Superoxide dismutase and catalase activities in the seminal plasma of normozoospermic and asthenozoospermic Beagles

We measured the blood plasma testosterone (T) levels and superoxide dismutase (SOD) and catalase activities in the seminal plasma of the ejaculates of 5 normal (2-5 years old) and 5 asthenozoospermic (AZ-) (3-5 years old) Beagles. Sperm ejaculated by AZ-dogs was incubated for 3 hr in Eagle's MEM only (controls) or Eagle's MEM containing 100 units/ml of SOD or catalase. Sperm motility was examined during incubation. The mean (+/- SE) plasma T level of the AZ-dogs (1.2 +/- 0.2 ng/ml) was significantly lower than in the normal dogs (2.5 +/- 0.2 ng/ml) (P<0.005). The mean (+/- SE) seminal plasma SOD and catalase activities (18.8 +/- 1.9 and 0.5 +/- 0.1 unit/g protein, respectively) were significantly lower in the AZ-dogs than in the normal dog (43.3 +/- 2.5 and 2.2 +/- 0.4 unit/g protein, respectively) (P<0.001 and 0.01, respectively). The motility of sperm incubated in Eagle's MEM containing SOD or catalase was significantly higher than that of control sperm incubated in only Eagle's MEM after 2 or 3 hr of incubation (P<0.05). The results of this study indicate that poor T secretion by the testes and low antioxidant enzyme activities are related to AZ in the dog.     

Doxorubicin affects the cardiac muscarinic system in the rat.

During the study on the mechanism of doxorubicin-induced cardiotoxicity, we observed that a long incubation (4 hr) with doxorubicin reduced the maximal negative inotropic effects of a muscarinic receptor agonist, carbachol. The mechanism responsible for this doxorubicin-induced reduction of the efficacy of carbachol was examined in isolated guinea pig hearts. In isolated left atrial muscle preparations, 1 hr incubation with 100 microM doxorubicin caused a parallel right-ward shift of the concentration-response curves for carbachol, but a longer (4 hr) incubation with this agent (30, 100 or 200 microM), caused a significant reduction of the magnitude of the negative inotropic effect of carbachol in addition to the concentration-dependent parallel right-ward shift. The 4-hr incubation with these concentrations of doxorubicin also reduced the maximal negative inotropic effect of an adenosine A1 receptor agonist, R-phenylisopropyl adenosine (R-PIA), without affecting the potency of this agonist. Doxorubicin (1 to 100 microM) reduced [3H]quinuclidinyl benzilate (QNB) binding in a concentration dependent manner, but failed to alter [3HIR-PIA binding. The decrease in the magnitude of the maximal negative inotropic effect by doxorubicin was caused by changes in the muscarinic system at steps common to the transduction of muscarinic and adenosine A1 receptor mechanisms.     

Protease-induced hyperactivation of canine spermatozoa associated with disappearance of lectin-binding glycoproteins on their surface

The relationship between the disappearance of glycoproteins from the surface of canine sperm and sperm capacitation was investigated in vitro. The protease (PR) concentration in flush fluids of the uterine horns and oviducts removed from 6 estrous, 5 diestrous, and 5 anestrous bitches was measured with a protease assay kit. Ejaculated sperm collected from 10 dogs were incubated for 4 hr in Eagle's MEM supplemented with 1 or 5 microg/ml PR, or to which no PR had been added (control). The glycoproteins on the surface of the sperm were stained with 4 different FITC-lectins (Con A, PHA-E, PNA, and WGA), and the percentages of hyperactivated (HA-) sperm and acrosome-reacted (AR-) sperm were evaluated. The mean PR concentration (5.95 microg/ml) in the flush fluid from the oviducts of the estrous bitches was significantly higher than in the fluid from their uterine horns (1.00 microg/ml; P<0.01). The PR concentrations of the flush fluids from the uterine horns and oviducts of both the diestrous and anestrous bitches were less than 0.05 microg/ml. Before incubation the acrosomal regions or entire heads of all sperm clearly stained with each FITC-lectin, but the percentages of sperm binding the 4 FITC-lectins decreased after incubation. The percentages of lectin-binding sperm in the MEM containing 5 microg/ml PR were significantly lower than in the control MEM (P<0.05 and 0.01). The mean percentages of motile sperm and HA-sperm after incubation in the MEM with PR were higher than in the control MEM, but there were no differences in the percentages of AR-sperm. The results indicate that HA-movement of sperm is induced by the disappearance of glycoproteins from the surface of canine sperm as a result of the action of PR in the oviductal fluid of estrous bitches.     

