bluuebunny/crossref_metadata_2025 huggingface dataset:
Water permeability of isolated endothelial cells at different temperatures
Authors: Garrick R. A. , Ryan U. S. , Chinard F. P.
Published: December 2017

The endothelial cells provide a potential pathway for water movement across the endothelium. The endothelial cell permeability to water can, therefore, be a factor in regulation of the rate of water movement out of the vasculature. Endothelial cells are isolated from calf pulmonary artery and cultured. The cells are removed from culture, and the diffusional water permeability is determined with the linear diffusion technique. The mean membrane permeability coefficients (PDS) determined at 20, 30, 37, and 41 degrees C are 160, 273, 304, and 387 x 10(-5) cm/s, respectively. The temperature dependence of PD is calculated with the Arrhenius equation to be 7.2 kcal/mol. If these values of PD are compared with values we have reported for the osmotic permeability coefficient (PF), the PF/PD is about one at each temperature. The values of PD in the isolated endothelial cells are compared with PD for endothelial cells estimated from whole organ studies and are similar to recently reported values.
K+ transport and membrane potentials in isolated rat parotid acini
Authors: Nauntofte B. , Dissing S.
Published: December 2017

42K+ transport properties of isolated rat parotid acini were characterized concomitant with measurements of membrane potentials (Em) by means of the fluorescent dye diSC3-(5). In unstimulated acini suspended in a 5 mM K+ buffer, Em was governed by the K+ and Cl- gradients and amounted to about -59 mV, a value that remained unaffected on cholinergic stimulation. In unstimulated acini, 42K+ influx was largely mediated by the Na+-K+ pump, and the residual influxes were mediated by a bumetanide-sensitive component (cotransport system) and by K+ channels. Efflux of 42K+ was largely mediated by a bumetanide-sensitive component and by K+ channels. In the unstimulated state, the cotransport system was mediating K+-K+ exchange without contributing to the net uptake of K+. Within 10 s after stimulation, a approximately 10-fold increase in the acinar K+ conductance (gK) occurred, resulting in a rapid net efflux of K+ that amounted to approximately 3.8 mmol.l cells-1.s-1. Measurements of 42K+ fluxes as a function of the external K+ concentration revealed that in the stimulated state gK increases when external K+ is raised from 0.7 to 10 mM, consistent with an activation of acinar gK by the binding of external K+ to the channel. 42K+ flux ratios as well as the effect of the K+ channel inhibitor from scorpion venom (LQV) suggest that approximately 90% of K+ transport in the stimulated state is mediated by "maxi" K+ channels.
RVD in principal and intercalated cells of rabbit cortical collecting tubule
Authors: Strange K.
Published: December 2017

Cells of the rabbit renal cortical collecting tubule possess significant regulatory volume decrease (RVD) capabilities. After a 100-mosmol/kg reduction in peritubular osmolality, principal and intercalated cells swell 40-45 and 30-35%, respectively, and immediately activate RVD mechanisms. Both cell types downregulate their volume to within 5-6% of control volume at initial rates of 3-6%/min. Return to isotonic saline causes both cell types to shrink (isotonic shrinkage) 25-35% below control volume due to the loss of osmotically active intracellular solutes during RVD. In most mammalian cells studied to date, RVD is mediated largely by passive KCl efflux via KCl cotransport, parallel K+ and Cl- channels, or parallel K+-H+ and Cl- -HCO3- exchange mechanisms. Peritubular application of 0.1 mM ouabain (0 Na+ lumen), bilateral CO2-HCO3- removal, or bilateral application of 0.02 mM bumetanide, 2.0 mM Ba2+, 2.0 mM anthracene-9-carboxylic acid, or 0.5 mM SITS had no significant effect on rates or magnitudes of RVD and isotonic shrinkage in either cell type. Bilateral elevation of K+ from 5 to 52.5 mM reverses or reduces the electrochemical gradient for K+ movement, causing accumulation of this ion in the cytoplasm, but had no effect on the rates or magnitude of principal and intercalated cell RVD. Principal and intercalated cells from K+- or Cl- -depleted tubules (1 h bilateral perfusion with K+- or Cl- -free saline at 37 degrees C) showed normal rates and magnitudes of RVD in K+- or Cl- -free hypotonic saline. Taken together, these results argue against a significant role of passive KCl efflux pathways in mediating principal and intercalated cell RVD.
Dexamethasone accelerates differentiation of A6 epithelia and increases response to vasopressin
Authors: Preston A. S. , Muller J. , Handler J. S.
Published: December 2017

When seeded heavily on a porous tissue culture dish, A6 cells, derived from the kidney of Xenopus laevis, form a highly differentiated epithelium within 4-6 days. When dexamethasone is added to the culture medium, morphological differentiation is completed by day 2, a time at which the control (untreated) is still a disorganized multilayer of cells. In addition to the morphologically evident monolayer of cuboidal cells, the accelerated differentiation is expressed as high transepithelial electrical resistance, short-circuit current, and adenylate cyclase response to vasopressin. When grown on impermeable plastic tissue culture dishes, A6 epithelia are less differentiated and do not respond to vasopressin. With the addition of dexamethasone at the time of seeding on a plastic tissue culture dish, vasopressin responsive adenylate cyclase activity is expressed, albeit at a slower rate than when grown on a porous surface. In addition, dexamethasone treatment of mature epithelia grown on a porous surface results, in hours, in an increase in the adenylate cyclase response to vasopressin. These results reveal two previously unrecognized interactions between adrenal steroid hormones and vasopressin, namely, accelerated differentiation and increased responsiveness of adenylate cyclase.
Role of aiNa in positive force-frequency staircase in guinea pig papillary muscle
Authors: Wang D. Y. , Chae S. W. , Gong Q. Y. , Lee C. O.
Published: December 2017

