Frances Plane
University of Alberta, Pharmacology, Faculty Member
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Research Interests: Medicine and ENDOTHELIUM
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ABSTRACT Triton X-100 (TX-100) is a nonionic detergent frequently used at millimolar concentrations to disrupt cell membranes and solubilize proteins. At low micromolar concentrations, TX-100 has been reported to inhibit the function of... more
ABSTRACT Triton X-100 (TX-100) is a nonionic detergent frequently used at millimolar concentrations to disrupt cell membranes and solubilize proteins. At low micromolar concentrations, TX-100 has been reported to inhibit the function of potassium channels. Here, we have used electrophysiological and functional techniques to examine the effects of TX-100 on another class of ion channels, L-type voltage-operated calcium channels (VOCCs). TX-100 (30 nmol·L(-1) to 3 μmol·L(-1)) caused reversible concentration-dependent inhibition of recombinant L-type VOCC (CaV 1.2) currents and of native L-type VOCC currents recorded from rat vascular smooth muscle cells and cardiac myocytes, and murine and human pancreatic β-cells. In functional studies, TX-100 (165 nmol·L(-1) to 3.4 μmol·L(-1)) caused concentration-dependent relaxation of rat isolated mesenteric resistance arteries prestimulated with phenylephrine or KCl. This effect was independent of the endothelium. TX-100 (1.6 μmol·L(-1)) inhibited depolarization-induced exocytosis in both murine and human isolated pancreatic β-cells. These data indicate that at concentrations within the nanomolar to low micromolar range, TX-100 significantly inhibits L-type VOCC activity in a number of cell types, an effect paralleled by inhibition of cell functions dependent upon activation of these channels. This inhibition occurs at concentrations below those used to solubilize proteins and may compromise the use of solutions containing TX-100 in bioassays.
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The sympathetic nervous system is a key regulator of arterial diameter and thus blood flow and pressure. Protease-activated receptors (PARs) are a novel class of G-protein coupled receptors activat...
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Nitric oxide (NO), an important endogenous signaling molecule released from vascular endothelial cells and nerves, activates the enzyme soluble guanylate cyclase to catalyze the production of cyclic guanosine monophosphate (cGMP) from... more
Nitric oxide (NO), an important endogenous signaling molecule released from vascular endothelial cells and nerves, activates the enzyme soluble guanylate cyclase to catalyze the production of cyclic guanosine monophosphate (cGMP) from guanosine triphosphate. cGMP, in turn, activates protein kinase G to phosphorylate a range of effector proteins in smooth muscle cells that reduce intracellular Ca2+ levels to inhibit both contractility and proliferation. The enzyme phosphodiesterase type 5 (PDE5) curtails the actions of cGMP by hydrolyzing it into inactive 5’-GMP. Small molecule PDE5 inhibitors (PDE5is), such as sildenafil, prolong the availability of cGMP and therefore, enhance NO-mediated signaling. PDE5is are the first-line treatment for erectile dysfunction but are also now approved for the treatment of pulmonary arterial hypertension (PAH) in adults. Persistent pulmonary hypertension in neonates (PPHN) is currently treated with inhaled NO, but this is an expensive option and arou...
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The endothelium, the single layer of cells lining all blood vessels,is a key regulator of blood flow and blood pressure within the body. Endothelial cells influence the contractile state of the surrounding smooth muscle cells via release... more
The endothelium, the single layer of cells lining all blood vessels,is a key regulator of blood flow and blood pressure within the body. Endothelial cells influence the contractile state of the surrounding smooth muscle cells via release of diffusible factors, such as nitric oxide (NO), and by direct electrical coupling via myoendothelial gap junctions (MEGJs). The majority of studies in this area have focused on chemical and physical stimuli that act directly on endothelial cells. It has long been held that arterial vasoconstriction is limited by the endothelium, but how contraction of smooth muscle cells leads to activation of the endothelium has not been addressed. We propose that inositol 1,4,5 trisphosphate (IP3) generated by vasoconstrictors crosses MEGJs, elicits Ca2+ release from IP3 receptors and activates intermediate conductance Ca2+-activated K+ (IKCa) channels to elicit a negative feedback response. Constriction and depolarization of rat mesenteric arteries to phenyleph...
