Relaxation of equine tracheal muscle in vitro by different adrenoceptor drugs

Strips of tracheal smooth muscle from 12 horses were contracted by carbachol in tissue baths under isometric conditions. This contraction (≅50% of maximum: EC₅₀) was relaxed completely with adrenoceptor drugs. The only exception was clenbuterol, where the degree of relaxation was ≅90%. In all horses the EC₅₀-value for isoprenaline (mean 1.6 × 10⁻⁸M) was less than that for adrenaline (mean 9.6 × 10^{− 8}M) and noradrenaline (mean 1. 8 × 10_{- 6}M). The potency ratio was 1 < 6 < 110 which indicates that the β₂-subtype dominates among the β-adrenoceptors of equine airways. All preparations were also very sensitive to the specific and potent β₂-receptor agonists clenbuterol (mean 5.7 × 10^{− 9}M) and procaterol (mean 3.6 × 10⁻¹⁰M). No differences in EC₅₀-values due to age, sex and breed were observed in this material. The standard deviation of the mean EC₅₀-values seems to be larger for the specific β₂-adrenoceptor agonists than for the unspecific. A reason for this could be differences in the pattern of the β-adrenoceptor population.     

Urinary Corticoid : Creatinine Ratios in Dogs with Pituitary-Dependent Hypercortisolism during Trilostane Treatment

Background: The adrenocorticotropic hormone (ACTH) stimulation test is used to evaluate trilostane treatment in dogs with hypercortisolism.
Hypothesis: The urinary corticoid : creatinine ratio (UCCR) is a good alternative to the ACTH stimulation test to determine optimal trilostane dose.
Animals: Eighteen dogs with pituitary-dependent hypercortisolism.
Methods: In this prospective study, the dose of trilostane was judged to be optimal on the basis of resolution of clinical signs of hypercortisolism and results of an ACTH stimulation test. The owners collected urine for determination of UCCR at 2-week intervals for at least 8 weeks after achieving the optimal trilostane dose.
Results: The UCCRs were significantly higher before treatment (11.5–202.0 × 10⁻⁶; median, 42.0 × 10⁻⁶) than at rechecks 2 months after optimal dosing, but they did not decrease below the upper limit of the reference range in the majority of dogs. The UCCRs of 11 dogs that initially were dosed insufficiently (range, 7.5–79.0 × 10⁻⁶; median, 31.0 × 10⁻⁶) did not differ significantly from UCCRs when the dosage was optimal (8.2–72.0 × 10⁻⁶; median, 33.0 × 10⁻⁶). Post-ACTH cortisol concentrations did not correlate significantly with UCCRs at rechecks during trilostane treatment. Long-term follow-up indicated that the decrease in UCCR below the upper limit of the reference was associated with hypocortisolism.
Conclusion and Clinical Importance: The UCCR cannot be used as an alternative to the ACTH stimulation test to determine the optimal dose of trilostane, but might be helpful in detecting dogs at risk for developing hypocortisolism during trilostane treatment.     

Expression of the μ Opioid Receptor and Effects of the Opioid Antagonist Naloxone on In Vitro Maturation of Oocytes Recovered from Anoestrous Bitches

The μ-opioid receptor (MOR) is expressed in bovine, human, equine and canine oocytes, and in seasonal breeders, it is expressed with higher intensity during the anoestrous phase. Supplementation of in vitro maturation (IVM) medium with opioid agents, agonists or antagonists, was shown to affect oocyte maturation in several species such as rat, bovine and equine. This study reports the effects of supplementing IVM medium with naloxone (Nx), an opioid antagonist, on nuclear and cytoplasmic maturation rate of oocytes recovered from anoestrous bitches. Cytoplasmic maturation was examined in terms of mitochondrial (mt) distribution. In order to confirm the receptor-mediated action of Nx, in oocytes of anoestrous bitches, MOR expression was analyzed by Western blot. Cumulus–oocyte complexes, recovered from the ovaries of bitches in anoestrous, were cultured in vitro and Nx was added at the concentrations of 1 × 10⁻⁶, 1 × 10⁻⁸ and 1 × 10⁻¹⁰  m . The rate of oocytes resuming meiosis after culture in presence of 1 × 10⁻⁶  m Nx (29%) was significantly higher than that of oocytes of control group (12%; p < 0.05). However, treatment with Nx did not affect mt distribution pattern. In denuded oocytes and in corresponding cumulus cells, a doublet of 65 and 50 kDa was observed. We conclude that, in oocytes of anoestrous bitches, MOR is expressed and Nx significantly improves nuclear maturation rate. Further studies should be performed to elucidate the expression of other opioid receptors, such as δ and κ, and possible interactive effects of their antagonists on canine oocyte maturation.     