In the ventricular papillary muscle of guinea pig heart, membrane potential, intracellular sodium activity (aiNa), and twitch force were measured simultaneously and continuously for many hours at stimulation rates of 0, 0.5, 1, 2, 3, 4, 5, and 6 Hz to investigate the relation of aiNa to twitch force and membrane potential both in the steady state and during the changes in these variables. After an increase in stimulation rate, both aiNa and twitch force increased progressively, reaching steady-state levels. The relation between twitch force and aiNa in the steady state was generally sigmoidal over the range of 0.5-5 Hz and steep in the 1- to 4-Hz range. After either increase or decrease in stimulation rate, the time course of change in aiNa was exponential and similar to that of change in twitch force. Moreover, the force-aiNa relation observed after increase in stimulation rate from 0.5 to 3 Hz resembled that observed after decrease in the rate from 3 to 0.5 Hz, indicating an absence of hysteresis in the relation. The results suggest that an increase in aiNa is an important factor involved in the force staircase. As stimulation rate was increased from 0.5 to higher rates (5 or 6 Hz) and then decreased back to 0.5 Hz, a hysteresis phenomenon was observed in the relation between twitch force and aiNa. This suggests that some secondary factor may alter the relation between twitch force and aiNa. As stimulation rate increased and aiNa rose, the steady-state diastolic membrane potential hyperpolarized. This result is consistent with the view that an increase in aiNa enhances the electrogenic Na+-K+ pump and hyperpolarizes the cell membrane.
Antibodies against purified epithelial sodium channel protein from bovine renal papilla
Authors: Sorscher E. J. , Accavitti M. A. , Keeton D. , Steadman E. , Frizzell R. A. , Benos D. J.
Published: December 2017

Monoclonal and polyclonal antibodies against amiloride-sensitive sodium channel protein purified from bovine renal papilla have been produced. These antibodies show considerable specificity in enzyme-linked immunosorbent assay (ELISA) and dot-blot assays and can immunoprecipitate radiolabeled channel protein. The polyclonal antibodies bind two sodium channel subunits on Western blots, namely, the 300- and 110-kDa polypeptides, and cross react with channel protein isolated from the amphibian A6 renal epithelial cell line. They can be used to immunoaffinity purify the channel in relatively high yield from a crude, detergent-solubilized bovine kidney homogenate. These antibodies should be useful in isolating the sodium channel gene, in studying the channel protein structure, in studying immunocytochemical localization, and in allowing purification of the channel from other tissue sources.
pH regulation in single glomerular mesangial cells. I. Acid extrusion in absence and presence of HCO3-
Authors: Boyarsky G. , Ganz M. B. , Sterzel R. B. , Boron W. F.
Published: December 2017

We have developed a technique to measure the fluorescence of a pH-sensitive dye (2,7-biscarboxyethyl-5(6)-carboxyfluorescein) in single glomerular mesangial cells in culture. The intracellular fluorescence excitation ratio of the dye was calibrated using the nigericin-high-K+ approach. In the absence of CO2-HCO3-, mesangial cells that are acid loaded by an NH+4 prepulse exhibit a spontaneous intracellular pH (pHi) recovery that is blocked either by ethylisopropylamiloride (EIPA) or removal of external Na+. This pHi recovery most probably reflects the activity of a Na+-H+ exchanger. When the cells are switched from a N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution to one containing CO2-HCO3-, there is an abrupt acidification due to CO2 entry, which is followed by a spontaneous recovery of pHi to a steady-state value higher than that prevailing in HEPES. Both the rate of recovery and the higher steady-state pHi imply that the application of CO2-HCO3- introduces an increase in net acid extrusion from the cell. One third of total net acid extrusion in CO2-HCO3- is EIPA sensitive and most likely is mediated by the Na+-H+ exchanger. The remaining two thirds of acid extrusion could be caused by a decrease in the background acid-loading rate and/or the introduction of a new, HCO3- -dependent acid-extrusion mechanism. The HCO3- -induced alkalinization cannot be accounted for by a HCO3- -induced reduction in the acid-loading rate. The latter can be estimated by applying EIPA in the absence of HCO3- and observing the rate of pHi decline. We found that this acid-loading rate is only about one fifth as great as the total net acid extrusion rate in the presence of HCO3-. Indeed, two thirds of net acid extrusion in HCO3- is blocked by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), an inhibitor of HCO3- -dependent transport. Furthermore, the effects of EIPA and SITS were additive. Thus, in the presence of CO2-HCO3-, a SITS-sensitive-HCO3- -dependent transporter is the dominant mechanism of acid extrusion. This mechanism also accounts for the increase in steady-state pHi on addition of CO2-HCO3-.
Expression of cardiac sarcolemmal Na+-Ca2+ exchange activity in Xenopus laevis oocytes
Authors: Longoni S. , Coady M. J. , Ikeda T. , Philipson K. D.
Published: December 2017

Injection of Xenopus laevis oocytes with rabbit heart poly(A)+RNA results in expression of Na+ inside (Nai+)-dependent Ca2+ uptake activity. The activity was measured by first loading the oocytes with Na+ using nystatin and then incubating the oocytes in K+ or Na+ medium containing 45Ca. The expressed Na+ gradient-dependent Ca2+ uptake was five to eight times that observed with water-injected oocytes or with poly(A)+RNA-injected oocytes for which the Na+ load step had been omitted. Induced activity was related to the amount of RNA injected and was insensitive to nifedipine. Fractionation of the poly(A)+RNA on a sucrose gradient determined that the active message had a size range between 3 and 5 kb. The properties of the Na+ gradient-dependent Ca2+ uptake indicated that Na+-Ca2+ exchange activity had been expressed in X. laevis oocytes. The result may be useful for cloning and identifying the molecular component responsible for Na+-Ca2+ exchange.
Diacylglycerol metabolism in isolated aortic smooth muscle cells
Authors: Severson D. L. , Hee-Cheong M.
Published: December 2017

