European Journal of Pharmacology (v.513, #1-2)

Adhesion molecules, which play a crucial role in the development of atherogenesis, are produced by endothelial cells following stimulation with various inflammatory cytokines. The current studies examined the effect of a potent water-soluble antioxidant, protocatechuic aldehyde (derived from the Chinese herb, Salvia miltiorrhiza), on the expression of adhesion molecules in human umbilical vein endothelial cells (HUVECs) stimulated with tumor necrosis factor-α (TNF-α).Protocatechuic aldehyde appeared to specifically downregulate the TNF-α-induced cell surface expression of vascular adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1) on HUVECs as well as the release of soluble VCAM-1and ICAM-1 from HUVECs in a dose–response manner at pharmacologically relevant concentrations (0.15–1.35 mM). We also observed a dose-dependent lowering of mRNA expression of VCAM-1 and ICAM-1 in the presence of protocatechuic aldehyde. Furthermore, protocatechuic aldehyde (0.15, 0.45, and 1.35 mM) notably inhibited TNF-α-induced upregulation of U937 cell adhesion to HUVECs to 83.7%, 60.9%, and 40.8%, respectively.A gel shift assay further showed that protocatechuic aldehyde inhibited the TNF-α-activated NF-κB and AP-1 DNA binding activities in a dose-dependent manner. Collectively, these results indicate that protocatechuic aldehyde inhibits TNF-α-stimulated VCAM-1 and ICAM-1expression in HUVECs through a mechanism that involves NF-κB and AP-1.
Keywords: Protocatechuic aldehyde; Endothelial cell; Adhesion; NF-κB (nuclear factor-κB); AP-1 (activator protein-1);

We used antagonists of cGMP-phosphodiesterases to examine the role of cGMP for light-scattering oscillations and cAMP-induced Ca2+-influx in Dictyostelium discoideum, however, SCH 51866 (cis-5,6a,7,8,9,9a-hexahydro-2-[4-(trifluoromethyl)phenylmethyl]-5-methyl-cyclopent[4,5]imidazo[2,1-b]purin-4(3H)-one) and sildenafil citrate (1-[[3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1-H-pyrazolo[4,3-d]pyrimidin-5-yl)-4-ethoxyphenyl]sulfonyl]-4-methylpiperazine citrate) were poor inhibitors of cGMP-hydrolysis. Instead, SCH 51866 (IC50  = 16 μM) and sildenafil, blocked chemoattractant (cAMP)-induced Ca2+-influx as determined with a Ca2+-specific electrode. SCH 51866 (150 μM) affected neither spontaneous cGMP transients during light-scattering-oscillations nor cAMP-mediated K+-efflux. SCH 51866 and sildenafil are competitive inhibitors of cGMP phosphodiesterases. However, the activity of cGMP-dependent protein kinase Iα (PKGIα) was not altered by SCH 51866 (150 μM). By contrast, patch-clamp measurements of bovine cone cGMP-gated-channels (cyclic-nucleotide-gated-channel, CNGA3), stably expressed in human embryonic kidney cells, HEK 293 cells, revealed reversible, competitive and dose-dependent inhibition of sodium currents by SCH 51866 (IC50  = 25 μM) and sildenafil, but not by another inhibitor of cGMP-phosphodiesterases, UK 114,542. The possibility that D. discoideum cells also express a cGMP-regulated channel is supported by our finding that LY 83583 (6-(phenylamino)-5,8-quinolinedione) (35 μM), known to inhibit cyclic-nucleotide-gated-channels as well as guanylyl-cyclases, reduced cAMP-induced Ca2+-influx in D. discoideum, but did not affect cAMP-induced cGMP accumulation. Utilizing a PDED null strain that exhibits a prolonged and elevated cGMP transient following receptor activation, we found that the inhibition of Ca2+-influx by SCH 51866 in the wildtype was absent in the mutant. Our results show that SCH 51866 and sildenafil are antagonists of a Ca2+-permeable channel (CNGA3) and that both compete with cGMP for a regulatory site of Ca2+-influx in D. discoideum.
Keywords: SCH 51866; Sildenafil citrate; Cyclic-nucleotide-gated-channel; cGMP-dependent protein kinase Iα; K+-efflux;

