicates that the fall in tension between sarcomere lengths of 2.0 and 1.7 micron is not due to length-dependent activation but is more likely to be the result of mechanical interference in the force-generating interaction between cross bridges and thin filament sites.
Transepithelial transport of guinea pig gastric mucous cell monolayers
Authors: Rutten M. , Rattner D. , Silen W.
Published: December 2017

Guinea pig gastric mucous epithelial cells were isolated, enriched, cultured in collagen cups, and put into Ussing chambers for electrophysiological studies. The cultured mucous cell monolayers, which retain the morphology of surface cells found in the intact tissue, had a maximal resistance (R) of 272 +/- 12 omega X cm2 and a potential difference (PD) of -3.8 +/- 0.4 mV (apical negative) between 4 and 10 days later (n = 33). The current-voltage and conductance-concentration relationships of the cultures were both nonlinear (n = 12). In addition, NaCl concentration gradients across the monolayer also gave asymmetrical and nonlinear dilution potentials, with the side of lower chemical potential always becoming electrically negative (n = 10). Calculation of the average Cl(-)-to-Na+ permeability ratio at pH 7.4 was 1.35, indicating a slightly greater conductance of anions over cations. Amiloride (0.1-1.0 microM) had no effect on PD or R when given from the apical or basal side (n = 18), but at higher concentrations (0.1-1.0 mM) there was a decrease in the PD. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid at 10(-4) M increased R from the apical side only (n = 14), and acetazolamide at 5 X 10(-4) M reduced the PD to -0.5 mV (n = 8). Only ouabain at 10(-4) M from the serosal side was effective in reducing the monolayer PD to zero. This culture preparation will prove useful for future studies in determining specific functions for this gastric cell type and how those functions relate to barrier function in the stomach.
Microelectrode measurements from oxyntic cells in intact Necturus gastric mucosa
Authors: Demarest J. R. , Machen T. E.
Published: December 2017

The electrical properties of oxyntic cells were measured in intact isolated Necturus fundic mucosa by dissecting away the serosal muscle and connective tissue and impaling the oxyntic cells across their basolateral membranes. Their properties under resting [i.e., not secreting acid (10(-4) M serosal cimetidine)] and stimulated (10(-4) M histamine) conditions were compared with those of surface cells impaled across their apical membranes in a separate set of experiments. Histamine hyperpolarized the transepithelial potential by 6-10 mV and reduced the transepithelial resistance by approximately 40%. The basolateral membrane potential (Vcs) of both cell types was significantly hyperpolarized by histamine, that of oxyntic cells from a resting value of -50 to -59 mV (P less than 0.001) and that of surface cells from -50 to -54 mV (P less than 0.05). Histamine also hyperpolarized the apical membrane potential (Vmc) of the oxyntic cells; however, the Vmc of surface cells was significantly depolarized. The ratio of the apical to basolateral cell membrane resistances Ra/Rb (delta Vmc/delta Vcs resulting from transepithelial current pulses) of resting oxyntic cells was 1.1 and that of surface cells was 3.6. Stimulation did not affect the Ra/Rb of either cell type. A tenfold increase in serosal K+ concentration depolarized Vcs and increased Ra/Rb of resting and stimulated oxyntic cells, indicating a significant basolateral K+ conductance. The results are consistent with a purely passive role for surface cells and indicate that stimulation results in a simultaneous decrease of both the apical and basolateral membrane resistances of the oxyntic cells.
Mitochondrial function and intracellular calcium in anoxic cardiac myocytes
Authors: Cheung J. Y. , Leaf A. , Bonventre J. V.
Published: December 2017

Mitochondrial dysfunction has been implicated as the cause of irreversible injury in the ischemic heart. To circumvent artifacts associated with organelle isolation, mitochondrial function was studied in intact isolated, Ca2+-tolerant rat ventricular myocytes. After 30 min of anaerobic incubation, myocyte viability decreased from 76 +/- 1 to 33 +/- 4%. Basal O2 consumption rates (nanoatoms . mg cell protein-1 . min-1) were 17.1 +/- 1.3 in aerobic cells and 51.0 +/- 9.8 in anoxic cells. Carbonylcyanide-p-trifluoromethoxyphenyl hydrazone (FCCP)-stimulated rates were 65.5 +/- 9.2 and 84.5 +/- 15.3 in aerobic and anoxic cells, respectively. Respiratory control ratio was lower in anoxic cells: 2.3 +/- 0.3 versus 4.2 +/- 0.4 in aerobic cells. These data suggest that early anoxic mitochondrial injury is due to increased permeability of the inner membrane. Addition of pyruvate, malate, and FCCP to cells made permeable by digitonin resulted in similar maximal O2 consumption rates: 276.5 +/- 31.8 in aerobic and 299.3 +/- 31.9 in anoxic cells, suggesting the electron transport chain is intact in anoxic cells. For purposes of investigating whether anoxic mitochondrial dysfunction is secondary to cellular or mitochondrial Ca2+ overload, total cell Ca2+, cytosolic free Ca2+ levels (measured by null-point titration), and mitochondrial Ca2+ contents (measured as FCCP-releasable Ca2+) were measured. There were no differences in these three parameters between aerobic and anoxic cells, suggesting that mitochondrial dysfunction and irreversible hypercontraction of isolated cardiac myocytes exposed to 30 min of anoxia are not related to Ca2+ overload.
Increased Ca2+ sensitivity of alpha 1-adrenoceptor-stimulated contraction in SHR caudal artery
Authors: Aqel M. B. , Sharma R. V. , Bhalla R. C.
Published: December 2017

