Paul M. Kerr

Heteromultimeric Kv1 Channels Contribute to Myogenic Control of Arterial Diameter

Inhibition of vascular smooth muscle (VSM) delayed rectifier K+ channels (KDR) by 4-aminopyridine (4-AP; 200 &mgr;mol/L) or correolide (1 &mgr;mol/L), a selective inhibitor of Kv1 channels, enhanced myogenic contraction of rat mesenteric arteries (RMAs) in response to increases in intraluminal pressure. The molecular identity of KDR of RMA myocytes was characterized using RT-PCR, real-time PCR, and immunocytochemistry. Transcripts encoding the pore-forming Kv&agr; subunits, Kv1.2, Kv1.4, Kv1.5, and Kv1.6, were identified and confirmed at the protein level with subunit-specific antibodies. Kv&bgr; transcript (&bgr;1.1, &bgr;1.2, &bgr;1.3, and &bgr;2.1) expression was also identified. Kv1.5 message was ≈2-fold more abundant than that for Kv1.2 and Kv1.6. Transcripts encoding these three Kv1&agr; subunits were ≈2-fold more abundant in 1st/2nd order conduit compared with 4th order resistance RMAs, and Kv&bgr;1 was 8-fold higher than Kv&bgr;2 message. RMA KDR activated positive to −50 mV, exhibited incomplete inactivation, and were inhibited by 4-AP and correolide. However, neither &agr;-dendrotoxin or &kgr;-dendrotoxin affected RMA KDR, implicating the presence of Kv1.5 in all channels and the absence of Kv1.1, respectively. Currents mediated by channels because of coexpression of Kv1.2, Kv1.5, Kv1.6, and Kv&bgr;1.2 in human embryonic kidney 293 cells had biophysical and pharmacological properties similar to those of RMA KDR. It is concluded that KDR channels composed of heteromultimers of Kv1 subunits play a critical role in myogenic control of arterial diameter.

WHAT'S WHERE AND WHY AT A VASCULAR MYOENDOTHELIAL MICRODOMAIN SIGNALLING COMPLEX

1 Modulation of vascular cell calcium is critical for the control of vascular tone, blood flow and pressure. 2 Specialized microdomain signalling sites associated with calcium modulation are present in vascular smooth muscle cells, where spatially localized channels and calcium store receptors interact functionally. Anatomical studies suggest that such sites are also present in endothelial cells. 3 The characteristics of these sites near heterocellular myoendothelial gap junctions (MEGJs) are described, focusing on rat mesenteric artery. The MEGJs enable current and small molecule transfer to coordinate arterial function and are thus critical for endothelium‐derived hyperpolarization, regulation of smooth muscle cell diameter in response to contractile stimuli and vasomotor conduction over distance. 4 Although MEGJs occur on endothelial cell projections within internal elastic lamina (IEL) holes, not all IEL holes have MEGJ‐related projections (approximately 0–50% of such holes have MEGJ‐related projections, with variations occurring within and between vessels, species, strains and disease). 5 In rat mesenteric, saphenous and caudal cerebellar artery and hamster cheek pouch arteriole, but not rat middle cerebral artery or cremaster arteriole, intermediate conductance calcium‐activated potassium channels (IKCa) localize to endothelial cell projections. 6 Rat mesenteric artery MEGJ connexins and IKCa are in close spatial association with endothelial cell inositol 1,4,5‐trisphosphate receptors and endoplasmic reticulum. 7 Data suggest a relationship between spatially associated endothelial cell ion channels and calcium stores in modulation of calcium release and action. Differences in spatial relationships between ion channels and calcium stores in different vessels reflect heterogeneity in vasomotor function, representing a selective target for the control of endothelial and vascular function.

Endothelial Feedback and the Myoendothelial Projection

Please cite this paper as: Kerr PM, Tam R, Ondrusova K, Mittal R, Narang D, Tran CHT, Welsh DG, Plane F. Endothelial feedback and the myoendothelial projection. Microcirculation 19: 416‐422, 2012.

