Robert Burnett

Chemerin Connects Fat to Arterial Contraction

Objective—Obesity and hypertension are comorbid in epidemic proportion, yet their biological connection is largely a mystery. The peptide chemerin is a candidate for connecting fat deposits around the blood vessel (perivascular adipose tissue) to arterial contraction. We presently tested the hypothesis that chemerin is expressed in perivascular adipose tissue and is vasoactive, supporting the existence of a chemerin axis in the vasculature. Approach and Results—Real-time polymerase chain reaction, immunohistochemistry, and Western analyses supported the synthesis and expression of chemerin in perivascular adipose tissue, whereas the primary chemerin receptor ChemR23 was expressed both in the tunica media and endothelial layer. The ChemR23 agonist chemerin-9 caused receptor, concentration-dependent contraction in the isolated rat thoracic aorta, superior mesenteric artery, and mesenteric resistance artery, and contraction was significantly amplified (more than 100%) when nitric oxide synthase was inhibited and the endothelial cell mechanically removed or tone was placed on the arteries. The novel ChemR23 antagonist CCX832 inhibited phenylephrine-induced and prostaglandin F2&agr;-induced contraction (+perivascular adipose tissue), suggesting that endogenous chemerin contributes to contraction. Arteries from animals with dysfunctional endothelium (obese or hypertensive) demonstrated a pronounced contraction to chemerin-9. Finally, mesenteric arteries from obese humans demonstrate amplified contraction to chemerin-9. Conclusions—These data support a new role for chemerin as an endogenous vasoconstrictor that operates through a receptor typically attributed to function only in immune cells.

Vascular reactivity, 5-HT uptake, and blood pressure in the serotonin transporter knockout rat

The handling of serotonin [5-hydroxytryptamine (5-HT)] depends on the serotonin transporter (SERT). A SERT knockout (KO) rat is a useful model to test the hypothesis that SERT is the primary mechanism for arterial 5-HT uptake and to investigate the impact of SERT removal on blood pressure. Wild-type (WT) and KO rats were used to measure 5-HT content (plasma, raphe, aorta, carotid, and mesenteric artery), aortic isometric contraction, and blood pressure. HPLC supported the lack of circulating 5-HT in plasma (ng/ml plasma, WT, 310 +/- 96; and KO, 1.0 +/- 0.5; P < 0.05). Immunohistochemistry and Western blot analyses validated the presence of the SERT protein in the WT rats and a lesser expression in the KO rat. The aorta isolated from KO rats had a normal contraction to phenylephrine and norepinephrine and a normal relaxation to the endothelium-dependent agonist acetylcholine compared with the aorta from WT. In contrast, the potency of 5-HT was increased in the aorta from KO rats compared with WT rats [-log EC(50) (M); WT, 5.71 +/- 0.08; and KO, 6.7 +/- 0.18] and maximum contraction was reduced [%phenylephrine (10 muM) contraction, WT, 113 +/- 6%; and KO, 52 +/- 12%]. 5-HT uptake was reduced but not abolished in arteries of the KO compared with the WT rats. Diurnal mean arterial blood pressure, heart rate, and locomotor activity level of the KO rats were similar to the WT rats. These data suggest that there are other mechanisms of 5-HT uptake in the arteries of the rat and that although the absence of circulating 5-HT and/or SERT function sensitizes arteries to 5-HT, SERT dysfunction does not impair normal blood pressure.

Serotonin (5-HT) in Veins: Not All in Vain

The circulatory system consists of veins and arteries. Compared with arteries, veins have been neglected in cardiovascular research. Although veins are significantly less muscular than similarly sized arteries, the contribution of veins to cardiovascular homeostasis cannot be left un-noted because veins accommodate 70% of the circulating blood. Circulating blood platelets contain the majority of systemic 5-HT (5-hydroxytryptamine; serotonin). Similar to venous function, the physiological role of 5-HT in the cardiovascular system is not well understood. Here, we present not only a review on 5-HT and veins but ways in which these two topics might intersect in a physiologically relevant manner. Here we show the novel findings that veins exhibit higher amounts of intracellular 5-HT than arteries. Moreover, we also show evidence that, similar to arteries, veins have the ability to uptake 5-HT. In this review, we introduce the venous system as a reservoir for 5-HT in the periphery, suggesting that veins, in addition to arteries, may represent an important target for drugs that interfere with the serotonergic system. In addition, the serotonergic system from synthesis to metabolism, 5-HT receptor activation and venous diseases will also be discussed.

