NO-releasing xanthine KMUP-1 bonded by simvastatin attenuates bleomycin-induced lung inflammation and delayed fibrosis.

BACKGROUND AND PURPOSE: Pulmonary fibrosis (PF) is a progressing lung injury initiated by pulmonary inflammation (PI). Bleomycin (BLM) is the most common pathogenesis of PF through early PI and extensive extracellular matrix deposition. This study is aimed to determine whether NO-releasing KMUP-1 inhibits PI and PF, and if so, the benefits of KMUP-1S resulted from simvastatin (SIM)-bonding to KMUP-1. EXPERIMENT APPROACH: C57BL/6 male mice were intra-tracheally administered BLM (4 U/kg) at day 0. KMUP-1 (1-5 mg/kg), KMUP-1S (2.5 mg/kg), SIM (5 mg/kg), Plus (KMUP-1 2.5 mg/kg + SIM 2.5 mg/kg), and clarithromycin (CAM, 10 mg/kg) were orally and daily administered for 7 and 28 days, respectively, to mice, sacrificed at day-7 and day-28 to isolate the lung tissues, for examining the inflammatory and fibrotic signaling and measuring the cell population and MMP-2/MMP-9 activity in broncholaveolar lavage fluid (BAL). KEY RESULTS: KMUP-1 and KUP-1S significantly decreased neutrophil counts in BAL fluid. Fibroblastic foci were histologically assessed by H&E and Masson's trichrome stain and treated with KMUP-1 and references. Lung tissues were determined the contents of collagen and the expressions of TGF-ß, α-SMA, HMGB1, CTGF, eNOS, p-eNOS, RhoA, Smad3, p-Smad3, MMP-2 and MMP-9 by Western blotting analyses, respectively. These changes areregulated by NO/cGMP and inhibited by various treatments. KMUP-1 and KMUP-1S predominantly prevented HMGB1/MMP-2 expression at day-7 and reduced TGF-ß/phosphorylated Smad3 and CTGF at day-28. CONCLUSIONS AND IMPLICATIONS: KMUP-1 and KMUP-S restore eNOS, inhibit iNOS/ROCKII/MMP-2/MMP-9, attenuate histologic collagen disposition and reduce BALF inflammatory cells, potentially useful for the treatment of BLM-lung PF.

NO donor KMUP-1 improves hepatic ischemia-reperfusion and hypoxic cell injury by inhibiting oxidative stress and pro-inflammatory signaling.

This study investigates whether KMUP-1 improves hepatic ischemia-reperfusion (I/R) and hypoxic cell injury via inhibiting Nox2- and reactive oxygen species (ROS)-mediated pro-inflammation. Rats underwent ischemia by occlusion of the portal vein and hepatic artery for 45 minutes. Reperfusion was allowed for 4 h. Serum was used for analysis of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). DNA extracted from liver homogenate was analyzed by electrophoresis to observe the fragmentation. Lipid peroxidation (LPO) was evaluated by measuring thiobarbituric acid-reactive substances (TBARS). NO and ROS contents were measured using Griess reagent and 2'-7'-dichlorofluorescein, respectively. Proteins levels were visualized by Western blotting. Liver damage was observed under a microscope. Intravenous KMUP-1 (0.25, 0.5 and 1 mg/kg) reduced I/R-induced ALT and AST levels, DNA fragmentation, ROS and malondialdehyde (MDA) and restored the NO levels of I/R rats. KMUP-1 protected the liver architecture from worsening of damage and focal sinusoid congestion, increased endothelium NO synthase (eNOS), guanosine 3', 5'cyclic monophosphate (cGMP), protein kinase G (PKG) and the B-cell lymphoma 2/Bcl-2-associated X protein (Bcl-2/Bax) ratio, attenuated phosphodiesterase 5A (PDE-5A) and cleaved caspase-3 expression in I/R-liver. In hypoxic HepG2 cells, KMUP-1 increased cGMP/PKG, restored peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and decreased matrix metalloproteinases-9 (MMP-9), Rho kinase II (ROCK II), hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelium growth factor (VEGF). KMUP-1 protects liver from I/R-injury and hypoxic hepatocytes from apoptosis-associated free radical generation and pro-inflammation by restoring/increasing NO/cGMP/PPAR-gamma, reducing ROS/Nox2 and inhibiting ROCKII/MMP-9.

