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1.  Biomarkers of necrotising soft tissue infections: aspects of the innate immune response and effects of hyperbaric oxygenation—the protocol of the prospective cohort BIONEC study 
BMJ Open  2015;5(5):e006995.
The mortality and amputation rates are still high in patients with necrotising soft tissue infections (NSTIs). It would be ideal to have a set of biomarkers that enables the clinician to identify high-risk patients with NSTI on admission. The objectives of this study are to evaluate inflammatory and vasoactive biomarkers as prognostic markers of severity and mortality in patients with NSTI and to investigate whether hyperbaric oxygen treatment (HBOT) is able to modulate these biomarkers. The overall hypothesis is that plasma biomarkers can be used as prognostic markers of severity and mortality in patients with NSTI and that HBOT reduces the inflammatory response.
Methods and analysis
This is a prospective, observational study being conducted in a tertiary referral centre. Biomarkers will be measured in 114 patients who have been operatively diagnosed with NSTI. On admission, baseline blood values will be obtained. Following surgery and HBOT, daily blood samples for measuring regular inflammatory and vasoactive biomarkers (pentraxin-3, interleukin-6 and nitrite) will be acquired. Samples will be analysed using validated ELISA assays, chemiluminescence and Griess reaction. Clinical data will be obtained during admission in the intensive care unit for a maximum of 7 days. The primary analysis will focus on pentraxin-3, interleukin-6 and nitrite as early markers of disease severity in patients with NSTI.
Ethics and dissemination
The study has been approved by the Regional Scientific Ethical Committee of Copenhagen (H-2–2014–071) and the Danish Data Protection Agency (J. no. 30–0900 and J. no. 30–1282). Results will be presented at national and international conferences and published in peer-reviewed scientific journals.
Trial registration
PMCID: PMC4431132  PMID: 25967993
2.  Cerebrospinal Fluid from Patients with Subarachnoid Haemorrhage and Vasospasm Enhances Endothelin Contraction in Rat Cerebral Arteries 
PLoS ONE  2015;10(1):e0116456.
Previous studies have suggested that cerebrospinal fluid from patients with subarachnoid hemorrhage (SAH) leads to pronounced vasoconstriction in isolated arteries. We hypothesized that only cerebrospinal fluid from SAH patients with vasospasm would produce an enhanced contractile response to endothelin-1 in rat cerebral arteries, involving both endothelin ETA and ETB receptors.
Intact rat basilar arteries were incubated for 24 hours with cerebrospinal fluid from 1) SAH patients with vasospasm, 2) SAH patients without vasospasm, and 3) control patients. Arterial segments with and without endothelium were mounted in myographs and concentration-response curves for endothelin-1 were constructed in the absence and presence of selective and combined ETA and ETB receptor antagonists. Endothelin concentrations in culture medium and receptor expression were measured.
Compared to the other groups, the following was observed in arteries exposed to cerebrospinal fluid from patients with vasospasm: 1) larger contractions at lower endothelin concentrations (p<0.05); 2) the increased endothelin contraction was absent in arteries without endothelium; 3) higher levels of endothelin secretion in the culture medium (p<0.05); 4) there was expression of ETA receptors and new expression of ETB receptors was apparent; 5) reduction in the enhanced response to endothelin after ETB blockade in the low range and after ETA blockade in the high range of endothelin concentrations; 6) after combined ETA and ETB blockade a complete inhibition of endothelin contraction was observed.
Our experimental findings showed that in intact rat basilar arteries exposed to cerebrospinal fluid from patients with vasospasm endothelin contraction was enhanced in an endothelium-dependent manner and was blocked by combined ETA and ETB receptor antagonism. Therefore we suggest that combined blockade of both receptors may play a role in counteracting vasospasm in patients with SAH.
PMCID: PMC4309584  PMID: 25629621
3.  Alterations of N-3 Polyunsaturated Fatty Acid-Activated K2P Channels in Hypoxia-Induced Pulmonary Hypertension 
Basic & clinical pharmacology & toxicology  2013;113(4):10.1111/bcpt.12092.
Polyunsaturated fatty acid (PUFA)-activated two-pore domain potassium channels (K2P) have been proposed to be expressed in the pulmonary vasculature. However, their physiological or pathophysiological roles are poorly defined. Here we tested the hypothesis that PUFA-activated K2P are involved in pulmonary vasorelaxation and that alterations of channel expression are pathophysiologically linked to pulmonary hypertension.
Expression of PUFA-activated K2P in the murine lung was investigated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), immunohistochemistry (IHC), by patch clamp (PC), and myography. K2P-gene expression was examined in chronic hypoxic mice.
QRT-PCR showed that the K2P2.1 and K2P6.1 were the predominantly expressed K2P in the murine lung. IHC revealed protein expression of K2P2.1 and K2P6.1 in the endothelium of pulmonary arteries and of K2P6.1 in bronchial epithelium. PC showed pimozide-sensitive K2P-like K+-current activated by docosahexaenoic acid (DHA) in freshly isolated endothelial cells as well as DHA-induced membrane hyperpolarization. Myography on pulmonary arteries showed that DHA-induced concentration-dependent and instantaneous relaxations that were resistant to endothelial removal and inhibition of NO and prostacyclin synthesis and to a cocktail of blockers of calcium-activated K+ channels but were abolished by high extracellular (30 mM) K+-concentration. Gene expression and protein of K2P2.1 were not altered in chronic hypoxic mice while K2P6.1 was up-regulated by fourfold.
In conclusion, the PUFA-activated K2P2.1 and K2P6.1 are expressed in murine lung and functional K2P-like channels contribute to endothelium-hyperpolarization and pulmonary artery relaxation. The increased K2P6.1-gene expression may represent a novel counter-regulatory mechanism in pulmonary hypertension, and suggest that arterial K2P2.1 and K2P6.1 could be novel therapeutic targets.
PMCID: PMC3835666  PMID: 23724868
endothelium; K2P2.1; K2P6.1; lung; polyunsaturated fatty acid; pulmonary hypertension; vasorelaxation
4.  Pulmonary Hypertension in Wild Type Mice and Animals with Genetic Deficit in KCa2.3 and KCa3.1 Channels 
PLoS ONE  2014;9(5):e97687.
In vascular biology, endothelial KCa2.3 and KCa3.1 channels contribute to arterial blood pressure regulation by producing membrane hyperpolarization and smooth muscle relaxation. The role of KCa2.3 and KCa3.1 channels in the pulmonary circulation is not fully established. Using mice with genetically encoded deficit of KCa2.3 and KCa3.1 channels, this study investigated the effect of loss of the channels in hypoxia-induced pulmonary hypertension.