Potassium and alpha-receptor-stimulated contractile responses of caudal artery rings of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were measured under conditions in which norepinephrine (NE) uptake and K+-induced NE release by nerve terminals were eliminated. The maximum isometric tension developed by SHR arterial rings was significantly more compared with WKY arterial rings when arteries were stimulated with NE but not when stimulated with K+. The Ca2+ sensitivity of NE-stimulated arterial rings was about twofold higher compared with WKY arterial rings. However, the Ca2+ sensitivity of K+-depolarized arterial rings was comparable between WKY and SHR. This increase in Ca2+ sensitivity was specifically due to changes in the alpha 1-receptor-mediated mechanisms in SHR. The 50% effective concentration (EC50) values for both NE and alpha 1-specific agonist, methoxamine hydrochloride, were comparable between WKY and SHR, suggesting that alpha 1-receptor sensitivity is not altered in SHR. The relative contributions of postsynaptic alpha 1- and alpha 2-receptors in caudal artery contractions as calculated from the experiments with alpha 1- and alpha 2-receptor agonist and antagonists were 80 and 20% in WKY and 95 and 5% in SHR, respectively. Nifedipine inhibition of caudal artery contractions was significantly greater (P less than 0.05) in SHR when stimulated with NE but not when stimulated with potassium. Our results indicate that the mechanisms involved in K+ depolarization-dependent contractions are not altered in SHR. However, the mechanisms involved in the coupling of alpha 1-adrenergic receptor and smooth muscle contractions may be altered in SHR caudal artery rings.
Rat skeletal muscle phosphorylase kinase: turnover and control of isozyme levels in culture
Authors: Salsgiver W. J. , Lawrence J. C.
Published: December 2017

The expression of phosphorylase kinase was investigated in rat skeletal muscle cells developing in vitro. The enzyme was immunoprecipitated from cells cultured in the presence of [35S]methionine, and the 35S-labeled alpha-, alpha'-, and beta-subunits of the kinase were resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Fusion of myoblasts into myotubes was associated with marked increases in the amounts of kinase activity and the three 35S-labeled subunits. In 2-wk-old myotubes, the net amount of alpha'-subunit represented less than 20% of the total alpha-subunits (alpha + alpha'); however, alpha'-subunits appeared to be synthesized at least as rapidly as alpha-subunits. That alpha'-subunits were degraded more rapidly was confirmed by pulse-chase experiments, which also indicated that alpha'-subunits were not formed by proteolytic processing of the larger alpha-subunit. Inhibition of the spontaneous contractile activity of the myotubes with lidocaine markedly increased both phosphorylase kinase activity and the amounts of the 35S-labeled subunits. The divalent cation ionophore, A23187, decreased the alpha-subunits by 60%, but did not change levels of the alpha'-subunits. Taken together, the present results indicate that rat myotubes synthesize the two isozymes of phosphorylase kinase, and that levels of both are controlled by differentiation and muscle activity.
Quantitative use of weak bases for estimation of cellular pH gradients
Authors: Montrose M. H. , Kimmich G. A.
Published: December 2017

An improvement in the usual procedure for estimating cellular H+ gradients by distribution of weak bases is described, which involves evaluation of and correction for the permeability of the ionic form of the sensor. In the case of methylamine, unidirectional uptake (influx) of the methylammonium ion is calculated by comparing the total influx of [14C]methylamine with the influx of the highly permeant ion [14C]tetraphenyl-phosphonium ([14C]TPP+) for two experimental situations in which the membrane potential differs. Comparison of the potential-dependent changes in unidirectional influx of methylamine and TPP+ allows calculation of the magnitude of influx for the methylammonium ion. This value can then be used to determine the error in the H+ gradient as estimated from the steady-state distribution of methylamine across the plasma membrane. By using ATP-depleted isolated small intestine cells from the chick as the test system, and imposed H+ gradients of defined magnitude, it can be shown that the observed error matches the calculated error.
Intrinsic, apparent, and effective affinities of co- and countertransport systems
Authors: Stein W. D.
Published: December 2017

