Vascular pharmacology of a novel cannabinoid-like compound, 3-(5-dimethylcarbamoyl-pent-1-enyl)-N-(2-hydroxy-1-methyl-ethyl)benzamide (VSN16) in the rat.

BACKGROUND AND PURPOSE: A putative novel cannabinoid receptor mediates vasorelaxation to anandamide and abnormal-cannabidiol and is blocked by O-1918 and by high concentrations of rimonabant. This study investigates VSN16, a novel water-soluble agonist, as a vasorelaxant potentially acting at non-CB1, non-CB2 cannabinoid receptors in the vasculature. EXPERIMENTAL APPROACH: VSN16 and some analogues were synthesized and assayed for vasodilator activity in the rat third generation mesenteric artery using wire myography. Also carried out with VSN16 were haemodynamic studies in conscious rats and binding studies to CB1 receptors of rat cerebellum. KEY RESULTS: VSN16 relaxed mesenteric arteries in an endothelium-dependent manner. The vasorelaxation was antagonized by high concentrations of the classical cannabinoid antagonists, rimonabant and AM 251, as well as by O-1918, an antagonist at the abnormal-cannabidiol receptor but not at CB1 or CB2 receptors. It did not affect [3H]CP55,940 binding to CB1 receptors in rat cerebellum. The vasorelaxation was not pertussis toxin-sensitive but was reduced by inhibition of nitric oxide synthesis, Ca(2+)-sensitive K+ channels (KCa) and TRPV1 receptors. In conscious rats VSN16 transiently increased blood pressure and caused a longer-lasting increase in mesenteric vascular conductance. Structure-activity studies on vasorelaxation showed a stringent interaction with the target receptor. CONCLUSIONS AND IMPLICATIONS: VSN16 is an agonist at a novel cannabinoid receptor of the vasculature. It acts on the endothelium to release nitric oxide and activate KCa and TRPV1. As it is water-soluble it might be useful in bringing about peripheral cannabinoid-like effects without accompanying central or severe cardiovascular responses.

GPR55 and the vascular receptors for cannabinoids.

CB1 and CB2 receptors mediate most responses to cannabinoids but not some of the cardiovascular actions of endocannabinoids such as anandamide and virodhamine, or those of some synthetic agents, like abnormal cannabidiol (abn-cbd). These agents induce vasorelaxation which is antagonised by rimonabant but only at high concentrations relative to those required to block CB1 receptors. Vasorelaxation to anandamide is sensitive to Pertussis toxin (though that to abn-cbd is not), and so is thought to be mediated by a G protein-coupled receptor through Gi/o. An orphan receptor, GPR55, apparently a cannabinoid receptor, is activated by abn-cbd, but is not the receptor mediating vasorelaxation to this agent, as the response persists in vessels from GPR55 knockout mice. However, the activity of anandamide in GPR55 knockout mice is not yet reported and so the role of GPR55 as a cannabinoid receptor mediating vascular responses has yet to be finalised.

Effect of polyunsaturated fatty acid (PUFA) supplementation on intestinal inflammation and necrotizing enterocolitis (NEC) in a neonatal rat model.

Inasmuch as long-chain polyunsaturated fatty acids (PUFA, metabolites of the essential n-3 and n-6 fatty acids) are known to modulate inflammation, we hypothesized that supplementation of formula with these compounds would prevent necrotizing enterocolitis (NEC) and intestinal inflammation in our neonatal rat model. Newborn rats were stressed with asphyxia and formula feeding, and randomly assigned to control formula, control with PUFA supplementation, and PUFA with nucleotides. Animals were followed for 72--96 h and assessed for death, gross and histologic NEC, intestinal apoptosis, endotoxemia, and intestinal mRNA synthesis of phospholipase A(2)-II (rate-limiting enzyme for platelet activating factor production), platelet activating factor receptor, and inducible nitric oxide synthase. We found that PUFA reduced the incidence of death and NEC compared with the other groups (NEC 8 of 24 versus 17 of 24 control and 13 of 23 PUFA + nucleotides, p < 0.05). Furthermore, PUFA reduced plasma endotoxemia at 48 h (25 +/- 4 EU/mL versus 276 +/- 39 EU/mL in control and 170 +/- 28 EU/mL in PUFA + nucleotide), intestinal phospholipase A(2)-II expression at 24 h, and platelet activating factor receptor expression at 48 h. Formula supplementation had no effect on apoptosis of intestinal epithelium or intestinal inducible nitric oxide synthase expression. Addition of nucleotides with PUFA abrogated the beneficial effects of PUFA on intestinal inflammation. We conclude that PUFA reduces the incidence of NEC and intestinal inflammation in a neonatal rat model.