Oxidized-low density lipoprotein inhibits cyclic AMP production by porcine luteal cells

Oxidized(OX)-low density lipoprotein (LDL) inhibits steroidogenesis by luteal cells (LC) from regressing porcine CL. The present study was designed to investigate the mechanism of inhibition by determining whether OX-LDL inhibits basal and agonist-stimulated cAMP production in regressing LC. Collagenase-dispersed porcine LC (n = 7 animals, estrous cycle Day 12-15) were cultured (2.5 x 10(5) cells/0.5 ml) in serum-free DMEM/Hams F-12 in duplicate wells at 37 degrees C. Approximately 18 hr after plating, media were replaced and LC were immediately treated with human LDL (0, 25, or 100 microg/ml) or OX-LDL (25 or 100 microg/ml). LC were incubated for 2 hr before addition of isobutylmethylxanthine (IBMX) to inhibit phosphodiesterase activity, immediately followed by hCG (100 ng/ml), cholera toxin (CT; 0.1 microM), forskolin (FS; 50 microM), or no further treatment (controls). LC were incubated for an additional 90 min. After removal of culture media, cells were extracted with 0.1 N HCl. Cell extracts were assayed for cAMP by enzyme immunoassay (EIA). HCG, CT, and FS increased (P < 0.05) cAMP production approximately four-, 10-, and 25-fold, respectively, relative to controls. OX-LDL (25 and 100 microg/ml) inhibited (P < 0.05) cAMP production by unstimulated, hCG-, and CT-stimulated LC, but not that by FS-stimulated LC. The highest concentration of OX-LDL (100 microg/ml) reduced cAMP formation by 39.8 +/- 6.6%, 44.7 +/- 10.5%, and 67.7 +/- 4.5% in unstimulated, hCG-, and CT-stimulated LC, respectively. In contrast, unmodified LDL (25 and 100 microg/ml) did not alter cAMP production. We conclude that OX-LDL can interfere with the cAMP signaling pathway in regressing luteal cells by acting at sites proximal to adenylate cyclase activation.     

Antisense oligonucleotide complementary to the BamHI-H gene family of Marek's disease virus induced growth arrest of MDCC-MSB1 cells in the S-phase.

DNA synthesis was effectively inhibited by antisense oligonucleotide A1 complementary to the BamHI-H gene family in Marek's disease virus (MDV)-derived lymphoblastoid MDCC-MSB1 cells. When a cell cycle distribution of a total cell population was analyzed by flow cytometry, the proportion of S-phase cells increased in the cell populations by treatment with oligonucleotide A1. Approximately 60-70% of the cells appeared in the S phase for 24 and 36 hr of incubation in the presence of oligonucleotide A1 (20-30% in the untreated control cells). The inhibition of cell cycle progression by treatment with oligonucleotide A1 was reversible. When the cells were treated with 5 microM aphidicolin for 12 hr, a similar pattern of cell cycle distribution was observed to that obtained after treatment with oligonucleotide A1. Aphidicolin is an inhibitor of cellular DNA polymerase alpha, and it halts progression of the cell cycle at the G1/S border or early S phase. When the cells were treated with aphidicolin for 12 hr and subsequently incubated with oligonucleotide A1, no significant difference was observed in the cycle phase distribution of cells in the presence and absence of oligonucleotide A1. In contrast, when the cells were treated with oligonucleotide A1 for 12 hr and subsequently incubated with aphidicolin, the cell cycle did not progress from the G1/S border or early S phase to the next phase.     

