The Role of O-GlcNAcylation in Perivascular Adipose Tissue Dysfunction of Offspring of High-Fat Diet-Fed Rats.

Perivascular adipose tissue (PVAT), which reduces vascular contractility, is dysfunctional in the male offspring of rats fed a high-fat diet (HFD), partially due to a reduced NO bioavailability. O-GlcNAcylation of eNOS decreases its activity, thus we investigated the role of O-GlcNAcylation in the prenatal programming of PVAT dysfunction. Female Sprague-Dawley rats were fed either a control (10% fat) or an obesogenic HFD (45% fat) diet for 12 weeks prior to mating, and throughout pregnancy and lactation. Offspring were weaned onto the control diet and were killed at 12 and 24 weeks of age. Mesenteric arteries from the 12-week-old offspring of HFD dams (HFDO) contracted less to U46619; these effects were mimicked by glucosamine in control arteries. PVAT from 12- and 24-week-old controls, but not from HFDO, exerted an anticontractile effect. Glucosamine attenuated the anticontractile effect of PVAT in the vessels from controls but not from HFDO. AMP-activated protein kinase (AMPK) activation (with A769662) partially restored an anticontractile effect in glucosamine-treated controls and HFDO PVAT. Glucosamine decreased AMPK activity and expression in HFDO PVAT, although phosphorylated eNOS expression was only reduced in that from males. The loss of anticontractile effect of HFDO PVAT is likely to result from increased O-GlcNAcylation, which decreased AMPK activity and, in males, decreased NO bioavailability.

Obesity-Related Perivascular Adipose Tissue Damage Is Reversed by Sustained Weight Loss in the Rat.

OBJECTIVE: Perivascular adipose tissue (PVAT) exerts an anticontractile effect in response to various vasoconstrictor agonists, and this is lost in obesity. A recent study reported that bariatric surgery reverses the damaging effects of obesity on PVAT function. However, PVAT function has not been characterized after weight loss induced by caloric restriction, which is often the first line treatment for obesity. APPROACH AND RESULTS: Contractility studies were performed using wire myography on small mesenteric arteries with and without PVAT from control, diet-induced obese, calorie restricted and sustained weight loss rats. Changes in the PVAT environment were assessed using immunohistochemistry. PVAT from healthy animals elicited an anticontractile effect in response to norepinephrine. This was abolished in diet-induced obesity through a mechanism involving increased local tumor necrosis factor-α and reduced nitric oxide bioavailability within PVAT. Sustained weight loss led to improvement in PVAT function associated with restoration of adipocyte size, reduced tumor necrosis factor-α, and increased nitric oxide synthase function. This was associated with reversal of obesity-induced hypertension and normalization of plasma adipokine levels, including leptin and insulin. CONCLUSIONS: We have shown that diet-induced weight loss reverses obesity-induced PVAT damage through a mechanism involving reduced inflammation and increased nitric oxide synthase activity within PVAT. These data reveal inflammation and nitric oxide synthase, particularly endothelial nitric oxide synthase, as potential targets for the treatment of PVAT dysfunction associated with obesity and metabolic syndrome.

Endothelium-derived hyperpolarising factors and associated pathways: a synopsis.

The term endothelium-derived hyperpolarising factor (EDHF) was introduced in 1987 to describe the hypothetical factor responsible for myocyte hyperpolarisations not associated with nitric oxide (EDRF) or prostacyclin. Two broad categories of EDHF response exist. The classical EDHF pathway is blocked by apamin plus TRAM-34 but not by apamin plus iberiotoxin and is associated with endothelial cell hyperpolarisation. This follows an increase in intracellular [Ca(2+)] and the opening of endothelial SK(Ca) and IK(Ca) channels preferentially located in caveolae and in endothelial cell projections through the internal elastic lamina, respectively. In some vessels, endothelial hyperpolarisations are transmitted to myocytes through myoendothelial gap junctions without involving any EDHF. In others, the K(+) that effluxes through SK(Ca) activates myocytic and endothelial Ba(2+)-sensitive K(IR) channels leading to myocyte hyperpolarisation. K(+) effluxing through IK(Ca) activates ouabain-sensitive Na(+)/K(+)-ATPases generating further myocyte hyperpolarisation. For the classical pathway, the hyperpolarising "factor" involved is the K(+) that effluxes through endothelial K(Ca) channels. During vessel contraction, K(+) efflux through activated myocyte BK(Ca) channels generates intravascular K(+) clouds. These compromise activation of Na(+)/K(+)-ATPases and K(IR) channels by endothelium-derived K(+) and increase the importance of gap junctional electrical coupling in myocyte hyperpolarisations. The second category of EDHF pathway does not require endothelial hyperpolarisation. It involves the endothelial release of factors that include NO, HNO, H(2)O(2) and vasoactive peptides as well as prostacyclin and epoxyeicosatrienoic acids. These hyperpolarise myocytes by opening various populations of myocyte potassium channels, but predominantly BK(Ca) and/or K(ATP), which are sensitive to blockade by iberiotoxin or glibenclamide, respectively.

