Kim A. Dora

Modulation of Endothelial Cell KCa3.1 Channels During Endothelium-Derived Hyperpolarizing Factor Signaling in Mesenteric Resistance Arteries

Arterial hyperpolarization to acetylcholine (ACh) reflects coactivation of KCa3.1 (IKCa) channels and KCa2.3 (SKCa) channels in the endothelium that transfers through myoendothelial gap junctions and diffusible factor(s) to affect smooth muscle relaxation (endothelium-derived hyperpolarizing factor [EDHF] response). However, ACh can differentially activate KCa3.1 and KCa2.3 channels, and we investigated the mechanisms responsible in rat mesenteric arteries. KCa3.1 channel input to EDHF hyperpolarization was enhanced by reducing external [Ca2+]o but blocked either with forskolin to activate protein kinase A or by limiting smooth muscle [Ca2+]i increases stimulated by phenylephrine depolarization. Imaging [Ca2+]i within the endothelial cell projections forming myoendothelial gap junctions revealed increases in cytoplasmic [Ca2+]i during endothelial stimulation with ACh that were unaffected by simultaneous increases in muscle [Ca2+]i evoked by phenylephrine. If gap junctions were uncoupled, KCa3.1 channels became the predominant input to EDHF hyperpolarization, and relaxation was inhibited with ouabain, implicating a crucial link through Na+/K+-ATPase. There was no evidence for an equivalent link through KCa2.3 channels nor between these channels and the putative EDHF pathway involving natriuretic peptide receptor-C. Reconstruction of confocal z-stack images from pressurized arteries revealed KCa2.3 immunostain at endothelial cell borders, including endothelial cell projections, whereas KCa3.1 channels and Na+/K+-ATPase α2/α3 subunits were highly concentrated in endothelial cell projections and adjacent to myoendothelial gap junctions. Thus, extracellular [Ca2+]o appears to modify KCa3.1 channel activity through a protein kinase A–dependent mechanism independent of changes in endothelial [Ca2+]i. The resulting hyperpolarization links to arterial relaxation largely through Na+/K+-ATPase, possibly reflecting K+ acting as an EDHF. In contrast, KCa2.3 hyperpolarization appears mainly to affect relaxation through myoendothelial gap junctions. Overall, these data suggest that K+ and myoendothelial coupling evoke EDHF-mediated relaxation through distinct, definable pathways.

Small‐ and Intermediate‐Conductance Calcium‐Activated K+ Channels Provide Different Facets of Endothelium‐Dependent Hyperpolarization in Rat Mesenteric Artery

Activation of both small‐conductance (SKCa) and intermediate‐conductance (IKCa) Ca2+‐activated K+ channels in endothelial cells leads to vascular smooth muscle hyperpolarization and relaxation in rat mesenteric arteries. The contribution that each endothelial K+ channel type makes to the smooth muscle hyperpolarization is unknown. In the presence of a nitric oxide (NO) synthase inhibitor, ACh evoked endothelium and concentration‐dependent smooth muscle hyperpolarization, increasing the resting potential (approx. −53 mV) by around 20 mV at 3 μm. Similar hyperpolarization was evoked with cyclopiazonic acid (10 μm, an inhibitor of sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA)) while 1‐EBIO (300 μm, an IKCa activator) only increased the potential by a few millivolts. Hyperpolarization in response to either ACh or CPA was abolished with apamin (50 nm, an SKCa blocker) but was unaltered by 1‐[(2‐chlorophenyl) diphenylmethyl]‐1H‐pyrazole (1 μm TRAM‐34, an IKCa blocker). During depolarization and contraction in response to phenylephrine (PE), ACh still increased the membrane potential to around −70 mV, but with apamin present the membrane potential only increased just beyond the original resting potential (circa−58 mV). TRAM‐34 alone did not affect hyperpolarization to ACh but, in combination with apamin, ACh‐evoked hyperpolarization was completely abolished. These data suggest that true endothelium‐dependent hyperpolarization of smooth muscle cells in response to ACh is attributable to SKCa channels, whereas IKCa channels play an important role during the ACh‐mediated repolarization phase only observed following depolarization.