Laminin binds to myostatin and attenuates its signaling

Myostatin is a growth and differentiation factor and acts as a negative regulator of skeletal muscle mass. Although the mechanism whereby myostatin controls muscle cell growth is mostly clarified, the regulation of myostatin activity after its secretion into the extracellular matrix (ECM) is still unclear. In the present study, we investigated the interaction between laminin and myostatin and the effect of laminin on myostatin signaling in vitro. The surface plasmon resonance assay showed that laminin bound to mature myostatin and activin receptor type IIB (ActRIIB), but did not bind to latency‐associated protein, which remains non‐covalently linked to mature myostatin. Furthermore, kinetic analysis demonstrated that the affinity of mature myostatin for laminin was similar to that for ActRIIB. Next, we examined the action of laminin on the myostatin signaling pathway using a conventional reporter assay. The luciferase activity of myostatin‐treated cells was repressed significantly (P < 0.05) by coincubation of laminin. These results suggest that laminin has a potential to regulate myostatin activity through binding to mature myostatin and/or its receptor ActRIIB.     

Consequences of Nitric Oxide Synthase Inhibition During Bovine Oocyte Maturation on Meiosis and Embryo Development

The importance of nitric oxide synthase (NOS) in bovine oocyte maturation was investigated. Oocytes were in vitro matured with the NOS inhibitor N^w- l -nitro-arginine methyl-ester (10⁻⁷, 10⁻⁵ and 10⁻³  m l -NAME) and metaphase II (MII) rates and embryo development and quality were assessed. The effect of l -NAME (10⁻⁷  m ) during pre-maturation and/or maturation on embryo development and quality was also assessed. l -NAME decreased MII rates (78–82%, p < 0.05) when compared with controls without l -NAME (96%). Cleavage (77–88%, p > 0.05), Day 7 blastocyst rates (34–42%, p > 0.05) and total cell numbers in blastocysts were similar for all groups (146–171 cells, p > 0.05). Day 8 blastocyst TUNEL positive cells (3–4 cells) increased with l -NAME treatment (p < 0.05). For oocytes cultured with l -NAME during pre-maturation and/or maturation, Day 8 blastocyst development (26–34%) and Day 9 hatching rates (15–22%) were similar (p > 0.05) to controls pre-matured and matured without NOS inhibition (33 and 18%, respectively), while total cell numbers (Day 9 hatched blastocysts) increased (264–324 cells, p < 0.05) when compared with the controls (191 cells). TUNEL positive cells increased when NOS was inhibited only during the maturation period (8 cells, p < 0.05) when compared with the other groups (3–4 cells). NO may be involved in meiosis progression to MII and its deficiency during maturation increases apoptosis in embryos produced in vitro . Nitric oxide synthase inhibition during pre-maturation and/or maturation affects embryo quality.     