The metabolism of the cell-permeable diacylglycerol (DG) analogue, 1,2-dioctanoyl-sn-glycerol (diC8), by isolated intact smooth muscle cells from rabbit aorta was determined. Radiolabeled diC8 was rapidly converted to monoacylglycerol, with smaller amounts of radioactivity recovered as free glycerol and in the total phospholipid fraction that included phosphatidic acid. The greater metabolism of diC8 by the lipase pathway (formation of monoacylglycerol and glycerol) as compared with the kinase pathway (formation of phosphatidic acid and other phospholipids) was observed consistently when experimental conditions were changed by varying the time of incubation (5-30 min), diC8 concentration (0.5-50 microM), and the content of smooth muscle cells in the incubation (0.5-5 X 10(5) cells). These results with the metabolism of exogenous diC8 by intact smooth muscle cells are consistent with in vitro determinations of DG kinase and lipase activities, where lipase activity (especially hydrolysis of the sn-1 position to yield the monoacylglycerol intermediate) was much greater than kinase activity measured with both soluble and particulate subcellular fractions from aortic smooth muscle cells. Both DG kinase and lipase activities were inhibited when 1-monoolein was added to the in vitro assay. Treatment of intact smooth muscle cells with 1-monoolein (200 microM), however, did not reduce the formation of monoacylglycerol from diC8.
Spermidine uptake by type II pulmonary epithelial cells in primary culture
Authors: Kameji R. , Rannels S. R. , Pegg A. E. , Rannels D. E.
Published: December 2017

The transport pathway for the polyamine spermidine (SPD) was characterized in primary isolates of type II pulmonary epithelial cells from rat lungs. [14C]spermidine was accumulated by type II cells via a temperature-, sodium-, and concentration-dependent saturable pathway, with an apparent Km of 0.48 microM and a maximum velocity (V max) of 0.32 pmol.microgram DNA-1.min-1. SPD uptake was inhibited by gramicidin and by reduced extracellular sodium but was unaffected by alpha-aminoisobutyric acid (AIB), which entered the cells by a similar saturable pathway. Uptake of SPD also was inhibited by the exogenous polyamines putrescine (PUTR) and spermine (SPM), as well as by the methylglyoxal bis(guanylhydrazone) (MGBG) and by paraquat (PQ). The order of potency of these inhibitors was SPM greater than PUTR = MGBG much greater than PQ. The absence of serum reduced the Vmax of the system slightly but had no effect on the apparent Km. In contrast, after 3 days in primary cell culture, the kinetics of SPD transport were altered by decreases in both the Km and Vmax of the uptake process. These observations indicate that type II pulmonary epithelial cells exhibit a pathway of polyamine uptake with general characteristics similar to those observed previously in intact lung tissue and other cell types.
Rates of synthesis and degradation of Na+-K+-ATPase during chronic ouabain treatment
Authors: Rayson B. M.
Published: December 2017

Chronic inhibition of Na+-K+-ATPase activity in outer medullary kidney tubules has previously been demonstrated to elicit a 60% increase in activity, measured under maximal velocity (Vmax) conditions (J. Biol. Chem. 260: 12740-12743, 1985). To investigate the cellular mechanism of this response, we measured the rates of Na+-K+-ATPase synthesis and degradation over its full time course. A transient increase in the rate of synthesis occurred after 12 h of ouabain treatment. After 24-h treatment, the rate of synthesis returned to a level not different from control levels. The relative degradation rate after 24-h treatment, however, was markedly lower in ouabain-treated cells than in control cells. Thus the augmentation of the number of Na+-K+-ATPase sites, elicited by the transient increase in synthesis described, was maintained under steady-state conditions by a reduction in apparent degradation rate constant.
Volume-sensitive, Cl-dependent K transport in resealed human erythrocyte ghosts
Authors: O'Neill W. C.
Published: December 2017

Potassium influx and efflux in Cl and NO3 media were measured in resealed ghosts prepared from human red cells. Cl-dependent K influx was three times that in intact cells and, as in intact cells, was partially supported by Br but not by thiocyanate (SCN). In other properties, this flux differed from that in intact cells: substitution of N-methylglucamine for Na did not decrease but rather increased Cl-dependent K influx, the affinity for external K was reduced, with a Km of 21.3 +/- 12.5 mM, and inhibition by furosemide and bumetanide was incomplete. Furosemide at 1 mM inhibited Cl-dependent influx by 26 and 51% at 4 and 20 mM K, respectively. Bumetanide inhibited Cl-dependent K influx by 0 and 55% at concentrations of 10 microM and 1 mM, respectively, in 4 mM K, with no further inhibition at 20 mM K. Neither the magnitude nor the properties of the flux were altered by preparing ghosts in the presence of 1,4-dithiothreitol, indicating that sulfhydryl oxidation was not responsible for the altered flux in ghosts. Treatment with N-ethylmaleimide (NEM) either before or after ghost preparation did not increase Cl-dependent K influx. However, Cl-dependent influx in ghosts could be augmented by increasing ghost volume or ATP content. Resealed human erythrocyte ghosts thus exhibit a volume- and ATP-sensitive, Cl-dependent K flux that differs substantially from the putative Na-K-Cl cotransport in intact cells in that it is independent of Na, is relatively resistant to furosemide and bumetanide, and has a low affinity for K.(ABSTRACT TRUNCATED AT 250 WORDS)
Regulation of intracellular calcium by cell pH in vascular smooth muscle cells
Authors: Siskind M. S. , McCoy C. E. , Chobanian A. , Schwartz J. H.
Published: December 2017

