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The objective of this study was to examine the effect of insulin resistance on endothelium-derived hyperpolarising factor (EDHF) and small mesenteric artery endothelial function using 25-week-old insulin-resistant obese Zucker rats (OZRs) and lean littermate control rats (LZRs). The involvement of gap junctions and their connexin subunits in the EDHF relaxation response was also assessed.Mesenteric arteries were evaluated using the following assays: (1) endothelial function by pressure myography, with internal diameter recorded using video microscopy; (2) connexin protein levels by western blotting; and (3) Cx mRNA expression by real-time PCR.Relaxations in response to acetylcholine were significantly smaller in mesenteric arteries from the OZRs than the LZRs, whereas there was no difference in relaxations in response to levcromakalim. Responses to acetylcholine were not altered by nitric oxide inhibitors, but were abolished by charybdotoxin in combination with apamin, which blocked the EDHF component of the response. 40Gap27 significantly attenuated the response to acetylcholine in the LZRs, but had no effect in the OZRs. Connexin 40 protein and Cx40 mRNA levels in mesenteric vascular homogenates were significantly smaller in the OZRs than in the LZRs, with no difference in connexin 43 or Cx43 mRNA levels.These findings demonstrate that endothelial dysfunction in mesenteric arteries from the insulin-resistant OZRs can be attributed to a defect in EDHF. The results also suggest that the defective EDHF is at least partly related to an impairment of connexin 40-associated gap junctions, through a decrease in connexin 40 protein and Cx40 mRNA expression in the OZRs.

A cDNA encoding palmitoyl-acyl carrier protein (ACP)-desaturase from cat's claw (Doxantha unguis-cati L.) was expressed in Arabidopsis thaliana and Brassica napus L. with the goal of decreasing the saturated fatty acid (FA) content of the seed oil. In general, transformation of Arabidopsis resulted in a greater change in the FA composition of the seed oil than for B. napus. An increase in palmitoleic acid (16:1cisΔ 9 ) was obtained in transgenic lines, suggesting that the 16:0-ACP-desaturase cDNA was expressed in the manner originally intended. Other effects on lipid metabolism, however, were observed in the seed of transgenic plants. In Arabidopsis, there was a large increase in the proportions of cis-vaccenic acid (18:1cisΔ 11 ) and cis-13-eicosenoic acid (20:1cisΔ 13 ), possibly generated through elongation of 16:1cisΔ 9 . Elongation of 18:1cisΔ 11 to 20:1cisΔ 13 , however, was not observed in B.napus indicating that certain aspects of lipid metabolism in the model plant, Arabidopsis, may not apply to B. napus. As well, the appearance of 18:1cisΔ 11 was accompanied by a decrease in the proportion of oleic acid (18:1cisΔ 9 ). Although the introduced ACP-desaturase resulted in synthesis of some unsaturated FAs, the overall saturated FA content was maintained at similar levels to the control or was enhanced. Increased levels of saturation were mainly associated with an increase in stearic acid, which unlike 16:0, is considered non-atherogenic. The results suggest that a mechanism exists further downstream in oil biosynthesis to counteract the decrease in saturation brought about by the 16:0-ACP-desaturase action.

S100A11 is a member of the S100 family of EF-hand Ca2+-binding proteins, which is expressed in smooth muscle and other tissues. Ca2+binding to S100A11 induces a conformational change that exposes a hydrophobic surface for interaction with target proteins. Affinity chromatography with immobilized S100A11 was used to isolate a 70-kDa protein from smooth muscle that bound to S100A11 in a Ca2+-dependent manner and was identified by mass spectrometry as annexin A6. Direct Ca2+-dependent interaction between S100A11 and annexin A6 was confirmed by affinity chromatography of the purified bacterially expressed proteins, by gel overlay of annexin A6 with purified S100A11, by chemical cross-linking, and by coprecipitation of S100A11 with annexin A6 bound to liposomes. The expression of S100A11 and annexin A6 in the same cell type was verified by RT-PCR and immunocytochemistry of isolated vascular smooth muscle cells. The site of binding of S100A11 on annexin A6 was investigated by partial tryptic digestion and deletion mutagenesis. The unique NH2terminal head region of annexin A6 was not required for S100A11 binding, but binding sites were identified in both NH2- and COOH-terminal halves of the molecule. We hypothesize that an agonist-induced increase in cytosolic free [Ca2+] leads to formation of a complex of S100A11 and annexin A6, which forms a physical connection between the plasma membrane and the cytoskeleton, or plays a role in the formation of signaling complexes at the level of the sarcolemma.

