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1.
Vnitr Lek ; 59(5): 344-51, 2013 May.
Article in Czech | MEDLINE | ID: mdl-23767446

ABSTRACT

INTRODUCTION: Lipoprotein associated phospholipase A2 (Lp PLA2) represent new cardiovascular risk factor and potential treatment target. We aimed to analyze the epidemiological situation of this factor in Czech population. METHODS AND RESULTS: The study population consisted from 1 962 subjects, a random samples of general population (postMONICA study), and from patients with manifest coronary or cerebrovascular disease (Czech samples of EUROASPIRE III survey). Lp PLA2 activity was estimated using commercial kits by diaDexus Inc. in frozen samples. Increased activity (by definition, i.e. > 195 nmol/ min/ ml) was observed in 21.1 % of sample, no apparent difference between subject with and without manifest vascular disease was found. Males showed higher Lp PLA2 activity, than females (179.6 vs 146, resp., p < 0.0001), while no substantial increase with age was observed. Taking Lp PLA2 activity > 195 as dependent variable, following independent variables entered the multiple logistic regression: male gender [with odds ratio 4.26 (3.26- 5.58)], low HDL cholesterol (i.e. < 1.0 mmol/ l in males or < 1.2 mmol/ l in females) [3.49 (2.62- 4.64)], LDLcholesterol > 2.5 mmol/ l [6.95 (4.79- 10.07)] and lipid  lowering treatment [0.59 (0.44- 0.79)]. In subject without manifest vascular disease, 6.3 % of them showed co incidence of markedly increased Lp PLA2 activity with high conventional risk (SCORE > 10 %). Expanding this group by intermediate risk subjects (ie. with Lp PLA2 activity 152- 194 and/ or SCORE 5- 9.9 %) leads to increase of this prevalence to 28.9 % of primary prevention subjects. CONCLUSION: Increased Lp PLA2 activity is in Czech population highly prevalent and with exception of lipid parameters, generally independent from conventional cardiovascular risk. However, up to 29 % of subject in primary prevention amalgamate increased Lp PLA2 activity with high conventional cardiovascular risk.


Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/blood , Cardiovascular Diseases/blood , Aged , Female , Humans , Male , Middle Aged , Risk Factors
2.
Diabetologia ; 51(12): 2325-32, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18825362

ABSTRACT

AIMS/HYPOTHESIS: Reduced bioavailability of nitric oxide (NO) is a hallmark of diabetes mellitus-induced vascular complications. In the present study we investigated whether a pharmacological increase of endothelial NO synthase (eNOS) production can restore the impaired hindlimb flow in a rat model of severe diabetes. METHODS: A model of diabetes mellitus was induced in male Sprague-Dawley rats by a single injection of streptozotozin. Rats were treated chronically with the eNOS transcription enhancer AVE3085 (10 mg [kg body weight](-1) day(-1); p.o.) or vehicle for 48 days and compared with controls. Endothelial function and arterial BP were investigated in vivo using an autoperfused hindlimb model and TIP-catheter measurement, respectively. Protein production of eNOS, total and phosphorylated vasodilator-stimulated phosphoprotein (VASP) were assessed in their quadriceps muscle tissue, whereas cyclic GMP (cGMP) concentrations were assessed in blood plasma. RNA levels of intracellular and vascular cell adhesion molecules (ICAM-1 and VCAM-1) were measured by real-time PCR. RESULTS: Untreated diabetic rats showed significantly reduced quadriceps muscle contents of eNOS (-64%) and phosphorylated VASP (-26%) protein associated with impaired vascular function (maximum vasodilatation: -30%, p < 0.05) and enhanced production of ICAM-1 (+121%) and VCAM-1 (+156%). Chronic treatment with AVE3085 did not alter arterial BP or severe hyperglycaemia, but did lead to significantly increased production of eNOS (+95%), cGMP (+128%) and VASP phosphorylation (+65%) as well as to improved vascular function (+36%) associated with reduced production of ICAM-1 (-36%) and VCAM-1 (-58%). CONCLUSIONS/INTERPRETATION: In a rat model of severe diabetes, pharmacological enhancement of impaired eNOS production and NO-cGMP signalling by AVE3085 restores altered hindlimb blood flow and prevents vascular inflammation.