Failure of neuropeptide y to modulate the release of LH and prolactin by cultured bovine pituitary cells

Possible direct effects of neuropeptide Y (NPY) on dispersed and cultured cells of the anterior lobe (AL) of the bovine pituitary were investigated. AL tissue from steers was enzymatically dissociated into individual cells, preincubated for 18 hr and then incubated in suspension cultures for 2 hr or 24 hr with either NPY, gonadotropin-releasing hormone (GnRH) or both. Release of luteinizing hormone (LH) and prolactin (PRL) into medium was quantified by radioimmunoassay and expressed as hormone released per 100,000 cells. Basal release of LH averaged 38 and 86 ng for 2 hr and 24 hr respectively while that of PRL averaged 118 and 438 ng for the same incubation periods. Addition of NPY did not alter (P>.05) basal release of LH or PRL for either duration of incubation. Also, NPY did not affect (P>.05) release of LH in response to GnRH. In summary, this study indicated that NPY, at in vitro dosages of .01 to 100nM, does not modulate the release of LH or PRL at the pituitary level in castrate cattle.     

Investigating the molecular mechanism of pamidronate‐induced in vitro cytotoxicity in canine osteosarcoma cells

Introduction: Pamidronate is used to treat metastatic bone lesions in human cancer patients. By directly inhibiting the mevalonate pathway, pamidronate disrupts GTPase‐binding protein prenylation, resulting in osteoclast apoptosis. Pamidronate has been demonstrated to exert dose‐ and time‐dependent cytotoxic effects in several canine malignant osteoblastic cell lines. However, the exact cytotoxic mechanism remains speculative. The purpose of this study was to investigate and characterize the molecular mechanism of pamidronate‐induced cytotoxicity in canine malignant osteoblasts. Methods: The involvement of farnesyl or geranylgeranyl pyrophosphate synthase inhibition was evaluated by performing rescue experiments with cells incubated for 48 hours with cytotoxic concentrations of pamidronate (50 and 100 μM), with or without farnesol (FOH) and geranylgeraniol (GGOH). Cell lysates were fluorometrically‐assessed for caspase‐3‐like activity (R&D Systems) following incubation with varying concentrations of pamidronate (control, 50 and 100 μM) for 48 hours. The expression of a prenylated GTPase protein, Rap1 A, was qualitatively evaluated with immunoblotting using a polyclonal goat antibody (Santa Cruz Biotechnology) in cells incubated for 48 hours with varying concentrations of pamidronate (0, 1, 10, 50, and 100 μM). Results: In cells treated with lower cytotoxic concentrations of pamidronate (50 μM) for 48 hours, the addition of GGOH, but not FOH, was able to diminish the degree cytotoxicity, p ≤ 0.05. However, cells incubated with higher cytotoxic concentrations of pamidronate (100 μM), neither GGOH nor FOH were able to reduce the level of pamidronate‐induced cytotoxicity. Caspase‐3‐like activity directly correlated with the degree of pamidronate‐induced cytotoxicity. Cells treated with pamidronate at 50 μM and 100 μM increased caspase‐3‐like activity by 5.3 and 7.1‐fold, respectively over untreated controls. The detection of Rap1A by immunoblotting requires further optimization. Conclusions: Pamidronate‐induced cytotoxicity in canine osteosarcoma cells may share a similar mechanism as has been demonstrated for osteoclasts. Inhibition of the mevalonate pathway appears to contribute to the observed cytotoxicity at lower doses of pamidronate. In addition, caspase‐3‐like activity contributes to the apoptotic process induced by pamidronate in malignant canine osteoblasts.     

Absorption of inorganic, trivalent and hexavalent chromium following oral and intrajejunal doses in rats

