bluuebunny/crossref_metadata_2025 huggingface dataset:
Preparation of individual human diploid fibroblasts and study of ion transport
Authors: Abraham E. H. , Breslow J. L. , Epstein J. , Chang-Sing P. , Lechene C.
Published: December 2017

A method for analyzing individual mammalian cells with electron probe microanalysis has been developed using human diploid fibroblasts. Cells were grown on the same support that is used for experimental manipulations and analysis. Steady-state cation and anion concentrations and kinetic processes during experimental perturbations could be measured on populations of less than 1,000 cells. Human diploid fibroblasts in normal tissue culture medium had the following intracellular concentrations (in mM): K, 168; Na, 25.0; Cl, 51.2; P, 84.1; S, 16.5; Ca, 6.04; and Mg, 10.0. The ratios of K to Na were equivalent when measured in the nuclear or cytoplasmic area of the cells. Serum in the incubation medium was found to increase the cellular effective permeability to Na by a factor of 2.5, while leaving the effective permeability to K unchanged. When returned to control medium after 7 h of incubation in K-free medium, the cells recovered normal K/Na in less than 1 h. In some experiments the coupling ratio of the ouabain-inhibitable cellular transport of Na to K was 3:2 and the ratio of Cl to K was 1:2. The sum of intracellular content (Na + K) (an estimate of cellular volume) did not change when the cells were placed in K-free medium and increased by less than 30% after ouabain treatment. After 5-7 h of ouabain treatment or of incubation in K-free medium, long after the intracellular K had been replaced by Na, the cellular chloride content had not changed significantly.
"On" and "off" responses of guinea pig ureter
Authors: Hong K. W. , Biancani P. , Weiss R. M.
Published: December 2017

The in vitro guinea pig ureter responds to 5-s trains of electrical stimuli with two contractions: the first, an “on” response, occurs within 0.1-0.3 s after the onset of the stimulus train; the second, an “off” response, occurs 0.2-1.0 s after the termination of the stimulus train. Force decreases between the two responses during a time when the stimulus is still being delivered. Longer duration and/or higher frequencies of stimuli within the train are required to elicit the off response than the on response. Neither the on nor the off response appears to be neurally mediated, since both responses are unchanged by tetrodotoxin, phentolamine, atropine, and pyrilamine. Decreasing temperature from 37 to 22 degrees C decreases the amplitude of the on response and increases the amplitude of the off response. Calcium-free solution, 2 mM ethylenediaminetetraacetic acid (EDTA), 1 mM Mn2+, and 1 microM verapamil abolish the on response at a time at which the off response continues to persist. Conversely, 0.5 mM caffeine and 0.1 mM theophylline abolish the off response, whereas they only slightly reduce the on response. These data suggest that the on response depends on extracellular free calcium, whereas the off response is more dependent on bound or stored calcium.
Adenosine metabolism in human skin fibroblasts
Authors: Holland M. J. , Murphy E. , Kelleher J. K.
Published: December 2017

When normal fibroblasts were incubated in media containing various initial concentrations of [8-14C]adenosine, ranging from 0.25 to 400 microM, under conditions where product formation was linear, greater than 90% of the intracellular label was found in adenine nucleotides, largely in the form of ATP, less than 1% of the intracellular label appeared in the nucleic acids, the remaining intracellular label was found in adenosine, inosine, and hypoxanthine, and the media contained two labeled products, inosine and hypoxanthine. Production of labeled inosine and hypoxanthine from adenosine was considerably lower in adenosine deaminase (ADA)-deficient cells than in normal cells and virtually eliminated in normal cells by the presence of 1 microM deoxycoformycin (a potent ADA inhibitor), suggesting that labeled inosine and hypoxanthine production requires ADA activity. Initial rates of deamination (inosine and hypoxanthine formation) and phosphorylation (adenine nucleotide formation) were estimated by examining the metabolic fate of [8-14C]adenosine in hypoxanthine phosphoribosyltransferase-deficient cells, which cannot recycle hypoxanthine. The estimate of the initial rate of phosphorylation exceeded that of deamination only at the lowest adenosine concentration examined (0.25 microM). The ratio of deamination to phosphorylation rose from approximately 1 at 0.41 microM to approximately 15 at 400 microM extracellular adenosine.
Human erythrocyte membrane proteins of zone 4.5 exist as families of related proteins
Authors: Whitfield C. F. , Coleman D. B. , Kay M. M. , Shiffer K. A. , Miller J. , Goodman S. R.
Published: December 2017

An analysis of the polypeptide composition of zone 4.5 of human erythrocyte membranes has been done by immunoautoradiographic and two-dimensional peptide mapping techniques. Results of these studies demonstrated that the Coomassie blue profile was constant, with 14 well-resolved bands present. Zone 4.5 polypeptides existed as at least four families of two or more components with closely related polypeptide backbones. The families could be distinguished on the basis of their extraction characteristics, immunological cross-reactivity, and two-dimensional peptide maps. One family was related to protein 4.1, one family was related to band 3, and two families were independent and not similar to other larger membrane proteins. The data show that all of the visualized bands in zone 4.5 do not have the same protein composition and that several closely related forms of some polypeptides are present.
Na-Ca exchange: stoichiometry and electrogenicity
Authors: Eisner D. A. , Lederer W. J.
Published: December 2017

