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• W1566775034 abstract "Gap junctions are important for coordination and transfer of signals between cells. Signals initiated in the vascular endothelium can spread through myoendothelial gap junctions and cause relaxation of coupled vascular smooth muscle cells. The cellular level of acidity is important for control of vascular function. The mechanisms linking disturbed acid–base balance to changes in vascular tone have not been understood in detail. We show that intracellular alkalinisation of endothelial cells in resistance arteries inhibits myoendothelial coupling and endothelium-dependent vasorelaxation. Although hyperpolarisations are generated in the endothelial cells under alkaline conditions, they are not transferred to the smooth muscle cells. Similarly, dye transfer between endothelial cells is inhibited during intracellular alkalinisation. These results support the suggestion that intracellular pH is important for control of gap junction conductivity and intercellular communication. We describe a potential new molecular mechanism for development of vascular dysfunction in pathologies (e.g. diabetes, hypertension) involving altered myoendothelial signalling. Abstract Gap junctions mediate intercellular signalling in arteries and contribute to endothelium-dependent vasorelaxation, conducted vascular responses and vasomotion. Considering its putative role in vascular dysfunction, mechanistic insights regarding the control of gap junction conductivity are required. Here, we investigated the consequences of endothelial alkalinisation for gap junction communication and endothelium-dependent vasorelaxation in resistance arteries. We studied mesenteric arteries from NMRI mice by myography, confocal fluorescence microscopy and electrophysiological techniques. Removing CO2/HCO3−, reducing extracellular [Cl−] or adding 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid inhibited or reversed Cl−/HCO3− exchange, alkalinised the endothelium by 0.2–0.3 pH units and inhibited acetylcholine-induced vasorelaxation. NO-synthase-dependent vasorelaxation was unaffected by endothelial alkalinisation whereas vasorelaxation dependent on small- and intermediate-conductance Ca2+-activated K+ channels was attenuated by ∼75%. The difference in vasorelaxation between arteries with normal and elevated endothelial intracellular pH (pHi) was abolished by the gap junction inhibitors 18β-glycyrrhetinic acid and carbenoxolone while other putative modulators of endothelium-derived hyperpolarisations – Ba2+, ouabain, iberiotoxin, 8Br-cAMP and polyethylene glycol catalase – had no effect. In the absence of CO2/HCO3−, addition of the Na+/H+-exchange inhibitor cariporide normalised endothelial pHi and restored vasorelaxation to acetylcholine. Endothelial hyperpolarisations and Ca2+ responses to acetylcholine were unaffected by omission of CO2/HCO3−. By contrast, dye transfer between endothelial cells and endothelium-derived hyperpolarisations of vascular smooth muscle cells stimulated by acetylcholine or the proteinase-activated receptor 2 agonist SLIGRL-amide were inhibited in the absence of CO2/HCO3−. We conclude that intracellular alkalinisation of endothelial cells attenuates endothelium-derived hyperpolarisations in resistance arteries due to inhibition of gap junction communication. These findings highlight the role of pHi in modulating vascular function. The vascular endothelium synthesises multiple vasoactive substances and activates vasomodulatory signalling cascades essential for regulation of active tension development. Production of nitric oxide (NO) by the endothelial NO-synthase (eNOS) and vasoactive prostanoids by cyclooxygenase (COX) are well-described catalytic steps involved in endothelium-dependent vasorelaxation. However, even following inhibition of eNOS and COX, endothelium-derived hyperpolarisation (EDH) and relaxation of vascular smooth muscle cells (VSMCs) is readily induced upon activation of the endothelium (Bellien et al. 2008). This NO- and prostanoid-independent vasorelaxant response has been known as the endothelium-derived hyperpolarising factor (EDHF) response and is prominent particularly in resistance arteries (Urakami-Harasawa et al. 1997) where it contributes to control of local tissue perfusion and systemic