Solutions to kinetic schemes for the simple carrier, the countertransporter (antiport, exchanger), and the rapid equilibrium cases of the cotransporter (symport) and co-chemiporter (cation-dependent ATPase) are listed. A distinction is made between the intrinsic, apparent, and effective affinities of the transporters for their substrates. Effective pumping requires that the active transporter binds the pumped substrate, at high affinity, realized at the “whence side” (from which pumping takes place) and, at low affinity, at the “whither side” (to which pumping takes place). It is demonstrated how effective pumping might be achieved by appropriate design of the transporter or chemiporter in terms of the energies of the intrinsic binding sites, the energies of the conformation changes that the pump protein undergoes, the dissociation constant of the chemical reaction that drives the co-chemiport, and the order of binding of the cosubstrates, appropriate at different prevailing levels of the driving substrate.
Potassium-chloride cotransport in resealed human red cell ghosts
Authors: Dunham P. B. , Logue P. J.
Published: December 2017

Furosemide-inhibitable K influx is threefold higher in resealed ghosts of human erythrocytes than in intact cells. The enhancement is specific for K in that furosemide-inhibitable Na influx is the same in resealed ghosts and intact cells. The enhanced K influx resembles K-Cl cotransport in intact cells in that it requires Cl but not Na. N-ethylmaleimide (NEM), which stimulates furosemide-inhibitable K influx in intact cells, is without effect (or slightly inhibitory) in resealed ghosts. The failure of NEM to enhance the flux was not due to low ATP in the ghosts. These findings suggest that enhancement of the K flux in ghosts occurs by oxidation of membrane protein sulfhydryl groups, known to occur with lysis, the same sulfhydryl groups at which NEM acts by alkylation. This conclusion is supported by two observations: dithiothreitol completely prevents the increase in K influx in ghosts; this agent inhibits both oxidation of sulfhydryl groups and alkylation of them by NEM; and K influx in resealed ghosts is sensitive to changes in cell volume, just as it is in NEM-treated intact cells.
Electroneutral secretion of bicarbonate by guinea pig gallbladder epithelium
Authors: Winterhager J. M. , Stewart C. P. , Heintze K. , Petersen K. U.
Published: December 2017

Transepithelial HCO3- movement in guinea pig gallbladder was investigated in vitro. Absorptive (JHCO3ms) and secretory (JHCO3sm) HCO3- fluxes, determined by use of the pH-stat method were approximately 1.0 and 2.1 mumol X cm-2 X h-1, respectively. The resultant net secretion equaled in magnitude, and balanced electrically, the excess in net absorption of Cl- over that of Na+ X JHCO3sm was dependent on luminal Cl- and serosal Na+; it was inhibited by mucosal 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS; 10(-3) M) and serosal ouabain (3 X 10(-5) M) but not by serosal amiloride (10(-3) M) and scarcely by bilateral methazolamide (10(-4) M). JHCO3ms was reduced by mucosal Cl- but enhanced by serosal Cl-; it was dependent on mucosal Na+ and inhibited by mucosal amiloride and bilateral methazolamide. Our findings are consistent with a model in which 1) serosal HCO3- enters the cell in cotransport with Na+ and is then extruded into the lumen by Cl-(-)HCO3- exchange at the apical membrane; 2) mucosal HCO3- enters the cell secondary to apical membrane Na+-H+ exchange and recycles into the lumen via Cl-(-)HCO3- exchange or is, to a lesser extent, absorbed across the basolateral membrane.
Role of prostaglandin release in the response of tight epithelia to Ca2+ ionophores
Authors: Erlij D. , Gersten L. , Sterba G. , Schoen H. F.
Published: December 2017

We have studied the effects of the Ca2+ ionophores A23187 and ionomycin on ion transport across amphibian skin and urinary bladder. Both A23187 and ionomycin stimulated transepithelial Na+ transport across the skin. Ionomycin also markedly increased the conductance of an amiloride-insensitive pathway. Both ionophores markedly stimulated the release of prostaglandin E2 (PGE2) into the solution bathing the serosal surface of the skin. Addition of indomethacin to the serosal bathing solution of the skin blocked both the stimulation of short-circuit current (Isc) and the release of prostaglandin caused by the ionophores. Acetylsalicylic acid also blocked the ionomycin-induced stimulation of Isc. These results suggest that the stimulation of Na+ transport caused by Ca2+ ionophores is mediated by the release of a product of the cyclooxygenase pathway, very likely PGE2. Ca2+ ionophores also stimulated the release of PGE2 in urinary bladders; however, they generally depressed Isc. Since the effect on Isc caused by the addition of exogenous PGE2 was different in urinary bladders than in skins, we suggest that at least part of the difference in the action of ionophores is due to the difference in the sensitivity of these epithelia to PGE2. Our results suggest that the heterogeneity of effects that Ca2+ ionophores cause in the physiological parameters of tight epithelia are not always the direct result of increased cytoplasmic Ca2+ but that they may be mediated by other tissue responses triggered by the addition of the ionophores.
Changes in intracellular sodium with chloride secretion in dog tracheal epithelium
Authors: Shorofsky S. R. , Field M. , Fozzard H. A.
Published: December 2017

