Christopher J. Garland

Spatial separation of endothelial small- and intermediate-conductance calcium-activated potassium channels (KCa) and connexins: possible relationship to vasodilator function?

Activation of endothelial cell small‐ (S) and intermediate‐ (I) conductance calcium‐activated potassium channels (KCa) and current or molecular transfer via myoendothelial gap junctions underlies endothelium‐derived hyperpolarization leading to vasodilation. The mechanism underlying the KCa component of vasodilator activity and the characteristics of gap junctions are targets for the selective control of vascular function. In the rat mesenteric artery, where myoendothelial gap junctions and connexin (Cx) 40 are critical for the transmission of the endothelial cell hyperpolarization to the smooth muscle, SKCa and IKCa provide different facets of the endothelium‐derived hyperpolarization response, being critical for the hyperpolarization and repolarization phases, respectively. The present study addressed the question of whether this functional separation of responses may be related to the spatial localization of the associated channels? The distribution of endothelial SKCa and IKCa and Cx subtype(s) were examined in the rat mesenteric artery using conventional confocal and high‐resolution ultrastructural immunohistochemistry. At the internal elastic lamina–smooth muscle cell interface at internal elastic lamina holes (as potential myoendothelial gap junction sites), strong punctate IKCa, Cx37 and Cx40 expression was present. SKCa, Cx37, Cx40 and Cx43 were localized to adjacent endothelial cell gap junctions. High‐resolution immunohistochemistry demonstrated IKCa and Cx37‐conjugated gold to myoendothelial gap junction‐associated endothelial cell projections. Clear co‐localization of KCa and Cxs suggests a causal relationship between their activity and the previously described differential functional activation of SKCa and IKCa. Such precise localizations may represent a selective target for control of vasodilator function and vascular tone.

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.

Myoendothelial gap junctions may provide the pathway for EDHF in mouse mesenteric artery

Endothelium-dependent hyperpolarization of vascular smooth muscle provides a major pathway for relaxation in resistance arteries. This can occur due to direct electrical coupling via myoendothelial gap junctions (MEGJs) and/or the release of factors (EDHF). Here we provide evidence for the existence of functional MEGJs in the same, defined branches of BALB/C mouse mesenteric arteries which show robust EDHF-mediated smooth muscle relaxation. Cyclopiazonic acid (CPA, 10 µM) was used to stimulate EDHF in arteries mounted under isometric conditions and constricted with phenylephrine. Simultaneous measurement of smooth muscle membrane potential and tension demonstrated that CPA caused a hyperpolarization of around 10 mV, reversing the depolarization to phenylephrine by 94% and the associated constriction by 66%. The relaxation to CPA was endothelium dependent, associated with the opening of Ca2+-activated K channels, and only in part due to the release of nitric oxide (NO). In the presence of the NO synthase inhibitor, L-NAME (100 µM), the relaxation to CPA could be almost completely inhibited with the putative gap junction uncoupler, carbenoxolone (100 µM). Inhibition of the synthesis of prostaglandins or metabolites of arachidonic acid had no effect under the same conditions, and small rises in exogenous K+ failed to evoke consistent or marked smooth muscle relaxation, arguing against a role for these molecules and ions as EDHF. Serial section electron microscopy revealed a high incidence of MEGJs, which was correlated with heterocellular dye coupling. Taken together, these functional and morphological data from a defined mouse resistance artery suggest that the EDHF response in this vessel may be explained by extensive heterocellular coupling through MEGJs, enabling spread of hyperpolarizing current.

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.

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.

Evidence for a Differential Cellular Distribution of Inward Rectifier K Channels in the Rat Isolated Mesenteric Artery

The distribution of functionally active, inwardly rectifying K (KIR) channels was investigated in the rat small mesenteric artery using both freshly isolated smooth muscle and endothelial cells and small arterial segments. In Ca2+-free solution, endothelial cells displayed a KIR current with a maximum amplitude of 190 ± 16 pA at –150 mV and sensitivity to block with 30 µM Ba2+ (n = 7). In smooth muscle cells, outward K current was activated at around –47 ± 3 mV, but there was no evidence of KIR current (n = 6). Furthermore, raising extracellular [K+] to either 60 or 140 mM, or applying the α1-adrenoceptor agonist phenylephrine (PE; 30 µM), failed to reveal an inwardly rectifying current in the smooth muscle cells, although PE did stimulate an iberiotoxin-sensitive outward K current (n = 4). Exogenous K+ (10.8–16.8 mM) both relaxed and repolarized endothelium-denuded segments of the mesenteric artery contracted with PE. These effects were depressed by 100 µM ouabain but unaffected by either 30 µM BaCl2 or 3 µM glibenclamide. These data suggest that functional, inwardly rectifying Ba2+-sensitive channels are restricted to the endothelial cell layer in the rat small mesenteric artery.

Thromboxane receptor stimulation associated with loss of SKCa activity and reduced EDHF responses in the rat isolated mesenteric artery

The possibility that thromboxane (TXA2) receptor stimulation causes differential block of the SKCa and IKCa channels which underlie EDHF‐mediated vascular smooth muscle hyperpolarization and relaxation was investigated in the rat isolated mesenteric artery. Acetylcholine (30 nM–3 μM ACh) or cyclopiazonic acid (10 μM CPA, SERCA inhibitor) were used to stimulate EDHF‐evoked smooth muscle hyperpolarization. In each case, this led to maximal hyperpolarization of around 20 mV, which was sensitive to block with 50 nM apamin and abolished by repeated stimulation of mesenteric arteries with the thromboxane mimetic, U46619 (30 nM–0.1 μM), but not the α1‐adrenoceptor agonist phenylephrine (PE). The ability of U46619 to abolish EDHF‐evoked smooth muscle hyperpolarization was prevented by prior exposure of mesenteric arteries to the TXA2 receptor antagonist 1 μM SQ29548. Similar‐sized smooth muscle hyperpolarization evoked with the SKCa activator 100 μM riluzole was also abolished by prior stimulation with U46619, while direct muscle hyperpolarization in response to either levcromakalim (1 μM, KATP activator) or NS1619 (40 μM, BKCa activator) was unaffected. During smooth muscle contraction and depolarization to either PE or U46619, ACh evoked concentration‐dependent hyperpolarization (to −67 mV) and complete relaxation. These responses were well maintained during repeated stimulation with PE, but with U46619 there was a progressive decline, so that during a third exposure to U46619 maximum hyperpolarization only reached –52 mV and relaxation was reduced by 20%. This relaxation could now be blocked with charybdotoxin alone. The latter responses could be mimicked with 300 μM 1‐EBIO (IKCa activator), an action not modified by exposure to U46619. An early consequence of TXA2 receptor stimulation is a reduction in the arterial hyperpolarization and relaxation attributed to EDHF. This effect appears to reflect a loss of SKCa activity.