Enhanced spontaneous Ca2+ events in endothelial cells reflect signalling through myoendothelial gap junctions in pressurized mesenteric arteries

Increases in global Ca(2+) in the endothelium are a crucial step in releasing relaxing factors to modulate arterial tone. In the present study we investigated spontaneous Ca(2+) events in endothelial cells, and the contribution of smooth muscle cells to these Ca(2+) events, in pressurized rat mesenteric resistance arteries. Spontaneous Ca(2+) events were observed under resting conditions in 34% of cells. These Ca(2+) events were absent in arteries preincubated with either cyclopiazonic acid or U-73122, but were unaffected by ryanodine or nicotinamide. Stimulation of smooth muscle cell depolarization and contraction with either phenylephrine or high concentrations of KCl significantly increased the frequency of endothelial cell Ca(2+) events. The putative gap junction uncouplers carbenoxolone and 18alpha-glycyrrhetinic acid each inhibited spontaneous and evoked Ca(2+) events, and the movement of calcein from endothelial to smooth muscle cells. In addition, spontaneous Ca(2+) events were diminished by nifedipine, lowering extracellular Ca(2+) levels, or by blockers of non-selective Ca(2+) influx pathways. These findings suggest that in pressurized rat mesenteric arteries, spontaneous Ca(2+) events in the endothelial cells appear to originate from endoplasmic reticulum IP(3) receptors, and are subject to regulation by surrounding smooth muscle cells via myoendothelial gap junctions, even under basal conditions.

Spreading dilatation in rat mesenteric arteries associated with calcium‐independent endothelial cell hyperpolarization

Both ACh and levcromakalim evoke smooth muscle cell hyperpolarization and associated relaxation in rat mesenteric resistance arteries. We investigated if they could evoke conducted vasodilatation along isolated arteries, whether this reflected spreading hyperpolarization and the possible mechanism involved. Focal micropipette application of either ACh, to stimulate endothelial cell muscarinic receptors, or levcromakalim, to activate smooth muscle KATP channels, each evoked a local dilatation (88 ± 14%, n= 6 and 92 ± 6% reversal of phenylephrine‐induced tone, n= 11, respectively) that rapidly spread upstream (at 1.5 mm 46 ± 19%, n= 6 and 57 ± 13%, n= 9) to dilate the entire isolated artery. The local dilatation to ACh was associated with a rise in endothelial cell [Ca2+]i (F/Ft = 0= 1.22 ± 0.33, n= 14) which did not spread beyond 0.5 mm (F/Ft = 0= 1.01 ± 0.01, n= 14), while the local dilatation to levcromakalim was not associated with any change in endothelial cell [Ca2+]i. In contrast, ACh and levcromakalim both stimulated local (12.7 ± 1.2 mV, n= 10 and 13.5 ± 4.7 mV, n= 10) and spreading (at 2 mm: 3.0 ± 1.1 mV, n= 5 and 4.1 ± 0.7 mV, n= 5) smooth muscle hyperpolarization. The spread of hyperpolarization could be prevented by cutting the artery, so was not due to a diffusible agent. Both the spreading dilatation and hyperpolarization were endothelium dependent. The injection of propidium iodide into either endothelial or smooth muscle cells revealed extensive dye coupling between the endothelial cells, but limited coupling between the smooth muscle cells. Some evidence for heterocellular spread of dye was also evident. Together, these data show that vasodilatation can spread over significant distances in mesenteric resistance arteries, and suggest this reflects an effective coupling between the endothelial cells to facilitate [Ca2+]i‐independent spread of hyperpolarization.

Activation of endothelial cell IK(Ca) with 1-ethyl-2-benzimidazolinone evokes smooth muscle hyperpolarization in rat isolated mesenteric artery.