Evidence for the presence of GPRC6A receptors in rat mesenteric arteries.

In this study, the presence of GPRC6A receptors in rat mesenteric artery was investigated. In artery homogenates, GPRC6A mRNA was detected and Western blotting showed the presence of GPRC6A protein. Immunohistochemical studies revealed GPRC6A in both endothelial cells and myocytes. In whole vessel segments, the GPRC6A activators, 300 microM l-ornithine and 100 microM Al(3+), induced endothelium-dependent myocyte hyperpolarizations sensitive to 10 microM TRAM-34, a blocker of intermediate conductance, Ca(2+)-sensitive K(+) channels (IK(Ca)). Activation of IK(Ca) with calindol (300 nM; a positive allosteric Ca(2+)-sensing receptor - CaR - modulator) was inhibited by 500 nM ouabain (inhibition of rat type 2 and type 3 Na(+)/K(+)-ATPases) but unaffected by 30 microM Ba(2+) (blockade of inwardly rectifying K(+) channels). Neither l-ornithine nor Al(3+) activated CaRs heterologously expressed in CHO or HEK293 cells. In the presence of 300 microM l-ornithine or 100 microM Al(3+), myocyte hyperpolarizations to calindol were potentiated whereas this potentiation and hyperpolarizations to l-ornithine were lost following incubation with an anti-GPRC6A antibody. It is concluded that GPRC6A receptors are present on mesenteric artery endothelial cells and myocytes and that their activation selectively opens IK(Ca) channels. This triggers a ouabain-sensitive myocyte hyperpolarization suggesting a close functional relationship between GPRC6A, the IK(Ca) channel and type 2 and/or type 3 Na(+)/K(+)-ATPases.

Evidence in favor of a calcium-sensing receptor in arterial endothelial cells: studies with calindol and Calhex 231.

Small increases in extracellular Ca2+ dilate isolated blood vessels. In the present study, the possibility that a vascular, extracellular Ca2+-sensing receptor (CaSR) could mediate these vasodilator actions was investigated. Novel ligands that interact with the CaSR were used in microelectrode recordings from rat isolated mesenteric and porcine coronary arteries. The major findings were that (1) raising extracellular Ca2+ or adding calindol, a CaSR agonist, produced concentration-dependent hyperpolarizations of vascular myocytes, actions attenuated by Calhex 231, a negative allosteric modulator of CaSR. (2) Calindol-induced hyperpolarizations were inhibited by the intermediate conductance, Ca2+-sensitive K+ (IKCa) channel inhibitors, TRAM-34, and TRAM-39. (3) The effects of calindol were not observed in the absence of endothelium. (4) CaSR mRNA and protein were present in rat mesenteric arteries and in porcine coronary artery endothelial cells. (5) CaSR and IKCa proteins were restricted to caveolin-poor membrane fractions. We conclude that activation of vascular endothelial CaSRs opens endothelial cell IKCa channels with subsequent myocyte hyperpolarization. The endothelial cell CaSR may have a physiological role in the control of arterial blood pressure.

Hydrogen peroxide and endothelium-dependent hyperpolarization in the guinea-pig carotid artery.

