Publications by authors named "Timothy V Murphy"

30 Publications

  • Page 1 of 1

Effect of β /β -adrenoceptor blockade on β -adrenoceptor activity in the rat cremaster muscle artery.

Br J Pharmacol 2021 Apr 27;178(8):1789-1804. Epub 2021 Feb 27.

Physiology, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia.

Background And Purpose: The physiological role of vascular β -adrenoceptors is not fully understood. Recent evidence suggests cardiac β -adrenoceptors are functionally effective after down-regulation of β /β -adrenoceptors. The functional interaction between the β -adrenoceptor and other β-adrenoceptor subtypes in rat striated muscle arteries was investigated.

Experimental Approach: Studies were performed in cremaster muscle arteries isolated from male Sprague-Dawley rats. β-adrenoceptor expression was assessed through RT-PCR and immunofluorescence. Functional effects of β -adrenoceptor agonists and antagonists and other β-adrenoceptor ligands were measured using pressure myography.

Key Results: All three β-adrenoceptor subtypes were present in the endothelium of the cremaster muscle artery. The β -adrenoceptor agonists mirabegron and CL 316,243 had no effect on the diameter of pressurized (70 mmHg) cremaster muscle arterioles with myogenic tone, while the β -adrenoceptor agonist SR 58611A and the nonselective β-adrenoceptor agonist isoprenaline caused concentration-dependent dilation. In the presence of β -adrenoceptor antagonists nadolol (10 μM), atenolol (1 μM) and ICI 118,551 (0.1 μM) both mirabegron and CL 316,243 were effective in causing vasodilation and the potency of SR 58611A was enhanced, while responses to isoprenaline were inhibited. The β -adrenoceptor antagonist L 748,337 (1 μM) inhibited vasodilation caused by β -adrenoceptor agonists (in the presence of β -adrenoceptor blockade), but L 748,337 had no effect on isoprenaline-induced vasodilation.

Conclusion And Implications: All three β-adrenoceptor subtypes were present in the endothelium of the rat cremaster muscle artery, but β -adrenoceptor mediated vasodilation was only evident after blockade of β -adrenoceptors. This suggests constitutive β -adrenoceptor activity inhibits β -adrenoceptor function in the endothelium of skeletal muscle resistance arteries.
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http://dx.doi.org/10.1111/bph.15398DOI Listing
April 2021

Agonist-evoked endothelial Ca signalling microdomains.

Curr Opin Pharmacol 2019 04 12;45:8-15. Epub 2019 Apr 12.

Physiology, School of Medical Sciences, University of New South Wales, Sydney, NSW 2052 Australia; Biomedical Science, School of Health and Sports Science, University of the Sunshine Coast, Maroochydore, Qld 4558 Australia.

Localized, oscillating Ca signals have been identified in discrete microdomains of vascular endothelial cells. At myoendothelial contacts (between endothelial and smooth muscle cells), both endothelial Ca pulsars (IP-mediated release of intracellular Ca) and Ca sparklets (extracellular Ca entry via TRP channels) contribute to endothelium-dependent hyperpolarization of smooth muscle, vasodilation, and feedback control of vasoconstriction. Ca sparklets occurring at close-contact domains between endothelial cells are possibly involved in conducted hyperpolarization and spreading vasodilation in arterial networks. This review summarizes these Ca signalling phenomena, examines the proposed mechanisms leading to their generation by G-protein-coupled receptor agonists, and explores the proposed physiological roles of these localized and specialized Ca signals.
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http://dx.doi.org/10.1016/j.coph.2019.03.005DOI Listing
April 2019

Transient receptor potential canonical type 3 channels: Interactions, role and relevance - A vascular focus.

Pharmacol Ther 2017 Jun 14;174:79-96. Epub 2017 Feb 14.

Inflammation and Healing Cluster, Faculty of Science, Health, Education and Engineering, University of the Sunshine Coast, Maroochydore, Qld 4558, Australia; Department of Physiology, School of Medical Sciences, University of New South Wales, Sydney, NSW 2052, Australia. Electronic address:

Transient receptor potential canonical type 3 channels (TRPC3) are expressed in neural, cardiac, respiratory and vascular tissues, with both similarities and differences between human and animal models for the same cell types. In common with all members of the six subfamilies of TRP channels, TRPC3 are non-voltage gated, non-selective cation channels that are mainly permeated by Ca, and have distinct molecular, biophysical, anatomical and functional properties. TRP channels are present in excitable and non-excitable cells where they sense and respond to a wide variety of physical and chemical stimuli. TRPC3 are expressed in the endothelium and/or smooth muscle of specific intact arteries, such as mesenteric, cerebral and myometrial, where they are critical for the control of vascular tone, and show altered activity in development and disease. In artery endothelium, TRPC3 contributes to endothelium-derived hyperpolarization and nitric oxide-mediated vasodilation. In artery smooth muscle, TRPC3 contributes to constrictor mechanisms. In both endothelium and smooth muscle, TRPC3 contributes to function via caveolae-caveolin dependent and independent mechanisms. In different cell types and states, like other TRP channels, TRPC3 can form complexes with other TRP proteins and associated channels and accessory proteins, including those associated with endo(sarco)plasmic reticulum (ER/SR), thereby facilitating Ca channel activation and/or refilling ER/SR Ca stores. The diversity of TRPC3 interactions with other vascular signaling components is a potential target for artery specific control mechanisms. This brief perspective highlights recent advances in understanding the functional diversity of TRPC3, with an emphasis on vascular health and disease.
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http://dx.doi.org/10.1016/j.pharmthera.2017.02.022DOI Listing
June 2017

TRPV3 expression and vasodilator function in isolated uterine radial arteries from non-pregnant and pregnant rats.

Vascul Pharmacol 2016 08 9;83:66-77. Epub 2016 Apr 9.

Department of Physiology, School of Medical Sciences, University of New South Wales, Sydney, NSW 2052, Australia; Inflammation and Healing Cluster, Faculty of Science, Health, Education and Engineering, University of the Sunshine Coast, Maroochydore, QLD 4558, Australia.

This study investigated the expression and function of transient receptor potential vanilloid type-3 ion channels (TRPV3) in uterine radial arteries isolated from non-pregnant and twenty-day pregnant rats. Immunohistochemistry (IHC) suggested TRPV3 is primarily localized to the smooth muscle in arteries from both non-pregnant and pregnant rats. IHC using C' targeted antibody, and qPCR of TRPV3 mRNA, suggested pregnancy increased arterial TRPV3 expression. The TRPV3 activator carvacrol caused endothelium-independent dilation of phenylephrine-constricted radial arteries, with no difference between vessels from non-pregnant and pregnant animals. Carvacrol-induced dilation was reduced by the TRPV3-blockers isopentenyl pyrophosphate and ruthenium red, but not by the TRPA1 or TRPV4 inhibitors HC-030031 or HC-067047, respectively. In radial arteries from non-pregnant rats only, inhibition of NOS and sGC, or PKG, enhanced carvacrol-mediated vasodilation. Carvacrol-induced dilation of arteries from both non-pregnant and pregnant rats was prevented by the IKCa blocker TRAM-34. TRPV3 caused an endothelium-independent, IKCa-mediated dilation of the uterine radial artery. NO-PKG-mediated modulation of TRPV3 activity is lost in pregnancy, but this did not alter the response to carvacrol.
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http://dx.doi.org/10.1016/j.vph.2016.04.004DOI Listing
August 2016

Activation of endothelial IKCa channels underlies NO-dependent myoendothelial feedback.