Approach and Result
Male wild type and KCa3.1−/−/KCa2.3T/T(+DOX) mice were exposed to chronic hypoxia for four weeks to induce pulmonary hypertension. The degree of pulmonary hypertension was evaluated by right ventricular pressure and assessment of right ventricular hypertrophy. Segments of pulmonary arteries were mounted in a wire myograph for functional studies and morphometric studies were performed on lung sections. Chronic hypoxia induced pulmonary hypertension, right ventricular hypertrophy, increased lung weight, and increased hematocrit levels in either genotype. The KCa3.1−/−/KCa2.3T/T(+DOX) mice developed structural alterations in the heart with increased right ventricular wall thickness as well as in pulmonary vessels with increased lumen size in partially- and fully-muscularized vessels and decreased wall area, not seen in wild type mice. Exposure to chronic hypoxia up-regulated the gene expression of the KCa2.3 channel by twofold in wild type mice and increased by 2.5-fold the relaxation evoked by the KCa2.3 and KCa3.1 channel activator NS309, whereas the acetylcholine-induced relaxation - sensitive to the combination of KCa2.3 and KCa3.1 channel blockers, apamin and charybdotoxin - was reduced by 2.5-fold in chronic hypoxic mice of either genotype.
Despite the deficits of the KCa2.3 and KCa3.1 channels failed to change hypoxia-induced pulmonary hypertension, the up-regulation of KCa2.3-gene expression and increased NS309-induced relaxation in wild-type mice point to a novel mechanism to counteract pulmonary hypertension and to a potential therapeutic utility of KCa2.3/KCa3.1 activators for the treatment of pulmonary hypertension.
PMCID: PMC4032241  PMID: 24858807
5.  Activation of endothelial and epithelial KCa2.3 calcium-activated potassium channels by NS309 relaxes human small pulmonary arteries and bronchioles 
British Journal of Pharmacology  2012;167(1):37-47.
Small (KCa2) and intermediate (KCa3.1) conductance calcium-activated potassium channels (KCa) may contribute to both epithelium- and endothelium-dependent relaxations, but this has not been established in human pulmonary arteries and bronchioles. Therefore, we investigated the expression of KCa2.3 and KCa3.1 channels, and hypothesized that activation of these channels would produce relaxation of human bronchioles and pulmonary arteries.
Channel expression and functional studies were conducted in human isolated small pulmonary arteries and bronchioles. KCa2 and KCa3.1 currents were examined in human small airways epithelial (HSAEpi) cells by whole-cell patch clamp techniques.
While KCa2.3 expression was similar, KCa3.1 protein was more highly expressed in pulmonary arteries than bronchioles. Immunoreactive KCa2.3 and KCa3.1 proteins were found in both endothelium and epithelium. KCa currents were present in HSAEpi cells and sensitive to the KCa2.3 blocker UCL1684 and the KCa3.1 blocker TRAM-34. In pulmonary arteries contracted by U46619 and in bronchioles contracted by histamine, the KCa2.3/ KCa3.1 activator, NS309, induced concentration-dependent relaxations. NS309 was equally potent in relaxing pulmonary arteries, but less potent in bronchioles, than salbutamol. NS309 relaxations were blocked by the KCa2 channel blocker apamin, while the KCa3.1 channel blocker, charybdotoxin failed to reduce relaxation to NS309 (0.01–1 µM).
KCa2.3 and KCa3.1 channels are expressed in the endothelium of human pulmonary arteries and epithelium of bronchioles. KCa2.3 channels contributed to endo- and epithelium-dependent relaxations suggesting that these channels are potential targets for treatment of pulmonary hypertension and chronic obstructive pulmonary disease.
PMCID: PMC3448912  PMID: 22506557
calcium-activated potassium channels; pulmonary arteries; bronchioles; KCa3.1; KCa2.3; NS309
6.  Pharmacological activation of KCa3.1/KCa2.3 channels produces endothelial hyperpolarization and lowers blood pressure in conscious dogs 
British Journal of Pharmacology  2012;165(1):223-234.
In rodents, the endothelial KCa channels, KCa3.1 and KCa2.3, have been shown to play a crucial role in initiating endothelium-derived hyperpolarizing factor (EDHF) vasodilator responses. However, it is not known to what extent these channels are involved in blood pressure regulation in large mammals, which would also allow us to address safety issues. We therefore characterized canine endothelial KCa3.1 and KCa2.3 functions and evaluated the effect of the KCa3.1/KCa2.3 activator SKA-31 on blood pressure and heart rate in dogs.
Canine endothelial KCa3.1/KCa2.3 functions were studied by patch-clamp electrophysiology and wire myography in mesenteric arteries. Systemic cardiovascular actions of acute SKA-31 administration were monitored in conscious, unstressed beagle dogs.
Mesenteric endothelial cells expressed functional KCa3.1 and KCa2.3 channels that were strongly activated by SKA-31. SKA-31 hyperpolarized the endothelial membrane and doubled endothelial hyperpolarization-dependent vasodilator responses in mesenteric arteries. SKA-31 (2 mg·kg−1, i.v.) rapidly decreased the MAP by 28 ± 6 mmHg; this response was transient (8 ± 1 s), and the initial drop was followed by a fast and pronounced increase in HR (+109 ± 7 beats min−1) reflecting baroreceptor activation. SKA-31 significantly augmented similar transient depressor responses elicited by ACh (20 ng·kg−1) and doubled the magnitude of the response over time.
Activation of endothelial KCa3.1 and KCa2.3 lowers arterial blood pressure in dogs by an immediate electrical vasodilator mechanism. The results support the concept that pharmacological activation of these channels may represent a potential unique endothelium-specific antihypertensive therapy.
PMCID: PMC3252979  PMID: 21699504
intermediate-conductance calcium-activated potassium channel; small-conductance calcium-activated potassium channel; endothelium-derived hyperpolarizing factor; SKA-31; blood pressure; hypertension
7.  Activation of Protease-Activated Receptor 2 Induces VEGF Independently of HIF-1 
PLoS ONE  2012;7(9):e46087.
Human adipose stem cells (hASCs) can promote angiogenesis through secretion of proangiogenic factors such as vascular endothelial growth factor (VEGF). In other cell types, it has been shown that induction of VEGF is mediated by both protease activated receptor 2 (PAR2) and hypoxia inducible factor 1(HIF-1). The present study hypothesized that PAR2 stimulation through activation of kinase signaling cascades lead to induction of HIF-1 and secretion of VEGF.