This study was designed to determine whether or not endothelium-dependent hyperpolarizations evoked by acetylcholine in the isolated guinea-pig carotid artery involve hydrogen peroxide. Membrane potential was recorded in the vascular smooth muscle cells of that artery. Under control conditions, acetylcholine induced endothelium-dependent hyperpolarization of the vascular smooth muscle cells which was not affected by the presence of catalase, superoxide dismutase or their combination. Neither the superoxide dismutase mimetic, tiron nor the thiol-reducing agent N-acetyl-L-cysteine modified the hyperpolarization evoked by 0.1 microM acetylcholine but each produced a partial and significant inhibition of the hyperpolarization induced by 1 microM acetylcholine. Neither 10 nor 100 microM hydrogen peroxide influenced the resting membrane potential of the smooth muscle cells and the higher concentration did not significantly influence the hyperpolarization elicited by acetylcholine. These data indicate that, in the guinea-pig isolated carotid artery, hydrogen peroxide is unlikely to contribute to the endothelium-dependent hyperpolarization evoked by acetylcholine.

Bradykinin-induced, endothelium-dependent responses in porcine coronary arteries: involvement of potassium channel activation and epoxyeicosatrienoic acids.

In coronary arteries, bradykinin opens endothelial intermediate- and small-conductance Ca2+-sensitive K+ channels (IK(Ca) and SK(Ca)) and, additionally, releases epoxyeicosatrienoic acids (EETs) from the endothelium. To clarify the involvement of these pathways in endothelium-dependent myocyte hyperpolarization, bradykinin-induced electrical changes in endothelial cells and myocytes of porcine coronary arteries (following nitric oxide (NO) synthase and cyclooxygenase inhibition) were measured using sharp microelectrodes. Hyperpolarization of endothelial cells by bradykinin (27.0 +/- 0.9 mV, n = 4) was partially inhibited (74%) by blockade of IK(Ca) and SK(Ca) channels using 10 microM TRAM-39 (2-(2-chlorophenyl)-2,2-diphenylacetonitrile) plus 100 nM apamin (leaving an iberiotoxin-sensitive component), whereas the response to substance P was abolished. After gap junction blockade with HEPES, (N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulphonic acid)) hyperpolarization of the endothelium by 100 nM bradykinin was abolished by TRAM-39 plus apamin, whereas myocyte hyperpolarization still occurred (12.9 +/- 1.0 mV, n=4). The residual hyperpolarizations to 100 nM bradykinin were antagonized by the EET antagonist, 14,15-EEZE (14,15-epoxyeicosa-5(Z)-enoic acid) (10 microM), and abolished by iberiotoxin. Bradykinin-induced myocyte hyperpolarizations were also reduced by 14,15-EEZE-mSI (14,15-EEZE-methylsulfonylimide) (5,6- and 14,15-EET antagonist), whereas those to exogenous 11,12-EET were unaffected. These data show that bradykinin-induced hyperpolarization of endothelial cells (due to the opening of IK(Ca) and SK(Ca) channels) is electrotonically transferred to the myocytes via gap junctions. Bradykinin (but not substance P) also hyperpolarizes myocytes by a mechanism (independent of endothelial cell hyperpolarization) which involves endothelial cell production of EETs (most likely 14,15- and/or 11,12-EET). These open endothelial IK(Ca) and SK(Ca) channels and also activate large-conductance calcium-sensitive K+ channels (BK(Ca)) on the surrounding myocytes.

Role of SK(Ca) and IK(Ca) in endothelium-dependent hyperpolarizations of the guinea-pig isolated carotid artery.