Vascul Pharmacol 2015 Nov 9;74:130-138. Epub 2015 Sep 9.

Cardiovascular Research Centre, University of Alberta, Edmonton, AlbertaT6G 2H7, Canada; Department of Pharmacology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AlbertaT6G 2H7, Canada. Electronic address:

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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http://dx.doi.org/10.1016/j.vph.2015.09.001DOI Listing
November 2015

Regulator of G protein signaling 5 is a determinant of gestational hypertension and preeclampsia.

Sci Transl Med 2015 Jun;7(290):290ra88

Harry Perkins Institute of Medical Research, Centre for Medical Research, The University of Western Australia, Nedlands, Western Australia 6009, Australia.

Preeclampsia is a systemic vascular disorder of pregnancy and is associated with increased sensitivity to angiotensin II (AngII) and hypertension. The cause of preeclampsia remains unknown. We identified the role of regulator of G protein (heterotrimeric guanine nucleotide-binding protein) signaling 5 (RGS5) in blood pressure regulation during pregnancy and preeclampsia. RGS5 expression in human myometrial vessels is markedly suppressed in gestational hypertension and/or preeclampsia. In pregnant RGS5-deficient mice, reduced vascular RGS5 expression causes gestational hypertension by enhancing vascular sensitivity to AngII. Further challenge by increasing AngII results in preeclampsia-like symptoms, namely, more severe hypertension, proteinuria, placental pathology, and reduced birth weight. In pregnant heterozygote null mice, treatment with peroxisome proliferator-activated receptor (PPAR) agonists normalizes vascular function and blood pressure through effects on RGS5. These findings highlight a key role of RGS5 at the interface between AngII and PPAR signaling. Because preeclampsia is refractory to current standard therapies, our study opens an unrecognized and urgently needed opportunity for treatment of gestational hypertension and preeclampsia.
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http://dx.doi.org/10.1126/scitranslmed.aaa5038DOI Listing
June 2015

Effect of diet-induced obesity on BK(Ca) function in contraction and dilation of rat isolated middle cerebral artery.

Vascul Pharmacol 2014 Apr 24;61(1):10-5. Epub 2014 Feb 24.

Department of Physiology, School of Medical Sciences, University of New South Wales, Sydney NSW 2052, Australia. Electronic address:

This study examined the effect of diet-induced obesity on the functional role of large-conductance Ca²⁺-activated K⁺ channels (BK(Ca)) in rat middle cerebral arteries. Male Sprague-Dawley rats were fed a control (chow) or high-fat diet for 16-20 weeks. Diet-induced obesity decreased maximum bradykinin-induced dilation of isolated, pressurized (80 mmHg) arteries, but vasodilation induced by sodium nitroprusside (SNP) was unaltered. Responses to bradykinin and SNP in arteries from both control and obese rats were abolished by combination of the nitric oxide synthase (NOS) and guanylate cyclase (sGC) inhibitors L-NAME (100 μmol/L) and ODQ (10 μmol/L) respectively, or by the BK(Ca) blocker iberiotoxin (IBTX, 0.1 μmol/L). Vasodilation induced by the PAR2 agonist SLIGRL in arteries from control-diet rats was abolished by L-NAME/ODQ, but unaffected by IBTX. Obesity greatly reduced the inhibitory effect of L-NAME/ODQ on SLIGRL-induced dilation, whereas IBTX alone now inhibited responses to SLIGRL. Neither obesity nor IBTX altered the responsiveness of the arteries to vasoconstrictors 5-hydroxytryptamine (5-HT) or angiotensin II (Ang II). Obesity had variable effects on the functional role of BK(Ca) in the middle-cerebral artery depending upon the agent used to stimulate the channel, reflecting the variety of mechanisms by which BK(Ca) may be activated.
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http://dx.doi.org/10.1016/j.vph.2014.02.002DOI Listing
April 2014

Pregnancy-induced remodelling and enhanced endothelium-derived hyperpolarization-type vasodilator activity in rat uterine radial artery: transient receptor potential vanilloid type 4 channels, caveolae and myoendothelial gap junctions.

J Anat 2013 Dec 16;223(6):677-86. Epub 2013 Oct 16.

Department of Physiology, School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia.

In pregnancy, the vasculature of the uterus undergoes rapid remodelling to increase blood flow and maintain perfusion to the fetus. The present study determines the distribution and density of caveolae, transient receptor potential vanilloid type 4 channels (TRPV4) and myoendothelial gap junctions, and the relative contribution of related endothelium-dependent vasodilator components in uterine radial arteries of control virgin non-pregnant and 20-day late-pregnant rats. The hypothesis examined is that specific components of endothelium-dependent vasodilator mechanisms are altered in pregnancy-related uterine radial artery remodelling. Conventional and serial section electron microscopy were used to determine the morphological characteristics of uterine radial arteries from control and pregnant rats. TRPV4 distribution and expression was examined using conventional confocal immunohistochemistry, and the contribution of endothelial TRPV4, nitric oxide (NO) and endothelium-derived hyperpolarization (EDH)-type activity determined using pressure myography with pharmacological intervention. Data show outward hypertrophic remodelling occurs in uterine radial arteries in pregnancy. Further, caveolae density in radial artery endothelium and smooth muscle from pregnant rats was significantly increased by ~94% and ~31%, respectively, compared with control, whereas caveolae density did not differ in endothelium compared with smooth muscle from control. Caveolae density was significantly higher by ~59% on the abluminal compared with the luminal surface of the endothelium in uterine radial artery of pregnant rats but did not differ at those surfaces in control. TRPV4 was present in endothelium and smooth muscle, but not associated with internal elastic lamina hole sites in radial arteries. TRPV4 fluorescence intensity was significantly increased in the endothelium and smooth muscle of radial artery of pregnant compared with control rats by ~2.6- and 5.5-fold, respectively. The TRPV4 signal was significantly higher in the endothelium compared with the smooth muscle in radial artery of both control and pregnant rats, by ~5.7- and 2.7-fold, respectively. Myoendothelial gap junction density was significantly decreased by ~37% in radial artery from pregnant compared with control rats. Pressure myography with pharmacological intervention showed that NO contributes ~80% and ~30%, and the EDH-type component ~20% and ~70% of the total endothelium-dependent vasodilator response in radial arteries of control and pregnant rats, respectively. TRPV4 plays a functional role in radial arteries, with a greater contribution in those from pregnant rats. The correlative association of increased TRPV4 and caveolae density and role of EDH-type activity in uterine radial artery of pregnant rats is suggestive of their causal relationship. The decreased myoendothelial gap junction density and lack of TRPV4 density at such sites is consistent with their having an integral, albeit complex, interactive role in uterine vascular signalling and remodelling in pregnancy.
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http://dx.doi.org/10.1111/joa.12127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3842208PMC
December 2013

Enhanced contractility in pregnancy is associated with augmented TRPC3, L-type, and T-type voltage-dependent calcium channel function in rat uterine radial artery.