Neuroprotective and anti-inflammatory activities of atorvastatin in a rat chronic constriction injury model.

Atorvastatin is an HMG-CoA reductase inhibitor used to treat hypercholesterolemic conditions associated with hypertension. This study aims to investigate the anti-inflammatory and neuroprotective effects of atorvastatin on peripheral neuropathic pain. Peripheral neuropathic pain was induced by chronic constriction injury (CCI) in Sprague-Dawley rats. Rats were divided into 3 groups including sham-operated, CCI, and atorvastatin-treated. Atorvastatin (10 mg/kg) or phosphate-buffered saline was orally administered for 2 weeks. All animals were assessed by neurobehavioral tests before surgery and at days 3, 7, 14 after surgery. Inflammatory and neuroprotective factors were evaluated by Western blot analysis. eNOS, COX2 and iNOS in the sciatic nerve were also studied using immunohistochemistry. Atorvastatin attenuated CCI-induced nociceptive sensitization and thermal hyperalgesia in a time-dependent manner. Atorvastatin improved CCI-induced neurobehavioral/inflammatory activity by inhibition of TGF-beta, pIkB/IkB, NFkB, COX2, iNOS, EP1 and EP4 in the sciatic nerve. Atorvastatin was also found to increase neuroprotection factors pAkt/Akt, eNOS and VEGF. Taken together, these data indicate that atorvastatin could protect the sciatic nerve against CCI-induced neuroinflammation and nociception.

Eugenolol and glyceryl-isoeugenol suppress LPS-induced iNOS expression by down-regulating NF-kappaB AND AP-1 through inhibition of MAPKS and AKT/IkappaBalpha signaling pathways in macrophages.

Eugenol and isoeugenol, two components of clover oil, have been reported to possess several biomedical properties, such as anti-inflammatory, antimicrobial and antioxidant effects. This study aims to examine the anti-inflammatory effects of eugenol, isoeugenol and four of their derivatives on expression of inducible nitric oxide synthase (iNOS) activated by lipopolysaccharide (LPS) in mouse macrophages (RAW 264.7), and to investigate molecular mechanisms underlying these effects. We found that two derivatives, eugenolol and glyceryl-isoeugenol, had potent inhibitory effects on LPS-induced upregulation of nitrite levels, iNOS protein and iNOS mRNA. In addition, they both suppressed the release of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) induced by LPS. Moreover, they both attenuated the DNA binding of NF-kB and AP-1, phosphorylation of inhibitory kB-alpha (IkB-alpha), and nuclear translocation of p65 protein induced by LPS. Finally, we demonstrated that glyceryl-isoeugenol suppressed the phosphorylation of ERK1/2, JNK and p38 MAPK, whereas eugenolol suppressed the phosphorylation of ERK1/2 and p38 MAPK. Taken together, these results suggest that that eugenolol and glyceryl-isoeugenol suppress LPS-induced iNOS expression by down-regulating NF-kB and AP-1 through inhibition of MAPKs and Akt/IkB-alpha signaling pathways. Thus, this study implies that eugenolol and glyceryl-isoeugenol may provide therapeutic benefits for inflammatory diseases.

KMUP-1 inhibits H441 lung epithelial cell growth, migration and proinflammation via increased NO/CGMP and inhibited RHO kinase/VEGF signaling pathways.