The intestinal absorption of trivalent and hexavalent chromium (Cr) given orally (experiment I) or infused in the intestine (experiment II) was investigated in rats. The nonabsorbable form of chromium (51Cr2O3) and water-soluble and more absorbable Na2(51)CrO4 (the hexavalent form of Cr) were compared. Total retention of chromium given orally ranged around 15 percent of the dose, regardless of the chromium compounds applied. The absorption rate of chromic oxide, which is considered a nonabsorbable compound, was 14.4 as a percentage of chromium intake. This result indicates that some loss of chromium has to be taken into account in metabolic trials made by the indicator method. In isolated rat intestine, from the injected Cr 2.5% of chromic oxide and 43.2% of sodium chromate were absorbed during an hour (experiment II). The absorbed chromium was transferred to the liver where the liver tissue retained 10.9% of chromic oxide and 51.1% of sodium chromate. Radioactivity of v. cava caudalis following intestinal injection of Na2CrO4 was thirtyfold greater than after Cr2O3 dosing. This phenomenon can be explained by the lower blood clearance of chromate. Different absorption rate of chromate depending on the route of administration could be due to the fact that the hexavalent form given orally was reduced to Cr3+ in the acidic environment of the stomach. When Na2CrO4 was infused directly in the intestine of rats, such reduction could not occur. This means that the acidic gastric juice might play a role in inhibiting the intestinal absorption of Na2CrO4 when this compound is given orally.     

Influence of canine cumulus oophorus on homologous sperm motility

Sperm ejaculated by 8 beagle dogs and the cumuli oophori collected from 3 estrous beagle bitches were co-incubated, and penetration of the sperm into cumuli was observed to investigate the influence of cumuli on homologous sperm.The percentages of hyperactivated sperm and acrosome-reacted sperm were calculated after incubation with homogenized cumuli. The hyaluronic acid content of the incubated cumuli was measured, and hyperactivation and the acrosome reaction of the sperm were evaluated in medium containing hyaluronic acid. The mean percentage of hyperactivated sperm (33.0%) and number (3.0) of sperm that had penetrated a cumulus among sperm incubated for 7 hr were significantly higher than the values for sperm incubated for 0.5 hr (P<0.01). Almost all sperm that had penetrated the cumuli had intact acrosome, as though they were hyperactivated. The percentages of motile sperm (77.3%) and hyperactivated sperm (23.6%) after 2 hr incubation in the medium containing homogenized cumuli were significantly higher than in control medium (P<0.01), but there was no difference between cumulus and control media in the percentages of acrosome-reacted sperm. The hyaluronic acid content of a cumulus increased after 24 hr incubation. After 2 and 4 hr of incubation the percentages of hyperactivated sperm in the medium containing hyaluronic acid were significantly higher than in the control medium (P<0.01). These results suggest that canine hyperactivated sperm with intact acrosome can penetrate homologous cumuli and that the sperm are able to pass through the cumulus because the hyperactivated movement is maintained by hyaluronic acid secreted by the cumulus cells.     

Cellular injury and lipid peroxidation induced by hexavalent chromium in isolated rat hepatocytes

In order to elucidate the relationship between cellular injury and lipid peroxidation induced by hexavalent chromium (CrVI), isolated rat hepatocytes treated with any one of scavengers of active oxygen species, antioxidants or antichromium agent were incubated with K2Cr2O7 as CrVI (1 mM Cr). After the incubation, the development of lipid peroxidation was determined as thiobarbituric acid (TBA)-reacting materials in total lipid extracts from the incubated hepatocytes. Cellular injury was observed as a leakage of lactate dehydrogenase (LDH) from hepatocytes into incubation medium. The contents of reduced glutathione (GSH) in hepatocytes were also assessed. Results obtained were as follows: (1) CrVI facilitated lipid peroxidation in isolated hepatocytes after 20 min of incubation. On the other hand, the cellular injury induced by CrVI was barely observed even after 60 min of incubation. (2) The CrVI-induced lipid peroxidation was inhibited by catalase and mannitol as scavengers of active oxygen species, or N,N'-diphenyl-p-phenylenediamine and alpha-tocopherol as antioxidants. However the cytotoxicity of CrVI could not be prevented by these chemicals. (3) CrVI depleted the contents of intracellular GSH and diminished the activities of glutathione reductase (GR) and glutathione-S-transferase (GST) except glutathione peroxidase. (4) The scavengers of active oxygen species and the antioxidants could not prevent the depletion of intracellular GSH induced by CrVI. (6) Ascorbic acid, antichromium agent, prevented all of the lipid peroxidation, the cellular injury and intracellular GSH depletion induced by CrVI.(ABSTRACT TRUNCATED AT 250 WORDS)     

The response of phospholipase C/protein kinase C and adenylyl cyclase/protein kinase A pathways in porcine theca interna cells to opioid agonist FK 33-824