Subject(s)
Diabetes Complications/enzymology , Diabetes Mellitus, Experimental/enzymology , Hindlimb/enzymology , Nitric Oxide Synthase Type III/metabolism , Vascular Diseases/enzymology , Animals , Cell Adhesion Molecules/metabolism , Cyclic GMP/blood , Diabetes Complications/blood , Diabetes Complications/genetics , Diabetes Mellitus, Experimental/blood , Diabetes Mellitus, Experimental/chemically induced , Diabetes Mellitus, Experimental/genetics , Gene Expression Regulation , Hindlimb/blood supply , Humans , Inflammation/blood , Inflammation/complications , Inflammation/enzymology , Inflammation/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Lipid Peroxidation , Male , Microfilament Proteins/metabolism , Muscles/metabolism , Nitric Oxide Synthase Type II/metabolism , Nitric Oxide Synthase Type III/genetics , Phosphoproteins/metabolism , Rats , Rats, Sprague-Dawley , Streptozocin/pharmacology , Vascular Cell Adhesion Molecule-1/metabolism , Vascular Diseases/blood , Vascular Diseases/complications , Vascular Diseases/genetics
3.
Biol Chem ; 382(10): 1483-90, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11727832

ABSTRACT

Bradykinin released by the endothelium is thought to play an important local role in cardiovascular regulation. However, the molecular identity of endothelial proteases liberating bradykinin from its precursors remained unclear. Using RT-PCR and Southern blotting techniques we detected mRNA for tissue kallikrein (KLK1) in human umbilical vein endothelial cells and in bovine aortic endothelial cells. Protein expression was confirmed by precipitation of KLK1 from lysates of endothelial cells pre-labeled with [35S]-cysteine/methionine. Partial purification of tissue kallikrein from total endothelial cell extracts resulted in a protein triplet of about 50 kDa in Western blots using specific anti-KLK1 antibodies. The immunodetection of tissue kallikrein antigen in the fractions from ion exchange chromatography correlated with the presence of amidolytic tissue kallikrein activity. Stimulation of endothelial cells with angiotensin II (ANG-II), which recently has been shown to activate the vascular kinin system and to cause vasodilation, resulted in the release of bradykinin and kallidin. ANG-II-dependent relaxation of pre-constricted rings from human umbilical veins was abolished in the presence of a specific tissue kallikrein inhibitor. We conclude that endothelial cells de novo express significant amounts of tissue kallikrein, which likely serves in the local generation of vasoactive kinins.


Subject(s)
Endothelium, Vascular/physiology , Tissue Kallikreins/metabolism , Umbilical Veins/physiology , Amino Acid Sequence , Angiotensin II/pharmacology , Animals , Cattle , Cells, Cultured , Humans , Kinins/metabolism , Molecular Sequence Data , Organ Culture Techniques , Umbilical Veins/drug effects , Vasodilation/physiology
4.
FASEB J ; 15(1): 79-89, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11149895

ABSTRACT

Production of nitric oxide (NO) in endothelial cells is regulated by direct interactions of endothelial nitric oxide synthase (eNOS) with effector proteins such as Ca2+-calmodulin, by posttranslational modifications such as phosphorylation via protein kinase B, and by translocation of the enzyme from the plasma membrane caveolae to intracellular compartments. Reversible acylation of eNOS is thought to contribute to the intracellular trafficking of the enzyme; however, protein factor(s) that govern the translocation of the enzyme are still unknown. Here we have used the yeast two-hybrid system and identified a novel 34 kDa protein, termed NOSIP (eNOS interacting protein), which avidly binds to the carboxyl-terminal region of the eNOS oxygenase domain. Coimmunoprecipitation studies demonstrated the specific interaction of eNOS and NOSIP in vitro and in vivo, and complex formation was inhibited by a synthetic peptide of the caveolin-1 scaffolding domain. NO production was significantly reduced in eNOS-expressing CHO cells (CHO-eNOS) that transiently overexpressed NOSIP. Stimulation with the calcium ionophore A23187 induced the reversible translocation of eNOS from the detergent-insoluble to the detergent-soluble fractions of CHO-eNOS, and this translocation was completely prevented by transient coexpression of NOSIP in CHO-eNOS. Immunofluorescence studies revealed a prominent plasma membrane staining for eNOS in CHO-eNOS that was abolished in the presence of NOSIP. Subcellular fractionation studies identified eNOS in the caveolin-rich membrane fractions of CHO-eNOS, and coexpression of NOSIP caused a shift of eNOS to intracellular compartments. We conclude that NOSIP is a novel type of modulator that promotes translocation of eNOS from the plasma membrane to intracellular sites, thereby uncoupling eNOS from plasma membrane caveolae and inhibiting NO synthesis.


