Chris Hillier

Endothelium-derived hyperpolarizing factor: Identification and mechanisms of action in human subcutaneous resistance arteries

BackgroundBoth a vascular endothelial cytochrome P450 (CYP450) product of arachidonic acid metabolism and the potassium ion (K+) have been identified as endothelium-derived hyperpolarizing factors (EDHFs) in animal vascular tissues. We studied the relative importance of EDHF, nitric oxide (NO), and prostacyclin (PGI2) as vasodilators in human subcutaneous arteries. We also examined the mechanisms underlying the vasodilator action of EDHF to elucidate its identity. Methods and ResultsSubcutaneous resistance arteries were obtained from 41 healthy volunteers. The contribution of EDHF to the vasodilation induced by acetylcholine was assessed by inhibiting production of NO, PGI2, and membrane hyperpolarization. The mechanisms underlying the relaxation evoked by K+ and EDHF were elucidated. EDHF was found to account for ≈80% of acetylcholine-mediated vasorelaxation. Its action was insensitive to the combination of barium and ouabain, whereas barium and ouabain reversed K+-mediated vasorelaxation. EDHF-mediated vasorelaxation, however, was sensitive to the phospholipase A2 inhibitor oleyloxyethyl phosphorylcholine and the CYP450 inhibitor ketoconazole. ConclusionsEDHF is the major contributor to endothelium-dependent vasorelaxation in human subcutaneous resistance arteries. A product of phospholipase A2/CYP450-dependent metabolism of arachidonic acid and not K+ is the likely identity of EDHF in human subcutaneous resistance arteries.

Neurohumoral effects of the new orally active renin inhibitor, aliskiren, in chronic heart failure

Suppression of the renin–angiotensin–aldosterone system (RAAS) is therapeutically valuable in chronic heart failure (CHF). RAAS inhibition can be achieved in a number of ways though an orally active renin inhibitor (RI) has never been studied before. We describe the neurohumoral effects of an RI.

Signalling mechanisms underlying the myogenic response in human subcutaneous resistance arteries.

OBJECTIVE In this study we have examined for the first time the signal transduction mechanisms involved in the generation of pressure-dependent myogenic tone in human small resistance arteries from the subcutaneous vascular bed. METHODS Myogenic responses and the subcellular mechanisms involved in the generation of this response were studied on a pressure myograph. RESULTS AND CONCLUSION Human subcutaneous resistance arteries constricted 14.1+/-1.1% in response to an increases in intraluminal pressure from 40 to 80 mmHg and a further 3.5+/-1.7% in response to the 80-120-mmHg pressure step. Ca(2+) depletion or nifedipine abolished this response, whereas BAY K 8644 increased this response to 20.6+/-2.1% (P<0.05, response vs. control). The phospholipase C inhibitor U-73122 reduced the myogenic response to 2.5+/-1.0% at 80 mmHg (P<0.01, response vs. control) and abolished it at 120 mmHg. Diacylglycerol lipase inhibition with RHC-80267 abolished all myogenic responses to pressure. The protein kinase C (PKC) activator phorbol 12,13-dibutyrate increased the maximal myogenic response to 20.9+/-1.8% (P<0.05, response vs. control), whereas the PKC inhibitor calphostin C abolished myogenic responses. These data show that the generation of pressure-dependent myogenic tone in human subcutaneous arteries is dependent on Ca(2+) influx via voltage operated Ca(2+) channels (VOCCs) and a concomitant requirement for the activation of phospholipase C (PLC), diacylglycerol, and PKC.

α1-Adrenoceptor subtypes involved in vasoconstrictor responses to exogenous and neurally released noradrenaline in rat femoral resistance arteries