Analysis of myostatin and its related factors in various porcine tissues

Myostatin is expressed in skeletal muscle tissue where it functions to suppress myoblast proliferation and myofiber hypertrophy. Recently, myostatin was detected in the tendon, mammary gland, and adipose tissue of mice. We sought to determine whether myostatin is expressed in the liver, spleen, lung, and kidney of pigs. Real-time PCR and Western blots demonstrated that myostatin, follistatin, decorin, and activin receptor IIB (ActRIIB) mRNA and proteins were expressed in skeletal muscle, heart muscle, and adipose tissue, and also in liver, spleen, lung, kidney, and cultured fibroblasts. The relative abundance of myostatin was closely related to follistatin and decorin in porcine tissues. Immunohistochemical analysis further demonstrated the presence of myostatin, follistatin, and decorin in the skeletal muscle, adipose tissue, heart muscle, liver, spleen, lung, and kidney of pigs. These results suggest that myostatin could be associated with certain functions of the internal organs, such as energy metabolism or fibrosis. We conclude that myostatin is a factor broadly expressed in the internal organs and muscle tissues of pigs.     

Retinol-Binding Protein in Serum and Urine of Hyperthyroid Cats before and after Treatment with Radioiodine

Background: Retinol-binding protein (RBP) is suggested as a clinically useful marker of renal function in cats.
Hypothesis: Serum and urinary RBP concentrations in hyperthyroid (HT) cats differ from those in healthy (H) cats; radioiodine (¹³¹I) treatment influences serum and urinary RBP concentrations in HT cats.
Animals: Ten HT and 8 H cats.
Methods: RBP concentration was evaluated in feline serum and urine samples from a prospective study.
Results: There was a significant ( P = .003) difference in the urinary RBP/creatinine (uRBP/c) ratios of H (−) and untreated HT (1.4 ± 1.5 × 10⁻² μg/mg) cats. Serum total thyroxine concentration (1.8 ± 1.9 μg/dL, 24 weeks) and uRBP/c (0.6 ± 1.0 × 10⁻² μg/mg, 24 weeks) decreased significantly ( P < .001) in HT cats at all time points after treatment with ¹³¹I, and these variables were significantly correlated with one another ( r = 0.42, P = .007). Serum RBP concentrations from HT cats (199 ± 86 μg/L) did not differ significantly ( P = .98) from those of H cats (174 ± 60) and did not change after treatment with ¹³¹I (182 ± 124 μg/L, P = .80).
Conclusion and Clinical Importance: The presence of urinary RBP in HT cats is a potential marker of tubular dysfunction that is correlated to thyroid status, although it is independent of circulating RBP concentrations. The decreased uRBP/c combined with the absence of changes in serum RBP after treatment suggests that the suspected tubular dysfunction was partly reversible with treatment of ¹³¹I.     

Vaso-reactivity of isolated bovine intra-mammary artery to endogenous prostanoids and nitric oxide

The modulatory role of locally produced cyclooxygenase products and endothelium-derived nitric oxide in controlling vascular tone was investigated in bovine intra-mammary artery. Vascular reactivity initiated by vasoactive compounds, endothelin-1 (ET-1), bradykinin (BK), and substance P (SP) was measured isometrically in an isolated tissue bath. The effects of a cyclooxygenase inhibitor, indomethacin (10⁻⁵ M) and an inhibitor of nitric oxide production, Nω-Nitro L-Arginine (L-NNA: 3 x 10⁻⁴ M) were determined during agonistmediated responses. Indomethacin alone markedly enhanced vascular contraction produced by ET-1, while L-NNA did not. Inhibition of endothelium-derived nitric oxide synthesis by L-NNA, however, significantly attenuated BK-and SP-induced vascular relaxations, whereas indomethacin had slight influence. The potentiation between indomethacin and L-NNA in regulating vasomotor tone was not observed in this vascular bed. Thus, it appeared that both the cyclooxygenase and endothelium-derived nitric oxide pathways participated in modifying vascular reactivity. Domination of one pathway over the other depended upon the agonist used to stimulate vascular tissue.     