In rat small mesenteric arteries contracted with phenylephrine, 1‐ethyl‐2‐benzimidazolinone (1‐EBIO; 3u2003–u2003300u2003μM) evoked concentration‐dependent relaxation that, above 100u2003μM, was associated with smooth muscle hyperpolarization. 1‐EBIO‐evoked hyperpolarization (maximum 22.1±3.6u2003mV with 300u2003μM, n=4) was endothelium‐dependent and inhibited by charybdotoxin (ChTX 100u2003nM; n=4) but not iberiotoxin (IbTX 100u2003nM; n=4). In endothelium‐intact arteries, smooth muscle relaxation to 1‐EBIO was not altered by either of the potassium channel blockers ChTX (100u2003nM; n=7), or IbTX (100u2003nM; n=4), or raised extracellular K+ (25u2003mM). Removal of the endothelium shifted the relaxation curve to the right but did not reduce the maximum relaxation. In freshly isolated mesenteric endothelial cells, 1‐EBIO (600u2003μM) evoked a ChTX‐sensitive outward K‐current. In contrast, 1‐EBIO had no effect on smooth muscle cell conductance whereas NS 1619 (33u2003μM) stimulated an outward current while having no effect on the endothelial cells. These data show that with concentrations greater than 100u2003μM, 1‐EBIO selectively activates outward current in endothelial cells, which presumably underlies the smooth muscle hyperpolarization and a component of the relaxation. Sensitivity to block with charybdotoxin but not iberiotoxin indicates this current is due to activation of IKCa. However, 1‐EBIO can also relax the smooth muscle by an undefined mechanism, independent of any change in membrane potential.

Spreading dilatation to luminal perfusion of ATP and UTP in rat isolated small mesenteric arteries

Levels of ATP achieved within the lumen of vessels suggest a key autacoid role. P2Y receptors on the endothelium may represent the target for ATP, leading to hyperpolarization and associated relaxation of vascular smooth muscle through the endothelium‐dependent hyperpolarizing factor (EDHF) pathway. EDHF signals radially from the endothelium to cause dilatation, and appears mechanistically distinct from the axial spread of dilatation, which we showed occurs independently of a change in endothelial cell Ca2+ in rat mesenteric arteries. Here we have investigated the potential of P2Y receptor stimulation to evoke spreading dilatation in rat resistance small arteries under physiological pressure and flow. Triple cannulation of isolated arteries enables focal application of purine and pyrimidine nucleotides to the endothelium, avoiding potential complicating actions of these agents on the smooth muscle. Nucleotides were locally infused through one branch of a bifurcation, causing near maximal local dilatation attributable to EDHF. Dilatation then spread rapidly into the adjacent feed artery and upstream against the direction of luminal flow, sufficient to increase flow into the feed artery. The rate of decay of this spreading dilatation was identical between nucleotides, and matched that to ACh, which acts only on the endothelium. In contrast, focal abluminal application of either ATP or UTP at the downstream end of cannulated arteries evoked constriction, which only in the case of ATP was also associated with modest spread of dilatation. The non‐hydrolysable ADP analogue, ADPβS, acting at P2Y1 receptors, caused robust local and spreading dilatation responses whether applied to the luminal or abluminal surface of pressurized arteries. Dilatation to nucleotides was sensitive to inhibition with apamin and TRAM‐34, selective blockers of small‐ and intermediate‐conductance Ca2+‐activated K+ channels, respectively. These data demonstrate that direct luminal stimulation of P2Y receptor on the endothelium of rat mesenteric arteries leads to marked spreading dilatation and thus suggests that circulating purines and pyrimidines may act as important regulators of blood flow.

A Role for Heterocellular Coupling and EETs in Dilation of Rat Cremaster Arteries

Objective: The authors probed endothelium‐dependent dilation and endothelial cell Ca2 + handling in myogenically active resistance arteries.