The α1‐adrenoceptor subtypes involved in responses to exogenous and neurally released noradrenaline in rat femoral resistance arteries were characterised using a small vessel myograph, with antagonists prazosin (nonsubtype selective), 5‐methyl‐urapidil (α1A‐selective), BMY 7378 (α1D‐selective) and the alkylating agent chloroethylclonidine (preferential for α1B‐). Prazosin and 5‐methyl‐urapidil produced rightward shifts of the exogenous noradrenaline concentration – response curve (CRC) with pA2 values of 9.2 and 9.1 respectively, in agreement with the presence of α1A‐adrenoceptors. BMY 7378 (1 μM) shifted the noradrenaline CRC with an apparent pKB of 6.7, in agreement with the presence of α1A‐, but not α1D‐, adrenoceptors. Chloroethylclonidine at 1 μM had no effect and at 10 μM produced only a small reduction (c. 20%) in the maximum response to noradrenaline, indicating little, if any, contribution from α1B‐adrenoceptors. Responses of the rat femoral resistance arteries to electrical field stimulation (EFS) at 5–30 Hz for 10 s and 0.05 ms pulse width were principally due to α1‐adrenoceptor stimulation. Prazosin and 5‐methyl‐urapidil inhibited EFS‐mediated responses with pIC50s of 9.3 and 8.2, respectively, consistent with the α1A‐adrenoceptor being the predominant subtype. Responses to EFS at 10–30 Hz were relatively insensitive to BMY 7378 (pIC50, 6.5–6.7), while responses to 5 Hz were inhibited with a significantly higher pIC50 of 8.02, suggesting the contribution of α1D‐adrenoceptors. Chloroethylclonidine had no effect on responses to EFS, ruling out the contribution of an α1B‐subtype. In the presence of cocaine, the predominant subtype involved in responses to EFS was the α1A‐adrenoceptor, with a contribution from α1D‐adrenoceptors at low frequency, as seen in the absence of cocaine. However, there was also a significant increase in the sensitivity to BMY 7378 at higher frequencies, suggesting that a further small α1D‐adrenoceptor component may be uncovered in the presence of cocaine. The present study has shown a predominant role of the α1A‐adrenoceptor in contractions due to exogenous noradrenaline and to neurally released noradrenaline in rat femoral resistance arteries. α1D‐Adrenoceptors are not involved in responses to exogenous noradrenaline but appear to be activated by neurally released noradrenaline at a low frequency of stimulation and at higher frequencies in the presence of neuronal‐uptake blockade.

Functional characterization of α1‐adrenoceptor subtypes in human skeletal muscle resistance arteries

α1‐adrenoceptor subtypes in human skeletal muscle resistance arteries were characterized using agonists noradrenaline (non‐selective) and A61603 (α1A‐selective), the antagonists prazosin (non‐selective), 5‐methyl‐urapidil (α1A‐selective) and BMY7378 (α1D‐selective) and the alkylating agent chloroethylclonidine (preferential for α1B). Small arteries were obtained from the non‐ischaemic skeletal muscle of limbs amputated for critical limb ischaemia and isometric tension recorded using wire myography. Prazosin antagonized responses to noradrenaline with a pA2 value of 9.18, consistent with the presence of α1‐adrenoceptors, although the Schild slope (1.32) was significantly different from unity. 5‐Methyl‐urapidil competitively antagonized responses to noradrenaline with a pKB value of 8.48 and a Schild slope of 0.99, consistent with the presence of α1A‐adrenoceptors. In the presence of 300 nM 5‐methyl‐urapidil, noradrenaline exhibited biphasic concentration response curves, indicating the presence of a minor population of a 5‐methyl‐urapidil‐resistant subtype. Contractile responses to noradrenaline were not affected by 1 μM chloroethylclonidine suggesting the absence of α1B‐adrenoceptors. Maximum responses to noradrenaline and A61603 were reduced to a similar extent by 10 μM chloroethylclonidine, suggesting an effect of chloroethylclonidine at α1A‐adrenoceptors at the higher concentration. BMY7378 (10 and 100 nM) had no effect on responses to noradrenaline. BMY7378 (1 μM) poorly shifted the potency of noradrenaline giving a pA2 of 6.52. These results rule out the presence of the α1D‐subtype. These results show that contractile responses to noradrenaline in human skeletal muscle resistance arteries are predominantly mediated by the α1A‐adrenoceptor subtype with a minor population of an unknown α1‐adrenoceptor subtype.

Multiparameter detection of apoptosis using red-excitable SYTO probes.

Functional assays allowing phenotypic characterization of different cell death parameters at a single‐cell level are important tools for preclinical anticancer drug screening. Currently, the selection of cytometric assays is limited by the availability of fluorescent probes with overlapping spectral characteristics. Following on our earlier reports on green and orange fluorescent SYTO probes, we provide herein further insights into applicability of novel red‐excitable SYTO stains (SYTO 17, 59–64) for multiparameter analysis of cell fate. In particular, SYTO 62 appears to be a spectrally favorable candidate. Using a correlative comparison between SYTO 16, Annexin V, YO‐PRO 1, and fluorescently labeled inhibitors of caspases (FLICA), we demonstrate the specificity of SYTO 62 in detection of apoptotic cell death. Used in conjunction with FLICA or Annexin V, SYTO 62 stain proved amenable for multivariate kinetic analysis of apoptotic events. Considering simplicity of staining protocols, low cost, and avoidance of spectral compensation problems, we expect that red‐excitable SYTO dyes will find a wide range of cytometric applications.