Opioids were found as factors affecting porcine ovarian steroidogenesis. The mechanism of opioid action, however, on porcine theca interna cells is completely unknown. Therefore, the present study was designed to investigate the possible involvement of two intracellular pathways, phospholipase C/protein kinase C and adenylyl cyclase/protein kinase A, in opioid signal transduction in porcine theca cells treated with mu opioid receptor agonist, FK 33-824. Incubation of the cells for 4 h with FK 33-824 at the dose 1 nM resulted in decreases in inositol phosphate accumulation as well as androstenedione (A(4)), testosterone (T), and estradiol (E(2)) secretions. Protein kinase C (PKC) inhibitors, staurosporine (1-100 nM), D-sphingosine (10-500 nM), and PKCi (100-2000 nM), both added alone and together with the opioid agonist, depressed release of the steroid hormones. PKC activator, phorbol ester (PMA, 1-100 nM), used alone was without effect on theca cell steroidogenesis, but added in combination with FK 33-824 abolished inhibitory influence of the opioid on A(4), T, and E(2) output. The steroid hormone secretion by PKC-deficient theca cells was inhibited by the opioid agonist. FK 33-824 also suppressed PKC activity reducing [(3)H]PDBu specific binding to theca cells, whereas ionomycin (a positive control) increased labeled phorbol ester binding to the cells. In the next experiment, cAMP release from theca cells during 2 and 4 h incubations with FK 33-824 (1-100 nM), naloxone (10 microM; opioid receptor antagonist), and LH (100 ng/mL; a positive control) was examined. FK 33-824 at the dose 1 nM inhibited cAMP secretion during 2 h incubation, but had no effect during longer incubation. LH in a manner independent on incubation time multiplied cAMP release. Protein kinase A inhibitor, PKAi (100-2000 nM), alone and in combination with FK 33-824 (1 nM), inhibited A(4), T, and E(2) secretions by theca cells. PKA activator, 8BrcAMP (10-1000 microM), stimulated the steroid hormone release, but this stimulatory effect was diminished in the presence of FK 33-824. The results allow to suggest that opioid peptides affect porcine theca cell steroidogenesis and their acute action on the cells is connected with the inhibition of phospholipase C/protein kinase C and adenylyl cyclase/protein kinase A signal transduction systems.     

Natural killer cell activity in untreated and treated dogs with lymphoma

Natural killer (NK) cell activity and function were determined for 11 untreated and treated dogs with lymphoma. Concurrent chromium release and single cell binding assays, methods used to measure overall cytotoxic activity and that from individual cells, respectively, were performed at effector-to-target cell ratios of 50:1 and 100:1, with incubation periods of 12 and 16 hours. Significant reduction was achieved in overall activity for untreated dogs, using a 16-hour incubation period and an effector-to-target ratio of 100:1 (P less than 0.05). Decreased activity (P less than 0.025) was also achieved for those dogs that were administered combination chemotherapy, consisting of such drugs as cyclophosphamide, vincristine, prednisone, and doxorubicin. There was no significant difference in binding or cytotoxic activity by individual cells in the untreated or treated dogs, compared with the healthy controls. Short- or long-term treatment with glucocorticoids did not influence overall NK cll activity or individual cell cytotoxicity. The overall cytotoxic activity in untreated dogs was reduced, but these dogs had relatively normal numbers of NK cells compared with paracontrols. This suggests that a defect in recycling or the ability to kill targets repetitively, may be involved. A similar defect was found in NK cells of dogs treated aggressively with combination chemotherapy.     

Mechanisms by which a phorbol ester and a diacylglycerol analog inhibit hen granulosa cell steroidogenesis

