Adrenomedullin facilitates reinnervation of phenol-injured perivascular nerves in the rat mesenteric resistance artery.

Our previous report showed that innervation of calcitonin gene-related peptide (CGRP)- and neuropeptide Y (NPY)-containing nerves in rat mesenteric resistance arteries was markedly reduced by topical application of phenol, and that nerve growth factor (NGF) facilitates the reinnervation of both nerves. We also demonstrated that a CGRP superfamily peptide, adrenomedullin, is distributed in perivascular nerves of rat mesenteric resistance arteries. In the present study, we investigated the influence of adrenomedullin on the reinnervation of mesenteric perivascular nerves following topical phenol treatment. Under pentobarbital-Na anesthesia, 8-week-old Wistar rats underwent in vivo topical application of phenol (10% phenol in 90% ethanol) to the superior mesenteric artery proximal to the bifurcation of the abdominal aorta. After the treatment, the animals were subjected to immunohistochemistry of the third branch of small arteries proximal to the intestine and to vascular responsiveness testing on day 7. Topical phenol treatment caused marked reduction of the density of NPY-like immunoreactive (LI)- and CGRP-LI nerve fibers in the arteries. Adrenomedullin (360 or 1000 ng/h) or NGF (250 ng/h), which was administered intraperitoneally for 7 days using an osmotic mini-pump immediately after topical phenol treatment, significantly increased the density of CGRP-LI- and NPY-LI nerve fibers compared with saline. Treatment with adrenomedullin (1000 ng/h) or NGF restored adrenergic nerve-mediated vasoconstriction and CGRP nerve-mediated vasodilation in the perfused mesenteric artery treated topically with phenol. These results suggest that adrenomedullin, like NGF, has a facilitatory effect on the reinnervation of perivascular nerves.

Angiotensin II type 2 receptors facilitate reinnervation of phenol-lesioned vascular calcitonin gene-related peptide-containing nerves in rat mesenteric arteries.

The present study was designed to investigate involvement of angiotensin II (Ang II) type 2 receptors (AT2 receptors) in restoration of perivascular nerve innervation injured by topical phenol treatment. Male Wistar rats underwent in vivo topical application of 10% phenol around the superior mesenteric artery. After phenol treatment, animals were subjected to immunohistochemistry of the third branch of small arteries, Western blot analysis of AT2 receptor protein expression in dorsal root ganglia (DRG) and studies of mesenteric neurogenic vasoresponsiveness. Ang II (750 ng/kg/day), nerve growth factor (NGF; 20 microg/kg/day) and PD123,319 (AT2 receptor antagonist; 10 mg/kg/day) were intraperitoneally administered for 7 days using osmotic mini-pumps immediately after topical phenol treatment. Losartan (AT1 receptor antagonist) was administered in drinking water (0.025%). Phenol treatment markedly reduced densities of both calcitonin gene-related peptide (CGRP)-like immunoreactivity (LI) and neuropeptide Y (NPY)-LI-containing fibers. NGF restored densities of both nerve fibers to the sham control level. Coadministration of Ang II and losartan significantly increased the density of CGRP-LI-fibers but not NPY-LI-fibers compared with saline control. The increase of the density of CGRP-LI-fibers by coadministration of Ang II and losartan was suppressed by adding PD123,319. Coadministration of Ang II and losartan ameliorated reduction of CGRP nerve-mediated vasodilation of perfused mesenteric arteries caused by phenol treatment. The AT2 receptor protein expression detected in DRG was markedly increased by NGF. These results suggest that selective stimulation of AT2 receptors by Ang II facilitates reinnervation of mesenteric perivascular CGRP-containing nerves injured by topical phenol application in the rat.

Innervation and functional changes in mesenteric perivascular calcitonin gene-related peptide- and neuropeptide Y-containing nerves following topical phenol treatment.