This review discusses the evidence concerning the stoichiometry of Na-Ca exchange. In particular we consider whether the Na-Ca exchange has been shown to transport more than two Na+ ions per Ca2+ ion and therefore whether it generates an electric current. The first part of this review discusses both direct and indirect evidence concerning the stoichiometry of the exchange and its possible voltage dependence. We find that, although there is some evidence suggesting that more than two Na+ ions may exchange for each Ca2+ ion, most of the available evidence is equivocal and cannot fix the stoichiometry precisely. Furthermore, using a simple and explicit circulating carrier model for the Na-Ca exchange, we show that the effect of membrane potential on the Na-Ca exchange may be considerably more complicated than is generally believed. In particular we find that both electrogenic and electroneutral exchanges will be affected by membrane potential. We therefore conclude that the demonstration of the voltage dependence of the Na-Ca exchange does not necessarily imply that it is electrogenic. Additionally, this analysis shows that, apart from a restricted range near thermodynamic equilibrium, it is impossible to predict either the magnitude or the direction of the effects of membrane potential on the exchange. In the second part of the review we consider whether any known membrane currents may be attributed to Na-Ca exchange. We show, in contrast to previous suggestions, that the Na-Ca exchange can theoretically produce a current that appears to be activated by intracellular Ca and that has a reversal potential. However, the experimental demonstration that a given current is produced by Na-Ca exchange is hampered by the existence of other Ca- and Na-dependent currents. In conclusion, we feel that there is no evidence that allows any particular membrane current to be unambiguously identified with the Na-Ca exchange.
Inositol trisphosphate and diacylglycerol as intracellular second messengers in liver
Authors: Williamson J. R. , Cooper R. H. , Joseph S. K. , Thomas A. P.
Published: December 2017

Receptor occupation by a variety of Ca2+-mobilizing hormones, such as alpha 1-adrenergic agents, vasopressin and angiotensin II, causes a rapid phosphodiesterase-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate in the plasma membrane with the production of the water soluble compound myo-inositol-1,4,5-trisphosphate (IP3) and the lipophilic molecule 1,2-diacylglycerol (DG). This review summarizes the recent evidence obtained in the liver that defines the roles of these products as intracellular messengers of hormone action. Intracellular Ca2+ mobilization is mediated by IP3, which releases Ca2+ from a subpopulation of the endoplasmic reticulum, resulting in a rapid increase of the cytosolic free Ca2+ concentration ( [Ca2+]i). Further effects of receptor occupancy are inhibition of the plasma membrane Ca2+-ATPase, despite net Ca2+ efflux, and an increased permeability of the plasma membrane to extracellular Ca2+. The activation of the phospholipid-dependent protein kinase C by DG does not alter Ca2+ fluxes across the plasma membrane. In contrast to some secretory cells, a synergism between protein kinase C activation and increased [Ca2+]i is not observed in liver. Activation of protein kinase C profoundly inhibits the response to alpha 1-adrenergic agonists, with only minimal effects on the vasopressin response. It is concluded that in liver the two inositol-lipid messenger systems, IP3 and DG, exert their effects by essentially separate pathways.
Potassium movements in denervated frog sartorius muscle
Authors: Venosa Roque A. , Kotsias Basilio A.
Published: December 2017

The movement of 42K+ across the sarcolemma and the resting membrane potential ( VM) of normal and denervated frog sartorius muscle were studied under several experimental conditions in preparations initially equilibrated in 100 mM K+ and 219 mM Cl-. The results can be summarized as follows. 1) In the absence of any driving force on K+, i.e., when the difference between VM and the K+ equilibrium potential ( EK) is zero ( VM - EK = 0), the K+ conductance ( gK) was 368 ΜS·cm-2 in control and 282 ΜS·cm-2 in denervated muscle. 2) The reduced gK of denervated muscles results from the addition of the opposite changes in the conductances of a Rb+-sensitive inward rectifying pathway ( gIR), which decreases, and a Rb+-insensitive linear channel ( gL), which increases. Thus in control muscles gK (368 ΜS·cm-2) equals gIR (359 ΜS·cm-2) plus gL (9 ΜS·cm-2), while in denervated muscles gK (282 ΜS·cm-2) equals gIR (198 ΜS·cm-2) plus gL (84 ΜS·cm-2). 3) Denervation significantly reduces the inward rectifying properties of the resting K+ permeability system. In the presence of outward driving forces on K+ ( VM - EK > 0) of 35-50 mV, the Rb+-sensitive inward rectifier channel appears to close completely in both control and denervated muscles. In the latter, however, the effect was not as well maintained as in the former, suggesting that its closing mechanism might be altered by denervation. 4) No changes were observed during the first 2 wk after denervation.

sarcolemma; resting potential; K+ equilibrium potential; K+ conductance

Submitted on July 25, 1983

Accepted on July 11, 1984
Altered norepinephrine uptake in neuronal cultures from spontaneously hypertensive rat brain
Authors: Sumners C. , Muther T. F. , Raizada M. K.
Published: December 2017

