A series of our studies on the dog venous system revealed that cholinergic excitatory innervation was localized in a group of veins: the portal, mesenteric, and hepatic veins and the middle segment of the inferior vena cava. Our studies on pharmacological responsiveness of dog veins also revealed that they could be divided into two groups: the visceral and somatic parts, and the cholinergic excitatory innervation localized to the visceral part. Considering these results and some relevant literature, a hypothesis is proposed on the classification of muscles of the cardiovascular system and some embryological aspects of the parasympathetic cholinergic innervation in the circulatory system are discussed. The embryonic circulatory system of vertebrates can be divided into two parts: somatic and visceral. The body of an embryo is regarded as a double tube and vessels of the visceral part and the heart belong to the inner tube. The muscle of these vessels and the heart are derived from visceral mesoderm, either the coelomic epithelium or mesenchymal cells, in common with muscle of the digestive tube; and thus the parasympathetic cholinergic nerves innervating the muscle of the digestive tube also distribute to these vessels and the heart. The heart and vascular muscles in the visceral part are structures developed early in the course of evolution in invertebrates. Their primary function is to propel the body fluid, and the chief structure containing them is the subintestinal circulatory channel (ventral aorta − heart − subintestinal vein). They exhibit spontaneous, rhythmic activity, showing characteristics of a single unit muscle, and receive parasympathetic cholinergic innervation. On the other hand, the vascular muscles in the somatic part are endothelium-associated muscles developed anew in the vertebrate; do not contract spontaneously, being classified as a multiunit muscle; and lack parasympathetic cholinergic innervation.

View full abstract

Although calmodulin binding to various sites of the Cav1.2 Ca²⁺ channel has been reported, the mechanism of the interaction is not fully understood. In this study we examined calmodulin binding to fragment channel peptides using a semi-quantitative pull-down assay. Calmodulin bound to the peptides with decreasing affinity order: IQ > preIQ > I-II loop > N-terminal peptide. A peptide containing both preIQ and IQ regions (Leu¹⁵⁹⁹ – Leu¹⁶⁶⁸) bound with approximately 2 mol of calmodulin per peptide. These results support the hypothesis that two molecules of calmodulin can simultaneously bind to the C-terminus of the Cav1.2 channel and modulate its facilitatory and inhibitory activities.

View full abstract

α-Viniferin, an oligostilbene of trimeric resveratrol, has been reported to have anti-inflammatory potential in carrageenin-induced paw edema or adjuvant-induced arthritis in animal models. However, little is known about the molecular basis. In this study, α-viniferin at 3 – 10 μM dose-dependently inhibited interferon (IFN)-γ–induced Ser⁷²⁷ phosphorylation of the signal transducer and activation of transcription-1 (STAT-1), a pivotal transcription factor controlling IFN-γ–targeted genes, in RAW 264.7 macrophages, and also IFN-γ–induced activation of the extracellular signal-regulated kinase (ERK)-1, a protein kinase upstream of the Ser⁷²⁷ phosphorylation of STAT-1. However, α-viniferin, only at a higher concentration of 10 μM, inhibited Janus kinase 2–mediated Tyr⁷⁰¹ phosphorylation of STAT-1 in the cells. To understand STAT-1–dependent inflammatory responses, we quantified nitric oxide (NO) or chemokines. α-Viniferin at 3 – 10 μM dose-dependently inhibited IFN-γ–induced production of NO, IFN-γ–inducible protein-10 (IP-10), or the monokine induced by IFN-γ (MIG) in RAW 264.7 cells and also that of NO in primary macrophages-derived from C57BL/6 mice. Furthermore, α-viniferin diminished IFN-γ–induced protein levels of inducible NO synthase (iNOS), attenuated mRNA levels of iNOS, IP-10, or MIG as well as inhibited promoter activity of the iNOS gene. In conclusion, this study proposes an anti-inflammatory mechanism of α-viniferin, down-regulating STAT-1–inducible inflammatory genes via inhibiting ERK-mediated STAT-1 activation in IFN-γ–stimulated macrophages.