We have previously shown that age-related reduction of innervation and function in mesenteric perivascular calcitonin gene-related peptide-containing vasodilator nerves takes place in spontaneously hypertensive rats. The present study was performed to investigate innervation and functional changes in perivascular calcitonin gene-related peptide- and adrenergic neuropeptide Y-containing nerves after topical treatment with phenol, which damages nerve fibers, around the rat superior mesenteric artery. Under pentobarbital-Na anesthesia, 8-week-old Wistar rats underwent in vivo topical application of phenol (10% phenol in 90% ethanol) or saline (sham rats) to the superior mesenteric artery proximal to the bifurcation of the abdominal aorta. After the treatment, the animals were subjected to immunohistochemistry of the 3rd branch of small arteries proximal to the intestine and to vascular responsiveness testing on day 3 through day 14. The innervation levels of calcitonin gene-related peptide-like immunoreactivity containing fibers and neuropeptide Y-like immunoreactivity containing fibers were markedly reduced on day 3 to day 14 and on day 5 to day 14 after the treatment, compared with those in sham-operated rats, respectively. In perfused mesenteric vascular beds isolated from phenol-treated rats, adrenergic nerve-mediated vasoconstriction and calcitonin gene-related peptide nerve-mediated vasodilation in response to periarterial nerve stimulation (2-12 Hz) were significantly decreased on day 3 and day 7. Neurogenic release of norepinephrine in phenol-treated rats on day 7 was significantly smaller that that in sham-operated rats. Nerve growth factor content in the mesenteric arteries of phenol-treated rats was significantly lower than that in sham-operated rats. Administration of nerve growth factor using osmotic mini-pumps for 7 days after the phenol treatment resulted in greater density of calcitonin gene-related peptide- and neuropeptide Y-like immunoreactivity fibers than in phenol-treated rats and restored decreased vascular responses to periarterial nerve stimulation. These results suggest that topical phenol-treatment of the mesenteric artery effectively induces functional denervation of perivascular nerves, which can be prevented or reversed by nerve growth factor treatment.

Distribution of adrenomedullin-containing perivascular nerves in the rat mesenteric artery.

Distribution of adrenomedullin (AM)-containing perivascular nerve fibers was studied in rat mesenteric arteries. Many fibers containing AM-like immunoreactivity (LI) were observed in the adventitia. AM-LI fibers were abolished by cold storage denervation or capsaicin but not 6-hydroxydopamine. Double immunostainings showed colocalization of AM-LI with calcitonin gene-related peptide (CGRP)-LI. The dorsal root ganglia had many AM-positive cells and AM mRNA detected by RT-PCR. Electron microscopy study revealed high proportions of immunogold labeling for AM and colocalization of both AM-LI and CGRP-LI in unmyelinated nerve axons. These results suggest that AM-containing perivascular nerves are distributed in the rat mesenteric artery.

Impaired metabolism of azathioprine in carbon tetrachloride-injured rats.

The metabolism of 6-(1-methyl-4-nitro-5-imidazolyl) thiopurine (azathioprine) in liver-injured rats was studied in vivo by measuring the clearance rate (K) of the drug from the blood, and its excretion in the urine. The K values in probenecid, diethyl maleate and carbon tetrachloride (CC14)-treated rats were much smaller than those in control rats. Probenecid, a known inhibitor of glutathione S-transferase, inhibited the urine excretion of azathioprine, and diethyl maleate produced a prompt depletion of hepatic reduced glutathione (GSH). Reduced levels of both GSH and glutathione S-transferase activity were observed in rats treated with CC14. Pretreatment with GSH, producing a slight and temporal increase of GSH in liver, resulted in no significant change of the K value. These findings, together with the reported kinetic data of the transferase, indicate that the conversion of azathioprine to 6-mercaptopurine (6-MP) in vivo may be catalyzed largely enzymatically by glutathione S-transferase in the liver.

Decreased disappearance rate of 1-(2-tetrahydrofuryl)-5-fluorouracil (FT-207) from the blood and its unresponsiveness to tocophreyl nicotinate and indomethacin in patients with primary hepatocellular carcinoma.

The disappearance rates(K) of FT-207 from the blood in patients with primary hepatoma and advanced cirrhosis of the liver were significantly lower than those in control patients with cancer but normal liver function. Pretreatment with tocopheryl nicotinate and indomethacin increased the K values in the control subjects, but was without effect on the K values in patients with primary hepatoma.

Determination of the disappearance rate of azathioprine from circulating rat blood by high-pressure liquid chromatography.

A rapid and specific method of high-pressure liquid chromatography for determining 6-(1-methyl-4-nitro-5-imidazole) mercaptopurine (azathioprine) in the blood was developed using mu Bondapak C18 as adsorbent and 30% aquous methanol as eluent. Azathioprine was completely separated from related compounds in blood. The standard curve showed a linear relationship at least between 0.6 and 30 micrograms per ml of serum. Recovery was satisfactory and the analytical time needed was less than 20 min. Disappearance rates of azathioprine from circulating blood in rats were 0.026 in probenecid-treated rats, and 0.103 in intact rats.

Demonstration of enzymatic activity converting azathioprine to 6-mercaptopurine.