Na-selective microelectrodes were employed to investigate the mechanism of Cl secretion by canine tracheal epithelium. In control tissues with a mean short-circuit current (Isc) of 30.1 microA/cm2, the intracellular Na activity (aiNa) was 10.7 mM. Following steady-state stimulation of Cl secretion with epinephrine (Isc = 126.4 microA/cm2), aiNa was 21.3 mM. These data indicate that there is sufficient energy in the Na gradient to drive Cl secretion by this tissue. When analyzed with simple kinetic models for the Na-K pump, they also suggest that the basolateral entry step involves the Na-K-2Cl cotransporter.
Serotonin uptake by bovine pulmonary artery endothelial cells in culture. I. Characterization
Authors: Lee S. L. , Fanburg B. L.
Published: December 2017

Serotonin (5-HT) uptake by bovine pulmonary artery endothelial cells in culture was measured at 5-HT concentrations of 1.5 X 10(-8) to 10(-3) M. The uptake increased linearly at concentrations of 10(-5) to 10(-3) M 5-HT, and the slope for uptake was 1.276 X 10(6) pmol 5-HT X 10(6) cells-1 X 30 min-1 X M concentration-1. The slope for uptake at 5-HT concentrations less than 10(-7) M was 7.10 X 10(6) pmol X 10(6) cells-1 X 30 min-1 X M concentration-1. With subtraction of the slope for linear uptake obtained at high concentrations of 5-HT from total uptake of 5-HT, a curve showing saturable uptake at concentrations greater than 10(-6) to 10(-5) M was obtained. Lineweaver-Burk analysis of the data yielded a saturated value of high-affinity transport of 6.25 pmol X 10(6) cells-1 X 30 min-1 and a Km of 10(-6) M. At 5-HT concentrations of 1.5 X 10(-8) to 5 X 10(-7) M, uptake was inhibited 75-80% by exposure to 4 degrees C or by absence of sodium in the medium; absence of sodium did not affect uptake at 5-HT concentrations greater than 10(-5) M, and 4 degrees C was less effective in inhibiting uptake at high 5-HT concentrations. Imipramine markedly inhibited uptake at low, but not at high, 5-HT concentrations. Ouabain only partially inhibited 5-HT uptake, but inhibition was more pronounced at low 5-HT concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)
Hypertonic cell volume regulation in mouse thick limbs. I. ADH dependency and nephron heterogeneity
Authors: Hebert S. C.
Published: December 2017

Differential interference contrast microscopy was used in combination with standard electrophysiological techniques in the in vitro perfused mouse medullary (mTALH) and cortical (cTALH) thick ascending limbs of Henle to evaluate the cell volume responses of these nephron segments to sudden increases in peritubular osmolality and to assess the role of antidiuretic hormone (ADH) and net NaCl absorption on hypertonic volume regulation. In the absence of CO2/HCO3- in external media, the cells of the mTALH behaved in a simple osmometric fashion, with an osmotic space equivalent to 70-80% of the total cell volume. However, in CO2/HCO3- -containing media, the cells of the mTALH, but not the cTALH, were able to increase their cell volume to the original volume after shrinkage in peritubular media made hypertonic with either NaCl or mannitol. This volume-regulatory increase response (VRI) in the mTALH was mediated by an increase in intracellular osmoles, and required peritubular ADH, at concentrations that stimulate maximally the rate of net NaCl absorption. This ADH effect on VRI could be mimicked by addition of dibutyryladenosine 3',5'-cyclic monophosphate to the bath in the absence of hormone. However, 10(-4) M luminal furosemide, a concentration that abolishes ADH-dependent NaCl absorption in the mTALH, had no effect on the VRI response. These results indicate that the cells of the mTALH, but not the cTALH, are capable of hypertonic volume regulation, that ADH (via adenosine 3',5'-cyclic monophosphate) is required for expression of the VRI response in the mTALH, and that the effects of ADH on net NaCl absorption and the VRI response in the mTALH are completely dissociable. Thus these results are consistent with a role for ADH in hypertonic VRI in the mammalian mTALH, which may operate to maintain constant cell volume in this nephron segment during antidiuresis.
Location of the chloride self-inhibitory site of the human erythrocyte anion exchange system
Authors: Knauf P. A. , Mann N. A.
Published: December 2017