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The vascular endothelium is a dynamic organ with secretory, synthetic, metabolic, and immunologic functions. Forming a continuous lining to every blood vessel in the body, endothelial cells play a key role in modulating vascular tone and... more
The vascular endothelium is a dynamic organ with secretory, synthetic, metabolic, and immunologic functions. Forming a continuous lining to every blood vessel in the body, endothelial cells play a key role in modulating vascular tone and permeability, angiogenesis, and in mediating haemostatic, inflammatory and reparative responses to local injury.
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Constriction of resistance arteries in response to global application of agonists is modulated by activation of the endothelium via myoendothelial feedback. Recent evidence supports a model of myoendothelial feedback in which generation... more
Constriction of resistance arteries in response to global application of agonists is modulated by activation of the endothelium via myoendothelial feedback. Recent evidence supports a model of myoendothelial feedback in which generation of localized InsP3-dependent Ca2+ transients leads to activation of intermediate conductance Ca2+-activated K+ (IKCa) channels to hyperpolarize the endothelium. We have now investigated the functional contribution of this pathway to limiting responses to two physiologically important vasoconstrictor stimuli, activation of sympathetic nerves and increases in intravascular pressure. In isolated segments of rat mesenteric resistance artery mounted in a wire or pressure myograph, pharmacological inhibition of IKCa channels significantly potentiated increases in tone elicited by global application agonists such as noradrenaline, but did not significantly alter increases in tone elicited by stimulation of sympathetic perivascular nerves, or myogenic reacti...
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The sympathetic nervous system is a key regulator of arterial diameter and thus blood flow and pressure. Protease-activated receptors (PARs) are a novel class of G-protein coupled receptors activat...
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Research Interests: Chemistry and ENDOTHELIUM
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Key points In isolated resistance arteries, endothelial modulation of vasoconstrictor responses to α1‐adrenoceptor agonists occurs via a process termed myoendothelial feedback: localized inositol trisphosphate (InsP3)dependent Ca2+... more
Key points In isolated resistance arteries, endothelial modulation of vasoconstrictor responses to α1‐adrenoceptor agonists occurs via a process termed myoendothelial feedback: localized inositol trisphosphate (InsP3)dependent Ca2+ transients activate intermediate conductance Ca2+activated K+ (IKCa) channels, hyperpolarizing the endothelial membrane potential to limit further reductions in vessel diameter. We demonstrate that IKCa channel‐mediated myoendothelial feedback limits responses of isolated mesenteric arteries to noradrenaline and nerve stimulation, but not to the thromboxane A2 mimetic U46619 or to increases in intravascular pressure. In contrast, in the intact mesenteric bed, although responses to exogenous noradrenaline were limited by IKCa channel‐mediated myoendothelial feedback, release of NO and activation of endothelial small conductance Ca2+activated K+ (SKCa) channels in response to increases in shear stress appeared to be the primary mediators of endothelial m...
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Hypercholesterolemia and atherosclerosis has been found to impair the vasodilation mediated by the release of endothelium-derived nitric oxide in human and animal arteries. We have previously found that native low-density lipoproteins... more
Hypercholesterolemia and atherosclerosis has been found to impair the vasodilation mediated by the release of endothelium-derived nitric oxide in human and animal arteries. We have previously found that native low-density lipoproteins (LDL) from healthy human donors inhibit acetylcholine (ACh) evoked relaxations in intact rabbit aortic rings precontracted with noradrenaline of serotonin in vitro Cu2+-oxidized LDLinhibits relaxations after preincubation for 30 min and the potency and reversibility of the inhibition depends on the donor (Jacobs et aL, 1990; Plane, F., et aL, 1992). Thus, native LDL from donors whose lipoproteins oxidize rapidly (fast oxidizers) inhibit potently and sometimes irreversibly whereas those which oxidize slowly (slow oxidizers) inhibit less potently and reversibly (Plane F., et al., 1992). We now show that antioxidants influence these effects. native LDL (2 mg protein/ml) caused a reduction in sensitively to ACh which was prevented by the addition of 10 uM probucol or 100 uM ascorbic acid, whereas, superoxide dismutase (20 U/ml) had no effect. This indicates that native LDL inhibit via a process involving free radicals but not superoxide. As expected, the antioxidants had no effect in vitro in the inhibition by oxidized LDL. Oxidized LDL prepared from the lipoproteins of selected volunteers (fast oxidizers), exhibited reduced and reversible inhibition of relaxation compared with the potent and irreversible inhibition prior to administration of the drug, resembling that of the slow oxidizers. We include that antioxidants such as probucol attenuate the inhibition of endothelium-dependent relaxation by LDL and may prevent the impairment resulting from hypercholesterolemia and atherosclerosis.