Am J Physiol Regul Integr Comp Physiol 2013 Oct 15;305(8):R917-26. Epub 2013 Aug 15.

Department of Physiology, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, Australia;

In pregnancy, α-adrenoceptor-mediated vasoconstriction is augmented in uterine radial arteries and is accompanied by underlying changes in smooth muscle (SM) Ca(2+) activity. This study aims to determine the Ca(2+) entry channels associated with altered vasoconstriction in pregnancy, with the hypothesis that augmented vasoconstriction involves transient receptor potential canonical type-3 (TRPC3) and L- and T-type voltage-dependent Ca(2+) channels. Immunohistochemistry showed TRPC3, L-type Cav1.2 (as the α1C subunit), T-type Cav3.1 (α1G), and Cav3.2 (α1H) localization to the uterine radial artery SM. Fluorescence intensity of TRPC3, Cav1.2, and Cav3.2 was increased, and Cav3.1 decreased in radial artery SM from pregnant rats. Western blot analysis confirmed increased TRPC3 protein expression in the radial artery from pregnant rats. Pressure myography incorporating pharmacological intervention to examine the role of these channels in uterine radial arteries showed an attenuation of phenylephrine (PE)-induced constriction with Pyr3 {1-[4-[(2,3,3-trichloro-1-oxo-2-propen-1-yl)amino]phenyl]-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic acid}-mediated TRPC3 inhibition or with nifedipine-mediated L-type channel block alone in vessels from pregnant rats; both effects of which were diminished in radial arteries from nonpregnant rats. Combined TRPC3 and L-type inhibition attenuated PE-induced constriction in radial arteries, and the residual vasoconstriction was reduced and abolished with T-type channel block with NNC 55-0396 in arteries from nonpregnant and pregnant rats, respectively. With SM Ca(2+) stores depleted and in the presence of PE, nifedipine, and NNC 55-0396, blockade of TRPC3 reversed PE-induced constriction. These data suggest that TRPC3 channels act synergistically with L- and T-type channels to modulate radial artery vasoconstriction, with the mechanism being augmented in pregnancy.
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http://dx.doi.org/10.1152/ajpregu.00225.2013DOI Listing
October 2013

Endothelial control of vasodilation: integration of myoendothelial microdomain signalling and modulation by epoxyeicosatrienoic acids.

Pflugers Arch 2014 Mar 8;466(3):389-405. Epub 2013 Jun 8.

Bristol Heart Institute, University of Bristol, Queens Building Level 7, Bristol Royal Infirmary, Upper Maudlin Street, Bristol, BS2 8HW, UK,

Endothelium-derived epoxyeicosatrienoic acids (EETs) are fatty acid epoxides that play an important role in the control of vascular tone in selected coronary, renal, carotid, cerebral and skeletal muscle arteries. Vasodilation due to endothelium-dependent smooth muscle hyperpolarization (EDH) has been suggested to involve EETs as a transferable endothelium-derived hyperpolarizing factor. However, this activity may also be due to EETs interacting with the components of other primary EDH-mediated vasodilator mechanisms. Indeed, the transfer of hyperpolarization initiated in the endothelium to the adjacent smooth muscle via gap junction connexins occurs separately or synergistically with the release of K(+) ions at discrete myoendothelial microdomain signalling sites. The net effects of such activity are smooth muscle hyperpolarization, closure of voltage-dependent Ca(2+) channels, phospholipase C deactivation and vasodilation. The spatially localized and key components of the microdomain signalling complex are the inositol 1,4,5-trisphosphate receptor-mediated endoplasmic reticulum Ca(2+) store, Ca(2+)-activated K(+) (KCa), transient receptor potential (TRP) and inward-rectifying K(+) channels, gap junctions and the smooth muscle Na(+)/K(+)-ATPase. Of these, TRP channels and connexins are key endothelial effector targets modulated by EETs. In an integrated manner, endogenous EETs enhance extracellular Ca(2+) influx (thereby amplifying and prolonging KCa-mediated endothelial hyperpolarization) and also facilitate the conduction of this hyperpolarization to spatially remote vessel regions. The contribution of EETs and the receptor and channel subtypes involved in EDH-related microdomain signalling, as a candidate for a universal EDH-mediated vasodilator mechanism, vary with vascular bed, species, development and disease and thus represent potentially selective targets for modulating specific artery function.
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http://dx.doi.org/10.1007/s00424-013-1303-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5846480PMC
March 2014

Advanced glycation end products acutely impair ca(2+) signaling in bovine aortic endothelial cells.

Front Physiol 2013 11;4:38. Epub 2013 Mar 11.

Department of Physiology, School of Medical Sciences, University of New South Wales Sydney, NSW, Australia.

Post-translational modification of proteins in diabetes, including formation of advanced glycation end products (AGEs) are believed to contribute to vascular dysfunction and disease. Impaired function of the endothelium is an early indicator of vascular dysfunction in diabetes and as many endothelial cell processes are dependent upon intracellular [Ca(2+)] and Ca(2+) signaling, the aim of this study was to examine the acute effects of AGEs on Ca(2+) signaling in bovine aortic endothelial cells (BAEC). Ca(2+) signaling was studied using the fluorescent indicator dye Fura-2-AM. AGEs were generated by incubating bovine serum albumin with 0-250 mM glucose or glucose-6-phosphate for 0-120 days at 37°C. Under all conditions, the main AGE species generated was carboxymethyl lysine (CML) as assayed using both gas-liquid chromatograph-mass spectroscopy and high-performance liquid chromatography. In Ca(2+)-replete solution, exposure of BAEC to AGEs for 5 min caused an elevation in basal [Ca(2+)] and attenuated the increase in intracellular [Ca(2+)] caused by ATP (100 μM). In the absence of extracellular Ca(2+), exposure of BAEC to AGEs for 5 min caused an elevation in basal [Ca(2+)] and attenuated subsequent intracellular Ca(2+) release caused by ATP, thapsigargin (0.1 μM), and ionomycin (3 μM), but AGEs did not affect extracellular Ca(2+) entry induced by the re-addition of Ca(2+) to the bathing solution in the presence of any of these agents. The anti-oxidant α-lipoic acid (2 μM) and NAD(P)H oxidase inhibitors apocynin (500 μM) and diphenyleneiodonium (1 μM) abolished these effects of AGEs on BAECs, as did the IP3 receptor antagonist xestospongin C (1 μM). In summary, AGEs caused an acute depletion of Ca(2+) from the intracellular store in BAECs, such that the Ca(2+) signal stimulated by the subsequent application other agents acting upon this store is reduced. The mechanism may involve generation of reactive oxygen species from NAD(P)H oxidase and possible activation of the IP3 receptor.
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http://dx.doi.org/10.3389/fphys.2013.00038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3593230PMC
March 2013

Increased caveolae density and caveolin-1 expression accompany impaired NO-mediated vasorelaxation in diet-induced obesity.