Experiments were performed with intact human red blood cells to determine whether the inhibitory effects of high Cl- concentrations on Cl- exchange are primarily due to interaction at the cytoplasmic or the external surface of the membrane. When internal Cl- was varied from 150 mM to 600 mM Cl- (using the nystatin technique), keeping external Cl- constant at 150 mM (with sucrose added to maintain osmotic balance), Cl- exchange was inhibited almost exactly as much as when both internal and external Cl- were increased from 150 mM to 600 mM. On the other hand, if internal Cl- was maintained constant at 600 mM, variation of external Cl- (with either sucrose, gluconate, or citrate-sucrose mixtures replacing Cl-) had no consistent effect on Cl- exchange. Even if internal Cl- was kept at 150 mM by substitution of gluconate for Cl-, an increase in external Cl- from 150 mM to 600 mM did not significantly inhibit Cl- exchange. Thus the self-inhibitory effects of Cl- seem to be caused primarily by binding to a site at the cytoplasmic side of the membrane. External Br-, on the other hand, did cause a significant inhibition of Cl- exchange. In contrast to the inhibitory effects of Cl- at neutral pH, at very high pH (around pH 11) there is an activation of Cl- exchange at very high Cl- concentrations. This effect, however, depends on binding of Cl- to an external site. Thus there seem to be at least two different low-affinity Cl- binding sites, one at the cytoplasmic side, which inhibits Cl- exchange, and one at the external side, which activates Cl- exchange at high external pH.
Quantitative requirement for ATP for active transport in isolated renal cells
Authors: Tessitore N. , Sakhrani L. M. , Massry S. G.
Published: December 2017

We investigated the quantitative relationship between cellular ATP concentration and Na+-K+-ATPase activity as measured by ouabain-sensitive 86Rb influx in rabbit proximal renal cells. Cellular ATP was reduced in a stepwise manner by rotenone (10(-7) to 10(-5) M) and was increased by 10 mM adenosine. During these maneuvers, ouabain-sensitive 86Rb influx was linearly related to cellular ATP and did not saturate up to 9.9 mM ATP. In contrast, Na+-K+-ATPase activity in membranes prepared from these cells saturated at 2.0 mM ATP at various sodium (10-100 mM) and potassium (4-100 mM) concentrations. Sodium-dependent phosphate uptake and alpha-methylglucoside (alpha-MG) uptake were both inhibited to a similar degree when cellular ATP was reduced. We conclude that 1) the ATP requirement for saturation of Na+-K+-ATPase is higher in intact renal cells than in the membranes, and 2) the uptake of phosphate and alpha-MG are similarly influenced by reduction in ATP. This effect of ATP on phosphate and AMG uptake is most likely an indirect one and is secondary to changes in the sodium gradient across the cell.
Active K transport across rabbit distal colon: relation to Na absorption and Cl secretion
Authors: Halm D. R. , Frizzell R. A.
Published: December 2017

We measured isotopic unidirectional fluxes of K to elucidate the mechanisms of active K transport across the distal colon of the rabbit. Separate pathways for active K absorption and active K secretion were detected using various transport inhibitors and stimulators. The rate and direction of net K transport depend on the activities of these two pathways. K absorption was reduced by orthovanadate (both solutions) or serosal Ba, consistent with ATPase-dependent uptake of K across the apical membrane and exit via a Ba-sensitive basolateral K conductance. K secretion was inhibited by serosal ouabain or mucosal Ba, indicating that K secretion involves basolateral uptake via the Na-K pump and apical exit via a Ba-sensitive K conductance. Active K secretion appears to be electrogenic, since inhibition by ouabain produced equivalent changes in the net K flux and short-circuit current. Addition of bumetanide to the serosal solution or the removal of either Na or Cl from the serosal solution inhibited K secretion; mucosal solution amiloride was without effect. These results indicate that this K secretory process is independent of electrogenic Na absorption but is mechanistically similar to Cl secretory processes. Both epinephrine and prostaglandin E2 (PGE2) stimulate K secretion, but only PGE2 also stimulates Cl secretion. The response to these secretogogues suggests that the mechanisms underlying K and Cl secretion are closely linked but can be regulated independently.
Passive and active membrane properties of canine gastric antral circular muscles
Authors: Bauer A. J. , Sanders K. M.
Published: December 2017

Experiments were performed to determine the mechanisms responsible for the gradient of electrical activity within circular muscle of the canine gastric antrum. Cable properties of canine gastric antral circular muscles were determined using the partitioned chamber technique of Abe and Tomita (J. Physiol. Lond. 196: 87-100, 1968). The length constant of the circular muscle near the myenteric plexus was 2.4 mm. This was significantly greater than the length constant of the circular muscle near the submucosa (1.7 mm). Membrane time constants were determined by two techniques. Although the time constant of the circular muscle near the myenteric plexus tended to be greater than that of the circular muscle near the submucosa, this difference was not statistically significant. The two regions of circular muscle also differed in their relative levels of excitability. Submucosal circular muscles demonstrated considerably more outward rectification on depolarization and were difficult to bring to threshold for slow waves. This study demonstrates that significant differences exist in the passive and active membrane properties of myenteric and submucosal circular muscle cells. The data help explain the gradient of electrical activity through the thickness of antral circular muscle.
Measurement of cytoplasmic free Ca2+ concentration in cultured muscle cells by aequorin and quin 2
Authors: James-Kracke M. R.
Published: December 2017