Endothelial dysfunction plays a role in the development of atherosclerosis and diabetes-associated vascular disease and, in the streptozotocin (STZ)-induced apoE-deficient diabetic mouse, we report that, when compared to the citrate (CIT)-treated nondiabetic apoE-deficient control, acetylcholine (Ach)-mediated endothelium-dependent relaxation was reduced in the small mesenteric arteries (SMA) and the plaque-prone regions of the aorta from the STZ-diabetic mouse. In the SMA the component of Ach-mediated relaxation that was attributed to nitric oxide (NO) from STZ-treated diabetic apoE-deficient mice was enhanced; however, the endothelium-derived hyperpolarizing factor (EDHF)-mediated component was reduced. The EDHF component was assessed by determining the component of the Ach-mediated response that was resistant to the combination of the NO synthase (NOS) inhibitor Nomega-nitro-L-arginine methyl ester, cyclooxygenase inhibitor, indomethacin, and soluble guanylate cyclase inhibitor, ODQ, and inhibited by the combination of the intermediate conductance KCa (IKCa) inhibitor TRAM-34 and the small-conductance KCa (SKCa) inhibitor apamin. Endothelial NOS was increased but SK2, SK3 and connexin (Cx) 37 mRNA expressions were significantly (P

1 Vascular reactivity to the alpha-adrenoceptor agonist phenylephrine (PE) was enhanced in small mesenteric arteries (SMA) from diabetic (db/db) mice under both high and low in vitro oxygen conditions. 2 Mechanical removal of the endothelium significantly attenuated the enhanced vascular reactivity of SMA from db/db mice. 3 Acute incubation of the SMA with sepiapterin, a precursor of tetrahydrobiopterin, and Nω-nitro L-arginine (L-NA), an inhibitor of nitric oxide (NO) synthase (NOS), resulted in no significant change in the enhanced vascular reactivity to PE in db/db mice. Endothelial nitric oxide synthase (eNOS) mRNA and protein levels in SMA were not different between db/+ and db/db mice. 4 Acute incubation of SMA with a combination of polyethylene glycol superoxide dismutase and catalase significantly reduced the enhanced contraction to PE in db/db mice. There were higher levels of malondialdehyde, a marker of lipid peroxidation and basal superoxide as measured by dihydroethidium staining, in SMA from db/db mice compared to db/+ mice. 5 Acute incubation with indomethacin, a nonselective inhibitor of cyclooxygenase, SQ 29548, a selective thromboxane receptor antagonist and furegrelate, a thromboxane synthesis inhibitor, significantly attenuated the enhanced contraction to PE in SMA from db/db mice. 6 This study demonstrates that the enhanced contractility of SMA from db/db mice to PE was endothelium dependent and involves elevated reactive oxygen species, cyclooxygenase activity and thromboxane synthesis, but not changes in the eNOS/NO pathway. British Journal of Pharmacology (2005) 144, 953–960. doi:10.1038/sj.bjp.0706121