This study investigates whether KMUP-1 protects soluble guanylate cyclase (sGC) and inhibits vascular endothelial growth factor (VEGF) expression in lung epithelial cells in hypoxia, therapeutically targeting epithelial proinflammation. H441 cells were used as a representative epithelial cell line to examine the role of sGC and VEGF in hypoxia and the anti-proinflammatory activity of KMUP-1 in normoxia. Human H441 cells were grown in hypoxia for 24-72 h. KMUP-1 (1, 10, 100 microM) arrested cells at the G0/G1 phase of the cell cycle, reduced cell survival and migration, increased p21/p27, restored eNOS, increased soluble guanylate cyclase (sGC) and PKG and inhibited Rho kinase II (ROCK-II). KMUP-1 (0.001-0.1 microM) concentration dependently increased eNOS in normoxia and did not inhibit phosphodiesterase-5A (PDE-5A) in hypoxic cells. Hypoxia-induced factor-1alpha (HIF-1alpha) and VEGF were suppressed by KMUP-1 but not by L-NAME (100 microM). The PKG inhibitor Rp-8-CPT-cGMPS (10 microM) blunted the inhibition of ROCK-II by KMUP-1. KMUP-1 inhibited thromboxane A2-mimetic agonist U46619-induced PDE-5A, TNF-alpha (100 ng/ml)-induced iNOS, and ROCK-II and associated phospho-p38 MAPK, suggesting multiple anti-proinflammatory activities. In addition, increased p21/p27 by KMUP-1 at higher concentrations might contribute to an increased Bax/Bcl-2 and active caspase-3/procaspase-3 ratio, concomitantly causing apoptosis. KMUP-1 inhibited ROCK-II/VEGF in hypoxia, indicating its anti-neoplastic and anti-inflammatory properties. KMUP-1 inhibited TNF-alpha-induced iNOS and U46619-induced PDE-5A and phospho-p38 MAPK in normoxia, confirming its anti-proinflammatory action. KMUP-1 could be used as an anti-proinflammatory to reduce epithelial inflammation.

Activation of BKCa channels via cyclic AMP- and cyclic GMP-dependent protein kinases by eugenosedin-A in rat basilar artery myocytes.

BACKGROUND AND PURPOSE: The study investigated whether eugenosedin-A, a 5-hydroxytryptamine and alpha/beta adrenoceptor antagonist, enhanced delayed-rectifier potassium (K(DR))- or large-conductance Ca(2+)-activated potassium (BK(Ca))-channel activity in basilar artery myocytes through cyclic AMP/GMP-dependent and -independent protein kinases. EXPERIMENTAL APPROACH: Cerebral smooth muscle cells (SMCs) were enzymatically dissociated from rat basilar arteries. Conventional whole cell, perforated and inside-out patch-clamp electrophysiology was used to monitor K(+)- and Ca(2+)-channel activities. KEY RESULTS: Eugenosedin-A (1 microM) did not affect the K(DR) current but dramatically augmented BK(Ca) channel activity in a concentration-dependent manner. Increased BK(Ca) current was abolished by charybdotoxin (ChTX, 0.1 microM) or iberiotoxin (IbTX, 0.1 microM), but not affected by a small-conductance K(Ca) blocker (apamin, 100 microM). BK(Ca) current activation by eugenosedin-A was significantly inhibited by an adenylate cyclase inhibitor (SQ 22536, 10 microM), a soluble guanylate cyclase inhibitor (ODQ, 10 microM), competitive antagonists of cAMP and cGMP (Rp-cAMP, 100 microM and Rp-cGMP, 100 microM), and cAMP- and cGMP-dependent protein kinase inhibitors (KT5720, 0.3 microM and KT5823, 0.3 microM). Eugenosedin-A reversed the inhibition of BK(Ca) current induced by the protein kinase C activator, phorbol myristyl acetate (PMA, 0.1 microM). Eugenosedin-A also prevented BK(Ca) current inhibition induced by adding PMA, KT5720 and KT5823. Moreover, eugenosedin-A reduced the amplitude of voltage-dependent L-type Ca(2+) current (I(Ca,L)), but without modifying the voltage-dependence of the current. CONCLUSIONS AND IMPLICATIONS: Eugenosedin-A enhanced BK(Ca) currents by stimulating the activity of cyclic nucleotide-dependent protein kinases. Physiologically, this activation would result in the closure of voltage-dependent calcium channels and thereby relax cerebral SMCs.

Effects of osmolytes on unfolding of chicken liver fatty acid synthase.

Urea-induced aggregation of chicken liver fatty acid synthase [acyl-CoA:malonyl-CoA C-acyltransferase (decarboxylating, oxoacyl- and enoyl-reducing and thioester-hydrolyzing), EC 2.3.1.85] was studied. The aggregation was facilitated at increased ionic strength. Methyl-beta-cyclodextrin and some osmolytes, such as glycerol, sucrose, proline, glycine, and heparin, could effectively prevent the aggregation, implying an artificial chaperone role of those substances during fatty acid synthase unfolding. The osmolytes also protected the enzyme from inactivation.

Unfolding and inactivation of fatty acid synthase from chicken liver during urea denaturation.