Ca2+ has been proposed to regulate expression of the gene for the Ca2+ pump of the sarcoplasmic reticulum in developing chicken myoblasts (A. N. Martonosi, L. Dux, R. L. Terjung, and D. Roufa. Ann. NY Acad. Sci. 402: 485-514, 1982. In the present study, intracellular free Ca2+ ([Ca2+]i) was measured with the photoprotein, aequorin, incorporated by a reversible hyperpermeabilization technique, and with the fluorescent probe, quin 2. Because aequorin reacts irreversibly with Ca2+ and is inactivated by heat, at 37 degrees C the active aequorin content declined markedly. The resting glow (1/10(6) of the maximum light) disappeared after a few hours, whereas the light from the total active aequorin remaining was still detectable when cells were lysed 3 days later. A new approach, which compared the rate of disappearance of aequorin in treated and control cells, was developed on the basis that aequorin would be inactivated more quickly in cells with higher [Ca2+]i. A23187, ionomycin, and trifluoperazine, all of which accelerated gene expression (A. N. Martonosi et al.), increased the rate of decay of active aequorin and therefore increased [Ca2+]i. "Ca shock", and ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid, which also increased the gene expression (A. N. Martonosi et al.), caused small increases of [Ca2+]i not detectable by aequorin but detectable by quin 2. X537A and ouabain had no effect on the gene expression (A. N. Martonosi et al.) and did not raise [Ca2+]i. The results support the proposal that Ca2+ may regulate expression of this gene.
Involvement of a cytoplasmic protein in calcium-dependent potassium efflux in red blood cells
Authors: Plishker G. A. , White P. H. , Cadman E. D.
Published: December 2017

The potassium permeability of the human red blood cell increases with the free intracellular calcium concentration. The efflux of potassium can be inhibited by iodoacetic acid. This inhibitory effect correlates directly with the carboxymethylation of a protein band found in both the hemolysate and membrane fractions. The present study provides two additional lines of evidence that this protein is involved directly with the calcium-dependent changes in potassium permeability: its association with the membrane is calcium dependent; and calcium-dependent potassium efflux from resealed ghost is inhibited by the incorporation of antibodies raised against this cytoplasmic protein.
Substance P and acetylcholine both suppress the same K+ current in dissociated smooth muscle cells
Authors: Sims S. M. , Walsh J. V. , Singer J. J.
Published: December 2017

The effect of substance P on freshly dissociated gastric smooth muscle cells was examined electrophysiologically. Substance P caused depolarization, associated with a membrane conductance decrease, which led to the generation of action potentials and contraction. When the membrane potential was held constant under voltage clamp, substance P induced a net inward current, also associated with a conductance decrease. The net inward current resulted from suppression of an outward K+ current, one which resembled the acetylcholine-sensitive M-current in these cells. When substance P maximally suppressed this outward K+ current, acetylcholine (ACh) had no additional effect. Conversely, when ACh fully suppressed the M-current, substance P was without additional effect. These results indicate that substance P suppresses the same outward K+ current affected by ACh. Suppression of M-current by substance P was observed in approximately half (44 of 85) of the cells studied in these experiments. In those cells that did not respond to substance P, ACh was nevertheless capable of suppressing the M-current. Thus both substance P and cholinergic agonists appear to exert their excitatory effects on smooth muscle cells by inhibiting a common K+ current.
Effects of tumor promoters on LLC-PK1 renal epithelial tight junctions and transepithelial fluxes
Authors: Mullin J. M. , O'Brien T. G.
Published: December 2017

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) irreversibly dissipates the electrical potential difference (PD) across LLC-PK1 renal epithelial cell sheets in a dose-dependent manner. The promoter is equally effective when presented to either cell surface. TPA is also equally effective at dissipating the apical-negative PD or the heretofore undescribed apical-positive PD, both of which can exist spontaneously across this monolayer. Diffusion potentials arising from imposed NaCl gradients across LLC-PK1 cell sheets are likewise reduced by TPA. The non-tumor-promoting parent compound, phorbol, is ineffective, but the more hydrophilic TPA analogue, phorbol 12,13-dibutyrate (PDBU) is as effective as TPA. Unlike TPA, the effects of PDBU are reversible. By observing the (paracellular) fluxes of D-mannitol and polyethylene glycol, these effects on PD are shown to be due to increased permeability of the tight junctions. This effect is immediately reversible once PDBU is removed. These findings are discussed in light of the ramifications of the breakdown of epithelial compartments in vivo and the role this could play in the regulation of epithelial cell growth.
Chronic stimulation of mammalian muscle: enzyme changes in individual fibers
Authors: Chi M. M. , Hintz C. S. , Henriksson J. , Salmons S. , Hellendahl R. P. , Park J. L. , Nemeth P. M. , Lowry O. H.
Published: December 2017

