Influence of oxytetracycline susceptibility as a first-line antibiotic on the clinical outcome in dairy cattle with acute Escherichia coli mastitis.

The purpose of this study was to clarify the therapeutic effects of oxytetracycline (OTC) as a first-line antibiotic in cattle with acute Escherichia coli mastitis and systemic signs. Drug susceptibility was determined by the minimum inhibitory concentration (MIC) of E. coli isolated from cows with acute E. coli mastitis (n=38). Cattle were divided into OTC-susceptible (S, n=30) and OTC-resistant (R, n=8) groups. They were further subdivided according to susceptibility to the antibiotic used as a second treatment, into susceptible-susceptible (SS, n=30), resistant-susceptible (RS, n=5), and resistant-resistant (RR, n=3) groups. Clinical signs on the day after initial treatment were compared between S and R groups as short-term indicators of treatment effects. The 28-day survival rate of cattle was then compared among SS, RS, and RR groups as a long-term indicator of treatment effects. There were no differences in clinical signs between S and R groups on the day after the first dose, but the 28-day survival rate was significantly greater in the SS group than in the RR group (P=0.04). The results demonstrated that an effective drug is essential for first-line treatment of acute coliform mastitis. However, anticipating the effectiveness of a first-line antibiotic based on clinical symptoms at the second day of treatment is impossible. It is important to build a picture of drug resistance trends in cattle herds for empirical selection of antibiotics to be administered.

Intranasal administration of vitamin D attenuates blood-brain barrier disruption through endogenous upregulation of osteopontin and activation of CD44/P-gp glycosylation signaling after subarachnoid hemorrhage in rats.

In this study, we investigated the role of vitamin D3 (VitD3) on endogenous osteopontin (OPN), a neuroprotective glycoprotein, after subarachnoid hemorrhage (SAH). The endovascular perforation SAH model in Sprague-Dawley rats was used to study the effect of intranasal VitD3 (30 ng/kg) before (Pre-SAH + VitD3) and after (Post-SAH + VitD3) subarachnoid hemorrhage. Vitamin D3 (30, 60, 120 ng/kg/day) increased more than one fold endogenous OPN expression in astrocytes and endothelial cells of rat brain. Vitamin D3 significantly decreased brain edema and Evans blue extravasation. In addition, neurobehavioral scores were significantly higher in Pre-SAH + VitD3, but partly higher in Post-SAH + VitD3, group compared with SAH group. These protective effects of vitamin D3 were completely attenuated by intracerebroventricular injection of transcription inhibitor Actinomycin D and significantly inhibited by small interfering ribonucleic acid (siRNA) for vitamin D receptor and OPN in Pre-SAH + VitD3 rats. OPN expression was significantly higher in Pre-SAH + VitD3 rats, specifically A and C, but not B, isomers were upregulated in the astrocytes, leading to CD44 splicing, and P-gp glycosylation in brain endothelial cells. The results show that intranasal vitamin D3 attenuates blood-brain barrier (BBB) disruption through endogenous upregulation of OPN and subsequent CD44 and P-gp glycosylation signals in brain endothelial cells. Furthermore, this study identifies a novel strategy for the cost-effective management of subarachnoid hemorrhage.

Effects of Krill-derived phospholipid-enriched n-3 fatty acids on Ca(2+) regulation system in cerebral arteries from ovariectomized rats.