We have previously reported that treatment of hen granulosa cells with the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), or the diacylglycerol analog, 1-oleoyl-2-acetylglycerol (OAG), attenuates the steroidogenic response to luteinizing hormone (LH) at sites both prior and distal to the formation of cyclic 3',5'-adenosine monophosphate (cAMP). The present study was designed to determine the site(s) of inhibition within the steroidogenic pathway by evaluating the effects of OAG and PMA on key enzyme systems involved in hen granulosa cell steroidogenesis: adenylyl cyclase, phosphodiesterase, the cholesterol-side-chain-cleavage (CSCC) complex and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). The adenylyl cyclase activator, forskolin (0.1 mM), stimulated a 3.3-fold increase in granulosa cell cAMP formation, and this increase was inhibited by the presence of OAG (2.5, 25 and 63 microM) in a dose-dependent manner. By contrast, a 1.8-fold increase in cAMP accumulation induced by the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX; 1.0 mM), was not altered by OAG at any dose (2.5, 25 and 63 microM). Inclusion of 25-hydroxycholesterol (2500 ng/tube) in the incubation medium in the presence of 1.0 microM cyanoketone resulted in a 10-fold increase in pregnenolone production. Increasing concentrations of OAG (2.5, 25 and 63 microM) caused a dose-dependent suppression of the conversion of 25-hydroxycholesterol to pregnenolone. On the other hand, granulosa cells incubated with 200 ng/tube pregnenolone increased progesterone production 100-fold, but this increase was not inhibited by either PMA (3.2, 32, 8.1 and 162 nM) or OAG (2.5, 25 and 63 microM). The results indicate that activation of protein kinase C can suppress the function of at least two key enzymes involved in hen granulosa cell steroidogenesis. Inhibition of adenylyl cyclase greatly reduces the steroidogenic response of granulosa cells to endocrine factors that act via increasing levels of cAMP (i.e. LH). Furthermore, a reduction in CSCC activity limits the availability of precursor required for progesterone production. These data provide additional evidence of a role for protein kinase C in modulating ovarian function in the domestic hen.     

Effects of antioxidants on induction of apoptosis in bursal cells of Fabricius during in vitro cultivation

After physically disrupting cell contacts, apoptosis of bursal cells of Fabricius was induced during in vitro cultivation. The percentage of apoptotic cells increased with incubation time and approximately 70% cells represented apoptosis after 6 hr of incubation. The induction of apoptosis was significantly inhibited by treatment of the cells with ascorbic acid (vitamin C), but not with trolox, a vitamin E analog. An intense DNA ladder pattern was shown at 6 hr post-isolation, which is a biochemical hallmark of apoptosis. Treatment of the cells with ascorbic acid inhibited the DNA fragmentation, but trolox did not. To monitor the intracellular production of reactive oxygen species (ROSs), the intensity of fluorescence emitted from DCFH-DA was measured. The intensity of fluorescence from cells incubated for 0.5-2 hr was approximately 2-fold higher than that from cells at 0 hr. The relative intensity of fluorescence decreased immediately after the addition of ascorbic acid to the cells. The intensity from the cells treated with ascorbic acid was 20-30% of that from the control cells at each incubation time. For trolox, the intensity was 50-70% of that from the control cells at each 1 to 2 hr incubation time. When ROSs-induced lipid peroxidation was assessed using cis-parinaric acid (PnA) as a monitor molecule, lipid peroxidation was found to occur in the control cells after isolation of the bursal cells. Treatment of the cells with trolox reduced lipid peroxidation, but treatment with ascorbic acid enhanced peroxidation.     

Effects of human recombinant interleukin 2 on in vitro tumor cytotoxicity in dogs.

In these studies, the effects of recombinant human interleukin 2 (rHuIL-2) were examined on in vitro tumor cytotoxicity by canine blood lymphocytes obtained from peripheral vessels through use of a chromium release microcytotoxicity assay. Cytotoxic activity by lymphokine-activated killer cells was significantly increased, compared with that by untreated lymphocytes in a dose-dependent manner. The maximal effect was attained with 300,000 IU of rHuIL-2/ml. Lymphokine-activated killing also was dependent on the duration of incubation with rHuIL-2. After 1 day of rHuIL-2 incubation, cytotoxicity was significantly increased, compared with that of untreated lymphocytes. Of the 3 times examined, cytotoxicity peaked after 3 days of rHuIL-2 incubation. High levels of cytotoxic activity were still present at 7 days of incubation. Numbers of granular lymphocytes increased over the times examined. These results demonstrate functional and morphologic changes in canine peripheral blood lymphocytes obtained from peripheral vessels after incubation with rHuIL-2 in a dose- and time-dependent manner.     