Subject(s)
Carrier Proteins/metabolism , Endothelium, Vascular/enzymology , Nitric Oxide Synthase/metabolism , Amino Acid Sequence , Animals , Binding Sites , CHO Cells , Calcimycin/pharmacology , Carrier Proteins/chemistry , Carrier Proteins/genetics , Caveolae/enzymology , Cricetinae , Gene Expression Profiling , Humans , Immunohistochemistry , Molecular Sequence Data , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/chemistry , Nitric Oxide Synthase/genetics , Precipitin Tests , Protein Binding , Protein Transport , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Alignment , Substrate Specificity , Two-Hybrid System Techniques , Ubiquitin-Protein Ligases
5.
Br J Pharmacol ; 128(6): 1316-22, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10578147

ABSTRACT

1 In this study we examined the endothelium-dependent effect of YC-1 - a benzyl indazole derivative which directly activates soluble guanylyl cyclase (sGC) - on vascular relaxation and nitric oxide (NO) and guanosine-3',5'-cyclic monophosphate (cyclic GMP) in endothelial cells. 2 In preconstricted rat aortic rings with intact endothelium, YC-1 produced a concentration-dependent relaxation. However, the concentration response curve was shifted rightward to higher concentrations of YC-1, when (i) the aortas were pre-treated with L-NG-nitroarginine methylester (L-NAME) or (ii) the endothelium was removed. 3 Incubation of bovine aortic endothelial cells (BAEC) with YC-1 produced a concentration-dependent NO synthesis and release as assessed using a porphyrinic microsensor. Pre-incubating cells with L-NAME or with 8-bromo-cyclic GMP decreased this effect indicating that the YC-1 stimulation of NO synthesis is due to an activation of nitric oxide synthase, but not to an elevation of cyclic GMP. No direct effect of YC-1 on recombinant endothelial constitutive NO synthase activity was observed. 4 The YC-1 stimulated NO release was reduced by 90%, when extracellular free calcium was diminished. 5 In human umbilical vein endothelial cells (HUVEC), YC-1 stimulated intracellular cyclic GMP production in a concentration- and time-dependent manner. Stimulation of cyclic GMP was greater with a maximum concentration of YC-1 compared to calcium ionophore A23187. Similar effects were observed in BAEC and rat microvascular coronary endothelial cells (RMCEC). 6 When HUVEC and RMCEC were pre-treated with L-NG-nitroarginine (L-NOARG), the maximum YC-1 stimulated cyclic GMP increase was reduced by >/=50%. 7 These results indicate, that beside being a direct activator of sGC, YC-1 stimulates a NO-synthesis and release in endothelial cells which is independent of elevation of cyclic GMP but strictly dependent on extracellular calcium. The underlying mechanism needs to be determined further.


Subject(s)
Endothelium, Vascular/drug effects , Guanylate Cyclase/metabolism , Indazoles/pharmacology , Nitric Oxide/metabolism , Animals , Aorta, Thoracic/drug effects , Aorta, Thoracic/physiology , Bradykinin/pharmacology , Calcimycin/pharmacology , Cattle , Cells, Cultured , Cyclic GMP/metabolism , Dose-Response Relationship, Drug , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Enzyme Activation/drug effects , Humans , In Vitro Techniques , Ionophores/pharmacology , Male , Muscle Relaxation/drug effects , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/physiology , NG-Nitroarginine Methyl Ester/pharmacology , Nitroarginine/pharmacology , Rats , Rats, Wistar , Solubility , Time Factors
6.
Proc Natl Acad Sci U S A ; 96(23): 13357-62, 1999 Nov 09.
Article in English | MEDLINE | ID: mdl-10557325