AIMS: To investigate the effects of n-3 polyunsaturated fatty acids on cerebral circulation, ovariectomized (OVX) rats were administered with phospholipids in krill oil (KPL) or triglycerides in fish oil (FTG); effects on the Ca(2+) regulating system in their basilar artery (BA) were then analyzed. MAIN METHODS: The rats were divided into 4 groups: control, OVX, OVX given KPL (OVXP), and OVX given FTG (OVXT) orally, daily for 2weeks. Time dependent relaxation (TDR) of contractile response to 5HT in BA was determined myographically, Na(+)/Ca(2+) exchanger (NCX) 1 mRNA expression was determined by real time PCR, and nucleotides were analyzed by HPLC. KEY FINDINGS: The level of TDR in OVX that was significantly lower in the control was inhibited by l-NAME and indomethacin; TEA inhibited TDR totally in the control but only partly in OVXP and OVXT. Relaxation induced by the addition of 5mM KCl to the BA pre-contracted with 5-HT was inhibited by TEA in the controls, OVXP and OVXT, but not in OVX. Overexpression of NCX1 mRNA in the BA from OVX was significantly inhibited by FTG. The ratio of ADP/ATP in cerebral arteries from OVX was significantly inhibited by KPL and FTG. Levels of triglyceride and arachidonic acid in the plasma of OVX increased, but were significantly inhibited by KPL and FTG. SIGNIFICANCE: Ovarian dysfunction affects Ca(2+) activated-, ATP-sensitive-K(+) channels and NCX1, which play crucial roles in the autoregulation of cerebral blood flow. Also, KPL may become as good a supplement as FTG for postmenopausal women.

Emulsification of coenzyme Q10 using gum arabic increases bioavailability in rats and human and improves food-processing suitability.

We evaluated the characteristics of a coenzyme Q(10) (CoQ(10)) formulation created with gum arabic. We defined the formulation's "modulus of inclusion," a reference index of the emulsified state, as the CoQ(10) not extracted by hexane as a percentage of the total CoQ(10) content of the formulation. The emulsified CoQ(10) formulation had a smaller particle size and larger modulus of inclusion value than the equivalent unemulsified formulation. In a kinetic study in rats, serum CoQ(10) levels were significantly greater with the emulsified CoQ(10) formulation than with the equivalent unemulsified formulation, which barely increased the levels. In a human study, oral intake of the emulsified formulation significantly increased plasma CoQ(10) levels, which peaked 6 h after intake, compared with the equivalent unemulsified formulation or CoQ(10) bulk powder. There was a significant positive correlation between baseline plasma CoQ(10) and total cholesterol levels, but no correlation was observed between absorption of CoQ(10) and baseline CoQ(10) levels. The emulsified CoQ(10) formulation was highly stable against heat and high humidity and in the presence of some materials (magnesium oxide, vitamin C, and vitamin E). In conclusion, emulsification of CoQ(10) using gum arabic increased bioavailability in both rats and humans and improved suitability for food processing.

Characterization of vasoconstrictor-induced relaxation in the cerebral basilar artery.

The vascular endothelium regulates vascular smooth muscle functions by releasing endothelium-derived vasoactive substances. To identify physiological mechanisms mediating the inhibitory effect of the endothelium on vasoconstrictors, the basilar arteries isolated from Wistar rats were used in an organ bath study. In the intact basilar artery (with endothelium), 100 nM serotonin (5-HT) induced phasic contraction (28.7+/-4.1% of 60 mM KCl-induced contraction) followed by profound time-dependent relaxation at 3 min (3.8+/-0.4%). In the denuded artery (without endothelium), the 5-HT-induced contraction was enhanced (51.7+/-16.1%), while the relaxation was abolished. In the intact basilar artery, the contraction was facilitated and the amplitude of the phasic contraction was significantly enhanced (70.1+/-10.3%), but time-dependent relaxation was still manifested at 3 min (25.7+/-10.0%) in the presence of Nomega-nitro-L-arginine methyl ester hydrochloride (L-NAME) and indomethacin. Time-dependent relaxation induced by 5-HT was abolished in Ca(2+)-free and in K(+)-free Krebs-Henseleit buffer (KHB). Furthermore, the 5-HT-induced contraction was enhanced by treatment with ouabain (105.6+/-11.8%), tetraethylammonium chloride (133.2+/-7.9%), charybdotoxin with apamin (145.4+/-6.4%) or BaCl(2) (72.2+/-13.8%) at 3 min; also, time-dependent relaxation was abolished by these blockers in the presence of L-NAME and indomethacin. U46619 (100 nM) induced sustained contraction without time-dependent relaxation in normal KHB, but charybdotoxin with apamin did not affect the contraction. The results suggest that time-dependent relaxation is modulated by endothelial sodium-potassium pump (Na(+)/K(+)-ATPase) and Ca(2+)-activated K(+) channel (K(Ca)) activity, especially small- and intermediate-conductance K(Ca)-prominent ionic mechanisms of the so-called endothelium-derived hyperpolarizing factor.

