Acute and chronic rejection: compartmentalization and kinetics of counterbalancing signals in cardiac transplants.

Acute and chronic rejection impact distinct compartments of cardiac allografts. Intramyocardial mononuclear cell infiltrates define acute rejection, whereas chronic rejection affects large arteries. Hearts transplanted from male to female C57BL/6 mice undergo acute rejection with interstitial infiltrates at 2 weeks that resolve by 6 weeks when large arteries develop arteriopathy. These processes are dependent on T cells because no infiltrates developed in T cell-deficient mice and transfer of CD4 T cells restored T cell as well as macrophage infiltrates and ultimately neointima formation. Markers of inflammatory macrophages were up-regulated in the interstitium acutely and decreased as markers of wound healing macrophages increased chronically. Programmed cell death protein, a negative costimulator, and its ligand PDL1 were up-regulated in the interstitium during resolution of acute rejection. Blocking PDL1:PD1 interactions in the acute phase increased interstitial T cell infiltrates. Toll-like receptor (TLR) 4 and its endogenous ligand hyaluronan were increased in arteries with neointimal expansion. Injection of hyaluronan fragments increased intragraft production of chemokines. Our data indicate that negative costimulatory pathways are critical for the resolution of acute interstitial infiltrates. In the arterial compartment recognition of endogenous ligands including hyaluronan by the innate TLRs may support the progression of arteriopathy.

Endogenous cannabinoids mediate hypotension after experimental myocardial infarction.

OBJECTIVES: We sought to determine whether endocannabinoids influence hemodynamic variables in experimental models of acute myocardial infarction (MI). BACKGROUND: Hypotension and cardiogenic shock are common complications in acute MI. Cannabinoids are strong vasodilators, and endocannabinoids are involved in hypotension in hemorrhagic and septic shock. METHODS: The early effect of left coronary artery ligation on hemodynamic variables was measured in rats pretreated with the selective cannabinoid(1) receptor (CB(1)) antagonist SR141716A (herein referred to as SR, 6.45 micromol/kg body weight intravenously) or vehicle. Endocannabinoids produced in monocytes and platelets were quantified by liquid chromatography/mass spectrometry (LC/MS), and their effects on blood pressure and vascular reactivity were determined. RESULTS: After MI, mean arterial pressure (MAP) dropped from 126 +/- 2 mm Hg to 76 +/- 3 mm Hg in control rats, whereas the decline in blood pressure was smaller (from 121 +/- 3 mm Hg to 108 +/- 7 mm Hg, p < 0.01) in rats pretreated with SR. SR increased the tachycardia that follows MI (change [Delta] in heart rate [HR] = 107 +/- 21 beats/min vs. 49 +/- 9 beats/min in control rats, p < 0.05). The MI sizes were the same in control rats and SR-treated rats. Circulating monocytes and platelets isolated 30 min after MI only decreased MAP when injected into untreated rats (DeltaMAP = -20 +/- 5 mm Hg), but not in SR-pretreated rats. The endocannabinoids anandamide and 2-arachidonyl glycerol were detected in monocytes and platelets isolated after MI, but not in cells from sham rats. Survival rates at 2 h after MI were 70% for control rats and 36% for SR-treated rats (p < 0.05). Endothelium-dependent arterial relaxation was attenuated in SR-treated rats (maximal relaxation: 44 +/- 3% [p < 0.01] vs. 70 +/- 3% in control rats) and further depressed by SR treatment (24 +/- 5%, p < 0.01 vs. MI placebo). CONCLUSIONS: Cannabinoids generated in monocytes and platelets contribute to hypotension in acute MI. Cannabinoid(1) receptor blockade restores MAP but increases 2-h mortality, possibly by impairing endothelial function.

Endocannabinoids acting at vascular CB1 receptors mediate the vasodilated state in advanced liver cirrhosis.

Advanced cirrhosis is associated with generalized vasodilation of unknown origin, which contributes to mortality. Cirrhotic patients are endotoxemic, and activation of vascular cannabinoid CB1 receptors has been implicated in endotoxin-induced hypotension. Here we show that rats with biliary cirrhosis have low blood pressure, which is elevated by the CB1 receptor antagonist SR141716A. The low blood pressure of rats with CCl4-induced cirrhosis was similarly reversed by SR141716A, which also reduced the elevated mesenteric blood flow and portal pressure. Monocytes from cirrhotic but not control patients or rats elicited SR141716A-sensitive hypotension in normal recipient rats and showed significantly elevated levels of anandamide. Compared with non-cirrhotic controls, in cirrhotic human livers there was a three-fold increase in CB1 receptors on isolated vascular endothelial cells. These results implicate anandamide and vascular CB1 receptors in the vasodilated state in advanced cirrhosis and indicate a novel approach for its management.