ABSTRACT

Hypertension is a side effect of systemically administered glucocorticoids, but the underlying molecular mechanism remains poorly understood. Ingestion of dexamethasone by rats telemetrically instrumented increased blood pressure progressively over 7 days. Plasma concentrations of Na(+) and K(+) and urinary Na(+) and K(+) excretion remained constant, excluding a mineralocorticoid-mediated mechanism. Plasma NO(2)(-)/NO(3)(-) (the oxidation products of NO) decreased to 40%, and the expression of endothelial NO synthase (NOS III) was found down-regulated in the aorta and several other tissues of glucocorticoid-treated rats. The vasodilator response of resistance arterioles was tested by intravital microscopy in the mouse dorsal skinfold chamber model. Dexamethasone treatment significantly attenuated the relaxation to the endothelium-dependent vasodilator acetylcholine, but not to the endothelium-independent vasodilator S-nitroso-N-acetyl-D,L-penicillamine. Incubation of human umbilical vein endothelial cells, EA.hy 926 cells, or bovine aortic endothelial cells with several glucocorticoids reduced NOS III mRNA and protein expression to 60-70% of control, an effect that was prevented by the glucocorticoid receptor antagonist mifepristone. Glucocorticoids decreased NOS III mRNA stability and reduced the activity of the human NOS III promoter (3.5 kilobases) to approximately 70% by decreasing the binding activity of the essential transcription factor GATA. The expressional down-regulation of endothelial NOS III may contribute to the hypertension caused by glucocorticoids.


Subject(s)
Dexamethasone/pharmacology , Down-Regulation , Hypertension/metabolism , Nitric Oxide Synthase/metabolism , Animals , Aorta/drug effects , Aorta/metabolism , Base Sequence , Cells, Cultured , DNA Primers , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Hypertension/chemically induced , Male , Nitrates/blood , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase Type III , Nitrites/blood , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Inbred WKY , Transcription Factors/metabolism , Vasodilation/drug effects
7.
Hypertension ; 34(2): 291-5, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10454456

ABSTRACT

Spontaneously hypertensive rats (SHR) begin to die from cardiovascular complications at approximately 15 months of age. We tested whether chronic ACE-inhibitor treatment would extend the lifespan of such old animals. We also studied cardiac hypertrophy and function, endothelial function and expression, and activity of NO synthase (eNOS). One hundred 15-month-old SHR were randomized into 3 groups, control (n=10), placebo-treated (n=45), and ramipril-treated with an antihypertensive dose of 1 mg. kg(-1). d(-1) in drinking water (n=45). Ex vivo experiments were performed after 15 months (control) and 21 months, when approximately 80% of the placebo group had died. Late treatment with ramipril significantly extended lifespan of the animals from 21 to 30 months. Fully established cardiac hypertrophy, observed in placebo-treated animals and in controls, was significantly reversed by ramipril treatment. In isolated working hearts, a significantly improved function associated with increased cardiac eNOS expression was seen versus placebo and control hearts. Endothelial dysfunction in isolated aortic rings from control and placebo-treated SHR was significantly improved by ACE inhibition and associated with enhanced NO release. Late treatment of SHR with the ACE inhibitor ramipril extended lifespan from 21 to 30 months, which is comparable to the lifespan of untreated normotensive Wistar-Kyoto rats. This lifespan extension, probably due to blood pressure reduction, correlated with increased eNOS expression and activity followed by a regression of left ventricular hypertrophy and cardiac and vascular dysfunction.


