Conducted dilatation to ATP and K+ in rat skeletal muscle arterioles.

AIM: During exercise in humans, circulating levels of ATP and K+ increase at a time when blood flow increases to satisfy metabolic demand. Both molecules can activate arteriolar K+ channels to stimulate vasodilatation; here, it is established whether conducted dilatation is observed in a skeletal muscle bed. METHODS: Isolated and cannulated rat cremaster arterioles were used to assess both local and conducted responses. Agents were either added to the bath, focally pulse-ejected to the downstream end of arterioles, or in triple-cannulated arterioles, luminally perfused into the downstream branches to assess both local and conducted responses. RESULTS: The endothelium-dependent agonist ACh and the KATP channel opener levcromakalim each stimulated both local and conducted vasodilatation. Focal, bolus delivery of ATP (10 µm) or KCl (33 mm) to the outside of arterioles stimulated a biphasic vasomotor response: rapid vasoconstriction followed by dilatation as each washed away. At lower concentrations of KCl (19 mm), constriction was avoided, and instead, Ba2+ -sensitive local dilatation and conducted dilatation were both observed. Luminal perfusion of ATP avoided constriction and activated P2Y1 receptors stimulating vasodilatation secondary to opening of KCa channels. In triple-cannulated arterioles, either ATP (10 µm) or K+ (15 mm) luminally perfused into daughter branches of a bifurcation stimulated local dilatation which conducted into the parent arteriole. CONCLUSION: The recognized physiological autocrine and paracrine mediators ATP and K+ each act to evoke both local and conducted vasodilatation in rat cremaster arterioles. Therefore, in situations when circulating levels are raised, such as during exercise, these agents can act as important regulators of blood flow.

EDH: endothelium-dependent hyperpolarization and microvascular signalling.

Endothelium-dependent hyperpolarizing factor (EDHF) is a powerful vasodilator influence in small resistance arteries and thus an important modulator of blood pressure and flow. As the name suggests, EDHF was thought to describe a diffusible factor stimulating smooth muscle hyperpolarization (and thus vasodilatation). However, this idea has evolved with the recognition that a factor can operate alongside the spread of hyperpolarizing current from the endothelium to the vascular smooth muscle (VSM). As such, the pathway is now termed endothelium-dependent hyperpolarization (EDH). EDH is activated by an increase in endothelial [Ca2+ ]i , which stimulates two Ca2+ -sensitive K channels, SKCa and IKCa . This was discovered because apamin and charybdotoxin applied in combination blocked EDHF responses, but iberiotoxin - a blocker of BKCa - was not able to substitute for charybdotoxin. SKCa and IKCa channels are arranged in endothelial microdomains, particularly within projections towards the adjacent smooth muscle, which are rich in IKCa channels and close to interendothelial gap junctions where SKCa channels, are prevalent. KCa activation hyperpolarizes endothelial cells, and K+ efflux through them can act as a diffusible 'EDHF' by stimulating VSM Na+ ,K+ -ATPase and inwardly rectifying K channels (KIR ). In parallel, hyperpolarizing current spreads from the endothelium to the smooth muscle through myoendothelial gap junctions located on endothelial projections. The resulting radial EDH is complemented by the spread of 'conducted' hyperpolarization along the endothelium of arteries and arterioles to affect conducted vasodilatation (CVD). Retrograde CVD effectively integrates blood flow within the microcirculation, but how the underlying hyperpolarization is sustained is unclear.

TRPM4 inhibitor 9-phenanthrol activates endothelial cell intermediate conductance calcium-activated potassium channels in rat isolated mesenteric artery.