Gene expression of energy and protein metabolism in hearts of hypertensive nitric oxide- or GSH-depleted mice by Helena Chon; Hans A.R. Bluyssen; Frank C.P. Holstege; Hein A. Koomans; Jaap A. Joles; Branko Braam (21-33).
Hypertension demands cardiac synthetic and metabolic adaptations to increased afterload. We studied gene expression in two models of mild hypertension without overt left ventricular hypertrophy using the NO synthase inhibitor nitro-l-arginine (l-NNA) and the glutathione depletor buthionine-S,R-sulfoximine (BSO). Mice were administered l-NNA, BSO, or water for 8 weeks. RNA of left ventricles was pooled per group, reverse transcribed, Cy3 and Cy5 labeled, and hybridized to cDNA microarrays. Normalized log2 Cy3/Cy5 ratios of ≥ 0.7 or ≤ −0.7 were considered significant. l-NNA and BSO both caused hypertension. Gene expression was regulated in cytoskeletal components in both models, protein synthesis in l-NNA-treated mice, and energy metabolism in BSO-treated mice. Energy metabolism genes shared several common transcription factor-binding sites such as Coup-Tf2, of which gene expression was increased in BSO-treated mice, and COMP-1. Characterization of the left ventricular adaptations as assessed with gene expression profiles reveals differential expression in energy and protein metabolism related to the pathogenetic background of the hypertension.
Keywords: Oxidative stress; Hypertension; Gene expression; Left ventricular hypertrophy; Energy metabolism;

Ligand binding and functional properties of human angiotensin AT1 receptors in transiently and stably expressed CHO-K1 cells by Minh Tam Le; Jean-Paul De Backer; László Hunyady; Patrick M.L. Vanderheyden; Georges Vauquelin (35-45).
Chinese Hamster Ovary Cells (CHO-K1) were transiently and stably transfected to express the human angiotensin AT1 receptor. Cell surface receptor expression was maximal 2 days after transient transfection. Their pharmacological and signalling properties differed from stably expressed receptors. Receptor reserve was significant in the transient cells but not in stable cells, explaining the higher potency of angiotensin II and the lower degree of insurmountable inhibition by candesartan in the transient cells. [Sar1Ile8]angiotensin II (sarile) is a potent angiotensin AT1 receptor antagonist for the stable cells but is a partial agonist, producing 19% of the maximal response by angiotensin II, in transient cells. Internalization of [3H]angiotensin II and [125I]sarile (i.e., acid-resistant binding) was more pronounced in stable cells. CHO-K1 cells were also transiently transfected with the enhanced green fluorescence-AT1 receptor gene. Confocal microscopy revealed rapid internalization induced by angiotensin II and sarile but not by candesartan. The above disparities may result from differences in receptor maturation and/or cellular surrounding.
Keywords: CHO-K1; Angiotensin II; Angiotensin AT1 receptor; Internalization; Receptor reserve;

Iron complexing activity of mangiferin, a naturally occurring glucosylxanthone, inhibits mitochondrial lipid peroxidation induced by Fe2+-citrate by Gilberto Pardo Andreu; René Delgado; Jesus A. Velho; Carlos Curti; Anibal E. Vercesi (47-55).
Mangiferin, a naturally occurring glucosylxanthone, has been described as having antidiabetic, antiproliferative, immunomodulatory and antioxidant activities. In this study we report for the first time the iron-complexing ability of mangiferin as a primary mechanism for protection of rat liver mitochondria against Fe2+-citrate induced lipid peroxidation. Thiobarbituric acid reactive substances and antimycin A-insensitive oxygen consumption were used as quantitative measures of lipid peroxidation. Mangiferin at 10 μM induced near-full protection against 50 μM Fe2+-citrate-induced mitochondrial swelling and loss of mitochondrial transmembrane potential (ΔΨ). The IC50 value for mangiferin protection against Fe2+-citrate-induced mitochondrial thiobarbituric acid reactive substance formation (9.02 ± 1.12 μM) was around 10 times lower than that for tert-butylhydroperoxide mitochondrial induction of thiobarbituric acid reactive substance formation. The xanthone derivative also inhibited the iron citrate induction of mitochondrial antimycin A-insensitive oxygen consumption, stimulated oxygen consumption due to Fe2+ autoxidation and prevented Fe3+ ascorbate reduction. Absorption spectra of mangiferin–Fe2+/Fe3+ complexes also suggest the formation of a transient charge transfer complex between Fe2+ and mangiferin, accelerating Fe2+ oxidation and the formation of a more stable Fe3+–mangiferin complex unable to participate in Fenton-type reaction and lipid peroxidation propagation phase. In conclusion, these results show that in vitro antioxidant activity of mangiferin is related to its iron-chelating properties and not merely due to the scavenging activity of free radicals. These results are of pharmacological relevance since mangiferin and its naturally contained extracts could be potential candidates for chelation therapy in diseases related to abnormal intracellular iron distribution or iron overload.
Keywords: Mangiferin; Lipid peroxidation; Mitochondria; Iron chelator;

