bluuebunny/crossref_metadata_2025 huggingface dataset:
A new method for determination of relative ion permeabilities in isolated cells
Authors: Kimmich G. A. , Randles J. , Restrepo D. , Montrose M.
Published: December 2017

The unidirectional influx of the lipophilic cation tetraphenylphosphonium (TPP+) into isolated epithelial cells is a function of the membrane potential that exists across the cellular plasma membrane. Because of the potential dependence, [14C]TPP+ influx can be used as a qualitative sensor of changes in the membrane potential induced by diffusion of ions after the experimental imposition of transmembrane ion gradients. This report describes a "crossover" procedure in which the influx of [14C]TPP+ during systematic changes in the ionic composition of incubation media is used to identify conditions in which no change in membrane potential occurs. The ion ratio at the crossover provides a measure of the relative permeabilities of the two test ions being compared. By using this approach, the ion permeabilities for intestinal epithelial cells prepared from White Rock chickens can be ranked relative to the permeability of Na+ (PNa), i.e., when PNa is equal to 1.0. The permeability sequence and relative values for ion permeability in this system are tris(hydroxymethyl)aminomethane-gluconate (less than 0.1) less than Li+ (0.3) less than Na+ (1.0) less than Cl- (2.0) less than K+ (6.0) = NO3- (6.0) less than SCN- (18) less than K+ + valinomycin (40). The procedure is general enough in principle to be of broad application to a wide variety of cell or membrane vesicle preparations.
Iodoacetic acid inhibition of calcium-dependent potassium efflux in red blood cells
Authors: Plishker G. A.
Published: December 2017

The metabolic inhibitor, iodoacetic acid (IAA), has commonly been used to increase Ca-dependent K efflux in red blood cells. It is thought that this effect of IAA involves the irreversible inhibition of glyceraldehyde-phosphate dehydrogenase (EC 1.2.1.12), resulting in the energy depletion of the cell. Without energy, active transport stops, and the K loss is enhanced both by increasing cellular Ca and by preventing K reuptake. The present study shows that in addition to this metabolic effect, which increases Ca-dependent K efflux, IAA also inhibits this efflux. This inhibition is irreversible and is not related to the ATP or Ca concentrations of the cells. The carboxymethylation of a specific protein band correlates with IAA inhibition of K efflux.
Chemical changes in rat leg muscle by phosphorus nuclear magnetic resonance
Authors: Kushmerick M. J. , Meyer R. A.
Published: December 2017

Phosphorus nuclear magnetic resonance spectra at 145 MHz were obtained with a surface coil from the gastrocnemius-plantaris muscles of anesthetized rats. Phosphocreatine (PCr) and inorganic phosphate (Pi) contents and intracellular pH were measured before, during, and after periods of contractile activity induced by twitch or tetanic stimulation of the sciatic nerve. Reduced levels of PCr, increased levels of Pi, and intracellular acidification were achieved in a graded fashion with increased rates of twitch stimulation from 2 to 10 Hz without detectable changes in the ATP content. In all cases, the decrease in PCr was matched by a stoichiometric increase in Pi content. The time constant of resynthesis of PCr averaged 30 s, was five times faster than the restoration of intracellular pH to control levels, and was independent of the degree of intracellular acidosis at the beginning of recovery.
Gap junctions and direct intercellular communication between rat uterine smooth muscle cells
Authors: Cole W. C. , Garfield R. E. , Kirkaldy J. S.
Published: December 2017

We have tested the hypothesis that an increase in direct intercellular communication accompanies the development of gap junctions (GJs) between rat uterine smooth muscle cells at parturition. Intercellular communication in these tissues was studied by exposing one portion of small strips of myometrium to 2-[3H]deoxy-D-glucose (2-DG) and determining the longitudinal distribution of tracer after a 5-h period of diffusion. The distribution of 2-DG was greater in parturient compared with ante- and postpartum tissues. Similarly, the apparent diffusion coefficient of 2-DG was almost 10-fold greater in delivering tissues (1.86 X 10(-6) cm2/s) than before (0.199 X 10(-6) cm2/s) or after (0.296 X 10(-6) cm2/s) parturition. Control experiments indicated that the redistribution of 2-DG was dependent on the presence of GJs and was the result of intracellular and direct cell-to-cell diffusion. The appearance of GJs is the myometrium at term facilitates direct intercellular communication between uterine smooth muscle cells during labor. This improved communication may be responsible for synchronizing and coordinating electrical, metabolic, and contractile activity in the uterine wall and, hence, the effective expulsion of fetuses.
Role of myosin phosphorylation in contractility of a platelet aggregate
Authors: Bromberg M. E. , Sevy R. W. , Daniel J. L. , Salganicoff L.
Published: December 2017

The relationship between tension and myosin 20,000-Da light chain phosphorylation in intact nonmuscle cells was investigated using a preparation of thrombin-activated, irreversibly aggregated platelets known as the platelet strip. Steady-state levels of tension generated by the platelet strip were found to be linearly related to the level of myosin phosphorylation. This relationship was observed during dose-dependent relaxation induced by the adenylate cyclase activators prostaglandin (PG) E1 and PGI2, and during contraction induced by ADP, epinephrine, and the prostaglandin endoperoxide analogue U-46619, which did not appreciably alter the basal level of adenosine 3',5'-cyclic monophosphate in the preparation. The fully relaxed platelet strip, in the absence of external Ca2+, was associated with a level of 12% light chain phosphorylation, which increased to 72% on maximal contraction. During both relaxation and contraction, changes in myosin phosphorylation were also found to precede or coincide with tension changes. Furthermore, steady-state contraction induced by ADP was associated with a maintained elevation in the level of myosin phosphorylation. These results support the concept that myosin phosphorylation is an important regulatory mechanism for contractility in platelets.
Effect of growth in low-Na+ medium on transport sites in cultured heart cells
Authors: Kim D. , Smith T. W.
Published: December 2017

