The influence of nitric oxide on the contractile activity of the isolated porcine ovarian and uterine arteries

The aim of the present study was to investigate the influence of nitric oxide (NO) on the contractile activity of the isolated porcine ovarian and uterine arteries. Segments of the vessels, obtained from the pigs on days 1-5, 8-13 and 17-20 of the oestrous cycle, were mounted in the organ bath with Krebs-Ringer solution and contractile activity changes of the vessels were measured using isometric transducers. In Experiment I the arteries pretreated with norepinephrine (NE; 10(-7) M) were treated with sodium nitroprusside (SNP, 10(-8)-10(-4) M), a NO donor. In Experiment II administration of NE (10(-7) M) was preceded by treatment with Nomega-nitro-L-arginine methyl ester (L-NAME, 10(-8)-10(-6) M), an inhibitor of NO synthase. Donor of NO at doses of 10(-8)-10(-7) M did not affect (P>0.05) the contractility, while at doses of 10(-5)-10(-4) M caused a dose-dependent relaxation (P<0.05) of both ovarian and uterine arteries in all periods examined. Moreover, SNP at doses of 10(-6)-10(-4) M it caused significantly higher (P<0.05) relaxation of the ovarian arteries collected on days 8-13 as compared to the vessels from days 1-5 of the cycle. Pretreatment of the vessels with L-NAME caused a dose-dependent, significant (P<0.05) increase in the vasocontractile action of NE in both the ovarian and uterine arteries as compared to contractile activity of NE administered alone. Moreover, L-NAME pretreatment at a dose of 10(-6) M caused significantly higher (P<0.05) intensification of NE action in ovarian and uterine arteries collected on days 8-13 as compared to the vessels from days 1-5 (P<0.05) and 17-20 (P<0.05) of the oestrous cycle. Obtained results indicate that NO plays an important role in the regulation of the contractile activity of the isolated porcine ovarian and uterine arteries. Our data suggest that this action may be, at least in a part, dependent on the hormonal status of the organism.     

Regional distribution of porcine brain blood flow during 50% nitrous oxide administration

Regional distribution of brain blood flow was examined in 6 healthy nonmedicated swine during inhalation of 50% O2 (+ 50% N2) and at 45 minutes of 50% end-tidal nitrous oxide administration. All animals were surgically prepared 10 to 12 days before the hemodynamic study. Catheters were implanted in the left atrium, ascending aorta, descending aorta, and pulmonary artery. Brain blood flow was determined, using 15-micron diameter radionuclide-labeled microspheres injected into the left atrium. Administration of 50% nitrous oxide markedly increased blood flow in all regions of the brain (except corpus callosum), even though the animals were not excited and the arterial blood pressure, arterial blood-gas tensions, pHa, and cardiac output were not different from respective control values. At 45 minutes of 50% nitrous oxide administration, cerebral, cerebellar, and brain-stem blood flows were 144%, 137%, and 137% of respective control values. It is concluded that 50% nitrous oxide administration caused marked vasodilatation in all regions of the porcine brain.     

Effect of the side of ovulation on the uterine morphometry,blood flow,progesterone, oestradiol and nitric oxide during spontaneous and induced oestrus in lactating dairy cows

This study investigates the changes in endometrial thickness between ipsilateral and contralateral uterine horns, diameter, blood flow area and hormonal concentrations in cows during natural and induced oestrus. In the induced group, six cows received a controlled internal device insert for 7 days. In the normal and induced oestrous group (n = 12), Doppler was performed day after day from day 5 till day 15. Oestradiol, progesterone and nitric oxide metabolites (NOMs) were measured. Results showed that dorsal, ventral and cross-section diameter of the ipsilateral horn increased during the induced oestrous group to reach a maximum on day 2 than during normal one. The total coloured area in both horns in a normal and induced group was also affected (p < .001), as the total coloured area in the ipsilateral horns, increased in the induced group, reach a maximum on day 2 after ovulation, while in normal it reaches a maximum on day of ovulation. Oestradiol increased during the induced cycle than during the normal one, but progesterone increased during days after ovulation reaches a higher value on day 10 in both groups. Nitric oxide showed two peaks, one on the day 3 and another peak on day 5. Conclusion: The ipsilateral uterine horn different diameters and thickness increased more rapidly in the induced oestrous group than the natural oestrus group. A complete analysis of variations in P4 and E2 concentrations and endometrial thickness suggested that decreases in P4 concentrations were related to an increase in endometrial thickness and that increases in E2 concentrations enhanced these endometrial changes.     

