Dibasic phosphorylation sites in the R domain of CFTR have stimulatory and inhibitory effects on channel activation

2004 ◽  
Vol 287 (3) ◽  
pp. C737-C745 ◽  
Author(s):  
Horia Vais ◽  
Rugang Zhang ◽  
William W. Reenstra
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Phosphorylation Sites ◽  
Dependent Manner ◽  
Channel Activity ◽  
Ovary Cells ◽  
Channel Opening ◽  
Excised Patches ◽  
A Site ◽  
R Domain

To better understand the mechanisms by which PKA-dependent phosphorylation regulates CFTR channel activity, we have assayed open probabilities ( Po), mean open time, and mean closed time for a series of CFTR constructs with mutations at PKA phosphorylation sites in the regulatory (R) domain. Forskolin-stimulated channel activity was recorded in cell-attached and inside-out excised patches from transiently transfected Chinese hamster ovary cells. Wild-type CFTR and constructs with a single Ser-to-Ala mutation as well as octa (Ser-to-Ala mutations at 8 sites) and constructs with one or two Ala-to-Ser mutations were studied. In cell-attached patches, Ser-to-Ala mutations at amino acids 700, 795, and 813 decreased Po, whereas Ser-to-Ala mutations at 737 and 768 increased Po. In general, differences in Po were due to differences in mean closed time. For selected constructs with either high or low values of Po, channel activity was measured in excised patches. With 1 mM ATP, Po was similar to that observed in cell-attached patches, but with 10 mM ATP, all constructs tested showed elevated Po values. ATP-dependent increases in Po were due to reductions in mean closed time. These results indicate that R-domain phosphorylation affects ATP binding and not the subsequent steps of hydrolysis and channel opening. A model was developed whereby R-domain phosphorylation, in a site-dependent manner, alters equilibrium between forms of CFTR with low and high affinities for ATP.

scholarly journals Recombinant human GM-CSF induces leukocytosis and activates peripheral blood polymorphonuclear neutrophils in nonhuman primates

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 206-213 ◽  
Author(s):  
P Mayer ◽  
C Lam ◽  
H Obenaus ◽  
E Liehl ◽  
J Besemer
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
White Blood Cells ◽  
Chinese Hamster ◽  
Coli Strain ◽  
Polymorphonuclear Neutrophils ◽  
Dependent Manner ◽  
Ovary Cells ◽  
Gm Csf ◽  
Cell Counts

The in vivo efficacy of glycosylated and nonglycosylated recombinant human granulocyte macrophage colony-stimulating factor (rh GM-CSF) expressed in Chinese hamster ovary cells and Escherichia coli respectively was studied in rhesus monkeys following a daily subcutaneous (SC; three times) or intravenous (IV; over six hours) dose for seven consecutive days. The monkeys responded to the rh GM-CSF with a prompt (within 24 hours) rise in circulating white blood cells (WBCs). Thereafter the total cell counts increased steadily in a dose- dependent manner with repeated dosing to numbers six times over the pretreatment levels. Overall, granulocyte counts increased fivefold, lymphocytes twofold to fourfold, and monocytes threefold to fourfold. Platelets and erythrocytes were unaffected. Within 1 week after the end of treatment the leukocytosis had disappeared. Of the two routes of treatment, SC (three times daily)-administered rh GM-CSF was more effective than the same dose given by a six-hour IV infusion. In addition to inducing leukocytosis, parenterally administered rh GM-CSF primed mature circulating granulocytes for enhanced oxidative metabolism and killing of an E coli strain. These results show that exogenously administered glycosylated or nonglycosylated rh GM-CSF is both an effective stimulator of leukocytosis and a potent activator of the phagocytic function of mature granulocytes in monkeys.