The inactivation and conformational changes of the multifunctional fatty acid synthase (acyl-CoA:malonyl-CoA C-acyltransferase (decarboxylating, oxoacyl- and enoyl-reducing and thioester-hydrolyzing), EC 2.3.1.85) from chicken liver have been studied in urea solution. The results show that complete inactivation of the fatty acid synthase occurs before obvious conformational changes with regard to the overall, beta-ketoacyl reduction and acetoacetyl-CoA reduction reactions. Significant conformational changes indicated by the changes of the intrinsic fluorescence emission and the circular dichroism spectra occurred at higher urea concentrations. The kinetic rate constants for the two phase inactivation and unfolding reactions were measured and semilogarithmic plots of the activity versus time gave curves which could be resolved into two straight lines, indicating that both the inactivation and unfolding processes consisted of fast and slow phases as a first-order reaction. The results from Lineweaver-Burk plots indicated that urea is a competitive inhibitor for acetyl-CoA and malonyl-CoA, with K(m) increasing with increasing urea concentrations. However, urea is a noncompetitive inhibitor for NADPH, the substrate of the overall reaction and beta-ketoacyl reduction reaction, and acetylacetate, the substrate of the beta-ketoacyl reduction reaction. Activation by low concentrations of urea was observed although this activation was only temporarily induced in an early stage of inactivation. The aggregation phenomenon of the fatty acid synthase in a certain concentration range of urea (3-4 M) was also observed during unfolding. This result shows that this multifunctional enzyme unfolds with competition with misfolding in the folding pathway. Comparison of inactivation and conformational changes of the enzyme as well as aggregation imply that unfolding intermediates may exist during urea denaturation. The possible unfolding pathway of fatty acid synthase is also discussed in this paper.

A xanthine-based KMUP-1 with cyclic GMP enhancing and K(+) channels opening activities in rat aortic smooth muscle.

1. KMUP-1 (1, 3, 5 mg kg(-1), i.v.), a xanthine derivative, produced dose-dependent sustained hypotensive and short-acting bradycardiac effects in anaesthetized rats. This hypotensive effect was inhibited by pretreatment with glibenclamide (5 mg kg(-1), i.v.). 2. In endothelium-intact or denuded aortic rings preconstricted with phenylephrine, KMUP-1 caused a concentration-dependent relaxation. This relaxation was reduced by endothelium removal, the presence of NOS inhibitor L-NAME (100 microM) and sGC inhibitors methylene blue (10 microM) and ODQ (1 microM). 3. The vasorelaxant effects of KMUP-1 was attenuated by pretreatment with various K(+) channel blockers TEA (10 mM), glibenclamide (1 microM), 4-AP (100 microM), apamin (1 microM) and charybdotoxin (ChTX, 0.1 microM). 4. Increased extracellular potassium levels (30 - 80 mM) caused a concentration-related reduction of KMUP-1-induced vasorelaxations. Preincubation with KMUP-1 (1, 10, 100 nM) increased the ACh-induced maximal vasorelaxations mediated by endogenous NO release, and enhanced the potency of exogenous NO-donor SNP. 5. The vasorelaxant responses of KMUP-1 (0.01, 0.05, 0.1 microM) together with a PDE inhibitor IBMX (0.5 microM) had an additive action. Additionally, KMUP-1 (100 microM) affected cyclic GMP metabolism since it inhibited the activity of PDE in human platelets. 6. KMUP-1 induced a dose-related increase in intracellular cyclic GMP levels in rat A10 vascular smooth muscle (VSM) cells, but not cyclic AMP. The increase in cyclic GMP content of KMUP-1 (0.1 - 100 microM) was almost completely abolished in the presence of methylene blue (10 microM), ODQ (10 microM), and L-NAME (100 microM). 7. In conclusion, these results indicate that KMUP-1 possesses the following merits: (1) stimulation of NO/sGC/cyclic GMP pathway and subsequent elevation of cyclic GMP, (2) K(+) channels opening, and (3) inhibition of PDE or cyclic GMP breakdown. Increased cyclic GMP display a prominent role in KMUP-1-induced VSM relaxations.

A new aspect of view in synthesizing new type beta-adrenoceptor blockers with ancillary antioxidant activities.