Histochem Cell Biol 2013 Feb 25;139(2):309-21. Epub 2012 Sep 25.

Department of Pharmacology, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, NSW 2052, Australia.

Diet-induced obesity induces changes in mechanisms that are essential for the regulation of normal artery function, and in particular the function of the vascular endothelium. Using a rodent model that reflects the characteristics of human dietary obesity, in the rat saphenous artery we have previously demonstrated that endothelium-dependent vasodilation shifts from an entirely nitric oxide (NO)-mediated mechanism to one involving upregulation of myoendothelial gap junctions and intermediate conductance calcium-activated potassium channel activity and expression. This study investigates the changes in NO-mediated mechanisms that accompany this shift. In saphenous arteries from controls fed a normal chow diet, acetylcholine-mediated endothelium-dependent vasodilation was blocked by NO synthase and soluble guanylyl cyclase inhibitors, but in equivalent arteries from obese animals sensitivity to these agents was reduced. The expression of endothelial NO synthase (eNOS) and caveolin-3 in rat saphenous arteries was unaffected by obesity, whilst that of caveolin-1 monomer and large oligomeric complexes of caveolins-1 and -2 were increased in membrane-enriched samples. The density of caveolae was increased at the membrane and cytoplasm of endothelial and smooth muscle cells of saphenous arteries from obese rats. Dissociation of eNOS from caveolin-1, as a prerequisite for activation of the enzyme, may be compromised and thereby impair NO-mediated vasodilation in the saphenous artery from diet-induced obese rats. Such altered signaling mechanisms in obesity-related vascular disease represent significant potential targets for therapeutic intervention.
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http://dx.doi.org/10.1007/s00418-012-1032-2DOI Listing
February 2013

Transient receptor potential canonical type 3 channels facilitate endothelium-derived hyperpolarization-mediated resistance artery vasodilator activity.

Cardiovasc Res 2012 Sep 21;95(4):439-47. Epub 2012 Jun 21.

Department of Physiology, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney 2052, Australia.

Aims: Microdomain signalling mechanisms underlie key aspects of artery function and the modulation of intracellular calcium, with transient receptor potential (TRP) channels playing an integral role. This study determines the distribution and role of TRP canonical type 3 (C3) channels in the control of endothelium-derived hyperpolarization (EDH)-mediated vasodilator tone in rat mesenteric artery.

Methods And Results: TRPC3 antibody specificity was verified using rat tissue, human embryonic kidney (HEK)-293 cells stably transfected with mouse TRPC3 cDNA, and TRPC3 knock-out (KO) mouse tissue using western blotting and confocal and ultrastructural immunohistochemistry. TRPC3-Pyr3 (ethyl-1-(4-(2,3,3-trichloroacrylamide)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate) specificity was verified using patch clamp of mouse mesenteric artery endothelial and TRPC3-transfected HEK cells, and TRPC3 KO and wild-type mouse aortic endothelial cell calcium imaging and mesenteric artery pressure myography. TRPC3 distribution, expression, and role in EDH-mediated function were examined in rat mesenteric artery using immunohistochemistry and western blotting, and pressure myography and endothelial cell membrane potential recordings. In rat mesenteric artery, TRPC3 was diffusely distributed in the endothelium, with approximately five-fold higher expression at potential myoendothelial microdomain contact sites, and immunoelectron microscopy confirmed TRPC3 at these sites. Western blotting and endothelial damage confirmed primary endothelial TRPC3 expression. In rat mesenteric artery endothelial cells, Pyr3 inhibited hyperpolarization generation, and with individual SK(Ca) (apamin) or IK(Ca) (TRAM-34) block, Pyr3 abolished the residual respective IK(Ca)- and SK(Ca)-dependent EDH-mediated vasodilation.

Conclusion: The spatial localization of TRPC3 and associated channels, receptors, and calcium stores are integral for myoendothelial microdomain function. TRPC3 facilitates endothelial SK(Ca) and IK(Ca) activation, as key components of EDH-mediated vasodilator activity and for regulating mesenteric artery tone.
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http://dx.doi.org/10.1093/cvr/cvs208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422079PMC
September 2012

Dietary obesity increases NO and inhibits BKCa-mediated, endothelium-dependent dilation in rat cremaster muscle artery: association with caveolins and caveolae.

Am J Physiol Heart Circ Physiol 2012 Jun 6;302(12):H2464-76. Epub 2012 Apr 6.

Department of Physiology, School of Medical Sciences, University of New South Wales, Sydney, Australia.

Obesity is a risk factor for hypertension and other vascular disease. The aim of this study was to examine the effect of diet-induced obesity on endothelium-dependent dilation of rat cremaster muscle arterioles. Male Sprague-Dawley rats (213 ± 1 g) were fed a cafeteria-style high-fat or control diet for 16-20 wk. Control rats weighed 558 ± 7 g compared with obese rats 762 ± 12 g (n = 52-56; P < 0.05). Diet-induced obesity had no effect on acetylcholine (ACh)-induced dilation of isolated, pressurized (70 mmHg) arterioles, but sodium nitroprusside (SNP)-induced vasodilation was enhanced. ACh-induced dilation of arterioles from control rats was abolished by a combination of the K(Ca) blockers apamin, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34), and iberiotoxin (IBTX; all 0.1 μmol/l), with no apparent role for nitric oxide (NO). In arterioles from obese rats, however, IBTX had no effect on responses to ACh while the NO synthase (NOS)/guanylate cyclase inhibitors N(ω)-nitro-L-arginine methyl ester (L-NAME; 100 μmol/l)/1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 μmol/l) partially inhibited ACh-induced dilation. Furthermore, NOS activity (but not endothelial NOS expression) was increased in arteries from obese rats. L-NAME/ODQ alone or removal of the endothelium constricted arterioles from obese but not control rats. Expression of caveolin-1 and -2 oligomers (but not monomers or caveolin-3) was increased in arterioles from obese rats. The number of caveolae was reduced in the endothelium of arteries, and caveolae density was increased at the ends of smooth muscle cells from obese rats. Diet-induced obesity abolished the contribution of large-conductance Ca(2+)-activated K(+) channel to ACh-mediated endothelium-dependent dilation of rat cremaster muscle arterioles, while increasing NOS activity and inducing an NO-dependent component.
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http://dx.doi.org/10.1152/ajpheart.00965.2011DOI Listing
June 2012

Calcium and endothelium-mediated vasodilator signaling.

Adv Exp Med Biol 2012 ;740:811-31

Department of Physiology, School of Medical Sciences, University of New South Wales, 2052 Sydney, NSW, Australia.