Increases in intracellular Na+ concentration ([Na+]i) lead to an increase in the number of Na+ pump sites in the cell membrane. To investigate further the role of [Na+]i in the regulation of Na+-K+-ATPase sites, we studied the effect of reduced [Na+]i on the number of Na+ pump sites and Na+-K+ pump activity using spontaneously beating cultured chick ventricular cells. Cells incubated in medium containing 60, 80, 100, or 140 mM Na+ for 24 or 48 h showed an extracellular [Na+]-dependent alteration in cellular Na+ content. The number of Na+ pump sites identified by [3H]ouabain binding binding declined with decreasing levels of Na+ in the medium in a time-dependent manner over 48 h, with a concomitant increase in cellular Na+ content. Verapamil (1 microM) or tetrodotoxin (1 microM) significantly reduced cellular Na+ content by 30 min of exposure and the number of Na+ pump sites by 48 h of incubation. Na+ pump activity determined from the ouabain-sensitive 42K+ uptake rate was significantly reduced in cells grown in low Na+ for 48 h, as was pump capacity, determined in Na+-loaded cells. These results support the view that [Na+]i exerts a long-term modulating effect on the number of physiologically functional Na+ pump sites.
Inhibition of cycling and noncycling cross bridges in skinned smooth muscle by vanadate
Authors: Nayler R. A. , Sparrow M. P.
Published: December 2017

Vanadate (Vi, 3-300 microM) reversibly inhibited force development elicited by micromolar Ca2+ in membrane-skinned fibers of smooth muscle from taenia coli and trachea of guinea pig. When relaxed fibers were preincubated with Vi, the contraction to Ca2+ was characterized by a peak response followed by a lower steady-state phase. The peak phase depended on the rate of contraction and the [Vi]and was absent after Vi incubation during a previous contraction. These observations were consistent with Vi binding to a site that was exposed during the cross-bridge cycle but absent in the relaxed state. The actin X myosin X ADP intermediate formed at the active site during the cross-bridge cycle is suggested as the site of action of Vi. A weak antagonism between Pi and Vi was demonstrated during contractions activated by myosin thiophosphorylation. High concentrations of Pi (6-12 mM) were needed to produce a small inhibition (10%) of maximal Ca2+-activated tension. Skinned fibers relaxed slowly after Ca2+ removal, and the absence of an active state suggested that tension was maintained by noncycling cross bridges. Both Vi and Pi increased the rate of tension loss by 10-fold, but Vi was 5-10 times more potent than Pi. It is suggested that Vi and Pi both act on the active site but that Pi has a more efficacious action on slowly cycling than rapidly cycling cross bridges.
Oxygen dependence of mitochondrial function in isolated rat cardiac myocytes
Authors: Kennedy F. G. , Jones D. P.
Published: December 2017

The O2 dependence of respiratory functions was studied in suspensions of isolated rat cardiac myocytes. Direct optical spectroscopy of the oxidation of cytochromes and oxygenation of Mb showed that cytochrome alpha 3 oxidation measured at 445-460 nm parallels Mb oxygenation; half-maximal values were at 8.0 and 8.5 microM, respectively. Thus there appears to be a close functional relationship between these components in the cells. The values are very high relative to comparable values for cytochrome alpha 3 oxidation in isolated rat heart mitochondria under state 3 conditions (0.43 microM) and isolated rat heart Mb (2.8 microM). Moreover, the measured values for half-maximal oxidation of cytochromes and oxygenation of Mb in the cells are sensitive to factors that alter the O2 consumption rate of the cells. These results indicate that mitochondrial respiration results in establishment of a gradient of O2 concentration from the suspending medium to the mitochondrial inner membrane. A portion of this gradient is between the suspending medium and the region occupied by Mb, and the remainder is between the region occupied by Mb and the inner mitochondrial membrane. The intracellular O2 gradient is an important factor in determining the O2 dependence of mitochondria in cells and hence may contribute to the O2 dependence of cardiac myocyte function in vivo.
Failure of extracellular digestive enzymes to alter in vitro secretion by rat pancreas
Authors: Saluja A. , Saluja M. , Powers R. E. , Houlihan M. J. , Steer M. L.
Published: December 2017

The basal rate of amylase secretion from rat pancreatic lobules was found to diminish with time. This decline, which is not due to depletion of amylase stores or loss of tissue integrity, has been attributed to the build-up of amylase in the extracellular space [Ho and Rothman, Am. J. Physiol. 245 (Cell Physiol. 14): C21-C27, 1983]. This accumulation of amylase would result in a decrease in the intracellular-extracellular amylase gradient, which, according to the so-called equilibrium hypothesis, is responsible for net amylase secretion under basal conditions. Using rat pancreatic lobules and acini, we have tested the validity of this hypothesis by adding to the incubation medium at the onset of incubation either collected secretory products or purified amylase. Based on the equilibrium hypothesis, one would have predicted that these additions would also reduce the concentration gradient favoring net secretion and would result in an apparent reduction in the initial rate of basal in vitro digestive enzyme secretion. The results obtained from these studies, however, were not in accord with those predictions, since these additions did not diminish the initial rate of basal amylase secretion. Our observations therefore indicate that the decrease in the rate of basal amylase secretion from rat pancreas with time is not due to the loss of a concentration gradient favoring efflux. These results argue against the validity of the equilibrium hypothesis.
Mapping subcellular distribution of Na+-K+-ATPase in rat parotid gland
Authors: Conteas C. N. , McDonough A. A. , Kozlowski T. R. , Hensley C. B. , Wood R. L. , Mircheff A. K.
Published: December 2017