A series of vanilloid-type beta-adrenoceptor blockers derived from antioxidant traditional Chinese herbal medicines were synthesized and tested for their antioxidant and adrenoceptor antagonistic activities. They all possessed significant beta-adrenoceptor blocking activities under in vitro experiments and radioligand binding assays. In addition, some compounds were further examined in in vivo tests and produced antagonist effects matching that of propranolol and labetalol by measurements of antagonism toward (-)isoproterenol-induced tachycardia and (-)phenylephrine-induced pressor responses in anesthetized rats. Furthermore, all of the compounds had antioxidant effects inherited from their original structures. In conclusion, compound 11 had the most potent beta-adrenoceptors blocking activity, 12 and 13 possessed high cardioselectivity, whereas 14, 15 and 16 possessed additional alpha-adrenoceptor blocking activity and 15 is the most effective antioxidant of all. The antioxidant activity may be due to their alpha and beta unsaturated side chain at position 1 and ortho-substituted methoxy moiety on 4-phenoxyethylamine.

Cutaneous blood flow and adrenoceptor response increase in segmental-type vitiligo lesions.

It has been proposed that two types of vitiligo exist from the physiological and clinical points of view. Nonsegmental-type vitiligo is associated with autoimmune diseases while segmental-type vitiligo results from the dysfunction of sympathetic nerves in the affected area. Using laser Doppler flowmetry and iontophoresis for cutaneous microcirculatory assessments, we evaluated these two types of vitiligo in regard to their physiological changes. Ten patients with facial stable stage segmental-type vitiligo and ten stable nonsegmental-type vitiligo patients were selected for this study. Our results revealed that a nearly threefold increase in cutaneous blood flow was noticed in segmental-type vitiligo as compared to contralateral normal skin. In contrast, a 1.4-1.5 times difference was found among nonsegmental-type vitiligo, lesion side clinically normal skin and contralateral normal skin. There was a significant increase in cutaneous alpha- and beta-adrenoceptor response in segmental-type vitiligo lesions. However, no change in plasma catecholamines or adrenoceptor densities on blood cells was noticed. Our findings suggest that a dysfunction of the sympathetic nerves exists in the affected skin and plays a role in the pathogenesis of segmental-type vitiligo.

A unique xanthine derivative KMCP-98 with activation of adenosine receptor subtypes.

KMCP-98 is a newly synthesized adenosine receptor agonist by alkylation at the 7-position of the xanthines nucleus. We first investigated the pharmacological activities of KMCP-98 under in vivo and in vitro conditions. Acute intravenous injection of KMCP-98 (1.0, 2.0 and 3.0 mg/kg) produced a temporary fall in blood pressure and heart rate, followed by a sustained fall in heart rate in pentobarbital-anesthetized Wistar rats. The hypotensive and bradycardiac responses were inhibited by pretreatment with an A(1) adenosine receptor antagonist 8-phenyltheophylline (8-PT, 0.5 mg/kg). Both KMCP-98 and adenosine (0.3-100 microM) produced negative inotropic activity in isolated guinea pig left atria. The negative inotropic activity of KMCP-98 was significantly blocked by pretreatment with A(1) receptor antagonists 8-PT (10 microM) and xanthine amine congener (XAC, 10 microM), a nonselective adenosine antagonist theophylline (10 microM), a K(+) channel blocker tetraethylammonium (TEA, 10 mM) and a K(ATP) channel blocker glibenclamide (1 microM). KMCP-98 (0.03-30 microM) produced concentration-dependent relaxations in carbachol (1 microM) precontracted guinea pig tracheal smooth muscle. The trachea relaxant response of KMCP-98 was markedly inhibited by A(2), A(2a) and A(2b) adenosine receptor antagonists 3,7-dimethyl-1-propargylxanthine (DMPX, 10 microM), 8-(3-chlorostyryl)caffeine (CSC, 10 microM) and alloxazine (10 microM), respectively, the nitric oxide synthase (NOS) inhibitor L-NAME (100 microM) and also by TEA and glibenclamide. In addition, KMCP-98 (0.03-30 microM) elicited relaxant response in norepinephrine (3 microM) precontracted rat thoracic aorta in a concentration-dependent manner. The thoracic aorta relaxant response of KMCP-98 was also significantly inhibited by DMPX, CSC, alloxazine, L-NAME, TEA and glibenclamide. Furthermore, the binding characteristics of KMCP-98, adenosine and 5'-N-ethylcarboxaminoadenosine (NECA) were evaluated in [(3)H]DPCPX and [(3)H]CGS 21680 binding to rat cortex and striatum, respectively. The K(i) values of KMCP-98 for predominate A(1) and A(2) adenosine receptor sites were 3908+/-952 and 158+/-10 nM, respectively. In conclusion, KMCP-98 was found to be a xanthine-based adenosine receptor agonist associated cardiac depression, tracheal and aortic smooth muscle relaxations.