This study examined whether hypertension attenuated cell-to-cell communication in skeletal muscle resistance arteries. Briefly, arteries feeding the retractor muscle of normotensive and hypertensive hamsters were cannulated, pressurized, and superfused with a physiological saline solution. Cell-to-cell communication was functionally assessed by application of vasoactive stimuli (via micropipette) to a small portion of a feed artery while diameter at sites distal to the point of agent application was monitored. In keeping with past observations, discrete application of a smooth muscle depolarizing agent (phenylephrine or KCl) elicited a localized vasoconstriction that conducted poorly along feed arteries from normotensive hamsters. In contrast, acetylcholine, an agent known to hyperpolarize endothelial cells, elicited a vasodilation in normotensive feed arteries that conducted with little decay. Whereas smooth muscle depolarizing agents continued to elicit a localized response, conduction of endothelium-dependent vasodilation was attenuated in hypertensive hamsters. This decrease occurred in the absence of changes in vessel reactivity to intravascular pressure or to global application of phenylephrine, U-46619, or acetylcholine. We propose, on the basis of these physiological observations, quantitative mRNA measurements of connexins 37, 40, 43, and 45, and analysis of the literature, that an increase in endothelial-to-endothelial or smooth muscle-to-endothelial coupling resistance is likely responsible for hypertension-induced impairment in vascular communication. We hypothesize that this attenuation could contribute to the rise in total peripheral resistance characteristically observed in hypertension.

The expression of diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) with predicted molecular mass of 56.9. kDa (BnDGAT1) was examined using microspore-derived cell suspension cultures of oilseed rape (Brassica napus L. cv Jet Neuf). As well, a recombinant histidine-tagged N-terminal fragment of BnDGAT1 [BnDGAT1(1–116)His6], which was relatively hydrophilic, was partially characterized. A temporal increase in DGAT activity occurred within a 24 h period following transfer of cells from 6% (w/v) sucrose to 14% (w/v) sucrose. Western blotting indicated that the abundance of BnDGAT1 protein was closely correlated with DGAT activity. BnDGAT1 mRNA also exhibited a temporal increase within the 24 h period following transfer of cells into higher sucrose concentrations, but the transcript level was not closely associated with DGAT activity as BnDGAT1 protein. The fragment BnDGAT1(1–116)His6, interacted with [1-14C] oleoyl-CoA, suggesting that the N-terminal region of BnDGAT1 may have a role in binding cellular acyl-CoA.

This study sought to define whether inward rectifying K+ (KIR) channels were modulated by vasoactive stimuli known to depolarize and constrict intact cerebral arteries. Using pressure myography and patch-clamp electrophysiology, initial experiments revealed a Ba2+-sensitive KIR current in cerebral arterial smooth muscle cells that was active over a physiological range of membrane potentials and whose inhibition led to arterial depolarization and constriction. Real-time PCR, Western blot, and immunohistochemical analyses established the expression of both KIR2.1 and KIR2.2 in cerebral arterial smooth muscle cells. Vasoconstrictor agonists known to depolarize and constrict rat cerebral arteries, including uridine triphosphate, U46619, and 5-HT, had no discernable effect on whole cell KIR activity. Control experiments confirmed that vasoconstrictor agonists could inhibit the voltage-dependent delayed rectifier K+ (KDR) current. In contrast to these observations, a hyposmotic challenge that activates mechanosensitive ion channels elicited a rapid and sustained inhibition of the KIR but not the KDR current. The hyposmotic-induced inhibition of KIR was 1) mimicked by phorbol-12-myristate-13-acetate, a PKC agonist; and 2) inhibited by calphostin C, a PKC inhibitor. These findings suggest that, by modulating PKC, mechanical stimuli can regulate KIR activity and consequently the electrical and mechanical state of intact cerebral arteries. We propose that the mechanoregulation of KIR channels plays a role in the development of myogenic tone.