Transient receptor potential vanilloid receptor-1 (TRPV1) is a sensory neuron-specific cation channel capable of integrating various noxious chemical and physical stimuli. The dog orthologue of TRPV1 was cloned using cDNA from nodose ganglia and heterologously expressed in HEK293OFF cells. At the amino acid level, dTRPV1 displays 85–89% sequence identity to other TRPV1 orthologues. Molecular pharmacological characterization of HEK293OFF cells expressing TRPV1 was assessed using a fluorescence imaging plate reader (FLIPR)-based calcium imaging assay. Dog TRPV1 was activated by various known TRPV1 agonists in a concentration-dependent manner: Ag23 = resiniferatoxin > olvanil ∼ arvanil > capsaicin > phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) >  N-oleoyldopamine (OLDA). In addition, select TRPV1 antagonists (capsazepine, I-resiniferatoxin and N-(-4-tertiarybutylphenyl)-4-(3-cholorpyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC)) were able to block the response of dTRPV1 to capsaicin. Furthermore, the dog TRPV1 lacked a conserved protein kinase A (PKA) phosphorylation site (117) found in other cloned orthologues, which may have physiological consequences on dog TRPV1 function. Taken together, these data constitute the first study of the cloning, expression and pharmacological characterization of dog TRPV1.
Keywords: TRPV1 (transient receptor potential vanilloid receptor-1); Cloning; Dog; Ganglia; FLIPR (fluorescence imaging plate reader); Capsaicin;

Residues stabilizing the heme moiety of the nitric oxide sensor soluble guanylate cyclase by Peter M. Schmidt; Christiane Rothkegel; Frank Wunder; Henning Schröder; Johannes-Peter Stasch (67-74).
Soluble guanylate cyclase, a heterodimer consisting of an α- and a heme-containing β-subunit, is the major receptor for the biological messenger nitric oxide (NO) and is involved in various signal transduction pathways. The heme moiety of the enzyme is bound between the axial heme ligand histidine105 and the recently identified counterparts of the heme propionic acids, tyrosine135 and arginine139. The latter residues together with an invariant serine137 form the unique heme binding motif Y-x-S-x-R. In this work, we show that replacement of the serine137 with alanine destabilizes the binding of the heme moiety and impairs NO-mediated soluble guanylate cyclase activation.
Keywords: Soluble guanylate cyclase; Heme; Nitric oxide; cGMP; BAY 58-2667; BAY 41-2272; YC-1;

The metabolism of tryptophan along the kynurenine pathway yields several neuroactive intermediates, including kynurenic acid, which is one of the few known endogenous N-methyl-d-aspartate receptor inhibitors; in parallel with this, it is an α7 nicotinic acetylcholinergic receptor antagonist. On the basis of these properties, kynurenic acid might therefore come into consideration as a therapeutic agent in certain neurobiological disorders. However, the use of kynurenic acid as a neuroprotective agent is practically excluded because kynurenic acid hardly crosses the blood–brain barrier. We recently synthetized a new compound, glucosamine-kynurenic acid, which is presumed to cross the blood–brain barrier more easily. In this study, the effects of systemically administered kynurenic acid and glucosamine-kynurenic acid on CA3 stimulation-evoked population spike activity in region CA1 of the rat hippocampus were compared. The effect of kynurenic acid or glucosamine-kynurenic acid was augmented by probenecid (200 mg/kg), which inhibits kynurenic acid excretion from the cerebrospinal fluid. The results showed that, while kynurenic acid administered i.p. or i.v. in doses of 17, 34, 68 or 136 μmol/kg did not cause any observable change in the animals, 136 μmol/kg glucosamine-kynurenic acid (either i.p. or i.v.) resulted in the sudden death of all the animals. The dose of 68 μmol/kg i.v., but not i.p., resulted in a sudden stoppage of breath, but the animals could be reanimated. As small a dose of glucosamine-kynurenic acid as 17 μmol/kg i.p. resulted in a reduction in population spike amplitudes; this effect was further augmented by probenecid, whereas neither 17 μmol/kg nor higher doses of pure kynurenic acid had a similar effect. The results presented here suggest that glucosamine-kynurenic acid passes the blood–brain barrier much more readily than does kynurenic acid.
Keywords: Kynurenic acid; Probenecid; Glucosamine-kynurenic acid; Neuroprotection; Hippocampus; (Rat);

