Gillian Edwards

Background and Purpose: Perivascular adipose tissue (PVAT) releases adipocyte‐derived hyperpolarizing factors (ADHFs) that may partly act by opening myocyte K⁺ channels. The present study in rat and mouse mesenteric arteries aimed to identify the myocyte K⁺ channel activated by PVAT and to determine whether adiponectin contributed to the hyperpolarizing effects of PVAT.Experimental Approach: Myocyte membrane potential was recorded from de‐endothelialized, non‐contracted rat and mouse mesenteric arteries in the presence and absence of PVAT.Key Results: The β₃‐adrenoceptor agonist, CL‐316,243 (10 μM), generated PVAT‐dependent, iberiotoxin‐sensitive myocyte hyperpolarizations resulting from BK_Ca channel opening and which were partially blocked by L‐NMMA (100 μM). Adiponectin (5 μg·mL⁻¹) also produced iberiotoxin‐sensitive hyperpolarizations in PVAT‐denuded arterioles. Activation of myocyte AMP‐activated protein kinase (AMPK) using 5 μM A‐769662 also induced BK_Ca‐mediated hyperpolarizations. Dorsomorphin abolished hyperpolarizations to CL‐316,243, adiponectin and A‐769662. In vessels from Adipo^−/− mice, hyperpolarizations to CL‐316,243 were absent whereas those to A‐769662 and adiponectin were normal. In rat vessels, adipocyte‐dependent hyperpolarizations were blocked by glibenclamide and clotrimazole but those to NS1619 (33 μM) were unaltered.Conclusions and Implications: Under basal, non‐contracted conditions, β₃‐adrenoceptor stimulation of PVAT releases an ADHF, which is probably adiponectin. This activates AMPK to open myocyte BK_Ca channels indirectly and additionally liberates NO, which also contributes to the observed PVAT‐dependent myocyte hyperpolarizations. Clotrimazole and glibenclamide each reversed hyperpolarizations to adiponectin and A‐769662, suggesting the involvement of myocyte TRPM4 channels in the ADHF‐induced myocyte electrical changes mediated via the opening of BK_Ca channels.

The extracellular calcium-sensing receptor (CaR) was first described in the parathyroid gland. Recent studies have shown that the CaR is also expressed in blood vessels, especially in the endothelial and adventitial layers but its physiological function is still not clear. However, an understanding of its possible role(s) in the vasculature (perivascular-neurones, heart and blood vessels) is important because of the use of synthetic positive allosteric CaR modulators in hyperparathyroidism and the potential importance of negative modulators in the treatment of osteoporosis. In this review, the effects of CaR activation and inhibition are detailed and the possible role of the CaR as both an amplifier and attenuator of myo-endothelial coupling in the vasculature is described.

Background and purpose:  Previous studies have shown that endothelium‐dependent hyperpolarization of myocytes is reduced in resistance arteries from spontaneously hypertensive rats (SHRs). The aim of the present study was to determine whether this reflects down‐regulation of endothelial K⁺ channels or their associated pathways.Experimental approach:  Changes in vascular K⁺ channel responses and expression were determined by a combination of membrane potential recordings and Western blotting.Key results:  Endothelium‐dependent myocyte hyperpolarizations induced by acetylcholine, 6,7‐dichloro‐1H‐indole‐2,3‐dione 3‐oxime (NS309) (opens small‐ and intermediate‐conductance calcium‐sensitive K⁺ channels, SK_Ca and IK_Ca, respectively) or cyclohexyl‐[2‐(3,5‐dimethyl‐pyrazol‐1‐yl)‐6‐methyl‐pyrimidin‐4‐yl]‐amine (SK_Ca opener) were reduced in mesenteric arteries from SHRs. After blocking SK_Ca channels with apamin, hyperpolarizations to acetylcholine and NS309 in SHR arteries were similar to those of controls. Hyperpolarization to 5 mM KCl was reduced in SHR arteries due to loss of the Ba²⁺‐sensitive, inward‐rectifier channel (K_IR) component; the contribution of ouabain‐sensitive, Na⁺/K⁺‐ATPases was unaffected. Protein expression of both SK_Ca and K_IR channels was reduced in SHR arteries; the caveolin‐1 monomer/dimer ratio was increased.Conclusions and implications:  In SHRs, the distinct pathway that generates endothelium‐dependent hyperpolarization in vascular myocyte by activation of IK_Ca channels and Na⁺/K⁺‐ATPases remains intact. The second pathway, initiated by endothelial SK_Ca channel activation and amplified by K_IR opening on both endothelial cells and myocytes is compromised in SHRs due to down‐regulation of both SK_Ca and K_IR and to changes in caveolin‐1 oligomers. These impairments in the SK_Ca–K_IR pathway shed new light on vascular control mechanisms and on the underlying vascular changes in hypertension.This article is commented on by Garland, pp. 833–835 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476‐5381.2010.00692.x