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The effect of native low‐density lipoproteins (LDL) and oxidized LDL (OXLDL) on the relaxations to endothelium‐derived relaxing factor (EDRF) in isolated, intact aortic rings of the rabbit were investigated. Native LDL induced a... more
The effect of native low‐density lipoproteins (LDL) and oxidized LDL (OXLDL) on the relaxations to endothelium‐derived relaxing factor (EDRF) in isolated, intact aortic rings of the rabbit were investigated. Native LDL induced a concentration‐dependent reversible inhibition of the relaxations elicited by acetylcholine (ACh) or A23187, in rings pre‐contracted by noradrenaline (NA), adrenaline (Ad) and 5‐hydroxytryptamine (5‐HT), but not phenylephrine (PE), which was not influenced by indomethacin. The inhibition was surmountable in the rings pre‐contracted with NA and Ad and only partially in those pre‐contracted with 5‐HT. OXLDL induced an inhibition of the relaxations elicited by ACh and A23187 which was independent of the contractile agonist. The extent of inhibition and its reversibility varied with the LDL from individual donors, but was unaffected by indomethacin. Native and oxidized LDL inhibited relaxations evoked by exogenous nitric oxide (NO) to the same extent. Higher conc...
Research Interests: Endocrinology, Chemistry, Interaction, Medicine, In Vitro, and 15 moreHumans, Internal Medicine, Nitric oxide, Animals, Acetylcholine, Oxidation, Rabbits, Phenylephrine, Aorta, Oxidation-Reduction, Muscle Relaxation, Nitroglycerin, Agonist, Pharmacology and pharmaceutical sciences, and In Vitro Techniques
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The vascular endothelium is one of the largest organs in the body and consists of a single layer of highly specialized cells with site-specific morphology and functions. Endothelial cells play a vital role in the regulation of vascular... more
The vascular endothelium is one of the largest organs in the body and consists of a single layer of highly specialized cells with site-specific morphology and functions. Endothelial cells play a vital role in the regulation of vascular tone in arterial, venous, microvascular, and lymphatic vascular beds. The endothelium also coordinates angiogenesis and controls cell adhesion, fluid homeostasis, and both innate and adaptive immunity. Fundamental research has shown that general and local anesthetics markedly modulate the biological activities of endothelial cells under aerobic and ischemia-reperfusion conditions, making the endothelium an important target of anesthetics in the cardiovascular system. Halogenated volatile anesthetics provide significant anti-inflammatory actions and protect the endothelium against ischemia-reperfusion injury, despite their inhibiting effects on endothelium-dependent vasorelaxation. They provide not only acute but also potential long-term, beneficial ef...
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Electrical communication and its role in blood flow regulation are built on an examination of charge movement in single, isolated vessels. How this process behaves in broader arterial networks remains unclear. This study examined the... more
Electrical communication and its role in blood flow regulation are built on an examination of charge movement in single, isolated vessels. How this process behaves in broader arterial networks remains unclear. This study examined the nature of electrical communication in arterial structures where vessel length and branching were varied. Analysis began with the deployment of an existing computational model expanded to form a variable range of vessel structures. Initial simulations revealed that focal endothelial stimulation generated electrical responses that conducted robustly along short unbranched vessels and to a lesser degree lengthened arteries or branching structures retaining a single branch point. These predictions matched functional observations from hamster mesenteric arteries and support the idea that an increased number of vascular cells attenuate conduction by augmenting electrical load. Expanding the virtual network to 31 branches revealed that electrical responses increasingly ascended from fifth- to first-order arteries when the number of stimulated distal vessels rose. This property enabled the vascular network to grade vasodilation and network perfusion as revealed through blood flow modeling. An elevation in endothelial-endothelial coupling resistance, akin to those in sepsis models, compromised this ascension of vasomotor/perfusion responses. A comparable change was not observed when the endothelium was focally disrupted to mimic disease states including atherosclerosis. In closing, this study highlights that vessel length and branching play a role in setting the conduction of electrical phenomenon along resistance arteries and within networks. It also emphasizes that modest changes in endothelial function can, under certain scenarios, impinge on network responsiveness and blood flow control.