Growth, weaning performance and blood indicators of humoral immunity in Holstein calves fed supplemental flavonoids

The primary objective was to test the hypothesis that flavonoids mediate immune response and affect calf performance. Twenty Holstein calves [7 ± 2 days age; 41.4 ± 0.7 kg body weight (BW)] were randomly assigned to four treatments of (i) no; (ii) low (7.3 × 10⁻⁵ g/kg BW); (iii) medium (7.3 × 10⁻⁴ g/kg BW); and (iv) high (3.6 × 10⁻³ g/kg BW) doses of flavonoids intake in a completely randomized design. Calves received the treatments as a tablet until weaning or a daily intake of 680 g starter. After weaning, calves received no supplemental flavonoids and monitored until 120 days of age. The flavonoids were extracted from propolis. Treatments did not affect body length, wither height and the severity of scours. At week 5 of age, BW was higher when calves fed the high compared to the low dose of flavonoids. At week 6, calves fed the high dose of flavonoids had higher BW than those fed no or low doses of flavonoids. The serum immunoglobulin G (IgG) concentrations remained lower at the first 3 weeks of the experiment when calves received the low but not the high doses of flavonoids. At week 4, both medium and low doses of flavonoids moderated serum IgG. At week 8, the medium and high but not the low doses of flavonoids lowered serum IgG. At week 6, calves fed high and medium flavonoids doses had lower blood immunoglobulin M (IgM) than control calves. Results suggest that flavonoids affect the humoral immune response and can improve growth in young calves. This response depended on calf age. Future studies are needed to further evaluate the premise that dietary forages or the main source of flavonoids are helpful for a less stressful weaning in the modern calf raising.     

Functional characterization of post-junctional adrenergic receptor subtypes in bovine intra-mammary arteries

We examined the functional role of adrenergic receptor subtypes (ARs) in bovine intra-mammary arteries (IMAs), 1.5–2.5 mm internal diameter. Norepinephrine (NE) and phenylephrine (PE) produced concentration-dependent increases in tone in segments maintained at a previously determined optimal basal tension in vitro . The sensitivity of the tissue to NE and PE, based on -log molar ED_50s was 6.87 ± 0.17 and 7.05 ± 0.35, respectively. In addition a Schild analysis yielded antagonist affinities for the receptor mediating contractile responses to NE (pA2 value) of 10.46 ± 0.85 for prazosin and 6.29 ± 0.18 for yohimbine. These data indicate a dominance of functional alpha 1 (α₁) over alpha 2 (α₂)-ARs in this tissue. Based on the inhibitory effects of chloroethylclonidine (CEC) on PE responses and the further reduction in sensitivity when nifedipine was added to the CEC, also in the presence of PE, we conclude that there is more than one α₁-AR subtype, with a predominant role for α_1B-ARs in phenylephrine responses. Stimulation of beta (β)-ARs, resulted in relatively small reductions in tone (the highest magnitude of response was 25.94 ± 6.46% of the papaverine maximum at 3×10⁻⁶ M isoproterenol); in addition, propranolol did not significantly alter tissue sensitivity to NE. Additional characterization of functional autonomic receptor populations in this circulatory bed will form a basis for future studies on circulatory dynamics in the mammary gland.     

Femtomolar bradykinin-induced relaxation of isolated bovine coronary arteries, mediated by endothelium-derived nitric oxide

We reported previously that bradykinin induces endothelium-dependent relaxation at nanomolar (n M ) concentrations in isolated bovine coronary arteries with an intact endothelium. Recently we have found that in the presence of 10 μ m indomethacin, femtomolar (f M ) concentrations of bradykinin induce endothelium-dependent relaxation in some bovine coronary arteries (≈ 10% of the coronary arteries examined). The present study was designed to characterize the relaxation induced by f M bradykinin. Relaxation was completely abolished by repeated application of f M bradykinin, by 100 μ m N^ω- nitro- l - arginine methyl ester and by 10 μ m methylene blue. Relaxation induced by n M bradykinin was partly affected by these treatments. Relaxation induced by both concentrations of bradykinin was inhibited by a B₂-kinin receptor antagonist, [Thi^5,8, D-Phe⁷]-bradykinin, in a concentration-dependent manner, but not by a B₁-kinin receptor antagonist, des-Arg⁹, [Leu⁸]-bradykinin. In the presence of 10 μ m captopril, an angiotensin-converting enzyme (ACE) inhibitor, all coronary arteries examined in this experiment showed endothelium-dependent relaxation to f M bradykinin. These results show that some bovine coronary arteries relax in response to f M bradykinin, and this response is mediated predominantly by the release of nitric oxide via stimulation of endothelial B₂-kinin receptors. The relaxation may be dependent on ACE activity.     