Intracellular calcium (Cai2+) and intracellular pH (pHi) are important regulators of a variety of intracellular processes. Cai2+ is a regulator of muscle contraction, but the role of pHi is unclear. The purpose of this study was to determine the effect of alterations of pHi on Cai2+. A7r5 vascular smooth muscle cells (VSMC) were grown to confluence on glass cover slips. Cai2+ was determined with the fluorescent probe fura-2 and pHi with 2,7-bis-carboxyethyl-5(6)-carboxy-fluorescein (BCECF). Alkalinization of the VSMC by exposure to 20 mM NH4Cl (delta pHi 0.41 +/- 0.07) resulted in a rise in Cai2+ from 99 +/- 8 to 146 +/- 13 nM (n = 5) in the presence of extracellular Ca2+ (Cao2+). In the absence of Cao2+, NH4Cl-induced alkalinization also resulted in a Cai2+ rise (delta Cai2+ = 26 +/- 4 nM, n = 5). Similar changes in Cai2+ were observed when cells were alkalinized by exposure to nigericin in a KCl buffer (pH 7.7). Neither 100 microM verapamil or 100 microM 8,8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate HCl (TMB-8) altered the alkaline-induced changes. After cellular Ca2+ stores were partially depleted by exposure to AVP in a Ca2+-free solution, subsequent cell alkalinization induced no changes in Cai2+. These results demonstrate that alkalinization of VSMCs leads to a rise in cytosolic Ca2+ via release of intracellular Ca2+ stores. The intracellular Ca2+ storage sites appear to be the same as those sites sensitive to AVP. Thus pHi may regulate Cai2+ and thereby play a role in the regulation of vascular smooth muscle tone.
Characterization of beta-adrenergic receptors in cultured bovine tracheal gland cells
Authors: Madison J. M. , Basbaum C. B. , Brown J. K. , Finkbeiner W. E.
Published: December 2017

We characterized the beta-adrenergic receptors that mediate secretory responses to isoproterenol in cultured bovine tracheal submucosal gland cells. Previous studies have shown that these cells have morphological and biochemical features characteristic of serous cells. Isoproterenol, epinephrine, and norepinephrine each stimulated the secretion of 35SO4-labeled macromolecules from these cultured serous cells with a rank order of potency (isoproterenol greater than epinephrine greater than norepinephrine) consistent with the presence of beta 2-adrenergic receptors. These functional studies were supported by radioligand-binding studies using [I125]-iodocyanopindolol (125I-CYP) to identify beta-adrenergic receptors. 125I-CYP binding to membrane particulates prepared from cultured serous cells was saturable and of high affinity (equilibrium dissociation constant 20 +/- 3 pM; mean +/- SE, n = 6) and was antagonized stereoselectively by propranolol. Adrenergic agonists competed for 125I-CYP-binding sites with a rank order of potency characteristic of the beta 2-adrenergic receptor subtype. A specific beta 2-adrenergic receptor antagonist, ICI 118.551, competed for a single class of 125I-CYP-binding sites with high affinity (inhibition constant 1.8 +/- 0.3 nM, n = 3). We concluded that the secretory response of cultured tracheal gland cells to isoproterenol is a response mediated by beta-adrenergic receptors of the beta 2 subtype.
Adaptation to Pi deprivation of cell Na-dependent Pi uptake: a widespread process
Authors: Escoubet B. , Djabali K. , Amiel C.
Published: December 2017

Phosphate enters kidney proximal tubular cells through an apical sodium-phosphate cotransport; this activity (Vmax) increases during phosphate deprivation (Kidney Int. 18: 36-47, 1980). This study investigated the mechanism of phosphate uptake and its adaptation to phosphate deprivation in cultured cells from different origins (kidney, LLC-PK1 and MDCK cells; liver, Fao cells; heart, myocyte primary cultures). All cells exhibited a sodium-dependent phosphate uptake that was reduced (greater than 75%) by external sodium substitution and inhibited by ouabain (35%) and 2,4-dinitrophenol or KCN (80%). Phosphate deprivation (exposure to phosphate-free medium) increased sodium-dependent phosphate uptake by 1.8- to 5.8-fold and decreased cell inorganic phosphate and ATP contents (70-80 and 17-30%, respectively). The stimulation of phosphate uptake resulted from an increase in Vmax without change in Km and was dependent on gene transcription and protein synthesis because it was inhibited by cycloheximide and 3-deoxyadenosine. Thus a deprivation-stimulated, sodium-dependent phosphate transport was demonstrated in cells originating from distal kidney tubules, liver, and heart. The findings suggest that in hypophosphatemic diseases, impairment of renal proximal phosphate reabsorption might be only one expression of a widespread alteration of cell phosphate regulation.
Insulin-induced hyperpolarization in mammalian skeletal muscle
Authors: Iannaccone S. T. , Li K. X. , Sperelakis N. , Lathrop D. A.
Published: December 2017

Insulin-induced hyperpolarization of up to 9 mV has been described in isolated frog [J. Physiol. Lond. 252: 43-58, 1975; Am. J. Physiol. 251 (Cell Physiol. 4): C249-C254, 1979] and mammalian (Molecular Basis of Insulin Action, New York: Plenum, 1985, p. 451-463; Am. J. Physiol. 197: 524-526, 1959; Am. J. Physiol. 198: 1066-1070, 1960) skeletal muscle. We have shown that a similar hyperpolarization occurs in situ after administration of insulin in anesthetized rats. In streptozotocin (STZ)-treated rats, insulin produced approximately 66-70% of the hyperpolarization observed in normal rat skeletal muscle in situ. Administration of ouabain in situ blocked the insulin-induced hyperpolarization in the normal group of rats and significantly blunted the effect in the STZ group. These results suggest that insulin-induced hyperpolarization in skeletal muscle results from direct activation of the Na+-K+-ATPase pump. In isolated skeletal muscle from normal and STZ rats, there was no difference in the amount of the insulin-induced hyperpolarization. There was an additive, but small, hyperpolarizing effect of insulin and isoproterenol when administered in combination, suggesting that the greater magnitude of the insulin-induced hyperpolarization observed in situ in normal rats may be due to an additive effect of injected insulin and endogenous release of epinephrine. Alternatively, STZ treatment may directly alter the Na+-K+ pump so that its response to insulin is lessened.
Hepatocyte beta-adrenergic responsiveness and guanine nucleotide-binding regulatory proteins
Authors: Garcia-Sainz J. A. , Huerta-Bahena M. E. , Malbon C. C.
Published: December 2017