Direct measurements of nitric oxide release in relation to expression of endothelial nitric oxide synthase in isolated porcine mitral valves

The aim of this study was to measure the direct release of nitric oxide (NO) from the porcine mitral valve using a NO microelectrode. Furthermore, the expression and localization of endothelial nitric oxide synthase (eNOS) in the mitral valve was studied using immunohistochemistry, Western blotting and RT-PCR. Results show that bradykinin increases NO release from mitral valves (DeltaBradykinin: 33.71 +/- 10.41 nm NO, P < 0.001, n = 10), whereas N-nitro-l-arginine methyl esther (l-NAME) decreases NO release when compared with basal level (Deltal-NAME: 82.69 +/- 15.66 nm NO, P < 0.005, n = 4). Both protein and mRNA expression of eNOS in mitral valves and in isolated valvular endothelial cells suggest that the NO release is mainly associated with the mitral valve endothelium. It is concluded that direct NO release from porcine mitral valves coincides with eNOS expression. This study documents useful techniques for investigations into the role of local NO release in mitral valve diseases.     

Use of ultrasonic Doppler waveforms to estimate changes in uterine artery blood flow and vessel compliance

Previous data from our laboratory have demonstrated that uterine blood flow (UBF) and uterine arterial smooth muscle tone vary regularly during the estrous cycle of the cow. Uterine blood flow is highest and uterine arterial tone is lowest at estrus, whereas UBF is lowest and uterine arterial tone is highest during the luteal phase of the cycle. Blood flow through arteries is highly pulsatile; changes in arterial wall properties affect shape of the velocity waveform. This study was conducted to evaluate changes in uterine arterial velocity waveforms throughout the estrous cycle of the cow and to relate these changes to fluctuations in UBF and concentrations of estrogen and(or) progesterone in systemic blood. Pulsatile velocity waveforms were obtained daily from pulsed-wave ultrasonic probes placed surgically on the middle uterine artery of five beef cows exhibiting estrous cycles of normal duration (d 0 = day of estrus). Velocity waveforms varied regularly during the estrous cycle of each cow in association with changes in UBF and steroid concentrations. Further, two distinct velocity waveform shapes were observed during the estrous cycle. The first waveform shape, which was observed during periods of high UBF (d -4 to +4 of the estrous cycle), was characteristic of a highly compliant vessel and was associated with a high estrogen:progesterone ratio. The second waveform shape, which was observed from d 7 to 14 of the estrous cycle, was characteristic of a less compliant vessel and was associated with a depressed estrogen:progesterone ratio. These data suggest that compliance of the uterine artery changes during the estrous cycle in association with the changing estrogen:progesterone ratio in blood.     

Expression of nitric oxide synthase-3 in porcine oocytes obtained at different follicular development

The present study was designed to determine the localization of nitric oxide synthase-3 (NOS-3) in porcine follicles during follicular development. A 130-kDa NOS-3 protein was found with greater frequency much in the oocytes than in the cumulus cells, as revealed by Western blotting analysis. The content of NOS-3 in the oocyte was higher in large follicles (> 7-mm diameter) than in small follicles (< 2-mm). The data by Western blotting showed the same pattern as the observations obtained from the immunohistochemical studies, in which the periphery of the oocyte stained strong positive. The inner surface cell layer of granulosa cells and cumulus cells were positive staining, especially in large antral follicles. In the primordial follicles, NOS-3 was restricted to the cytoplasm of oocytes, and no stained product was observed in the nucleus of oocytes or granulosa cells. A significant synthesis of NO by oocytes was observed in the presence of ionomycin, but not in the absence of ionomycin, indicating that oocyte NOS-3 functions in response to transient elevations in the intracellular calcium level. We concluded that NOS-3 is expressed in the oocyte from the primordial follicular stage to antral follicular stage, and that it is functional at least in the antral follicles.     

