NH3 permeation through the apical membrane of MDCK cells is via a lipid pathway

1988 ◽  
Vol 255 (1) ◽  
pp. F135-F141
Author(s):  
K. Golchini ◽  
I. Kurtz
Keyword(s):  
Activation Energy ◽  
Acetic Acid ◽  
Plasma Membrane ◽  
Time Course ◽  
Apical Membrane ◽  
Mdck Cells ◽  
Disulfonic Acid ◽  
Dependent Manner ◽  
Cell Monolayers ◽  
Dose Dependent

The pathway for NH3 permeation across the apical membrane of MDCK cells was determined by measuring the effect of membrane fluidizing agents, protein reactive agents, and temperature on cellular NH3 influx. The rate of NH3 influx was calculated from the time course of increase in intracellular pH (pHi), measured with 2,7-biscarboxyethyl-5(6)-carboxyfluorescein, when MDCK cell monolayers were exposed to NH4Cl. The apical membrane NH3 permeability was 7.13 +/- 0.37 x 10(-3) cm/s (n = 12) at 37 degrees C and 1.23 +/- 0.07 x 10(-3) cm/s (n = 7) at 18 degrees C. In comparison, apical membrane permeability at 37 degrees C to the weak acids, valeric acid and acetic acid, were 1.39 +/- 0.11 x 10(-2) cm/s (n = 4) and 6.93 +/- 0.11 x 10(-3) cm/s (n = 4), respectively. The activation energy for NH3 permeation was 15.0 +/- 1.0 kcal/mol (17.5 degrees C-37.5 degrees C). In the presence of the membrane fluidizing agents, heptanol or chloroform, NH3 permeability increased in a dose-dependent manner. Heptanol (15 mM) significantly decreased the activation energy for NH3 permeation to 4.4 +/- 0.6 kcal/mol, P less than 0.001. The carboxyl reactive agent (1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluensulfonic acid 1 mM), aminoreactive agents (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid 50 microM; picrylsulphonic acid 1 mM), the sulphydryl reactive agent (p-chloromercuriphenylsulfonic acid 1 mM), and the nonspecific membrane protein cleaving agent pronase (1 mg/ml) had no effect on the NH3 influx. The results suggest that NH3 permeates the plasma membrane of MDCK cells via a lipid pathway.

scholarly journals Apical Membrane Targeting of Nedd4 Is Mediated by an Association of Its C2 Domain with Annexin Xiiib

2000 ◽  
Vol 149 (7) ◽  
pp. 1473-1484 ◽  
Author(s):  
Pamela J. Plant ◽  
Frank Lafont ◽  
Sandra Lecat ◽  
Paul Verkade ◽  
Kai Simons ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Apical Membrane ◽  
Mdck Cells ◽  
Immunogold Electron Microscopy ◽  
Apical Plasma Membrane ◽  
Apical Region ◽  
Apical Surface ◽  
Dependent Manner ◽  
Interacting Proteins ◽  
C2 Domain

Nedd4 is a ubiquitin protein ligase (E3) containing a C2 domain, three or four WW domains, and a ubiquitin ligase HECT domain. We have shown previously that the C2 domain of Nedd4 is responsible for its Ca2+-dependent targeting to the plasma membrane, particularly the apical region of epithelial MDCK cells. To investigate this apical preference, we searched for Nedd4-C2 domain-interacting proteins that might be involved in targeting Nedd4 to the apical surface. Using immobilized Nedd4-C2 domain to trap interacting proteins from MDCK cell lysate, we isolated, in the presence of Ca2+, a ∼35–40-kD protein that we identified as annexin XIII using mass spectrometry. Annexin XIII has two known isoforms, a and b, that are apically localized, although XIIIa is also found in the basolateral compartment. In vitro binding and coprecipitation experiments showed that the Nedd4-C2 domain interacts with both annexin XIIIa and b in the presence of Ca2+, and the interaction is direct and optimal at 1 μM Ca2+. Immunofluorescence and immunogold electron microscopy revealed colocalization of Nedd4 and annexin XIIIb in apical carriers and at the apical plasma membrane. Moreover, we show that Nedd4 associates with raft lipid microdomains in a Ca2+-dependent manner, as determined by detergent extraction and floatation assays. These results suggest that the apical membrane localization of Nedd4 is mediated by an association of its C2 domain with the apically targeted annexin XIIIb.