Increased α1- and α2-adrenoceptor-mediated contractile responses of human skeletal muscle resistance arteries in chronic limb ischemia

Objective: Recently, we have shown augmented contractile responses of skeletal muscle resistance arteries to noradrenaline in patients with critical limb ischemia. We investigated whether this increased sensitivity in skeletal muscle resistance arteries is due to either α1- or α2-adrenoceptor-mediated responses or both. Methods: Skeletal muscle resistance arteries were isolated from the proximal (non-ischemic) and distal (ischemic) parts of limbs amputated for critical limb ischemia and mounted on a small vessel wire myograph. Cumulative concentration response curves of the vessel segments to noradrenaline, phenylephrine and brimonidine were obtained in the presence or the absence of the selective antagonists, prazosin and RS79948. Results: Noradrenaline and phenylephrine produced almost equal maximal contractile responses. Brimonidine responses were smaller and were almost abolished by 0.1 μM RS 79948 while those of phenylephrine and noradrenaline were not affected. Prazosin reduced the maximum responses to brimonidine, shifted the concentration response curves of noradrenaline and phenylephrine rightwards giving p K B values of 9.86 and 9.33, respectively. Maximum responses produced by all three agonists in distal vessels were significantly higher than those obtained in proximal vessels. Conclusions: Noradrenaline contractile responses in skeletal muscle resistance arteries are predominantly mediated by α1-adrenoceptors. Both α1- and α2-adrenoceptor-mediated responses are increased in the arteries from ischemic regions that may aggravate the decreased blood flow to the limbs due to arterial occlusion.

The α1A-adrenoceptor subtype mediates contraction in rat femoral resistance arteries

In this study, alpha(1)-adrenoceptor subtypes were characterised in rat femoral resistance arteries mounted on a small vessel myograph. A-61603 was found to be more potent than noradrenaline and phenylephrine in these arteries. Brimonidine (UK 14304) could not evoke any contractile responses and the sensitivity to noradrenaline and phenylephrine was not affected by (8aR,12aS,13aS)-5,8,8a,9,10,11,12,12a,13a-decahydro-3-methoxy-12-(ethylsulphonyl)-6H-isoquino[2,1-g][1,6]-naphthyridine (RS 79948), ruling out the presence of alpha(2)-adrenoceptors. Prazosin, 5-methyl-urapidil and 2-([2,6-dimethoxyphenoxyethyl]aminomethyl)-1,4-benzodioxane (WB 4101) produced rightward shifts in the sensitivity to noradrenaline, giving pA(2) values of 9.6, 9.4 and 10.4, respectively, in agreement with the presence of alpha(1A)-adrenoceptors. (8-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione (BMY 7378; 1 microM) produced a small shift in the sensitivity of noradrenaline giving a pK(B) of 7.2. In the presence of 300 nM 5-methyl-urapidil, sensitivity to noradrenaline was not further shifted by 1 microM BMY 7378. Responses to noradrenaline were unaffected by the alpha(1B)-adrenoceptor alkylating agent chloroethylclonidine (1 microM). These results suggest alpha(1A)-adrenoceptors mediate contractile responses to noradrenaline in rat femoral resistance arteries.

Endothelin mediated contraction of equine laminar veins

REASONS FOR PERFORMING STUDY Endothelin-1 (ET-1) may be a key mediator in the pathogenesis of laminitis, but endothelin-mediated responses in the venous microcirculation of the equine foot have yet to be fully characterised. OBJECTIVES To characterise the response of equine laminar veins to ET-1 and evaluate the ET-1 receptor subtypes that mediate this response. METHODS Small veins (150-500 microns) draining the equine digital laminae from healthy horses and ponies subjected to euthanasia at an abattoir were investigated using wire myography. Concentration response curves were constructed for ET-1 in the presence of ETA (BQ123) and ETB (BQ788) receptor antagonists, and L-NAME, a nitric oxide synthase blocker. The selective ETB receptor agonist BQ3020 was investigated alone and following incubation with L-NAME, with or without BQ788. RESULTS Endothelin-1 contraction of laminar veins was significantly inhibited by BQ123 but not by BQ788. In the presence of L-NAME, sensitivity of laminar veins to ET-1 was enhanced 4-fold, and further addition of BQ788 did not alter this increased sensitivity. BQ3020 induced no venoconstriction; however, in the presence of L-NAME, it caused contraction of veins with approximately 30% of the efficacy of ET-1. The action of BQ3020 in the presence of L-NAME was abolished by BQ788. CONCLUSIONS Both ETA and ETB receptors are involved in the net tonic response to ET-1 in normal laminar veins. A population of ETB receptors may be present on the vascular endothelium and on smooth muscle of laminar veins, and the action of ET-1 at these 2 sites is likely to be approximately equal and opposite. POTENTIAL RELEVANCE Our results clarify the function of the ET-1 receptor subtypes in laminar veins from healthy horses. Further study of ET-1 receptors in laminitic horses is therefore warranted.

Endothelial function is preserved in pregnant women with well‐controlled type 1 diabetes

Objective Pregnant women with diabetes mellitus have a higher incidence of adverse pregnancy outcomes. Vascular, and in particular, endothelial function may be significantly modified in diabetes resulting in impaired endothelium‐dependent relaxation. This study aims to investigate endothelium‐dependent relaxation in pregnant women with pre‐existing type 1 diabetes mellitus.