blood pressure. Several molecular mechanisms have been proposed to mediate EDH, including endothelial production of diffusible factors such as C-type natriuretic peptide (CNP) (Chauhan et al. 2003), 11,12-epoxyeicosatrienoic acid (11,12-EET) (Fisslthaler et al. 1999) and H2O2 (Shimokawa & Matoba, 2004) or generation of a local increase in extracellular [K+] leading to activation of inward rectifier K+ channels (Kir) and the Na+/K+-ATPase (Edwards et al. 1998). Most commonly, however, EDH is ascribed to activation of endothelial K+ channels of small (SK) and intermediate (IK) conductance (Busse et al. 2002; Bellien et al. 2008). This induces endothelial cell (EC) hyperpolarisation, and subsequent electrotonic spread of charge through myoendothelial gap junctions hyperpolarises and relaxes the coupled VSMCs (Edwards et al. 1999; Goto et al. 2002). Numerous studies have shown that EDH is altered during pathological conditions such as diabetes, hypertension and ischaemia/reperfusion injury (Feletou & Vanhoutte, 2004). Currently, however, not much is known about specific disease mechanisms that modify myoendothelial signalling, and further mechanistic insights are desired to elucidate the link between disease pathogenesis and altered EDH. One potential factor that modifies gap junction communication – and hence possibly EDH – is intracellular pH (pHi). Several studies have shown that gap junction communication is inhibited at low pHi (Turin & Warner, 1977; Spray et al. 1981); and more recently, a bell-shaped relationship between the junctional resistance and pHi has been reported in isolated cardiomyocyte cell pairs with maximal conduction observed around pHi 7.0 (Swietach et al. 2007). As this pH optimum is slightly below resting physiological pHi, gap junction conductivity would be predicted to be particularly sensitive to alkaline shifts in pHi and recently the inhibitory effect of intracellular alkalinisation on gap junction communication was confirmed using cell pairs of the mouse neuroblastoma cell line N2A heterologously expressing connexin 36 (Gonzalez-Nieto et al. 2008). Despite these interesting findings based on isolated cells, the consequences of pHi-mediated changes in gap junction resistance for physiological functions in general, and vascular functions in particular, have not previously been addressed. Acid–base disturbances are frequently encountered in clinical practice, particularly in patients with renal, pulmonary or metabolic disease. Furthermore, several studies have shown that membrane transport of acid–base equivalents is altered in cardiovascular disease (Boedtkjer & Aalkjaer, 2012). Although it has been realised for more than a century that extracellular acid–base disturbances greatly influence vascular function, the vasomotor effects of intracellular acid–base disturbances have only been addressed more recently (Boedtkjer & Aalkjaer, 2012). We have shown that EC acidification inhibits acetylcholine-induced NO-mediated vasorelaxation (Boedtkjer et al. 2011, 2012) consequent to a strong pH dependency of the eNOS (Fleming et al. 1994; Boedtkjer et al. 2011). In contrast, the vascular consequences of EC alkalinisation have not previously been determined. Cellular base extrusion in the vascular wall is predominantly mediated by Cl−/HCO3− exchangers (also known as anion exchangers, AEs), which under physiological conditions perform electroneutral exchange of extracellular Cl− for intracellular HCO3− and thus contribute in particular to the recovery of pHi during intracellular alkalinisation. In addition to the dependency on Cl− and HCO3−, the AEs are inhibited by 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS). Both AE2 (slc4a2) and AE3 (slc4a3) have been detected in arteries (Brosius et al. 1997). Under normal circumstances, EC steady-state pHi is established as a balance between the metabolic acid–base production, the net acid-loading activity of the AEs and the net acid-extruding activities of the Na+/H+-exchanger NHE1 and the Na+,HCO3−-cotransporter NBCn1 (Boedtkjer & Aalkjaer, 2012). In the present study, we used ion substitution and pharmacological inhibition to modify AE activity and investigate functional effects of EC alkalinisation on endothelium-dependent vasorelaxation. Male NMRI mice (7–12 weeks old) obtained from Taconic Europe (Ry, Denmark) were