Stationarity of the heart rate variability by acceleration plethysmography: short-term measurements of healthy young males in daily life.

The acceleration plethysmography (APG) is a useful tool to analyze the heart rate variability (HRV) with the merits of being portable and simple in operation. To use it clinically, 'stationarity' of the HRV measurements in resting state should be justified as a fundamental premise. Therefore, the purpose of this research was to investigate the stationarity of the short-term HRV derived from APG in resting state in daily life. The sample size was determined on the basis of alpha = 0.05 and beta = 0.20. The HRV measurements of 44 healthy male subjects were done twice during a one-week period by the APG system. The HRV parameters were examined by the assessments of distribution, equality of the mean and variance and internal consistency reliability. Concerning spectral components represented as proportion, normalized units and the logarithm of the rate of each component, the normal distribution and the homogeneity of variance in two measurements were confirmed. Significant difference was not found between the mean values in two measurements. The internal consistency reliability evaluated by Cronbach's alpha of 0.6-0.7 was acceptable for every parameter. It is concluded that for healthy young males, the parameters of short-term HRV derived from APG measured in resting state in the same time zone in daily life remain stationary within a certain range regardless of the measurement day.

Characteristics of contractile activity in the renal artery of ovariectomized rats.

The incidence of cardiovascular disease is markedly lower in cycling, pre-menopausal women and post-menopausal women receiving estrogen than in men or untreated post-menopausal women. Clinical studies demonstrate a protective role of estrogen in hormone replacement therapy in terms of reducing cardiovascular risk. However, the benefits of hormone replacement therapy in cardiovascular disease remain unclear. We investigated the effects of estrogen on the contractile responses of the renal artery of ovariectomized Wistar rats (OVX) compared to both ovariectomized 17 beta-estradiol-treated rats (OVXE) and sham-operated (control) rats. Isometric contraction of renal artery was recorded with a strain gauge transducer. The maximum contractile response of the renal artery smooth muscle to KCl (80 mM) in the OVXE group was significantly higher than that in both the control and OVX groups. The phenylephrine (PE) concentration-response curves in all three groups indicated a greater sensitivity at lower concentrations of PE following treatment with 100 microM L-arginine methyl ester (L-NAME). The EC50 values for PE in the three groups were 2 times lower in the presence of L-NAME than those lacking exposure to L-NAME. The EC50 value for PE in the OVX group was approximately 3 times lower in the presence of L-NAME than in those lacking exposure to L-NAME and 100 nM BMY 7378, an alpha 1D-adrenoceptor antagonist. The rate of relaxation of the PE-induced contraction (T1/2) was significantly reduced in the OVX group relative to both the control and OVXE groups. T1/2 values after treatment with 100 microM L-NAME were slower than those lacking exposure to L-NAME in all groups. Further, the T1/2 value of the OVX group was 2 times greater than that of the control; this change was reversed in the OVXE group. In conclusion, our results suggest that estrogen regulates contraction and relaxation in the renal artery via NO synthase activity and alteration of the Ca2+ transport systems.

A novel approach for the determination of contractile and calcium responses of the basilar artery employing real-time confocal laser microscopy.