Perivascular adipose tissue contains functional catecholamines

The sympathetic nervous system and its neurotransmitter effectors are undeniably important to blood pressure control. We made the novel discovery that perivascular adipose tissue (PVAT) contains significant concentrations of catecholamines. We hypothesized that PVAT contains sufficient releasable catecholamines to affect vascular function. High‐pressure liquid chromatography, isometric contractility, immunohistochemistry, whole animal approaches, and pharmacology were used to test this hypothesis. In normal rat thoracic aorta and superior mesenteric artery, the indirect sympathomimetic tyramine caused a concentration‐dependent contraction that was dependent on the presence of PVAT. Tyramine stimulated release of norepinephrine (NA), dopamine (DA) and the tryptamine serotonin (5‐hydroxytryptamine [5‐HT]) from PVAT isolated from both arteries. In both arteries, tyramine‐induced concentration‐dependent contraction was rightward‐shifted and reduced by the noradrenaline transporter inhibitor nisoxetine (1 μmol/L), the vesicular monoamine transporter inhibitor tetrabenazine (10 μmol/L), and abolished by the α adrenoreceptor antagonist prazosin (100 nmol/L). Inhibitors of the DA and 5‐HT transporter did not alter tyramine‐induced, PVAT‐dependent contraction. Removal of the celiac ganglion as a neuronal source of catecholamines for superior mesenteric artery PVAT did not significantly reduce the maximum or shift the concentration‐dependent contraction to tyramine. Electrical field stimulation of the isolated aorta was not affected by the presence of PVAT. These data suggest that PVAT components that are independent of sympathetic nerves can release NA in a tyramine‐sensitive manner to result in arterial contraction. Because PVAT is intimately apposed to the artery, this raises the possibility of local control of arterial function by PVAT catecholamines.

A Serotonergic System in Veins: Serotonin Transporter-Independent Uptake

We hypothesized that the 5-hydroxytryptamine (5-HT; serotonin) system is present and functional in veins. In vena cava (VC), the presence of the 5-HT synthesis rate-limiting enzyme tryptophan hydroxylase-1 mRNA and accumulation of the 5-HT synthesis intermediate 5-hydroxytryptophan after incubation with tryptophan supported the ability of veins to synthesize 5-HT. The presence of 5-HT and its metabolite 5-hydroxyindole acetic acid was measured by high-performance liquid chromatography in VC and jugular vein (JV), and it was compared with similarly sized arteries aorta (RA) and carotid (CA), respectively. In rats treated with the monoamine oxidase-A (MAO-A) inhibitor pargyline to prevent 5-HT metabolism, basal 5-HT levels were higher in veins than in arteries. 5-HT uptake was observed after exposure to exogenous 5-HT in all vessels. The presence of MAO-A and the 5-HT transporter (SERT) in VC was observed by immunohistochemistry and Western analysis. However, 5-HT uptake was not inhibited by the SERT inhibitors fluoxetine and/or fluvoxamine in VC and JV, as opposed to the inhibition in RA and CA. Moreover, studies performed in VC from mutant rats lacking SERT showed no differences in 5-HT uptake compared with VC from wild type. These data suggest the SERT is not functional under physiological conditions in veins. The differences in 5-HT handling between veins and arteries may represent alternative avenues for targeting the 5-HT system in the peripheral circulation for controlling vascular tone.

Indoleamine 2,3-diooxygenase in periaortic fat: mechanisms of inhibition of contraction.