Luteotrophic and luteolytic effects of nitric oxide in sheep are dose-dependent in vivo

It has been suggested that nitric oxide (NO) acts in either an anti-luteolytic or in a luteolytic manner, but the mechanism for these opposing roles is unclear. We hypothesized that NO may act in a dose-dependent manner to regulate luteal function, whereby low concentrations of NO might stimulate luteal progesterone production (i.e. luteotrophic) and high concentrations of NO might reduce concentrations of plasma progesterone (i.e. luteolytic). To test this hypothesis we infused increasing concentrations of the fast-acting NO donor, dipropylenetriamine NONOate (DPTA), into the arterial supply of sheep with ovarian transplants bearing a corpus luteum (CL). Infusions were performed on sheep with CL 11 days of age (n=9) or over 30 days of age (n=15). We measured changes in the concentration of progesterone in ovarian venous plasma during the 1-h infusion and for 24h after the infusion, and then compared the mean concentration of progesterone between treatment groups for effects by dose and dose by period interactions. Compared with saline-treated controls (n=6), the highest dose of 1000 microg/min DPTA (n=6) reduced (P0.05) in sheep infused with the lowest dose of 1 microg/min DPTA (n=6) compared with controls. We conclude that NO regulates luteal function in a dose-dependent manner in sheep in vivo.     

Collateral uterine blood flow in Holstein cows during the third trimester of pregnancy

Blood flow to the gravid and nongravid uterine horns of four multiparous Holstein cows (mean +/- SD, BW=641.8 +/- 95.4 kg; age=4.8 +/- 1.2 years; parity=3.0 +/- 1.2) was measured on days 225, 248, and 266 of gestation. Surgery was conducted on day 214.5 +/- 4.0 of gestation through the flank of the standing cows. Transit-time ultrasonic flow probes (diameter 12 or 14 mm) were fitted surgically around the uterine arteries of each cow. Surgery was completed within two hours of anesthesia, and the animals recovered rapidly following surgery. Uterine blood flow (UBF, l/min) was recorded at 10 sec intervals for approximately 23.5 hours; these values were averaged to determine UBF. The mean gravid UBF was significantly (P<0.05) greater than the nongravid UBF in this study. The range of the gravid and nongravid UBFs varied from 3.61 to 14.05 and 0.72 to 6.54 l/min, respectively. There were no changes (P>0.1) in the mean gravid and nongravid UBFs from day 225 to 266 of gestation.     

Effects of topical nitroglycerine patches and ointment on digital venous plasma nitric oxide concentrations and digital blood flow in healthy conscious horses

OBJECTIVE: To measure and compare palmar digital venous plasma nitric oxide (NO) concentrations and digital arterial blood flow after application of topical nitroglycerine (NTG). STUDY DESIGN: Experimental study. ANIMALS: Healthy adult horses (n=8). METHODS: Digital blood flow was measured by an ultrasonic Doppler flow probe surgically implanted around the medial palmar digital artery. Blood was collected from a catheter placed in the medial palmar digital vein for quantification of NO. NTG patches, NTG ointment or control patches were placed over the palmar digital vessels at the level of the fetlock. Two horses had an intra-arterial infusion of an NTG solution into the medial palmar digital artery in a pilot study. RESULTS: Digital arterial blood flow did not change significantly with application of the NTG patches, NTG ointment, or control patches. There were no statistically significant or biologically important changes in digital venous NO concentrations across time or between treated and control horses. In the pilot study, digital arterial blood flow and palmar digital venous NO concentrations increased with intra-arterial infusion of NTG. CONCLUSIONS: In clinically healthy horses, digital arterial blood flow and digital venous plasma NO concentrations did not change significantly with application of the NTG patches/ointment. These treatments are unlikely to have an effect on the digital vasculature of laminitic horses, however, further investigation is warranted. CLINICAL RELEVANCE: Although NTG patches have been used as a method of decreasing vasomotor tone and improving digital blood flow in horses with laminitis, this study provides evidence in healthy conscious horses that this treatment is not effective in altering digital blood flow.     