Lipid solubility of vasodilatory vanilloid-type beta-blockers on the functional and binding activities of beta-adrenoceptor subtypes.

Various vanilloid-type beta-adrenoceptor blockers were studied on guinea pig right atrium and trachea and rat colon. In addition, we also investigated their beta(1)-, beta(2)-, and beta(3)-adrenoceptor binding affinities. All these beta-adrenergic antagonists inhibited (-)isoproterenol-induced positive chronotropic effects of the right atrium and tracheal relaxation responses in a concentration-dependent manner. Some of these agents prevented the inhibition of rat colon spontaneous motility by (-)isoproterenol. Of the agents tested, we found that ferulidilol, eugenodilol, eugenolol, isoeugenolol, and ferulinolol, as well as propranolol and metoprolol, possessed beta(3)-adrenoceptor blocking activities, others were nearly without effectiveness. Furthermore, the binding characteristics of vanilloid-type beta-adrenergic antagonists were evaluated in [3H]CGP-12177, a beta(1)/beta(2)-adrenoceptor blocker and a beta(3)-adrenoceptor agonist, binding to beta(1)-, beta(2)-, and beta(3)-adrenoceptor sites in rat ventricle, lung, and interscapular brown adipose tissue (IBAT) membranes, respectively. Eugenodilol, eugenolol, metoprolol, isoeugenolol, and ferulinolol were less potent than both propranolol and ferulidilol in competing for the beta(3)-adrenoceptor binding sites. From the results of in vitro functional and binding studies, we suggested that propranolol, ferulidilol, eugenodilol, eugenolol, metoprolol, isoeugenolol, and ferulinolol all possessed beta(3)-adrenoceptor blocking activities. On the other hand, we also found that eugenodilol, eugenolol, metoprolol, isoeugenolol, and ferulinolol had a low lipid solubility in comparison with propranolol and ferulidilol. In conclusion, we proposed that beta(3)-adrenoceptor antagonistic actions of these vanilloid-type beta-blockers were positively correlated with their lipid solubility.

Expression and mutagenesis studies of cobrotoxin from Taiwan cobra.

The cDNA encoding cobrotoxin was constructed from the cellular RNA isolated from the venom glands of Naja naja atra (Taiwan cobra). The cDNA was subcloned into the expression vector pET20b(+) and transformed into BL21(DE3) Escherichia coli strain. Expressed cobrotoxin was isolated from inclusion bodies of E. coli and subjected to refolding into its folded structure. The refolded cobrotoxin was purified by high-performance liquid chromatography and exhibited a neurotoxicity in inhibiting acetylcholine-induced muscle contractions. Recombinant cobrotoxin showed a tendency to isomerize its disulfide bonds as that observed with native cobrotoxin. An appreciable decrease in the rate of isomerization reaction was observed when Glu-38 was replaced with Gln-38 or Lys-47 was replaced with Glu-47 or Gln-47. These results reflect that the element in controlling the disulfide isomerization of cobrotoxin is closely associated with the charged side chains in the cobrotoxin molecule.

Nitrated nonivamide displaying a drawback of proton's role in capsaicin-associated sensory and neuronal activities.

Nitrated nonivamide (NVANO)-induced triad hypotension, and biphasic bradycardia at 0.25-1.0 mg/kg (IV) was inhibited by capsazepine (1.0 mg/kg, IV), atropine (1.0 mg/kg, IV), and vagotomy in rats. NVANO also elicited a hypotensive spinal reflex at 5.0 mg/kg (IA). In the isolated rat vagus, NVANO (10.0-100.0 microM) revealed a sensory C-spike inhibition and membrane depolarization. NVANO (5.0 microM)-induced calcium influx in the isolated rat dorsal root ganglion cells (DRGs) was diminished by capsazepine (10.0 microM). In the isolated guinea pig atria, NVANO (1.0-50.0 microM)-induced positive inotropic and chronotropic activities were antagonized by capsazepine (1.0-10.0 microM) and human calcitonin gene-related peptide(8-37) (hCGRP(837); 0.1-1.0 microM).