Indoleamine 2,3-dioxygenase (IDO) metabolizes L-tryptophan to L-kynurenine, promotes immunosuppression, and has been described as a consumer of superoxide. We discovered IDO expression in periaortic fat and tested the hypothesis that periarterial IDO functionally reduces agonist-induced contraction. Our model was the thoracic aorta, abdominal aorta, and superior mesenteric artery of the male Sprague-Dawley rat. Periaortic fat from the thoracic aorta stained intensely for IDO, the brown fat marker uncoupling protein-1, and oil red O as a general lipid marker. White fat around the mesenteric artery and abdominal aorta stained less for IDO; brown fat was less abundant. IDO activity (kynurenine-to-tryptophan ratio via HPLC) was detected in visceral and mesenteric artery fat (ratio: ∼4) but was highest in perithoracic aortic fat (ratio: 10 ± 1.1). In isometric contractile experiments, periadventitial fat reduced ANG II-induced thoracic aortic (with fat: 34% of without fat) and mesenteric artery (with fat: 63% of without fat) maximal contraction. In contrast, periadventitial fat did not reduce agonist-induced contraction in the abdominal aorta. The IDO inhibitor 1-L-methyltryptophan (1-MT) reversed the fat-induced reduction of ANG II-induced contraction in the thoracic aorta but not in the mesenteric artery. The IDO metabolite kynurenine relaxed the thoracic aorta only at high (9 mM) concentrations, whereas the downstream metabolite quinolinic acid (1 mM) relaxed the contracted thoracic aorta (∼80%). 1-MT did not correct the reduction in basal superoxide levels observed in the presence of perithoracic aortic fat. We conclude that IDO is an enzyme active primarily in brown fat surrounding the thoracic aorta and depresses aortic contractility.

Body distribution of infused serotonin in rats.

1 Our goal was to investigate the body distribution of serotonin (5‐hydroxytryptamine; 5‐HT) in rats infused with 5‐HT (25 µg/kg per min) for 7 days and the contribution of the 5‐HT transporter (SERT) for 5‐HT uptake into the tissues. 2 Mini‐osmotic pumps containing 5‐HT or vehicle were implanted in rats knocked out for SERT (SERT‐KO) or in wild‐type (WT) rats. On the 8th day, tissues were harvested for measurements of 5‐HT by high‐performance liquid chromatography (HPLC). The 5‐HT metabolite 5‐hydroxyindole acetic acid (5‐HIAA) was also measured by HPLC, because an increase in 5‐HIAA in tissues from rats receiving 5‐HT reflects 5‐HT uptake followed by metabolism. 3 In WT rats infused with 5‐HT, an increase in 5‐HT or 5‐HIAA was observed in the heart, pancreas, thyroid, adrenal gland, kidney, seminal vesicle, bladder, prostate, liver, oesophagus, stomach, femur, trachea, lung and spleen compared with vehicle‐infused rats. An increase in 5‐HT and 5‐HIAA was not observed in aorta, vena cava and jejunum. In tissues from SERT‐KO rats infused with 5‐HT, the content of 5‐HT or 5‐HIAA was decreased in most of the tissues studied compared with 5‐HT‐infused WT rats. Although 5‐HT uptake in the kidney, seminal vesicle, prostate, jejunum and trachea is SERT dependent, it is SERT independent in the pancreas. The remaining tissues display SERT‐dependent and ‐independent mechanisms for 5‐HT uptake. 4 Altogether, tissues from different systems, such as the cardiovascular, endocrine, genitourinary and gastrointestinal, accumulate 5‐HT mainly via SERT and, thus, these systems are potential targets for drugs that interfere with 5‐HT homeostasis.