1. This study was designed to determine whether the endothelium-dependent hyperpolarizations evoked by acetylcholine in guinea-pig carotid artery involve a cytochrome P450 metabolite and whether they are linked to the activation of two distinct populations of endothelial K(Ca) channels, SK(Ca) and IK(Ca.) 2. The membrane potential was recorded in the vascular smooth muscle cells of the guinea-pig isolated carotid artery. All the experiments were performed in the presence of N(omega)-L-nitro arginine (100 microM) and indomethacin (5 microM). 3. Under control conditions (Ca(2+): 2.5 mM), acetylcholine (10 nM to 10 muM) induced a concentration- and endothelium-dependent hyperpolarization of the vascular smooth muscle cells. Two structurally different specific blockers of SK(Ca), apamin (0.5 microM) or UCL 1684 (10 microM), produced a partial but significant inhibition of the hyperpolarization evoked by acetylcholine whereas charybdotoxin (0.1 microM) and TRAM-34 (10 microM), a nonpeptidic and specific blocker of IK(Ca), were ineffective. In contrast, the combinations of apamin plus charybdotoxin, apamin plus TRAM-34 (10 microM) or UCL 1684 (10 microM) plus TRAM-34 (10 microM) virtually abolished the acetylcholine-induced hyperpolarization. 4. In the presence of a combination of apamin and a subeffective dose of TRAM-34 (5 microM), the residual hyperpolarization produced by acetylcholine was not inhibited further by the addition of either an epoxyeicosatrienoic acid antagonist, 14,15-EEZE (10 microM) or the specific blocker of BK(Ca), iberiotoxin (0.1 microM). 5. In presence of 0.5 mM Ca(2+), the hyperpolarization in response to acetylcholine (1 microM) was significantly lower than in 2.5 mM Ca(2+). The EDHF-mediated responses became predominantly sensitive to charybdotoxin or TRAM-34 but resistant to apamin. 6. This investigation shows that the production of a cytochrome P450 metabolite, and the subsequent activation of BK(Ca), is unlikely to contribute to the EDHF-mediated responses in the guinea-pig carotid artery. Furthermore, the EDHF-mediated response involves the activation of both endothelial IK(Ca) and SK(Ca) channels, the activation of either one being able to produce a true hyperpolarization.

Potassium and potassium clouds in endothelium-dependent hyperpolarizations.

A small increase in extracellular K(+) acts as a local, physiological regulator of blood flow to certain vascular beds. The K(+) derives from active tissues such as contracting skeletal muscle and brain and increases blood supply to these organs by the activation of Na(+)/K(+)-ATPases and/or inwardly-rectifying K(+) channels on the vascular myocytes. K(+) liberated from the vascular endothelium also acts as an endothelium-derived hyperpolarizing and relaxing factor within blood vessels. The K(+) effluxes from endothelial cell intermediate- and small-conductance, Ca(2+)-sensitive K(+) channels which open in response to stretch and local hormones. In many vessels, endothelium-derived hyperpolarizing factor (EDHF) seems identical to the K(+) derived from endothelial cells; it activates Na(+)/K(+)-ATPases (particularly those containing alpha2 and alpha3 subunits) and inward rectifiers (particularly Kir2.1) located on the vascular myocytes. Vasospastic agents generate "potassium clouds" around vascular smooth muscle cells via the efflux of this ion through large conductance, Ca(2+)-sensitive K(+) channels on the myocytes. These potassium clouds can reduce the hyperpolarizing actions of endothelium-derived K(+) by effectively saturating the Na(+)/K(+)-ATPases and inward rectifiers on the muscle cells and they may be of clinical significance in vasospastic conditions.

[Communication between endothelial and smooth muscle cells]. / Dialogue entre cellules endothéliales et cellules musculaires lisses.

Vascular endothelial cells play a fundamental role in the control of vascular tone, and therefore in the control of local blood flow, by releasing various contracting (endothelin, prostaglandins) and relaxing (prostacycline, NO) factors. An additional mechanism involving the hyperpolarization of the vascular smooth muscle cells is observed mainly in the coronary vascular bed and in the periphery. This phenomenon was attributed to an elusive endothelial factor called endothelium-derived hyperpolarizing factor (EDHF). This mechanism is now better understood. It involves first an increase in the endothelial intracellular concentration of calcium, the activation of endothelial potassium channels and the resulting hyperpolarization of the endothelial cells. The hyperpolarization of the endothelial cells is transmitted to the smooth muscle cells by different pathways. This hyperpolarization propagates along the vessels not only via the smooth muscle cells but also via the endothelial cells. Therefore, the endothelial layer can also be considered as a conducting tissue. The discovery of specific inhibitors of the endothelial cell hyperpolarization allows the assessment of the contribution of EDHF-mediated responses in the control of vascular tone.

Characterization of a charybdotoxin-sensitive intermediate conductance Ca2+-activated K+ channel in porcine coronary endothelium: relevance to EDHF.