BACKGROUND AND PURPOSE: Smooth muscle transient receptor potential melastatin 4 (TRPM4) channels play a fundamental role in the development of the myogenic arterial constriction that is necessary for blood flow autoregulation. As TRPM4 channels are present throughout the vasculature, we investigated their potential role in non-myogenic resistance arteries using the TRPM4 inhibitor 9-phenanthrol. EXPERIMENTAL APPROACH: Pressure and wire myography were used to assess the reactivity of rat arteries, the latter in combination with measurements of smooth muscle membrane potential. Immunohistochemistry (IHC) and endothelial cell (EC) calcium changes were assessed in pressurized vessels and patch clamp measurements made in isolated ECs. KEY RESULTS: The TRPM4 inhibitor 9-phenanthrol reversibly hyperpolarized mesenteric arteries to circa EK and blocked α1 -adrenoceptor-mediated vasoconstriction. Hyperpolarization was abolished and vasoconstriction re-established by damaging the endothelium. In mesenteric and cerebral artery smooth muscle, 9-phenanthrol hyperpolarization was effectively blocked by the KCa 3.1 inhibitor TRAM-34. 9-Phenanthrol did not increase mesenteric EC [Ca(2+)]i , and Na(+) substitution with N-methyl-D-glucamine only increased the muscle resting potential by 10 mV. Immunolabelling for TRPM4 was restricted to the endothelium and perivascular tissue. CONCLUSIONS AND IMPLICATIONS: These data reveal a previously unrecognized action of the TRPM4 inhibitor 9-phenanthrol - the ability to act as an activator of EC KCa 3.1 channels. They do not indicate a functionally important role for TRPM4 channels in the reactivity of non-myogenic mesenteric arteries.

ß1-Adrenoceptor stimulation suppresses endothelial IK(Ca)-channel hyperpolarization and associated dilatation in resistance arteries.

BACKGROUND AND PURPOSE: In small arteries, small conductance Ca²âº-activated K⁺ channels (SK(Ca)) and intermediate conductance Ca²âº-activated K⁺ channels (IK(Ca)) restricted to the vascular endothelium generate hyperpolarization that underpins the NO- and PGI2-independent, endothelium-derived hyperpolarizing factor response that is the predominate endothelial mechanism for vasodilatation. As neuronal IK(Ca) channels can be negatively regulated by PKA, we investigated whether ß-adrenoceptor stimulation, which signals through cAMP/PKA, might influence endothelial cell hyperpolarization and as a result modify the associated vasodilatation. EXPERIMENTAL APPROACH: Rat isolated small mesenteric arteries were pressurized to measure vasodilatation and endothelial cell [Ca²âº]i , mounted in a wire myograph to measure smooth muscle membrane potential or dispersed into endothelial cell sheets for membrane potential recording. KEY RESULTS: Intraluminal perfusion of ß-adrenoceptor agonists inhibited endothelium-dependent dilatation to ACh (1 nM-10 µM) without modifying the associated changes in endothelial cell [Ca²âº]i . The inhibitory effect of ß-adrenoceptor agonists was mimicked by direct activation of adenylyl cyclase with forskolin, blocked by the ß-adrenoceptor antagonists propranolol (non-selective), atenolol (ß1) or the PKA inhibitor KT-5720, but remained unaffected by ICI 118 551 (ß2) or glibenclamide (ATP-sensitive K⁺ channels channel blocker). Endothelium-dependent hyperpolarization to ACh was also inhibited by ß-adrenoceptor stimulation in both intact arteries and in endothelial cells sheets. Blocking IK(Ca) {with 1 µM 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34)}, but not SK(Ca) (50 nM apamin) channels prevented ß-adrenoceptor agonists from suppressing either hyperpolarization or vasodilatation to ACh. CONCLUSIONS AND IMPLICATIONS: In resistance arteries, endothelial cell ß1-adrenoceptors link to inhibit endothelium-dependent hyperpolarization and the resulting vasodilatation to ACh. This effect appears to reflect inhibition of endothelial IK(Ca) channels and may be one consequence of raised circulating catecholamines.

Vascular hyperpolarization to ß-adrenoceptor agonists evokes spreading dilatation in rat isolated mesenteric arteries.

BACKGROUND AND PURPOSE: ß-Adrenoceptor stimulation causes pronounced vasodilatation associated with smooth muscle hyperpolarization. Although the hyperpolarization is known to reflect K(ATP) channel activation, it is not known to what extent it contributes to vasodilatation. EXPERIMENTAL APPROACH: Smooth muscle membrane potential and tension were measured simultaneously in small mesenteric arteries in a wire myograph. The spread of vasodilatation over distance was assessed in pressurized arteries following localized intraluminal perfusion of either isoprenaline, adrenaline or noradrenaline. KEY RESULTS: Isoprenaline stimulated rapid smooth muscle relaxation associated at higher concentrations with robust hyperpolarization. Noradrenaline or adrenaline evoked a similar hyperpolarization to isoprenaline if the α(1)-adrenoceptor antagonist prazosin was present. With each agonist, glibenclamide blocked hyperpolarization without reducing relaxation. Focal, intraluminal application of isoprenaline, noradrenaline or adrenaline during block of α(1)-adrenoceptors evoked a dilatation that spread along the entire length of the isolated artery. This response was endothelium-dependent and inhibited by glibenclamide. CONCLUSIONS AND IMPLICATIONS: Hyperpolarization is not essential for ß-adrenoceptor-mediated vasodilatation. However, following focal ß-adrenoceptor stimulation, this hyperpolarization underlies the ability of vasodilatation to spread along the artery wall. The consequent spread of vasodilatation is dependent upon the endothelium and likely to be of physiological relevance in the coordination of tissue blood flow.