View full abstract

In guinea-pig cardiomyocytes, a cAMP-dependent Cl⁻ current (I_Cl,cAMP) flows through a cardiac isoform of the cystic fibrosis transmembrane conductance regulator (CFTR), which belongs to a family of the ATP-binding cassette (ABC) proteins. Although several K⁺-channel openers and sulfonylurea ATP-sensitive K⁺ (K_ATP)–channel blockers reportedly inhibit I_Cl,cAMP, effects of nicorandil on the Cl⁻ current have not been evaluated. This study was conducted to examine the effects of nicorandil on I_Cl,cAMP in isolated guinea-pig ventricular cells using patch clamp techniques. Nicorandil in concentrations higher than 300 μM enhanced the I_Cl,cAMP preactivated by 0.1 μM isoproterenol. The isoproterenol-induced I_Cl,cAMP was inhibited by 100 μM glibenclamide, but not by 100 μM pinacidil. SNAP (S-nitroso-N-acetyl-D,L-penicillamine, 10 μM), a nitric oxide (NO) donor, similarly enhanced the isoproterenol-induced I_Cl,cAMP. However, SG-86, a denitrated metabolite possessing K⁺ channel–opening action, failed to enhance the Cl⁻ current. When the I_Cl,cAMP was activated by 3-isobutyl-1-methylxanthine (IBMX, 30 μM), either nicorandil or SNAP failed to enhance the isoproterenol-induced I_Cl,cAMP. Thus, nicorandil enhances I_Cl,cAMP in guinea-pig cardiomyocytes through an increase in intracellular cGMP, although direct modulation of I_Cl,cAMP by NO cannot be completely excluded.

View full abstract

G protein–coupled receptors, in particular, Ca²⁺-mobilizing G_q-coupled receptors have been reported to be targets for anesthetics. Opioids are commonly used analgesics in clinical practice, but the effects of anesthetics on the opioid μ-receptors (μOR) have not been systematically examined. We report here an electrophysiological assay to analyze the effects of anesthetics and ethanol on the functions of μOR in Xenopus oocytes expressing a μOR fused to chimeric Gα protein G_qi5 (μOR-G_qi5). Using this system, the effects of halothane, ketamine, propofol, and ethanol on the μOR functions were analyzed. In oocytes expressing μOR-G_qi5, the μOR agonist DAMGO ([D-Ala²,N-MePhe⁴,Gly-ol]-enkephalin) elicited Ca²⁺-activated Cl⁻ currents in a concentration-dependent manner (EC₅₀ = 0.24 μM). Ketamine, propofol, halothane, and ethanol themselves did not elicit any currents in oocytes expressing μOR-G_qi5, whereas ketamine and ethanol inhibited the DAMGO-induced Cl⁻ currents at clinically equivalent concentrations. Propofol and halothane inhibited the DAMGO-induced currents only at higher concentrations. These findings suggest that ketamine and ethanol may inhibit μOR functions in clinical practice. We propose that the electrophysiological assay in Xenopus oocytes expressing μOR-G_qi5 would be useful for analyzing the effects of anesthetics and analgesics on opioid receptor function.

View full abstract

Angiostatin (AS), a proteolytic fragment of plasminogen, is a potent antiangiogenic factor. It was reported that AS attenuates the vasodilatory response to vascular endothelial growth factor (VEGF) in isolated interventricular arterioles. Here, we investigated the effect of AS on nitric oxide (NO) production in human umbilical vein endothelial cells (HUVECs). AS inhibited VEGF-stimulated NO production in a dose-dependent manner, whereas AS alone did not affect basal NO production. Disruption of kringle structures by reduction of disulfide bonds resulted in the loss of the inhibitory effect of AS on VEGF-stimulated NO production. To elucidate how AS might impair VEGF activation of endothelial NO synthase (eNOS), we further examined whether AS would affect Ca²⁺-dependent and -independent pathways of eNOS activation. AS had no effect on the transient increase in cytosolic Ca²⁺ levels elicited by VEGF. In contrast, AS prevented VEGF-potentiated eNOS phosphorylation at Ser1177. These results clearly indicate that AS inhibits VEGF-stimulated NO production in HUVECs without affecting basal NO production. The kringle structures of AS are required for this effect, and impairment of Ser1177 phosphorylation of eNOS might be involved in the inhibition of VEGF-stimulated NO production by AS.