Vascular tone refers to the balance between arterial constrictor and dilator activity. The mechanisms that underlie tone are critical for the control of haemodynamics and matching circulatory needs with metabolism, and thus alterations in tone are a primary factor for vascular disease etiology. The dynamic spatiotemporal control of intracellular Ca(2+) levels in arterial endothelial and smooth muscle cells facilitates the modulation of multiple vascular signaling pathways. Thus, control of Ca(2+) levels in these cells is integral for the maintenance of tone and blood flow, and intimately associated with both physiological and pathophysiological states. Hence, understanding the mechanisms that underlie the modulation of vascular Ca(2+) activity is critical for both fundamental knowledge of artery function, and for the development of targeted therapies. This brief review highlights the role of Ca(2+) signaling in vascular endothelial function, with a focus on contact-mediated vasodilator mechanisms associated with endothelium-derived hyperpolarization and the longitudinal conduction of responses over distance.
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http://dx.doi.org/10.1007/978-94-007-2888-2_36DOI Listing
September 2012

Myoendothelial contacts, gap junctions, and microdomains: anatomical links to function?

Microcirculation 2012 Jul;19(5):403-15

Department of Pharmacology, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia.

In several species and in many vascular beds, ultrastructural studies describe close contact sites between the endothelium and smooth muscle of <∼20nm. Such sites are thought to facilitate the local action of signaling molecules and/or the passage of current, as metabolic and electrical coupling conduits between the arterial endothelium and smooth muscle. These sites have the potential for bidirectional communication between the endothelium and smooth muscle, as a key pathway for coordinating vascular function. The aim of this brief review is to summarize the literature on the ultrastructural anatomy and distribution of key components of MECC sites in arteries. In addition to their traditional role of facilitating electrical coupling between the two cell layers, data on the role of MECC sites in arteries, as signaling microdomains involving a spatial localization of channels, receptors and calcium stores are highlighted. Diversity in the density and specific characteristics of MECC sites as signaling microdomains suggests considerable potential for functional diversity within and between arteries in health and disease.
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http://dx.doi.org/10.1111/j.1549-8719.2011.00146.xDOI Listing
July 2012

Differential effects of diet-induced obesity on BKCa {beta}1-subunit expression and function in rat skeletal muscle arterioles and small cerebral arteries.

Am J Physiol Heart Circ Physiol 2011 Jul 2;301(1):H29-40. Epub 2011 May 2.

Dept. of Physiology, School of Medical Sciences, Univ. of New South Wales, Sydney, NSW 2052 Australia.

Mechanisms underlying obesity-related vascular dysfunction are unclear. This study examined the effect of diet-induced obesity on expression and function of large conductance Ca(2+)-activated potassium channel (BK(Ca)) in rat pressurized small resistance vessels with myogenic tone. Male Sprague-Dawley rats fed a cafeteria-style high fat diet (HFD; ∼30% energy from fat) for 16-20 wk were ∼30% heavier than controls fed standard chow (∼13% fat). Obesity did not alter BK(Ca) α-subunit function or α-subunit protein or mRNA expression in vessels isolated from the cremaster muscle or middle-cerebral circulations. In contrast, BK(Ca) β(1)-subunit protein expression and function were significantly reduced in cremaster muscle arterioles but increased in middle-cerebral arteries from obese animals. Immunohistochemistry showed α- and β(1)-subunits were present exclusively in the smooth muscle of both vessels. Cremaster muscle arterioles from obese animals showed significantly increased medial thickness, and media-to-lumen ratio and pressurized arterioles showed increased myogenic tone at 30 mmHg, but not at 50-120 mmHg. Myogenic tone was not affected by obesity in middle-cerebral arteries. The BK(Ca) antagonist iberiotoxin constricted both cremaster muscle and middle-cerebral arterioles from control rats; this effect of iberiotoxin was abolished in cremaster muscle arteries only from obese rats. Diet-induced obesity has contrasting effects on BK(Ca) function in different vascular beds, through differential effects on β(1)-subunit expression. However, these alterations in BK(Ca) function had little effect on overall myogenic tone, suggesting that the mechanisms controlling myogenic tone can be altered and compensate for altered BK(Ca) expression and function.
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http://dx.doi.org/10.1152/ajpheart.00134.2011DOI Listing
July 2011

Endothelium-dependent vasodilation in human mesenteric artery is primarily mediated by myoendothelial gap junctions intermediate conductance calcium-activated K+ channel and nitric oxide.

J Pharmacol Exp Ther 2011 Mar 20;336(3):701-8. Epub 2010 Dec 20.

Department of Pharmacology, School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia.

Myoendothelial microdomain signaling via localized calcium-activated potassium channel (K(Ca)) and gap junction connexins (Cx) is critical for endothelium-dependent vasodilation in rat mesenteric artery. The present study determines the relative contribution of NO and gap junction-K(Ca) mediated microdomain signaling to endothelium-dependent vasodilation in human mesenteric artery. The hypothesis tested was that such activity is due to NO and localized K(Ca) and Cx activity. In mesenteric arteries from intestinal surgery patients, endothelium-dependent vasodilation was characterized using pressure myography with pharmacological intervention. Vessel morphology was examined using immunohistochemical and ultrastructural techniques. In vessel segments at 80 mm Hg, the intermediate (I)K(Ca) blocker 1-[(2-chlorophenyl)diphenyl-methyl]-1H-pyrazole (TRAM-34; 1 μM) inhibited bradykinin (0.1 nM-3 μM)-induced vasodilation, whereas the small (S) K(Ca) blocker apamin (50 and 100 nM) had no effect. Direct IK(Ca) activation with 1-ethyl-2-benzimidazolinone (1-EBIO; 10-300 μM) induced vasodilation, whereas cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (1-30 μM), the SK(Ca) activator, failed to dilate arteries, whereas dilation induced by 1-EBIO (10-100 μM) was blocked by TRAM-34. Bradykinin-mediated vasodilation was attenuated by putative gap junction block with carbenoxolone (100 μM), with remaining dilation blocked by N-nitro l-arginine methyl ester (100 μM) and [1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one] (10 μM), NO synthase and soluble guanylate cyclase blockers, respectively. In human mesenteric artery, myoendothelial gap junction and IK(Ca) activity are consistent with Cx37 and IK(Ca) microdomain expression and distribution. Data suggest that endothelium-dependent vasodilation is primarily mediated by NO, IK(Ca), and gap junction Cx37 in this vessel. Myoendothelial microdomain signaling sites are present in human mesenteric artery and are likely to contribute to endothelium-dependent vasodilation via a mechanism that is conserved between species.
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http://dx.doi.org/10.1124/jpet.110.165795DOI Listing
March 2011

Under pressure - Kv channels and myogenic control of cerebral blood flow.

J Physiol 2010 Oct;588(Pt 19):3635-6

Department of Pharmacology, School of Medical Sciences, University of New South Wales, Sydney, NSW, 2052, Australia.

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http://dx.doi.org/10.1113/jphysiol.2010.197996DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2998215PMC
October 2010

Endothelium-dependent vasodilation in myogenically active mouse skeletal muscle arterioles: role of EDH and K(+) channels.

Microcirculation 2009 Jul 7;16(5):377-90; 1 p following 390. Epub 2009 May 7.

School of Medical Sciences, RMIT University, Victoria, Australia.