Desensitization of endothelial P2Y1 receptors by PKC-dependent mechanisms in pressurized rat small mesenteric arteries.

BACKGROUND AND PURPOSE: Extracellular nucleotides play a crucial role in the regulation of vascular tone and blood flow. Stimulation of endothelial cell P2Y1 receptors evokes concentration-dependent full dilatation of resistance arteries. However, this GPCR can desensitize upon prolonged exposure to the agonist. Our aim was to determine the extent and nature of P2Y1 desensitization in isolated and pressurized rat small mesenteric arteries. EXPERIMENTAL APPROACH: The non-hydrolyzable selective P2Y1 agonist ADPbetaS (3 microM) was perfused through the lumen of arteries pressurized to 70 mmHg. Changes in arterial diameter and endothelial cell [Ca(2+)](i) were obtained in the presence and absence of inhibitors of protein kinase C (PKC). KEY RESULTS: ADPbetaS evoked rapid dilatation to the maximum arterial diameter but faded over time to a much-reduced plateau closer to 35% dilatation. This appeared to be due to desensitization of the P2Y1 receptor, as subsequent endothelium-dependent dilatation to acetylcholine (1 microM) remained unaffected. Luminal treatment with the PKC inhibitors BIS-I (1 microM) or BIS-VIII (1 microM) tended to augment concentration-dependent dilatation to ADPbetaS (0.1-3 microM) and prevented desensitization. Another PKC inhibitor, Gö 6976 (1 microM), was less effective in preventing desensitization. Measurements of endothelial cell [Ca(2+)](i) in pressurized arteries confirmed the P2Y1 receptor but not M(3) muscarinic receptor desensitization. CONCLUSIONS AND IMPLICATIONS: These data demonstrate for the first time the involvement of PKC in the desensitization of endothelial P2Y1 receptors in pressurized rat mesenteric arteries, which may have important implications in the control of blood flow by circulating nucleotides.

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.

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 microM. Similar hyperpolarization was evoked with cyclopiazonic acid (10 microM, an inhibitor of sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA)) while 1-EBIO (300 microM, 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 microM 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.

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 (K(IR)) channels was investigated in the rat small mesenteric artery using both freshly isolated smooth muscle and endothelial cells and small arterial segments. In Ca(2+)-free solution, endothelial cells displayed a K(IR) current with a maximum amplitude of 190 +/- 16 pA at -150 mV and sensitivity to block with 30 microM Ba(2+) (n = 7). In smooth muscle cells, outward K current was activated at around -47 +/- 3 mV, but there was no evidence of K(IR) current (n = 6). Furthermore, raising extracellular [K(+)] to either 60 or 140 mM, or applying the alpha(1)-adrenoceptor agonist phenylephrine (PE; 30 microM), 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 microM ouabain but unaffected by either 30 microM BaCl(2) or 3 microM glibenclamide. These data suggest that functional, inwardly rectifying Ba(2+)-sensitive channels are restricted to the endothelial cell layer in the rat small mesenteric artery.

A mechanosensitive cation channel in endothelial cells and its role in vasoregulation.

Ca2+ is an important intracellular second messenger in signal transduction of endothelial cells. It has long been recognized that a mechanosensitive Ca2+-permeable channel is present in vascular endothelial cells. The activity of this channel may increase intracellular Ca2+ level in endothelial cells. A recent finding is that the activity of this channel may be regulated by cGMP through a protein kinase G-dependent pathway. Inhibition of the channel by cGMP abolishes the Ca2+ influx elicited by flow. Several inhibitors of the cation channel including Gd3+, Ni2+, and SK&F-96365 also inhibit the Ca2+ influx due to flow stimulation. These data suggest that a mechanosensitive cation channel is the primary pathway mediating the flow-induced Ca2+ entry in vascular endothelial cells. Another important finding is that the opening of this mechanosensitive channel by KT5823 leads to endothelium-dependent vascular dilation. Therefore, it appears that this channel may play a crucial role in the regulation of vascular tone.