Recent subcellular fractionation studies have raised the possibility that Na+-K+-ATPase might be present in both the apical and the basal-lateral membranes of exocrine gland acinar cells. Analytical fractionation and immunofluorescence microscopy studies of rat parotid glands were performed to confirm this interpretation. The distributions of biochemical markers after analyses based on differential sedimentation, equilibrium density-gradient centrifugation, and partitioning in an aqueous polymer two-phase system defined a total of 15 physically and biochemically distinct membrane populations. Among these populations, it was possible to select one (designated population i) with the characteristics expected of acinar cell basal-lateral plasma membranes. It contained Na+-K+-ATPase enriched 33-fold, and gamma-glutamyl transpeptidase enriched 23-fold with respect to the initial homogenate. A second population (designated population c) had the characteristics expected of acinar cell apical plasma membranes; it contained Na+-K+-ATPase enriched 28-fold, and gamma-glutamyl transpeptidase enriched 53-fold with respect to the initial homogenate. Although the identification of population c remains provisional, immunofluorescence studies verified that Na+-K+-ATPase is present in both the apical and the basal-lateral acinar cell plasma membranes. In view of these results, it is likely that the apical Na+-K+-ATPase would participate in series with basal-lateral sodium- and chloride-entry pathways in driving the secretory electrolyte fluxes.
Role of protein kinase C in inhibition of renin release caused by vasoconstrictors
Authors: Kurtz A. , Pfeilschifter J. , Hutter A. , Buhrle C. , Nobiling R. , Taugner R. , Hackenthal E. , Bauer C.
Published: December 2017

It was the aim of the present study to get insight into some of the intracellular mechanisms by which the vasoconstrictor hormones angiotensin II (ANG II), arginine vasopressin (AVP), and norepinephrine (NE) inhibit renin release from renal juxtaglomerular cells. To this end a primary cell culture from rat renal cortex was established that consisted of 50% juxtaglomerular cells. The cultured juxtaglomerular cells contained prominent renin granules closely resembling those in the intact kidney and responded to a number of stimuli of renin release. By using these cultures, we found that ANG II (10(-7) M), AVP (10(-6) M), and NE (10(-5) M) inhibited renin release and increased the calcium permeability of the plasma membrane of the cultured cells. Both the effects on renin release and on calcium permeability could be diminished or even be abolished by the calcium channel blocker verapamil (Vp) (10(-5) M). ANG II, AVP, and NE led to an increased formation of diacylglycerol (DAG), a well-known stimulator of protein kinase C (PKC). Moreover, a direct stimulation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) (10(-8)-10(-6) M) also inhibited renin release and increased the calcium permeability of the cell membrane. Similar to ANG II, AVP, and NE, the effects of TPA on calcium permeability and renin release could be diminished by Vp. In conclusion, these results point toward a common mechanism by which vasoconstrictors inhibit renin release from renal juxtaglomerular cells: ANG II, AVP, and NE activate a phospholipase C, which generates DAG.(ABSTRACT TRUNCATED AT 250 WORDS)
Calmodulin activation and calcium regulation of parotid gland adenylate cyclase
Authors: Piascik M. T. , Babich M. , Jacobson K. L. , Watson E. L.
Published: December 2017

The effect of Ca2+ on the adenylate cyclase activity associated with membranes prepared from mouse parotid gland has been examined. Ca2+ stimulated then inhibited adenylate cyclase activity, with values for half-maximal stimulation and inhibition of 0.6 and 10 microM, respectively. Maximal activation (1.4-fold) was observed at 2 microM free Ca2+. These membranes contained 1.2 microgram calmodulin/mg protein. Exogenous calmodulin (0.2-1.2 microgram) activated, in a concentration-dependent manner, adenylate cyclase activity, with maximal activation being 2.5-fold at 12 micrograms calmodulin. Preparation of membranes in 2 mM ethyleneglycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacet ic acid (EGTA) resulted not only in a significant decrease in calmodulin levels (0.5 microgram calmodulin/mg protein) but also in a loss of the ability of Ca2+ to stimulate the enzyme. Exogenous calmodulin restored the ability of Ca2+ to stimulate the adenylate cyclase activity associated with EGTA-treated membranes. Trifluoperazine (50 microM) blocked the ability of Ca2+ to activate adenylate cyclase activity in control membranes. The effect of trifluoperazine could be reversed by exogenous calmodulin (0.5 or 5.0 micrograms). These data indicate that calmodulin mediates the activation of parotid gland adenylate cyclase by Ca2+ and that Ca2+, at concentrations which stimulate and inhibit amylase secretion, can activate and inhibit adenylate cyclase activity.
Role of intracellular calcium in cellular volume regulation
Authors: Wong S. M. , Chase H. S.
Published: December 2017