Hepatocytes isolated from hypothyroid, adrenalectomized, or partially hepatectomized rats display an enhanced beta-adrenergic responsiveness as compared with cells from control animals. The enhanced beta-adrenergic responsiveness is evidenced by both increased ureagenesis and adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in response to isoproterenol. The role of stimulatory guanine nucleotide-binding protein (Gs) and inhibitory guanine nucleotide-binding protein (Gi) in the enhanced responsiveness was studied. It was observed, contrary to what would have been anticipated, that the level of Gs [as reflected by cholera toxin-catalyzed ADP ribosylation, 5'-guanosine gamma-thiotriphosphate (GTP gamma S)-stimulated adenylate cyclase activity, and a functional reconstitution assay] was decreased in liver membranes from adrenalectomized and partially hepatectomized rats as compared with the controls. Furthermore, the level of Gi was increased in these conditions as reflected by pertussis toxin-catalyzed ADP ribosylation. The data suggest that changes in beta-adrenergic receptor levels rather than the levels of guanine nucleotide-binding (G) regulatory proteins predominate in regulation of hepatic beta-adrenergic responses by hypothyroidism, adrenalectomy, or partial hepatectomy.
Internal pH-sensitive site couples Cl-(-)HCO3- exchange to Na+-H+ antiport in lymphocytes
Authors: Mason M. J. , Smith J. D. , Garcia-Soto J. J. , Grinstein S.
Published: December 2017

Parallel exchange of Na+-H+ and Cl-(-)HCO3- is thought to be central to the translocation of electrolytes and water during cell volume regulation and in transepithelial transport. Coupling between these transporters is thought to be indirect, through changes in the concentration of HCO3-, which result from alterations in cytosolic pH (pHi). The possibility of a more direct, HCO3-(-)independent interaction between Cl-(-)HCO3- exchange and the Na+-H+ antiport was studied in rat thymic lymphocytes. Measurements of radioactive Cl- flux and of HCO3-(-)dependent pHi changes demonstrated the presence of a Na+-independent, 4,4'diisothiocyanostilbene-2,2'-disulfonic acid-sensitive Cl-(-)HCO3- exchanger. At constant external pH, the rate of Cl-(-)HCO3- exchange was markedly accelerated by increasing pHi between 7.0 and 7.4. This activation was not related to variations in the concentration of HCO3- and is likely caused by a direct effect of intracellular H+ (OH-) on the exchanger. Osmotic shrinking of the cells induced a cytoplasmic alkalinization, due to activation of the Na+-H+ antiport. Concomitantly, the rate of anion exchange also increased. The stimulation of Cl-(-)HCO3- exchange was eliminated when the alkalinization caused by Na+-H+ exchange was precluded. These observations suggest that the exquisite pHi sensitivity of the Cl-(-)HCO3- exchange system provides a mechanism whereby the rates of cation and anion transport are closely coupled.
Membrane site of action of CO2 on colonic sodium absorption
Authors: Charney A. N. , Egnor R. W.
Published: December 2017

Increases in ambient CO2 tension increase colonic sodium absorption by increasing mucosal to serosal sodium flux. We examined the membrane site of CO2 action by utilizing the polyene antibiotic nystatin to create aqueous pores in the apical membrane. Under these conditions, the basolateral rather than the apical membrane is rate limiting for sodium absorption. Pairs of stripped rat distal colonic segments were mounted in modified Ussing chambers in a Ringer-HCO3 solution gassed with either 3% CO2-97% O2 or 11% CO2-89% O2. Mucosal-to-serosal 22Na and 36Cl fluxes were measured under short-circuited conditions, and ouabain-sensitive absorption was calculated before and after the addition of mucosal nystatin 300 U/ml. Ouabain-sensitive sodium absorption was fivefold greater at 11% CO2 than at 3% CO2 before nystatin addition. Nystatin increased short-circuit current (Isc), transcolonic conductance (Gt) and ouabain-sensitive sodium absorption at 3% CO2 but only increased Isc and Gt at 11% CO2. The levels of sodium absorption at 3% and 11% CO2 after nystatin were equal and identical to the level measured at 11% CO2 in the absence of nystatin. Ouabain-sensitive chloride absorption was similar at 3% and 11% CO2 in the absence of nystatin and was not affected by nystatin addition. These findings suggest that ambient CO2 tension affects colonic sodium absorption by a selective action at the apical membrane.
Density-dependent hyperpolarization in cultured aortic smooth muscle cells
Authors: Blennerhassett M. G. , Kannan M. S. , Garfield R. E.
Published: December 2017