1. This study characterizes the K(+) channel(s) underlying charybdotoxin-sensitive hyperpolarization of porcine coronary artery endothelium. 2. Two forms of current-voltage (I/V) relationship were evident in whole-cell patch-clamp recordings of freshly-isolated endothelial cells. In both cell types, iberiotoxin (100 nM) inhibited a current active only at potentials over +50 mV. In the presence of iberiotoxin, charybdotoxin (100 nM) produced a large inhibition in 38% of cells and altered the form of the I/V relationship. In the remaining cells, charybdotoxin also inhibited a current but did not alter the form. 3. Single-channel, outside-out patch recordings revealed a 17.1+/-0.4 pS conductance. Pipette solutions containing 100, 250 and 500 nM free Ca(2+) demonstrated that the open probability was increased by Ca(2+). This channel was blocked by charybdotoxin but not by iberiotoxin or apamin. 4. Hyperpolarizations of intact endothelium elicited by substance P (100 nM; 26.1+/-0.7 mV) were reduced by apamin (100 nM; 17.0+/-1.8 mV) whereas those to 1-ethyl-2-benzimidazolinone (1-EBIO, 600 microM, 21.0+/-0.3 mV) were unaffected (21.7+/-0.8 mV). Substance P, bradykinin (100 nM) and 1-EBIO evoked charybdotoxin-sensitive, iberiotoxin-insensitive whole-cell perforated-patch currents. 5 A porcine homologue of the intermediate-conductance Ca(2+)-activated K(+) channel (IK1) was identified in endothelial cells. 6. In conclusion, porcine coronary artery endothelial cells express an intermediate-conductance Ca(2+)-activated K(+) channel and the IK1 gene product. This channel is opened by activation of the EDHF pathway and likely mediates the charybdotoxin-sensitive component of the EDHF response.

EDHF: bringing the concepts together.

Endothelial cells synthesize and release vasoactive mediators in response to various neurohumoural substances (e.g. bradykinin or acetylcholine) and physical stimuli (e.g. cyclic stretch or fluid shear stress). The best-characterized endothelium-derived relaxing factors are nitric oxide and prostacyclin. However, an additional relaxant pathway associated with smooth muscle hyperpolarization also exists. This hyperpolarization was originally attributed to the release of an endothelium-derived hyperpolarizing factor (EDHF) that diffuses to and activates smooth muscle K(+) channels. More recent evidence suggests that endothelial cell receptor activation by these neurohumoural substances opens endothelial cell K(+) channels. Several mechanisms have been proposed to link this pivotal step to the subsequent smooth muscle hyperpolarization. The main concepts are considered in detail in this review.

Endothelium-dependent hyperpolarization to acetylcholine in carotid artery of guinea pig: role of lipoxygenase.

This study was designed to determine whether lipoxygenase-dependent metabolites of arachidonic acid are involved in the endothelium-dependent hyperpolarization of the guinea pig carotid artery. The membrane potential of vascular smooth muscle cells was measured with intracellular microelectrodes and potassium channels were studied on freshly isolated cells with the patch-clamp technique. Acetylcholine-induced hyperpolarizations were not affected by arachidonyl trifluoromethyl ketone (AACOCF3), quinacrine (phospholipase A inhibitors), or eicosatetraenoic acid (nonspecific inhibitor of lipoxygenase, cytochrome P450, and cyclooxygenase). In contrast, cinnamyl-3,4 dihydroxy-alpha-cyanocinnamate (CDC) and AA861 (lipoxygenase inhibitors) as well as 1-(6-(17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino) hexyl)-1H-pyrrole-2,5-dione (U-73122) (phospholipase C inhibitor) produced a significant inhibition of the hyperpolarization. An opener of intermediate conductance calcium-activated potassium channels, 1-ethyl-2-benzamidazolinone (1-EBIO), induced a hyperpolarization that was unaffected by AACOCF3, CDC, AA861, or U-73122 but was inhibited by charybdotoxin. (+/-)12-hydroxy-eicosatetraenoic acid (12-HETE) and 12(S)-hydroperoxy-eicosatetraenoic acid (12(S)-HpETE) did not induce any significant changes in membrane potential. CDC inhibited the voltage-gated potassium current and increased the large conductance calcium-activated potassium current whereas AA861 inhibited both potassium currents. These results confirm that, in the isolated carotid artery of the guinea pig, stimulation of endothelial muscarinic receptors involves phospholipase C activation and indicate that the activation of phospholipase A2 and the release of lipoxygenase metabolites is unlikely to explain endothelium-dependent hyperpolarization.