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

1. In rat small mesenteric arteries contracted with phenylephrine, 1-ethyl-2-benzimidazolinone (1-EBIO; 3-300 microM) evoked concentration-dependent relaxation that, above 100 microM, was associated with smooth muscle hyperpolarization. 2. 1-EBIO-evoked hyperpolarization (maximum 22.1+/-3.6 mV with 300 microM, n=4) was endothelium-dependent and inhibited by charybdotoxin (ChTX 100 nM; n=4) but not iberiotoxin (IbTX 100 nM; n=4). 3. In endothelium-intact arteries, smooth muscle relaxation to 1-EBIO was not altered by either of the potassium channel blockers ChTX (100 nM; n=7), or IbTX (100 nM; n=4), or raised extracellular K(+) (25 mM). Removal of the endothelium shifted the relaxation curve to the right but did not reduce the maximum relaxation. 4. In freshly isolated mesenteric endothelial cells, 1-EBIO (600 microM) evoked a ChTX-sensitive outward K-current. In contrast, 1-EBIO had no effect on smooth muscle cell conductance whereas NS 1619 (33 microM) stimulated an outward current while having no effect on the endothelial cells. 5. These data show that with concentrations greater than 100 microM, 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 IK(Ca). However, 1-EBIO can also relax the smooth muscle by an undefined mechanism, independent of any change in membrane potential.

Cell-cell communication in the vessel wall.

Intercellular communication between cells within the blood vessel wall plays an important role in the control of artery diameter. The endothelial cells lining the lumen of arteries can evoke smooth muscle hyperpolarization both by the release of a factor (EDHF) and by direct cell-cell coupling through gap junctions. Hyperpolarizing current can spread rapidly to cause widespread vasodilatation, and thus increase blood flow to that segment. In addition to the spread of current, small molecules, such as Ca2+, can also pass between cells, but at a much reduced rate. Instead of co-ordinating changes in diameter, intercellular Ca2+ signalling acts to amplify and, in special cases, modulate vascular responses. Together, direct cell-cell communication enables the blood vessel wall to act as a functional syncytium, which is influenced by surrounding tissues and nerves, and blood constituents.

Properties of smooth muscle hyperpolarization and relaxation to K+ in the rat isolated mesenteric artery.

Smooth muscle membrane potential and tension in rat isolated small mesenteric arteries (inner diameter 100-200 microm) were measured simultaneously to investigate whether the intensity of smooth muscle stimulation and the endothelium influence responses to exogenous K+. Variable smooth muscle depolarization and contraction were stimulated by titration with 0.1-10 microM phenylephrine. Raising external K+ to 10.8 mM evoked correlated, sustained hyperpolarization and relaxation, both of which were inhibited as the smooth muscle depolarized and contracted to around -38 mV and 10 mN, respectively. At these higher levels of stimulation, raising the K+ concentration to 13.8 mM still hyperpolarized and relaxed the smooth muscle. Relaxation to endothelium-derived hyperpolarizing factor, released by ACh, was not altered by the level of stimulation. In endothelium-denuded arteries, the concentration-relaxation curve to K+ was shifted to the right but was not depressed. In denuded arteries, relaxation to K+ was unaffected by the extent of prior stimulation and was blocked with 0.1 mM ouabain but not with 30 microM Ba2+. The ability of K+ to stimulate simultaneous hyperpolarization and relaxation in the mesenteric artery is consistent with a role as an endothelium-derived hyperpolarizing factor activating inwardly rectifying K+ channels on the endothelium and Na+-K+-ATPase on the smooth muscle cells.

Endothelial cell protein kinase G inhibits release of EDHF through a PKG-sensitive cation channel.

The release of dilator agents from vascular endothelial cells is modulated by changes in cytosolic Ca(2+) concentration ([Ca(2+)](i)). In this study, we demonstrate the presence of a Ca(2+)-permeable cation channel in inside-out membrane patches of endothelial cells isolated from small mesenteric arteries. The activity of the channel is increased by KT-5823, a highly selective inhibitor of protein kinase G (PKG), while it is decreased by direct application of active PKG. Application of KT-5823 induces Ca(2+) influx in the endothelial cells isolated from small mesenteric arteries, and it also causes endothelium-dependent relaxations in isolated small mesenteric arteries. KT-5823-induced relaxations in small mesenteric arteries are greatly reduced by 35 mM K(+) or 50 nM charybdotoxin + 50 nM apamin, suggesting that endothelium-derived hyperpolarizing factor (EDHF) is the participating dilator. The involvement of EDHF is further supported by experiments in which the relaxations of small mesenteric arteries are shown to be accompanied by membrane repolarization. These data strongly argue for a major role of a PKG-sensitive cation channel in modulating the release of EDHF from endothelial cells in rat small mesenteric arteries.