killed by cervical dislocation. The whole mesenteric bed was excised and first- or second-order mesenteric arteries were dissected free of surrounding tissue. All animal procedures were approved by the Danish Animal Care and Use Committee. Arteries were mounted in wire myographs (DMT, Aarhus, Denmark) and normalised to 90% of the internal diameter corresponding to a transmural pressure of 100 mmHg as previously described (Mulvany & Halpern, 1977). Following precontraction with noradrenaline to 60–80% of maximal active tension, the response to acetylcholine was investigated. When drugs with reversible actions were used, or consequences of ion substitution were investigated, effects of time were eliminated by alternating the order of the protocol applied to two or four arteries from the same mouse. The average response observed in these arteries was used to represent that animal. When drugs with irreversible actions were employed, arteries were run in parallel as time and vehicle controls. Endothelial pHi and [Ca2+]i responses were evaluated by fluorescence confocal microscopy of mouse mesenteric arteries. Endothelial pHi was measured in 2′,7′-bis-(carboxypropyl)-5(6)-carboxyfluorescein (BCPCF)-loaded inverted arteries as previously described (Boedtkjer & Aalkjaer, 2009). Intracellular [Ca2+] measurements from ECs were performed similar to previous reports (Boedtkjer et al. 2011), using a combination of Calcium Green-1 and Fura Red loaded preferentially into ECs by perfusion of the fluorophores through the lumen of the arteries. Resting endothelial [Ca2+]i levels in the presence and absence of CO2/HCO3− were investigated in paired experiments. Inhibition of the endoplasmic reticulum Ca2+-ATPase by cyclopiazonic acid (CPA) induces a uniform [Ca2+]i rise in the ECs (data not shown), and we analysed the proportion of ECs responding to acetylcholine with an increase in the [Ca2+]-dependent fluorescence ratio of more than 20% of the response to 10 μmol l−1 CPA. We also investigated the effect of CO2/HCO3− on the magnitude of the [Ca2+]i response in ECs that responded to acetylcholine application. For these paired analyses, the order of the protocol (i.e. whether the response to acetylcholine was investigated first under CO2/HCO3−-free or CO2/HCO3−-containing conditions) was alternated between preparations to avoid potential effects of time. The change in [Ca2+]-dependent fluorescence ratio was calculated from the average ratio level measured during the 20 s preceding the application of acetylcholine and during the last 45 s of the 2 min acetylcholine exposure. Arteries were mounted in a wire myograph (DMT) and normalised as described above. Membrane potential (Vm) measurements were performed using aluminium silicate microelectrodes (WPI, Hitchin, UK) with a resistance of 40–120 MΩ when backfilled with 3 mol l−1 KCl and recorded with an Intra-767 amplifier (WPI), visualised on an oscilloscope (Gould-Nicolet Technologies, Loughton, UK) and continuously stored with a PowerLab system (ADInstruments, Dunedin, New Zealand). Electrode entry into cells resulted in an abrupt drop in voltage followed by a sharp return to baseline upon retraction. To measure membrane potentials from VSMCs, arteries were mounted in normal configuration and the electrode was advanced into the vessel wall from the adventitial side. Membrane potentials from ECs were measured using inverted arteries allowing the electrode to be inserted into ECs from the luminal surface of the arteries. To confirm that these respective methods resulted in measurements from the expected cell types, 5% propidium iodide was included in the electrode solution and the impaled cell visualised at the end of the experiment by confocal microscopy (excitation at 514 nm, emission recorded at wavelengths >560 nm), a principle previously employed by others (Siegl et al. 2005; Haddock et al. 2006; Behringer et al. 2012). If the ECs are dye coupled, the propidium iodide spreads from the impaled EC into neighbouring ECs, which also become fluorescently stained although at a somewhat reduced intensity. Disregarding fluorescence signals more than 50 μm from the nearest stained nucleus, we estimated the degree of cellular coupling by analysing the total number of stained nuclei. The composition of the CO2/HCO3−-containing physiological salt