As smooth muscle cell (SMC) membrane potential (E(m)) is critical for vascular responsiveness, and arteriolar SMCs are depolarized at physiological intraluminal pressures, we hypothesized that myogenic tone impacts on dilation mediated by endothelium-derived hyperpolarization (EDH). Studies were performed on cannulated mouse cremaster arterioles [diameter, 33+/-2 microm (n=23) at 60 mmHg; SMC Em -34.6+/-1.2 mV (n=7)]. Myogenic activity was assessed as tone developed in response to intraluminal pressure. Functional observations were related to mRNA, protein expression, and anatomy. Acetylcholine concentration-response curves showed a modest shift following indomethacin (10 microM) and L-NAME (100 microM), although maximal vasodilation was achieved. Residual dilation was removed by apamin (1 microM) in combination with TRAM-34 (1 microM) or charybotoxin (0.1 microM), indicating the requirement of small (S) and intermediate (I) calcium-activated potassium channels (K(Ca)). Charybdotoxin, but not TRAM-34, caused vasoconstriction, presumably through the inhibition of SMC BK(Ca). Expression of SK3 and IK1 was confirmed by immunohistochemistry and polymerase chain reaction, while myoendothelial junctions were common, suggesting a high degree of cell coupling. Also consistent with a role for endothelial K(Ca) channels, acetylcholine increased endothelium [Ca(2 +)](i). Apamin and TRAM-34 similarly blocked EDH-mediated dilation at intraluminal pressures of 30 and 90 mmHg, suggesting that in mouse arterioles, SK(Ca -) and IK(Ca -) mediated mechanisms predominate and operate independently of physiological levels of myogenic activation.
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http://dx.doi.org/10.1080/10739680902804042DOI Listing
July 2009

Heterogeneity in function of small artery smooth muscle BKCa: involvement of the beta1-subunit.

J Physiol 2009 Jun 9;587(Pt 12):3025-44. Epub 2009 Apr 9.

Dalton Cardiovascular Research Center and Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, MO 65211, USA.

Arteriolar myogenic vasoconstriction occurs when increased stretch or membrane tension leads to smooth muscle cell depolarization and opening of voltage-gated Ca2+ channels. To prevent positive feedback and excessive pressure-induced vasoconstriction, studies in cerebral artery smooth muscle have suggested that activation of large conductance, Ca2+-activated K+ channels (BKCa) provides an opposing hyperpolarizing influence reducing Ca2+ channel activity. We have hypothesized that this mechanism may not equally apply to all vascular beds. To establish the existence of such heterogeneity in vascular reactivity, studies were performed on rat vascular smooth muscle (VSM) cells from cremaster muscle arterioles and cerebral arteries. Whole cell K+ currents were determined at pipette [Ca2+] of 100 nM or 5 microM in the presence and absence of the BKCa inhibitor, iberiotoxin (IBTX; 0.1 microM). Similar outward current densities were observed for the two cell preparations at the lower pipette Ca2+ levels. At 5 microM Ca2+, cremaster VSM showed a significantly (P < 0.05) lower current density compared to cerebral VSM (34.5 +/- 1.9 vs 45.5 +/- 1.7 pA pF(-1) at +70 mV). Studies with IBTX suggested that the differences in K+ conductance at 5 microM intracellular [Ca2+] were largely due to activity of BKCa. 17beta-Oestradiol (1 microM), reported to potentiate BKCa current via the channel's beta-subunit, caused a greater effect on whole cell K+ currents in cerebral vessel smooth muscle cells (SMCs) compared to those of cremaster muscle. In contrast, the alpha-subunit-selective BKCa opener, NS-1619 (20 microM), exerted a similar effect in both preparations. Spontaneously transient outward currents (STOCs) were more apparent (frequency and amplitude) and occurred at more negative membrane potentials in cerebral compared to cremaster SMCs. Also consistent with decreased STOC activity in cremaster SMCs was an absence of detectable Ca2+ sparks (0 of 76 cells) compared to that in cerebral SMCs (76 of 105 cells). Quantitative PCR showed decreased mRNA expression for the beta1 subunit and a decrease in the beta1:alpha ratio in cremaster arterioles compared to cerebral vessels. Similarly, cremaster arterioles showed a decrease in total BKCa protein and the beta1:alpha-subunit ratio. The data support vascular heterogeneity with respect to the activity of BKCa in terms of both beta-subunit regulation and interaction with SR-mediated Ca2+ signalling.
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http://dx.doi.org/10.1113/jphysiol.2009.169920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2718259PMC
June 2009

Membrane cholesterol depletion with beta-cyclodextrin impairs pressure-induced contraction and calcium signalling in isolated skeletal muscle arterioles.

J Vasc Res 2007 30;44(4):292-302. Epub 2007 Mar 30.

Microvascular Biology Group, School of Medical Sciences, RMIT University, Bundoora, Australia.

Objective: Given evidence for clustering of signalling molecules and ion channels in cholesterol-rich membrane domains, the involvement of such structures in arteriolar smooth muscle mechanotransduction was examined.

Method: To determine the contribution of smooth muscle cholesterol-rich membrane domains to the myogenic response, isolated arterioles were exposed to the cholesterol-depleting agent beta-cyclodextrin (1-10 mM) in the absence and presence of excess exogenous cholesterol.

Results: beta-Cyclodextrin significantly impaired pressure-induced vasoconstriction, while excess cholesterol attenuated this effect. Impaired myogenic constriction was evident in de-endothelialized vessels, indicating an action at the level of smooth muscle. beta-Cyclodextrin treatment uncoupled increases in intracellular Ca(2+) from myogenic constriction and depleted intracellular Ca(2+) stores consistent with a loss of connectivity between plasma membrane and sarcoplasmic reticulum signalling. However, beta-cyclodextrin-treated arterioles showed unaltered constrictor responses to KCl and phenylephrine. Electron microscopy verified that beta-cyclodextrin caused a decrease in caveolae, while confirmation of smooth muscle containing caveolae was obtained by immunostaining for caveolin-1. Viability of beta-cyclodextrin-treated arterioles was confirmed by agonist sensitivity and propidium iodide nuclear staining.

Conclusion: The data suggest that smooth muscle cholesterol-rich membrane domains contribute to the myogenic response. Further studies are required to determine whether this relates to specific mechanosensory events or generalized alterations in membrane function.
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http://dx.doi.org/10.1159/000101451DOI Listing
July 2007

Arteriolar myogenic signalling mechanisms: Implications for local vascular function.

Clin Hemorheol Microcirc 2006 ;34(1-2):67-79

Department of Physiology and Pharmacology, University of New South Wales, Sydney, New South Wales 2052, Australia.