Isoeugenolol: a selective beta1-adrenergic antagonist with tracheal and vascular smooth muscle relaxant properties.

Isoeugenolol (1.0, 3.0, 5.0 mg/kg, i.v.) produced a dose-dependent bradycardia and a decrease in blood pressure in anesthetized Wistar rats. Isoeugenolol inhibited the tachycardia effects induced by (-)isoproterenol, but had no blocking effect on the arterial pressor responses induced by (-)phenylephrine. In isolated guinea pig tissues, isoeugenolol antagonized (-)isoproterenol-induced positive inotropic and chronotropic effects on the atria and tracheal relaxations in a concentration-dependent manner. The apparent pA2 values for isoeugenolol on right atria, left atria and trachea were 7.63+/-0.03, 7.89+/-0.12 and 6.12+/-0.05, respectively, indicating that isoeugenolol was a highly selective beta1-adrenoceptor blocker. On the other hand, isoeugenolol produced a mild direct cardiac depression at high concentration and was without intrinsic sympathomimetic activity (ISA). In isolated rat thoracic aorta, isoeugenolol relaxed more potently the contractions induced by (-)phenylephrine (10 microM) and 5-HT (10 microM) than those by high K+ (75 mM). In isolated guinea pig trachea, isoeugenolol attenuated the carbachol (1 microM)-con-tracted trachea more significantly than those contracted with high K+. Furthermore, the binding characteristics of isoeugenolol and various beta-adrenoceptor antagonists were evaluated in [3H]CGP-12177 binding to rat ventricle, lung and interscapular brown adipose tissue (IBAT) membranes. The -log IC50 values of isoeugenolol for predominate beta1-, beta2- and beta3-adrenergic receptor sites were 5.82+/-0.09, 4.74+/-0.05 and 4.73+/-0.12, respectively. In conclusion, isoeugenolol was found to be a highly selective beta1-adrenoceptor antagonist with tracheal and vascular smooth muscle relaxant activities, but was devoid of alpha-adrenoceptor-blocking action.

Eugenodilol: a third-generation beta-adrenoceptor blocker, derived from eugenol, with alpha-adrenoceptor blocking and beta2-adrenoceptor agonist-associated vasorelaxant activities.

Eugenodilol, derived from natural eugenol, was first investigated with in vivo and in vitro models. In our in vivo study, eugenodilol (0.5, 1.0, and 1.5 mg/kg, i.v.) produced dose-dependent hypotensive and bradycardic responses in pentobarbital-anesthetized Wistar rats. Eugenodilol also inhibited the tachycardia and arterial pressor effects induced by (-)isoproterenol and phenylephrine, respectively. In our in vitro study, eugenodilol competitively antagonized (-)isoproterenol-induced positive inotropic and chronotropic effects and tracheal-relaxation responses on isolated guinea pig tissues in a concentration-dependent manner. The apparent pA2 values were 7.88+/-0.12 for right atria, 7.52+/-0.05 for left atria, and 7.33+/-0.15 for trachea, indicating that eugenodilol was a nonselective beta-adrenoceptor blocker. In thoracic aorta experiments, the apparent pA2 values of alpha-adrenoceptor blockade were 7.05+/-0.25 and 6.87+/-0.08 for eugenodilol and labetalol, respectively. In addition, eugenodilol produced cumulative relaxation responses on isolated guinea pig tracheal strips. The effects were competitively antagonized by ICI 118,551 (10(-8)-10(-6) M), a relatively selective beta2-adrenoceptor antagonist. In the radioligand-binding assay, the Ki values of [3H]CGP-12177 binding to rat ventricle and lung membranes were 9.72 and 48.29 nM, respectively, and the value of [3H]prazosin binding to rat brain membrane was 38.72 nM. These results further confirmed the alpha/beta-adrenoceptors-blocking activities of eugenodilol reported in the functional studies. We conclude that eugenodilol is a novel third-generation beta-adrenoceptor blocker with ancillary blocking activity at alpha-adrenoceptors and weak sympathomimetic activity at beta2-adrenoceptors.

A highly selective beta1-adrenergic blocker with partial beta2-agonist activity derived from ferulic acid, an active component of Ligusticum wallichii Franch.