Uptake of [3H]norepinephrine (NE) has been characterized and compared in neuronal cultures prepared from the brains of 1-day-old normotensive (Wistar-Kyoto, WKY) and spontaneously hypertensive (SH) rats. In cultures from both strains total [3H]NE uptake consisted of a sodium-dependent portion and a sodium-independent portion. The sodium-dependent [3H]NE uptake was inhibited by NE uptake blockers such as maprotiline or desmethylimipramine (both at 0.5-100 microM). This sodium-dependent, NE uptake blocker-sensitive portion of the uptake was also stereospecific, preferring the l-isomer of NE. In contrast, the sodium-independent uptake was not sensitive to maprotiline or desmethylimipramine. Autoradiograms of cultures incubated with [3H]NE showed label concentrated in certain, but not all, neurites and in a few neuronal cell bodies. Cultures incubated with label in sodium-free buffer did not show any such localization of grains but instead showed a diffuse pattern. Incubation of neuronal WKY or SH brain cultures with various concentrations of l-[3H]NE and unlabeled l-NE in the presence or absence of sodium enabled the construction of saturation curves for sodium-dependent uptake in each culture type. In WKY cultures, Km and maximal velocity of uptake (Vmax) values of 0.37-0.45 microM and 0.58-0.69 pmol X mg protein-1 X min-1, respectively, were obtained for sodium-dependent uptake. In contrast, the Km and Vmax values for [3H]NE uptake in SH neuronal cultures were 1.4 microM and 1.31 pmol X mg protein-1 X min-1, respectively. Kinetic analyses of the results show that in SH neuronal cultures the [3H]NE uptake sites are of lower affinity but higher capacity compared with those in WKY neuronal cultures.
Primary culture of duck salt gland. I. Morphology of confluent cell layers
Authors: Lowy R. J. , Schreiber J. H. , Dawson D. C. , Ernst S. A.
Published: December 2017

Dissociated avian salt gland secretory cells were maintained in primary culture after plating on hydrated collagen gels. When seeded at 3 X 10(6) cells/cm2, confluent cell sheets formed within 2-3 days, whereas cultures seeded at lower densities formed a complex reticulum of cell aggregates, which remained nonconfluent even after 7 days. Scanning electron microscopy showed that the free surface of 3-day confluent cultures consisted of intermixed convex and flattened cell membranes with prominent junctional boundaries and abundant microvilli. Transmission electron microscopy indicated that these cultures were multilayers of 1-4 cells in thickness. The plasma membranes of the superficial cells were polarized into apical and basolateral regions displaying, respectively, microvilli and interdigitating lateral membrane folds. These membrane domains were separated by shallow occluding junctions, which consisted of both single strands and simple net-like arrays in freeze-fracture images. Underlying epithelial cells retained lateral membrane folds and formed desmosomal contacts with superficial and neighboring cells. These cultures, unlike the intact tissue, allow direct access to the apical and basolateral cell surfaces for electrophysiological analysis of transmural active ion transport.
Agonist-induced isotonic contraction of cultured mesangial cells after multiple passage
Authors: Venkatachalam M. A. , Kreisberg J. I.
Published: December 2017

Contractile cells under conditions of prolonged culture lose their ability to contract in the usual manner (i.e., isotonically). One explanation for this may be that contraction is prevented by tight cell-to-substrate adhesion. Two models in which substrate adhesiveness was expected to be diminished were used to test this hypothesis. In one, cells were seeded onto collagen-coated dishes and used within 40 min of plating. In the other, cells were plated onto dishes coated with poly-2-hydroxyethyl methacrylate (poly-HEMA) and used, depending on thickness of the poly-HEMA substrate, up to periods of 1 wk. Cells plated onto such substrates contracted when challenged with either PGE2 (2 X 10(-6) and 2 X 10(-9) M), arginine vasopressin (AVP, 10(-6)-10(-9) ), or the calcium ionophore A23187 (5 micrograms/ml). Contraction took place within 5-15 min at 37 degrees C. The contraction seen with AVP was due to its pressor action because 1-desamino-8-D-arginine vasopressin (dDAVP), the antidiuretic analogue, did not cause contraction and the anti-pressor analogue [1-(beta-mercapto-beta beta-cyclopentamethylene propionic acid)-4-valine 8-D-arginine]-vasopressin [d(CH2)5-VDAVP] blocked contraction by AVP. The contraction seen with AVP was dependent on extracellular calcium, whereas that observed with prostaglandin E2 (PGE2) was not.(ABSTRACT TRUNCATED AT 250 WORDS)
Very small fat cell populations determined by a modified osmium tetroxide-urea method
Authors: DeMartinis F. D.
Published: December 2017

To facilitate investigations on very small fat cell (VSFC) populations in adipose tissue, an alternate method of preparing fat tissue samples was explored. The osmium tetroxide-8M urea method, modified by addition of a 95% ethanol step in tissue processing, centrifugation between steps, and final resuspension in 55% glycerol in 0.01% Triton-saline, was compared with the collagenase method for determination of VSFC populations in Fischer 344 epididymal and Sprague-Dawley retroperitoneal adipose depots. For each method and in both depots, the average histogram of 300 adipocyte diameters, measured by microscopy, was bimodal with the nadir between 30 and 40 micron diameter. The average histogram of fat cells less than 35 micron in diameter showed a separate population of VSFC existed in each depot. The modified osmium-urea method gave better results and was easier to perform than the collagenase method. It has confirmed our earlier results and raises anew questions concerning a role for the natural existence of a VSFC population in the adipose depot.
Aldosterone-induced proteins: characterization using lectin-affinity chromatography
Authors: Blazer-Yost B. , Cox M.
Published: December 2017