Organic cation transporter 3 contributes to norepinephrine uptake into perivascular adipose tissue

Perivascular adipose tissue (PVAT) reduces vasoconstriction to norepinephrine (NE). A mechanism by which PVAT could function to reduce vascular contraction is by decreasing the amount of NE to which the vessel is exposed. PVATs from male Sprague-Dawley rats were used to test the hypothesis that PVAT has a NE uptake mechanism. NE was detected by HPLC in mesenteric PVAT and isolated adipocytes. Uptake of NE (10 μM) in mesenteric PVAT was reduced by the NE transporter (NET) inhibitor nisoxetine (1 μM, 73.68 ± 7.62%, all values reported as percentages of vehicle), the 5-hydroxytryptamine transporter (SERT) inhibitor citalopram (100 nM) with the organic cation transporter 3 (OCT3) inhibitor corticosterone (100 μM, 56.18 ± 5.21%), and the NET inhibitor desipramine (10 μM) with corticosterone (100 μM, 61.18 ± 6.82%). Aortic PVAT NE uptake was reduced by corticosterone (100 μM, 53.01 ± 10.96%). Confocal imaging of mesenteric PVAT stained with 4-[4-(dimethylamino)-styrl]-N-methylpyridinium iodide (ASP(+)), a fluorescent substrate of cationic transporters, detected ASP(+) uptake into adipocytes. ASP(+) (2 μM) uptake was reduced by citalopram (100 nM, 66.68 ± 6.43%), corticosterone (100 μM, 43.49 ± 10.17%), nisoxetine (100 nM, 84.12 ± 4.24%), citalopram with corticosterone (100 nM and 100 μM, respectively, 35.75 ± 4.21%), and desipramine with corticosterone (10 and 100 μM, respectively, 50.47 ± 5.78%). NET protein was not detected in mesenteric PVAT adipocytes. Expression of Slc22a3 (OCT3 gene) mRNA and protein in PVAT adipocytes was detected by RT-PCR and immunocytochemistry, respectively. These end points support the presence of a transporter-mediated NE uptake system within PVAT with a potential mediator being OCT3.

Interaction between GABA and norepinephrine in interleukin-1β-induced suppression of the luteinizing hormone surge

Interleukin-1beta (IL-1beta), a cytokine that is closely associated with inflammation and immune stress, is known to interfere with reproductive functions. Earlier studies have demonstrated that IL-1beta inhibits the luteinizing hormone (LH) surge during the afternoon of proestrus in female rats. We have shown that this effect is most probably mediated through a reduction in norepinephrine (NE) levels in the medial preoptic area (MPA) of the hypothalamus. However, the mechanism by which IL-1beta decreases NE levels in the MPA is unclear. We hypothesized that the inhibitory neurotransmitter, GABA could play a role in decreasing NE levels in the MPA. To test this, ovariectomized, steroid-primed rats were injected (i.p.) with either PBS-BSA (control) or 5 microg of IL-1beta, alone or in combination with i.c.v. administration of GABA-A and GABA-B receptor antagonists, Bicuculline and CGP 35348 (CGP) respectively. Animals were subjected to push-pull perfusion of the MPA and perfusates collected at 30 min intervals were analyzed for both NE and GABA levels using HPLC-EC. Simultaneously, serial plasma samples were obtained through jugular catheters and were analyzed for LH levels using RIA. Compared to control rats, NE levels decreased significantly in the MPA in IL-1beta-treated rats (p<0.05). Concurrently, there was a significant increase in GABA levels in the MPA (p<0.05). The GABA-A receptor antagonist, bicuculline, was able to reverse the effect of IL-1beta on NE and LH, while the GABA-B receptor antagonist, CGP 35348 was without any effect. This leads us to conclude that the IL-1beta-induced suppression of the LH surge is most probably mediated through an increase in GABA levels in the MPA which causes a reduction in NE levels. This is probably one of the mechanisms by which IL-1beta inhibits reproductive functions.

Effects of butyrate precursors on electron relocation when methanogenesis is inhibited in ruminal mixed cultures

Aims:  Inhibition of ruminal methanogenesis often causes accumulation of H2, formate and ethanol, which are not energy substrates for ruminants. It was hypothesized that the addition of butyrate precursors would avoid the formation of these products and relocate electrons into butyrate.