Activation of endothelial IKCa channels underlies NO-dependent myoendothelial feedback

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.

Endothelial calcium-activated potassium channels as therapeutic targets to enhance availability of nitric oxide

The vascular endothelium plays a critical role in vascular health by controlling arterial diameter, regulating local cell growth, and protecting blood vessels from the deleterious consequences of platelet aggregation and activation of inflammatory responses. Circulating chemical mediators and physical forces act directly on the endothelium to release diffusible relaxing factors, such as nitric oxide (NO), and to elicit hyperpolarization of the endothelial cell membrane potential, which can spread to the surrounding smooth muscle cells via gap junctions. Endothelial hyperpolarization, mediated by activation of calcium-activated potassium (K(Ca)) channels, has generally been regarded as a distinct pathway for smooth muscle relaxation. However, recent evidence supports a role for endothelial K(Ca) channels in production of endothelium-derived NO, and indicates that pharmacological activation of these channels can enhance NO-mediated responses. In this review we summarize the current data on the functional role of endothelial K(Ca) channels in regulating NO-mediated changes in arterial diameter and NO production, and explore the tempting possibility that these channels may represent a novel avenue for therapeutic intervention in conditions associated with reduced NO availability such as hypertension, hypercholesterolemia, smoking, and diabetes mellitus.

High fructose consumption in pregnancy alters the perinatal environment without increasing metabolic disease in the offspring

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.

Modulation of resistance artery tone by the trace amine β-phenylethylamine: dual indirect sympathomimetic and α1-adrenoceptor blocking actions

The trace amine β-phenylethylamine (PEA) is normally present in the body at low nanomolar concentrations but can reach micromolar levels after ingestion of drugs that inhibit monoamine oxidase and primary amine oxidase. In vivo, PEA elicits a robust pressor response, but there is no consensus regarding the underlying mechanism, with both vasodilation and constriction reported in isolated blood vessels. Using functional and biochemical approaches, we found that at low micromolar concentrations PEA (1–30 μM) enhanced nerve-evoked vasoconstriction in the perfused rat mesenteric bed but at a higher concentration (100 μM) significantly inhibited these responses. The α2-adrenoceptor antagonist rauwolscine (1 µM) also enhanced nerve-mediated vasoconstriction, but in the presence of both rauwolscine (1 µM) and PEA (30 µM) together, nerve-evoked responses were initially potentiated and then showed time-dependent rundown. PEA (10 and 100 μM) significantly increased noradrenaline outflow from the mesenteric bed as determined by high-pressure liquid chromatography coupled with electrochemical detection. In isolated endothelium-denuded arterial segments, PEA (1 µM to 1 mM) caused concentration-dependent reversal of tone elicited by the α1-adrenoceptor agonists noradrenaline (EC50 51.69 ± 10.8 μM; n = 5), methoxamine (EC50 68.21 ± 1.70 μM; n = 5), and phenylephrine (EC50 67.74 ± 16.72 μM; n = 5) but was ineffective against tone induced by prostaglandin F2α or U46619 (9,11-dideoxy-9α,11α-methanoepoxyprostaglandin F2α). In rat brain homogenates, PEA displaced binding of both [3H]prazosin (Ki ≈ 25 μM) and [3H]rauwolscine (Ki ≈ 1.2 μM), ligands for α1- and α2-adrenoceptors, respectively. These data provide the first demonstration that dual indirect sympathomimetic and α1-adrenoceptor blocking actions underlie the vascular effects of PEA in resistance arteries.

Triton X-100 inhibits L-type voltage-operated calcium channels

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.

Vasoconstrictor stimulus determines the functional contribution of myoendothelial feedback to mesenteric arterial tone

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 modulation of vasoconstriction. We propose that (1) the functional contribution of myoendothelial feedback to arterial tone is determined by the nature of the vasoconstrictor stimulus, and (2) although IKCa channel‐mediated myoendothelial feedback may contribute to local control of arterial diameter, in the intact vascular bed, increases in shear stress may be the major stimulus for engagement of the endothelium during vasoconstriction.