Aldosterone-stimulated Na+ transport in toad urinary bladder is associated with the synthesis of a specific group of proteins whose induction appears to be related to the natriferic effect of the hormone. These aldosterone-induced proteins (AIPs) occur in two slightly different molecular weight classes (around 70 kDa), each class being composed of several proteins with discrete isoelectric points (range, 5.5-6.0). Because glycosylation is a common cause of such electrophoretic polymorphism and microheterogeneity, we examined whether these proteins are glycoproteins. Tunicamycin (a specific inhibitor of N-linked glycosylation) inhibited aldosterone-stimulated Na+ transport and AIP synthesis without affecting overall protein synthesis. The vast majority of epithelial cell proteins did not bind to the mannose-specific lectin, concanavalin A-sepharose. In contrast, both classes of AIPs bound to concanavalin A-sepharose, but the affinities of the higher and lower molecular weight proteins were markedly different: the former were readily eluted with 0.2 M alpha-methyl-D-mannoside alone, whereas the latter could only be eluted with 0.4 M alpha-methyl-D-mannoside in combination with high concentrations of NaCl (2.5-5.0 M). These studies indicate that 1) glycosylation is important in the natriferic response to aldosterone, 2) the AIPs are N-linked mannose-containing glycoproteins, and 3) the electrophoretic polymorphism of the AIPs is due, at least in part, to differences in glycosylation. Furthermore, concanavalin A-affinity chromatography provides a simple means for the partial purification of these putative "effectors" of the cellular action of aldosterone.
Vasopressin stimulates DNA synthesis and ion transport in quiescent epithelial cells
Authors: Reznik V. M. , Shapiro R. J. , Mendoza S. A.
Published: December 2017

The mitogenic effect of vasopressin was studied in subconfluent quiescent renal epithelial cells (MDCK). Vasopressin stimulated DNA synthesis in the presence of low concentrations of serum. Vasopressin increased the entry of Na into the cells and increased ouabain-sensitive 86Rb uptake, a measure of Na-K pump activity. Because the activity of the Na-K pump in MDCK cells is steeply dependent on intracellular Na, it is likely that stimulation of the Na-K pump by vasopressin was mediated by the increase in Na entry into the cells. Thus both serum and vasopressin stimulate Na uptake and Na-K pump activity in quiescent MDCK cells with a subsequent increase in DNA synthesis. It is concluded that growth regulation in epithelial cells may be mediated in part by changes in monovalent ion transport.
The platelet strip. II. Pharmacomechanical coupling in thrombin-activated human platelets
Authors: Salganicoff L. , Sevy R. W.
Published: December 2017

A model of contracted, irreversibly aggregated thrombin-activated human platelets relaxes when treated with ethyleneglycol-bis(beta-aminoethylether-N,N'-tetraacetic acid (EGTA) in the presence of Mg2+. Inhibition of the cyclooxygenase or blockade of the thromboxane A2 receptor decreases the tension partially, but EGTA treatment is needed for full relaxation. After a stable relaxation has been achieved (3-4 h), Ca2+ addition in a cumulative manner does not reinduce contraction. Whether in the absence or presence of external Ca2+, the relaxed preparation contracts when stimulated with ADP, epinephrine, thromboxane A2 or its analogues, or thrombin. At supramaximal doses, each of the agonists activates only a partial amount of the total tension capable of being generated. Addition of an agonist of a different class to the partially contracted preparation further increases its force. The contractile responses are reversible on washout, with kinetics dependent on the class of agonist and time of contact with the preparation. The contraction induced by the prolonged simultaneous stimulation with ADP, arachidonate, and thrombin reverts very slowly on washout of the agonists and for all practical purposes reproduces the initial state of irreversible platelet contraction.
Complete response to vasopressin requires epithelial organization in A6 cells in culture
Authors: Lang M. A. , Muller J. , Preston A. S. , Handler J. S.
Published: December 2017

Arginine vasopressin (AVP) stimulates adenylate cyclase activity in A6 epithelia grown on filters but not in A6 epithelia grown on plastic culture dishes. When A6 cells are subcultured from culture dishes and seeded at high density on filters, stimulation of adenylate cyclase by AVP is not evident for approximately 3 days. Peak stimulation by AVP occurs between 5 and 10 days. The time course for development of responsiveness to AVP corresponds to the development of an ordered epithelium with polarized cells, tight junctions, and a high transepithelial resistance. A similar correlation is seen when mature filter-grown epithelia that respond to AVP are dissociated by chelation of calcium and subcultured as single cells onto another filter. Stimulation of adenylate cyclase is not evident until an epithelium with significant electrical resistance is reformed. Filters seeded at 1.6 X 10(6) cells/cm2 require 1-2 days to show AVP sensitivity and electrical resistance; filters seeded at 0.4 X 10(6) cells/cm2 require 4 days. Experiments to test the function of the subunits of adenylate cyclase with forskolin (catalytic subunit) and with cholera toxin (Ns, guanine nucleotide-binding subunit) indicate that they are functional during the entire time and that it is the receptor for AVP that is not functional until an ordered epithelium has been formed.
Synthetic atrial peptide inhibits intracellular calcium release in smooth muscle
Authors: Meisheri K. D. , Taylor C. J. , Saneii H.
Published: December 2017

The effects of a synthetic atrial peptide (atriopeptin II; AP II) on the agonist-induced intracellular Ca2+ release was examined in the isolated rabbit aorta. The agonist-induced phasic contraction in a Ca2+-free physiological salt solution containing 2 mM ethyleneglycol-bis(beta-aminoethyl-ether)-N,N'-tetraacetic acid (EGTA-PSS) was used as an indicator of the intracellular Ca2+ release. The addition of AP II (10(-9)-10(-7) M) for 15 min to the tissue during the EGTA-PSS exposure caused a dose-dependent inhibition of norepinephrine (NE; 10(-6) M)-induced phasic contraction. The half-maximal inhibiting concentration of AP II was 3 X 10(-9) M, with 10(-7) M AP II causing 91% inhibition. This was confirmed by studying the inhibitory effect of AP II (10(-7) M) on NE-stimulated 45Ca efflux. Furthermore, the internal Ca2+ release by histamine (10(-5) M) and caffeine (25 mM), both of which share this internal Ca2+ pool with NE, was also inhibited by AP II. Thus AP II appears to be a potent inhibitor of the intracellular Ca2+ release that is utilized by various agonists for the activation of vascular smooth muscle. This may be an important mechanism by which AP II produces relaxation of blood vessels.
Passive permeability of human red blood cells to calcium
Authors: McNamara M. K. , Wiley J. S.
Published: December 2017