Endothelium-derived hyperpolarizing factor (EDHF), notably in the microcirculation, plays an important role in the regulation of vascular tone. The cellular events that mediate EDHF are critically dependent, in a vessel dependent manner, on small conductance calcium-activated potassium channels (SK) and intermediate conductance calcium-activated potassium channels (IK) as well as the presence of the gap junction connexins 37, 40, and 43. We hypothesized that the expression levels of SK, IK, as well as vascular connexins, notably 37, 40 and 43 but, potentially, connexin 45, would show correlation with the contribution of EDHF to acetylcholine-mediated vasodilatation as well as, in the absence of endothelial-derived NO, higher expression levels in eNOS(-/-) mice. Wire myograph studies were performed to confirm the contribution of EDHF to endothelium-dependent relaxation in 1st, 2nd and 3rd order small mesenteric arteries from C57BL/6J eNOS-expressing (eNOS(+/+)) and eNOS-deficient C57BL/6J (eNOS(-/-)) mice. Small mesenteric arteries, as well as the branch points between 1st and 2nd and 2nd and 3rd order vessels, were analysed for the expression of mRNA for SK1, SK2, SK3, IK and large conductance calcium-activated potassium channels (BK) and comparable studies were performed for connexins 37, 40, 43 and 45. Although the contribution of EDHF to endothelium-dependent relaxation was significantly greater in the 3rd order vessels from the eNOS(+/+) the real-time (RT) polymerase chain reaction (PCR) data showed no differences for the expression levels of mRNA for any of the channel subtypes or the connexins within the small mesenteric arteries from either the eNOS(+/+) or eNOS(-/-) mice, nor, based on RT PCR analysis, were there differences in expression of the potassium channels studied in the branch points versus 1st, 2nd or 3rd order vessels. These data suggest that neither the gene expression of calcium-activated potassium channels nor vascular connexins are modulated by NO; however, their functional contribution to endothelium-dependent relaxation may be modulated by other physiological parameters.

Endothelial dysfunction plays a role in the development of atherosclerosis and diabetes-associated vascular disease and, in the streptozotocin (STZ)-induced apoE-deficient diabetic mouse, we report that, when compared to the citrate (CIT)-treated nondiabetic apoE-deficient control, acetylcholine (Ach)-mediated endothelium-dependent relaxation was reduced in the small mesenteric arteries (SMA) and the plaque-prone regions of the aorta from the STZ-diabetic mouse. In the SMA the component of Ach-mediated relaxation that was attributed to nitric oxide (NO) from STZ-treated diabetic apoE-deficient mice was enhanced; however, the endothelium-derived hyperpolarizing factor (EDHF)-mediated component was reduced. The EDHF component was assessed by determining the component of the Ach-mediated response that was resistant to the combination of the NO synthase (NOS) inhibitor Nomega-nitro-L-arginine methyl ester, cyclooxygenase inhibitor, indomethacin, and soluble guanylate cyclase inhibitor, ODQ, and inhibited by the combination of the intermediate conductance KCa (IKCa) inhibitor TRAM-34 and the small-conductance KCa (SKCa) inhibitor apamin. Endothelial NOS was increased but SK2, SK3 and connexin (Cx) 37 mRNA expressions were significantly (P0.05) decreased in the SMA from STZ-treated apoE-deficient mice compared to the CIT-treated controls. There was no difference in the IKCa expression or in Cx 40, 43 and 45 mRNA levels between STZ- and CIT-treated mice. The microvasculature of STZ-induced apoE-deficient mice developed endothelial dysfunction, which may be linked to a decrease in the contribution of the EDHF component due to a decrease in SK2 and 3 and Cx 37 expression.