Evaluation of the induction of vasoactive mediators from equine digital vein endothelial cells by endotoxin

OBJECTIVE: To determine the effect of endotoxin (lipopolysaccharide [LPS]) on vasoactive mediator production by cultured equine digital vein endothelial cells (EDVECs). SAMPLE POPULATION: EDVECs obtained from forelimb digital veins of 7 healthy adult horses. PROCEDURES: EDVECs were incubated with or without LPS (1 microg/mL) for 0, 2, 4, 6, 22, and 24 hours. The EDVECs were incubated for 18 hours with LPS (10 pg/mL to 1 microg/mL) with or without ibuprofen, cycloheximide, or L-nitroarginine methyl ester. Medium concentrations of prostacyclin, cyclic guanosine monophosphate, endothelin-1, and thromboxane A(2) were determined. Changes in inducible nitric oxide synthase and cyclooxygenase-2 expression were determined. RESULTS: LPS stimulated mean 4.2- and 14.1-fold increases in EDVEC prostacyclin and cyclic guanosine monophosphate production, respectively, after 22 hours. These effects were LPS concentration-dependent (LPS concentrations that induced a response halfway between the maximum response and baseline of 1.50 and 1.22 ng/mL, respectively). The LPS-induced cyclic guanosine monophosphate production was significantly inhibited (to basal concentrations) by L-nitroarginine methyl ester, and prostacyclin production was inhibited by cycloheximide and ibuprofen. Production of thromboxane A(2) by EDVECs was not detected. Endothelin-1 accumulated in the medium, but LPS did not enhance its production. Inducible nitric oxide synthase expression in EDVECs was not detected with the available antibodies, whereas LPS stimulated cyclooxygenase-2 expression in a time- and concentration-dependent manner. CONCLUSIONS AND CLINICAL RELEVANCE: LPS stimulated vasoactive mediator production by equine endothelial cells, which may play a role in LPS-induced digital hypoperfusion.     

The multiplication of transmissible gastroenteritis viruses in several cell lines originated from porcine kidney and effects of trypsin on the growth of the viruses

Plaque formation, replication and related cytopathic function of 9 strains of transmissible gastroenteritis (TGE) virus were examined in primary cells and cell lines such as CPK, IB-RS-2, ESK, and PK-15 originated from porcine kidney and the effects of trypsin on the replication of TGE virus were examined in CPK cells. All strains produced a cytopathic effect and grew well in CPK cells as well as in primary porcine kidney cells. The effect of trypsin on the plaque formation was different from strains. The number of plaques produced by strains TO-163, Ukiha and Niigata increased from 2.6 to 3.52 times when trypsin was present in the medium during incubation at 37 degrees C for 1 hr after adsorption of the virus at 4 degrees C for 2 hr. The plaque sizes of TO-163, h-5, Ukiha and Niigata became larger from 1.4 to 1.7 times, when trypsin was present in the agar MEM overlay.     

In vitro effects of 5-hydroxytryptamine and cisapride on the circular smooth muscle of the jejunum of horses

OBJECTIVE: To determine effects of cisapride and 5-hydroxytryptamine (5-HT) on the jejunum of horses. SAMPLE POPULATION: Jejunal muscle strips from 8 horses. PROCEDURE: Muscle strips were suspended in isolated muscle baths. Isometric stress responses to 5-HT and cisapride, with and without specific antagonists, were determined. RESULTS: Muscle strips incubated with atropine and tetrodotoxin responded to 5-HT and cisapride with an increase in contractile force. The 5-HT caused a concentration-dependent increase in contractile amplitude, with a maximum response (Emax) of 1,151+/-214 g/cm2 and a molar concentration that induces contractile force equal to 50% of maximum response (EC50) of 0.028+/-0.002 microM. Prior incubation with the 5-HT2 antagonist ketanserin decreased the Emax (626 +/-147 g/cm2) and potency (EC50, 0.307+/-0.105 microM) of 5-HT Prior incubation with the 5-HT3 antagonist tropisetron decreased the efficacy (Emax, 894+/-184 g/cm2) to 5-HT Cisapride also caused a concentration-dependent increase in contractile amplitude, with an Emax of 331+/-82 g/cm2 and an EC50 of 0.302+/-0.122 microM. Prior incubation with ketanserin decreased the Emax (55+/-17 g/cm2) and potency (EC50, 0.520+/-0.274 microM) of cisapride. CONCLUSION AND CLINICAL RELEVANCE: Stimulatory effects of 5-HT and cisapride on circular smooth muscle of equine jejunum are mediated primarily through a noncholinergic effect. The effects of 5-HT are mediated, at least partially, by 5-HT2 and 5-HT3 receptors, whereas the effects of cisapride are mediated primarily by 5-HT2 receptors. This may impact treatment of horses with postoperative ileus.     