Leptin-regulated endocannabinoids are involved in maintaining food intake.

Leptin is the primary signal through which the hypothalamus senses nutritional state and modulates food intake and energy balance. Leptin reduces food intake by upregulating anorexigenic (appetite-reducing) neuropeptides, such as alpha-melanocyte-stimulating hormone, and downregulating orexigenic (appetite-stimulating) factors, primarily neuropeptide Y. Genetic defects in anorexigenic signalling, such as mutations in the melanocortin-4 (ref. 5) or leptin receptors, cause obesity. However, alternative orexigenic pathways maintain food intake in mice deficient in neuropeptide Y. CB1 cannabinoid receptors and the endocannabinoids anandamide and 2-arachidonoyl glycerol are present in the hypothalamus, and marijuana and anandamide stimulate food intake. Here we show that following temporary food restriction, CB1 receptor knockout mice eat less than their wild-type littermates, and the CB1 antagonist SR141716A reduces food intake in wild-type but not knockout mice. Furthermore, defective leptin signalling is associated with elevated hypothalamic, but not cerebellar, levels of endocannabinoids in obese db/db and ob/ob mice and Zucker rats. Acute leptin treatment of normal rats and ob/ob mice reduces anandamide and 2-arachidonoyl glycerol in the hypothalamus. These findings indicate that endocannabinoids in the hypothalamus may tonically activate CB1 receptors to maintain food intake and form part of the neural circuitry regulated by leptin.

Endocannabinoids as cardiovascular modulators.

Cannabinoids, the bioactive constituents of the marijuana plant and their synthetic and endogenous analogs cause not only neurobehavioral, but also cardiovascular effects. The most important component of these effects is a profound decrease in blood pressure and heart rate. Although multiple lines of evidence indicate that the hypotensive and bradycardic effects of anandamide and other cannabinoids are mediated by peripherally located CB1 cannabinoid receptors, anandamide can also elicit vasodilation in certain vascular beds, which is independent of CB1 or CB2 receptors. Possible cellular mechanisms underlying these effects and the cellular sources of vasoactive anandamide are discussed.

Cardiovascular effects of 2-arachidonoyl glycerol in anesthetized mice.

Cannabinoids, including the endogenous ligand anandamide, elicit pronounced hypotension and bradycardia through the activation of CB1 cannabinoid receptors. A second endogenous cannabinoid, 2-arachidonoyl glycerol (2-AG), has been proposed to be the natural ligand of CB1 receptors. In the present study, we examined the effects of 2-AG on mean arterial pressure and heart rate in anesthetized mice and assessed the role of CB1 receptors through the use of selective cannabinoid receptor antagonists and CB1 receptor knockout (CB1(-/-)) mice. In control ICR mice, intravenous injections of 2-AG or its isomer 1-AG elicit dose-dependent hypotension and moderate tachycardia that are unaffected by the CB1 receptor antagonist SR141716A. The same dose of SR141716A (6 nmol/g IV) completely blocks the hypotensive effect and attenuates the bradycardic effect of anandamide. 2-AG elicits a similar hypotensive effect, resistant to blockade by either SR141716A or the CB2 antagonist SR144528, in both CB1(-/-) mice and their homozygous (CB1(+/+)) control littermates. In ICR mice, arachidonic acid (AA, 15 nmol/g IV) elicits hypotension and tachycardia, and indomethacin (14 nmol/g IV) inhibits the hypotensive effect of both AA and 2-AG. Synthetic 2-AG incubated with mouse blood is rapidly (<2 minutes) and completely degraded with the parallel appearance of AA, whereas anandamide is stable under the same conditions. A metabolically stable ether analogue of 2-AG causes prolonged hypotension and bradycardia in ICR mice, and both effects are completely blocked by SR141716A, whereas the same dose of 2-AG-ether does not influence blood pressure and heart rate in CB1(-/-) mice. These findings are interpreted to indicate that exogenous 2-AG is rapidly degraded in mouse blood, probably by a lipase, which masks its ability to interact with CB1 receptors. Although the observed cardiovascular effects of 2-AG probably are produced by an arachidonate metabolite through a noncannabinoid mechanism, the CB1 receptor-mediated cardiovascular effects of a stable analogue of 2-AG leaves open the possibility that endogenous 2-AG may elicit cardiovascular effects through CB1 receptors.

Anandamide amidohydrolase of porcine brain: cDNA cloning, functional expression and site-directed mutagenesis(1).