The membrane potential (Em) of cultured aortic smooth muscle cells from Sprague-Dawley (SD), Wistar-Kyoto (WKY), and spontaneously hypertensive (SHR) rats was measured in proliferating primary cultures. Em of SD cells in high-density cultures was -51 to -58 mV, whereas that of low-density cultures (1-2 days) was -30 mV. This difference was due to a continuous process of hyperpolarization during proliferation in culture. Em of WKY and SHR hyperpolarized similarly, from -12 to -42 and -38 mV, respectively. Hyperpolarization of Em of SD, WKY, and SHR cells was related to cell density rather than time in culture. Em may be a sensitive and significant indicator of the changes in the differentiated state expressed by proliferating smooth muscle in vitro.
Two distinct mechanisms are responsible for single K channel block by internal tetraethylammonium ions
Authors: Yamamoto D. , Suzuki N.
Published: December 2017

Tetraethylammonium (TEA) ions blocked the unitary currents through the delayed rectifier potassium channels of Drosophila neurons from the cytoplasmic side of inside-out membrane patches by two distinct mechanisms. First, TEA attenuated the single-channel conductance, probably by producing very rapid block-unblock reactions at the inner mouth of the potassium pore. Second, TEA markedly enhanced the slow inactivation, making the incidence of channel openings highly nonrandom; blank traces with no channel openings during repetitive depolarizations showed a significant tendency to be clustered in the presence of TEA. This second action accounts for almost half of the reduction of average current produced by 10 mM internal TEA.
Measurement and simulation of noninactivating Ca current in smooth muscle cells
Authors: Imaizumi Y. , Muraki K. , Takeda M. , Watanabe M.
Published: December 2017

An attempt was made to obtain electrophysiological evidence for continuous influx of Ca ion through voltage-dependent Ca channel (VDCC) in smooth muscle during long depolarization, for example in high K solution. Noninactivated Ca current [ICa(ni)] remaining after the accomplishment of voltage-dependent inactivation by prolonged depolarization for approximately 1 min was detected by three means under whole cell voltage clamp in several types of smooth muscle cells. The measurement of ICa(ni) was performed by micropuff application of Cd2+ or Ca2+ in the presence or absence of 5 mM extracellular Ca, respectively, or jump of extracellular Ca concentration [( Ca]o). The current-voltage relationship of ICa(ni) evaluated by these means had a peak at approximately -10 mV. The peak amplitude ranged from 5 to 25 pA, depending on whether the cells were isolated from guinea pig urinary bladder, ureter, vas deferens, taenia caecum, or rabbit portal vein. The ICa(ni) may be large enough to explain sustained contraction in high K solution, at least in these smooth muscle tissues. A window current simulated from the steady-state activation and inactivation curves and the maximum conductance of Ca current (ICa) in these cells suggests a theoretical basis for the observed ICa(ni).
Na+ and Cl- transport across rabbit nonciliated bronchiolar epithelial (Clara) cells
Authors: Van Scott M. R. , Davis C. W. , Boucher R. C.
Published: December 2017

Radioisotopic flux measurements were performed on rabbit Clara cell epithelium cultured in serum-free hormone-supplemented medium to identify the major ion transport pathways in the cell type. Clara cells cultured in serum-free hormone-supplemented medium exhibit a large short-circuit current compared with cells maintained in serum-containing medium (45 microA/cm2 vs. 15 microA/cm2). The responses to amiloride and isoproterenol, however, are similar for cells grown in the two media. A net amiloride-sensitive movement of Na+ in the mucosal (M)-to-serosal (S) direction undershort- and open-circuit conditions is detected (1.48 and 0.67 mueq.h-1.cm-2, respectively). No statistically significant difference in the unidirectional fluxes of Cl- is apparent in the basal state, but a net flux of Cl- in the S-to-M direction is observed after exposure of the apical membrane to amiloride (0.93 mueq.h-1.cm-2). The partial ionic conductances for Na+ and Cl- estimated from the fluxes measured in the passive directions (JNaS----M, JClM----S) exceed the total tissue conductance by 20%. Ussing flux ratio analyses of Cl- movements at clamped potentials between -60 and +20 mV show that Cl- movements are not strictly through passive conductive pathways at negative potentials. The movement of Cl- can be modeled by passive diffusion combined with Cl- -Cl- exchange equal to 20% of total passive fluxes of Na+ and Cl-. These observations indicate that 1) Na+ absorption is the major active ion transport pathway across cultured Clara cells, 2) active Cl- secretion is minimal in the basal state, and 3) approximately 20% of the unidirectional Cl- fluxes occur via nonconductive pathways.
Cellular Cl- transport in cultured cystic fibrosis airway epithelium
Authors: Willumsen N. J. , Davis C. W. , Boucher R. C.
Published: December 2017

Cultured human nasal epithelia derived from cystic fibrosis (CF) patients were studied with double-barreled, Cl- -selective microelectrodes to measure membrane potentials and intracellular Cl- activity (aClc). The aClc of CF cultures was 46.5 +/- 2.5 mM (n = 28), a value not significantly different from aClc of normal human nasal cells. Reduction of the luminal [Cl-] from 120 to 3 mM failed to reveal any apical Cl- permeability (conductive or nonconductive) in CF cultures. Bumetanide (10(-4) M, serosal) led to a 10 mM decrease in aClc without affecting the electrical parameters of the cells. Reduction of serosal [Cl-] led to a marked decrease in aClc (from 58.0 +/- 6.7 to 26.8 +/- 2.9 mM) that could partly be blocked by bumetanide. Reduction of serosal [Cl-] led to a rapid depolarization (5.4 +/- 0.7 mV) of the basolateral membrane potential (Vb), a decrease of the fractional apical membrane resistance (0.03 +/- 0.01), and an increase (34 +/- omega.cm2) in the transepithelial resistance (Rt). We conclude that 1) the apical membrane of CF airway epithelia is impermeable to Cl-, and 2) Cl- transport across the basolateral membrane occurs mainly through a bumetanide-inhibitable cotransport system but also through a Cl- conductance, neither of which appears to be affected by CF.
Shunt resistance and ion permeabilities in normal and cystic fibrosis airway epithelia
Authors: Willumsen N. J. , Boucher R. C.
Published: December 2017