Ca2+ influx was measured into human erythrocytes in which efflux was blocked by either introduction of an intracellular Ca2+ chelator, introduction of the Ca2+ chelator followed by ATP depletion, or depletion of the Ca2+ pump cofactors ATP and Mg2+. The Ca2+ influx under all three conditions was 14-20 mumol . 1 cells-1 . h-1, which is an order of magnitude higher than the influx previously reported for cells depleted of either ATP or Mg2+ separately. The difference between the two values was explained by the finding of substantial Ca2+ efflux from the Ca2+-loaded ATP-depleted cells, whereas this efflux was insignificant from cells loaded with quin 2 and then ATP depleted. Under these latter conditions Ca2+ influx estimates the unidirectional permeability to this cation. Studies using this technique showed that Ca2+ influx was the same in media of isotonic sodium, potassium, lithium, choline, or magnesium chlorides. Moreover the dependence of Ca2+ influx on external Ca2+ concentration was well described by the sum of saturable and nonsaturable (linear) components.
Electrophysiological characterization of single pregnant rat myometrial cells in short-term primary culture
Authors: Mollard P. , Mironneau J. , Amedee T. , Mironneau C.
Published: December 2017

Smooth muscle cells were isolated from the longitudinal layer of pregnant rat myometrium (18-19 days) and studied either freshly dissociated or during short-term primary culture (until 30 h) using intracellular microelectrode techniques and direct microscopic observation. The isolated myometrial cells excluded trypan blue vital stain and could repetitively contract in response to various stimuli. Electrophysiological studies at 37 degrees C showed normal resting potential (-54.5 +/- 7.5 mV, n = 71). Action potentials with overshoot (+7.8 +/- 4.6 mV, n = 71) could be elicited by intracellular stimulation. Moreover, the membrane potential was largely dependent on the external K+ concentration. The action potential was suppressed in a Ca2+-free solution [with 0.1 mM ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid], and the overshoot amplitude was clearly Ca2+ dependent. The action potential was inhibited by Mn2+ ions (1 mM), Co2+ ions (1 mM), and D 600 (1 microM) but was unaffected by tetrodotoxin (2 microM) and external Na+ removal. Tetraethylammonium chloride (TEA, 10 mM) and 4-aminopyridine (4-AP, 10 mM) increased both overshoot amplitude and duration of the electrical responses. When the cell surface area was measured with light microscopy, the mean specific membrane resistance was 14.8 +/- 4.6 k omega . cm2 (n = 14), and the mean specific membrane capacitance was 2.3 +/- 0.7 microF/cm2 (n = 14). Outward-going rectification was consistently observed in all cells examined. This was either inhibited by TEA and 4-AP (10 mM each) or reduced in the presence of 1 mM Mn2+.(ABSTRACT TRUNCATED AT 250 WORDS)
Calcium-induced transient potassium efflux in human red blood cells
Authors: Adorante J. S. , Macey R. I.
Published: December 2017

Human red blood cells pretreated with low-ionic-strength solutions and resuspended in saline respond biphasically to extracellular Ca. At first, addition of Ca causes a large transient K efflux of as much as 600 mM . liter cell H2O-1 . h-1; this is followed by a decrease in K flux below control levels. The first phase (phase I) resembles the Gardos effect in several respects. It is inhibited by oligomycin, by external K, and by increased exposure time to Ca. Further, the K permeability of phase I is similar to that of the Gardos effect (5 X 10(-8)-9 X 10(-8) cm/s), and the cells hyperpolarize in a low-K medium when Ca2+ is added. However, phase I is not identical to the Gardos phenomenon. For example, La, which prevents the Gardos response, is ineffective on phase I. Moreover, external Ba prevents the development of phase I but not the Gardos response, whereas internal Ba prevents the Gardos response. Attempts to demonstrate a Ca leak or pump failure during phase I have failed; passive Ca movements of both treated and normal cells are similar. The results suggest that low-ionic-strength solution exposes Ca-sensitive sites to the external medium; these sites are maintained when the cells are returned to saline.
Isolation and characterization of plasma membranes from bovine carotid arteries
Authors: Sharma R. V. , Bhalla R. C.
Published: December 2017