View full abstract

Novel agents that inhibit nerve growth factor signaling are required for the treatment of inflammatory pain. The present study investigated the effect of local administration of inhibitory peptide of TrkA (IPTRK3), a synthetic cell-penetrating peptide that antagonizes TrkA function, in complete Freund’s adjuvant (CFA)–induced hyperalgesia in rats. Three hours after subcutaneous injection of CFA into the plantar surface of the rat’s left hind paw, 10 mM IPTRK3 was injected at the same site. Thermal and mechanical hyperalgesia were tested in the ipsilateral hind paw until 7 days after CFA injection. The ipsilateral dorsal root ganglion (DRG) was dissected out for immunohistochemical analysis of transient receptor potential vanilloid subfamily member 1 (TRPV1) channels and TrkA. Local injection of this peptide significantly suppressed both thermal and mechanical hyperalgesia produced by CFA and also significantly reduced TRPV1 expression at the DRG. These results suggest that local administration of IPTRK3 is likely effective in the treatment of inflammatory pain in rats.

View full abstract

Muscarinic acetylcholine receptors (mAChRs) of rat cerebral cortex were evaluated using a tissue segment radioligand binding assay. [³H]-Quinuclidinyl benzilate (QNB, a hydrophobic ligand) specifically bound to mAChRs in the cortex segments. The total mAChRs level was approximately 2,000 fmol/mg protein, which was estimated after incubation for 120 min at 37°C or for 8 h at 4°C. These mAChRs were a mixture of high- and low-affinity sites for N-methylscopolamine (NMS) in a 70:30 ratio. In contrast, only a single high-affinity site for NMS was detected following incubation for 30 min at 37°C, whose abundance was about 70% of that of the total mAChRs. Atropine showed a single affinity for mAChRs under all conditions. These indicate that mAChRs are constitutively expressed not only on plasma membrane sites but also at intracellular sites in rat cerebral cortex and that the receptors at both sites have different affinities for NMS. Acetylcholine completely inhibited [³H]-QNB binding to both mAChRs without any change in the subcellular distribution, suggesting the possibility that acetylcholine can access, and bind to, both mAChRs in intact tissue. Two different affinity states for acetylcholine were detected only in plasma membrane mAChRs at 37°C. The present study demonstrates a unique subcellular distribution, and distinct pharmacological profiles, of mAChRs in rat cerebral cortex.

View full abstract

ProTx-I peptide, a venom toxin of the tarantula Thrixopelma pruriens, has been reported to interact with voltage-gated ion channels. ProTx-I reduced Ba²⁺ currents through recombinant human T-type voltage-gated Ca²⁺ channels, Ca_v3.1 (hCa_v3.1), with roughly 160-fold more potency than through hCa_v3.2 channels. Chimeric channel proteins (hCa_v3.1/S3S4 and hCa_v3.2/S3S4) were produced by exchanging fourteen amino acids in the hCa_v3.1 domain IV S3-S4 linker region and the corresponding region of hCa_v3.2 between each other. The ProTx-I sensitivity was markedly reduced in the hCa_v3.1/S3S4 chimera as compared to the original hCa_v3.1 channel, while the hCa_v3.2/S3S4 chimera exhibited greater ProTx-I sensitivity than the original hCa_v3.2 channel. These results suggest that the domain IV S3-S4 linker in the hCa_v3.1 channel may contain residues involved in the interaction of ProTx-I with T-type Ca²⁺ channels.

View full abstract

Based on radioligand binding and signal transduction assays in our previous study, we have determined the binding pattern and functional efficacy of the constitutively active mutant N111G of angiotensin II type 1 (AT₁) receptor. We have also shown that the N111G mutant induces homologous internalization through mediation of the AT₁-receptor antagonist valsartan. In this study we demonstrated that other AT₁-receptor antagonists, candesartan, losartan, and telmi-sartan, also stimulate internalization of N111G mutant receptor to the same extent. We further showed that the internalization pattern is also similar for all the AT₁-receptor antagonists.