The effect of xylazine on the isolated sheep trachea

The effect of xylazine on the isolated sheep trachea and its possible interactions with the α₂-adrenergic antagonist, atipamezole, and the anticholinergic agent, atropine, was studied. The mechanical responses of the tracheal preparations were recorded after exposing each one to cumulatively increasing concentrations of xylazine alone or in the presence of atipamezole or atropine.
Xylazine exerted a concentration-dependent contractile effect, with a threshold concentration of 10^--⁷M while the maximum activity was produced at a concentration of 10^--⁵M (EC₅₀= 2.3 × 10^--⁷). This xylazine-induced contractile effect was inhibited by atipamezole, but not significantly modified by atropine. Thus, it is concluded that α₂-adrenoceptors exist in the sheep trachea and it is suggested that α₂-adrenoceptor agonists may act on airways in sheep directly through stimulation of peripheral α₂-adrenergic receptors and indirectly via central α₂-adrenergic receptor activation of parasympathetic tone.     

Effects of Alendronate on A Disintegrin and Metalloproteinase with Thrombospondin Motifs Expression in the Developing Epiphyseal Cartilage in Rats

A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) have been reported to play a role in the degradation of aggrecan, a major component of cartilage. This study was performed to examine the effects of alendronate on the expression of ADAMTS in developing femoral epiphyseal cartilage. Primary cultured chondrocytes from this cartilage were treated with alendronate in vitro and postnatal day 1 rats were injected subcutaneously with alendronate (1 mg/kg) every second day in vivo . The number of cultured chondrocytes and their aggrecan mRNA levels were unaffected by the alendronate treatment at 10⁻⁶ to 10⁻⁴  m concentrations. The mRNA levels of ADAMTS-1, -2 and -9 in chondrocytes were also unaffected. However, the levels of ADAMTS-5 and -4 were reduced significantly by the same treatment. The thickness of the proliferating chondrocyte layers and the aggrecan mRNA levels in the epiphysis were unaffected by the alendronate treatment in vivo . However, the hypertrophied chondrocyte layers became significantly thicker, and the size of the secondary ossification centre was reduced significantly by the same treatment ( P  < 0.05). Both ADAMTS-4 and -5 mRNA expressions were also reduced significantly in vivo . The immunoreactivity against ADAMTS-4 was seen in hypertrophied chondrocytes and reduced significantly by the alendronate treatment. These results suggested that alendronate can inhibit the degradation of aggrecan in the articular cartilage by downregulating the expression of matrix enzymes such as ADAMTS-4 and -5.     

Concentrations of thyroid hormones and iodothyronine binding proteins in serum of the koala (Phascolarctos cinereus)

Objective To examine circulating total and free thyroid hormone (T₃ and T₄) concentrations, determine serum iodothyronine binding characteristics and estimate thyroid stimulating hormone (TSH) activity in sera of coastal and inland koalas.
Design A prospective study.
Procedure Koala serum T₃ and T₄ were measured by radioimmunoassay. T₄ binding parameters were determined by radioligand binding and electrophoresis. Koala TSH values were determined by bioassay.
Results Mean total T₄ concentrations were 3.2 ± 2.1 nM although values were significantly higher in inland-dwelling females in comparison to coastal-dwelling males. Free T₄ was 3.3 ± 2.1 pM. Total and free T₃ were 0.4 ± 0.2 nM and 1.4 ± 0.9 pM respectively, although these values were at the lower end of the assay detection limit and should be viewed with reservation. Electrophoresis of [¹²⁵I]-T₄-labelled serum revealed only two proteins of electrophoretic mobility similar to human transthyretin (TTR) and albumin. Scatchard analysis of T₄ binding to serum gave a curvilinear plot, which could be resolved into two binding sites with affinities identical to that of TTR and albumin but both of low concentration. The bioactivity of the TSH present in the sera was measured using a cell line (JP09) transfected with the human TSH receptor. The mean level of stimulation found in the sera corresponded to a bovine TSH activity of less than 10 mU/L.
Conclusion These results suggest that the serum concentrations of free and total thyroid hormones in koalas are low compared to other marsupials and very low compared to eutherian mammals. The mechanism of maintenance of euthyroidism in this species remains to be determined.     