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The relative functional importance of potassium channels and cGMP-dependent pathways in the relaxation of vascular smooth muscle to the novel nitric oxide donor, diethylamine NONOate (DEA NONOate), was investigated in a resistance artery.... more
The relative functional importance of potassium channels and cGMP-dependent pathways in the relaxation of vascular smooth muscle to the novel nitric oxide donor, diethylamine NONOate (DEA NONOate), was investigated in a resistance artery. The contribution from cGMP-dependent signalling pathways was examined by exposing arteries to 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of soluble guanylyl cyclase, while the contribution through potassium channels was assessed with different sub-type-selective potassium channel blockers. DEA NONOate (3 nM-10 microM) evoked sustained relaxation in isolated segments of the rat small mesenteric artery contracted with phenylephrine (pEC50=6.7+/-0.2; n=11). The relaxation was attenuated significantly by either ODQ (10 microM; pEC50=5.8+/-0.4; n=7) or charybdotoxin (ChTX; 50 nM; pEC50=6.3+/-0.2; n=4), a peptide blocker of large conductance, calcium-activated potassium channels (BK(Ca)). The inhibitory effects of ODQ and ChTX were additive (pEC50=5.1+/-0.4; n=9). The selective inhibitor of BK(Ca) channels, iberiotoxin (IbTX; 30 nM), and 4-aminopyridine (4-AP; 1 mM), an inhibitor of voltage-gated potassium channels (Kv), failed to modify DEA NONOate-evoked relaxation. However, in the combined presence of both ODQ and either IbTX or 4-AP the relaxation was attenuated significantly (n=3). The blocker of ATP-modulated potassium channels (K(ATP)), glibenclamide (10 microM), and of small conductance calcium-activated potassium channels (SK(Ca)), apamin (30 nM), each failed to affect ODQ-sensitive or -resistant relaxations to DEA NONOate (n=3). In conclusion, relaxation to DEA NONOate in the rat isolated, small mesenteric artery can occur via both cGMP-dependent (ODQ-sensitive) and -independent (ODQ-resistant) mechanisms. However, the contribution made to relaxation by potassium channels appears to be unmasked following pharmacological attenuation of cGMP-dependent signalling pathways. The inhibitory action of ChTX suggests part of the cGMP-insensitive component involves the activation of potassium channels, a suggestion supported by the inhibitory actions of 4-AP and IbTX in the absence of cGMP.
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Agonist-induced vasoconstriction triggers a negative feedback response whereby movement of charged ions through gap junctions and/or release of endothelium-derived (NO) limit further reductions in diameter, a mechanism termed... more
Agonist-induced vasoconstriction triggers a negative feedback response whereby movement of charged ions through gap junctions and/or release of endothelium-derived (NO) limit further reductions in diameter, a mechanism termed myoendothelial feedback. Recent studies indicate that electrical myoendothelial feedback can be accounted for by flux of inositol trisphosphate (IP3) through myoendothelial gap junctions resulting in localized increases in endothelial Ca(2+) to activate intermediate conductance calcium-activated potassium (IKCa) channels, the resultant hyperpolarization then conducting back to the smooth muscle to attenuate agonist-induced depolarization and tone. In the present study we tested the hypothesis that activation of IKCa channels underlies NO-mediated myoendothelial feedback. Functional experiments showed that block of IP3 receptors, IKCa channels, gap junctions and transient receptor potential canonical type-3 (TRPC3) channels caused endothelium-dependent potentiation of agonist-induced increase in tone which was not additive with that caused by inhibition of NO synthase supporting a role for these proteins in NO-mediated myoendothelial feedback. Localized densities of IKCa and TRPC3 channels occurred at the internal elastic lamina/endothelial-smooth muscle interface in rat basilar arteries, potential communication sites between the two cell layers. Smooth muscle depolarization to contractile agonists was accompanied by IKCa channel-mediated endothelial hyperpolarization providing the first demonstration of IKCa channel-mediated hyperpolarization of the endothelium in response to contractile agonists. Inhibition of IKCa channels, gap junctions, TRPC3 channels or NO synthase potentiated smooth muscle depolarization to agonists in a non-additive manner. Together these data indicate that rather being distinct pathways for the modulation of smooth muscle tone, NO and endothelial IKCa channels are involved in an integrated mechanism for the regulation of agonist-induced vasoconstriction.