We investigated the role of intracellular calcium in epithelial cell volume regulation using cells isolated from the toad urinary bladder. A suspension of cells was prepared by treatment of the bladder with collagenase followed by ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid. The cells retained their ion-transporting capabilities: ouabain (1 mM) and amiloride (10 microM) inhibited cellular uptake of 86Rb and 22Na, respectively. Using a Coulter counter to measure cellular volume, we found that we could swell cells either by reducing the extracellular osmolality or by adding the permeant solute urea (45 mM) isosmotically. Under both conditions, cells first swelled and then returned to their base-line volume, in spite of the continued presence of the stimulus to swell. Volume regulation was inhibited when cells were swelled at low extracellular [Ca] (100 nM) and was retarded in cells preloaded with the calcium buffer quin 2. Swelling increased the intracellular free calcium concentration ([Ca]i), as measured by quin 2 fluorescence: [Ca]i increased 35 +/- 9 nM (n = 6) after hypotonic swelling and 42 +/- 3 nM (n = 3) after urea swelling. Reducing extracellular [Ca] to less than 100 nM prevented the swelling-induced increase in [Ca]i, suggesting that the source of the increase in [Ca]i was extracellular. This result was confirmed in measurements of cellular uptake of 45Ca: the rate of uptake was significantly higher in swollen cells compared with control (1.1 +/- 0.2 vs. 0.4 +/- 0.1 fmol . cell-1 X 5 min-1). Our experiments provide the first demonstration that cellular swelling increases [Ca]i. This increase is likely to play a critical role in cellular volume regulation.
Na+-K+ regulation in cultured vascular smooth muscle cell of the spontaneously hypertensive rat
Authors: Tamura H. , Hopp L. , Kino M. , Tokushige A. , Searle B. M. , Khalil F. , Aviv A.
Published: December 2017

Na+-K+ passive transport and activity of the Na+ pump were examined in serially passed cultured vascular smooth muscle cells originating from spontaneously hypertensive (SH), Wistar-Kyoto (WKY), and Wistar (W) rats. Measurements included 22Na+ and 86Rb+ (K+ analogue) uptake and washout rate constants as well as intracellular Na+ and K+ levels. The aforementioned variables were studied in cells subjected to either 2 mM Ca2+ or Ca2+-deficient media. In 2 mM Ca2+ medium, SH rat cells demonstrated the highest exchange (uptake and washout) rate constants for Na+ and Rb+ (K+) among cells of the three rat strains. At this extracellular Ca2+ concentration, the Na+ pump activity of SH rat cells was higher than that of WKY rat cells and was not different from that of W rat cells. Incubation in Ca2+-deficient medium resulted in increased magnitudes of Rb+ washout and Na+ uptake rate constants in all cell preparations associated with elevated intracellular Na+ concentrations and augmented activity of the Na+ pump. Under this condition, cells derived from SH rats showed the highest Na+ uptake and Rb+ washout rate constants associated with the highest Na+ pump activity. The increase in intracellular Na+ level in Ca2+-deficient medium was the highest in SH rat cells. These findings show that innate membrane defects and the response of the Na+ pump to these abnormalities can be demonstrated in in vitro-grown vascular smooth muscle cells of the SH rat.
K+, Na+, and Cl- activities in ventricular myocytes isolated from rabbit heart
Authors: Desilets M. , Baumgarten C. M.
Published: December 2017

Intracellular K+, Na+, and Cl- activities (aiK, aiNa and aiCl) were measured in ventricular myocytes enzymatically isolated from adult rabbit heart. The activities in normal Tyrode solution containing 2.5 mM Ca2+ were the following (in mM): aiK = 100.0 +/- 3.5 (n = 9); aiNa = 8.4 +/- 1.5 (n = 6); and aiCl = 17.9 +/- 1.5 (n = 11) (mean +/- SE). Membrane potential was -81.6 +/- 0.7 mV (n = 26). These values were determined after correction for changes of junction and tip potential at the reference electrode, estimated to be 4.9 +/- 0.6 mV (n = 7) for 0.15 M KCl-filled electrodes; and intracellular interference detected by the Cl- ion-selective electrode, 11.2 +/- 0.6 mM (n = 4). Extended-tip shunting was avoided by fabricating Na+ ion-selective microelectrodes from aluminosilicate rather than borosilicate glass. These results show that isolated cardiac cells can maintain normal intracellular ion activities. Diffusion of electrolyte from the reference electrode can rapidly alter the intracellular milieu, however. After 10 min of impalement with 0.15 M KCl-filled microelectrodes (resistance approximately equal to 25 M omega), aiK increased by 8.7 +/- 2.0 mM and aiCl by 10.3 +/- 3.1 mM. In contrast, aiNa did not significantly change during the double impalement.
Effect of altered arterial O2 tensions on muscle metabolism in dog skeletal muscle during fatiguing work
Authors: Hogan M. C. , Welch H. G.
Published: December 2017