A plasma membrane fraction from bovine carotid arteries has been isolated by extraction of a crude microsomal fraction with a low-ionic-strength buffer containing ATP and Ca2+. This step was followed by sucrose-density-gradient centrifugation in the presence of 0.6 M KCl. The plasma membrane vesicles were enriched 60- to 80-fold in Na+-K+-adenosinetriphosphatase, 5'-nucleotidase, and phosphodiesterase I activities. The final yields of these marker enzymes were 12-18% of the total activities in the postnuclear supernatant, and the protein yield was 100-120 micrograms/g wet wt of carotid arteries. Contamination of the plasma membrane fraction by mitochondria and sarcoplasmic reticulum was low as judged by low activities of succinate--cytochrome-c reductase and NADPH--cytochrome-c reductase, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoprecipitation with smooth muscle-specific actin antibodies showed that the plasma membrane fraction was substantially free from myosin and actin contamination. The plasma membrane vesicles accumulated Ca2+ in the presence of ATP, and the accumulation was increased by calmodulin. Ca2+ accumulated in the presence or absence of calmodulin could be released almost completely from the vesicles by the addition of the Ca2+ ionophore A23187 but not by ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid, indicating that Ca2+ uptake in the presence of ATP is intravesicular. The effects of phosphate and oxalate on Ca2+ uptake in the plasma membranes were different from one another. Phosphate increased Ca2+ uptake in a concentration- and time-dependent manner, and the increase in Ca2+ uptake could be observed as early as 1 min. On the other hand, oxalate at concentrations up to 5 mM did not increase Ca2+ uptake significantly during the 30-min incubation. These plasma membranes can prove useful for the study of ion transport across plasma membranes, hormone binding, characterization of calcium channels, and preparation of antibodies against plasma membrane proteins.
Sodium requirement for insulin release: putative role in regulation of intracellular pH
Authors: Biden T. J. , Janjic D. , Wollheim C. B.
Published: December 2017

The effects on insulin release of Na+ removal, alteration of extracellular pH, and inhibition of acid extrusion processes were examined using freshly isolated and 46-h cultured islets of Langerhans. Na+ removal inhibited the secretory response to 16.7 mM glucose specifically in fresh islets but stimulated the low-glucose response in cultured islets. These divergent effects of Na+ depletion were perfectly mimicked by amiloride, an inhibitor of Na+-H+ exchange, and could be overcome in a raised HCO-3, pH 8.0 medium. Simply raising extracellular pH at constant HCO-3 had no effect. Na+ removal also inhibited the secretory responses to other nutrient secretagogues such as D-glyceraldehyde and L-leucine but not to agents like 3-isobutyl-1-methylxanthine or 12-O-tetradecanoylphorbol-13-acetate. The results show that Na+ is not itself essential for the secretory process but rather suggest that it plays a permissive role in the regulation of intracellular pH via Na+-H+ exchange. Such a regulation appears important mainly for nutrient-stimulated insulin release, which is associated with the generation of acidic metabolites.
Alterations in activities of calmodulin and heat-stable calmodulin-binding protein in rat testis
Authors: Koide Y. , Ono T. , Ishinami C. , Yamashita K.
Published: December 2017

Effects of postnatal development, surgical cryptorchidism, and hypophysectomy on the activity of calmodulin and of a 32,000-molecular weight (32) calmodulin-binding protein (Ono et al., J. Biol. Chem. 259: 9011-9016, 1984) were examined in rat testis. In adult rats, approximately 95% of their activities distributed in seminiferous tubule preparation, and most of them were in the 105,000-g supernatant. Both activities were low early in the development, but the calmodulin activity increased between 25 and 45 days of age, and the 32 K calmodulin-binding protein activity increased between 35 and 45 days of age. In contrast, surgical cryptorchidism caused a rapid decline in their activities in the abdominal testis, whereas their activities in a scrotal testis did not decline. Hypophysectomy similarly resulted in a gradual decline in their activities. Replacement of follicle-stimulating hormone or testosterone for 7 days from 30 days after hypophysectomy did not quantitatively restore either the testes weight or the activities of the proteins. The results indicate a primary distribution of both proteins in mature germ cells.
Role of cell replication in regulation of Na-coupled hexose transport in LLC-PK1 epithelial cells
Authors: Moran A. , Handler J. S. , Hagan M.
Published: December 2017

The glucose concentration in growth medium has been shown to regulate the number of sodium-coupled glucose transporters in LLC-PK1 epithelial cells. Epithelia grown in high concentrations of glucose express fewer transporters than epithelia grown in low concentrations of glucose. In the present work, the effect of a dose of ionizing radiation sufficient to block the incorporation of thymidine was examined in order to gauge the importance of cell replication in the hexose transport regulatory process. The low rate of thymidine incorporation in the plateau phase was completely eliminated by ionizing radiation. Under conditions of irradiation that completely blocked thymidine incorporation, down-regulation, namely the loss of alpha-methylglucoside-concentrating capacity, brought about by switching the epithelium from low to high glucose-containing medium, is independent of the irradiation and therefore most likely is also independent of cell replication. In contrast, the up-regulatory phenomenon is strongly impaired by radiation. This impairment may be due to specific radiation impairment of gene expression necessary for the up-regulatory process. It is apparent from the dose-response data that up-regulation is not inhibited by irradiation in a simple manner and is not inhibited at the same radiation dose as cell replication.
Effect of adenosine 3',5'-cyclic monophosphate on volume and cytoskeleton of MDCK cells
Authors: Mills J. W. , Lubin M.
Published: December 2017

We examined the effect of N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP) on the volume and cytoskeleton of confluent cultures of Madin-Darby canine kidney (MDCK) cells. A 90-min exposure to 1 mM DBcAMP resulted in a 20% reduction in volume as measured by [14C]-urea water space. The volume in cells exposed to isobutylmethylxanthine (IBMX, 0.1 mM) was reduced by 24%. In control cultures F-actin, revealed by staining with nitrobenzoxadiazole-phallacidin, was found at the base of the cell as fibers, at the junctional region as a circumferential band, and on the apical cell surface as a mottled fluorescence. A dense pattern of microtubules, revealed by indirect immunofluorescence, was seen throughout the cell. Exposure to DBcAMP for 90 min resulted in a change of F-actin fibers into dense bundles near the periphery of the cell. This effect was even more striking when cells were exposed to IBMX. Cytochalasin B disrupted F-actin and resulted in a volume reduction similar to that in DBcAMP. Neither DBcAMP nor IBMX affected the distribution of microtubules. Moreover, colchicine, which completely disrupted the microtubules, did not change MDCK cell volume. The results suggest that DBcAMP and F-actin play a role in volume control in MDCK cells.
Voltage-dependent aminoglycoside blockade of the sarcoplasmic reticulum K+ channel
Authors: Oosawa Y. , Sokabe M.
Published: December 2017