Intercellular Ca2+ signalling: the artery wall.

The fact that many cells in the cardiovascular system are coupled via gap junctions enables direct intercellular Ca2+ signalling. Ca2+ ions and/or inositol trisphosphate (IP3) can pass through these aqueous pores. Cell-cell coupling can occur between cells of the same type, or via special junctions between adjacent cells of different types. Homocellular coupling acts to amplify and prolong Ca2+ signals, whereas heterocellular coupling allows complex interactions between the cells, including the modulation of their respective responses. This review will focus on the interactions between cells that form the blood vessel wall, illustrating how cell-cell communication defines important physiological functions.

Microvascular dilation in response to occlusion: a coordinating role for conducted vasomotor responses.

In rat cremasteric microcirculation, mechanical occlusion of one branch of an arteriolar bifurcation causes an increase in flow and vasodilation of the unoccluded daughter branch. This dilation has been attributed to the operation of a shear stress-dependent mechanism in the microcirculation. Instead of or in addition to this, we hypothesized that the dilation observed during occlusion is the result of a conducted signal originating distal to the occlusion. To test this hypothesis, we blocked the ascending spread of conducted vasomotor responses by damaging the smooth muscle and endothelial cells in a 200-microm segment of second- or third-order arterioles. We found that a conduction blockade eliminated or diminished the occlusion-associated increase in flow through the unoccluded branch and abolished or strongly attenuated the vasodilatory response in both vessels at the branch. We also noted that vasodilations induced by ACh (10(-4) M, 0.6 s) spread to, but not beyond, the area of damage. Taken together, these data provide strong evidence that conducted vasomotor responses have an important role in coordinating blood flow in response to an arteriolar occlusion.

The involvement of intracellular Ca(2+) in 5-HT(1B/1D) receptor-mediated contraction of the rabbit isolated renal artery.

5-Hydroxytryptamine(1B/1D) (5-HT(1B/1D)) receptor coupling to contraction was investigated in endothelium-denuded rabbit isolated renal arteries, by simultaneously measuring tension and intracellular [Ca(2+)], and tension in permeabilized smooth muscle cells. In intact arterial segments, 1 nM - 10 microM 5-HT failed to induce contraction or increase the fura-2 fluorescence ratio (in the presence of 1 microM ketanserin and prazosin to block 5-HT(2) and alpha(1)-adrenergic receptors, respectively). However, in vessels pre-exposed to either 20 mM K(+) or 30 nM U46619, 5-HT stimulated concentration-dependent increases in both tension and intracellular [Ca(2+)]. 1 nM - 10 microM U46619 induced concentration-dependent contractions. In the presence of nifedipine (0.3 and 1 microM) the maximal contraction to U46619 (10 microM) was reduced by around 70%. The residual contraction was abolished by the putative receptor operated channel inhibitor, SKF 96365 (2 microM). With 0.3 microM nifedipine present, 100 nM U46619 evoked similar contraction to 30 nM U46619 in the absence of nifedipine, but contraction to 5-HT (1 nM - 10 microM) was abolished. In permeabilized arterial segments, 10 mM caffeine, 1 microM IP(3) or 100 microM phenylephrine, each evoked transient contractions by releasing Ca(2+) from intracellular stores, whereas 5-HT had no effect. In intact arterial segments pre-stimulated with 20 mM K(+), 5-HT-evoked contractions were unaffected by 1 microM thapsigargin, which inhibits sarco- and endoplasmic reticulum calcium-ATPases. In vessels permeabilized with alpha-toxin and then pre-contracted with Ca(2+) and GTP, 5-HT evoked further contraction, reflecting increased myofilament Ca(2+)-sensitivity. Contraction linked to 5-HT(1B/1D) receptor stimulation in the rabbit renal artery can be explained by an influx of external Ca(2+) through voltage-dependent Ca(2+) channels and sensitization of the contractile myofilaments to existing levels of Ca(2+), with no release of Ca(2+) from intracellular stores.

An indirect influence of phenylephrine on the release of endothelium-derived vasodilators in rat small mesenteric artery.