Vitamin D3 upregulates plasma membrane Ca2+-ATPase expression and potentiates apico-basal Ca2+ flux in MDCK cells

2004 ◽  
Vol 286 (2) ◽  
pp. F363-F369 ◽  
Author(s):  
Sertac N. Kip ◽  
Emanuel E. Strehler
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Apical Plasma Membrane ◽  
Dependent Manner ◽  
Functional Studies ◽  
Physiological Relevance ◽  
Ubiquitous System ◽  
Dose Dependent

Plasma membrane Ca2+-ATPases (PMCAs) are a ubiquitous system for the expulsion of Ca2+ from eukaryotic cells. In tight monolayers of polarized Madin-Darby canine kidney (MDCK) cells representing a distal kidney tubule model, PMCAs are responsible for about one-third of the vectorial Ca2+ transport under resting conditions, with the remainder being provided by the Na+/Ca2+ exchanger. Vitamin D3 (VitD) is known to increase PMCA expression and activity in Ca2+-transporting tissues such as the intestine, as well as in osteoblasts and Madin-Darby bovine kidney epithelial cells. We found that VitD upregulated the expression of the PMCAs (mainly PMCA4b) in MDCK cell lysates at the RNA and protein level in a time- and dose-dependent manner. Interestingly, VitD caused a decrease of the PMCAs in the apical plasma membrane fraction and a concomitant increase of the pumps in the basolateral membrane. Functional studies demonstrated that transcellular 45Ca2+ flux from the apical-to-basolateral compartment was significantly enhanced by VitD. These findings demonstrate that VitD is a positive regulator of the PMCAs in MDCK epithelial cells. The correlation of decreased apical/increased basolateral expression of the PMCAs with an increase in transcellular Ca2+ flux from the apical (urine) toward the basolateral (blood) compartment indicates the physiological relevance of VitD function in kidney tubular Ca2+ reabsorption.

Intracerebroventricular injection of prostaglandin E2 increases splenic sympathetic nerve activity in rats

1995 ◽  
Vol 269 (3) ◽  
pp. R662-R668 ◽  
Author(s):  
T. Ando ◽  
T. Ichijo ◽  
T. Katafuchi ◽  
T. Hori
Keyword(s):  
Prostaglandin E2 ◽  
Sympathetic Nerve ◽  
Time Course ◽  
Sympathetic Nerve Activity ◽  
Dependent Manner ◽  
Central Administration ◽  
Nerve Activity ◽  
High Doses ◽  
Alpha Chloralose ◽  
Dose Dependent

The effects of central administration of prostaglandin E2 (PGE2) and its selective agonists on splenic sympathetic nerve activity (SNA) were investigated in urethan- and alpha-chloralose-anesthetized rats. An intra-third-cerebroventricular (13V) injection of PGE2 (0.1-10 nmol/kg) increased splenic SNA in a dose-dependent manner. An I3V injection of an EP1 agonist, 17-phenyl-omega-trinor PGE2 (1-30 nmol/kg), also resulted in a dose-dependent increase in splenic SNA, with a time course similar to that of PGE2-induced responses. In contrast, EP2 agonists, butaprost (10-100 nmol/kg I3V) and 11-deoxy-PGE1 (10-100 nmol/kg I3V), had no effect on splenic SNA. An I3V injection of M & B-28767 (an EP3/EP1 agonist, EP3 >> EP1) increased splenic SNA only at high doses (10-100 nmol/kg). Pretreatment with an EP1 antagonist, SC-19220 (200 and 500 nmol/kg), completely blocked the responses of splenic SNA to PGE2 (0.1 nmol/kg) and M & B-28767 (10 nmol/kg), respectively. These findings indicate that brain PGE2 increases splenic SNA through its action on EP1 receptors.

scholarly journals Sequestration of phosphoinositides by mutated MARCKS effector domain inhibits stimulated Ca2+mobilization and degranulation in mast cells

2011 ◽  
Vol 22 (24) ◽  
pp. 4908-4917 ◽  
Author(s):  
Deepti Gadi ◽  
Alice Wagenknecht-Wiesner ◽  
David Holowka ◽  
Barbara Baird
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Time Course ◽  
Fluorescent Protein ◽  
Immunoglobulin E ◽  
Dependent Manner ◽  
Effector Domain ◽  
Granule Exocytosis ◽  
Kinase C