Bifidobacterial supplementation reduces the incidence of necrotizing enterocolitis in a neonatal rat model.

BACKGROUND & AIMS: Neonatal necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease of premature infants partly caused by intestinal bacterial proliferation. Because bifidobacteria are thought to reduce the risk for intestinal disturbances associated with pathogenic bacterial colonization, we hypothesized that exogenous bifidobacterial supplementation to newborn rats would result in intestinal colonization and a reduction in the incidence of neonatal NEC. METHODS: Newborn rat pups were given Bifidobacterium infantis (10(9) organisms per animal daily), Escherichia coli, or saline control and exposed to the NEC protocol consisting of formula feeding (Esbilac; 200 cal. kg(-1). day(-1)) and asphyxia (100% N(2) for 50 seconds followed by cold exposure for 10 minutes). Outcome measures included stool and intestinal microbiological evaluation, gross and histological evidence of NEC, plasma endotoxin concentration, intestinal phospholipase A(2) expression, and estimation of intestinal mucosal permeability. RESULTS: Bifidobacterial supplementation resulted in intestinal colonization by 24 hours and appearance in stool samples by 48 hours. Bifidobacteria-supplemented animals had a significant reduction in the incidence of NEC compared with controls and E. coli-treated animals (NEC, 7/24 B. infantis vs. 19/27 control vs. 16/23 E. coli; P < 0.01). Plasma endotoxin and intestinal phospholipase A(2) expression were lower in bifidobacteria-treated pups than in controls, supporting the role of bacterial translocation and activation of the inflammatory cascade in the pathophysiology of NEC. CONCLUSIONS: Intestinal bifidobacterial colonization reduces the risk of NEC in newborn rats.

Interactions in indices of vitamin A, zinc and copper status when these nutrients are fed to rats at adequate and increased levels.

The purpose of the present study was to determine the effects of feeding nutritionally adequate and increased levels of vitamin A (retinyl acetate at 1.4, 34.4, and 206.4 mg/kg diet) in combination with adequate or increased Zn (12 and 240 mg/kg) and Cu (5 and 50 mg/kg) on serum and tissue concentrations of retinol and retinyl palmitate and on indices of Cu and Zn status in female Sprague-Dawley rats, and to measure interactive effects of such nutrient imbalances. Rats fed on diets containing 34.4 and 206.4 mg vitamin A/kg had higher feed intakes and relative liver weights than those fed on diets containing 1.4 mg vitamin A/kg. An interaction between dietary Cu and Zn and an independent effect of vitamin A affected serum ceruloplasmin oxidase (EC 1.16.3.1) activity. Rats fed on high Zn, adequate-Cu diets (240 and 5 mg Zn and Cu/kg respectively) had lower serum ceruloplasmin oxidase levels than rats fed on adequate-Zn, adequate-Cu diets (12 and 5 mg Zn and Cu/kg respectively). This effect was not observed in rats fed on high-Zn, high-Cu diets (240 and 50 mg Zn and Cu/kg respectively). Alterations in dietary levels of Cu and vitamin A independently affected haemoglobin levels. Serum cholesterol concentration was affected by interactions between Zn and vitamin A and Cu and vitamin A. Levels of retinol and retinyl palmitate in liver and kidney were significantly higher in rats fed on diets with increased dietary vitamin A than in those fed on diets with adequate vitamin A. Three-way interactions among Cu, Zn, and vitamin A affected levels of retinol in serum and liver. Two-way interactions between Cu and vitamin A affected liver retinyl palmitate and the sum of liver retinol+retinyl palmitate. An independent effect of dietary Zn on these variables was also observed. Interactions between Cu and vitamin A affected levels of Cu in liver and kidney, while Fe and Zn in kidney were affected by interactions between Cu and Zn. This study demonstrates that differing interactions among variables of vitamin A metabolism and mineral status occur with higher dietary levels of vitamin A, Zn and Cu in the rat.

Sodium, potassium and chloride utilization by rats given various inorganic anions.