Subject(s)
Angiotensin-Converting Enzyme Inhibitors/therapeutic use , Antihypertensive Agents/therapeutic use , Hypertension/drug therapy , Hypertension/mortality , Ramipril/therapeutic use , Age Factors , Animals , Aorta, Thoracic/drug effects , Aorta, Thoracic/metabolism , Blood Pressure/drug effects , Blotting, Western , Body Weight , Cardiomegaly/complications , Data Interpretation, Statistical , Endothelium, Vascular/cytology , Endothelium, Vascular/enzymology , Endothelium, Vascular/metabolism , Heart/drug effects , Heart/physiology , Heart Ventricles/metabolism , Hypertension/complications , In Vitro Techniques , Luminescent Measurements , Male , Myocardium/metabolism , Nitric Oxide/analysis , Nitric Oxide/metabolism , Nitric Oxide Synthase/analysis , Nitric Oxide Synthase/metabolism , Placebos , Random Allocation , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Superoxides/analysis , Time Factors
9.
Mol Pharmacol ; 53(4): 630-7, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9547352

ABSTRACT

In primary human umbilical vein endothelial cells (HUVECs), incubation with phorbol-12-myristate-13-acetate (PMA) enhanced basal and bradykinin-stimulated nitric oxide production. In the HUVEC-derived cell line EA.hy 926, PMA and phorbol-12,13-dibutyrate stimulated endothelial nitric oxide synthase (NOS III) mRNA expression in a concentration- and time-dependent manner. Maximal mRNA expression (3.3-fold increase) was observed after 18 hr. NOS III protein and activity were increased to a similar extent. The specific protein kinase C (PKC) inhibitors bisindolylmaleimide I (1 microM), Gö 6976 [12-(2 cyanoethyl)-6,7,12, 13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo-[3, 4-c]carbazole] (1 microM), Ro-31-8220 [3-[1-[3(amidinothio)propyl-1H-inoyl-3-yl]3-(1-methyl-1H- indoyl-3-yl) maleimide methane sulfonate] (1 microM), and chelerythrine (3 microM) did not change NOS III expression when applied alone, but they all prevented the up-regulation of NOS III mRNA produced by PMA. Of the PKC isoforms expressed in EA.hy 926 cells (alpha, beta I, delta, epsilon, eta, zeta, lambda, and mu), only PKC alpha and PKC epsilon showed changes in protein expression after PMA treatment. Incubation of EA.hy 926 cells with PMA for 2-6 hr resulted in a translocation of PKC alpha and PKC epsilon from the cytosol to the cell membrane, indicating activation of these isoforms. After 24 hr of PMA incubation, both isoforms were down-regulated. The time course of activation and down-regulation of these two PKC isoforms correlated well with the PMA-stimulated increase in NOS III expression. When human endothelial cells (ECV 304 or EA.hy 926) were transiently or stably transfected with a 3.5-kb fragment of the human NOS III promoter driving a luciferase reporter gene, PMA stimulated promoter activity up to 2.5-fold. On the other hand, PMA did not change the stability of the NOS III mRNA. These data indicate that stimulation of PKC alpha, PKC epsilon, or both by active phorbol esters represents an efficacious pathway activating the human NOS III promoter in human endothelium.


Subject(s)
Endothelium, Vascular/enzymology , Gene Expression Regulation , Isoenzymes/metabolism , Nitric Oxide Synthase/genetics , Protein Kinase C/metabolism , Transcription, Genetic , Biological Transport/drug effects , Biological Transport/genetics , Bradykinin/pharmacology , Cells, Cultured , Cyclic GMP/biosynthesis , Down-Regulation/drug effects , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Enzyme Activation/drug effects , Enzyme Activation/genetics , Enzyme Inhibitors/pharmacology , Enzyme Stability/drug effects , Enzyme Stability/genetics , Gene Expression Regulation/drug effects , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/biosynthesis , Isoenzymes/genetics , Nitric Oxide Synthase/biosynthesis , Promoter Regions, Genetic/drug effects , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/biosynthesis , Protein Kinase C-alpha , Protein Kinase C-epsilon , RNA, Messenger/biosynthesis , RNA, Messenger/drug effects , Tetradecanoylphorbol Acetate/pharmacology , Time Factors , Transfection/drug effects , Umbilical Veins , Up-Regulation/drug effects , Up-Regulation/genetics
10.
J Pharmacol Exp Ther ; 280(2): 1109-16, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9023330