Protein kinase C β (PKCβ) participates in antigen-stimulated mast cell degranulation mediated by the high-affinity receptor for immunoglobulin E, FcεRI, but the molecular basis is unclear. We investigated the hypothesis that the polybasic effector domain (ED) of the abundant intracellular substrate for protein kinase C known as myristoylated alanine-rich protein kinase C substrate (MARCKS) sequesters phosphoinositides at the inner leaflet of the plasma membrane until MARCKS dissociates after phosphorylation by activated PKC. Real-time fluorescence imaging confirms synchronization between stimulated oscillations of intracellular Ca2+concentrations and oscillatory association of PKCβ–enhanced green fluorescent protein with the plasma membrane. Similarly, MARCKS-ED tagged with monomeric red fluorescent protein undergoes antigen-stimulated oscillatory dissociation and rebinding to the plasma membrane with a time course that is synchronized with reversible plasma membrane association of PKCβ. We find that MARCKS-ED dissociation is prevented by mutation of four serine residues that are potential sites of phosphorylation by PKC. Cells expressing this mutated MARCKS-ED SA4 show delayed onset of antigen-stimulated Ca2+mobilization and substantial inhibition of granule exocytosis. Stimulation of degranulation by thapsigargin, which bypasses inositol 1,4,5-trisphosphate production, is also substantially reduced in the presence of MARCKS-ED SA4, but store-operated Ca2+entry is not inhibited. These results show the capacity of MARCKS-ED to regulate granule exocytosis in a PKC-dependent manner, consistent with regulated sequestration of phosphoinositides that mediate granule fusion at the plasma membrane.

Differential regulation of H+-ATPases in MDCK-C11 cells by aldosterone and vasopressin

2009 ◽  
Vol 87 (9) ◽  
pp. 653-665 ◽  
Author(s):  
Priscilla M.C. Dos Santos ◽  
Fabio P. Freitas ◽  
Jeane Mendes ◽  
Ana Lucia Tararthuch ◽  
Ricardo Fernandez
Keyword(s):  
Atpase Activity ◽  
Mdck Cells ◽  
Collecting Duct ◽  
Colorimetric Method ◽  
Proton Pumps ◽  
Ph Optimum ◽  
Cell Monolayers ◽  
Dose Dependent Fashion ◽  
Specific Antagonist ◽  
Dose Dependent

The objective of the present work was to characterize the biochemical activity of the proton pumps present in the C11 clone of Madin–Darby canine kidney (MDCK) cells, akin to intercalated cells of the collecting duct, as well as to study their regulation by hormones like aldosterone and vasopressin. MDCK-C11 cells from passages 78 to 86 were utilized. The reaction to determine H+-ATPase activity was started by addition of cell homogenates to tubes contained the assay medium. The inorganic phosphate (Pi) released was determined by a colorimetric method modified from that described by Fiske and Subbarow. Changes in intracellular calcium concentration in the cells was determined using the Ca2+-sensing dye fluo-4 AM. Homogenates of MDCK-C11 cells present a bafilomycin-sensitive activity (vacuolar H+-ATPase), and a vanadate-sensitive activity (H+/K+-ATPase). The bafilomycin-sensitive activity showed a pH optimum of 6.12. ATPase activity was also stimulated in a dose-dependent fashion as K+ concentration was increased between 0 and 50 mmol·L–1, with an apparent Km for the release of Pi of 0.13 mmol·L–1 and Vmax of 22.01 nmol·mg–1·min–1. Incubation of cell monolayers with 10−8 mol·L–1 aldosterone for 24 h significantly increased vacuolar H+-ATPase activity, an effect prevented by 10−5 mol·L–1 spironolactone. Vacuolar H+-ATPase activity was also stimulated by 10−11 mol·L–1 vasopressin, an effect prevented by a V1 receptor-specific antagonist. This dose of vasopressin determined a sustained rise of cytosolic ionized calcium. We conclude that (i) homogenates of MDCK-C11 cells present a bafilomycin-sensitive (H+-ATPase) activity and a vanadate-sensitive (H+/K+-ATPase) activity, and (ii) vacuolar H+-ATPase activity is activated by aldosterone through a genomic pathway and by vasopressin through V1 receptors.

scholarly journals Endogenous H2S is required for hypoxic sensing by carotid body glomus cells

2012 ◽  
Vol 303 (9) ◽  
pp. C916-C923 ◽  
Author(s):  
Vladislav V. Makarenko ◽  
Jayasri Nanduri ◽  
Gayatri Raghuraman ◽  
Aaron P. Fox ◽  
Moataz M. Gadalla ◽  
...  
Keyword(s):  
Carotid Body ◽  
Time Course ◽  
Channel Blocker ◽  
Primary Site ◽  
Dependent Manner ◽  
Free Medium ◽  
Adult Rats ◽  
Glomus Cells ◽  
Dose Dependent ◽  
Carotid Body Glomus Cells