Functional polymorphisms in the α-subunit of the human epithelial Na+ channel increase activity

2006 ◽  
Vol 290 (4) ◽  
pp. F821-F827 ◽  
Author(s):  
Qiusheng Tong ◽  
Anil G. Menon ◽  
James D. Stockand
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Severe Form ◽  
Na Channel ◽  
Channel Activity ◽  
Wild Type ◽  
Α Subunit ◽  
Ovary Cells ◽  
Functional Polymorphisms ◽  
Black Populations

Activity of the epithelial Na+ channel (ENaC) is limiting for Na+ reabsorption at the distal nephron. Gain-of-function mutations in ENaC cause Liddle's syndrome: a severe form of inheritable hypertension. Several polymorphisms in α-hENaC possibly associated with abnormal Na+ handling by the kidney and the salt-sensitive hypertension prevalent in black populations have been reported. The functional effects of α-hENaC polymorphisms on channel activity, however, remain controversial and have not been directly tested in a mammalian background. We ask here whether polymorphisms at positions 334, 618, and 663 in α-hENaC influence channel activity. Activity of wild-type (A334, C618, A663) and polymorphic ENaC expressed in Chinese hamster ovary cells was assessed with patch-clamp electrophysiology. While the A334T polymorphism had little effect on macroscopic ENaC currents, the C618F and A663T polymorphisms significantly increased ENaC activity >3.3- and 1.6-fold, respectively. Similarly, polymorphic ENaC had greater activity compared with wild-type channels in excised patches with activity of C618F and A663T channels increased 3.8- and 2.6-fold, respectively. Unitary channel conductances and reversal potentials were not different for polymorphic and wild-type ENaC. Increases in activity resulted primarily from increases in the apparent number of active (polymorphic) channels in the plasma membrane. Moreover, addition of a reducing agent to the cytosol significantly increased activity of wild-type ENaC equal to that of C618F polymorphic channels but had no effect on these latter channels. These results are consistent with the C618F and A663T polymorphisms leading to elevated ENaC activity with the possibility that they facilitate altered Na+ handling by the kidney.

Stereoselective Interaction of Ketamine with Recombinant [micro sign], [small kappa, Greek], and [small delta, Greek] Opioid Receptors Expressed in Chinese Hamster Ovary Cells 

1999 ◽  
Vol 90 (1) ◽  
pp. 174-182 ◽  
Author(s):  
Kazuyoshi Hirota ◽  
Hirobumi Okawa ◽  
Balraj L. Appadu ◽  
David K. Grandy ◽  
Lakshmi A. Devi ◽  
...  
Keyword(s):  
Opioid Receptors ◽  
Room Temperature ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Dependent Manner ◽  
Ovary Cells ◽  
Racemic Ketamine ◽  
Dose Dependent

Background The authors examined the interaction of ketamine with recombinant mu, kappa, and delta opioid receptors and recombinant orphan opioid receptors expressed in Chinese hamster ovary cells (CHO-mu, CHO-kappa, CHO-delta, and CHO(ORL1), respectively). Methods CHO-mu, CHO-kappa, and CHO-delta membranes were incubated with the opioid receptor radioligand [3H]diprenorphine at room temperature. Ketamine (racemic, R(-) and S(+)) was included at concentrations covering the clinical range. CHO(ORL1) membranes were incubated with [125I]Tyr(14)nociceptin and racemic ketamine at room temperature. The effects of racemic ketamine and selective opioid receptor agonists (mu: [D-Ala2, MePhe4, Gly(ol)5] enkephalin (DAMGO); kappa: spiradoline or delta: [D-pen2, D-pen5] enkephalin (DPDPE)) on forskolin-stimulated cyclic adenosine monophosphate formation also were examined. Data are mean +/- SEM. Results Racemic ketamine increased the radioligand equilibrium dissociation constant for [3H]diprenorphine from 85+/-5 to 273+/-11, 91+/-6 to 154+/-16, and 372+/-15 to 855+/-42 pM in CHO-mu, CHO-kappa, and CHO-delta, respectively. The concentration of radioligand bound at saturation was unaffected. In CHO-mu and CHO-kappa cells, racemic ketamine did not slow the rate of naloxone-induced [3H]diprenorphine dissociation. Ketamine and its isomers also displaced [3H]diprenorphine binding to mu, kappa, and delta receptors in a dose-dependent manner, with pKi values for racemic ketamine of 4.38+/-0.02, 4.55+/-0.04, and 3.57+/-0.02, respectively. S(+)-ketamine was two to three times more potent than R(-)-ketamine at mu and kappa receptors. Racemic ketamine displaced [125I]Tyr(14)nociceptin with an estimated affinity constant of 0.5 mM. Racemic ketamine inhibited the formation of cyclic adenosine monophosphate (naloxone insensitive) in a dose-dependent manner (concentration producing 50% inhibition approximately 2 mM) in all cell lines, including untransfected CHO cells. Ketamine (100 microM) reversed DAMGO (mu) and spiradoline (kappa) inhibition of formation of cyclic adenosine monophosphate. Conclusions Ketamine interacts stereoselectively with recombinant mu and kappa opioid receptors.