Possible Role for K+ in Endothelium-Derived Hyperpolarizing Factor–Linked Dilatation in Rat Middle Cerebral Artery

Background and Purpose— Endothelium-derived hyperpolarizing factor (EDHF) and K+ are vasodilators in the cerebral circulation. Recently, K+ has been suggested to contribute to EDHF-mediated responses in peripheral vessels. The EDHF response to the protease-activated receptor 2 ligand SLIGRL was characterized in cerebral arteries and used to assess whether K+ contributes as an EDHF. Methods— Rat middle cerebral arteries were mounted in either a wire or pressure myograph. Concentration-response curves to SLIGRL and K+ were constructed in the presence and absence of a variety of blocking agents. In some experiments, changes in tension and smooth muscle cell membrane potential were recorded simultaneously. Results— SLIGRL (0.02 to 20 &mgr;mol/L) stimulated concentration and endothelium-dependent relaxation. In the presence of NG-nitro-l-arginine methyl ester, relaxation to SLIGRL was associated with hyperpolarization and sensitivity to a specific inhibitor of IKCa, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (1&mgr;mol/L), reflecting activation of EDHF. Combined inhibition of KIR with Ba2+ (30&mgr;mol/L) and Na+/K+-ATPase with ouabain (1 &mgr;mol/L) markedly attenuated the relaxation to EDHF. Raising extracellular [K+] to 15 mmol/L also stimulated smooth muscle relaxation and hyperpolarization, which was also attenuated by combined application of Ba2+ and ouabain. Conclusions— SLIGRL evokes EDHF-mediated relaxation in the rat middle cerebral artery, underpinned by hyperpolarization of the smooth muscle. The profile of blockade of EDHF-mediated hyperpolarization and relaxation supports a pivotal role for IKCa channels. Furthermore, similar inhibition of responses to EDHF and exogenous K+ with Ba2+ and ouabain suggests that K+ may contribute as an EDHF in the middle cerebral artery.

Depletion of intracellular Ca2+ stores enhances flow-induced vascular dilatation in rat small mesenteric artery.

The effect of depleting intracellular Ca2+ stores on flow‐induced vascular dilatation and the mechanism responsible for the vasodilatation were examined in rat isolated small mesenteric arteries. The arteries were pressurized to 50u2003mmHg and preconstricted with phenylephrine. Intraluminal flow reversed the effect of phenylephrine, resulting in vasodilatation. Flow dilatation consisted of an initial transient peak followed by a sustained plateau phase. The magnitude of dilatation was markedly reduced by removing Ca2+ from the intraluminal flow medium. Depletion of intracellular Ca2+ stores with either cyclopiazonic acid (CPA, 2u2003μM) or 1,4‐dihydroxy‐2,5‐di‐tert‐butylbenzene (BHQ, 10u2003μM) significantly augmented the magnitude of flow dilatation. Flow‐induced endothelial cell Ca2+ influx was also markedly enhanced in arteries pretreated with CPA or BHQ. Flow‐induced dilatation was insensitive to Nw‐nitro‐L‐arginine methyl ester (100u2003μM) plus indomethacin (3u2003μM) or to oxyhemoglobin (3u2003μM), but was markedly reduced by 30u2003mM extracellular K+ or 2u2003mM tetrabutylammonium (TBA), suggesting an involvement of EDHF. Catalase at 1200u2003Uu2003ml−1 abolished the flow‐induced dilatation, while the application of exogenous H2O2 (90–220u2003μM) induced relaxation in phenylephrine‐preconstricted arteries. Relaxation to exogenous H2O2 was blocked in the presence of 30u2003mM extracellular K+, and H2O2 (90u2003μM) hyperpolarized the smooth muscle cells, indicating that H2O2 can act as an EDHF. In conclusion, flow‐induced dilatation in rat mesenteric arteries can be markedly enhanced by prior depletion of intracellular Ca2+ stores. Furthermore, these data are consistent with a role for H2O2 as the vasodilator involved.

Evidence against C-type natriuretic peptide as an arterial 'EDHF'.

C‐type natriuretic peptide (CNP) is found in and released from vascular endothelial cells. Recently, a novel role has been suggested for this peptide, that of an endothelium‐derived hyperpolarizing factor or EDHF. Implicit in this proposal is a widespread role for CNP as a key mediator of vascular dilatation. In this issue of the British Journal of Pharmacology, Leuranguer et al. compare the profile of membrane potential changes evoked with this putative EDHF or with endogenous EDHF (activated with ACh) in small carotid arteries. Marked differences between the two profiles lead them to discount a possible role for CNP as an EDHF.