A method for determination of shunt resistance (Rs) and absolute conductive ion permeabilities of the apical membrane in epithelia from steady-state data is described. The method assumes that the currents are satisfactorily described by the Goldman-Hodgkin-Katz regime. Its application requires measurements of standard transepithelial electrophysiological parameters and of one or more intracellular ion activities. It is applicable under both open- and short-circuit conditions. The method was tested in an electrophysiological analysis of cultured normal and cystic fibrosis (CF) human nasal epithelium. In 15 normal and 10 CF preparations with mean transepithelial resistances of 338 and 427 omega.cm2, Rs was 412 and 623 omega.cm2, respectively. The Rs values determined with the present method were strongly correlated (r = 0.94) with those obtained with another method available in the electrophysiological literature but were as a mean 20% lower. Amiloride increased Rs by 25% in CF and by 8% in normal preparations. In normal preparations, the apical Cl permeability (PCla) was 3.6 x 10(-6) cm/s, and the apical Na permeability (PNaa) was 1.6 x 10(-6) cm/s. In CF preparations, PCla was reduced to a maximum of 2.3 x 10(-7) cm/s, whereas PNaa was increased to 6.2 x 10(-6) cm/s. The apical membrane electromotive force was -1 mV in normal and 43 mV in CF preparations. It is concluded that the method can be used to calculate Rs, apical membrane ion permeabilities, and electromotive forces from steady-state electrophysiological data.
8-BrcAMP-induced capacitance and transport of H2O and Na in skin and urinary bladder of urodele amphibians
Authors: Silver R. B. , Palmer L. G.
Published: December 2017

The effects of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) on capacitance (C), osmotic water flow (Jv), and amiloride-sensitive short-circuit current (INa) were studied in bladder and skin derived from the tiger salamander (aquatic and postmetamorphosed terrestrial phase). 8-BrcAMP-dependent increases in C, measured from the transepithelial voltage response to constant current pulses, occurred in aquatic (delta C = 44%) and terrestrial (delta C = 61%) bladders and terrestrial skin (delta C = 19%). Jv (200-mosM gradient, mucosal side hypotonic) was observed in the bladders and was further enhanced by addition of 8-BrcAMP [10(-3) M; delta Jv = 0.42 microliter.min-1.microF-1 (aquatic) and 0.32 microliter.min-1.microF-1 (terrestrial)]. The aquatic and terrestrial skins were relatively impermeable to water, but the terrestrial skin showed a small response to 8-BrcAMP (delta Jv = 0.04 microliter.min-1.microF-1). 8-BrcAMP-mediated natriferic responses were observed in aquatic bladder (delta INa = 62%) and terrestrial skin (delta INa = 105%). Antidiuretic hormone (ADH)-induced Jv was also observed in the aquatic bladder (delta Jv = 0.33 microliter.min-1.microF-1) and was similar to the 8-BrcAMP-mediated Jv measured in this tissue. The terrestrial bladder displayed a more vigorous response to 8-BrcAMP than to ADH (delta JvADH = 0.09 microliter.min-1.microF-1 and delta Jv8-BrcAMP = 0.32 microliter.min-1.microF-1), suggesting that diminished sensitivity to ADH accompanies the transition from water to land in this species.
Local cerebral glucose utilization during status epilepticus in newborn primates
Authors: Fujikawa D. G. , Dwyer B. E. , Lake R. R. , Wasterlain C. G.
Published: December 2017

The effect of bicuculline-induced status epilepticus (SE) on local cerebral metabolic rates for glucose (LCMRglc) was studied in 2-wk-old ketamine-anesthetized marmoset monkeys, using the 2-[14C]-deoxy-D-glucose autoradiographical technique. To estimate LCMRglc in cerebral cortex and thalamus during SE, the lumped constant (LC) for 2-deoxy-D-glucose (2-DG) and the rate constants for 2-DG and glucose were calculated for these regions. The control LC was 0.43 in frontoparietal cortex, 0.51 in temporal cortex, and 0.50 in thalamus; it increased to 1.07 in frontoparietal cortex, 1.13 in temporal cortex, and 1.25 in thalamus after 30 min of seizures. With control LC values, LCMRglc in frontoparietal cortex, temporal cortex, and dorsomedial thalamus appeared to increase four to sixfold. With seizure LC values, LCMRglc increased 1.5- to 2-fold and only in cortex. During 45-min seizures, LCMRglc in cortex and thalamus probably increases 4- to 6-fold initially and later falls to the 1.5- to 2-fold level as tissue glucose concentrations decrease. Together with our previous results demonstrating depletion of high-energy phosphates and glucose in these regions, the data suggest that energy demands exceed glucose supply. The long-term effects of these metabolic changes on the developing brain remain to be determined.
Effect of higher aortic pressure on ribosome formation and cAMP content in rat heart
Authors: Watson P. A. , Haneda T. , Morgan H. E.
Published: December 2017

Elevation of aortic perfusion pressure from 60 to 120 mmHg in beating and arrested rat hearts raised cAMP content and increased rates of ribosome formation but had no effect on total protein synthesis during 1 h of perfusion. The activity of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase increased as perfusion pressure was elevated in arrested hearts. A regulatory link between increased cAMP content and accelerated ribosome formation was hypothesized to exist. When hearts were arrested with tetrodotoxin and exposed to 0.2 mM methacholine, a muscarinic-cholinergic agonist that blocked pressure-induced increases in cAMP content, elevation of aortic pressure to 120 mmHg failed to increase the rate of ribosome formation. When aortic pressure was maintained at 60 mmHg, exposure of beating hearts to glucagon increased cAMP content and mimicked the effect of elevated aortic pressure to accelerate rates of ribosome formation. These studies support the hypothesis that increased aortic pressure preferentially accelerates rates of ribosome formation by a cAMP-dependent mechanism.
cAMP delays disappearance of gap junctions between pairs of rat hepatocytes in primary culture
Authors: Säez J. A. , Gregory W. A. , Watanabe T. , Dermietzel R. , Hertzberg E. L. , Reid L. , Bennett M. V. L. , Spray D. C.
Published: December 2017