Influence of infused beta-adrenergic agonists on porcine blood metabolites and catecholamines

Anesthetized pigs were infused sequentially with increased concentrations of beta-adrenergic agonists. At selected times during infusion, blood pressure, heart rate and plasma concentrations of free fatty acids (FFA), glycerol, glucose, lactate, norepinephrine, epinephrine and dopamine were measured. Azaperone, a drug used to calm the pigs before anesthesia, caused hypotension and bradycardia but did not affect plasma metabolites. Infusion of norepinephrine, epinephrine, isoproterenol or clenbuterol produced changes in plasma metabolites and plasma catecholamines. These changes during norepinephrine infusion were attributed to the infused agonist, whereas those during epinephrine infusion might have resulted to some extent from release of norepinephrine. Plasma isoproterenol was not quantified because it interfered with the assay of epinephrine and dopamine so that it was not possible to distinguish between infused isoproterenol and release of endogenous epinephrine and dopamine. Infusion of clenbuterol caused a small increase in plasma norepinephrine so that some of the increase in plasma FFA, glycerol and lactate during clenbuterol infusion may result from release of endogenous norepinephrine.     

Upregulation of cyclooxygenase-2 expression in porcine macula densa with chronic nitric oxide synthase inhibition

The objective of this study was to investigate the effects of chronic inhibition of nitric oxide synthase (NOS) on cyclooxygenase-2 (COX-2) expression in the macula densa (MD) of swine, as well as the effects on expression of related proteins. Adult female Yucatan swine were given either tap water (control, n = 6) or water with N (G)-nitro-L-arginine methyl ester (L-NAME, 100 mg/liter, n = 5) for a minimum of 30 days. Duplicate samples of kidney were fixed or snap frozen. There was a significant (P = .0082) upregulation of COX-2 mRNA expression in the MD of L-NAME, as well as an apparent increase in COX-2 protein. Plasma renin activity also increased with L-NAME treatment (control, 0.34 ± 0.08 ng/ml; L-NAME, 1.26 ± 0.03 ng/ml; P = .00000003). There were no differences between groups in expression of either inducible NOS or renin protein or in serum electrolyte concentrations. In conclusion, with chronic inhibition of NOS, COX-2 in MD is upregulated, perhaps to compensate for loss of nitric oxide. Increases in COX-2 products may counteract renal arteriolar constriction and sustain renin release.     

In vitro effects of Actinobacillus pleuropneumoniae on inducible nitric oxide synthase and cyclooxygenase-2 in porcine alveolar macrophages

OBJECTIVE: To determine the amount of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) activity in alveolar macrophages in response to Actinobacillus pleuropneumoniae (APP) by determining nitric oxide (NO) and prostaglandin E2 (PGE2) concentrations. SAMPLE POPULATION: Freshly isolated porcine alveolar macrophages. PROCEDURE: Alveolar macrophages were incubated for 48 hours with APP (1 X 10(4) colony-forming units/mL), interleukin-1beta, (IL-1beta; 5 U/mL), tumor necrosis factor-alpha (TNFalpha; 500 U/mL), interferon-gamma (IFN-gamma, 100 U/mL), or lipopolysaccharide (LPS; 10 microg/mL). In a second experiment, alveolar macrophages were incubated with fresh medium (negative control), APP alone, or APP with 1 of the following: IL-1beta, TNF-alpha, or IFN-gamma. In a third experiment, alveolar macrophages were incubated with fresh medium (negative control), LPS (positive control), APP alone, or APP with 1 of the following: an iNOS inhibitor (3.3 microM), a COX-2 inhibitor (10 microM); or both the iNOS and COX-2 inhibitors. Supernatant was obtained at 0, 3, 6, 9, 12, 24, and 48 hours after treatment for determination of NO and PGE2 production. RESULTS: The addition of APP to alveolar macrophages resulted in significant increases in NO and PGE2 production. The addition of APP and IFN-gamma synergistically induced NO production. Inhibition of iNOS and COX-2 decreased NO and PGE2 production, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: In vitro activation of alveolar macrophages by APP results in increased production of NO and PGE2. Nitric oxide and PGE2 production appears to be largely dependent on iNOS and COX-2 activity. Pharmacologic modulation of iNOS and COX-2 activity may represent a therapeutic target for pigs with pleuropneumonia.     