Peripheral and spinal mechanisms of antinociceptive action of lumiracoxib by Jair Lozano-Cuenca; Gilberto Castañeda-Hernández; Vinicio Granados-Soto (81-91).
The possible participation of the nitric oxide (NO)-cyclic GMP-K+ channel pathway, serotonergic or opioidergic system on lumiracoxib-induced local or intrathecal antinociception was assessed in the formalin test. Local or intrathecal administration of lumiracoxib dose-dependently produced antinociception in the second phase of the test. Moreover, local or intrathecal pretreatment with N G-l-nitro-arginine methyl ester (l-NAME, NO synthesis inhibitor), 1H-(1,2,4)-oxadiazolo(4,2-a)quinoxalin-1-one (ODQ, guanylyl cyclase inhibitor), glibenclamide (ATP-sensitive K+ channel blocker), charybdotoxin and apamin (large- and small-conductance Ca2+-activated-K+ channel blockers, respectively) or margatoxin (voltage-dependent K+ channel blocker), but not N G-d-nitro-arginine methyl ester (d-NAME) or vehicle, significantly prevented lumiracoxib-induced antinociception. The intrathecal injection of methiothepin (serotonin receptor antagonist) reduced lumiracoxib-induced intrathecal antinociception. Local peripheral or intrathecal naloxone did not modify either local or intrathecal lumiracoxib-induced antinociception. Results suggest that lumiracoxib activates the NO-cyclic GMP-K+ channels to produce local and intrathecal antinociception. Data also suggest that lumiracoxib activates the intrathecal serotonergic system, but not opioid receptors either at peripheral or spinal sites.
Keywords: Lumiracoxib; K+ channel; NO-cyclic GMP pathway; Charybdotoxin; Apamin; Margatoxin; Serotonergic system; Opioid receptor;

Enhanced reactivity to vasopressin in rat basilar arteries during vasospasm after subarachnoid hemorrhage by Tsuyoshi Nishihashi; Cristina C. Trandafir; Aimin Wang; Xu Ji; Kazuyoshi Kurahashi (93-100).
Subarachnoid hemorrhage increases the plasma level of vasopressin, a well-known vasoconstrictor. We examined the sensitivity to vasopressin in rat basilar artery after subarachnoid hemorrhage using a rat subarachnoid hemorrhage model. Vasospasm was observed 1–2 days after subarachnoid hemorrhage induction, and the contractile response to vasopressin in rat basilar arteries was assessed. The concentration–response curve for vasopressin in subarachnoid hemorrhage (1 day) rats shifted leftward compared with that of control rats. The concentration–response curve for vasopressin V1 receptor agonist also shifted leftward and upward compared with that of control rats. The concentration–response curve for vasopressin was inhibited not by vasopressin V2 receptor antagonist but by vasopressin V1 receptor antagonist. Thus, it was demonstrated that the vasoconstricting effect of vasopressin was significantly enhanced in the vasospasm phase after subarachnoid hemorrhage.
Keywords: Subarachnoid hemorrhage; Rat basilar artery; Vasopressin; Vasopressin V1 receptor; Endothelium-dependent contraction;