Arterioles typically exist in a state of partial constriction that is related to the level of intraluminal pressure. This vasomotor response is a function of the vascular smooth muscle and occurs independently of neurohumoral and endothelial input. The physiological relevance of myogenic constriction relates to the setting of peripheral resistance, provision of a level of tone that vasodilators can access, and a contribution to control of capillary pressure. Despite its importance in the regulation of microvascular haemodynamics the exact cellular mechanisms linking intraluminal pressure to myogenic constriction remain uncertain. Studies using isolated, cannulated arteriole techniques, and freshly dispersed smooth muscle cells, have shown that increased intraluminal pressure/cell stretch leads to smooth muscle cell membrane depolarisation, the opening of L-type voltage-gated Ca2+ channels (VGCC), Ca2+-dependent activation of myosin light chain kinase and actomyosin-based contraction. Questions remain as to how the initial stimulus is detected and how these events lead to membrane depolarisation. A candidate pathway for the mechanosensory events involves the link between extracellular matrix proteins, cell surface integrins and the subsequent activation of intracellular signalling events. Membrane depolarisation may occur through the involvement of various ion channels, including non-selective cation channels (possibly themselves mechanosensitive) that predominantly pass Na+ from the extracellular space. Evidence suggests that this may involve TRP-like channels, possibly TRPM4 or TRPC6 isoforms that are modulated by diacylglycerol and protein kinase C. In addition, the exact roles played by various Ca2+ pools, including those occurring in spatially-restricted domains, and Ca2+ sensitisation, remain uncertain despite the clearly important role of VGCC. Similarly, while a change in intraluminal pressure is associated with the generation of a number of second messengers and the activation of various protein kinases, their roles in myogenic contraction versus long-term adaptive responses, such as tissue remodelling, are still to be defined.
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July 2006

Approaches for introducing peptides into intact and functional arteriolar smooth muscle: manipulation of protein kinase-based signalling.

Clin Exp Pharmacol Physiol 2003 Sep;30(9):653-8

Microvascular Biology Group, School of Medical Sciences, RMIT University, Bundoora, Victoria 3083, Australia.

1. An exact understanding of signal transduction pathways within intact and functional arteriolar smooth muscle is made difficult by limited access to the intracellular environment due to the cell membrane. The aim of the present studies was to determine the feasibility of using polycationic lipids and reverse permeabilization for the introduction of peptide inhibitors into smooth muscle cells of the intact arteriolar wall. 2. Isolated cannulated arterioles were exposed to polycationic lipid preparations together with varying concentrations of the protein beta-galactosidase (30-90 microg/mL). Similar experiments were also performed using cultured smooth muscle cells. Staining for the chromogenic substrate of beta-galactosidase (5-bromo-4-chloro-3-indolyl-beta-d-galactosidase; X-gal) demonstrated incorporation of the protein into cultured cells but not intact arteriolar smooth muscle. Similarly, polycationic lipid treatment did not enable loading of arteriolar smooth muscle (as assessed by cAMP-mediated vasodilation) with the protein kinase (PK) A inhibitory peptide PKI. 3. In contrast, reverse permeabilization, using high ATP concentrations in the presence of EGTA enabled introduction of PKI and inhibition of forskolin-mediated vasodilatation. Furthermore, arterioles maintained full viability following reverse permeabilization, as demonstrated by an ability to develop spontaneous myogenic tone. 4. Reverse permeabilization provides a method for introducing peptide inhibitors into functional arteriolar smooth muscle and manipulating signal transduction. Protein transfection using polycationic lipids appears to be limited by the barrier provided by the adventitia or inherent differences between cells under cultured conditions compared within the intact arteriole.
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http://dx.doi.org/10.1046/j.1440-1681.2003.03892.xDOI Listing
September 2003

Intraluminal pressure stimulates MAPK phosphorylation in arterioles: temporal dissociation from myogenic contractile response.

Am J Physiol Heart Circ Physiol 2003 Oct 12;285(4):H1764-73. Epub 2003 Jun 12.

Microvascular Biology Group, School of Medical Sciences, RMIT University, Plenty Rd., Bundoora, Victoria 3083, Australia.

Members of the MAPK family of enzymes, p42/44 and p38, have been implicated in both the regulation of contractile function and growth responses in vascular smooth muscle. We determined whether such kinases are activated during the arteriolar myogenic response after increases in intraluminal pressure. Particular emphasis was placed on temporal aspects of activation to determine whether such phosphorylation events parallel the known time course for myogenic contraction. Experiments used single cannulated arterioles isolated from the cremaster muscle of rats with some vessels loaded with the fluorescent Ca2+-sensitive dye fura 2 (2 microM). The p42/44 inhibitor PD-98059 (50 microM) caused vasodilation but did not prevent pressure-induced myogenic constriction. The vasodilator response was accompanied by decreased smooth muscle intracellular Ca2+. Western blotting revealed a significant increase in the level of phosphorylation of p42/44 15 min after the application of a 30- to 100-mmHg pressure step. Phosphorylation of p42/44 was a late event that appeared to be temporally dissociated from contraction, which was complete within 1-5 min. EGF (80 nM) caused marked phosphorylation of p42/44 but only acted as a weak vasoconstrictor. The p38 inhibitor SB-203580 (10 microM) did not alter baseline diameter, nor did it prevent myogenic vasoconstriction. Consistent with these observations, SB-203580 did not cause a measurable change in intracellular Ca2+. The results demonstrate activation of the p42/44 class of MAPK resulting from increased transmural pressure. Such activation is, however, dissociated from the acute pressure-induced vasoconstrictor response in terms of time course and may represent the activation of compensatory, but parallel, pathways, including those related to growth and remodeling.
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http://dx.doi.org/10.1152/ajpheart.00468.2003DOI Listing
October 2003

Matrix protein glycation impairs agonist-induced intracellular Ca2+ signaling in endothelial cells.

J Cell Physiol 2002 Oct;193(1):80-92

Microvascular Biology Group, School of Medical Sciences, RMIT University, Bundoora, Victoria, Australia.

Studies have shown diabetes to be associated with alterations in composition of extracellular matrix and that such proteins modulate signal transduction. The present studies examined if non-enzymatic glycation of fibronectin or a mixed matrix preparation (EHS) alters endothelial cell Ca(2+) signaling following agonist stimulation. Endothelial cells were cultured from bovine aorta and rat heart. To glycate proteins, fibronectin (10 microg/ml), or EHS (2.5 mg/ml) were incubated (37 degrees C, 30 days) with 0.5 M glucose-6-phosphate. Matrix proteins were coated onto cover slips after which cells (10(5) cells/ml) were plated and allowed to adhere for 16 h. For measurement of intracellular Ca(2+), cells were loaded with fura 2 (2 microM) and fluorescence intensity monitored. Bovine cells on glycated EHS showed decreased ability for either ATP (10(-6) M) or bradykinin (10(-7) M) to increase Ca(2+) (i). In contrast, glycated fibronectin did not impair agonist-induced increases in Ca(2+) (i). In the absence of extracellular Ca(2+), ATP elicited a transient increase in Ca(2+) (i) consistent with intracellular release. Re-addition of Ca(2+) resulted in a secondary rise in Ca(2+) (i) indicative of store depletion-mediated Ca(2+) entry. Both phases of Ca(2+) mobilization were reduced in cells on glycated mixed matrix; however, as the ratio of the two components was similar in all cells, glycation appeared to selectively impair Ca(2+) release from intracellular stores. Thapsigargin treatment demonstrated an impaired ability of cells on glycated EHS to increase cytoplasmic Ca(2+) consistent with decreased endoplasmic reticulum Ca(2+) stores. Further support for Ca(2+) mobilization was provided by increased baseline IP(3) levels in cells plated on glycated EHS. Impaired ATP-induced Ca(2+) release could be induced by treating native EHS with laminin antibody or exposing cells to H(2)O(2) (20-200 microM). Glycated EHS impaired Ca(2+) signaling was attenuated by treatment with aminoguanidine or the antioxidant alpha-lipoic acid. The results demonstrate that matrix glycation impairs agonist-induced Ca(2+) (i) increases which may impact on regulatory functions of the endothelium and implicate possible involvement of oxidative stress.
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http://dx.doi.org/10.1002/jcp.10153DOI Listing
October 2002

Cellular signalling in arteriolar myogenic constriction: involvement of tyrosine phosphorylation pathways.