solution (PSS) used in this study was as follows (in mmol l−1): NaCl 114, Hepes 10, NaHCO3 25, MgSO4 1.20, KCl 4.70, glucose 5.50, EDTA 0.026, KH2PO4 1.18, CaCl2 1.60. In CO2/HCO3−-free solutions, NaHCO3 was substituted with an equimolar amount of NaCl. We used two different low [Cl−] solutions. In the first, NaCl was substituted with an equimolar amount of sodium gluconate while CaCl2 was substituted with 8.5 mmol l−1 calcium gluconate to compensate for the Ca2+-chelating action of gluconate, as suggested by others (Swietach et al. 2007). In the other solution, NaCl and KCl were substituted with equimolar amounts of non-chelating sodium methanesulphonate and potassium methanesulphonate, respectively. In all solutions, pH was adjusted to 7.40 at 37°C after bubbling with 5% CO2 balance air (CO2/HCO3−-containing solutions) or nominally CO2-free atmospheric air (CO2/HCO3−-free solutions). Data are expressed as mean ± SEM. Paired two-tailed Student's t test was used when two interventions on the same arteries were compared. When more than two interventions performed on the same arteries were compared, one-way ANOVA for repeated measures was employed followed by Bonferroni post-hoc tests. When successive interventions were compared between groups, two-way ANOVA for repeated measures was used followed by a Bonferroni post-hoc test. When measurements from arteries isolated from different mice were compared, unpaired statistical tests were employed. The number of stained nuclei in dye coupling experiments under different conditions was compared by a non-parametric Mann–Whitney test. Concentration–response relationships were analysed by sigmoidal curve fits and the derived parameters (logEC50 and maximum values) were compared by extra sum-of-squares F-tests. P < 0.05 was considered statistically significant; n equals number of mice (one to four arteries per mouse). Statistical analyses were performed using GraphPad Prism 5.03 software. Steady-state pHi in mesenteric artery ECs was 7.24 ± 0.09 (n= 19) in the presence of CO2/HCO3−. To study the effects of EC alkalinisation on vasomotor responses, we investigated changes in pHi caused by manoeuvres that inhibit or reverse Cl−/HCO3− exchange. Removing CO2/HCO3−, reducing extracellular [Cl−] to 4.7 mmol l−1 (by substitution with gluconate) or 3.2 mmol l−1 (by substitution with methanesulphonate) or adding 200 μmol l−1 DIDS alkalinised the ECs around 0.2–0.3 pH units (Fig. 1A). Prior application of 200 μmol l−1 DIDS completely abolished the alkalinisation observed when bath [Cl−] was reduced to 4.7 mmol l−1 by substitution with gluconate (ΔpHi=−0.05 ± 0.02, n= 4, P < 0.01 vs. gluconate substitution without DIDS; Supplementary Fig. 1). This is consistent with low bath [Cl−] reversing the DIDS-sensitive AE activity. Manoeuvres that inhibit or reverse Cl−/HCO3− exchange cause intracellular alkalinisation of ECs. The alkalinisation induced by omission of CO2/HCO3− can be reversed by simultaneous inhibition of Na+/H+-exchange A, mean steady-state pHi changes (obtained 15–30 min after the change of bath solution or addition of drug; n= 4–12) induced by omission of CO2/HCO3−, or by – in the presence of CO2/HCO3−– reducing extracellular [Cl−] or adding 200 μmol l−1 DIDS. Extracellular Cl− was substituted with either methanesulphonate or gluconate. The Ca2+-chelating action of gluconate was compensated for by increasing total [Ca2+] to 8.5 mmol l−1. B, mean steady-state pHi change (n= 4) induced by addition of 1 μmol l−1 of the NHE1-specific inhibitor cariporide in the absence of CO2/HCO3−. Statistical significance was determined by one-sample t tests. *P < 0.05, **P < 0.01, ***P < 0.001 vs. 0. In the absence of CO2/HCO3−, EC steady-state pHi was 7.53 ± 0.05 (n= 39). The pHi change caused by omission of CO2/HCO3− was reversible upon restoration of CO2/HCO3− (Supplementary Fig. 2A and B). Both omission and restoration of CO2/HCO3− elicited a fast transient change in pHi of large magnitude which within 5–10 min resulted in a stable steady-state change in pHi (Supplementary Fig. 2A and B) of the magnitude reported in Fig. 1A. Upon addition of 1 μmol l−1 of the NHE1-specific inhibitor cariporide in the absence of CO2/HCO3−, EC pHi declined and within approximately 5 min reached a new steady-state level close to that seen in the presence of CO2/HCO3− (Fig. 1B