Relaxations to oestrogen receptor subtype selective agonists in rat and mouse arteries by Khalid Al Zubair; Adibah Razak; Sotiria Bexis; James R. Docherty (101-108).
It has been recently reported that the oestrogen receptor α agonist PPT (4,4′,4ʺ-(4-propyl-[1H]-pyrazole-1,3,5-triyl) tris-phenol) is more potent than the oestrogen receptor β agonist DPN (2,3-bis(4-hydroxyphenyl)-propionitrile) at producing relaxations in rat mesenteric artery. We have investigated the relaxant actions of PPT and DPN in rat and mouse aorta and mesenteric artery. In rat aortic rings contracted with KCl (40 mM), the oestrogen receptor β agonist DPN produced significantly greater relaxations than the oestrogen receptor α agonist PPT. In wild-type (WT) mouse aorta, the same result was found, but in WT mouse mesenteric artery, as in rat mesenteric artery, DPN was significantly less potent than PPT in females but had similar potency to PPT in males. Relaxations to DPN also occurred in aorta from nitric oxide synthase-3-knockout (NOS-3-KO) mice, and in denuded aorta from both mouse and rat. Hence, in the mouse mesenteric artery, as in the rat mesenteric artery, PPT is at least as potent as DPN at producing relaxations; however, DPN was much more potent than PPT in the rat and mouse aorta. Effects of oestrogen receptor subtype selective agonists are tissue dependent. In addition, actions are largely endothelium-independent.
Keywords: Oestrogen receptor α; Oestrogen receptor β; Oestrogen receptor; PPT (4,4′,4ʺ-(4-propyl-[1H]-pyrazole-1,3,5-triyl) tris-phenol); DPN (2,3-bis(4-hydroxyphenyl)-propionitrile); Mouse aorta; Mouse mesenteric artery;

Evidence for two atypical conformations of beta-adrenoceptors and their interaction with Gi proteins by Iraídes N. Santos; Marie Sumitame; Viviane M. Caceres; Marilia F. Moreira; Marta H. Krieger; Regina C. Spadari-Bratfisch (109-118).
In this study, we investigated whether the responses of right atria from sinoaortic denervated rats to CGP12177 (4(3-t-butylamino-2-hydroxypropoxy benzidimidazole-2 one, hydrochloride)), isoprenaline and norepinephrine desensitized in parallel and whether CGP12177 interacted with distinct conformations of β-adrenoceptors. Right atria from rats 48 h after sinoaortic denervation were subsensitive to isoprenaline, norepinephrine and CGP12177. One week after sinoaortic denervation, the sensitivity to CGP12177 had recovered whereas the responses to isoprenaline and norepinephrine were still subsensitive, suggesting that the binding sites for these molecules showed independent behavior. In atria from 48 h sinoaortic-denervated rats, propranolol or 3 μM CGP20712A (2-hydroxy-5(2-((2-hydroxy-3-(4-((methyl-4-trifluormethyl)1H imidazole-2-yl)-phenoxypropyl) amino) ethoxy)-benzamide monomethane sulphonate)) blocked the responses to 10 nM–1 μM CGP12177 and steepened the curves. The concentration–response curves to CGP12177 in the presence of ICI118,551 (erythro-dl-1(-methylindan-4-yloxy)-3-isopropylamino-butan-2-ol) were biphasic, suggesting that CGP12177 interacted with at least two conformations of β-adrenoceptors that showed negative cooperativism, one acting through β2-adrenoceptor-Gi and the other via β1-adrenoceptor-Gs. This hypothesis was confirmed in right atria from sinoaortic-denervated rats treated with pertussis toxin.
Keywords: β1-Adrenoceptor; β4-Adrenoceptor; Sinoaortic denervation; CGP12177;

Formation of releasable NO stores by S-nitrosoglutathione in arteries exhibiting tolerance to glyceryl-trinitrate by Mamadou Sarr; Irina Lobysheva; Aminata S. Diallo; Jean-Claude Stoclet; Valérie B. Schini-Kerth; Bernard Muller (119-123).
S-Nitrosating nitric oxide (NO) donors like S-nitrosoglutathione (GSNO) induce a persistent inhibition of vascular tone, through the formation of releasable NO stores. In this study, we investigate whether GSNO also induces NO stores-related effects in vessels exhibiting tolerance to glyceryl-trinitrate. Rat aortic rings treated with glyceryl-trinitrate (100 μM for 1 h) exhibited increased level of superoxide and a decrease in NO elevation and relaxation induced by subsequent addition of glyceryl-trinitrate. In glyceryl-trinitrate-treated rings as in controls, pre-exposure to GSNO (1 μM for 30 min) induced a persistent hyporesponsiveness to noradrenaline and a relaxant response to N-acetylcysteine (a low molecular weight thiol which can displace NO from NO stores), both of which being inhibited by guanylyl-cyclase or cyclic GMP-dependent protein kinase inhibitors. These data indicate that GSNO can promote the formation of releasable NO stores in arteries exhibiting increased superoxide level and tolerance to glyceryl-trinitrate. Formation of releasable NO stores is of potential interest to restore the protective effect of NO in organic nitrate-tolerant blood vessels.
Keywords: N-Acetylcysteine; Nitric oxide; Nitric oxide store; Organic nitrate; Superoxide anion; Tolerance;