Single fibers of rabbit fast-twitch tibialis anterior (TA) muscles were analyzed after continuous low-frequency stimulation for up to 8 wk. After 2-5 wk, every fiber showed higher levels of citrate synthase, hexokinase, and 3-oxoacid CoA-transferase than any control fiber; in some cases these levels were 2-10 times higher (well above any found even in the control soleus, a slow-twitch muscle). Average levels of malate dehydrogenase and alanine transaminase also rose dramatically, but peak single fiber levels were not much above the highest in controls. These differential effects confirm at the single fiber level that chronic stimulation can alter mitochondrial composition. Lactate dehydrogenase, fructose-bisphosphatase, and adenylate kinase declined to levels far below those of any control TA fiber, and, in the case of fructose-bisphosphatase, to within the activity range of control soleus fibers. According to their staining reaction for myofibrillar ATPase, TA fibers were initially 23% type IIA, and 74% type IIB, but by 5 wk these had been converted to a mixture of type I, IIA, and IIC fibers. At 5 wk, levels of lactate dehydrogenase, adenylate kinase, and malate dehydrogenase were characteristic of their (new) ATPase type, but 3-oxoacid CoA transferase had increased to levels 6-15 times higher than in control fibers of the same type.
Direct control of contraction force of single frog atrial cells by extracellular ions
Authors: Shepherd N. , Kavaler F.
Published: December 2017

We describe a method by which the ionic surround of an isolated frog heart cell can be changed within a small fraction of a contraction cycle while continuously measuring contraction force. With this method, we have investigated the effect on force development of changing the extracellular concentrations of Ca [( Ca]o) and Na [( Na]o) in the period between electrically driven contractions and during the rising phase of a contraction. Raising or lowering either [Ca]o or [Na]o more than 300 ms prior to a stimulus caused peak force of the next contraction to be changed 100% of the way to the steady-state value characteristic of the new ionic concentrations. Similar maneuvers at later times relative to the stimulus caused progressively smaller changes. Lowering [Ca]o from 2 to 1 mM or raising [Na]o from 78 to 110 mM 100 ms after stimulation brought twitch force 35 and 67% of the way to the new steady states, respectively. We conclude that extracellular Ca is the source of activator Ca in these cells and that extracellular Na plays a role in regulation of the intracellular Ca concentration early in the contraction cycle.
Antioxidant defense mechanisms of endothelial cells: glutathione redox cycle versus catalase
Authors: Suttorp N. , Toepfer W. , Roka L.
Published: December 2017

The importance of the glutathione (GSH) redox cycle and of catalase as intracellular antioxidant defense systems in cultured endothelial cells against an extracellular flux of H2O2, a critical mediator of polymorphonuclear leukocyte-induced oxidant injury of endothelial cells, was examined. The activities of different parts of the GSH redox cycle were impaired by 1,3-bis(2-chloroethyl)-1-nitrosourea, buthionine sulfoximine, diethyl maleate and 2-cyclohexene-1-one. Catalase activity was inhibited by 3-amino-1,2,4-triazole. After an impairment of the GSH redox cycle, but not of catalase, the susceptibility of pulmonary artery endothelial cells to an attack by H2O2 was dramatically increased independent of the source of extracellularly generated hydrogen peroxide (i.e., glucose oxidase or stimulated polymorphonuclear leukocytes). Exogenous catalase, d-alpha-tocopherol, and particularly Trolox, the chroman compound of tocopherol, but not phytol, the fatty acid side chain of tocopherol, provided almost complete protection of the endothelial cells against a H2O2-mediated attack. Additional fluorometric studies suggested that H2O2 is scavenged by the antioxidants before it hits the target cells.
Status epilepticus-induced changes in primate cortical energy metabolism
Authors: McCandless D. W. , DeFrance J. F. , Dworsky S. , Presley-Zimmer E.
Published: December 2017

The objective of this study was to evaluate changes in cortical energy metabolism in experimentally induced seizures in the primate. Cynamologus fascicularis monkeys were anesthetized, and a craniotomy was performed. Small samples from the motor cortex were removed for measurement of energy metabolites just prior to intravenous bicuculline infusion (0.6 mg/kg), 20 min after the onset of seizures, and 2 h after the second sample. Samples were also taken for electron microscopy. Results showed decreased phosphocreatine values 20 min after the onset of seizures, whereas ATP levels were normal. Two hours after the onset of seizures, phosphocreatine had returned to normal, but ATP levels were below normal. Examination of tissue by electron microscopy showed evidence of cell damage 2 h, 20 min after the onset of seizures. These findings are consistent with the concept that sustained seizures may lead to irreversible cell damage and that alterations in energy metabolism may contribute to the cell death.
Use of monoclonal antibodies to culture rat proximal tubule cells
Authors: Stanton R. C. , Mendrick D. L. , Rennke H. G. , Seifter J. L.
Published: December 2017