Short-term injection of ferulinolol (0.1, 0.5, and 1.0 mg/kg, i.v.) produced dose-dependent bradycardia responses in pentobarbital-anesthetized Wistar rats, whereas it had no significant effects on the blood pressure. Ferulinolol markedly inhibited the tachycardia effects induced by (-)isoproterenol but did not show any blocking effect on the arterial pressor responses induced by (-)phenylephrine. These findings clearly suggested that ferulinolol had a beta-adrenergic blocking activity; nevertheless, it did not involve an alpha-adrenergic blocking action. In isolated guinea pig tissues, ferulinolol competitively antagonized (-)isoproterenol-induced positive inotropic and chronotropic effects of the atria and tracheal relaxation responses. The parallel shift to the right of the concentration-response curve of (-)isoproterenol suggested that ferulinolol was a beta-adrenoceptor-competitive antagonist. The apparent pA2 values for ferulinolol on right atria, left atria, and trachea were 7.62 +/- 0.05, 7.54 +/- 0.01, and 6.28 +/- 0.11, respectively. Ferulinolol was more potent on the atria than on tracheal tissues, demonstrating that it possessed beta1-adrenoceptor selectivity. The intrinsic sympathomimetic activity (ISA) of ferulinolol and propranolol were determined on isolated atria and trachea from reserpine-treated guinea pig. Propranolol caused significantly negative inotropic and chronotropic effects at > or =1 microM, whereas ferulinolol possessed fewer cardiodepressant activities than propranolol. In reserpine-treated tracheal strips, ferulinolol produced dose-dependent relaxant responses, but propranolol was without effectiveness. Preincubating the preparations with ICI 118,551 (0.1, 1.0, and 10 nM), a beta2-adrenoceptor antagonist, significantly shifted the concentration-relaxation curves of ferulinolol to a region of higher concentrations. These results implied that ferulinolol had a partial beta2-agonist activity. Further, binding characteristics of ferulinolol and various beta-adrenoceptor antagonists were evaluated in [3H]CGP-12177 binding to rat ventricular or lung membranes. The Ki values of ferulinolol, atenolol, metoprolol, and (-)propranolol were 103, 262, 123, and 0.23 nM, respectively, in ventricular membranes, and 2,412, 7,539, 2,186, and 0.72 nM, respectively, in lung membranes. In conclusion, ferulinolol was found to be a highly selective beta1-adrenoceptor antagonist with partial beta2-agonist activity but was devoid of alpha-adrenoceptor blocking action.

Characterization and cloning of long neurotoxin homolog from Naja naja atra.

The cDNA encoding a long neurotoxin homolog was constructed from the cellular RNA isolated fom the venom glands of Naja naja atra (Taiwan cobra) by reverse transcription-polymerase chain reaction. BLAST searches for sequence similarity in the GenBank databases reveal that the cDNA sequence of the long neurotoxin homolog is not highly homologous with long and short neurotoxins. Although the long neurotoxin homolog exhibited an activity to inhibit acetylcholine-induced muscle contractions as Naja naja atra cobrotoxin, the degree of inhibition caused by the addition of long neurotoxin homolog was only approximately 35% of that observed with the addition of cobrotoxin. Moreover, the primary structure of the long neurotoxin homolog did not fulfill the characteristic features of long or short neurotoxins. Together with long neurotoxin homologs from other snake species, they probably represent an evolutionary divergence between long and short neurotoxins.

Capsazocaine: a capsaicin-sensitive functional antagonist displays an argument on sensory capsaicin receptor.

1. Intravenous infusion of capsazocaine (CAPBZ), a molecular fusion product of irritant synthetic capsaicin and local analgesic benzocaine, at 100 micrograms/kg/min for 15 min inhibited capsaicin (10 micrograms/kg, IV)-induced spinal release of substance P-like immunoreactivity and vagus reflex responses in blood pressure and heart rate changes in rats. 2. Intrathecal perfusion of CAPBZ (1.0 nM) also reversed retrograde epigastric intraarterial capsaicin (10 micrograms/kg)-induced hypotensive spinal reflex. 3. In isolated guinea pig tissues, CAPBZ (1.0-100.0 microM) inhibited capsaicin (1.0 microM)-sensitive sensory and functional activities, including cardiatonic, bronchial, tracheal and ileal contractilities. CAPBZ is suggested to be a capsaicin antagonist.