In this study we investigated the ability of aerosolized substance P to induce either cough or bronchoconstriction in guinea-pigs. We have also examined whether pre-treatment, by the inhaled route, of animals with a combination of the neutral endopeptidase inhibitor, phosphoramidon (10−3 M), and the diaminopeptidase IV inhibitor, diprotin A (10−3 M), enhances the airway response to substance P. Moreover, we also assessed whether aerosol pre-treatment of guinea-pigs with either substance P or bradykinin, at 10−4 M, affects the citric acid-induced cough and/or bronchoconstriction. Challenge of guinea-pigs with substance P only at 10−3 M resulted in significant bronchconstriction but only a weak and variable cough response (1.1±0.6; P>0.05). Pre-treatment of guinea-pigs with both phosphoramidon and diprotin A resulted in a small non-significant increase in the cough response (2.8±0.9 vs. 1.1±0.6; P>0.05) but significantly enhanced substance P-induced bronchoconstriction (P<0.05). Moreover, exposure of guinea-pigs to substance P (10−4 M) prior to citric acid challenge (0.6M) resulted in a significant (P<0.05) enhancement of the citric acid-induced bronchoconstriction but not the citric acid-induced cough (11.7±1.8 vs. 12.8±1.5; P>0.05). In contrast, exposure of guinea-pigs to bradykinin (10−4 M) prior to the citric acid challenge resulted in a significant enhancement of the cough response (9.2±1.9 vs. 25.8±2.5; P<0.05) but not the bronchoconstriction (P>0.05). These data do not support a major peripheral role for substance P in the cough reflex, although bradykinin is able to sensitize the cough reflex. Furthermore, these data suggest that bronchoconstriction, induced by citric acid, is not responsible for the cough associated with this irritant.
Keywords: Substance P; Bradykinin; Citric acid and cough;

To clarify the function of P2 receptor subtypes in mouse stomach, the motor responses to ATP, α,β-methyleneATP (α,β-MeATP), P2X receptor agonist, 2-methylthioATP (2-MeSATP), P2Y receptor agonist, and the effects of the desensitisation of P2X receptors with α,β-MeATP and of P2Y receptors with ADPβS were analysed recording the endoluminal pressure from whole-organ. ATP-induced relaxation was antagonised by suramin, non-selective P2 receptor antagonist, by desensitisation of P2Y receptors with ADPβS, and increased by desensitisation of P2X receptors with α,β-MeATP. α,β-MeATP produced biphasic responses: relaxation, reduced by P2X- or P2Y desensitisation, and contraction, almost abolished by P2X desensitisation and potentiated by P2Y desensitisation. 2-MeSATP induced relaxation, which was antagonised by P2Y desensitisation and increased by P2X desensitisation. Tetrodotoxin increased the relaxation induced by purines and deeply antagonised the contraction to α,β-MeATP. Our results suggest that in mouse stomach are present muscular P2Y receptors, subserving relaxation, and neuronal presynaptic P2X receptors, mediating contraction.
Keywords: ATP; P2X receptor; P2Y receptor; Mouse stomach; Contraction; Relaxation;

γ-Globulin-induced modulation with necrotic-like morphology of peripheral blood neutrophils by Kenichi Sugita; Junichi Hirao; Osamu Arisaka; Mitsuoki Eguchi (141-144).
To determine the effect of intravenous immunoglobulin-administration on neutrophil function, we obtained neutrophils from patients with an acute phase of Kawasaki disease. In vitro IgG-induced modulation of neutrophils into Annexin-V-positive and propidium iodide-negative cells was observed in 20 of 28 patients in the presence of more than 300 μg/ml IgG and showed necrosis-like changes in morphologic features. However, we could not find any patients showing promotion of the sub-G1 cell fraction on DNA content analysis. The modulatory effect of in vitro IgG was not observed in neutrophils from healthy volunteers and was significantly correlated with the antifebrile effect of in vivo IgG.
Keywords: Neutrophils; Apoptosis; Necrotic morphological change; IgG; Kawasaki disease;

