Narumi Hobara

Neuronal Nitric-Oxide Synthase Inhibition Facilitates Adrenergic Neurotransmission in Rat Mesenteric Resistance Arteries

The effects of nonselective nitric-oxide synthase (NOS) inhibitors [N-ω-nitro-l-arginine methyl ester (l-NAME) and N-ω-nitro-l-arginine (l-NNA)] and specific neuronal NOS (nNOS) inhibitor [vinyl-l-N-5-(1-imino-3-butenyl)-l-ornithine (l-VNIO)] on adrenergic nerve-mediated vasoconstriction were studied in rat perfused mesenteric vascular beds without endothelium. Perfusion of l-NAME, l-NNA, or l-VNIO markedly augmented vasoconstrictor responses to periarterial nerve stimulation (PNS; 2-8 Hz) without affecting vasoconstriction induced by exogenously injected norepinephrine (NE). Addition of l-arginine, a precursor for the synthesis of nitric oxide (NO), reversed the augmentation of the PNS response by l-NAME. The PNS (8 Hz)-evoked NE release in the perfusate was increased by l-NAME perfusion. In preparations treated with capsaicin [a depleter of calcitonin gene-related peptide (CGRP)-containing nerves], l-NAME did not augment vasoconstrictor responses to PNS or NE injection. Combined perfusion of CGRP(8-37) (a CGRP receptor antagonist) and l-NAME induced additive augmentation of the vasoconstrictor response to PNS but did not affect the response to NE injection. In preparations with active tone produced by methoxamine and in the presence of guanethidine, l-NAME perfusion did not affect the vasodilator response induced by PNS. Immunostaining of the mesenteric artery showed the presence of nNOS-like immunopositive nerve fibers, which were absent in arteries pretreated with capsaicin. These findings suggest that NO, which is released from perivascular capsaicin-sensitive nerves, presynaptically inhibits neurogenic NE release to modulate adrenergic neurotransmission.

Long-Term Inhibition of Angiotensin Prevents Reduction of Periarterial Innervation of Calcitonin Gene-Related Peptide (CGRP)-Containing Nerves in Spontaneously Hypertensive Rats

The aim of this study was to investigate age-related changes in the density of calcitonin gene-related peptide (CGRP)-containing nerve fibers in spontaneously hypertensive rats (SHR) and the effects of long-term inhibition of the renin-angiotensin system on these changes. The density of immunocytochemically stained nerve fibers in the mesenteric artery was quantified by computer-assisted image processing. An age-related decrease in the density of CGRP-like immunoreactive (LI)-containing nerve fivers but not neuropeptide Y (NPY)-LI-containing sympathetic nerve fibers was found in the mesenteric artery of SHR but not Wistar Kyoto rats (WKY). The density of NPY-LI-containing sympathetic nerve fibers was significantly greater in SHR than in WKY. SHR were treated for 7 weeks with angiotensin converting enzyme inhibitor (0.005% temocapril), angiotensin II type-1 (AT1) receptor antagonist (0.025% losartan) or vasodilator (0.01% hydralazine) in their drinking water. Each drug treatment significantly lowered the systolic blood pressure measured by tail-cuff method. Long-term treatment of SHR with temocapril and losartan significantly increased the density of CGRP-LI-containing nerve fibers in mesenteric arteries. However, the density after hydralazine treatment was similar to the level in non-treated SHR. The density of NPY-LI-containing nerve fibers was not increased by any of the drug treatments. These results suggest that long-term inhibition of the renin-angiotensin system in SHR prevents remodeling of CGRPergic nerve fibers and prevents the reduction of CGRPergic nerve function.

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.

