An N-terminal fragment of mouse DGAT1 binds different acyl-CoAs with varying affinity.

A histidine-tagged recombinant N-terminal fragment of type-1 mouse liver diacylglycerol acyltransferase (DGAT; EC 2.3.1.20), MmDGAT1(1-95)His6, was expressed in Escherichia coli, and used to investigate possible acyl-CoA-binding properties. Analysis of the purified fragment by MALDI-TOF mass spectrometry revealed a polypeptide with molecular mass of about 11 kDa which was consistent with the calculated molecular mass based on the deduced amino acid sequence. Lipidex-1000 binding assays indicated that MmDGAT1(1-95)His(6) interacted with long chain fatty acyl-CoAs similar to observations on DGAT1 from oilseed rape (Brassica napus). Binding, as a function of acyl-CoA concentration, differed for palmitoyl (16:0), stearoyl (18:0), and erucoyl (cisDelta(13)22:1)-CoA. Binding of stearoyl- or erucoyl-CoA to MmDGAT1(1-95)His(6) as a function of acyl-CoA concentration, however, was sigmoid and displayed positive cooperativity suggesting that MmDGAT1 may be subject to allosteric modulation by acyl-CoAs. An intra-polypeptide segment within the N-terminal region of MmDGAT1 contained remnants of an acyl-CoA-binding signature initially identified in plant DGAT1. The acyl-CoA-binding site in mammalian DGAT1 could represent a potential target for therapeutic interventions for disorders such as type-2 diabetes and obesity.

Calcium-activated potassium channel and connexin expression in small mesenteric arteries from eNOS-deficient (eNOS-/-) and eNOS-expressing (eNOS+/+) mice.

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.

Acyl-CoA-binding and self-associating properties of a recombinant 13.3 kDa N-terminal fragment of diacylglycerol acyltransferase-1 from oilseed rape.

BACKGROUND: 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. RESULTS: 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 (22:1cisDelta13)-CoA over oleoyl (18:1cisDelta9)-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. CONCLUSION: 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.

Enhanced vascular reactivity of small mesenteric arteries from diabetic mice is associated with enhanced oxidative stress and cyclooxygenase products.

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.

Endothelial dysfunction in the streptozotocin-induced diabetic apoE-deficient mouse.

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<0.05) 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 endothelium in health and disease--a target for therapeutic intervention.

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.

Hyposmotic challenge inhibits inward rectifying K+ channels in cerebral arterial smooth muscle cells.

This study sought to define whether inward rectifying K(+) (K(IR)) 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 Ba(2+)-sensitive K(IR) 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 K(IR)2.1 and K(IR)2.2 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 K(IR) activity. Control experiments confirmed that vasoconstrictor agonists could inhibit the voltage-dependent delayed rectifier K(+) (K(DR)) current. In contrast to these observations, a hyposmotic challenge that activates mechanosensitive ion channels elicited a rapid and sustained inhibition of the K(IR) but not the K(DR) current. The hyposmotic-induced inhibition of K(IR) 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 K(IR) activity and consequently the electrical and mechanical state of intact cerebral arteries. We propose that the mechanoregulation of K(IR) channels plays a role in the development of myogenic tone.

Identification of a novel interaction between the Ca(2+)-binding protein S100A11 and the Ca(2+)- and phospholipid-binding protein annexin A6.

S100A11 is a member of the S100 family of EF-hand Ca(2+)-binding proteins, which is expressed in smooth muscle and other tissues. Ca(2+) 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 Ca(2+)-dependent manner and was identified by mass spectrometry as annexin A6. Direct Ca(2+)-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 NH(2) terminal head region of annexin A6 was not required for S100A11 binding, but binding sites were identified in both NH(2)- and COOH-terminal halves of the molecule. We hypothesize that an agonist-induced increase in cytosolic free [Ca(2+)] 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.

A nonthiazolidinedione peroxisome proliferator-activated receptor gamma agonist reverses endothelial dysfunction in diabetic (db/db-/-) mice.

We have previously reported that endothelium-dependent relaxation to acetylcholine is impaired in small mesenteric arteries from spontaneously diabetic (db/db) mice. The objective of the present study was to examine the effects of treatment of the db/db and the insulin-resistant ob/ob mice with the PPARgamma agonist 2-(2-(4-phenoxy-2-propylphenoxy)ethyl)indole-5-acetic acid (COOH). In the db/db model, an 8-week treatment with COOH (30 mg/kg/day) reduced plasma glucose from 48.0 +/- 2.5 (untreated) to 12.6 +/- 1.1 mM. In contrast, plasma glucose was not elevated in untreated ob/ob mice. Relaxation of small mesenteric arteries mediated by acetylcholine was impaired in the untreated db/db diabetic mice (51.7 +/- 7.4% maximal relaxation, n = 6) but not in the ob/ob mice (70.8 +/- 8.6% maximal relaxation, n = 3). This impairment was reversed with COOH treatment (86.9 +/- 0.4% maximal relaxation, n = 5). Malondialdehyde was elevated in plasma from diabetic db/db mice (13.9 +/- 1.1 versus 12.0 +/- 0.7 micromol/ml); however, when normalized to total cholesterol, no significant differences in the ratio of lipid peroxidation in plasma were identified. Western blot analysis and quantitative polymerase chain reaction for eNOS was performed on the isolated mesenteric vessels and revealed no differences in the relative levels of eNOS expression in diabetic and control animals; in addition, treatment with COOH had no significant effect on eNOS levels in either group. In summary, endothelial dysfunction and hyperglycemia were completely normalized in COOH-treated db/db mice. In contrast, nonhyperglycemic ob/ob mice exhibited normal vasodilatory responses to acetylcholine and, consequently, COOH treatment had no effect on endothelial function.

Hypertension attenuates cell-to-cell communication in hamster retractor muscle feed arteries.

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.

Endothelial dysfunction in Type 2 diabetes correlates with deregulated expression of the tail-anchored membrane protein SLMAP.

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 alpha1-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-gamma 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.

Pyrimidine nucleotides suppress KDR currents and depolarize rat cerebral arteries by activating Rho kinase.

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 Ca(2+) 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(+) (K(DR)) current was isolated and shown to consist of a slowly inactivating 4-aminopyridine (4-AP)-sensitive and an -insensitive component. The 4-AP-sensitive K(DR) current 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 K(DR) and 2) PKC inhibitors (calphostin C or bisindolylmaleimide I) could not prevent the suppression of K(DR) by UTP. The Rho kinase inhibitor Y-27632 abolished the ability of UTP to inhibit the K(DR) current, 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 Ca(2+) influx through voltage-operated Ca(2+) channels.