and Supplementary Fig. 3). To investigate the consequences of EC alkalinisation for endothelium-dependent regulation of artery tone, we studied the response of noradrenaline-precontracted mouse mesenteric arteries to acetylcholine. The effects of the interventions were investigated when pHi after an intervention (e.g. solution change or addition of drug) had reached a new steady-state level (see, for example, Supplementary Fig. 2). Under control conditions, arteries investigated in the presence of CO2/HCO3− relaxed around 70–80% when exposed to 0.5 or 3 μmol l−1 acetylcholine (Fig. 2A). This acetylcholine-induced vasorelaxation was reduced to around 50% in the absence of CO2/HCO3− (Fig. 2A). The difference between arteries investigated in the presence and absence of CO2/HCO3− was maintained after eNOS inhibition with 100 μmol l−1 N-nitro-l-arginine methyl ester (l-NAME; Fig. 2A). In contrast, the l-NAME-sensitive component of the vasorelaxant response to acetylcholine was unaffected by omission of CO2/HCO3− (Fig. 2A). After combined inhibition of COX (with 3 μmol l−1 indomethacin) and eNOS, the difference in vasorelaxation to acetylcholine was still observed between arteries investigated in the presence and absence of CO2/HCO3− (Fig. 2B and C). Representative traces of the l-NAME- and indomethacin-insensitive vasorelaxation to acetylcholine are shown in Fig. 2B and its sensitivity to IK and SK inhibitors (50 nmol l−1 apamin plus 50 nmol l−1 charybdotoxin or 1 μmol l−1 TRAM-34) is shown in Fig. 2D and E. Together, these results demonstrate that the EDH-type vasorelaxation in mouse mesenteric arteries is inhibited in the absence of CO2/HCO3−. The effect of removing CO2/HCO3− on endothelium-dependent vasorelaxation was reversible upon restoration of CO2/HCO3− and the order of the protocol was alternated between preparations to eliminate potential effects of time (Supplementary Fig. 2C). EDH-type vasorelaxation is reduced by omission of CO2/HCO3− in mesenteric arteries A, vasorelaxation to acetylcholine was attenuated in the absence of CO2/HCO3− both before and after treatment with 100 μmol l−1 l-NAME (n= 5). The effect of omitting or restoring CO2/HCO3− was investigated 30 min after the buffer change to allow a new steady-state pHi to be reached (Supplementary Fig. 2A and B). In this and the following panels, the black bars and traces represent experiments performed in the presence of CO2/HCO3−, while the red bars and traces represent experiments performed under CO2/HCO3−-free conditions. B, representative force recordings showing the relaxation of noradrenaline (NA)-precontracted arteries to acetylcholine (ACh) in the presence of 100 μmol l−1 l-NAME and 3 μmol l−1 indomethacin with or without CO2/HCO3−. C, vasorelaxation to acetylcholine after treatment with 100 μmol l−1 l-NAME and 3 μmol l−1 indomethacin was reduced in the absence of CO2/HCO3− (n= 12). The effect of omitting or restoring CO2/HCO3− was investigated 30 min after the buffer change to allow a new steady-state pHi to be reached. D, vasorelaxation to acetylcholine in the presence of 100 μmol l−1 l-NAME and 3 μmol l−1 indomethacin was abolished by 50 nmol l−1 apamin and 1 μmol l−1 TRAM-34 (n= 5). E, vasorelaxation to acetylcholine in the presence of 100 μmol l−1 l-NAME and 3 μmol l−1 indomethacin was abolished by 50 nmol l−1 apamin and 50 nmol l−1 charybdotoxin (n= 5). Comparisons were made with two-way ANOVA followed by Bonferroni post-hoc tests. *P < 0.05, **P < 0.01, ***P < 0.001, NS: not significantly different vs. CO2/HCO3−. #P < 0.05, ##P < 0.01, ###P < 0.001 vs. CO2/HCO3− in C. We hypothesised that EC alkalinisation was the cause of the attenuated EDH-type vasorelaxation and investigated whether other manoeuvres that alkalinise the endothelium (Fig. 1A) could replicate the effect of removing CO2/HCO3−. Indeed, the response to acetylcholine was greatly attenuated when bath [Cl−] was reduced to 4.7 mmol l−1 by substitution with gluconate (Fig. 3A) or 3.2 mmol l−1 by substitution with methanesulphonate (Fig. 3B). Also, 200 μmol l−1 DIDS, a non-specific AE inhibitor, reduced EDH-type vasorelaxation (Fig. 3C). To further support that pHi is the mechanistic link between omission of CO2/HCO3− and reduced EDH-type vasorelaxation, we investigated whether restoring EC pHi in the absence of CO2/HCO3− would restore the