These experiments were conducted to determine whether changes in arterial O2 tension are related to changes in muscle metabolism during fatiguing contractions. Arterial and venous circulation to the gastrocnemius muscle (n = 8) was isolated, and the Achilles tendon was attached to a force transducer. Each muscle was electrically stimulated through the sciatic nerve for three 2-min periods of fatiguing contractions separated by 8 min of rest. The arterial O2 tensions were altered for each work period (mean PO2 = 44, 72, 391 Torr). Arterial and venous samples were drawn to measure lactate, O2 and CO2 concentrations (Van Slyke analysis), and [H+]. Muscle biopsies were taken to measure muscle [H+] (homogenate method) and lactate. Fatigue was evaluated as the decline in tension from peak initial tension. At the end of the contraction periods, values were significantly different (P less than 0.05) between the low arterial O2 tension and the high for flow [84 +/- 6 (mean +/- SE) vs. 70 +/- 8 ml X 100 g-1 X min-1], muscle lactate contraction (44 +/- 10 vs. 26 +/- 4 mmol/kg dry wt), and lactate release (122 +/- 12 vs. 57 +/- 14 mumol X 100 g-1 X min-1). O2 uptake and the rate of fatigue were not different among treatments during contractions. Muscle [H+] increased (work [H+] minus rest [H+]) to a significantly greater extent during low arterial O2 tensions compared with high (P less than 0.05). We conclude that alterations in arterial O2 tension during fatiguing contractions induce changes in blood and muscle acid-base status and in muscle metabolism that are independent of O2 uptake.
Evidence for physiological regulation of myometrial gap junction permeability
Authors: Cole W. C. , Garfield R. E.
Published: December 2017

We have investigated whether direct intercellular communication between uterine smooth muscle cells of delivering rats is influenced by intracellular adenosine 3',5'-cyclic monophosphate concentration ([cAMPi]) and agents relevant to the hormonal control of pregnancy and parturition. The rate of diffusion of phosphorylated 2-[3H]deoxy-D-glucose (2-DG) in strips of longitudinal myometrium from rats in labor, indicated by the apparent diffusion coefficient for this molecule, was observed to be significantly reduced in tissues with elevated [cAMPi] (after treatment with dibutyryl cAMP, 8-bromo cAMP, forskolin, and theophylline) in the absence of any change in the area of gap junctions (GJs). Similarly, several agonists that elevate [cAMPi] in this tissue (e.g., isoproterenol, relaxin, carbacyclin, prostaglandin E2) also reduced 2-DG diffusion. These data suggest that the permeability of GJs in uterine smooth muscle may be regulated by [cAMPi] and physiologically relevant agonists. Control of GJ permeability may be important for the physiological regulation of intercellular communication and the extent of synchronous contractile activity in the uterine wall during pregnancy and parturition.
Resolution of apical from basolateral membrane of shark rectal gland
Authors: Dubinsky W. P. , Monti L. B.
Published: December 2017

Membrane fractions were isolated from the rectal gland of Squalus acanthias using differential centrifugation and a sucrose gradient run in the presence of 1 M KBr. Using the basolateral membrane marker Na+-K+-ATPase, we obtained a sixfold purification with the most highly purified fraction from the gradient (sp act = 336 +/- 37 mumol X mg protein-1 X h-1). Electrogenic Br- transport was used as a marker activity of the apical membrane, which enabled the identification and purification of a membrane fraction that is highly resolved from the basolateral membrane. The most active fraction was purified approximately 50-fold compared with the crude homogenate. In this fraction, the specific activity of electrogenic anion transport was 296 +/- 87 nmol X mg protein-1 X min-1, whereas the ATPase was only 17.6 +/- 5.7 mumol X mg protein-1 X h-1, representing about a 4-5% contamination of the apical fraction with the basolateral membrane.
Forskolin, a tool for rat parotid secretion studies: 45Ca efflux is not related to cAMP
Authors: Dreux C. , Imhoff V. , Huleux C. , Busson S. , Rossignol B.
Published: December 2017

In the present work, we investigated, by use of forskolin, whether adenosine 3',5'-cyclic monophosphate (cAMP) level and Ca movements were modulated sequentially or in parallel by the activation of the beta-adrenergic receptor in the rat parotid gland. Forskolin-induced [3H]protein secretion was dependent on external Ca, whereas isoproterenol-induced secretion was not. This effect was not due to a requirement of adenylate cyclase for Ca, since the cAMP level increase induced by forskolin was not Ca dependent. Furthermore isoproterenol induced 45Ca efflux, whereas forskolin did not. 45Ca efflux was correlated neither to cAMP nor to secretion, since when there was a massive augmentation of cAMP there was no change in 45Ca efflux, and forskolin, which induced much secretion, was unable to induce Ca efflux. Carbachol potentiated the secretion induced by forskolin in the absence of Ca, whereas it did not potentiate the isoproterenol-induced response. From these results we suggest that beta-adrenergic receptor activation would lead to two parallel events, cAMP accumulation and Ca movements, which together would lead to maximal secretion.
D-glucose transport and concentration in tumoral insulin-producing cells
Authors: Giroix M. H. , Sener A. , Malaisse W. J.
Published: December 2017

The uptake of D-[U-14C]glucose and D-[5-3H]glucose by tumoral insulin-producing cells of the RINm5F line was measured over 3-30 min of incubation at 7-37 degrees C. The apparent distribution space of the hexose ranged from values as low as the L-[1-14C]glucose space to values 10 times higher than the 3H2O space. Although a major fraction of the radioactivity recovered in the cellular pellet corresponded to the metabolites of D-glucose, the results suggested that the transport of D-glucose into the tumoral cells represents a saturable and temperature-dependent process. When D-glucose was measured by an enzymic procedure in cells incubated for 30 min at 37 degrees C in the presence of 50.0 mM D-glucose, the apparent distribution space of the hexose remained lower than the intracellular water space. These results indicate that the RINm5F cells have lost an essential attribute of the glucose-sensing device in normal insulin-producing cells, namely the ability to ensure the equilibration of D-glucose concentrations across the plasma membrane.
Calcium binding to cardiac sarcolemmal vesicles: potential role as a modifier of contraction
Authors: Bers D. M. , Allen L. A. , Kim Y.
Published: December 2017