INTRODUCTION: Intracellular calcium concentration ([Ca(2+)](i)) modifications in endothelial and smooth muscle cells represent a key element in the pathogenesis of cerebral artery vasospasm. Therefore, the present study documented potential application of confocal laser microscopy in the determination of contractile and [Ca(2+)](i) responses in basilar artery. METHODS: Experiments were performed on the rat isolated basilar artery. Changes in [Ca(2+)](i) were determined by ratiometry involving Fluo-4/AM and Fura Red/AM. Contractile function was calculated from the change in fluorescent area by Fluo-4/AM. RESULTS: KCl (50 mM) elicited an increase in [Ca(2+)](i) and contraction in basilar artery; moreover, nearly well maintained responses were evident for at least 120 min following the first application. 10 microM 5-hydroxytryptamine (5-HT), 10 microM alpha,beta-methyleneadenosine 5'-triphosphate (alpha,beta-meATP) and 10 nM vasopressin (VP) also induced increases in [Ca(2+)](i) and contraction dose-dependently. Additionally, 10 microM acetylcholine elicited a transient [Ca(2+)](i) decrease and sustained relaxation. In individual cells, rhythmical changes in [Ca(2+)](i) were observed after 10 microM 5-HT. VP (10 nM) evoked modest Ca(2+) oscillation in individual cells; however, Ca(2+) oscillation was not detectable with 10 microM alpha,beta-meATP. DISCUSSION: These results indicate that this method offers reproducibility and quantifiable effects. Imaging technology may therefore be applied to the estimation of [Ca(2+)](i) responses at the tissue level as well as at the level of the individual cell. Thus, confocal laser microscopy is a suitable tool for estimation of small artery function.

Histamine type 1 receptor deficiency reduces airway inflammation in a murine asthma model.

BACKGROUND: Histamine plays an important role in immediate and late immune responses. The histamine type 1 (H1) receptor is expressed on several immune cell populations, but its role in a murine model of asthma remains unclear. The present study evaluated the role of histamine H1 receptors in airway allergic inflammation by comparing the development of bronchial asthma in histamine H1 receptor gene knockout (H1RKO) and wild-type mice. METHODS: H1RKO and wild-type mice were sensitized by intraperitoneal injection of ovalbumin (OVA) or saline, and then challenged with aerosolized OVA or saline. Ventilatory timing in response to inhaled methacholine was measured, and samples of blood, bronchoalveolar lavage, and lung tissues were taken 24 h after the last OVA challenge. RESULTS: OVA-treatedwild-type mice showed significantly increased airway eosinophilic infiltration, and airway response to methacholine compared to OVA-treated H1RKO mice. The serum level of immunoglobulin E and levels of interleukin (IL)-4, IL-5, IL-13, and TGF-beta1 in bronchoalveolar lavage fluid were lower in OVA-treated H1RKO mice than in OVA-treated wild-type mice, but there was no significant difference in interferon-gamma expression. Overall, deletion of histamine H1 receptors reduced allergic responses in a murine model of bronchial asthma. CONCLUSION: Histamine plays an important role via H1 receptors in the development of T helper type 2 responses to enhance airway inflammation.

Current topics in pharmacological research on bone metabolism: inhibitory effects of bisphosphonates on the differentiation and activity of osteoclasts.

Despite the extensive use of bisphosphonates (BPs) in the treatment of metabolic bone diseases associated with increased osteoclastic bone resorption, the precise mechanism of their action on bone metabolism is still unclear. To clarify at which stages of osteoclast differentiation and activation that BPs influence, we examined the osteoclasts generated from mononuclear precursors and osteoclasts in the calvaria by laser scanning confocal microscopy. The studies showed that BPs inhibit lipopolysaccharide- or parathyroid hormone-induced osteoclast differentiation, fusion, attachment, actin ring formation, and activation and that both beta3 integrin and osteopontin have an important role in cytoskeletal rearrangements associated with cell attachment and resorption in osteoclasts.

Functional differences of Na+/Ca2+ exchanger expression in Ca2+ transport system of smooth muscle of guinea pig stomach.