Influence of conditioned media from bovine cotyledon tissue cultures on function of bovine neutrophils.

Bovine fetal placental (cotyledon) tissue obtained from pregnant cows on days 255, 265, and 275 of gestation, as well as immediately after parturition (n = 5) was incubated in media for 48 hours, and the incubation media were collected. Neutrophils from 4 ovariectomized nonpregnant cows were incubated for 2 hours with conditioned media from placental tissue cultures or medium (control). Immediately after incubation, the neutrophils were subjected to the following leukocyte function assays: chemotaxis against zymosan-activated serum, chemotaxis against undiluted conditioned media (only neutrophils that were incubated in medium only), random migration, ingestion of 125I-iododeoxyuridine Staphylococcus aureus (125I-IdUR-S aureus), iodination of proteins, cytochrome C reduction, and antibody-independent and -dependent cell-mediated cytotoxicity. Conditioned media from cultured cotyledon tissue was chemoattractant for bovine neutrophils, and increased chemotactic response of neutrophils against zymosan-activated serum by 13%. The following neutrophil functions were decreased: random migration by 25%, iodination of proteins by 44%, cytochrome C reduction by 13%, and antibody-dependent cell-mediated cytotoxicity by 5%. Ingestion of 125I-IdUR-S aureus and antibody-independent cell-mediated cytotoxicity were not influenced by coincubation of neutrophils and conditioned media. Time of gestation did not alter the effects of conditioned media on neutrophil function. It was concluded that chemotactic properties of cotyledon tissue extracts, as has been reported earlier, may be attributable to substances released by fetal placental tissue. Those substances might also locally or systemically influence the oxygen-dependent antimicrobial system of neutrophils, thereby causing an increased susceptibility to bacterial infections in the peripartum period.     

Mechanisms of NO-resistant relaxation induced by acetylcholine in rabbit renal arteries

The effects of K+ channel blockers and P2Y receptor agonist/antagonist on the vasorelaxation mediated by endothelium-derived hyperpolarizing factor (EDHF) were investigated in the rabbit renal artery. Acetylcholine (ACh, 1 nM-10 microM) induced endothelium-dependent relaxation of arterial rings precontracted with norepinephrine (NE, 1 microM) in a concentration-dependent manner. NG-nitro-L-arginine (L-NAME. 0.1 mM), an inhibitor of NO synthase, partially inhibited the ACh-induced endothelium-dependent relaxation. The ACh-induced relaxation was only partially inhibited by L-NAME whereas combined addition of L-NAME and 30 mM KCl completely inhibited the relaxation. The ACh-induced relaxation observed in the presence of L-NAME was significantly reduced by a combination of iberiotoxin (0.1 microM) and apamin (1 microM), and almost completely blocked by 4-aminopyridine (5 mM). The ACh-induced relaxation was antagonized by P2Y receptor antagonist, cibacron blue (10 and 100 microM) in a concentration-dependent manner. Furthermore, ADPbetaS, a potent P2Y agonist, induced the endothelium-dependent relaxation, and this relaxation was markedly reduced by either the combination of iberiotoxin and apamin or by cibacron blue alone. In conclusion, ACh may activate the release of ATP from endothelial cells which in turn activates a P2Y receptor on the endothelial cells followed by a release of EDHF, resulting in a vasorelaxation via a mechanism that involves activation of both the voltage-gated K+ channels and the Ca2+-activated K+ channels. EY WORDS: ATP, K+ channel, rabbit renal artery.