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.

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.

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.

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.

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.

Vaninolol: a new selective beta 1-adrenoceptor antagonist derived from vanillin.

The beta-adrenoceptor blocking properties of vaninolol ((+/-)4-[4'-(2-hydroxy-3-tert-butyl-aminopropoxy)-3'-methoxyphenyl]- 3-buten-2-one), derived from vanillin, were first investigated under in vivo and in vitro conditions. Vaninolol (0.1, 0.5, 1.0 mg/kg, i.v.), as well as propranolol, produced a dose-dependent bradycardia response and a sustained pressor action in urethane-anesthetized normotensive rats. Vaninolol inhibited the tachycardia effects induced by (-)isoproterenol, but had no blocking effect on the arterial pressor responses induced by phenylephrine. These findings suggested that vaninolol possessed beta-adrenergic blocking activity, but was without alpha-adrenergic blocking activity. In isolated guinea-pig tissues, vaninolol antagonized (-)isoproterenol-induced positive inotropic and chronotropic effects of the atria and tracheal relaxation responses in a concentration-dependent manner. The parallel shift to the right of the concentration-response curve of (-)isoproterenol suggested that vaninolol was a beta-adrenoceptor competitive antagonist. The effect of vaninolol was more potent on the atria than on tracheal tissues, indicating it had some beta 1-adrenoceptor selectivity. On the other hand, the order of the hydrophilicity was atenolol >> vaninolol > propranolol. In addition, vaninolol had a mild direct cardiac depression at high concentrations and was without intrinsic sympathomimetic activity (ISA). Furthermore, binding characteristics of vaninolol and other beta-adrenoceptor antagonists were evaluated in [3H]dihydroalprenolol binding to guinea-pig ventricular membranes. The order of potency of beta-adrenoceptor antagonists in competing for the binding sites was (-)propranolol >> vaninolol > or = atenolol. In conclusion, vaninolol was found to be a selective beta 1-adrenoceptor antagonist with relatively low lipophilicity in comparison with propranolol, devoid of ISA, and had a mild myocardial depressant effect.

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 novel neurotoxin, cobrotoxin b, from Naja naja atra (Taiwan cobra) venom: purification, characterization, and gene organization.

A novel neurotoxin, cobrotoxin b, was isolated from Naja naja atra (Taiwan cobra) venom by successive chromatographies on gel filtration and SP-Sephadex C-25 columns. The yield of this novel toxin was 5% of that of cobrotoxin from the same venom. Its neurotoxicity determined as the inhibition of acetylcholine-induced muscle contractions was approximately 50% of that of cobrotoxin. Cobrotoxin b consists of 61 amino acid residues including 8 cysteine residues. Moreover, there are 12 amino acid substitutions between cobrotoxin b and cobrotoxin. The genomic DNA, with a size of 2,386bp, encoding the precursor of cobrotoxin b was isolated from the liver of N. naja atra. The gene consists of three exons separated by two introns. This exon/intron structure is essentially the same as that reported for the cobrotoxin gene. Moreover, the nucleotide sequences of the two neurotoxin genes exhibit 92% identity. These results highly suggest that the cobrotoxin b and cobrotoxin genes are derived from a common ancestor. Comparative analyses of cobrotoxin b and cobrotoxin precursors showed that the protein-coding regions of the exons are more diverse than introns, except for in the signal peptide domain. This indicates that the protein-coding regions may have arised via accelerated evolution. BLAST searches for sequence similarity in the GeneBank databases showed that intron 1 of the cobrotoxin b and cobrotoxin genes encodes a small nucleolar RNA (snoRNA). However, the snoRNA gene is absent from the gene encoding the Laticauda semifasciata erabutoxin c precursor (L. semifasciata and N. naja atra are sea and land snakes, respectively). Since previous studies suggested the potential mobility of snoRNA genes during evolution, we propose that intron insertions or deletions of snoRNA genes occurred with the evolutionary divergence between the sea snake and land snake neurotoxins.

Postweaning copper restriction and behavior in the Long-Evans rat.

A variety of behaviors was assessed in Long-Evans male rats placed on either a low copper diet, a marginal copper diet, or an adequate copper diet at weaning. Rats in the low copper group had slightly, but significantly, enlarged hearts and gained less weight than rats fed diets containing higher copper levels. Treatment effects were not detected in measurements of muricide, open-field activity, water intake, shock sensitivity, and shock avoidance and memory.

C-fiber-evoked autonomic cardiovascular effects after injection of Piper betle inflorescence extracts.

Piper betle inflorescence extracts contain eugenol (6.2%) and safrole (78.9%). Intravenous injections of water extracts of P. betle inflorescence (PBE), eugenol, and safrole in rats induced hypotensive and bradycardiac effects, whereas both intraarterial and intrathecal injections of PBE, eugenol and safrole resulted in hypotensive and tachycardiac effects. Moreover, the effects of intravenous injections of PBE were reversed or inhibited by the pretreatment with bilateral vagotomy, atropine (1 mg/kg, i.p.) and capsaicin (100 mg/kg, s.c.). Effects of intraarterial injections of PBE on blood pressure were inhibited by the pretreatment with substance P (SP) antagonist (1 nmol, i.t.) and clonidine (2.5 micrograms, i.t.), while heart rate was only inhibited by the pretreatment with SP antagonist (1 nmol, i.t.). In addition, the tachycardia resulting from intrathecal injections of PBE was inhibited by pretreatment with propranolol (0.3 mg/kg, i.v.). Eugenol and safrole induced the same pattern on blood pressure and heart rate changes as PBE in rats after various treatments. This report suggests that acute administration of betel inflorescence extracts by different routes may activate C-fiber-evoked parasympathetic and sympathetic cardiovascular reflexes in rats.