The plasma membrane ATP-dependent Ca2+ pump and the Na+/Ca2+ exchanger (NCX) are the major means of Ca2+ extrusion in smooth muscle. However, little is known regarding distribution and function of the NCX in guinea pig gastric smooth muscle. The expression pattern and distribution of NCX isoforms suggest a role as a regulator of Ca2+ transport in cells. Na+ pump inhibition and the consequent to removal of K+ caused gradual contraction in fundus. In contrast, the response was significantly less in antrum. Western blotting analysis revealed that NCX1 and NCX2 are the predominant NCX isoforms expressed in stomach, the former was expressed strongly in antrum, whereas the latter displayed greater expression in fundus. Isolated plasma membrane fractions derived from gastric fundus smooth muscle were also investigated to clarify the relationship between NCX protein expression and function. Na+-dependent Ca2+ uptake increased directly with Ca2+ concentration. Ca2+ uptake in Na+-loaded vesicles was markedly elevated in comparison with K+-loaded vesicles. Additionally, Ca2+ uptake by the Na+- or K+-loaded vesicles was substantially higher in the presence of A23187 than in its absence. The result can be explained based on the assumption that Na+ gradients facilitate downhill movement of Ca2+. Na+-dependent Ca2+ uptake was abolished by the monovalent cationic ionophore, monensin. NaCl enhanced Ca2+ efflux from vesicles, and this efflux was significantly inhibited by gramicidin. Results documented evidence that NCX2 isoform functionally contributes to Ca2+ extrusion and maintenance of contraction-relaxation cycle in gastric fundus smooth muscle.

A traditional herbal medicine, rikkunshi-to (TJ-43), prevents intracellular signaling disorders in gastric smooth muscle of diabetic rats.

Prevention of diabetic gastrointestinal dysfunction is of utmost importance. The present study demonstrated that diacylglycerol kinase (DGK) activity in diabetic gastric smooth muscle in the resting state was approximately 3.5-fold greater than that in controls. However, oral administration of TJ-43 (1% of food intake) or subcutaneous insulin injection (12 units/kg/day) in streptozotocin-induced diabetic rats (DM) for 2 weeks prevented DGK abnormalities based on the control level. Increased DGK activity in the resting state of DM was inhibited significantly by R59022, neomycin or staurosporine; in contrast, these drugs did not affect DGK activity in controls, insulin-treated DM or TJ-43-treated DM. In controls, the endogenous phosphatidic acid (PA) level was inhibited significantly by R59022 or neomycin but not affected by staurosporine. On the other hand, these three drugs significantly inhibited endogenous PA levels in DM, and neomycin significantly inhibited endogenous PA levels in insulin-treated and TJ-43-treated DM. This suggests that TJ-43 could prevent alteration of DGK activity and PA formation without reduction of blood glucose levels. Moreover, these effects were greater than those of insulin treatment. Results suggested that TJ-43 treatment influenced the hyperreactivity of DGK and DAG formation via phospholipase C activity. In conclusion, TJ-43 can be recommended with respect to enhancement of the quality of life in patients displaying diabetic gastrointestinal complications.

Novel diacylglycerol kinase inhibitor selectively suppressed an U46619-induced enhancement of mouse portal vein contraction under high glucose conditions.

1. Diacylglycerol kinase (DG kinase) is a key enzyme in vascular contraction; however, alterations of the regulatory mechanisms in vascular dysfunction are poorly understood. In this study, the effect of a novel DG kinase inhibitor, stemphone, on vascular contraction was investigated. 2. The conventional DG kinase inhibitor, 6-[2-(4-[(4-fluorophenyl)phenyl-methylene]-1-piperidinyl)ethyl]-7-methyl-5H-thiazolo [3,2-alpha] pyrimidine-5-one (R59022) (0.1-30 microm), inhibited thromboxane A(2) analogue 9,11-dideoxy-11alpha,9alpha-epoxymethanoprostaglandin F(2alpha) (U46619)-induced sustained contractions in mouse aorta and porcine coronary artery in a dose-dependent manner. Treatment with stemphone did not affect contractions in these tissues. However, stemphone significantly inhibited (>0.3 microm) U46619-induced spontaneous phasic contraction in mouse portal vein. This inhibitory effect was not detected following R59022 treatment in portal vein. Therefore, stemphone demonstrated selectivity in terms of portal vein contraction. 3. Under high glucose (22.2 mm) conditions, U46619-induced contraction was enhanced in these three types of vascular tissue. Inhibitory effects of R59022 were attenuated under these conditions; however, effects of stemphone were observed. These results indicated that stemphone could inhibit portal vein contraction under high glucose conditions, for example, diabetes. These data suggested the possibility that DG kinase may be a target of hyperportal pressure. 4. Total mass of DG was enhanced under high glucose conditions. DG was derived from incorporated glucose via de novo synthesis in the absence of phospholipase C pathway mediation. This enhanced DG under high glucose conditions activated a calcium-independent protein kinase C (PKC). This PKC was associated with calcium-independent DG kinase activation. Treatment with stemphone also inhibited calcium-independent DG kinase. These signal transduction pathways were distinguishable from a DG-PKC pathway under normal glucose conditions. 5. The present investigation suggested that stemphone selectively inhibited overcontraction of portal vein induced by high glucose levels. This phenomenon was attributable to inhibition of calcium-independent DG kinase activation that occurred under high glucose conditions mediated by both DG synthesized from glucose and calcium-independent PKC activation.