Single-channel conductance of the K+ channel from sarcoplasmic reticulum (SR) was reduced by aminoglycoside antibiotics such as neomycin and ribostamycin and also by n-hexylamine from either side of the membrane in a dose- and voltage-dependent manner. K+ channels were incorporated into an artificial phospholipid bilayer. This inhibition follows a single-site titration curve. The voltage dependence of the inhibition is explained by assuming that these drugs bind to the open state of a single channel on one site located approximately 40% of the way through the membrane from the cis side (the side to which SR vesicles are added) when drugs are added to the cis side and bind on another site located approximately 40% of the way through the membrane from the trans side (the opposite side to the cis side) when drugs are added to the trans side.
Ba2+ inhibition of VIP- and A23187-stimulated Cl- secretion by T84 cell monolayers
Authors: Mandel K. G. , McRoberts J. A. , Beuerlein G. , Foster E. S. , Dharmsathaphorn K.
Published: December 2017

Addition of either 10(-8) M vasoactive intestinal polypeptide (VIP) or 10(-6) M A23187 to T84 cell monolayers, grown on permeable supports and mounted in Ussing chambers, stimulated net Cl- secretion. The effect of 10(-6) M A23187 on Cl- flux was consistently smaller than that observed with 10(-8) M VIP. In both cases the increase in net Cl- secretion accounted for the entire change in the observed short-circuit current (Isc). Since Cl- enters the cells through a basolaterally localized Na+-K+-Cl(-)-cotransport system (J. Clin. Invest. 75: 462, 1985), the fate of K+, which is cotransported with Cl- during VIP, and A23187-mediated Cl- secretion was explored. Unidirectional and net transepithelial 42K+ flux rates were negligible compared with 36Cl- flux rates (less than 4% of Cl- flux), indicating that little K+ was secreted along with Cl-. K+ recycling across the basolateral membrane was suggested from experiments in which 86Rb+ efflux (as a tracer for K+) was measured across the apical and basolateral membranes of 86Rb+ -preloaded monolayers under voltage-clamped conditions. In the absence of secretagogues, 86Rb+ efflux was 10-fold higher across the basolateral membrane than across the apical membrane. 86Rb+ efflux across the basolateral membrane was accelerated two- to threefold by addition of either VIP or A23187. In each case accelerated efflux was inhibited by 5 mM Ba2+. Cl- secretion induced by VIP or A23187 was also inhibited by serosal addition of Ba2+.(ABSTRACT TRUNCATED AT 250 WORDS)
Calcium dependence of myosin phosphorylation and airway smooth muscle contraction and relaxation
Authors: Gerthoffer W. T.
Published: December 2017

The time course and the steady-state calcium dependence of myosin phosphorylation and isotonic shortening velocity were studied during contraction and relaxation of canine tracheal smooth muscle. Dephosphorylation of myosin coincided with the decay of isotonic shortening velocity during rapid relaxation following agonist washout. However, the decay of shortening velocity preceded dephosphorylation during a slow relaxation induced by Ca2+-free physiological salt solution (PSS). Carbachol dose-response curves for isometric stress development and myosin phosphorylation were superimposable but shifted to the left of the shortening velocity dose-response. The steady-state Ca2+ dependence of myosin phosphorylation was defined using carbachol and K+ as agonists. There was a significant dissociation of dephosphorylation and relaxation following a stepwise reduction of extracellular CaCl2 concentration. This result was related to muscarinic activation because the dissociation of relaxation and dephosphorylation was reduced by atropine in muscles stimulated with K+. Myosin phosphorylation was completely dissociated from contraction when muscles were stimulated with carbachol in Ca2+-free PSS and contracted by readmission of CaCl2. Mechanisms in addition to myosin phosphorylation appear to regulate airway muscle tone and shortening velocity, and two possibilities are discussed.
Evidence for separate cellular origins of sodium and acid-base transport in the turtle bladder
Authors: Durham J. H. , Nagel W.
Published: December 2017

Transmembrane electrical parameters of the epithelial cells in short-circuited turtle bladders were measured to determine whether those cells participating in Na reabsorption also participate in electrogenic transepithelial acidification and alkalinization. Amiloride-induced increases in intracellular potential (Vsca), apical fractional resistance (FRa), and concomitant decreases in short-circuit current (Isc) denote the participation of the impaled cells in Na reabsorption. In bladders from postabsorptive turtles, amiloride increased Vsca by -45 mV, increased FRa by 37%, and decreased Isc from 36 to -10 microA/cm2. In bladders from NaHCO3-loaded turtles, amiloride increased Vsca by -21 mV, FRa by 21%, and decreased Isc from 22 to 0 microA/cm2. Neither the subsequent inhibition of the negative acidification current in postabsorptive bladders, nor stimulation of positive alkalinization current in bladders from NaHCO3-loaded turtles was associated with any transmembrane electrical change that could be attributed to changes in those transport processes. It is concluded that the electrogenic luminal acidification and alkalinization processes of the turtle bladder are not produced by, or electrically coupled to, those cells that are involved in Na reabsorption.
Role of monosaccharide transporter in vitamin C uptake by placental membrane vesicles
Authors: Ingermann R. L. , Stankova L. , Bigley R. H.
Published: December 2017