Vaninolol: a novel compound for the treatment of glaucoma and ischemic retinopathy.

Vaninolol has been confirmed as a selective beta 1-adrenergic blocking agent. The new compound was studied for its effects on intraocular pressure (IOP), the ocular blood flow and the retinal function in this report. Vaninolol showed marked delay in IOP recovery demonstrating that the agent possessed an ocular hypotensive action. This effect is equipotent or slightly more active than L-timolol. In addition, effects of vaninolol on the ocular blood flow of ocular hypertensive eyes were determined using the colored microspheres technique. It was found that vaninolol improved the ocular blood flow in ciliary body and retina, but not in iris and choroid. Further, vaninolol is able to improve the b-wave recovery in electroretinography significantly, indicating that it is possible to develop a new agent for the treatment of ischemic retinopathy.

Effects of xanthone glycoside on ephedrine-induced biting behavior and motor activity.

1-Hydroxy-3,4,7,8-tetramethoxyxanthone, beta-sitosterol, uvaol-3-palmitate, and sweroside have been isolated from the fresh whole plant of Tripterospermum lanceolatum (Hayata) Haraex Satake (Gentianaceae). Our results on the pharmacological studies of xanthone glycoside and lanceoside suggested that they have a CNS depressant effect.

Vanidil: a newly synthesized antiangina nonvolatile organic solid nitrate.

Newly synthesized Vanidil (4-o-(1,2-dinitroglyceryl)-6-nitrovanillic acid) is a nonvolatile organic solid nitrate, mp: 85 degrees C. In in vitro tests, Vanidil can inhibit 3,4-diaminopyridine (2 x 10(-2) M) induced pig coronary rhythmic vasocontraction, a prelude to coronary vasospasm, and nonrhythmic tonic vasocontraction. Vanidil is suggested as a potential antiangina agent.

A new beta-adrenoceptor blocking agent derived from dehydrozingerone.

1. Dehydrozingeronolol (DZPN; 0.1, 0.5, 1.0 mg/kg, i.v.) produced a dose-dependent bradycardia response and a sustained pressor action in urethane-anesthetized normotensive rats. DZPN inhibited the tachycardia effects by (-)isoproterenol, but had no blocking effect on the arterial pressor responses induced by phenylephrine. 2. In in vitro study, DZPN antagonized (-)isoproterenol-induced positive chronotropic effects in guinea-pig isolated right atria and relaxation responses in rat isolated uterus horns. 3. DZPN causes mild direct cardiac depression at high concentrations without intrinsic sympathomimetic activity (ISA). 4. The order of potency of beta-adrenoceptor antagonists in competing for the [3H]dihydroalprenolol binding sites was (-)propranolol >> DZPN > or = atenolol.

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.

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.

Vasomolol: an ultra short-acting and vasorelaxant vanilloid type beta 1-adrenoceptor antagonist.

The ultra-short-acting and vasorelaxant beta 1-adrenoceptor blocking activities of vasomolol, a guaiacoxypropanolamine derivative of vanillic acid ethyl ester, were studied. Vasomolol (0.5, 1.0, 3.0 mg/kg intravenously, i.v.) produced a dose-dependent bradycardia response and demonstrated particularly a hypotensive action with an ultra-short-acting property in pentobarbital-anesthetized normotensive rats. Vasomolol's steady state of beta-blockade was attained < or = 10 min after initial infusion, and a rapid recovery from blockade occurred after discontinuation of the infusion, although intravenous infusion of vasomolol (300 micrograms/kg/min) could not inhibit pressor responses induced by (-)phenylephrine (10 micrograms/kg i.v.). In isolated rat thoracic aorta, vasomolol (1-10 microM) inhibited vascular smooth muscle contractions induced by both (-)phenylephrine (10(-5) M) and high K+ (75 mM) concentration dependently. This inhibitory effect of vasomolol was more sensitive on K(+)-induced than on (-)phenylephrine-induced contractions, suggesting that the block of Ca2+ influx may involve the major mechanism of vasorelaxation. In isolated guinea pig tissues, vasomolol (0.01-10 microM) antagonized the (-)isoproterenol (ISO)-induced positive inotropic and chronotropic effects of the atria and tracheal relaxation responses in a concentration-dependent manner. The parallel shift to the right of the concentration-response curve of (-)ISO suggested that vasomolol was a beta-adrenoceptor competitive antagonist. The effect of vasomolol was more potent on atria than on tracheal tissues, indicating that it possesses beta 1-adrenoceptor selectivity. In addition, vasomolol did not show intrinsic sympathomimetic activity (ISA). Moreover, the binding characteristics of vasomolol were evaluated in [3H]dihydroalprenolol ([3H]DHA) binding to porcine ventricular membranes. Vasomolol was an ultra-short-acting and highly selective beta 1-adrenoceptor antagonist with vasorelaxant activity and is devoid of ISA.