Glucose-dependent enhancement of spontaneous phasic contraction is suppressed in diabetic mouse portal vein: association with diacylglycerol-protein kinase C pathway.

We investigated portal vein (PV) contractility in diabetes using a mouse model (ob/ob mouse) of spontaneous noninsulin-dependent diabetic mellitus. Spontaneous phasic contraction in control mice (C57Bl) was increased in the presence of the thromboxane A(2) analog 9,11-dideoxy-11alpha, 9alpha-epoxymethanoprostaglandin F(2)alpha (U46619) in a time- and concentration-dependent manner. This response was enhanced under high glucose conditions (22.2 mM). Diacylglycerol (DG) was synthesized from glucose and was not affected by phospholipase C (PLC) inhibition under resting conditions in normal glucose. Inhibition of DG-induced PKC activation with 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo-(2,3-alpha)pyrrolo(3,4-c)-carbazole (Gö6976), a calcium-dependent protein kinase C (PKC) inhibitor, was only observed under normal glucose conditions. High glucose levels enhanced PLC-independent DG formation followed by an induction of total phosphatidylinositol turnover via calcium-independent PKC activation in C57Bl mice. In ob/ob mice, the high glucose-induced enhancement of PV contraction in response to U46619 was suppressed. These findings suggest that these differences are associated with long-term exposure of tissue to a hyperglycemic state. Under high glucose conditions, DG derived from glucose fell below 50% in C57Bl mice. Moreover, the DG-related calcium-independent PKC was desensitized in ob/ob mice. These results suggest that suppression of the glucose-induced enhancement of PV contraction involves both a decrease in glucose-derived DG formation and reduction of the glucose sensitivity of DG-related PKC.

High-glucose enhances a thromboxane A2-induced aortic contraction mediated by an alteration of phosphatidylinositol turnover.

The effect of the thromboxane A(2) analogue U46619 (9,11-dideoxy-11alpha,9alpha-epoxymethanoprostaglandin F(2)(alpha)) on sustained contraction in the mouse aorta was investigated. U46619 induced concentration-dependent (1 - 100 nM) increases in contraction. These contractile responses were enhanced significantly under high-glucose-physiological salt solution (HG-PSS) (2-fold greater than normal-PSS) conditions. This hyperactivation may be associated with aortic dysfunction in diabetes. However, the mechanisms remain unclear. HG-PSS enhanced U46619-induced accumulation of endogenous diacylglycerol (DG). Phospholipase C inhibitor (U73122) suppressed DG accumulation under normal conditions; however, suppression was not observed under high-glucose conditions. The HG-PSS-induced enhancement of contraction was inhibited by protein kinase C (PKC) inhibitor (calphostin C). This result indicated that accumulated DG might increase PKC activity, which then stimulates DG kinase activation as a feedback mechanism. DG kinase inhibition also suppressed HG-PSS-induced enhancement of contraction. Increased myo-inositol incorporation was detected under high-glucose conditions, indicating an acceleration of phosphatidylinositol (PI)-turnover. Moreover, rho kinase inhibitor (Y27632) suppressed U46619-induced contraction exclusively in normal-PSS. These findings indicated that HG-PSS treatment increases DG synthesis derived from incorporated glucose, PKC and DG kinase activation, and enhances the U46619-induced contraction via acceleration of PI-turnover. This series of responses may be involved in the dysfunction of aorta under high-glucose conditions occurring in association with diabetes.