Possible involvement of phosphatidylinositol 3-kinase in the maintenance of metaphase II attest in porcine oocytes matured in vitro

It has been reported that phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB) pathway plays a crucial role in the meiotic resumption and progression to the metaphase II (MII) stage of oocytes. However, the role of this pathway in meiotic arrest at the MII stage (cytostatic activity) is not well understood. In this study the effect of a PI3K inhibitor, LY294002, on the MAPK and p34^cdc2 kinase activities of matured porcine oocytes was examined. After maturation culture, both the MAPK and p34^cdc2 kinase activities in the oocytes were gradually decreased in a time-dependent manner. Although 25 µmol/L LY294002 did not affect either the MAPK or p34^cdc2 kinase activities, 50 µmol/L LY294002 suppressed the PKB phosphorylation and slightly decreased MAPK activity, but not the p34^cdc2 kinase activity. Therefore the effect of 10 µmol/L Ca²⁺ ionophore which was reported as inducing a transient decrease of p34^cdc2 kinase but not MAPK activities, was also examined in LY294002-treated oocytes. By additional treatment with LY294002 after Ca²⁺ ionophore, both the MAPK and p34^cdc2 kinase activities were decreased in a time-dependent manner, concomitantly with improvement of pronuclear formation. Therefore, we concluded that PI3K is involved in the maintenance of MAPK activity in matured porcine oocytes.     

Effect of oral administration of 3,3',4,4',5-pentachlorobiphyenl on the intestinal microbiota of Sprague–Dawley rats

The effect of the endocrine-disrupting chemical 3,3',4,4',5-pentachlorobiphyenl (PCB 126) on intestinal microbiota after oral administration, and the improvement of intestinal microbiota and feces quantity by the subsequent administration of Lactobacillus acidophilus or Lactobacillus reuteri was investigated. All the rats were given 100 μg/kg bodyweight of PCB 126. The changes in bacterial counts were confirmed using a culture method. The administration of PCB 126 tended to decrease the bacterial counts of lactobacilli (10^9.6−10^10.2 to 10^8.8−10^9.2) and bifidobacteria (10^5.3−10^6.1 to 10^3.6−10^4.2), and to increase those of Enterobacteriaceae (10^8.2−10^9.1 to 10^9.4−10^10.3) and staphylococci (10^6.6−10^7.4 to 10^7.2−10^8.4) compared to no PCB 126 administration. After administration of PCB 126, L. acidophilus or L. reuteri orally administered to rats caused Enterobacteriaceae and staphylococci counts to decrease, suggesting that the intestinal microbiota was improved by the lactobacilli. The administration of L. acidophilus and L. reuteri improved the balance of intestinal microbiota, and defecation volume returned to its normal level. L. acidophilus and L. reuteri have a remedial effect on intestinal microbiota affected by PCB 126 and can function to lessen accumulated PCB 126 volume.     

Metabolites of Monascus ruber in silages

A total of 233 silages were examined and found that Monascus ruber was present in 43 samples with counts between 1 × 10³ and 9 × 10⁶ colony-forming units (CFU)/g (mean: 2 × 10⁵ CFU/g). Monacolin K_L and the hydroxy acid monacolin K_A were detected by liquid chromatography-mass spectrometry in 45 and 50 of 233 samples at levels ranging from 25–15 600 and 28–65 400  μ g/kg, respectively. Citrinin was found with high-performance liquid chromatography-fluorescence detection (FLD) in 14 (6%) samples, the concentrations varied between 2.4 and 64.2  μ g/kg. The concentrations of citrinin were low and toxic effects are not anticipated. Monacolin K_A and monacolin K_L occur frequently and in considerable amounts in silages. These metabolites are believed to influence the metabolic activity of rumen anaerobic fungi resulting in a poorer digestion of crude fibre.     