Methodology/Principal Findings
Immunohistochemistry revealed the expression of PAR2 receptors on the surface of hASCs. Blocking the PAR2 receptors with a specific antibody prior to trypsin treatment showed these receptors are involved in trypsin-evoked increase in VEGF secretion from hASCs. Blocking with specific kinase inhibitors suggested that that activation of MEK/ERK and PI3-kinase/Akt pathways are involved in trypsin-eveoked induction of VEGF. The effect of the trypsin treatment on the transcription of VEGF peaked at 6 hours after the treatment and was comparable to the activation observed after keeping hASCs for 24 hours at 1% oxygen. In contrast to hypoxia, trypsin alone failed to induce HIF-1 measured with ELISA, while the combination of trypsin and hypoxia had an additive effect on both VEGF transcription and secretion, results which were confirmed by Western blot.
In hASCs trypsin and hypoxia induce VEGF expression through separate pathways.
PMCID: PMC3457954  PMID: 23049945
8.  Non-endothelial endothelin counteracts hypoxic vasodilation in porcine large coronary arteries 
BMC Physiology  2011;11:8.
The systemic vascular response to hypoxia is vasodilation. However, reports suggest that the potent vasoconstrictor endothelin-1 (ET-1) is released from the vasculature during hypoxia. ET-1 is reported to augment superoxide anion generation and may counteract nitric oxide (NO) vasodilation. Moreover, ET-1 was proposed to contribute to increased vascular resistance in heart failure by increasing the production of asymmetric dimethylarginine (ADMA). We investigated the role of ET-1, the NO pathway, the potassium channels and radical oxygen species in hypoxia-induced vasodilation of large coronary arteries.
In prostaglandin F2α (PGF2α, 10 μM)-contracted segments with endothelium, gradual lowering of oxygen tension from 95 to 1% O2 resulted in vasodilation. The vasodilation to O2 lowering was rightward shifted in segments without endothelium at all O2 concentrations except at 1% O2. The endothelin receptor antagonist SB217242 (10 μM) markedly increased hypoxic dilation despite the free tissue ET-1 concentration in the arterial wall was unchanged in 1% O2 versus 95% O2. Exogenous ET-1 reversed hypoxic dilation in segments with and without endothelium, and the hypoxic arteries showed an increased sensitivity towards ET-1 compared to the normoxic controls. Without affecting basal NO, hypoxia increased NO concentration in PGF2α-contracted arteries, and an NO synthase inhibitor, L-NOARG,(300 μM, NG-nitro-L-Arginine) reduced hypoxic vasodilation. NO-induced vasodilation was reduced in endothelin-contracted preparations. Arterial wall ADMA concentrations were unchanged by hypoxia. Blocking of potassium channels with TEA (tetraethylammounium chloride)(10 μM) inhibited vasodilation to O2 lowering as well as to NO. The superoxide scavenger tiron (10 μM) and the putative NADPH oxidase inhibitor apocynin (10 μM) leftward shifted concentration-response curves for O2 lowering without changing vasodilation to 1% O2. PEG (polyethylene glycol) catalase (300 u/ml) inhibited H2O2 vasodilation, but failed to affect vasodilation to O2 lowering. Neither did PEG-SOD (polyethylene glycol superoxide dismutase)(70 u/ml) affect vasodilation to O2 lowering. The mitochondrial inhibitors rotenone (1 μM) and antimycin A (1 μM) both inhibited hypoxic vasodilatation.
The present results in porcine coronary arteries suggest NO contributes to hypoxic vasodilation, probably through K channel opening, which is reversed by addition of ET-1 and enhanced by endothelin receptor antagonism. These latter findings suggest that endothelin receptor activation counteracts hypoxic vasodilation.
PMCID: PMC3118136  PMID: 21575165
9.  Pressure Load: The Main Factor for Altered Gene Expression in Right Ventricular Hypertrophy in Chronic Hypoxic Rats 
PLoS ONE  2011;6(1):e15859.
The present study investigated whether changes in gene expression in the right ventricle following pulmonary hypertension can be attributed to hypoxia or pressure loading.
Methodology/Principal Findings
To distinguish hypoxia from pressure-induced alterations, a group of rats underwent banding of the pulmonary trunk (PTB), sham operation, or the rats were exposed to normoxia or chronic, hypobaric hypoxia. Pressure measurements were performed and the right ventricle was analyzed by Affymetrix GeneChip, and selected genes were confirmed by quantitative PCR and immunoblotting. Right ventricular systolic blood pressure and right ventricle to body weight ratio were elevated in the PTB and the hypoxic rats. Expression of the same 172 genes was altered in the chronic hypoxic and PTB rats. Thus, gene expression of enzymes participating in fatty acid oxidation and the glycerol channel were downregulated. mRNA expression of aquaporin 7 was downregulated, but this was not the case for the protein expression. In contrast, monoamine oxidase A and tissue transglutaminase were upregulated both at gene and protein levels. 11 genes (e.g. insulin-like growth factor binding protein) were upregulated in the PTB experiment and downregulated in the hypoxic experiment, and 3 genes (e.g. c-kit tyrosine kinase) were downregulated in the PTB and upregulated in the hypoxic experiment.
Pressure load of the right ventricle induces a marked shift in the gene expression, which in case of the metabolic genes appears compensated at the protein level, while both expression of genes and proteins of importance for myocardial function and remodelling are altered by the increased pressure load of the right ventricle. These findings imply that treatment of pulmonary hypertension should also aim at reducing right ventricular pressure.
PMCID: PMC3016335  PMID: 21246034
10.  Combination of Ca2+-activated K+ channel blockers inhibits acetylcholine-evoked nitric oxide release in rat superior mesenteric artery 
British Journal of Pharmacology  2006;149(5):560-572.
Background and purpose:
The present study investigated whether calcium-activated K+ channels are involved in acetylcholine-evoked nitric oxide (NO) release and relaxation.
Experimental approach:
Simultaneous measurements of NO concentration and relaxation were performed in rat superior mesenteric artery and endothelial cell membrane potential and intracellular calcium ([Ca2+]i) were measured.
Key results.