ABSTRACT

In bovine aortic endothelial cells (BAECs), we previously demonstrated B1 and B2 kinin receptor-mediated increases in intracellular guanosine-3',5'-cyclic monophosphate (cGMP). In this study, the B2 kinin receptor agonist bradykinin increased cGMP in rat microvascular coronary endothelial cells (RMCECs) and human umbilical vein endothelial cells (HUVECs), which could be prevented with the specific B2 kinin receptor antagonist icatibant but not with the B1 kinin receptor antagonist des-Arg9-[Leu8]bradykinin or with the nonpeptide kinin receptor antagonist WIN 64338. B2 kinin receptor mRNA could be detected in all three cell types using reverse transcription-polymerase chain reaction and subsequent Southern blotting. The B1 kinin receptor agonist des-Arg9-bradykinin increased cGMP in RMCECs but not in HUVECs. The response in RMCECs could be prevented by des-Arg9-[Leu8]bradykinin as well as by WIN 64338 but not by icatibant. In BAECs, the B1 kinin receptor-mediated cGMP synthesis could be prevented by icatibant and desensitized by preincubation with des-Arg9-bradykinin as well as bradykinin. We detected B1 kinin receptor mRNA in RMCECs and HUVECs but not in BAECs. In HUVECs, the detection of B1 kinin receptor mRNA is in contradiction to the cGMP measurements. In BAECs, the atypical B1 kinin receptor pharmacology, the heterologous desensitization of the receptor and the failure to detect B1 kinin receptor mRNA cannot be explained by a typical B1 kinin receptor subtype. Thus, B2 kinin receptors with similar pharmacology are constitutively expressed in each of the three endothelial cell types. However, the endothelial cell types are heterogeneous in the expression of typical B1 kinin receptors and the pharmacology of the B1 kinin receptor-mediated responses.


Subject(s)
Bradykinin/analogs & derivatives , Bradykinin/pharmacology , Cyclic GMP/metabolism , Endothelium, Vascular/physiology , Naphthalenes/pharmacology , Organophosphorus Compounds/pharmacology , Receptors, Bradykinin/physiology , Transcription, Genetic/drug effects , Animals , Aorta , Bradykinin/metabolism , Bradykinin Receptor Antagonists , Cattle , Cells, Cultured , Coronary Vessels , DNA Primers , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Humans , Microcirculation , Naphthalenes/metabolism , Organophosphorus Compounds/metabolism , Polymerase Chain Reaction , Rats , Receptor, Bradykinin B1 , Receptors, Bradykinin/biosynthesis , Species Specificity , Umbilical Veins
11.
J Pharmacol Exp Ther ; 271(3): 1611-5, 1994 Dec.
Article in English | MEDLINE | ID: mdl-7996475

ABSTRACT

Despite a wealth of data, the mechanism of the direct dilator effect of furosemide on the systemic arterial and venous systems is far from being satisfactorily understood. Therefore, we investigated whether furosemide is capable of stimulating the production of the endogenous vasodilators nitric oxide and prostacyclin in primary cultured bovine aortic endothelial cells by an enhanced synthesis and release of endothelium-derived kinins. Nitric oxide production was assessed in terms of intracellular guanosine cyclic-3',5' monophosphate accumulation; kinin and prostacyclin release were determined by specific radioimmunoassays. Furosemide concentration- and time-dependently increased the formation of nitric oxide and prostacyclin. Maximal increases of both autacoids were already obtained after a 5-min incubation with 3 x 10(-7) to 10(-6) mol/l of furosemide. In the same concentration range, furosemide led to an enhanced release of kinins into the supernatant of the cells. This observation was supported by the inhibitory effect of the specific B2 kinin receptor antagonist icatibant (Hoe 140) on the furosemide-induced increase of nitric oxide and prostacyclin. Thus the hemodynamic effects, and in particular the direct early dilator effect, of furosemide may be explained in part by an enhanced endothelial synthesis and release of bradykinin and related kinins, which in turn stimulates endothelial autacoid formation via B2 kinin receptor activation.