In vivo effects of histidine and carnosine against diabetic deterioration in diabetic Balb/cA mice were studied. Histidine and carnosine at 0.5, 1 g/l were added into drinking water. After 4 weeks intake of these agents, the content of histidine and carnosine in plasma, heart and liver significantly elevated (P<0.05). The intake of these agents significantly decreased plasma glucose and fibronectin levels (P<0.05); however, only 1 g/l histidine and carnosine treatments significantly increased insulin level (P<0.05) in diabetic mice. Triglyceride level in heart and liver was dose-dependently reduced by histidine or carnosine treatments (P<0.05); however, only 1 g/l histidine and carnosine treatments significantly reduced cholesterol level in heart and liver (P<0.05). The administration of histidine or carnosine significantly enhanced catalase activity and decreased lipid oxidation levels in kidney and liver (P<0.05); however, only 1 g/l histidine and carnosine treatments significantly increased glutathione peroxidase activity (P<0.05). The increased interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha in diabetic mice were significantly suppressed by the intake of histidine or carnosine (P<0.05). In human low density lipoprotein, histidine or carnosine showed dose-dependently suppressive effect in glucose-induced oxidation and glycation (P<0.05). These data suggest that histidine and carnosine are potential multiple-protective agents for diabetic complications prevention or therapy.
Keywords: Diabetes; Histidine; Carnosine; Hyperlipidemia; Cytokine;

The present study used thyroidectomized insulin-resistant, type 2 diabetic Goto–Kakizaki (GK) rats to assess whether insulin resistance and hypothyroidism modulate ovarian physiology. Animals were treated with daily injections of 5 IU equine chorionic gonadotropin for 5 days starting 1 week after thyroidectomy. Control groups included rats of GK and control (Wistar) strains treated only with equine chorionic gonadotropin or thyroidectomy, or with no treatment (intact). In Wistar rats, equine chorionic gonadotropin injections tended to increase the serum concentrations of luteinizing hormone (LH) and testosterone more in the thyroidectomy group than in intact rats. Similar changes in LH and testosterone were observed in the thyroidectomy + equine chorionic gonadotropin and equine chorionic gonadotropin groups of GK rats, but the LH and testosterone levels in the thyroidectomy + equine chorionic gonadotropin group were significantly higher in GK rats. Expression of ovarian LH receptor messenger RNA (mRNA) was enhanced by thyroidectomy. The LH receptor mRNA levels were significantly higher in the thyroidectomy + equine chorionic gonadotropin group of GK rats than in the corresponding group of control rats. These results indicate that hypothyroidism in animals with insulin resistance and type 2 diabetes promotes LH and testosterone secretions, and suggests that the enhanced-testosterone levels is partially mediated by the enhancement of LH receptor expression and an increase in the serum level of LH.
Keywords: Insulin resistance; Ovarian hormone; The luteinizing hormone receptor;

Combined effect of probucol and insulin on cataracts of diabetic rats fed a high cholesterol diet by Masumi Yoshida; Hitoshi Kimura; Kouhei Kyuki; Mikio Ito (159-168).
We investigated the effects of long-term treatment with probucol, a hypolipidemic agent with antioxidative action, insulin, or their combination on cataracts of streptozotocin-induced diabetic rats fed a high cholesterol diet. Each rat was checked for cataracts at 0, 1, 2, 4, 8, 12 and 15 weeks after streptozotocin injection. Cataracts were observed from 8 weeks in untreated hypercholesterolemic and diabetic rats and the incidence of catarats increased to 100% by 15 weeks. The incidence of cataracts in rats treated with probucol, insulin and their combination was first seen at 12, 12 and 15 weeks, respectively, and was 86%, 63% and 33%, respectively, at 15 weeks.The preventive effects of both agents alone and their combination on the cataracts were confirmed by histopathological evaluation of eyeballs. The combined treatment with both agents markedly improved hyperglycemia, hyperlipidemia and increased serum lipid peroxide levels. These results indicate that the combined treatment with probucol and insulin is useful in preventing the development and progression of diabetic cataracts.
Keywords: Diabetes; Cataract; Hyperglycemia; Hyperlipidemia; Lipid peroxide;