EDH. Indeed, addition of 1 μmol l−1 cariporide not only returned EC pHi to near-normal levels (Fig. 1 and Supplementary Fig. 3), it also rescued the EDH-type vasorelaxation (Fig. 3D). DIDS and low extracellular [Cl−] inhibit EDH-type vasorelaxation, while the Na+/H+ exchange inhibitor cariporide, which restores EC pHi, also rescues the EDH-type vasorelaxation in the absence of CO2/HCO3− A, reduction of extracellular [Cl−] to 4.7 mmol l−1 by substitution with gluconate inhibits EDH-type vasorelaxation (n= 4). The effect of reducing or restoring extracellular [Cl−] was investigated 15 min after the solution change to allow pHi to reach a new steady-state level (Supplementary Fig. 1). B, reduction of extracellular [Cl−] to 3.2 mmol l−1 by substitution with methanesulphonate inhibits EDH-type vasorelaxation (n= 5). The effect of reducing or restoring extracellular [Cl−] was investigated 15 min after the solution change to allow pHi to reach a new steady-state level (Supplementary Fig. 1). C, addition of 200 μmol l−1 DIDS inhibits EDH-type vasorelaxation. The relaxation relative to a pre-treatment control relaxation is shown together with a time control run in parallel (n= 5). The effect of DIDS was investigated 20 min after addition of the drug to allow time for pHi to reach a new steady-state level. D, EDH-type vasorelaxation evoked by 3 μmol l−1 acetylcholine was restored by addition of 1 μmol l−1 cariporide in the absence of CO2/HCO3− (n= 8). The effect of cariporide was investigated 15 min after addition of the drug, when a new steady-state pHi level had been reached (Supplementary Fig. 3). Comparisons were made with a two-way ANOVA followed by a Bonferroni post-hoc test. *P < 0.05, **P < 0.01, ***P < 0.001 vs. CO2/HCO3− or as specified. NS, not significantly different. To confirm that the attenuated EDH-type vasorelaxation was not due to a general inhibition of VSMC relaxation, we finally investigated the response to the endothelium-independent vasodilator S-nitroso-N-acetylpenicillamine (SNAP). We found no significant difference in the sensitivity (logEC50=−5.78 ± 0.06 with CO2/HCO3− and logEC50=−5.80 ± 0.10 without CO2/HCO3−; n= 6; P= 0.81, extra sum-of-squares F-test) or maximum response (maximum relative relaxation was 102 ± 4% with CO2/HCO3− and 94 ± 6% without CO2/HCO3−; n= 6; P= 0.30, extra sum-of-squares F-test) to the NO donor SNAP between arteries investigated in the presence and nominal absence of CO2/HCO3− (Supplementary Fig. 4). To investigate the mechanistic background for the altered vascular responsiveness to acetylcholine during endothelial alkalinisation, we investigated whether it was associated with changes in VSMC membrane potential. The resting VSMC membrane potential was similar in the presence (Vm=−47.1 ± 1.5 mV; n= 11) and absence (Vm=−46.3 ± 1.1 mV; n= 9) of CO2/HCO3−. We showed also in paired experiments that omission of CO2/HCO3− did not change the VSMC membrane potential (ΔVm=−0.2 ± 2.5 mV; n= 5; P= 0.93, one-sample two-tailed t test). We next measured VSMC membrane potential responses to acetylcholine in the presence of 100 μmol l−1 l-NAME and 3 μmol l−1 indomethacin. In the presence of CO2/HCO3−, application of 3 μmol l−1 acetylcholine hyperpolarised VSMCs by 10.8 ± 2.0 mV (Fig. 4A and B). In contrast, when arteries were investigated in the absence of CO2/HCO3−, the VSMC membrane potential was unaffected by application of acetylcholine (ΔVm=−1.0 ± 1.0 mV; Fig. 4A and B). EDH-type VSMC hyperpolarisations to acetylcholine and SLIGRL-amide are attenuated by omission of CO2/HCO3− while the response to pinacidil is unaffected A, average traces (n= 4–5) of the VSMC membrane potential response to acetylcholine in the presence and absence of CO2/HCO3−. All experiments were performed in the presence of 100 μmol l−1 l-NAME and 3 μmol l−1 indomethacin. Vertical grey lines represent SEM. Membrane potentials were measured at least 30 min after the change of bath solution to allow pHi to reach a new steady-state level. B, average effect (n= 4–5) of 3 μmol l−1 acetylcholine on VSMC membrane potential in the presence and absence of CO2/HCO3−. All experiments were performed in the presence of 100 μmol l−1 l-NAME and 3 μmol l−1 indomethacin. C, original traces showing the VSMC membrane potential response to 10 μmol l−1 SLIGRL-amide in the presence and absence of