A combination of apamin plus charybotoxin, which are, respectively, blockers of small-conductance and of intermediate- and large-conductance Ca2+-activated K channels abolished acetylcholine (10 μM)-evoked hyperpolarization of endothelial cell membrane potential. Acetylcholine-evoked NO release was reduced by 68% in high K+ (80 mM) and by 85% in the presence of apamin plus charybdotoxin. In noradrenaline-contracted arteries, asymmetric dimethylarginine (ADMA), an inhibitor of NO synthase inhibited acetylcholine-evoked NO release and relaxation. However, only further addition of oxyhaemoglobin or apamin plus charybdotoxin eliminated the residual acetylcholine-evoked NO release and relaxation. Removal of extracellular calcium or an inhibitor of calcium influx channels, SKF96365, abolished acetylcholine-evoked increase in NO concentration and [Ca2+]i. Cyclopiazonic acid (CPA, 30 μM), an inhibitor of sarcoplasmic Ca2+-ATPase, caused a sustained NO release in the presence, but only a transient increase in the absence, of extracellular calcium. Incubation with apamin and charybdotoxin did not change acetylcholine or CPA-induced increases in [Ca2+]i, but inhibited the sustained NO release induced by CPA.
Conclusions and Implications:
Acetylcholine increases endothelial cell [Ca2+]i by release of stored calcium and calcium influx resulting in activation of apamin and charybdotoxin-sensitive K channels, hyperpolarization and release of NO in the rat superior mesenteric artery.
PMCID: PMC2014669  PMID: 16967048
acetylcholine; endothelium; superior mesenteric artery; K+ channels; nitric oxide
11.  Neuropeptide Y2 receptors are involved in enhanced neurogenic vasoconstriction in spontaneously hypertensive rats 
British Journal of Pharmacology  2006;148(5):703-713.
The present study addressed the role of neuropeptide (NPY) Y2 receptors in neurogenic contraction of mesenteric resistance arteries from female spontaneously hypertensive rats (SHR). Arteries were suspended in microvascular myographs, electrical field stimulation (EFS) was performed, and protein evaluated by Western blotting and immunohistochemistry.In vasopressin-activated endothelium-intact arteries, NPY and fragments with selectivity for Y1 receptors, [Leu31,Pro34]NPY, Y2 receptors, NPY(13–36), and rat pancreatic polypeptide evoked more pronounced contractions in segments from SHR than in Wistar Kyoto (WKY) arteries, even in the presence of the Y1 receptor antagonist, BIBP3226 (0.3 μM, (R)-N(2)-(diphenacetyl)-N-[(4-hydroxyphenyl)methyl]D-arginineamide).In the presence of prazosin and during vasopressin activation, EFS-evoked contractions were larger in arteries from SHR compared to WKY. EFS contractions were enhanced by the Y2 receptor selective antagonist BIIE0246TF (0.5 μM, (S)-N2-[[1-[2-[4-[(R,S)-5,11-dihydro-6(6h)-oxodibenz[b,e]azepin-11-y1]-1-piperazinyl]-2-oxoethyl]cyclo-pentyl-N-[2-[1,2-dihydro-3,5 (4H)-dioxo-1,2-diphenyl-3H-1,2,4-triazol-4-yl]ethyl]-argininamide), reduced by BIBP3226, and abolished by the combination of BIBP3226 and BIIE0246TF.Immunoblotting showed NPY Y1 and Y2 receptor expression to be similar in arteries from WKY and SHR, although a specific Y2 receptor band at 80 kDa was detected only in arteries from WKY.Immunoreaction for NPY was enhanced in arteries from SHR. In contrast to arteries from WKY, BIIE0246TF increased NPY immunoreactivity in EFS-stimulated arteries from SHR.The present results suggest that postjunctional neuropeptide Y1 and Y2 receptors contribute to neurogenic contraction of mesenteric small arteries. Moreover, both enhanced NPY content and altered neuropeptide Y1 and Y2 receptor activation apparently contribute to the enhanced neurogenic contraction of arteries from SHR.
PMCID: PMC1751866  PMID: 16715120
BII0246TF; hypertension; mesenteric small arteries; spontaneously hypertensive rat; Wistar Kyoto rat; neuropeptide Y (NPY); receptors
12.  The superoxide dismutase mimetic, tempol, blunts right ventricular hypertrophy in chronic hypoxic rats 
British Journal of Pharmacology  2003;141(1):105-113.
The purpose of this study was to investigate whether a membrane-permeable superoxide dismutase mimetic, tempol, added either alone or in combination with the nitric oxide (NO) donor molsidomine, prevents the development of pulmonary hypertension (PH) in chronic hypoxic rats.Chronic hypobaric hypoxia (10% oxygen) for 2 weeks increased the right ventricular systolic pressure (RVSP), right ventricle and lung wet weight. Relaxations evoked by acetylcholine (ACh) and the molsidomine metabolite SIN-1 were impaired in isolated proximal, but not distal pulmonary arteries, from chronic hypoxic rats.Treatment with tempol (86 mg kg−1 day−1 in drinking water) normalized RVSP and reduced right ventricular hypertrophy, while systemic blood pressure, lung and liver weights, and blunted ACh relaxation of pulmonary arteries were unchanged.Treatment with molsidomine (15 mg kg−1 day−1 in drinking water) had the same effects as tempol, except that liver weight was reduced, and potassium and U46619-evoked vasoconstrictions in pulmonary arteries were increased. Combining tempol and molsidomine did not have additional effects compared to tempol alone. ACh relaxation in pulmonary arteries was not normalized by these treatments.The media to lumen diameter ratio of the pulmonary arteries was greater for the hypoxic rats compared to the normoxic rats, and was not reversed by treatment with tempol, molsidomine, or the combination of tempol and molsidomine.We conclude that tempol, like molsidomine, is able to correct RVSP and reduce right ventricular weight in the rat hypoxic model. Functional and structural properties of pulmonary small arteries were little affected. The results support the possibility that superoxide dismutase mimetics may be a useful means for the treatment of PH.
PMCID: PMC1574166  PMID: 14656807
Molsidomine; tempol; chronic hypoxic pulmonary hypertension; proximal pulmonary arteries; distal resistance pulmonary arteries
13.  Mechanisms involved in the early increase of serotonin contraction evoked by endotoxin in rat middle cerebral arteries 
British Journal of Pharmacology  2003;140(4):671-680.