1. The possibility that stimulation of smooth muscle alpha(1)-adrenoceptors modulates contraction via the endothelium was examined in rat small mesenteric arteries. 2. N(omega)-nitro-L-arginine methyl ester, (L-NAME, 100 microM to inhibit NO synthase) increased contraction to single concentrations of phenylephrine (1 - 3 microM) by approximately 2 fold (from a control level of 14.2+/-3.0 to 34. 1+/-4.2% of the maximum contraction of the artery, n=20). The action of L-NAME was abolished by disrupting the endothelium. 3. The subsequent addition of apamin (to inhibit small conductance Ca(2+)-activated K(+) channels, 50 nM) further augmented phenylephrine contractions, in an endothelium-dependent manner, to more than 3 fold above control (50.4+/-5.3% of the maximum contraction, n=11). 4.Charybdotoxin (non-selective inhibitor of large conductance Ca(2+)-activated K(+) channels, BK(Ca), 50 nM) plus L-NAME augmented the level of phenylephrine contraction to 4 - 5-fold above control (64.1+/-3.1%, n=5), but this effect was independent of the endothelium. The potentiation of contraction by charybdotoxin could be mimicked with the selective BK(Ca) inhibitor, iberiotoxin,. 5. Apamin together with L-NAME and charybdotoxin further significantly increased the phenylephrine contraction by 5 - 6-fold, to 79.9+/-3.5% of the maximum contraction of the artery (n=13). 6. Phenylephrine failed directly to increase the intracellular Ca(2+) concentration in endothelial cells freshly isolated from the small mesenteric artery. 7. Stimulation of smooth muscle alpha(1)-adrenoceptors in the mesenteric artery induces contraction that is markedly suppressed by the endothelium. The attenuation of contraction appears to reflect both the release of NO from the endothelium and the efflux of K(+) from both endothelial and smooth muscle cells. This suggests that the release of NO and endothelium-derived hyperpolarizing factor can be evoked indirectly by agents which act only on the smooth muscle cells.

Role of heterocellular Gap junctional communication in endothelium-dependent smooth muscle hyperpolarization: inhibition by a connexin-mimetic peptide.

A synthetic connexin-mimetic peptide (Gap 27 peptide) was used to evaluate the contribution of gap junctional communication to smooth muscle responses mediated by the endothelium-dependent agonist acetylcholine (ACh) in rabbit mesenteric arteries. Hyperpolarizations and relaxations to 0.1 and 1 microM ACh observed in the presence of nitric oxide synthase and cyclooxygenase inhibition were markedly attenuated by the peptide at a concentration of 300 microM, whereas the hyperpolarizing response to levcromakalim, a KATP channel opener, was unaffected. The peptide also attenuated intercellular transfer of Lucifer yellow in confluent cultures of COS-7 cells, thus confirming its ability to modulate the permeability of gap junctions. The findings demonstrate that heterocellular gap junctional communication contributes to NO- and prostanoid-independent mechanisms of vasorelaxation that are widely attributed to an endothelium-derived hyperpolarizing factor.

K+ is an endothelium-derived hyperpolarizing factor in rat arteries.

In arteries, muscarinic agonists such as acetylcholine release an unidentified, endothelium-derived hyperpolarizing factor (EDHF) which is neither prostacyclin nor nitric oxide. Here we show that EDHF-induced hyperpolarization of smooth muscle and relaxation of small resistance arteries are inhibited by ouabain plus Ba2+; ouabain is a blocker of Na+/K+ ATPase and Ba2+ blocks inwardly rectifying K+ channels. Small increases in the amount of extracellular K+ mimic these effects of EDHF in a ouabain- and Ba2+-sensitive, but endothelium-independent, manner. Acetylcholine hyperpolarizes endothelial cells and increases the K+ concentration in the myoendothelial space; these effects are abolished by charbdotoxin plus apamin. Hyperpolarization of smooth muscle by EDHF is also abolished by this toxin combination, but these toxins do not affect the hyperpolarizaiton of smooth muscle by added K+. These data show that EDHF is K+ that effluxes through charybdotoxin- and apamin-sensitive K+ channels on endothelial cells. The resulting increase in myoendothelial K+ concentration hyperpolarizes and relaxes adjacent smooth-muscle cells by activating Ba2+-sensitive K+ channels and Na+/K+ ATPase. These results show that fluctuations in K+ levels originating within the blood vessel itself are important in regulating mammalian blood pressure and flow.