Subject(s)
Epoprostenol/biosynthesis , Furosemide/pharmacology , Kinins/biosynthesis , Nitric Oxide/biosynthesis , Animals , Cattle , Cells, Cultured , Cyclic GMP/biosynthesis , Endothelium, Vascular/metabolism
12.
J Biol Chem ; 267(1): 644-8, 1992 Jan 05.
Article in English | MEDLINE | ID: mdl-1370452

ABSTRACT

Peptides corresponding to amino acids 321-339 (peptide GS21) and 416-431 (peptide GS31) of the cGMP-gated channel from bovine rod photoreceptors were synthesized and used as antigens for the preparation of polyclonal antibodies. After affinity purification, both antipeptide antibodies were found to bind specifically to the channel protein after Western blotting, but only the antibody against GS21 gave satisfactory results on enzyme-linked immunosorbent assay and electron microscopy. Using immunocytochemistry, we were able to localize amino acids 321-339 to the extracellular side of the rod photoreceptor plasma membrane. By synthesizing heptapeptides corresponding to amino acids 324-330 (peptide GS2s) and 420-426 (peptide GS3s), we were able to affinity purify antibodies specific for two N-glycosylation consensus sites in the channel protein. As assessed by Western blotting, antibodies against GS3s were found to bind to both the glycosylated and deglycosylated channel proteins, whereas antibodies against GS2s only bound to the channel protein after enzymatic deglycosylation. Together, these results allow the refinement of folding models for the cGMP-gated channel and implicate Asn-327 as being the sole site of N-glycosylation.


Subject(s)
Cyclic GMP/metabolism , Ion Channels/metabolism , Peptides/immunology , Photoreceptor Cells/metabolism , Rod Cell Outer Segment/metabolism , Amino Acid Sequence , Animals , Asparagine/metabolism , Blotting, Western , Cattle , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Chromatography, Affinity , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Glycosylation , Immunohistochemistry , Ion Channel Gating , Ion Channels/chemistry , Ion Channels/immunology , Microscopy, Electron , Molecular Sequence Data , Peptides/chemical synthesis , Protein Conformation , Rod Cell Outer Segment/ultrastructure
13.
Biochim Biophys Acta ; 1061(2): 247-52, 1991 Jan 30.
Article in English | MEDLINE | ID: mdl-1998696

ABSTRACT

The K(+)-dependence of the rod photoreceptor sodium-calcium exchanger was investigated using the Ca2(+)-sensitive dye arsenazo III after reconstitution of the purified protein into proteoliposomes. The uptake of Ca2+ by Na(+)-loaded liposomes was found to be greatly enhanced by the presence of external K+ (EC50 approximately 1 mM) in a Michaelis-Menten manner, suggesting that one K+ ion is involved in the transport of one Ca2+ ion. We also found a minimal degree of Ca2+ uptake in the total absence of K+. Other alkali cations, notably Rb+ and, to a lesser extent, Cs+, were also able to stimulate Na(+)-Ca2+ exchange. We also investigated the K(+)-dependence of the photoreceptor Na(+)-Ca2+ exchanger by determining the effects of electrochemical K+ gradients on the Na(+)-activated Ca2+ efflux from proteoliposomes. We found that, under conditions of membrane voltage clamp with FCCP, inwardly directed electrochemical K+ gradients (i.e., K0+ greater than Ki+) inhibited, whereas an outwardly directed electrochemical K+ gradient (i.e., Ki+ greater than K0+) enhanced, Na(+)-dependent Ca2+ efflux, consistent with the notion that K+ is cotransported in the same direction as Ca2+. The investigation of the reconstituted exchanger at physiological (i.e. Ki+ = 110 mM, K0+ = 2.5 mM) potassium concentrations revealed that the Na(+)-dependence of Ca2(+)-efflux was highly cooperative (n = 3.01 from Hill plots), indicating that at least three, but possibly four, Na+ ions are exchanged for one Ca2+ ion. Under these conditions the reconstituted exchanger showed a Km for Na+ of 26.1 mM, and a turnover number of 115 Ca2+.s-1 per exchanger molecule. Our results with the purified and reconstituted sodium-calcium exchanger from rod photoreceptors are therefore consistent with previous reports (Cervetto, L., Lagnado, L., Perry, R.J., Robinson, D.W. and McNaughton, P.A. (1989) Nature 337, 740-743; Schnetkamp, P.P.M., Basu, D.K. and Szerencsei, R.T. (1989) Am. J. Physiol. 257, C153-C157) that the sodium-calcium exchanger of rod photoreceptors cotransports K+ under physiological conditions with a stoichiometry of 4 Na+:1 Ca2+, 1K+.