Influence of vitamin E on mast cell mediator release

Abstract We investigated the influence of vitamin E on mediator activity and release in a canine mastocytoma cell line (C2) as a model for canine atopic dermatitis. Cells were incubated without and with vitamin E (100 µ m ) for 24 h. The histamine and prostaglandin D₂ (PGD₂) release as well as the chymase and tryptase activity were measured. To stimulate the PGD₂ and histamine release, cells were incubated with the wasp venom peptide mastoparan (50 µ m ) for 30 or 45 min. Nonstimulated as well as mastoparan-stimulated histamine and PGD₂ release was reduced significantly in vitamin E-treated cells. The activity of chymase tended to decrease, but the tryptase activity of C2 cells was not influenced by vitamin E. These results indicate that vitamin E decreased the production and release of inflammatory mediators in C2 cells, suggesting that vitamin E might have a possible beneficial effect in inflammatory diseases.     

Binding of sulphamethazine to pig plasma proteins and albumin

The interaction of sulphamethazine (SMZ) with pig plasma proteins and albumin was studied by ultrafiltration and equilibrium dialysis. Binding to pig plasma proteins was monophasic (affinity ˜9.0 mol/L × 10³) and the main binding protein was albumin. At 37ΩC and pH 7.4, the affinity of SMZ for albumin was about 8.0 mol/L x 10³ and the number of binding sites was estimated as 1.4. Increasing the temperature from 4 to 45ΩC resulted in a sevenfold decrease in affinity, and increasing pH from 6.0 to 8.0 enhanced affinity for pig albumin ten-fold. The free energy of binding (-ΔG) and enthalpy change (-ΔH) were around 5.5 and 5.1 Kcal/mol, respectively. The total entropy change (ΔS) was small and positive, around 2 cal/mol/°K. Studies with the fluorescent probes warfarin and dansylsarcosine, suggest that these bind to separate sites on porcine albumin. SMZ displaced both probes and inhibited the deacetylation of p-nitrophenyl acetate by pig albumin. We conclude that: (1) binding of SMZ to pig plasma proteins and albumin is weak; (2) the interaction with albumin is exothermic and enthalpy driven, and (3) pig albumin, like other mammalian albumins, appears to possess discrete binding sites for warfarin and dansylsarcosine. SMZ interacts with both these loci.     

The role of thromboxane A2 in regulating porcine basilar arterial tone

The aim of the present study was to clarify the participation of endogenous arachidonic acid (AA) metabolites in regulating porcine basilar, coronary, pulmonary and mesenteric arterial tones in vitro . A cyclooxygenase inhibitor, indomethacin, relaxed basilar artery but not other arteries examined. Quinacrine (a phospholipase A₂ inhibitor), OKY-046 (a thromboxane (TX) A₂ synthetase inhibitor) and ONO-3708 (a TXA₂/prostaglandin H₂ receptor antagonist) produced relaxation in basilar arteries with intact endothelium. Nordihydroguaiaretic acid (a lipoxygenase inhibitor) had no effect on the tone. The amount of TXB₂ (a stable metabolite of TXA₂) spontaneously released from porcine basilar arteries was 6–10 fold more than those from other arteries. Indomethacin and OKY-046 mostly inhibited the production of TXB₂. Endothelial denudation decreased indomethacin-induced relaxation and the amount of TXB₂. These results suggest that a vasoconstricting substance(s) is released from endothelial cells and possibly smooth muscle cells in porcine basilar arteries in vitro . The main constricting substance is proposed to be TXA₂. On the other hand, several arteries from peripheral vascular beds did not release this vasoconstricting substance.