Passive Ca binding to cardiac sarcolemmal vesicles isolated from rabbit ventricles was measured under ionic conditions similar to intracellular and extracellular media. The first of two main goals was to evaluate whether certain agents induce changes in Ca binding at the external sarcolemmal surface that might contribute to the overall effect of these agents on cardiac muscle contraction. The agents studied were ouabain, verapamil, nifedipine, Bay K 8644, caffeine, ryanodine, and milrinone over a broad range of concentrations, including concentrations at which these agents exert strong effects on cardiac contractile performance. None of these agents produced significant alterations in Ca binding, such that it is unlikely that any part of their actions can be attributed to changes in Ca binding to the external sarcolemmal surface. In contrast, when [Na] is reduced from 140 mM, sarcolemmal Ca binding increases or decreases depending on what replacement is used to avoid changes of osmolarity. Thus the possible effect of Na reduction on surface Ca must be considered in physiological experiments where extracellular [Na] is changed. The second main goal was to evaluate the effects of membrane potential, Na and Mg on Ca bound to the inner surface of the sarcolemma under ionic conditions similar to those expected intracellularly (e.g., [Ca] = 0.3-5.0 microM). Ca binding was inhibited by physiological concentrations of Na and Mg and was sensitive to membrane potential such that depolarization of a normally polarized cell would cause Ca to be released from these sarcolemmal sites. From a quantitative standpoint, it is not clear whether the effect of depolarization would be to contribute sarcolemmal Ca to the activation of the myofilaments or merely to limit the ability of the inner sarcolemmal surface to buffer the rise in intracellular [Ca] associated with contraction.
Determinants of Ca2+-dependent stress maintenance in skinned swine carotid media
Authors: Moreland R. S. , Murphy R. A.
Published: December 2017

Ca2+-dependent stress maintenance without proportional myosin light chain (MLC) phosphorylation was demonstrated in the detergent-skinned swine carotid media when [Ca2+] was reduced from high to intermediate concentrations (Chatterjee, M., and R. A. Murphy. Science Wash. DC 221: 464-466, 1983). In this study of the same preparation, we examined the influence of the initial [Ca2+] and MLC phosphorylation levels on the formation of a stress maintaining state (the "latch" state). Fibers were contracted with 0.82, 1.3, 2.1, or 7.2 microM Ca2+ and then exposed to a lower [Ca2+] to determine the magnitude and Ca2+ sensitivity of stress maintenance. MLC phosphorylation levels were measured in all strips. The magnitude of the stress that rapidly developed was dependent on the initial activating [Ca2+]. The Ca2+ sensitivity of stress maintenance appeared to be independent of the initial levels of stress and MLC phosphorylation. However, the magnitude of the maintained stress was dependent on the initial levels of Ca2+. Only two values for half-maximal responses were evinced in all Ca2+-dependent stress curves: 1.4 X 10(-6) M Ca2+ for rapid stress development and 3.1 X 10(-7) M for stress maintenance. Cumulative [Ca2+]-response curves were shown to result in a decreased maximal stress response and an increase in the apparent Km compared with curves determined by responses to single [Ca2+] exposures. This was associated with a time- and stress-related deterioration of the preparation. The latter was not prevented by exogenous calmodulin or leupeptin (an effective inhibitor of an endogenous Ca2+-dependent protease shown to disrupt the cellular cytoskeleton).(ABSTRACT TRUNCATED AT 250 WORDS)
Effects of TPA on short-circuit current across frog skin
Authors: Mauro T. , O'Brien T. G. , Civan M. M.
Published: December 2017

TPA (12-O-tetradecanoylphorbol-13-acetate) is an effective tumor promoter that affects a variety of ion transport processes. To examine the relationship between effects on transport and growth and differentiation, we have been studying the actions of TPA on frog skin, a particularly well-characterized epithelium. We have reported that high concentrations of TPA stimulate base-line short-circuit current (ISC) and inhibit the subsequent natriferic action of vasopressin. The current study of 89 preparations extends those findings. The Km of the stimulatory effect of TPA is approximately 3 nM; this high affinity indicates that the transport phenomenon does not simply reflect a nonspecific interaction of phorbol ester with the plasma membranes. TPA acts largely or entirely at the mucosal surface of both split and whole skins; thus the sidedness of the effect does not arise from adsorption onto the underlying connective tissue when TPA is applied to the serosal surface of whole skin. Amiloride, an inhibitor of apical Na+ entry, abolishes ISC across frog skins pretreated with TPA. The phorbol ester also increases ISC across split skins, preparations which do not produce net Cl-transport. Indomethacin (1 microM) blocks PGE1 release, but does not alter the response to TPA at a fivefold lower concentration than previously used. NDGA (nordihydroguaretic acid, 10 microM), an inhibitor of the lipoxygenase pathway, partially inhibited the responses of ISC to 8 nM TPA. The present results indicate that frog skin is highly responsive to TPA at concentrations known to activate protein kinase C in broken-cell preparations.(ABSTRACT TRUNCATED AT 250 WORDS)
Band 3 is the basolateral anion exchanger of dark epithelial cells of turtle urinary bladder
Authors: Drenckhahn D. , Oelmann M. , Schaaf P. , Wagner M. , Wagner S.
Published: December 2017