Pages C1–C11: J. A. Sáez, W. A. Gregory, T. Watanabe, R. Dermietzel, E. L. Hertzberg, L. Reid, M. V. L. Bennett, and D. C. Spray. “cAMP delays disappearance of gap junctions between pairs of rat hepatocytes in primary culture.” Page C8: Fig. 8B should appear as follows:
Volume-dependent K+ transport in rabbit red blood cells comparison with oxygenated human SS cells
Authors: al-Rohil N. , Jennings M. L.
Published: December 2017

In this study the volume-dependent or N-ethylmaleimide (NEM)-stimulated, ouabain-insensitive K+ influx and efflux were measured with the tracer 86Rb+ in rabbit red blood cells. The purpose of the work was to examine the rabbit as a potential model for cell volume regulation in human SS red blood cells and also to investigate the relationship between the NEM-reactive sulfhydryl group(s) and the signal by which cell swelling activates the transport. Ouabain-resistant K+ efflux and influx increase nearly threefold in cells swollen hypotonically by 15%. Pretreatment with 2 mM NEM stimulates efflux 5-fold and influx 10-fold (each measured in an isotonic medium). The ouabain-resistant K+ efflux was dependent on the major anion in the medium. The anion dependence of K+ efflux in swollen or NEM-stimulated cells was as follows: Br- greater than Cl- much greater than NO3- = acetate. The magnitudes of both the swelling- and the NEM-stimulated fluxes are much higher in young cells (density separated but excluding reticulocytes) than in older cells. Swelling- or NEM-stimulated K+ efflux in rabbit red blood cells was inhibited 50% by 1 mM furosemide, and the inhibitory potency of furosemide was enhanced by extracellular K+, as is known to be true for human AA and low-K+ sheep red blood cells. The swelling-stimulated flux in both rabbit and human SS cells has a pH optimum at approximately 7.4. We conclude that rabbit red blood cells are a good model for swelling-stimulated K+ transport in human SS cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Sodium and chloride transport across the isolated porcine gallbladder
Authors: O'Grady S. M. , Wolters P. J.
Published: December 2017

Porcine gallbladder, stripped of serosal muscle, mounted in Ussing chambers, and bathed in plasma-like Ringer solution generates a serosal positive transepithelial potential of 4-7 mV and a short-circuit current (Isc) of 50-120 microA/cm2. Substitution of Cl with gluconate or HCO3 with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) results in a 50% decrease in Isc. Treatment with 1 mM amiloride (mucosal side) or 0.1 mM acetazolamide (both sides) causes 25-27% inhibition of the Isc. Mucosal addition of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibits the Isc by 17%. Serosal addition of 0.1 mM bumetanide inhibits the Isc by 28%. Amiloride (1 mM) inhibits the net transepithelial fluxes of Na and Cl by 55 and 41%, respectively. Substitution of Cl with gluconate inhibits the net Na flux by 50%, whereas substitution of HCO3 with HEPES inhibits 85-90% of the net Na flux and changes Cl absorption to net secretion. Based on these results, it is hypothesized that Na and Cl transport across the apical membrane is mediated by two pathways, Na-H/Cl-HCO3 exchange and Na-HCO3 cotransport. Partial recycling of Cl and HCO3 presumably occurs through a Cl conductive pathway and Cl-HCO3 exchange, respectively, in the apical membrane. This results in net Na absorption, which accounts for most of the Isc observed under basal conditions. The effect of bumetanide on the basolateral membrane and the fact that Cl secretion occurs when HCO3 is absent suggests that Cl secretion involves a basolateral NaCl or Na-K-Cl cotransport system arranged in series with a Cl conductive pathway in the apical membrane.
Mechanism of chloride uptake in rabbit corneal epithelium
Authors: Bonanno J. A. , Klyce S. D. , Cragoe E. J.
Published: December 2017

The mechanism of chloride uptake at the basal membrane (stromal side) of rabbit corneal epithelium was examined by observing the effects of ion transport inhibitors and ion concentrations on the stimulated epithelial short-circuit current (Isc). Loop diuretics inhibited the theophylline-stimulated peak and sustained Isc. Treatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 0.2 mM) and/or 5-(N,N-dimethyl)amiloride (0.1 mM) as well as the potent anion exchange inhibitor, 5c(+)[(2,3,9,9a-tetrahydro-1H-fluoren-7-yl)oxy]acetic acid (0.01 mM), had no significant effect on Isc. These results are consistent with Cl- uptake by a Na+-Cl- or Na+-K+-2Cl- cotransport mechanism rather than Cl(-)-HCO3(-)-OH- exchange coupled to Na+-H+ exchange. Incubation in low [Na+] or [Cl-] before stimulation with forskolin (0.1 mM) reduced both peak and sustained Isc, and saturation kinetics were exhibited. Hill coefficients for [Na+] and [Cl-] were 0.99 and 1.04, respectively, for peak Isc and 0.66 and 1.18, respectively, for sustained Isc. Apparent ion affinities for Na+ and Cl- were 13.5 and 18 mM, respectively, for peak Isc and 15 and 22 mM, respectively, for sustained Isc. These results favor Cl- uptake by a 1 Na+:1 Cl- cotransport mechanism for the rabbit corneal epithelium, but involvement of K+ in this process has not been eliminated.