The present study investigated the mechanisms involved in the increased 5-hydroxytryptamine (5-HT) vasoconstriction observed in rat middle cerebral arteries exposed in vitro to lipopolysaccharide (LPS, 10 μg ml−1) for 1–5 h. Functional, immunohistochemical and Western blot analysis and superoxide anion measurements by ethidium fluorescence were performed.LPS exposure increased 5-HT (10 μM) vasoconstriction only during the first 4 h. In contrast to control tissue, indomethacin (10 μM), the COX-2 inhibitor NS 398 (10 μM), the TXA2/PGH2 receptor antagonist SQ 29,548 (1 μM) and the TXA2 synthase inhibitor furegrelate (1 μM) reduced 5-HT contraction of LPS-treated arteries from hour one. The iNOS inhibitor aminoguanidine (0.1 mM) increased 5-HT contraction from hour three of LPS incubation.The superoxide anion scavenger superoxide dismutase (SOD, 100 U ml−1) and the H2O2 scavenger catalase (1000 U ml−1), as well as the respective inhibitors of NAD(P)H oxidase and xanthine oxidase, apocynin (0.3 mM) and allopurinol (0.3 mM), reduced 5-HT contraction after LPS incubation. LPS induced an increase in superoxide anion levels that was abolished by PEG-SOD.Subthreshold concentrations of the TXA2 analogue U 46619, xanthine/xanthine oxidase and H2O2 potentiated, whereas those of sodium nitroprusside inhibited, the 5-HT contraction.COX-2 expression was increased at 1 and 5 h of LPS incubation, while that of iNOS, Cu/Zn-SOD and Mn-SOD was only increased after 5 h. All the three vascular layers expressed COX-2 and Cu/Zn-SOD. iNOS expression was detected in the endothelium and adventitia after LPS.In conclusion, increased production of TXA2 from COX-2, superoxide anion and H2O2 enhanced vasoconstriction to 5-HT during the first few hours of LPS exposure; iNOS and SOD expression counteracted that increase at 5 h. These changes can contribute to the disturbance of cerebral blood flow in endotoxic shock.
PMCID: PMC1574084  PMID: 14534151
Lipopolysaccharide; middle cerebral artery; nitric oxide; thromboxane; oxygen radical
14.  Different modulation by Ca2+-activated K+ channel blockers and herbimycin of acetylcholine- and flow-evoked vasodilatation in rat mesenteric small arteries 
British Journal of Pharmacology  2003;138(8):1562-1570.
The present study addressed whether endothelium-dependent vasodilatation evoked by acetylcholine and flow are mediated by the same mechanisms in isolated rat mesenteric small arteries, suspended in a pressure myograph for the measurement of internal diameter.In pressurized arterial segments contracted with U46619 in the presence of indomethacin, shear stress generated by the flow evoked relaxation. Thus, in endothelium-intact segments low (5.1±0.6 dyn cm−2) and high (19±2 dyn cm−2) shear stress evoked vasodilatations that were reduced by, respectively, 68±11 and 68±8% (P<0.05, n=7) by endothelial cell removal. Acetylcholine (0.01–1 μM) evoked concentration-dependent vasodilatation that was abolished by endothelial cell removal.Incubation with indomethacin alone did not change acetylcholine and shear stress-evoked vasodilatation, while the combination of indomethacin with the nitric oxide (NO) synthase inhibitor, NG,NG-asymmetric dimethyl-L-arginine (ADMA 1 mM), reduced low and high shear stress-evoked vasodilatation with, respectively, 52±15 and 58±10% (P<0.05, n=9), but it did not change acetylcholine-evoked vasodilatation.Inhibition of Ca2+-activated K+ channels with a combination of apamin (0.5 μM) and charybdotoxin (ChTX) (0.1 μM) did not change shear stress- and acetylcholine-evoked vasodilatation. In the presence of indomethacin and ADMA, the combination of apamin (0.5 μM) and ChTx (0.1 μM) increased contraction induced by U46619, but these blockers did not change the vasodilatation evoked by shear stress. In contrast, acetylcholine-evoked vasodilatation was abolished by the combination of apamin and charybdotoxin.In the presence of indomethacin, the tyrosine kinase inhibitor, herbimycin A (1 μM), inhibited low and high shear stress-evoked vasodilatation with, respectively, 32±12 and 68±14% (P<0.05, n=8), but it did not change vasodilatation induced by acetylcholine. In the presence of indomethacin and ADMA, herbimycin A neither changed shear stress nor acetylcholine-evoked vasodilatation.The present study suggests that Ca2+-activated K+ channels sensitive for the combination of apamin and ChTx are involved in acetylcholine-evoked, mainly non-NO nonprostanoid factor-mediated, vasodilatation, while an Src tyrosine kinase plays a role for flow-evoked NO-mediated vasodilatation in rat mesenteric small arteries.
PMCID: PMC1573811  PMID: 12721112
Flow; acetylcholine; endothelium; resistance arteries; rat; herbimycin A
15.  Characterization of the 5-hydroxytryptamine receptors mediating contraction in the pig isolated intravesical ureter 
British Journal of Pharmacology  2003;138(1):137-144.
This study was designed to investigate the effect of 5-hydroxytryptamine (5-HT) and to characterize the 5-HT receptors involved in 5-HT responses in the pig intravesical ureter.5-HT (0.01–10 μM) concentration-dependently increased the tone of intravesical ureteral strips, whereas the increases in phasic contractions were concentration-independent. The 5-HT2 receptor agonist α-methyl 5-HT, mimicked the effect on tone whereas weak or no response was obtained with 5-CT, 8-OH-DPAT, m-chlorophenylbiguanide and RS 67333, 5-HT1, 5-HT1A, 5-HT3 and 5-HT4 receptor agonists, respectively. 5-HT did not induce relaxation of U46619-contracted ureteral preparations. Pargyline (100 μM), a monoaminooxidase A/B activity inhibitor, produced leftward displacements of the concentration-response curves for 5-HT.5-HT-induced tone was reduced by the 5-HT2 and 5-HT2A receptor antagonists ritanserine (0.1 μM) and spiperone (0.2 μM), respectively. However, 5-HT contraction was not antagonized by cyanopindolol (2 μM), SDZ–SER 082 (1 μM), Y-25130 (1 μM) and GR 113808 (0.1 μM), which are respectively, 5-HT1A/1B, 5-HT2B/2C, 5-HT3, and 5-HT4 selective receptor antagonists.Removal of the urothelium did not modify 5-HT-induced contractions. Blockade of neuronal voltage-activated sodium channels, α-adrenergic receptors and adrenergic neurotransmission with tetrodotoxin (1 μM), phentolamine (0.3 μM) and guanethidine (10 μM), respectively, reduced the contractions to 5-HT. However, physostigmine (1 μM), atropine (0.1 μM) and suramin (30 μM), inhibitors of cholinesterase activity, muscarinic- and purinergic P2-receptors, respectively, failed to modify the contractions to 5-HT.These results suggest that 5-HT increases the tone of the pig intravesical ureter through 5-HT2A receptors located at the smooth muscle. Part of the 5-HT contraction is indirectly mediated via noradrenaline release from sympathetic nerves.