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Maternal carbohydrate intake is one important determinant of fetal body composition, but whether increased exposure to individual sugars has long-term adverse effects on the offspring is not well established. Therefore, we examined the... more
Maternal carbohydrate intake is one important determinant of fetal body composition, but whether increased exposure to individual sugars has long-term adverse effects on the offspring is not well established. Therefore, we examined the effect of fructose feeding on the mother, placenta, fetus and her offspring up to 6 months of life when they had been weaned onto a standard rodent diet and not exposed to additional fructose. Dams fed fructose were fatter, had raised plasma insulin and triglycerides from mid-gestation and higher glucose near term. Maternal resistance arteries showed changes in function that could negatively affect regulation of blood pressure and tissue perfusion in the mother and development of the fetus. Fructose feeding had no effect on placental weight or fetal metabolic profiles, but placental gene expression for the glucose transporter GLUT1 was reduced, whereas the abundance of sodium-dependent neutral amino acid transporter-2 was raised. Offspring born to fructose-fed and control dams were similar at birth and had similar post-weaning growth rates, and neither fat mass nor metabolic profiles were affected. In conclusion, raised fructose consumption during reproduction results in pronounced maternal metabolic and vascular effects, but no major detrimental metabolic effects were observed in offspring up to 6 months of age.
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In rat mesenteric artery, endothelium-derived hyperpolarizing factor (EDHF) is blocked by a combination of apamin and charybdotoxin (ChTX). The site of action of these toxins has not been established. We compared the effects of ChTX and... more
In rat mesenteric artery, endothelium-derived hyperpolarizing factor (EDHF) is blocked by a combination of apamin and charybdotoxin (ChTX). The site of action of these toxins has not been established. We compared the effects of ChTX and apamin applied selectively to the endothelium and to the smooth muscle. In isometrically mounted arteries, ACh (0.01-10 micrometers), in the presence of indomethacin (2.8 microM) and Nomega-nitro-L-arginine methyl ester (L-NAME) (100 microM), concentration dependently relaxed phenylephrine (PE)-stimulated tone (EC50 50 nM; n = 10). Apamin (50 nM) and ChTX (50 nM) abolished this relaxation (n = 5). In pressurized arteries, ACh (10 microM), applied intraluminally in the presence of indomethacin (2.8 microM) and L-NAME (100 microM), dilated both PE-stimulated (0.3-0.5 microM; n = 5) and myogenic tone (n = 3). Apamin (50 nM ) and ChTX (50 nM) applied intraluminally abolished ACh-induced dilatations. Bath superperfusion of apamin and ChTX did not affect A...
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Acetylcholine-evoked relaxation of noradrenaline-stimulated segments of the rabbit basilar artery was accompanied by a small, transient hyperpolarization of the smooth muscle cell membrane which was diminished by repeated exposure to the... more
Acetylcholine-evoked relaxation of noradrenaline-stimulated segments of the rabbit basilar artery was accompanied by a small, transient hyperpolarization of the smooth muscle cell membrane which was diminished by repeated exposure to the agonist. In the presence of glibenclamide (10 microM) or high concentrations of potassium chloride (65 mM), the acetylcholine-evoked smooth muscle hyperpolarization was abolished, whereas the relaxation response was unaffected. Nitric oxide (NO gas in solution; 0.5-15 microM) evoked dose-dependent relaxation in noradrenaline contracted arterial segments, but had no effect on the smooth muscle membrane potential, even at a saturated concentration (150 microM), which was 10 times higher than required to stimulate maximal relaxation. Additionally, NO-evoked relaxations were unaffected by glibenclamide (10 microM), but the responses were significantly attenuated in the presence of 65 mM potassium chloride. These data show that, as in the rabbit middle cerebral artery, acetylcholine-evoked hyperpolarization in the rabbit basilar artery is mediated by glibenclamide-sensitive potassium channels. However, in contrast to the middle cerebral artery and to other vessels such as the rat mesenteric artery, the change in smooth muscle membrane potential does not make an important contribution to the relaxation evoked either by this agonist or by NO.