Subject(s)
Carrier Proteins/metabolism , Photoreceptor Cells/metabolism , Potassium/metabolism , Animals , Biological Transport , Cattle , Cesium/pharmacology , Kinetics , Liposomes/metabolism , Photoreceptor Cells/drug effects , Rubidium/pharmacology , Sodium-Calcium Exchanger
14.
Biochim Biophys Acta ; 1022(3): 283-90, 1990 Mar.
Article in English | MEDLINE | ID: mdl-1690570

ABSTRACT

We have characterised the spectroscopic properties of the metallochromic dye dichlorophosphonazo III and describe its use for the determination of changes of Mg2+ concentration in the micromolar range. Using a previously described reconstitution procedure, we incorporated the cGMP-gated channel from bovine rod photoreceptors into magnesium-containing liposomes and used the dye to monitor cGMP-activated Mg2(+)-efflux. The Km and cooperativity of the cGMP-dependence were identical regardless of whether Mg2+ or Ca2+ was the transported ion, however, the vmax for Ca2+ was more than 2-fold higher than that for Mg2+. We thereby determined a channel selectivity (Ca2+:Mg2+) of 1.0:0.44 in the presence of symmetrical (30 mM) K+. We also describe conditions where Mg2+ or Ca2+ effluxes can be selectively monitored in the presence of each other. This allowed the demonstration that magnesium ions can flow through the cGMP-gated channel even in the presence of an identically directed calcium gradient. Together these results indicate that magnesium ions may enter the photoreceptor rod outer segment cytosol through the cGMP-gated channel under dark conditions, thereby alluding to the existence of an as yet unknown Mg2(+)-extrusion mechanism, distinct from the Na+/Ca2(+)-exchanger, in these cells.


Subject(s)
Azo Compounds , Cyclic GMP/pharmacology , Ion Channel Gating/drug effects , Ion Channels/metabolism , Magnesium/metabolism , Naphthalenesulfonates , Photoreceptor Cells/metabolism , Animals , Calcium/metabolism , Cattle , Coloring Agents , Hydrogen-Ion Concentration , Potassium/pharmacology , Rod Cell Outer Segment/analysis , Spectrophotometry
15.
J Biol Chem ; 264(35): 20934-9, 1989 Dec 15.
Article in English | MEDLINE | ID: mdl-2480349

ABSTRACT

In order to investigate the lectin-binding properties of the photoreceptor cGMP-gated channel, solubilized and purified channel protein was incubated with immobilized lectins followed by reconstitution of unbound proteins. Of the lectins tested, only concanavalin A (ConA) was able to specifically sediment channel activity. A 240-kDa protein, which copurifies with the 63-kDa channel protein but does not bind ConA, was also found to be sedimented by the ConA-affinity matrix, thereby implicating that it is associated with the channel complex. Treatment of the purified channel protein with the enzyme glycopeptidase F in the presence of the denaturing detergent sodium dodecyl sulfate resulted in a rapid reduction of the apparent molecular mass by 1.90 kDa, and the abolition of ConA-binding. No intermediate molecular weight species were observed, suggesting that the channel protein is N-glycosylated at one site only. Under nondenaturing conditions, the kinetics of deglycosylation were distinctly two-phased: 50-60% deglycosylation was achieved rapidly; however, prolonged incubation was required to arrive at complete deglycosylation. Reconstitution experiments showed that deglycosylation had no significant effect on the kinetics of channel protein activation by cGMP.


Subject(s)
Eye Proteins/metabolism , Ion Channels/metabolism , Lectins , Photoreceptor Cells/metabolism , Rod Cell Outer Segment/metabolism , Amidohydrolases , Animals , Cattle , Concanavalin A/metabolism , Cyclic GMP/pharmacology , Cyclic Nucleotide-Gated Cation Channels , Electrophoresis, Polyacrylamide Gel , Eye Proteins/isolation & purification , Kinetics , Molecular Weight , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase , Thermodynamics
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