Background and purpose: The extracellular calcium‐sensing receptor (CaR) in vascular endothelial cells activates endothelial intermediate‐conductance, calcium‐sensitive K⁺ channels (IK_Ca) indirectly leading to myocyte hyperpolarization. We determined whether CaR expression and function was modified in a rat model of type II diabetes.Experimental approach: Pressure myography, western blotting, sharp microelectrode and K⁺‐selective electrode recordings were used to investigate the functional expression of the CaR and IK_Ca in rat mesenteric arteries.Key results: Myocyte hyperpolarization to the CaR activator calindol was inhibited by Calhex 231. U46619‐induced vessel contraction elevated the extracellular [K⁺] around the myocytes, and inhibition of this ‘K⁺ cloud’ by iberiotoxin was needed to reveal calindol‐induced vasodilatations. These were antagonized by Calhex 231 and significantly smaller in Zucker diabetic fatty rat (ZDF) vessels than in Zucker lean (ZL) controls. Myocyte hyperpolarizations to calindol were also smaller in ZDF than in ZL arteries. In ZDF vessels, endothelial cell CaR protein expression was reduced; IK_Ca expression was also diminished, but IK_Ca‐generated hyperpolarizations mediated by 1‐EBIO were unaffected.Conclusions and implications: The reduced CaR‐mediated hyperpolarizing and vasodilator responses in ZDF arteries result from a decrease in CaR expression, rather than from a modification of IK_Ca channels. Detection of CaR‐mediated vasodilatation required the presence of iberiotoxin, suggesting a CaR contribution to vascular diameter, that is, inversely related to the degree of vasoconstriction. Compromise of the CaR pathway would favour the long‐term development of a higher basal vascular tone and could contribute to the vascular complications associated with type II diabetes.British Journal of Pharmacology (2008) 154, 652–662; doi:10.1038/bjp.2008.108; published online 14 April 2008

Background and purpose: The small and intermediate conductance, Ca²⁺‐sensitive K⁺ channels (SK_Ca and IK_Ca, respectively) which are pivotal in the EDHF pathway may be differentially activated. The importance of caveolae in the functioning of IK_Ca and SK_Ca channels was investigated.Experimental approach: The effect of the caveolae‐disrupting agent methyl‐β‐cyclodextrin (MβCD) on IK_Ca and SK_Ca localization and function was determined.Key results: EDHF‐mediated, SK_Ca‐dependent myocyte hyperpolarizations evoked by acetylcholine in rat mesenteric arteries (following blockade of IK_Ca with TRAM‐34) were inhibited by MβCD. Hyperpolarizations evoked by direct SK_Ca channel activation (using NS309 in the presence of TRAM‐34) were also inhibited by MβCD, an effect reversed by cholesterol. In contrast, IK_Ca‐dependent hyperpolarizations (in the presence of apamin) were unaffected by MβCD. Similarly, in porcine coronary arteries, EDHF‐mediated, SK_Ca‐dependent (but not IK_Ca‐dependent) endothelial cell hyperpolarizations evoked by substance P were inhibited by MβCD. In mesenteric artery homogenates subjected to sucrose‐density centrifugation, caveolin‐1 and SK3 (SK_Ca) proteins but not IK1 (IK_Ca) protein migrated to the buoyant, caveolin‐rich fraction. MβCD pretreatment redistributed caveolin‐1 and SK3 proteins into more dense fractions. In immunofluorescence images of porcine coronary artery endothelium, SK3 (but not IK1) and caveolin‐1 were co‐localized. Furthermore, caveolin‐1 immunoprecipitates prepared from native porcine coronary artery endothelium contained SK3 but not IK1 protein.Conclusions and Implications: These data provide strong evidence that endothelial cell SK_Ca channels are located in caveolae while the IK_Ca channels reside in a different membrane compartment. These studies reveal cellular organisation as a further complexity in the EDHF pathway signalling cascade.British Journal of Pharmacology (2007) 151, 332–340; doi:10.1038/sj.bjp.0707222

In coronary arteries, bradykinin opens endothelial intermediate‐ and small‐conductance Ca²⁺‐sensitive K⁺ channels (IK_Ca and SK_Ca) and, additionally, releases epoxyeicosatrienoic acids (EETs) from the endothelium. To clarify the involvement of these pathways in endothelium‐dependent myocyte hyperpolarization, bradykinin‐induced electrical changes in endothelial cells and myocytes of porcine coronary arteries (following nitric oxide (NO) synthase and cyclooxygenase inhibition) were measured using sharp microelectrodes. Hyperpolarization of endothelial cells by bradykinin (27.0±0.9 mV, n=4) was partially inhibited (74%) by blockade of IK_Ca and SK_Ca channels using 10 μM TRAM‐39 (2‐(2‐chlorophenyl)‐2,2‐diphenylacetonitrile) plus 100 nM apamin (leaving an iberiotoxin‐sensitive component), whereas the response to substance P was abolished. After gap junction blockade with HEPES, (N‐(2‐hydroxyethyl)piperazine‐N′‐(2‐ethanesulphonic acid)) hyperpolarization of the endothelium by 100 nM bradykinin was abolished by TRAM‐39 plus apamin, whereas myocyte hyperpolarization still occurred (12.9±1.0 mV, n=4). The residual hyperpolarizations to 100 nM bradykinin were antagonized by the EET antagonist, 14,15‐EEZE (14,15‐epoxyeicosa‐5(Z)‐enoic acid) (10 μM), and abolished by iberiotoxin. Bradykinin‐induced myocyte hyperpolarizations were also reduced by 14,15‐EEZE‐mSI (14,15‐EEZE‐methylsulfonylimide) (5,6‐ and 14,15‐EET antagonist), whereas those to exogenous 11,12‐EET were unaffected. These data show that bradykinin‐induced hyperpolarization of endothelial cells (due to the opening of IK_Ca and SK_Ca channels) is electrotonically transferred to the myocytes via gap junctions. Bradykinin (but not substance P) also hyperpolarizes myocytes by a mechanism (independent of endothelial cell hyperpolarization) which involves endothelial cell production of EETs (most likely 14,15‐ and/or 11,12‐EET). These open endothelial IK_Ca and SK_Ca channels and also activate large‐conductance calcium‐sensitive K⁺ channels (BK_Ca) on the surrounding myocytes. British Journal of Pharmacology (2005) 145, 775–784. doi:10.1038/sj.bjp.0706256