The enzymatic conversion of azathioprine to 6-mercaptopurine was detected at pH 6.5 with rat liver supernatants, although the non-enzymatic reaction predominated at pH 7.0 and 7.5. Glutathione S-transferase may catalize this conversion. Activities of the enzyme in liver with both zathioprine and 1,2-dichloro-4-nitrobenzene as substrate decreased upon carbon tetrachloride-induced hepatic injury. These results may explain an ineffectiveness of azathioprine in patients with severe hepatic damage.

Biochemical and morphological study on hepatotoxicity of azathioprine in rat.

Sprague-Dawley rats given azathioprine in the diet for 3 to 4 weeks developed severe liver damage. Elevations of serum alkaline phosphatase and gamma-glutamyl transpeptidase activities were associated with increased hepatic glucose 6-phosphate dehydrogenase levels and decreased liver glucose 6-phosphatase activities, i.e., conditions which were commonly observed in various hepatotoxin-induced liver injuries. Light and electron microscopic observations revealed centrolobular necrosis with large scars and the proliferation of the mitochondria and rough endoplasmic reticulum. This model could be used to study the mechanisms of azathioprine-induced liver damage and its prevention.

Nephrotoxicity of pyrroloquinoline quinone in rats.

When pyrroloquinoline quinone (PQQ) was intraperitoneally injected into rats daily for 4 days at a dose of 11.5 mg/kg body weight/injection, functional and morphologic changes of the kidneys were clearly observed. The most prominent finding was necrotic and degenerative changes of the proximal tubular epithelium as well as hematuria and an elevation of serum creatinine concentration.

Paracrine control of mesenteric perivascular axo-axonal interaction.

Immunohistochemical study of rat mesenteric arteries showed dense innervation of adrenergic nerves, calcitonin gene-related peptide (CGRP)-containing nerves (CGRPergic nerves), nitric oxide-containing nerves (nitrergic nerves). Double-immunostaining revealed that most CGRPergic or nitrergic nerves were in close contact with adrenergic nerves. CGRPergic and transient receptor potential vanilloid-1 (TRPV1)-immunopositive nerves appeared in the same neurone. In rat perfused mesenteric vascular beds without endothelium and with active tone, perfusion of nicotine, or bolus injection of capsaicin and acetylcholine and periarterial nerve stimulation (PNS) lowered pH levels of out flowed perfusate concomitant with vasodilation. Cold-storage denervation of preparations abolished pH lowering induced by nicotine and PNS. Guanethidine inhibited PNS- and nicotine-, but not acetylcholine- and capsaicin-, induced pH lowering. Pharmacological analysis showed that protons were released not only from adrenergic nerves but also from CGRPergic nerves. A study using a fluorescent pH indicator demonstrated that nicotine, acetylcholine and capsaicin applied outside small mesenteric artery lowered perivascular pH levels, which were not observed in Ca(2+) free medium. Exogenously injected hydrochloric acid in denuded preparations induced pH lowering and vasodilation, which was inhibited by denervation, TRPV1 antagonists and capsaicin without affecting pH lowering. These results suggest that excitement of adrenergic nerves releases protons to activate TRPV1 in CGRPergic nerves and thereby induce vasodilation. It is also suggested that CGRPergic nerves release protons with exocytosis to facilitate neurotransmission via a positive feedback mechanism.

A ketolide antibiotic, telithromycin, inhibits vascular adrenergic neurotransmission in the rat mesenteric vascular bed.

BACKGROUND AND PURPOSE: A ketolide antibiotic, telithromycin, has side effects including temporary loss of consciousness in clinical use, but the underlying mechanisms remain unclear. This study investigated the effects of telithromycin on perivascular nerve function in rat mesenteric arteries, in comparison with those of macrolide (erythromycin and clarithromycin) and new quinolone antibiotics (levofloxacin and gatifloxacin). EXPERIMENTAL APPROACH: In vitro, vascular responses and release of noradrenaline induced by periarterial nerve stimulation (PNS) of rat perfused mesenteric vascular beds were measured in the presence of each antibiotic. In vivo blood pressure measurement was performed in Wistar rats. KEY RESULTS: In mesenteric preparations with resting tone, telithromycin (10 nM-10 microM) markedly inhibited PNS (4-12 Hz)-induced adrenergic nerve- and exogenous noradrenaline-mediated vasoconstriction, whereas the other antibiotics slightly inhibited PNS-induced responses without affecting noradrenaline-induced responses. Telithromycin significantly reduced PNS (12 Hz)-evoked noradrenaline release in the perfusate. In pre-constricted preparations with or without endothelium, telithromycin (0.1 nM-10 microM) caused a concentration-dependent vasodilation. Telithromycin (10 nM) inhibited calcium-induced vasoconstriction in high KCl and calcium-free medium. None of the antibiotics used affected PNS (0.5-2 Hz)-induced calcitonin gene-related peptide (CGRP) nerve- and exogenous CGRP-mediated vasodilation. Intravenous injection of telithromycin significantly lowered blood pressure in anaesthetized rats. CONCLUSIONS AND IMPLICATIONS: These results suggest that telithromycin causes not only strong inhibition of perivascular adrenergic neurotransmission but also a vasodilator action in mesenteric vascular beds and hypotension. It is thus possible that telithromycin increases visceral blood flow, consequently reducing cerebral blood flow and resulting in a temporary loss of consciousness.