scholarly journals A Real-Time, Fluorescence-Based Assay for Measuring µ-Opioid Receptor Modulation of Adenylyl Cyclase Activity in Chinese Hamster Ovary Cells

2013 ◽  
Vol 19 (2) ◽  
pp. 223-231 ◽  
Author(s):  
Alisa Knapman ◽  
Fe Abogadie ◽  
Peter McIntrye ◽  
Mark Connor
Keyword(s):  
Adenylyl Cyclase ◽  
Real Time ◽  
Opioid Receptor ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Dependent Manner ◽  
Simultaneous Application ◽  
Ovary Cells ◽  
Receptor Modulation

Inhibition of adenylyl cyclase (AC) activity is frequently used to measure µ-opioid receptor (MOR) activation. We sought to develop a simple, rapid assay of AC activity in whole cells that could be used to study MOR signaling. Chinese hamster ovary cells expressing human MOR (CHO-MOR cells) were grown in 96-well plates and loaded with membrane potential–sensitive fluorescent dye. CHO-MOR cells were treated with the AC activator forskolin (FSK), with or without simultaneous application of MOR agonists, and the resulting change in fluorescence was measured. CHO-MOR cells hyperpolarized in response to application of FSK ( pEC50, 7.3) or calcitonin ( pEC50, 9.4). A submaximally effective concentration of FSK (300 nM) caused a 52% ± 2% decrease in fluorescence. Simultaneous application of the opioids DAMGO ( pEC50, 7.4; Emax, 56%), morphine ( pEC50, 7.0; Emax, 61%); and buprenorphine ( pEC50, 8.6; Emax, 24%) inhibited the FSK response in a dose-dependent manner while having no effect by themselves. The effects of DAMGO were blocked by pertussis toxin. This assay represents a simple, robust method for real-time observation of AC inhibition by MOR in CHO cells. It represents an appealing alternative to end-point assays that rely on cAMP accumulation and can avoid potential confounds associated with rapid desensitization of MOR signaling.

scholarly journals Genetic instability at the adenine phosphoribosyltransferase locus in mouse L cells.

1982 ◽  
Vol 2 (3) ◽  
pp. 250-257 ◽  
Author(s):  
J A Tischfield ◽  
J J Trill ◽  
Y I Lee ◽  
K Coy ◽  
M W Taylor
Keyword(s):  
Chinese Hamster Ovary ◽  
Hot Spot ◽  
Chinese Hamster Ovary Cells ◽  
Human Fibroblasts ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Adenine Phosphoribosyltransferase ◽  
Ovary Cells ◽  
L Cells ◽  
A Site

Resistance to adenine analogs such as 2,6-diaminopurine occurs at a rate of approximately 10(-3) per cell per generation in mouse L cells. This resistance is associated with a loss of detectable adenine phosphoribosyltransferase activity. Other genetic loci in L cells have the expected mutation frequency (approximately 10(-6)). Transformation of L cell mutants with Chinese hamster ovary cell DNA results in transformants with adenine phosphoribosyltransferase activity characteristic of Chinese hamster ovary cells. No activation of the mouse gene occurs on hybridization with human fibroblasts. That this high frequency event is the result of mutation rather than an epigenetic event is supported by antigenic and reversion studies of the 2,6-diaminopurine-resistant clones. These results are consistent with either a mutational hot-spot, a locus specific mutator gene, or a site of integration of an insertion sequence.