Current renal cell culture techniques are limited by either a low yield of cells or by heterogeneity of cell types. We have used monoclonal antibodies to microvillus membrane proteins to isolate and culture a pure population of proximal tubule cells. The cells were characterized as proximal tubule cells by phase microscopy, enzyme histochemistry for alkaline phosphatase, butyrate esterase, and gamma-glutamyltransferase, electron microscopy, and specific reactivity with a variety of monoclonal antibodies for proximal tubule cells. Growth over 2-7 days yielded cell numbers up to 1,000-fold greater than obtained by single tubule microdissection. Dome formation was observed, suggesting intact fluid transport. In addition, Na+-H+ exchange and Na+-dependent D-hexose transport, known transport processes of the proximal tubule, were demonstrated by microfluorimetry of single cells and methyl-alpha-D-glucopyranoside uptake, respectively. Our results indicate that large numbers of homogeneous, cultured rat proximal tubule cells that maintain characteristics of in vivo proximal tubule cells can be obtained using a monoclonal antibody technique of isolation.
Voltage-activated Na+ currents and their suppression by phorbol ester in clonal insulin-producing RINm5F cells
Authors: Rorsman P. , Arkhammar P. , Berggren P. O.
Published: December 2017

The whole-cell configuration of the patch-clamp technique was applied on the clonal insulin-producing cell line RINm5). Thus attempts were made to characterize voltage-activated inward and outward membrane currents and to examine to what extent these were affected by both long-term and acute exposure to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Current responses to voltage-clamp steps up to -40 mV were small. A pulse to -28 mV evoked an inward current, and slowly activating outward currents developed at potentials above -20 mV. The inward current had a V-shaped current-voltage relationship, reaching a peak between -10 and 0 mV, whereas the outward current increased linearly at potentials beyond -20 mV. It was demonstrated that the inward currents are carried primarily by Ca2+ and Na+ and the outward current by K+. After long-term exposure to TPA, there was a suppression of Na+ currents in one-third of the cells, whereas the Ca2+ and K+ currents were unaffected. Acute exposure to the phorbol ester increased the Ca2+ currents with little effect on the Na+ currents. The extent to which the differences in effects on membrane currents initiated by respective acute and long-term exposure to TPA may reflect two separate mechanisms of protein kinase C activation, the latter related to regulation of differentiation of the RINm5F cells, merits further investigation.
Hormonal regulation of Na+-K+-ATPase in cultured epithelial cells
Authors: Johnson John P. , Jones Doris , Wiesmann William P.
Published: December 2017

Page C186: John P. Johnson, Doris Jones, and William P. Wiesmann. “Hormonal regulation of Na+-K+-ATPase in cultured epithelial cells.” Page C187—C189: the appropriate units for the activity of Na+-K+-ATPase should be micromoles per milligram protein per hour, not micromoles per microgram protein per hour for Table 1, Figure 3, and text.
Structural effects of exposure of smooth muscle in sucrose gap apparatus
Authors: Daniel E. E. , Posey-Daniel V. , Jager L. P. , Berezin I. , Jury J.
Published: December 2017

Structural changes were studied over time of a smooth muscle tissue (opossum esophagus circular muscle) exposed in various compartments of a functioning single sucrose gap. The tissues in the Krebs-perfused compartment were qualitatively normal, possessing nerve profiles with little damage, normal appearing smooth muscle and interstitial cells of Cajal as well as gap junctions between muscle cells and interstitial cells. However, in the sucrose compartment tissues had serious damage to smooth muscle, interstitial cells and nerves, and normal gap junctions disappeared. There was also damage to all these structures in tissues exposed to KCl, but most striking was the disappearance of most interstitial cells. These studies raise serious questions about the assumptions underlying the basis for functioning of multicellular tissues in the sucrose gap.
High-speed ultrasensitive instrumentation for myofibril mechanics measurements
Authors: Iwazumi T.
Published: December 2017

A novel instrument for measuring the mechanics of a single myofibril is described. The principle of the transducer operation is to attach a myofibril to a very fine wire suspended in a magnetic field. Feedback circuits pass current through the wire to maintain the length constant when the myofibril contracts. The wire position is measured optoelectronically at a resolution below 1 A. The myofibril measurement system consists of two independent transducers and is capable of resolving tension down to 0.5 ng/square root Hz and controlling the myofibril length with a 10-microseconds rise time. Optical and electronic designs of the system and calibration and adjustment procedures are described. Experimental chamber design, a flow controller, and an environmental noise cancellation scheme are also discussed.