PMCID: PMC1573645  PMID: 12522083
5-hydroxytryptamine; smooth muscle contraction; 5-HT agonists and antagonists; 5-HT2A receptors; pig intravesical ureter
16.  Nitric oxide, prostanoid and non-NO, non-prostanoid involvement in acetylcholine relaxation of isolated human small arteries 
British Journal of Pharmacology  2000;129(1):184-192.
The main purpose of the study was to clarify to which extent nitric oxide (NO) contributes to acetylcholine (ACh) induced relaxation of human subcutaneous small arteries.Arterial segments were mounted in myographs for recording of isometric tension, NO concentration and smooth muscle membrane potential.In noradrenaline-contracted arteries, ACh induced endothelium-dependent relaxations. The NO synthase inhibitor, NG-nitro-L-arginine (L-NOARG) had a small significant effect on the concentration-response curves for ACh, and in the presence of L-NOARG, indomethacin only caused a small additional rightward shift in the ACh relaxation.The NO scavenger, oxyhaemoglobin attenuated relaxations for ACh and for the NO donor S-nitroso-N-acetylpenicillamine (SNAP). Inhibition of guanylyl cyclase with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), and inhibition of protein kinase G with β-phenyl-1, N2-etheno-8-bromoguanosine- 3′, 5′- cyclic monophosphorothioate, Rp-isomer, slightly attenuated ACh relaxation, but abolished SNAP induced relaxation.ACh induced relaxation without increases in the free NO concentration. In contrast, for equivalent relaxation, SNAP increased the NO concentration 32±8 nM.ACh hyperpolarized the arterial smooth muscle cells with 11.4±1.3 mV and 10.5±1.3 mV in the absence and presence of L-NOARG, respectively. SNAP only elicited a hyperpolarization of 1.6±0.9 mV.In the presence of indomethacin and L-NOARG, ACh relaxation was almost unaffected by lipoxygenase inhibition with nordihydroguaiaretic acid, or cytochrome P450 inhibition with 17-octadecynoic acid or econazole. ACh relaxation was strongly reduced by the combination of charybdotoxin and apamin, but small increments in the extracellular potassium concentration induced no relaxations.The study demonstrates that the NO/L-arginine pathway is present in human subcutaneous small arteries and to a limited extent is involved in ACh induced relaxation. The study also suggests a small contribution of arachidonic acid metabolites. However, ACh relaxation is mainly dependent on a non-NO, non-prostanoid endothelium dependent hyperpolarization.
PMCID: PMC1621136  PMID: 10694219
Acetylcholine; endothelium; human artery; nitric oxide; membrane potential; NO sensitive microelectrode
17.  A new experimental approach in endothelium-dependent pharmacological investigations on isolated porcine coronary arteries mounted for impedance planimetry 
British Journal of Pharmacology  1999;128(1):165-173.
The aim of this study was to investigate whether the balloon-based impedance planimetry technique could be a useful tool in endothelium-dependent investigations.Porcine large coronary arteries contracted with prostaglandin F2α (PGF2α, 10 μM) did not relax to bradykinin (0.1 nM–0.1 μM), but did relax to sodium nitroprusside (SNP, 10 μM). However, after eversion of the segments, bradykinin induced relaxations with pD2 values and maximal responses of 8.78±0.09 and 75±2% (n=6), respectively.Incubation with captopril (1 μM) did not reveal a relaxation to bradykinin in the normal vessel configuration and had no influence on the concentration-relaxation relationship in everted segments.Lowering the luminal pressure in contracted segments from 131±5 mmHg (isometric, n=5) to 60 mmHg (isobaric, n=5) did not facilitate the action of bradykinin.Eversion of segments did not influence the concentration-response relationship for K+ (4.7–125 mM), PGF2α (0.3–30 μM), and SNP (30 nM–30 μM), although the time-courses of responses were faster when the agents were added from the intimal compared to the adventitial side of the preparation.In the same everted segment contracted with PGF2α, the concentration-response relationship for bradykinin was not different under isometric and isobaric conditions.These results indicate that, (1) reduced endothelium-dependent relaxations to adventitially administered substances can be ascribed to a diffusion barrier in the vessel wall, while enzymatic degradation, luminal pressure and precontractile responses seem not to play a role, (2) impedance planimetry applied to everted cylindrical segments could be a useful experimental approach in pharmacological studies of endothelium-dependent responses under isobaric and isometric conditions.
PMCID: PMC1571598  PMID: 10498848
Bradykinin; diffusion barrier; endothelium; eversion; impedance planimetry; isobaric and isometric conditions; large coronary artery; luminal pressure; pig; time-course
18.  Contribution of K+ channels and ouabain-sensitive mechanisms to the endothelium-dependent relaxations of horse penile small arteries 
British Journal of Pharmacology  1998;123(8):1609-1620.