The purpose of the present studies was to examine the effect of ingestion of sodium and potassium salts of various fixed anions on blood pressure, and to assess interactions among electrolytes. In the first study, Sprague-Dawley rats fed on purified diets supplemented with Na salts of chloride, sulphate, bisulphate, carbonate and bicarbonate for 7 weeks developed higher blood pressures than rats fed on the basal diet. In a second study, rats fed on Na or K salts of HSO4, HCO3 or Cl had higher blood pressures than rats fed on the basal diet. Blood pressure measurements were not correlated with plasma volume, plasma renin activity, or plasma atrial natriuretic peptide concentrations at 7 weeks. Plasma renin activity was depressed in rats fed on supplemental Na and even more in rats fed on supplemental K salts rather than the basal diet. Generally, rats fed on supplemental Na excreted Na in urine and absorbed Na in the gut more efficiently than rats fed on the basal diet or diets supplemented with K, but the anions fed also altered Na absorption and excretion. In a third study, rats fed on diets supplemented with any Cl salt, but especially KCl, absorbed K more efficiently than those fed on the basal diet. In studies 1 and 2, the efficiency of urinary excretion of K was greatest when HCO3 and CO3 salts were fed and least when HSO4 salts were fed. Despite large variations in the efficiency of absorption and excretion of Na and K, tissue levels of the electrolytes remained constant.

Calcium, magnesium and phosphorus utilization by rats fed sodium and potassium salts of various inorganic anions.

We hypothesized that urinary excretion of calcium would be affected by both urinary excretion of acid and of total fixed anions. Calcium, magnesium and phosphorus utilization was examined in rats fed semipurified diets supplemented with approximately 0.4 mol sodium/kg diet or approximately 0.4 mol potassium/kg diet as chloride, sulfate, bisulfate, carbonate or bicarbonate salts in two studies. The ingestion of supplemental fixed anions (chloride, sulfate or bisulfate) increased urinary excretion of calcium, magnesium and phosphorus. It made no difference whether the anions were ingested as sodium or potassium salts. In Study 1, 80% of the variation in urinary calcium excretion could be explained on the basis of urinary excretion of sulfate, ammonia and total anions. In Study 2, 77% of the variation in urinary calcium excretion could be predicted on the basis of urinary excretion of total anions and sulfate. Although bone and plasma calcium concentrations were not responsive to these dietary changes, less magnesium was retained in bones of rats fed any of the supplemental salts.

Blood pressure, fluid compartments and utilization of chloride in rats fed various chloride diets.

The effects of various levels and types of dietary chloride salts on blood pressure were examined in three studies. Weanling Sprague-Dawley rats were fed semipurified diets that contained moderate (1.9 mg Cl/g diet) and supplemental (15.6 mg Cl/g diet) chloride as NaCl, KCl, lysine monohydrochloride with or without CaCO3, or MgCl2 for 56 or 119 d. Rats fed excess chloride excreted more than 84% of the chloride in urine, excreted increased urine volumes (from 3 to 7 wk), tended to consume more fluids (especially if NaCl was fed), had significantly increased blood pressure (7 and 13 wk), had hypertrophied kidneys (8 and 17 wk) and had altered levels of sodium and potassium in their kidneys (17 wk), but experienced no changes in the size of fluid compartments, such as plasma volume or bromine space. Altogether, 56% of the variance in blood pressure measurements at wk 7 could be predicted on the basis of urinary chloride excretion during wk 7 and kidney weight as a percentage of body weight and kidney sodium concentration, but only 30% of the variance in blood pressure measurements at wk 16 could be predicted on the basis of urinary chloride excretion during wk 16 and kidney sodium concentrations.

Effect of various chloride salts on the utilization of phosphorus, calcium, and magnesium.

Only part of the effect of dietary protein on urinary calcium excretion can be ascribed to sulfur amino acids. We hypothesized that chloride, another factor often associated with isolated proteins, and another amino acid, lysine, affect utilization of calcium. The effects of supplemental dietary chloride, inorganic or organic, on calcium, phosphorus, and magnesium utilization were studied in two rat studies. Weanling Sprague-Dawley rats were fed semi-purified diets that contained moderate (1.8 mg Cl/g diet) or supplemental (15.5 mg Cl/g diet) chloride as sodium chloride, potassium chloride, or lysine monohydrochloride with or without calcium carbonate for 56 or 119 days. Rats fed supplemental sodium chloride or potassium chloride had higher urinary phosphorus excretion, more efficient phosphorus absorption, but unchanged tissue phosphorus levels after 7 and 16 weeks of dietary treatment as compared to rats fed moderate chloride. Rats fed supplemental sodium chloride or potassium chloride excreted more calcium in urine at 7 weeks and absorbed calcium less efficiently at 16 weeks. Tissue calcium concentrations were unaffected, but total tibia magnesium and plasma magnesium concentrations were lower in rats fed supplemental sodium chloride or potassium chloride than those fed moderate chloride. Lysine chloride with or without additional calcium elevated urinary calcium excretion even more than sodium chloride and potassium chloride ingestion. Rats fed lysine chloride with supplemental calcium had smaller apparent absorption and urinary losses of phosphorus and magnesium after 16 weeks and lower tibia and plasma magnesium concentrations than rats fed lysine chloride.