The Type 2 diabetic db/ db mouse experiences vascular dysfunction typified by changes in the contraction and relaxation profiles of small mesenteric arteries (SMAs). Contractions of SMAs from the db/ db mouse to the α1-adrenoceptor agonist phenylephrine (PE) were significantly enhanced, and acetylcholine (ACh)-induced relaxations were significantly depressed. Drug treatment of db/ db mice with a nonthiazolidinedione peroxisome prolifetor-activated receptor-γ agonist and insulin sensitizing agent 2-[2-(4-phenoxy-2-propylphenoxy)ethyl]indole-5-acetic acid (COOH) completely prevented the changes in endothelium-dependent relaxation, but, with the discontinuation of therapy, endothelial dysfunction returned. Dysfunctional SMAs were found to specifically upregulate the expression of a 35-kDa isoform of sarcolemmal membrane-associated protein (SLMAP), which is a component of the excitation-contraction coupling apparatus and implicated in the regulation of membrane function in muscle cells. Real-time PCR revealed high SLMAP mRNA levels in the db/ db microvasculature, which were markedly downregulated during COOH treatment but elevated again when drug therapy was discontinued. These data reveal that the microvasculature in db/ db mice undergoes significant changes in vascular function with the endothelial component of vascular dysfunction specifically correlating with the overexpression of SLMAP. Thus changes in SLMAP expression may be an important indicator for microvascular disease associated with Type 2 diabetes.

Vascular reactivity to the alpha-adrenoceptor agonist phenylephrine (PE) was enhanced in small mesenteric arteries (SMA) from diabetic (db/db) mice under both high and low in vitro oxygen conditions. Mechanical removal of the endothelium significantly attenuated the enhanced vascular reactivity of SMA from db/db mice. Acute incubation of the SMA with sepiapterin, a precursor of tetrahydrobiopterin, and N(omega)-nitro L-arginine (L-NA), an inhibitor of nitric oxide (NO) synthase (NOS), resulted in no significant change in the enhanced vascular reactivity to PE in db/db mice. Endothelial nitric oxide synthase (eNOS) mRNA and protein levels in SMA were not different between db/+ and db/db mice. Acute incubation of SMA with a combination of polyethylene glycol superoxide dismutase and catalase significantly reduced the enhanced contraction to PE in db/db mice. There were higher levels of malondialdehyde, a marker of lipid peroxidation and basal superoxide as measured by dihydroethidium staining, in SMA from db/db mice compared to db/+ mice. Acute incubation with indomethacin, a nonselective inhibitor of cyclooxygenase, SQ 29548, a selective thromboxane receptor antagonist and furegrelate, a thromboxane synthesis inhibitor, significantly attenuated the enhanced contraction to PE in SMA from db/db mice. This study demonstrates that the enhanced contractility of SMA from db/db mice to PE was endothelium dependent and involves elevated reactive oxygen species, cyclooxygenase activity and thromboxane synthesis, but not changes in the eNOS/NO pathway.

In this review we discuss the contribution of NO, prostacyclin and endothelium-derived relaxing factor--endothelium-derived hyperpolarizing factor, or EDHF, to vascular function. We also explore the hypotheses (1): that tissues can store NO as nitrosothiols (RSNOs) and (2) that such RSNO stores can be modulated by physiological and pathophysiological processes. Notably in the microcirculation, EDHF appears to play an important role in the regulation of vascular tone. Leading candidates for EDHF include extracellular potassium (K+), an epoxygenase product, hydrogen peroxide and/or a contribution from myoendothelial gap junctions. Data from our laboratory indicate that in mouse vessels, different endothelium-dependent vasodilators, such as acetylcholine and protease-activated receptor (PAR) agonists, release different endothelium-derived relaxing factors. The combination of two K-channel toxins, apamin and charybdotoxin, inhibits EDHF activity in most protocols. Endothelial dysfunction is considered as the major risk factor and a very early indicator of cardiovascular disease including the cardiovascular complications of type I & types II diabetes. Impaired endothelium-dependent vasodilatation results primarily from a decreased synthesis of endothelium-derived nitric oxide (NO) and/or an increase in the production of reactive oxygen species such as superoxide. We have shown that the administration of tetrahydrobiopterin, an important co-factor for nitric oxide synthase (NOS) partially restores endothelial function (1) in leptin-deficient mice (db/db) with spontaneous type II diabetes, as well as (2) in human vascular tissue harvested for coronary artery bypass grafting (CABG). These data suggest that a deficiency in the availability of tetrahydrobiopterin plays an important role in vascular dysfunction associated with Type II diabetes. In addition, changes in the contribution of EDHF occur in vascular tissue from the db/db mice suggesting a compensatory increase in EDHF production; whether this alteration in EDHF production is physiological or pathophysiological remains controversial.