Clin Exp Pharmacol Physiol 2002 Jul;29(7):612-9

Microvascular Biology Group, School of Medical Sciences, RMIT University, Melbourne, Victoria, Australia.

1. An increase in transmural pressure in arterioles results in a shortening of vascular smooth muscle cells, with subsequent constriction of the vessel. The mechanisms underlying this myogenic contraction are not fully understood; however, the obligatory role of increases in intracellular [Ca(2+)] and myosin light chain phosphorylation have been demonstrated. 2. The myogenic response shows a relationship with smooth muscle cell membrane potential and influx of extracellular Ca(2+) through voltage-operated Ca(2+) channels (VOCC). Mechanically sensitive channels and possibly release of Ca(2+) from intracellular stores may play a role. However, there are other components of myogenic contraction that cannot be explained by a Ca(2+)-MLCK mechanism, for example the initial sensing of alterations in transmural pressure, whether sustained myogenic constriction involves myofilament Ca(2+) sensitization or remodelling of the vessel wall in response to a maintained increase in transmural pressure. 3. In an attempt to investigate these areas, recent studies have examined a role for tyrosine phosphorylation pathways in pressure-induced contraction of arterioles. In rat pressurized cremaster arterioles, tyrosine kinase inhibitors dilated vessels showing spontaneous myogenic tone and tyrosine phosphatase inhibitors caused vasoconstriction. However, pressure-induced myogenic constriction of vessels persisted in the presence of these agents. Biochemical studies revealed that phosphotyrosine formed at a relatively slow rate (significant after 5 min, with maximal increase after approximately 15 min) in response to increased vessel transmural pressure, in contrast with myosin light chain phosphorylation or the time-course of myogenic constriction itself (maximum within 1 min). 4. Taken together, these observations support the idea of a role for tyrosine phosphorylation pathways in longer-term responses to increased transmural pressure rather than acute myogenic constriction. Phosphotyrosine formation was also more closely correlated to vessel wall tension (pressure x diameter) than the diameter of the arterioles alone. The identity of the tyrosine-phosphorylated proteins requires further investigation; however, there is some evidence supporting roles for cSrc-type tyrosine kinases and p44 mitogen-activated protein kinase. The longer-term responses of blood vessels to increased transmural pressure that may involve tyrosine phosphorylation pathways include maintenance of myogenic constriction and vessel wall remodelling.
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http://dx.doi.org/10.1046/j.1440-1681.2002.03698.xDOI Listing
July 2002

Mechanisms underlying pervanadate-induced contraction of rat cremaster muscle arterioles.

Eur J Pharmacol 2002 May;442(1-2):107-14

Microvascular Biology Group, School of Medical Sciences, RMIT University, PO Box 71, 3083 Bundoora, Victoria, Australia.

The current study examined the role of extracellular Ca2+, calmodulin and myosin light-chain kinase (MLCK) in pervanadate-induced constriction of cannulated, pressurized rat cremaster arterioles. Pervanadate (0.03-100 microM) induced a concentration-dependent constriction of arterioles that was significantly attenuated (P<0.05) by the tyrosine kinase inhibitor tyrphostin 47 (30 microM). The L-type voltage-sensitive Ca2+ channel antagonists verapamil (10 microM) and nifedipine (1 microM) dilated vessels possessing myogenic tone but had no demonstrable effect on pervanadate constriction, while a higher concentration of nifedipine (10 microM) reduced constriction by approximately 50%. Pervanadate-induced contractions were reduced by the calmodulin inhibitor W-7 (N-(6-aminohexyl)-chloro-1-naphtalene sulphonamide, 50 microM) and the MLCK inhibitor ML-7 (1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine, 10 or 30 microM). Removal of extracellular Ca2+ abolished the contractile effect of pervanadate. Measurement of changes in arteriolar wall [Ca2+] using the Ca2+ sensitive dye Fura-2 showed that pervanadate did not increase [Ca2+] during arteriolar constriction. These observations suggest that pervanadate-induced contraction of smooth muscle in the cremaster arteriole involves Ca2+/calmodulin-dependent myosin phosphorylation and possibly sensitization of the contractile apparatus to Ca2+.
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http://dx.doi.org/10.1016/s0014-2999(02)01498-xDOI Listing
May 2002

Capacitative Ca(2+) entry in vascular endothelial cells is mediated via pathways sensitive to 2 aminoethoxydiphenyl borate and xestospongin C.

Br J Pharmacol 2002 Jan;135(1):119-28

Microvascular Biology Group, School of Medical Sciences, RMIT University, Bundoora, Victoria 3083, Australia.

1. Agonists increase endothelial cell intracellular Ca(2+), in part, by capacitative entry, which is triggered by the filling state of intracellular Ca(2+) stores. It has been suggested that depletion of endoplasmic reticulum (ER) Ca(2+) stores either leads to a physical coupling between the ER and a plasma membrane channel, or results in production of an intracellular messenger which affects the gating of membrane channels. As an axis involving the IP(3) receptor has been implicated in a physical coupling mechanism the aim of this study was to examine the effects of the putative IP(3) receptor antagonists/modulators, 2 aminoethoxydiphenyl borate (2APB) and xestospongin C, on endothelial cell Ca(2+) entry. 2. Studies were conducted in fura 2 loaded cultured bovine aortic endothelial cells and endothelial cells isolated from rat heart. 3. 2APB (30 - 300 microM) inhibited Ca(2+) entry induced by both agonists (ATP 1 microM, bradykinin 0.1 microM) and receptor-independent mechanisms (thapsigargin 1 microM, ionomycin 0.5 and 5 microM). 2APB did not diminish endothelial cell ATP-induced production of IP(3) nor effect in vitro binding of [(3)H]-IP(3) to an adrenal cortex binding protein. Capacitative Ca(2+) entry was also blocked by disruption of the actin cytoskeleton with cytochalasin (100 nM) while the initial Ca(2+) release phase was unaffected. 4. Similarly to 2APB, xestospongin C (3 - 10 microM) inhibited ATP-induced Ca(2+) release and capacitative Ca(2+) entry. Further, xestospongin C inhibited capacitative Ca(2+) entry induced by thapsigargin (1 microM) and ionomycin (0.5 microM). 5. The data are consistent with a mechanism of capacitative Ca(2+) entry in vascular endothelial cells which requires (a) IP(3) receptor binding and/or an event distal to the activation of the ER receptor and (b) a spatial relationship, dictated by the cytoskeleton, between Ca(2+) release and entry pathways.
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http://dx.doi.org/10.1038/sj.bjp.0704465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1573124PMC
January 2002