Lipid absorption and intestinal tumour incidence in rats fed on varying levels of calcium and butterfat.

The purpose of the 2 x 2 factorial study was to determine the effect of varying levels of dietary calcium (2.5 and 10 g/kg) and butterfat (50 and 200 g/kg) on lipid utilization and on development of colon tumours in animals initiated with 1,2-dimethylhydrazine dihydrochloride. Among rats fed on 200 g butterfat/kg, the fourfold increase in Ca intake induced more than a sevenfold increase in faecal excretion of total lipids and almost a fortyfold increase in faecal excretion of acid-extractable lipid. Among rats fed on 50 g butterfat/kg, the ingestion of supplemental Ca had a less dramatic effect and induced only a twofold increase in faecal excretion of total lipids and a threefold increase in acid-extractable lipid. The volume of intestinal adenocarcinomas was correlated with the excretion of acid-extractable lipid in faeces (R 0.369, P less than 0.02). Caecal enzymic activity was not correlated with tumour incidence or size or faecal lipid excretion. Overall, the fourfold increase in Ca intakes decreased total lipid absorption significantly but by less than 6%.

Calcium and magnesium utilization in rats: effect of dietary butterfat and calcium and of age.

This factorial study was designed to examine the effect of short- and long-term ingestion of dietary calcium (0.25% and 1.0%) and butterfat (5% and 20%) and treatment with dimethylhydrazine (DMH) in rats. Calcium, magnesium and phosphorus absorption and, to a lesser extent, total diet digestibility decreased as the rats aged from 2 to 8 mo. Increased ingestion of butterfat had no effect on apparent absorption of calcium among young rats but tended to decrease calcium absorption in mature rats. The weak effect (P less than 0.05) of butterfat on calcium absorption reflected the small amount of calcium (less than 2% of fecal calcium) associated with lipids in feces. Ingestion of 10 vs. 2.5 mg Ca/g diet reduced the efficiency of apparent absorption of calcium, magnesium and phosphorus of young and mature rats and resulted in slightly, but significantly, greater retention of calcium in bone after 27 wk. The total amounts of calcium retained in tibias were correlated with the amounts of calcium absorbed by rats after 4, but not after 27, wk of dietary treatments. Rats dosed with DMH exhibited improved efficiency of calcium absorption, but the incidence of intestinal tumors did not affect mineral utilization.

Changes in intestinal function of rats initiated with DMH and fed varying levels of butterfat, calcium, and magnesium.

The effects of dietary calcium, magnesium, and butterfat on intestinal function and flora in rats initiated with 1,2-dimethylhydrazine (DMH) were studied. Male weanling rats were assigned to six isocaloric diets that varied in their levels of calcium and magnesium (0.25% Ca with 0.05% Mg, 1.0% Ca with 0.05% Mg, or 0.625% Ca with 0.50% Mg) and butterfat (5% or 20%). One-half of the rats in each treatment were injected subcutaneously with DMH weekly for four weeks. This short-term exposure to DMH increased colonic ornithine decarboxylase (ODC) activity and the mass of cecal contents. Ingestion of the high levels of either calcium or magnesium depressed colonic ODC activity and depressed apparent absorption of organic matter, calcium, magnesium, and phosphorus. Ingestion of excess magnesium increased the mass of the cecal contents by twofold, caused hypertrophy of cecal walls, and increased the total amount of protein and total nitroreductase and beta-glucuronidase activity in the ceca of rats. Ingestion of supplemental calcium had less dramatic effects and increased the mass of cecal contents by only 28% and decreased the total amount of protein in the ceca. On the basis of their different effects on cecal microflora, magnesium appears to have less potential than does calcium as a protective agent against colon cancer.