In vitro relaxation of vascular smooth muscle by atropine: involvement of K+ channels and endothelium.

Cumulative addition of atropine to the organ bath containing endothelium-intact (+E) rat aorta, which was precontracted with phenylephrine (PE, 1 microM) and subsequently relaxed with carbachol (1 microM), caused biphasic changes in the vascular contractility of +E rat aortic rings. Low concentrations of atropine (10 nM-1.0 microM) caused progressive restoration of contraction to PE; whereas at higher concentrations (1-100 microM), atropine caused progressive relaxation. Atropine-induced aortic relaxation was significantly inhibited upon endothelium removal by either rubbing or saponin treatment, but considerable relaxation still persisted in the range of 30-100 microM atropine. Similar findings were also obtained when the nitric oxide (NO) generation was inhibited with 300 microM NO synthase inhibitor, L-NAME. Atropine-induced relaxation was also observed when 5-hydroxytryptamine (5-HT) was used as the agonist and the atropine-relaxation was more potent at lower concentrations of PE and 5-HT. However, atropine had no effect on the contraction elicited by KCl or prostaglandin F(2 alpha). Also, atropine-induced relaxation was not affected by indomethacin (1-10 microM), nicotine (10-100 microM) or hexamethonium (30 microM). Pretreatment of +E aorta with tetraethylammonia (TEA, 3-10 mM) or 4-aminopyridine (4-AP, 1-3 mM) showed prominent inhibitory effect on atropine-induced relaxation; on the other hand, preincubation with glibenclamide (1-10 microM), BaCl(2) (1-30 microM) or 2 microM charybdotoxin and apamin, had little effect on the relaxation induced by atropine. When added to tissues after relaxation to atropine, TEA and 4-AP concentration-dependently reversed the relaxation in -E aorta, whereas in +E aorta, TEA up to 30 mM and 4-AP up to 10 mM only partially affected atropine-induced relaxation. Although TEA and 4-AP potentiated the PE-contraction, such potentiation is unlikely to contribute to the change in sensitivity to atropine-induced relaxation, since in the presence of 15 mM KCl, which also potentiated PE-contraction to a comparable extent, the atropine-relaxation remains unchanged. Scopolamine also acts like atropine, except that the effect of scopolamine was smaller than that of atropine and is primarily endothelium-dependent. Atropine-induced relaxation also occurs in medium artery (renal artery) and small muscular artery (mesenteric artery). In conclusion, atropine-relaxation is mediated in part via voltage-dependent K(+) channels in both smooth muscle and endothelium and forms the mechanistic basis for the observed vasodilation, reduced blood pressure and facial flushing following atropine overdose.

Dysfunction of aorta involves different patterns of intracellular signaling pathways in diabetic rats.

Rat models of insulin-dependent (streptozotocin-induced) and independent (Otsuka Long-Evans Tokushima Fatty (OLETF)) diabetes had sustained and transient increases in blood glucose levels. Over-contraction due to norepinephrine was seen exclusively in streptozotocin rat aorta. Contraction was enhanced under high-glucose conditions in OLETF rats. In order to understand the association between these patterns of changes, total diacylglycerol was measured as a key element of phosphatidylinositol-turnover due to the conversion of some incorporated glucose into diacylglycerol. Streptozotocin rats had enhanced basal diacylglycerol. Both diacylglycerol kinase (metabolic enzyme of diacylglycerol) and total phosphatidylinositol turnover activities also increased on norepinephrine stimulation, independent of extracellular glucose level. On the other hand, diacylglycerol, diacylglycerol kinase and phosphatidylinositol turnover in OLETF rats increased under high glucose conditions in the absence of norepinephrine treatment. These results indicated that diacylglycerol and diacylglycerol kinase-mediated phosphatidylinositol turnover acceleration was influenced by an increase in glucose levels in OLETF rats or by receptor-mediated signals in streptozotocin rats including glucose desensitization based on submaximal incorporation. We suggest that the alteration of vascular dysfunction is induced by different factors in each type of diabetes.