The turtle urinary bladder serves as a model for collecting duct functions in the mammalian kidney. The epithelium of both the turtle bladder and the mammalian collecting duct can generate a steep gradient for H+ ions between blood and urine. Secretion of H+ into the urine is coupled to a basolateral efflux of HCO-3 that appears to be exchanged mainly against Cl-. Here we show that approximately 80% of the dark cells of the bladder contain a 110,000 relative molecular weight (Mr) analogue of the turtle erythrocyte anion exchanger, band 3. The band 3 analogue is confined to the basolateral cell surface and is absent from the apical membrane. A minor population of the dark cells (approximately 20%), which have been previously suggested to represent reverse cells that are involved in HCO-3 secretion rather than absorption, appears not to express a band 3-like anion exchanger, at either the apical or the basolateral membrane. The bladder band 3 protein is colocalized with actin and isoforms of ankyrin (200,000 Mr) and spectrin (230,000 Mr) along the basolateral membrane. Linkage of band 3 via ankyrin to the spectrin-actin lattice may restrict this anion exchanger to the basolateral membrane surface. In view of our previous observation of a band 3-like anion exchanger in the collecting duct epithelium of the rat kidney, these findings point to a common molecular basis for acid-base transport in the mammalian collecting duct and the reptilian urinary bladder.
Norepinephrine metabolism in neuronal cultures is increased by angiotensin II
Authors: Sumners C. , Shalit S. L. , Kalberg C. J. , Raizada M. K.
Published: December 2017

In this study we have examined the actions of angiotensin II (ANG II) on catecholamine metabolism in neuronal brain cell cultures prepared from the hypothalamus and brain stem. Neuronal cultures prepared from the brains of 1-day-old Sprague-Dawley rats exhibit specific neuronal uptake mechanisms for both norepinephrine (NE) and dopamine (DA), and also monoamine oxidase (MAO) and catechol O-methyltransferase (COMT) activity. Separate neuronal uptake sites for NE and DA were identified by using specific neuronal uptake inhibitors for each amine. In previous studies, we determined that ANG II (10 nM-1 microM) stimulates increased neuronal [3H]NE uptake by acting at specific receptors. We have confirmed these results here and in addition have shown that ANG II (1 nM-10 microM, 10-120 min) has no significant effects on neuronal [3H]DA uptake. These results suggest that the actions of ANG II are restricted to the NE transporter in neuronal cultures. It is possible that ANG II stimulates the intraneuronal metabolism of at least part of the NE that is taken up, because the peptide stimulates MAO activity, an effect mediated by specific ANG II receptors. ANG II had no effect on COMT activity in neuronal cultures. Therefore, the use of neuronal cultures of hypothalamus and brain stem we have determined that ANG II can specifically alter NE metabolism in these areas, while apparently not altering DA metabolism.
Time-dependent apical membrane K+ and Na+ selectivity in cultured kidney cells
Authors: Thomas S. R. , Mintz E.
Published: December 2017

Intracellular microelectrodes were used to study apical membrane selectivity to Na+ and K+ of cultured toad kidney cells (A6) grown on permeable supports. Membrane selectivity was tested by responses of apical membrane potential to replacement of Na+ by K+ or tetraethylammonium and by addition of amiloride to perfusion solutions. The A6 epithelia fell into two groups: those with K+-selective apical membranes, lack of amiloride sensitivity, and near-zero transepithelial potential (group I); and those with Na+-selective apical membranes and a serosa-positive, amiloride-sensitive transepithelial potential (Vm----s; group II). The transition from group I to group II behavior appeared definitive and time dependent, occurring approximately 10 days after plating onto filters. Transepithelial measurements under sterile conditions showed that overnight incubation with aldosterone (10(-7) M), after development of tight junctions (transepithelial resistance elevated) but before development of significant Vm----s, induced the switch from group I to group II behavior. Apical addition of Ba2+, a known blocker of K+ channels, unexpectedly reduced transepithelial resistance (Rm----s) in group I and group II A6, suggesting that it not only blocked K+ channels (when they are present) but may also open a parallel conductive pathway. In summary, after approximately 10 days in culture, apical membranes of A6 epithelia undergo a switch from K+ to Na+ selectivity, overnight incubation with aldosterone can trigger this change, and finally, Ba2+ may open a paracellular conductive pathway.
Prevention of alpha 2-adrenergic inhibition on ADH action by pertussis toxin in rabbit CCT
Authors: Ribeiro C. P. , Ribeiro-Neto F. , Field J. B. , Suki W. N.
Published: December 2017

The present studies were performed to investigate the mechanism whereby alpha 2-adrenergic receptor occupancy inhibits the hydrosmotic action of antidiuretic hormone (ADH) in isolated cortical collecting tubules (CCT). The ADH-ribosyltransferase activity of pertussis toxin (PT) was used to promote covalent modification in CCT Ni, the inhibitory regulatory protein of adenylate cyclase, which presumably mediates the alpha 2-adrenergic inhibition of water flow. Tubules preincubated with PT were studied after the addition of ADH and then after the superimposition of clonidine. In these studies, the inhibition of Jv (water absorption, nl X mm-1 X min-1) and Pf (water permeability coefficient, cm/s), by the addition of 10(-4) M clonidine to the bath, was attenuated by PT in a concentration-dependent manner. Reversal of the inhibitory action of clonidine was accomplished with a concentration of 1.0 micrograms/ml PT. To further elucidate the molecular basis of Ni-mediated transduction of the alpha 2-adrenergic signal, ADP-ribosylation studies were undertaken in membrane preparations of dissected CCT segments. PT ADP ribosylated a 40,000 Mr peptide which was proportional to the amount of membrane protein added. Furthermore, pretreatment of CCT during dissection with 0.5 micrograms/ml PT dramatically decreased the susceptibility of the subunit of Ni (alpha i) to be subsequently ADP ribosylated by PT, when compared with CCT preparations not previously treated with PT. Cholera toxin ADP ribosylated a 42,000 Mr peptide from CCT membranes and PT pretreatment did not interfere with the reaction. We conclude that CCT segments have both the pertussis and cholera toxin substrates and the effect of clonidine to attenuate ADH action is mediated through Ni.
Potentiation of contraction in bullfrog ventricle strips by manganese and nickel
Authors: Kavaler F. , Brommundt G.
Published: December 2017