Anandamide (arachidonoylethanolamide) is an endogenous ligand for cannabinoid receptors, and its cannabimimetic activities are lost when the compound is hydrolyzed to arachidonic acid and ethanolamine by an enzyme referred to as anandamide amidohydrolase. We cloned a cDNA for the enzyme of porcine brain, and the cDNA encoded a protein of 579 amino acids with a molecular mass of 62.9 kDa. The amino acid sequence was 81, 80 and 85% identical with the enzymes previously cloned from the liver of rat, mouse, and human, respectively. When the enzyme protein was overexpressed in COS-7 cells, the particulate fraction of the cells showed an anandamide hydrolyzing activity and also catalyzed the reverse reaction synthesizing anandamide from arachidonic acid and ethanolamine both with a specific activity of 0. 2-0.3 micromol/min/mg protein at 37 degrees C. The brain enzyme exhibited a wide substrate specificity hydrolyzing oleamide, 2-arachidonoylglycerol, and methyl arachidonate. The point mutation of Ser-217, Asp-237, Ser-241, or Cys-249 completely abolished the hydrolyses of all the above-mentioned substrates as well as the synthesis of anandamide in the reverse reaction.

Enzymes of porcine brain hydrolyzing 2-arachidonoylglycerol, an endogenous ligand of cannabinoid receptors.

Anandamide and 2-arachidonoylglycerol (2-AG) are two endogenous ligands for the cannabinoid receptors, and their cannabimimetic activities are lost when they are hydrolyzed enzymatically. Cytosol and particulate fractions of porcine brain exhibited a high 2-AG hydrolyzing activity of 100 nmol/min/mg protein. Most of the activity could be attributed to a monoacylglycerol lipase-like enzyme that did not hydrolyze anandamide. It was separated by hydroxyapatite chromatography from anandamide amidohydrolase, which is also capable of hydrolyzing 2-AG as well as anandamide. Thus, porcine brain has at least two enzymes capable of hydrolyzing 2-AG. The 2-AG hydrolase activities of both the cytosolic and particulate enzymes were irreversibly and time-dependently inhibited by methyl arachidonyl fluorophosphonate with IC50 values as low as 2-3 nM.

A hydrolase enzyme inactivating endogenous ligands for cannabinoid receptors.

Cannabinoids are psychoactive components of marijuana, and bind to specific G protein-coupled receptors in the brain and other mammalian tissues. Anandamide (arachidonoylethanolamide) was discovered as an endogenous agonist for the cannabinoid receptors. Hydrolysis of anandamide to arachidonic acid and ethanolamine results in the loss of its biological activities. The enzyme responsible for this hydrolysis was solubilized, partially purified from the microsomes of porcine brain, and referred to as anandamide amidohydrolase. In addition to the anandamide hydrolysis, the enzyme preparation catalyzed anandamide synthesis by the condensation of arachidonic acid with ethanolamine. Several lines of enzymological evidence suggested that a single enzyme catalyzes both the hydrolysis and synthesis of anandamide. This reversibility was confirmed by the use of a recombinant enzyme of rat liver overexpressed in COS-7 cells. However, in consideration of the high Km value for ethanolamine as a substrate for the anandamide synthesis, the enzyme was presumed to act as a hydrolase rather than a synthase under physiological conditions. The recombinant enzyme acted not only as an amidase hydrolyzing anandamide and other fatty acid amides but also as an esterase hydrolyzing methyl ester of arachidonic acid. 2-Arachidonoylglycerol, which was found recently to be another endogenous ligand, was also efficiently hydrolyzed by the esterase activity of the same enzyme. The anandamide hydrolase and synthase activities were detected in a variety of rat organs, and liver showed by far the highest activities. A high anandamide hydrolase activity was also detected in small intestine but only after the homogenate was precipitated with acetone to remove endogenous lipids inhibiting the enzyme activity. The distribution of mRNA of the enzyme was in agreement with that of the enzyme activity.

Anandamide amidohydrolase reacting with 2-arachidonoylglycerol, another cannabinoid receptor ligand.

Two endogenous ligands for cannabinoid receptors, anandamide (arachidonylethanolamide) and 2-arachidonoylglycerol, lose their biological activities by enzymatic hydrolysis. A cDNA for a rat liver enzyme hydrolyzing anandamide as well as oleamide was overexpressed in COS-7 cells. When the particulate fraction was allowed to react with 2-arachidonoylglycerol, arachidonic acid was produced. In contrast, this hydrolytic reaction did not occur with the control cells. The hydrolysis of 2-arachidonoylglycerol proceeded about 4-fold faster than the anandamide hydrolysis with a Km value as low as 6 microM and an optimal pH of 10. Phenylmethylsulfonyl fluoride and methyl arachidonyl fluorophosphonate inhibited the hydrolysis of both anandamide and 2-arachidonoylglycerol in parallel. Furthermore, the hydrolysis of [14C]2-arachidonoylglycerol was inhibited by anandamide dose-dependently. These results suggest that anandamide and 2-arachidonoylglycerol can be inactivated by the same enzyme.