Altered pharmacodynamics of 1-(2-tetrahydrofuryl)-5-fluorouracil by pretreatment of rats with phenobarbital, indomethacin, diclofenac sodium and carbon tetrachloride.

The disappearance curves of 1-(2-tetrahydrofuryl)-5-fluorouracil (FT-207) from the circulating blood were divided into three different phases, K1, K2 and K3 after its intravenous administration to rats. The relationship between the activity of liver microsomal drug-metabolizing enzymes and K3 values, which were obtained by measuring FT-207 levels between 5 and 24 h after the injection, was observed with phenobarbital, indomethacin, diclofenac sodium and carbon tetrachloride-pretreated rats. Therefore, the blood disappearance rate of K3 was suggested to be an effective parameter for estimating FT-207 metabolism in the liver.

Activation and inhibition of yeast aldehyde dehydrogenase activity by pantethine and its metabolites.

D-Pantethine-related metabolites, such as taurine, D-pantetheine, coenzyme A and D-pantothenate, activated yeast aldehyde dehydrogenase in vitro. D-Pantethine and cysteamine hydrochloride, however, strongly inhibited the activity of this enzyme.

Effect of various central nervous system-acting drugs on ethanol and acetaldehyde metabolism in rats.

Blood ethanol levels following intravenous ethanol administration were much higher during anesthesia with diethyl ether (ether), phenobarbital and pentobarbital than in unanesthetized control rats. Blood acetaldehyde levels were significantly lower during anesthesia with ether. Ether inhibited alcohol dehydrogenase (ADH) activities in vitro in an uncompetitive fashion; Ki, ether concentration which produced 50% inhibition of NADH formation, was 9.7 mM. Pentobarbital produced slight elevations of ADH activities in vitro and phenobarbital had no effects. Acetaldehyde levels during anesthesia with phenobarbital and pentobarbital were slightly higher than in unanesthetized animals. Phenobarbital and pentobarbital inhibited aldehyde dehydrogenase (ALDH) activities in vitro in a noncompetitive fashion; Ki were 29 and 37 mM, respectively. Ether did not influence ALDH activities in vitro. Thus, pentobarbital was suggested as the most appropriate anesthetic agent in such animal experiments.

Lowering of liver acetaldehyde but not ethanol concentrations by pretreatment with taurine in ethanol-loaded rats.

A rise in blood and liver acetaldehyde concentrations following ethanol loading (1.5 g/kg b.wt) was significantly reduced when rats were pretreated orally with taurine (0.5 g/kg), a potent in vitro activator of yeast aldehyde dehydrogenase. This taurine pretreatment produced a 4-fold increase in liver taurine content.

Quinone derivatives lower blood and liver acetaldehyde but not ethanol concentrations following ethanol loading to rats.

A rise in blood and liver acetaldehyde concentrations following an intragastric administration of ethanol to rats was significantly inhibited when the quinone derivatives 2,3-dimethoxy-5-methyl-6-decaprenyl-1,4-benzoquinone (ubidecarenone, coenzyme Q10), 4,5-dihydro-4,5-dioxo-1H-pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylic acid (pyrroloquinoline quinone, PQQ) and 6-(10-hydroxydecyl)-2,3-dimethoxy-5-methyl-1,4-benzoquinone (idebenone) were injected intraperitoneally, prior to ethanol load, at a dose of 10, 11.5 and 30 mg/kg of body weight, respectively. When acetaldehyde was incubated in vitro with 1,4-benzoquinone (3.7-13.0 mM) or PQQ (1.4-4.9 mM) at 0 and 40 degrees C, the acetaldehyde concentrations slowly decreased with incubation time at 40 degrees C. The results suggest that low acetaldehyde concentrations following ethanol load are due to an accelerated oxidation of acetaldehyde by PQQ in the liver and the circulating blood.