Sodium-lack contractures by strips of bullfrog ventricle were found to be increased in the presence of manganous ion (1–10 mM). In addition, peak force development was usually attained earlier in the presence of manganous ion and the rate of relaxation was decreased by nickel (0.7–2.0 mM), although the latter cation did not potentiate contractural force. Both manganese and nickel had only depressant effects on potassium-excess contractures, as well as on electrically stimulated twitches. Depressant effects of manganese and nickel on sodium-lack contractures were also found. These were smaller, the lower the extracellular sodium concentration and the higher the extracellular calcium concentration. When these well-known negative inotropic effects of the divalent cations were suppressed in sodium-free, calcium-rich media, their potentiating effects were unmasked, resulting in marked augmentation by these cations of potassium-excess contractures and of twitches, along with slowing of relaxation. Experimental maneuvers that have been reported to bring about entry of manganese into these cardiac cells did not increase the observed positive inotropic effect. It thus seems probable that these ions act on the membrane of the ventricle fiber. Also, in view of earlier evidence that they neither affect myofibrillar function nor induce calcium release from mitochondria, it is provisionally concluded that the mechanism of their potentiating effect on mechanical activation is due to their inhibition of calcium extrusion from the fibers, described in the accompanying paper.
Effect of length and cross-bridge attachment on Ca2+ binding to cardiac troponin C
Authors: Hofmann P. A. , Fuchs F.
Published: December 2017

The sensitivity of skinned cardiac muscle bundles to Ca2+ is a function of sarcomere length. Ca2+ sensitivity is increased as fiber length is extended along the ascending limb of the force-length curve and it has been suggested that this phenomenon makes a major contribution to the steep force-length relationship that exists in living cardiac muscle. To gain greater insight into the mechanism behind the length dependence of Ca2+ sensitivity isotopic measurements of Ca2+ binding to detergent-extracted bovine, ventricular muscle bundles were made under conditions in which troponin C was the only major Ca2+ binding species. Experiments were designed to determine whether 1) Ca2+-troponin C affinity varies in the sarcomere length range corresponding to the ascending limb of the force-length curve, and 2) Ca2+ binding correlates with length per se or with changes in the number of length-dependent cross-bridge attachments. Measurements were made of Ca2+ binding in the rigor and relaxed states. The latter state was produced by suppressing actin-myosin interaction with the phosphate analogue, sodium vanadate. After vanadate treatment it is possible to obtain a complete Ca2+ saturation curve in the presence of physiological MgATP concentrations and at constant sarcomere length. The results show that the binding of Ca2+ to the regulatory site of cardiac troponin C is length dependent but this length dependence is actually a dependence on the number of attached cross bridges.
Gap junctions and synchronization of polarization process during epithelial reorganization
Authors: Rabito C. A. , Jarrell J. A. , Scott J. A.
Published: December 2017

The formation of intercellular communication during epithelial reorganization of LLC-PK1 cells was studied with a new, nondisruptive flow cytometric technique. The assembly of these junctions, as demonstrated by cell-to-cell transfer of the fluorescent dye carboxy dimethyl fluorescein, occurs very early during epithelial reorganization. Close cell-to-cell interaction is required for the assembly to occur. Low temperature, treatment with 10(-3) M ouabain, or treatment with the Ca2+ ionophore A23187 inhibits assembly of these junctions. Removal of Ca2+ from the incubation medium, on the other hand, has no effect. Cycloheximide (10(-6) M) is also without effect, suggesting that protein synthesis is not required during the assembly of the junctions and that either the trypsin used to disperse the cells does not affect the junctional components or the cells have a rather extensive reserve pool of junctional precursors. The concomitant delay in the development of intercellular communication and the reorganization of the epithelial membrane, as previously observed with cells in asynchronous growth [Am. J. Physiol. 251 (Renal Fluid Electrolyte Physiol. 20): F978-F987, 1986], is consistent with the theory that junctional intercellular communication coordinates cellular activity during epithelial reorganization.
Ryanodine modifies conductance and gating behavior of single Ca2+ release channel
Authors: Rousseau E. , Smith J. S. , Meissner G.
Published: December 2017

Ryanodine affects excitation-contraction coupling in skeletal and cardiac muscle by specifically interacting with the sarcoplasmic reticulum (SR) Ca2+ release channel. The effect of the drug at the single channel level was studied by incorporating skeletal and cardiac SR vesicles into planar lipid bilayers. The two channels were activated by micromolar free Ca2+ and millimolar ATP and inhibited by Mg2+ and ruthenium red. Addition of micromolar concentrations of ryanodine decreased about twofold the unit conductance of the Ca2+- and ATP-activated skeletal and cardiac channels. A second effect of ryanodine was to increase the open probability (Po) of the channels in such a way that Po was close to unity under a variety of activating and inactivating conditions. The effects of ryanodine were long lasting in that removal of ryanodine by perfusion did not return the channels into their fully conducting state.