Dehydroascorbic acid (DHA), the reversibly oxidized form of vitamin C, was taken up much more rapidly than L-glucose into membrane vesicles prepared from the maternal face of the human placental syncytiotrophoblast. DHA uptake was sensitive to inhibition by cytochalasin B and was independent of a sodium concentration gradient. At equilibrium, the concentration of DHA in the vesicles did not exceed that of the medium. DHA and the D-glucose analogue, 3-O-methyl-D-glucose (3-O-MG) appeared to compete with one another for the transporter. The 3-O-MG and DHA inhibitory constants were indistinguishable. Vesicles loaded with a high concentration of 3-O-MG and suspended in low 3-O-MG displayed a marked, transitory enhancement of DHA, but not L-glucose uptake. These findings suggest that DHA is taken into the first cellular boundary of the placenta between maternal and fetal circulations by the sodium-independent monosaccharide transporter. In contrast to DHA, L-ascorbic acid, the reversibly reduced form of vitamin C, was taken into these vesicles much more slowly. This uptake was not affected by cytochalasin B nor by a sodium concentration gradient; it appeared to occur by simple diffusion.
Polycations reduce vasopressin-induced water flow by endocytic removal of water channels
Authors: Beauwens R. , te Kronnie G. , Snauwaert J. , in't Veld P. A.
Published: December 2017

Several polycations added to the luminal solution were found to inhibit the vasopressin (ADH)-induced water flow in toad urinary bladder but not the ADH-induced increase in sodium transport or in urea permeability. Ultrastructural studies were conducted to evaluate the uptake of cationized ferritin. It was found that endocytosis of cationized ferritin by luminal cells was strikingly enhanced on exposure to ADH; this increased endocytosis was concomitant with inhibition of transepithelial ADH-induced water flow. Various maneuvers preventing endocytosis were also found to counteract the polycation-induced inhibition of the ADH effect. It is suggested that polycations are endocytosed in vesicles whose walls contain the water channels but not the urea or sodium channels.
Effects of insulin, adenosine, and prostaglandin on alpha-adrenergic-stimulated respiration in brown adipocytes
Authors: Schimmel R. J. , McCarthy L.
Published: December 2017

The present study compared the effects of insulin, 2-chloroadenosine, and prostaglandin E2 as inhibitors of respiration in hamster brown adipocytes stimulated with either isoproterenol or phenylephrine. Addition of 2-chloroadenosine or prostaglandin E2 strongly antagonized isoproterenol-stimulated respiration; phenylephrine-stimulated respiration was also partially inhibited by 2-chloroadenosine and prostaglandin E2, but the extent of inhibition caused by these agents was not as great as when isoproterenol was used. Isoproterenol-stimulated respiration was inhibited by insulin, but phenylephrine-stimulated respiration was insensitive to the inhibitory effect of insulin. When brown adipocytes were pretreated with pertussis toxin, isoproterenol-stimulated respiration was enhanced, but phenylephrine-stimulated respiration was not significantly affected. The inhibitory effects of 2-chloroadenosine and prostaglandin E2 on isoproterenol-stimulated respiration were completely blocked by pertussis toxin, indicating that the mode of action of these inhibitory hormones was secondary to inhibition of adenylate cyclase and resultant inhibition of lipolysis. Prostaglandin E2 inhibition of phenylephrine-stimulated respiration was also abolished by pertussis toxin. In contrast, 2-chloroadenosine inhibition of phenylephrine-stimulated respiration persisted in adipocytes treated with pertussis toxin. These data suggest that phenylephrine stimulates respiration through a mechanism that is not altered by pertussis toxin and further that 2-chloroadenosine inhibition of isoproterenol- or phenylephrine-stimulated respiration can be dissociated.
Muscle ammonia metabolism during isometric contraction in humans
Authors: Katz A. , Sahlin K. , Henriksson J.
Published: December 2017

The changes in ammonia (NH3) and amino acid contents in human skeletal muscle during isometric exercise (2/3 maximal voluntary contraction force) to fatigue have been investigated. Biopsies from musculus quadriceps femoris were obtained at rest, fatigue, and 1 and 4 min recovery. Muscle NH3 (n = 9) increased from 1.3 +/- 0.3 (mean +/- SE) mmol/kg dry muscle (dm) at rest to 3.6 +/- 0.6 at fatigue (P less than 0.01) and remained elevated during recovery, whereas the lactate increase after contraction decreased rapidly during recovery. Total adenine nucleotide (TAN) content decreased from 28.7 +/- 0.5 mmol/kg dm at rest to 25.1 +/- 0.6 at fatigue (P less than 0.001). Muscle glutamine did not change after contraction (P greater than 0.05), whereas glutamate decreased (P less than 0.001), and alanine increased (P less than 0.001). In vivo AMP deaminase activity (measured by the rate of TAN decrease) was positively correlated with the percentage of fast-twitch fibers (r = 0.92; P less than 0.001) and the ATP turnover rate (r = 0.75; P less than 0.001) but was not related to the muscle lactate content (r = 0.27; P greater than 0.05). Phosphocreatine decreased to 6.1 +/- 0.7 mmol/kg dm (range = 1-11) after contraction. It is concluded that during exercise activation of AMP deaminase in vivo occurs when a high ATP turnover rate is coupled with a low phosphocreatine level, muscle pH is of minor importance for direct activation of AMP deaminase in vivo, and increases in NH3 do not have an important influence on glycolysis.