Penile small arteries (effective internal lumen diameter of 300–600 μm) were isolated from the horse corpus cavernosum and mounted in microvascular myographs in order to investigate the mechanisms underlying the endothelium-dependent relaxations to acetylcholine (ACh) and bradykinin (BK).In arteries preconstricted with the thromboxane analogue U46619 (3–30 nM), ACh and BK elicited concentration-dependent relaxations, pD2 and maximal responses being 7.71±0.09 and 91±1% (n=23), and 8.80±0.07 and 89±2% (n=24) for ACh and BK, respectively. These relaxations were abolished by mechanical endothelial cell removal, attenuated by the nitric oxide (NO) synthase (NOS) inhibitor, NG-nitro-L-arginine (L-NOARG, 100 μM) and unchanged by indomethacin (3 μM). However, raising extracellular K+ to concentrations of 20–30 mM significantly inhibited the ACh and BK relaxant responses to 63±4% (P<0.01, n=7) and to 59±4% (P<0.01, n=6), respectively. ACh- and BK-elicited relaxations were abolished in arteries preconstricted with K+ in the presence of 100 μM L-NOARG.In contrast to the inhibitor of ATP-sensitive K+ channels, the blockers of Ca2+-activated K+ (KCa) channels, charybdotoxin (30 nM) and apamin (0.3 μM), each induced slight but significant rightward shifts of the relaxations to ACh and BK without affecting the maximal responses. Combination of charybdotoxin and apamin did not cause further inhibition of the relaxations compared to either toxin alone. In the presence of L-NOARG (100 μM), combined application of the two toxins resulted in the most effective inhibition of the relaxations to both ACh and BK. Thus, pD2 and maximal responses for ACh and BK were 7.65±0.08 and 98±1%, and 9.17±0.09 and 100±0%, respectively, in controls, and 5.87±0.09 (P<0.05, n=6) and 38±11% (P<0.05, n=6), and 8.09±0.14 (P<0.01, n=6) and 98±1% (n=6), respectively, after combined application of charybdotoxin plus apamin and L-NOARG.The selective inhibitor of guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 5 μM) did not alter the maximal responses to either ACh or BK, but slightly decreased the sensitivity to both agonists, δpD2 being 0.25±0.07 (P<0.05, n=6) and 0.62±0.12 (P<0.01, n=6) for ACh and BK, respectively. Combined application of ODQ and charybdotoxin plus apamin produced further inhibition of the sensitivity to both ACh (δpD2=1.39±0.09, P<0.01, n=6) and BK (1.29±0.11, P<0.01, n=6), compared to either ODQ or charybdotoxin plus apamin alone.Exogenous nitric oxide (NO) present in acidified solutions of sodium nitrite (NaNO2) and S-nitroso-cysteine (SNC) both concentration-dependently relaxed penile resistance arteries, pD2 and maximal responses being 4.84±0.06 and 82±3% (n=12), and 6.72±0.07 and 85±4% (n=19), respectively. Charybdotoxin displaced to the right the dose-relaxation curves for both NO (δpD2 0.38±0.06, P<0.01, n=6) and SNC (δpD2 0.50±0.10, P<0.01, n=5), whereas apamin only reduced sensitivity (δpD2=0.35±0.12, P<0.05, n=5) and maximum response (65±9%, P<0.05, n=6) to SNC. ODQ shifted to the right the dose-relaxation curves to both NO and SNC. The relaxant responses to either NO or SNC were not further inhibited by a combination of ODQ and charybdotoxin or ODQ and charybdotoxin plus apamin, respectively, compared to either blocker alone.In the presence of 3 μM phentolamine, 5 μM ouabain contracted penile resistance arteries by 50±6% (n=17) of K-PSS, but did not significantly change the relaxant responses to either ACh, BK or NO. However, in the presence of L-NOARG ouabain reduced the ACh- and BK-elicited relaxation from 94±3% to 16±5% (P<0.0001, n=6), and from 98±2% to 13±3% (P<0.0001, n=5), respectively. Combined application of ODQ and ouabain inhibited the relaxations to NO from 92±2% to 26±3% (P<0.0001, n=6).The present results demonstrate that the endothelium-dependent relaxations of penile small arteries involve the release of NO and a non-NO non-prostanoid factor(s) which probably hyperpolarize(s) smooth muscle by two different mechanisms: an increased charybdotoxin and apamin-sensitive K+ conductance and an activation of the Na+-K+ATPase. These two mechanisms appear to be independent of guanylate cyclase stimulation, although NO itself can also activate charybdotoxin-sensitive K+ channels and the Na+-K+ pump through both cyclic GMP-dependent and independent mechanisms, respectively.
PMCID: PMC1565334  PMID: 9605568
Endothelium; acetylcholine; bradykinin; nitric oxide; K+-channels; charybdotoxin; apamin; cyclic GMP; ouabain; penile small arteries
19.  Mechanisms of relaxations of bovine isolated bronchioles by the nitric oxide donor, GEA 3175 
British Journal of Pharmacology  1998;123(5):895-905.
The present study was designed to investigate the effects and mechanisms of relaxation induced by the nitric oxide (NO) donor, GEA 3175 (a 3-aryl-substituted oxatriazole derivative) on bovine bronchioles (effective lumen diameter 200–800 μm) suspended in microvascular myographs for isometric tension recording.In segments of bovine bronchioles contracted to 5-hydroxytryptamine, GEA 3175 (10−8–10−4 M) induced concentration-dependent reproducible relaxations. These relaxations were slow in onset compared to other NO-donors such as 3-morpholinosydonimine-hydrochloride (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP).In 5-hydroxytryptamine-contracted preparations the order of relaxant potency (pD2) was: salbutamol (7.80)>GEA 3175 (6.18)>SIN-1 (4.90)>SNAP (3.55). In segments contracted to acetylcholine, the relaxant responses were reduced and GEA 3175 relaxed the bronchioles with pD2=4.41±0.12 and relaxations of 66±10% (n=4), while SNAP and salbutamol caused relaxations of 19±6% (n=4) and 27±6% (n=8) at the highest concentration used, respectively.Oxyhaemoglobin (10−5 M), the scavenger of nitric oxide, caused rightward shifts of the concentration-relaxation curves to GEA 3175 and NO. 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 3×10−6 M) and LY 83583 (10−6 M), the inhibitors of soluble guanylate cyclase, also reduced the relaxations induced by GEA 3175 and nitric oxide. However, ODQ did not affect salbutamol-evoked relaxation in the bovine small bronchioles.GEA 3175-induced relaxations were reduced in potassium-rich (60 mmol l−1 K+) solution. Glibenclamide (10−6 M) markedly inhibited the relaxations induced by the opener of ATP-sensitive K+ channels, levcromakalim (3×10−8–10−5 M), but it did not modify the relaxations induced by GEA 3175 or salbutamol. Apamin (5×10−7 M), a blocker of the small Ca2+-activated K+-channels did not affect the relaxations to GEA 3175. In contrast, blockers of large Ca2+-activated K+-channels, charybdotoxin (3×10−8–10−7 M) and iberiotoxin (10−8 M), did inhibit the relaxations to GEA 3175. The combination of apamin and charybdotoxin did not induce an additional inhibitory effect on the relaxations to GEA 3175 compared to charybdotoxin alone.In preparations where a concentration-response curve to GEA 3175 or NO was first obtained in the presence of LY 83583, incubation with charybdotoxin (10−7 M) did produce an additional inhibitory effect of the relaxations. However, in the presence of ODQ (3×10−6 M), iberiotoxin (10−8 M) did not produce additional reduction of the NO- or GEA 3175-induced relaxations.The present results suggest that the slow-releasing NO-donor GEA 3175 is more potent than the traditional NO donors in inducing relaxations of bovine bronchioles. GEA 3175, as for exogenously added NO, elicits relaxations through a cyclic GMP-dependent mechanism followed by opening of large conductance Ca2+-activated K+-channels.