This study examined whether, and by what signaling and ionic mechanisms, pyrimidine nucleotides constrict rat cerebral arteries. Cannulated cerebral arteries stripped of endothelium and pressurized to 15 mmHg constricted in a dose-dependent manner to UTP. This constriction was partly dependent on the depolarization of smooth muscle cells and the activation of voltage-operated Ca2+channels. The depolarization and constriction induced by UTP were unaffected by bisindolylmaleimide I, a PKC inhibitor that abolished phorbol ester (PMA)-induced constriction in cerebral arteries. In contrast, the Rhokinase inhibitor Y-27632 attenuated the ability of UTP to both constrict and depolarize cerebral arteries. With patch-clamp electrophysiology, a voltage-dependent delayed rectifying K+(KDR) current was isolated and shown to consist of a slowly inactivating 4-aminopyridine (4-AP)-sensitive and an -insensitive component. The 4-AP-sensitive KDRcurrent was potently suppressed by UTP through a mechanism that was not dependent on PKC. This reflects observations that demonstrated that 1) a PKC activator (PMA) had no effect on KDRand 2) PKC inhibitors (calphostin C or bisindolylmaleimide I) could not prevent the suppression of KDRby UTP. The Rho kinase inhibitor Y-27632 abolished the ability of UTP to inhibit the KDRcurrent, as did inhibition of RhoA with C3 exoenzyme. Cumulatively, these observations indicate that Rho kinase signaling plays an important role in eliciting the cerebral constriction induced by pyrimidine nucleotides. Moreover, they demonstrate for the first time that Rhokinase partly mediates this constriction by altering ion channels that control membrane potential and Ca2+influx through voltage-operated Ca2+channels.

Human acylation stimulating protein (ASP) and bovine serum albumin (BSA) have been shown to stimulate diacylglycerol acyltransferase activity in microsomes from cultures of Brassica napus (Weselake et al., 2000; Little et al., 1994). In the current study, B. napus was transformed with cDNA encoding ASP or BSA to evaluate possible effects on seed oil biosynthesis.

Diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the acyl-CoA-dependent acylation of sn-1, 2-diacylglycerol to generate triacylglycerol and CoA. The deduced amino acid sequence of cDNAs encoding DGAT1 from plants and mammals exhibit a hydrophilic N-terminal region followed by a number of potential membrane-spanning segments, which is consistent with the membrane-bound nature of this enzyme family. In order to gain insight into the structure/function properties of DGAT1 from Brassica napus (BnDGAT1), we produced and partially characterized a recombinant polyHis-tagged N-terminal fragment of the enzyme, BnDGAT1(1–116)His6, with calculated molecular mass of 13,278 Da. BnDGAT1(1–116)His6 was highly purified from bacterial lysate and plate-like monoclinic crystals were grown using this preparation. Lipidex-1000 binding assays and gel electrophoresis indicated that BnDGAT1(1–116)His6 interacts with long chain acyl-CoA. The enzyme fragment displayed enhanced affinity for erucoyl (22:1cisΔ13)-CoA over oleoyl (18:1cisΔ9)-CoA, and the binding process displayed positive cooperativity. Gel filtration chromatography and cross-linking studies indicated that BnDGAT1(1–116)His6 self-associated to form a tetramer. Polyclonal antibodies raised against a peptide of 15 amino acid residues representing a segment of BnDGAT1(1–116)His6 failed to react with protein in microsomal vesicles following treatment with proteinase K, suggesting that the N-terminal fragment of BnDGAT1 was localized to the cytosolic side of the ER. Collectively, these results suggest that BnDGAT1 may be allosterically modulated by acyl-CoA through the N-terminal region and that the enzyme self-associates via interactions on the cytosolic side of the ER.