Vanilloid receptors mediate adrenergic nerve- and CGRP-containing nerve-dependent vasodilation induced by nicotine in rat mesenteric resistance arteries

Previous studies showed that nicotine induces adrenergic nerve‐dependent vasodilation that is mediated by endogenous calcitonin gene‐related peptide (CGRP) released from CGRP‐containing (CGRPergic) nerves. The mechanisms underlying the nicotine‐induced vasodilation were further studied. Rat mesenteric vascular beds without endothelium were contracted by perfusion with Krebs solution containing methoxamine, and the perfusion pressure was measured with a pressure transducer. Perfusion of nicotine (1–100 μM) for 1 min caused concentration‐dependent vasodilation. Capsazepine (vanilloid receptor‐1 antagonist; 1–10 μM) and ruthenium red (inhibitor of vanilloid response; 1–30 μM) concentration‐dependently inhibited the nicotine‐induced vasodilation without affecting the vasodilator response to exogenous CGRP. Nicotine‐induced vasodilation was not inhibited by treatment with 3,4‐dihydroxyphenylalanine (DOPA) receptor antagonist (L‐DOPA cyclohexyl ester; 0.001–10 μM), dopamine D1 receptor‐selective antagonist (SCH23390; 1–10 μM), dopamine D2 receptor antagonist (haloperidol; 0.1–0.5 μM), ATP P2x receptor‐desensitizing agonist (α,β‐methylene ATP; 1–10 μM), adenosine A2 receptor antagonist (8(p‐sulfophenyl)theophylline; 10–50 μM) or neuropeptide Y (NPY)‐Y1 receptor antagonist (BIBP3226; 0.1–0.5 μM). Immunohistochemical staining of the mesenteric artery showed dense innervation of CGRP‐ and vanilloid receptor‐1‐positive nerves, with both immunostainings appearing in the same neuron. The mesenteric artery was also densely innervated by NPY‐positive nerves. Double immunostainings showed that both NPY and CGRP immunoreactivities appeared in the same neuron of the artery. These results suggest that nicotine acts on presynaptic nicotinic receptors to release adrenergic neurotransmitter(s) or related substance(s), which then stimulate vanilloid receptor‐1 on CGRPergic nerves, resulting in CGRP release and vasodilation.

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.

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.

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.

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

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).

Investigation of low levels of plasma valproic acid concentration following simultaneous administration of sodium valproate and rizatriptan benzoate

Drug interaction between rizatriptan benzoate, an anti‐migraine agent, and sodium valproate (VPA‐Na), an anticonvulsant, was studied in rats. When rizatriptan benzoate was administered orally immediately after VPA‐Na oral administration, the pharmacokinetic parameters, such as plasma valproic acid (VPA) and area under the plasma concentration‐time curve up to 3 h (AUC0–3), were significantly decreased compared with those in the control group. However, when rizatriptan benzoate was administered intraperitoneally immediately after VPA‐Na orally, these parameters were not changed. In addition, when benzoic acid was administered orally immediately after VPA‐Na orally, these were significantly lower compared with the control values. Therefore, it might be possible that VPA transport by monocarboxylate transporter was competitively inhibited by rizatriptan benzoate and thus absorption of VPA was decreased.

Adrenomedullin release in the rat mesenteric resistance artery.

Adrenomedullin (AM) is a potent vasodilator peptide whose major source is the vascular wall. In the present study, the mechanism of release of AM was investigated in the rat mesenteric resistance artery. The isolated mesenteric vascular bed was perfused with Krebs solution at a constant flow rate (5 ml/min) and AM in the perfusate was measured by a highly sensitive enzyme immunoassay (Immunoenzymometric assay; IEMA) method. In preparations without endothelium, spontaneous release of AM was detected in the perfusate (68.7+/-5.8 fmol/ml, n=45). Periarterial nerve stimulation (PNS, 4 and 8 Hz) caused 11.4+/-3.9% (4 Hz) and 9.1+/-3.5% (8 Hz) decreases in the spontaneous release of AM. Removal of Ca2+ from the medium did not affect the spontaneous AM release, but abolished the PNS-induced inhibition of spontaneous AM release. Perfusion of 10nM calcitonin gene-related peptide (CGRP) or 0.1 microM capsaicin (inducer of CGRP release) inhibited significantly the spontaneous AM release. PNS (8 Hz)-induced inhibition of spontaneous AM release was antagonized by CGRP(8-37) (CGRP receptor antagonist). These results suggest that AM is mainly released from vascular smooth muscle cells of the rat mesenteric artery and endogenous or exogenous CGRP inhibits AM release.