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Activation of protein kinase C by phorbol esters inhibits the endothelium-dependent relaxations evoked by certain stimuli. The release of endothelium-derived relaxing factor can be evoked by a number of distinct subcellular processes,... more
Activation of protein kinase C by phorbol esters inhibits the endothelium-dependent relaxations evoked by certain stimuli. The release of endothelium-derived relaxing factor can be evoked by a number of distinct subcellular processes, including activation of a pertussis toxin-sensitive G-protein. The aim of the present study was to determine whether or not the inhibitory effect of phorbol esters on endothelial function was associated with inhibition of the pertussis toxin-sensitive pathway. Rings of canine coronary artery were suspended for isometric tension recording in organ chambers filled with modified Krebs-Ringer bicarbonate solution, gassed with 95% O2-5% CO2 (37 degrees C). Treatment of arterial rings with pertussis toxin (100 ng/ml) or with phorbol myristate acetate (PMA, 10(-8) M) inhibited the endothelium-dependent relaxations produced by UK 14,304, an alpha-2 adrenergic agonist, leukotriene C4 or by NaF, a direct activator of G-proteins, but did not affect the endotheliu...
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The relative functional importance of potassium channels and cGMP-dependent pathways in the relaxation of vascular smooth muscle to the novel nitric oxide donor, diethylamine NONOate (DEA NONOate), was investigated in a resistance artery.... more
The relative functional importance of potassium channels and cGMP-dependent pathways in the relaxation of vascular smooth muscle to the novel nitric oxide donor, diethylamine NONOate (DEA NONOate), was investigated in a resistance artery. The contribution from cGMP-dependent signalling pathways was examined by exposing arteries to 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of soluble guanylyl cyclase, while the contribution through potassium channels was assessed with different sub-type-selective potassium channel blockers. DEA NONOate (3 nM-10 microM) evoked sustained relaxation in isolated segments of the rat small mesenteric artery contracted with phenylephrine (pEC50=6.7+/-0.2; n=11). The relaxation was attenuated significantly by either ODQ (10 microM; pEC50=5.8+/-0.4; n=7) or charybdotoxin (ChTX; 50 nM; pEC50=6.3+/-0.2; n=4), a peptide blocker of large conductance, calcium-activated potassium channels (BK(Ca)). The inhibitory effects of ODQ and ChTX were additive (pEC50=5.1+/-0.4; n=9). The selective inhibitor of BK(Ca) channels, iberiotoxin (IbTX; 30 nM), and 4-aminopyridine (4-AP; 1 mM), an inhibitor of voltage-gated potassium channels (Kv), failed to modify DEA NONOate-evoked relaxation. However, in the combined presence of both ODQ and either IbTX or 4-AP the relaxation was attenuated significantly (n=3). The blocker of ATP-modulated potassium channels (K(ATP)), glibenclamide (10 microM), and of small conductance calcium-activated potassium channels (SK(Ca)), apamin (30 nM), each failed to affect ODQ-sensitive or -resistant relaxations to DEA NONOate (n=3). In conclusion, relaxation to DEA NONOate in the rat isolated, small mesenteric artery can occur via both cGMP-dependent (ODQ-sensitive) and -independent (ODQ-resistant) mechanisms. However, the contribution made to relaxation by potassium channels appears to be unmasked following pharmacological attenuation of cGMP-dependent signalling pathways. The inhibitory action of ChTX suggests part of the cGMP-insensitive component involves the activation of potassium channels, a suggestion supported by the inhibitory actions of 4-AP and IbTX in the absence of cGMP.