PMCID: PMC1565239  PMID: 9535018
Bovine bronchioles; GEA 3175; nitric oxide; K+-channels; ODQ; iberiotoxin; charybdotoxin; glibenclamide; apamin
20.  Involvement of a glibenclamide-sensitive mechanism in the nitrergic neurotransmission of the pig intravesical ureter 
British Journal of Pharmacology  1997;120(4):609-616.
The present study was designed to investigate whether potassium (K+) channels are involved in the relaxations to nitric oxide (NO) of pig intravesical ureteral preparations suspended in organ baths for isometric tension recordings. In ureteral strips treated with guanethidine (10−5 M) and atropine (10−7 M) to block adrenergic neurotransmission and muscarinic receptors, respectively, NO was either released from nitrergic nerves by electrical field stimulation (EFS, 0.5–10 Hz, 1 ms duration, 20 s trains), or exogenously-applied as an acidified solution of sodium nitrite (NaNO2, 10−6–10−3 M).Incubation with an inhibitor of guanylate cyclase activation by NO, methylene blue (10−5 M) did not change the basal tension of intravesical ureteral strips but inhibited the relaxation induced by EFS or exogenous NO on ureteral preparations contracted with the thromboxane analogue U46619 (10−7 M).Incubation with charybdotoxin (3×10−8 M) and apamin (5×10−7 M), which are inhibitors of large and small conductance calcium (Ca2+)-activated K+ channels, respectively, did not modify basal tension or the relaxations induced by EFS and exogenous NO. Treatment with charybdotoxin or apamin plus methylene blue (10−5 M) significantly reduced the relaxations to EFS and exogenous NO. However, in both cases the reductions were similar to the inhibition evoked by methylene blue alone. The combined addition of charybdotoxin plus apamin did not change the relaxations to EFS or exogenously added NO of the porcine intravesical ureter.Cromakalim (10−8–3×10−6 M), an opener of ATP-sensitive K+ channels, evoked a dose-dependent relaxation with a pD2 of 7.3±0.2 and maximum relaxant effect of a 71.8±4.2% of the contraction induced by U46619 in the pig intravesical ureter. The blocker of ATP-sensitive K+ channels, glibenclamide (10−6 M), inhibited markedly the relaxations to cromakalim.Glibenclamide (10−6 M) had no effect on the basal tone of ureteral preparations but significantly reduced the relaxations induced by both EFS and exogenous NO. Combined treatment with methylene blue (10−5 M) and glibenclamide (10−6 M) did not exert an effect greater than that of methylene blue alone on either EFS- or NO-evoked relaxations of the pig ureter.The present results suggest that NO acts as an inhibitory neurotransmitter in the pig intravesical ureter and relaxes smooth muscle through a guanylate cyclase-dependent mechanism which seems to favour the opening of glibenclamide-sensitive K+ channels.
PMCID: PMC1564511  PMID: 9051298
Pig intravesical ureter; electrical field stimulation; nitric oxide; methylene blue; charybdotoxin; apamin; glibenclamide; cromakalim; KATP channels
21.  Involvement of ATP in the non-adrenergic non-cholinergic inhibitory neurotransmission of lamb isolated coronary small arteries 
British Journal of Pharmacology  1997;120(3):411-420.
The involvement of non-adrenergic non-cholinergic (NANC) transmitters, such as nitric oxide (NO) and adenosine 5′-triphosphate (ATP), in the neurogenic relaxation of lamb coronary small arteries was investigated in vessel segments with an internal lumen diameter of 200–550 μm, isolated from the left ventricle of the heart, and suspended for isometric tension recording in microvascular myographs.In both endothelium-intact and -denuded coronary small arteries treated with phentolamine (3×10−6 M), propranolol (3×10−6 M), and atropine (10−6 M) and contracted to 3×10−7 M of the thromboxane analogue U46619, electrical field stimulation (EFS) evoked frequency-dependent relaxations, which were markedly reduced in the presence of tetrodotoxin (10−6 M).Exogenous NO added as acidified sodium nitrite (10−6–10−3 M) and L-nitrosocysteine induced potent relaxations of lamb coronary small arteries. However, both inhibition of NO synthase with NG-nitro-L-arginine (L-NOARG, 3×10−5 M), and mechanical endothelial cell removal increased rather than inhibited relaxations to EFS. In small arteries processed for NADPH-diaphorase histochemistry, activity was only observed within endothelial cells.In arteries contracted to U46619, exogenously added ATP caused concentration-dependent relaxations with pD2 and maximum responses of 4.72±0.12 and 89.6±3.8% (n=12), respectively. ADP and the P2Y-agonist, 2-methylthio-ATP, induced relaxations equipotent to ATP, while the P2X-agonist, α, β-methylene ATP (10−9–10−4 M), and the P2U-agonist, UTP (10−9–10−4 M) only caused small transient relaxations at the highest concentrations (10−4 and 10−3 M).ATP and EFS-induced relaxations were unchanged in the presence of the P1-purinoceptor antagonist, 8-phenyltheophylline (10−5 M), while this antagonist inhibited the concentration-dependent relaxations to adenosine. In contrast, the P2-purinoceptor antagonist, suramin (3×10−5 M), markedly reduced the relaxations to EFS.After desensitization of P2X-purinoceptors with α, β-methylene ATP (2×10−5 M), the relaxations to exogenous added ATP were enhanced, but this procedure did not influence the relaxations to EFS. In contrast, the P2Y-purinoceptor antagonist, basilen blue E-3G (3×10−5 M, earlier named reactive blue 2) significantly inhibited the concentration-relaxation curves to ATP and almost abolished the EFS-induced relaxations.Mechanical removal of the endothelium significantly inhibited ATP-induced maximal relaxations without affecting sensitivity, pD2 and maximum relaxations being 4.72±0.12 and 89.7±3.8% (n=10), and 5.45±0.38 and 48.0±8.6% (P<0.05, paired t test, n=10) in endothelium-intact and -denuded coronary small arteries, respectively. However, incubation with L-NOARG did not change relaxations elicited by ATP.The present study suggests that in NANC conditions neurogenic relaxations of coronary small arteries are mediated by ATP, which relaxes coronary small arteries through P2Y-purinoceptors. A prejunctional modulation of these relaxations by endothelial-derived NO cannot be excluded.
PMCID: PMC1564479  PMID: 9031744
ATP; coronary small arteries; electrical field stimulation; endothelium; nitric oxide

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