CO2/HCO3−. The experiments were performed in the presence of 100 nmol l−1 iberiotoxin, 100 μmol l−1 l-NAME and 3 μmol l−1 indomethacin. Membrane potentials were measured at least 30 min after the change of bath solution to allow pHi to reach a new steady-state level. D, average effect (n= 4) of 10 μmol l−1 SLIGRL-amide on VSMC membrane potential in the presence and absence of CO2/HCO3−. All experiments were performed in the presence of 100 nmol l−1 iberiotoxin, 100 μmol l−1 l-NAME and 3 μmol l−1 indomethacin. E, average effect (n= 3) of 10 μmol l−1 SLIGRL-amide on VSMC membrane potential in the presence and absence of 50 nmol l−1 apamin and 1 μmol l−1 TRAM-34. All experiments were performed in the presence of CO2/HCO3−, 100 μmol l−1 l-NAME, 3 μmol l−1 indomethacin and 100 nmol l−1 iberiotoxin. F, original trace of the VSMC membrane potential response to 3 μmol l−1 acetylcholine and 10 μmol l−1 pinacidil in the absence of CO2/HCO3−. G, average VSMC membrane potential responses (n= 2–3) to 10 μmol l−1 pinacidil applied in the presence or absence of CO2/HCO3−. Membrane potentials were measured at least 30 min after the change of bath solution to allow pHi to reach a new steady-state level. The comparisons were performed by paired or unpaired Student's t tests. *P < 0.05, **P < 0.01, NS: not significantly different vs. CO2/HCO3−. To investigate whether another agonist which activates EDH shows a similar dependency on CO2/HCO3−, we measured the VSMC membrane potential response to 10 μmol l−1 of the proteinase-activated receptor-2 (PAR2) agonist SLIGRL-amide (McGuire et al. 2002). Initial experiments indicated that SLIGRL-amide in addition to endothelium-dependent effects acted directly on the VSMCs through activation of large conductance (BK) Ca2+-activated K+ channels; and hence, all experiments in addition to 100 μmol l−1 l-NAME and 3 μmol l−1 indomethacin were performed in the presence of 100 nmol l−1 of the BK-selective antagonist iberiotoxin. As shown in Fig. 4C and D, SLIGRL-amide induced a strong VSMC hyperpolarisation in the presence of CO2/HCO3− (ΔVm=−21.5 ± 3.3 mV). This response was completely abolished in the nominal absence of CO2/HCO3− (ΔVm=−1.0 ± 1.8 mV; Fig. 4C and D). Supporting the EDH-type nature of the response to SLIGRL-amide under the given conditions, we showed that the hyperpolarisation to 10 μmol l−1 SLIGRL-amide in the presence of CO2/HCO3− was completely inhibited after application of 50 nmol l−1 apamin and 1 μmol l−1 TRAM-34 (Fig. 4E). Importantly, although no hyperpolarisation was seen to the endothelium-dependent agonists acetylcholine and SLIGRL-amide in the absence of CO2/HCO3−, a normal hyperpolarisation was evoked when pinacidil (10 μmol l−1) was applied under the same conditions to open ATP-sensitive K+ channels (KATP channels) in the VSMCs and thus alter VSMC membrane potential independently of the endothelium (Fig. 4F and G). As previously described, the mechanism responsible for EDH remains controversial, and probably varies between vascular beds. Thus, we investigated which mechanism(s) was responsible for the acetylcholine-induced EDH-type vasorelaxation in the investigated mouse mesenteric arteries (Fig. 5). No effect of the Kir channel inhibitor Ba2+ (30 μmol l−1; Fig. 5A), the BK channel-specific inhibitor iberiotoxin (100 nmol l−1; Fig. 5B) or the H2O2 scavenging enzyme polyethylene glycol (PEG)-catalase (250 U ml−1; Fig. 5C) was seen. The EDH-type vasorelaxation, however, was inhibited by application of the gap junction inhibitors carbenoxolone (100 μmol l−1; Fig. 5D) or 18β-glycyrrhetinic acid (30 μmol l−1; Fig. 5E). Although these widely used gap junction inhibitors have additional unspecific effects (Matchkov et al. 2004; Behringer et al. 2012), our findings are consistent with the EDH-type vasorelaxation in mouse mesenteric small arteries being medi" @default.
• W1566775034 created "2016-06-24" @default.
• W1566775034 creator A5003682621 @default.
• W1566775034 creator A5009773928 @default.
• W1566775034 creator A5035755730 @default.
• W1566775034 date "2013-02-11" @default.
• W1566775034 modified "2023-09-24" @default.
• W1566775034 title "Endothelial alkalinisation inhibits gap junction communication and endothelium-derived hyperpolarisations in mouse mesenteric arteries" @default.
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