View full abstract

Nerve growth factor (NGF) facilitates reinnervation of perivascular nerves that regulate vascular tone and blood flow. This study investigated whether NGF prevents tumor growth by promoting neuronal regulation of tumor blood flow. The growth rate of DU145 prostate carcinoma cells subcutaneously implanted into nude mice was significantly inhibited by subcutaneous NGF administration. Significant suppression of tumor growth continued after withdrawing NGF. NGF increased vascular smooth muscle cells in tumor tissues, but had no cytotoxic action on tumor cells in vitro. These results suggest that NGF prevents tumor growth via an indirect effect, probably innervation or maturation of the tumor neovasculature.

View full abstract

RhoA upregulation has been suggested in bronchial smooth muscles (BSMs) of asthmatic rats. Here, we cloned/characterized the 5′-promoter region of the rat rhoA. A transcription-initiation site was identified at 66-bp upstream of the reference sequence, GenBank-BC061732. Luciferase assay using interleukin-13 (IL-13)–stimulated cells revealed a significant promoter activity at 238- to 166-bp upstream of the transcription-initiation site, which contains a signal transducer and activation of transcription (STAT) 6–binding region. The IL-13–induced increase in luciferase activity was inhibited by a STAT6 inhibitor, AS1517499, or a Janus kinases (JAKs) inhibitor, JAK Inhibitor-I, but not by tyrphostin-AG490, WHI-P131, or tyrphostin-AG9 (selective JAK2, JAK3, and Tyk2 inhibitors, respectively). Thus, rat BSM rhoA expression may have causal relation to the IL-13–JAK1–STAT6 signaling.

View full abstract

Oxaliplatin is a platinum-based chemotherapy drug characterized by the development of acute and chronic peripheral neuropathies. Mexiletine, an orally available Na⁺-channel blocker, has widely been used in patients with chronic painful diabetic neuropathy. In the present study, we examined the effect of mexiletine on oxaliplatin-induced neuropathic pain in rats. Mexiletine (100, but not 10 and 30, mg/kg, p.o.) completely reversed both mechanical allodynia and cold hyperalgesia induced by oxaliplatin (4 mg/kg, i.p., twice a week). Lidocaine (30, but not 3 and 10, mg/kg, i.p.) also significantly relieved both pain behaviors. These results suggest that mexiletine may be effective in relieving the oxaliplatin-induced neuropathic pain clinically.

View full abstract

The role of platelet-derived growth factor (PDGF)-BB / PDGF receptor (PDGFR)-β signal in inhibition of synaptic transmission by hypoxia is unclear. In the nucleus tractus solitarius neurons, hypoxia with N₂ or NaCN decreased the amplitude of excitatory postsynaptic currents (EPSCs) similarly in wild type (WT) and PDGFR-β gene–knockout (KO) mice. Recovery of EP SCs after a high concentration of NaCN in KO mice was significantly faster than that in WT mice, while recovery after a low concentration of NaCN or N₂ was not different between both mice. These results suggest that the PDGF-BB / PDGFR-β signal modulates the excitatory synaptic transmission during hypoxia.

View full abstract

We examined the role of aldosterone-sensitive neurons in the nucleus tractus solitarius (NTS) in the arterial baroreceptor reflex (baroreflex) function. Baroreflex sensitivity was induced by phenylephrine in high sodium–loaded rats and was significantly reduced. This baroreflex sensitivity was reversed by microinjection of the mineralocorticoid receptor (MR) antagonist eplerenone into the NTS. 11β-Hydroxysteroid dehydrogenase type 2 neurons and MR were also identified in the NTS. These data suggest that the aldosterone-sensitive neurons in the NTS may have an important role in baroreflex function.

View full abstract

We recently demonstrated that SA13353, a transient receptor potential vanilloid 1 (TRPV1) agonist, reduced the severity of the symptoms of kidney injury, arthritis, and encephalomyelitis in disease models. Here, we investigated the effects of orally administered SA13353 on leukocyte infiltration in lipopolysaccharide (LPS)-induced acute lung injury and ovalbumin-induced allergic airway inflammation. In LPS-induced lung injury, SA13353 attenuated neutrophil infiltration and the increase of TNF-α and CINC-1 levels. In allergic airway inflammation, SA13353 tended to inhibit leukocyte infiltration and attenuated the increase of IL-4 and IL-12p40. These results suggest that somatosensory TRPV1 may play an anti-inflammatory role in lung inflammation.

View full abstract