H2S generated by the enzyme cystathionine-γ-lyase (CSE) has been implicated in O2 sensing by the carotid body. The objectives of the present study were to determine whether glomus cells, the primary site of hypoxic sensing in the carotid body, generate H2S in an O2-sensitive manner and whether endogenous H2S is required for O2 sensing by glomus cells. Experiments were performed on glomus cells harvested from anesthetized adult rats as well as age and sex-matched CSE+/+ and CSE−/− mice. Physiological levels of hypoxia (Po2 ∼30 mmHg) increased H2S levels in glomus cells, and dl-propargylglycine (PAG), a CSE inhibitor, prevented this response in a dose-dependent manner. Catecholamine (CA) secretion from glomus cells was monitored by carbon-fiber amperometry. Hypoxia increased CA secretion from rat and mouse glomus cells, and this response was markedly attenuated by PAG and in cells from CSE−/− mice. CA secretion evoked by 40 mM KCl, however, was unaffected by PAG or CSE deletion. Exogenous application of a H2S donor (50 μM NaHS) increased cytosolic Ca2+ concentration ([Ca2+]i) in glomus cells, with a time course and magnitude that are similar to that produced by hypoxia. [Ca2+]i responses to NaHS and hypoxia were markedly attenuated in the presence of Ca2+-free medium or cadmium chloride, a pan voltage-gated Ca2+ channel blocker, or nifedipine, an L-type Ca2+ channel inhibitor, suggesting that both hypoxia and H2S share common Ca2+-activating mechanisms. These results demonstrate that H2S generated by CSE is a physiologic mediator of the glomus cell's response to hypoxia.

Effects of pinacidil on coronary Ca2+-myosin phosphorylation in cold potassium cardioplegia model

2000 ◽  
Vol 279 (3) ◽  
pp. H882-H888 ◽  
Author(s):  
Naruto Matsuda ◽  
Kathleen G. Morgan ◽  
Frank W. Sellke
Keyword(s):  
Time Course ◽  
Dependent Manner ◽  
Coronary Smooth Muscle ◽  
Intracellular Ca ◽  
No Signaling ◽  
Synthase Inhibitor ◽  
Mlc Phosphorylation ◽  
Dose Dependent ◽  
Cardioplegic Solutions

The effects of the potassium (K+) channel opener pinacidil (Pin) on the coronary smooth muscle Ca2+-myosin light chain (MLC) phosphorylation pathway under hypothermic K+cardioplegia were determined by use of an in vitro microvessel model. Rat coronary arterioles (100–260 μm in diameter) were subjected to 60 min of simulated hypothermic (20°C) K+cardioplegic solutions (K+= 25 mM). We first characterized the time course of changes in intracellular Ca2+concentration, MLC phosphorylation, and diameter and observed that the K+cardioplegia-related vasoconstriction was associated with an activation of the Ca2+-MLC phosphorylation pathway. Supplementation with Pin effectively suppressed the Ca2+accumulation and MLC phosphorylation in a dose-dependent manner and subsequently maintained a small decrease in vasomotor tone. The ATP-sensitive K+(KATP)-channel blocker glibenclamide, but not the nitric oxide (NO) synthase inhibitor Nω-nitro-l-arginine methyl ester, significantly inhibited the effect of Pin. K+cardioplegia augments the coronary Ca2+-MLC pathway and results in vasoconstriction. Pin effectively prevents the activation of this pathway and maintains adequate vasorelaxation during K+cardioplegia through a KATP-channel mechanism not coupled with the endothelium-derived NO signaling cascade.

scholarly journals Evaluation of Analgesic and Anti-inflammatory Activities of Polyalthia simiarum (Hook. F. &Thomson)

2019 ◽  
Vol 5 (1) ◽  
pp. 18-23
Author(s):  
Selina Kabir ◽  
Ronok Zahan ◽  
Abdullah Mohammad Sarwaruddin Chowdhury ◽  
Choudhury Mahmood Hasan ◽  
Mohammad Abdur Rashid
Keyword(s):  
Acetic Acid ◽  
Analgesic Activity ◽  
Biological Activities ◽  
Dependent Manner ◽  
Paw Edema ◽  
Immersion Method ◽  
Ongoing Research ◽  
Tail Immersion ◽  
Anti Inflammatory ◽  
Dose Dependent