Molecular cloning and characterization of avian bombesin-like peptide receptors: new tools for investigating molecular basis for ligand selectivity.

(1) Bombesin (BN), originally isolated from amphibians, is structurally related to a family of BN-like peptides found in mammals, which include gastrin-releasing peptide (GRP) and neuromedin B (NMB). These peptides have important effects on secretion, smooth muscle contraction, metabolism and behavior. Here we report cloning and characterization of two subtypes of BN-like peptide receptors in Aves. (2) The amino-acid sequence of chick GRP-R (chGRP-R) is highly identical with mammalian and amphibian GRP-R, and this receptor showed high affinity for GRP, BN and synthetic bombesin agonist, [D-Phe(6), beta-Ala(11), Phe(13), Nle(14)]bombesin(6-14) ([FAFNl]BN(6-14)). The chGRP-R gene was localized to chicken chromosome 1q23distal-q24proximal, where chick homologs of other human X-linked genes have also been mapped. (3) ChBRS-3.5, having sequence similarities to both mammalian bombesin-like peptide receptor subtype-3 and amphibian bombesin-like peptide receptor subtype-4, showed high affinity for [FAFNl]BN(6-14), moderate affinity for BN, but low affinity for both GRP and NMB. (4) Expression of both receptors was detected in brain, but only chGRP-R was expressed in gastrointestinal (GI) tissues. (5) When expressed in Chinese hamster ovary K1 cells, these receptors mediate intracellular calcium mobilization upon agonist stimulation. These results suggest that a novel BN peptide may occur in Aves as an endogenous ligand for chBRS-3.5. (6) The receptor sequences responsible for ligand selectivities were discussed and this knowledge about avian BN-like peptide receptors will help us to understand the molecular basis for agonist sensitivities of BN-like peptide receptors.

Enhancement effect under high-glucose conditions on U46619-induced spontaneous phasic contraction in mouse portal vein.

The effect of the thromboxane A(2) analog 9,11-dideoxy-11alpha, 9alpha-epoxymethanoprostaglandin F(2alpha) (U46619) on spontaneous phasic contractions in the mouse portal vein was studied. U46619 induced concentration-dependent (1-100 nM) increases in amplitude, frequency, and contractile period (ON-time) of the contraction. Both amplitude and ON-time were enhanced significantly under high-glucose (HG; 4-fold greater than normal) conditions. This hyperactivation may be associated with portal vein dysfunction in diabetes. However, the mechanisms remain unclear. HG enhanced the U46619-induced accumulation of endogenous diacylglycerol (DG). Phospholipase C inhibition suppressed accumulation under normal conditions; however, this suppression was not observed under HG conditions. The HG-induced enhancement of U46619-induced contraction was inhibited by protein kinase C (PKC) inhibition. This finding indicated that accumulated DG might increase PKC activity. Activated PKC stimulated DG kinase activation as a feedback mechanism. DG kinase inhibition also suppressed the HG-induced enhancement of contraction. Increased myo-inositol incorporation was detected under HG conditions, indicating an acceleration of phosphatidylinositol (PI) turnover. This acceleration was inhibited by PKC and DG kinase inhibitors. These findings indicated that HG treatments increased DG synthesis derived from incorporated glucose, PKC, and DG kinase activation. These responses induce hyperactivation of the amplitude and contractile period of contraction mediated by acceleration of PI turnover. This series of responses may be involved in the dysfunction of the portal vein under the HG conditions occurring with diabetes.

Suppression by Hydrangeae Dulcis Folium of D-galactosamine-induced liver injury in vitro and in vivo.

Hydrangeae Dulcis Folium, the fermented and dried leaves of Hydrangea macrophylla SER. var. thunbergii MAKINO, suppressed D-galactosamine-induced liver injury by 85.2% when added to the diet at 1% and fed to rats for fifteen days. The hepatoprotective effect is more potent than that of a milk thistle extract and turmeric powder. Some fractionated extracts showed hepatoprotective activity in the D-galactosamine-induced in vitro liver injury model.