Genetic instability at the adenine phosphoribosyltransferase locus in mouse L cells

1982 ◽  
Vol 2 (3) ◽  
pp. 250-257
Author(s):  
J A Tischfield ◽  
J J Trill ◽  
Y I Lee ◽  
K Coy ◽  
M W Taylor
Keyword(s):  
Chinese Hamster Ovary ◽  
Hot Spot ◽  
Chinese Hamster Ovary Cells ◽  
Human Fibroblasts ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Adenine Phosphoribosyltransferase ◽  
Ovary Cells ◽  
L Cells ◽  
A Site

Resistance to adenine analogs such as 2,6-diaminopurine occurs at a rate of approximately 10(-3) per cell per generation in mouse L cells. This resistance is associated with a loss of detectable adenine phosphoribosyltransferase activity. Other genetic loci in L cells have the expected mutation frequency (approximately 10(-6)). Transformation of L cell mutants with Chinese hamster ovary cell DNA results in transformants with adenine phosphoribosyltransferase activity characteristic of Chinese hamster ovary cells. No activation of the mouse gene occurs on hybridization with human fibroblasts. That this high frequency event is the result of mutation rather than an epigenetic event is supported by antigenic and reversion studies of the 2,6-diaminopurine-resistant clones. These results are consistent with either a mutational hot-spot, a locus specific mutator gene, or a site of integration of an insertion sequence.

scholarly journals Antithrombotic effect of recombinant human thrombomodulin on thrombin- induced thromboembolism in mice

Blood ◽  
1990 ◽  
Vol 75 (7) ◽  
pp. 1396-1399 ◽  
Author(s):  
K Gomi ◽  
M Zushi ◽  
G Honda ◽  
S Kawahara ◽  
O Matsuzaki ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Lethal Effect ◽  
Dependent Manner ◽  
Antithrombotic Effect ◽  
Concentration Dependent Manner ◽  
Ovary Cells ◽  
Recombinant Thrombomodulin

Abstract Antithrombotic effect of recombinant human thrombomodulin in mice, both in vitro and in vivo, was studied. The soluble recombinant human thrombomodulin was expressed in Chinese hamster ovary cells and purified from the conditioned medium by a modification of the conventional method. Recombinant thrombomodulin prolonged thrombin clotting time for mouse plasma in a dose-dependent manner. Thrombin was injected into the lateral tail vein of mice and caused acute thromboembolism. All mice injected with thrombin died of thromboembolism; however, preinjection with recombinant human thrombomodulin neutralized the lethal effect of thrombin in a concentration-dependent manner. Histologic examination showed that fibrin deposits were found in all large and small arteries in the lung from mice injected with thrombin; however, fibrin deposits were not detected in any large arteries from the mouse preinjected with thrombomodulin.

scholarly journals Sodium-dependent inactivation of sodium/calcium exchange in transfected Chinese hamster ovary cells

2008 ◽  
Vol 295 (4) ◽  
pp. C872-C882 ◽  
Author(s):  
Olga Chernysh ◽  
Madalina Condrescu ◽  
John P. Reeves
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cytosolic Ph ◽  
Ovary Cells ◽  
Dependent Inactivation ◽  
Excised Patches ◽  
Calcium Exchange ◽  
High Concentrations

High concentrations of cytosolic Na+ ions induce the time-dependent formation of an inactive state of the Na+/Ca2+ exchanger (NCX), a process known as Na+-dependent inactivation. NCX activity was measured as Ca2+ uptake in fura 2-loaded Chinese hamster ovary (CHO) cells expressing the wild-type (WT) NCX or mutants that are hypersensitive (F223E) or resistant (K229Q) to Na+-dependent inactivation. As expected, 1) Na+-dependent inactivation was promoted by high cytosolic Na+ concentration, 2) the F223E mutant was more susceptible than the WT exchanger to inactivation, whereas the K229Q mutant was resistant, and 3) inactivation was enhanced by cytosolic acidification. However, in contrast to expectations from excised patch studies, 1) the WT exchanger was resistant to Na+-dependent inactivation unless cytosolic pH was reduced, 2) reducing cellular phosphatidylinositol-4,5-bisphosphate levels did not induce Na+-dependent inactivation in the WT exchanger, 3) Na+-dependent inactivation did not increase the half-maximal cytosolic Ca2+ concentration for allosteric Ca2+ activation, 4) Na+-dependent inactivation was not reversed by high cytosolic Ca2+ concentrations, and 5) Na+-dependent inactivation was partially, but transiently, reversed by an increase in extracellular Ca2+ concentration. Thus Na+-dependent inactivation of NCX expressed in CHO cells differs in several respects from the inactivation process measured in excised patches. The refractoriness of the WT exchanger to Na+-dependent inactivation suggests that this type of inactivation is unlikely to be a strong regulator of exchange activity under physiological conditions but would probably act to inhibit NCX-mediated Ca2+ influx during ischemia.

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