Background: Polyalthia simiarum (Hook. F. &Thomson) exhibits different effects in human body. Objective: As a part of ongoing research on medicinal plants of Bangladesh, the present study is focused to investigate the analgesic and anti-inflammatory activities of stem bark of Polyalthia simiarum (Annonaceae). Methodology: The ethyl acetate (EA) and petroleum ether (PE) extracts were subjected to qualitative chemical investigation for the identification of different phytoconstituents. The analgesic activity was determined for its central and peripheral pharmacological actions using tail immersion method and acetic acid-induced writhing test. The anti-inflammatory activity was evaluated by carrageenan induced paw edema in rats. Analgesic and anti-inflammatory data were evaluated statistically analysed by Dunnett’s-T test. Result: Both extracts at the dose of 50- and 100 mg/kg b.w., produced significant increase in pain threshold in tail immersion method whereas significantly reduced the writhing caused by acetic acid in a dose dependent manner. The EA and PE extracts showed anti-inflammatory activities at 50- and 100 mg/kg body weight. Among all the extracts, the EA extract showed a dose dependent and comparable analgesic activity in all the tested methods and also reduced the paw edema considerably (27.5% and 39.1% inhibition after 4h), in dose dependent manner when compared to carrageenan induced control rat. Conclusion: Therefore, the EA and PE extracts of Polyalthia simiarum were capable to exhibit moderate analgesic and anti-inflammatory activities. This is the first report of analgesic and anti-inflammatory potential of Polyalthia simiarum and can be further investigated to isolate the active compounds responsible for the biological activities. Journal of National Institute of Neurosciences Bangladesh, 2019;5(1): 18-23

Effect of cocaine and methylecgonidine on intracellular Ca2+ and myocardial contraction in cardiac myocytes

1997 ◽  
Vol 273 (2) ◽  
pp. H893-H901 ◽  
Author(s):  
L. Huang ◽  
J. H. Woolf ◽  
Y. Ishiguro ◽  
J. P. Morgan
Keyword(s):  
Structural Damage ◽  
Time Course ◽  
Crack Cocaine ◽  
Pyrolysis Product ◽  
Myocardial Contraction ◽  
Dependent Manner ◽  
Response Curves ◽  
Inotropic Effects ◽  
Cell Shortening ◽  
Dose Dependent

We evaluated the cardiac effects of the principle pyrolysis product of crack cocaine smoking, methylecgonidine (MEG), in comparison with cocaine. Peak cell shortening and intracellular Ca2+, as detected by the Ca2+ indicator indo 1, were recorded in enzymatically isolated ferret myocytes. Both cocaine and MEG decreased peak cell shortening and peak intracellular Ca2+ concentration ([Ca2+]i) in a dose-dependent manner (10(-8)-10(-4) M). MEG shifted the peak [Ca2+]i-to-peak shortening relationship downward and was more potent than cocaine. Atropine (10(-6) M) upwardly shifted the dose-response curves of MEG, cocaine, and carbachol but not of procaine. The negative inotropic effects of MEG were inhibited by methoctramine, a selective M2 receptor blocker but not by M1 (pirenzepine) or M3 (4-diphenylacetoxy-N-methylpiperidine methiodide) blocking agents. In contrast to cocaine, the effects of large doses of MEG were irreversible over the time course of our experiments, raising the possibility of structural damage. We conclude that MEG acts primarily on M2 cholinergic receptors in the heart to produce acute cardiac intoxication and, in contrast to cocaine, may decrease the myofilament Ca2+ responseness and cause structural damage to myocytes by a direct toxic effect.

scholarly journals Microtubule perturbation inhibits intracellular transport of an apical membrane glycoprotein in a substrate-dependent manner in polarized Madin-Darby canine kidney epithelial cells.

1990 ◽  
Vol 1 (12) ◽  
pp. 921-936 ◽  
Author(s):  
M J van Zeijl ◽  
K S Matlin
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Intracellular Transport ◽  
Mdck Cells ◽  
Sodium Dodecyl ◽  
Apical Plasma Membrane ◽  
Dependent Manner ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

The effects of microtubule perturbation on the transport of two different viral glycoproteins were examined in infected Madin-Darby canine kidney (MDCK) cells grown on both permeable and solid substrata. Quantitative biochemical analysis showed that the microtubule-depolymerizing drug nocodazole inhibited arrival of influenza hemagglutinin on the apical plasma membrane in MDCK cells grown on both substrata. In contrast, the microtubule-stabilizing drug taxol inhibited apical appearance of hemagglutinin only when MDCK cells were grown on permeable substrata. On the basis of hemagglutinin mobility on sodium dodecyl sulfate gels and its sensitivity to endo H, it was evident that nocodazole and taxol arrested hemagglutinin at different intracellular sites. Neither drug caused a significant increase in the amount of hemagglutinin detected on the basolateral plasma membrane domain. In addition, neither drug had any noticeable effect on the transport of the vesicular stomatitis virus (VSV)-G protein to the basolateral surface. These results shed light on previous conflicting reports using this model system and support the hypothesis that microtubules play a role in the delivery of membrane glycoproteins to the apical, but not the basolateral, domain of epithelial cells.

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