Significance of nitric oxide concentration in plasma and uterine secretes with puerperal endometritis in dairy cows

Endometritis is an inflammation of the endometrial lining of the uterus without systemic signs, which is associated with chronic postpartum infection of the uterus with pathogenic bacteria. Nitric oxide (NO) is an inflammatory mediator that among other effects causes smooth muscle relaxation and mediated cytoimmunity and inflammation toxicity. To see if the nitric oxide concentration in plasma and uterine secrets is related with postpartum endometritis, NO concentrations in plasma and uterine secrets were measured in dairy cows with puerperal endometritis (clinical endometritis (n = 60) and subclinical endometritis (n = 58)). Cows with clinical or subclinical endometritis showed higher concentrations of NO in both plasma and uterine secrets when compared with normal cows and the highest concentrations of NO in plasma and uterine secrets were found in dairy cows with clinical endometritis. Expression level of NOS2 mRNA in endometrial biopsies from cows with puerperal endometritis was also higher and the highest expression of NOS2 mRNA was found in cows with clinical endometritis. The results showed that concentrations of NO in plasma and uterine fluid are related with the degree of endometritis which may be useful to diagnose the endometritis in dairy cows.     

The effect of intravenous insulin infusion on renal blood flow in conscious sheep is partially mediated by nitric oxide but not by prostaglandins

To test the effect of insulin on renal perfusion and the participation of NO and PG as mediators of this response, renal blood flow (RBF) was measured in sheep (n = 8) implanted with ultrasonic flow probes around renal arteries and with a systemic arterial pressure (SAP, n = 4) telemetry device. Three protocols were performed: 1) RBF and SAP were recorded (0800 to 1800 h) in fed and fasted sheep, with the latter receiving intravenous (i.v.) infusions (0.5 mL/min) of insulin at 2 or 6 mU/(kg·min); 2) fasted sheep received i.v. infusions of either an inhibitor of NO synthesis (N(G)-nitro-L-arginine methyl ester, L-NAME) alone [0.22 mg/(kg·min), 1000 to 1200 h] or L-NAME (1000 to 1200 h) + insulin during the second hour (6 mU/(kg·min), 1100 to 1200 h); and 3) the same protocol was followed as in protocol 2, substituting L-NAME with ketoprofen [0.2 mg/(kg·min)], a cyclooxygenase inhibitor. In all protocols, plasma insulin and glucose were determined. During insulin administration, euglycemia was maintained and hypokalemia was prevented by infusing glucose and KCl solutions. After the onset of meals, a long-lasting 18% increase in RBF and a 48% insulin increase were observed (P < 0.05), without changes in SAP. Low- and high-dose insulin infusions increased RBF by 19 and 40%, respectively (P < 0.05). As after meals, the increases in RBF lasted longer than the insulin increase (P < 0.05). The L-NAME infusion decreased RBF by 15% (P < 0.05); when insulin was added, RBF increased to preinfusion values. Ketoprofen decreased RBF by 9% (P < 0.05); when insulin was added, RBF increased to 13% above preinfusion values (P < 0.05). In no case was a modification in SAP or glucose noted during the RBF changes. In conclusion, insulin infusion mimics the meal-dependent increase in RBF, independent of SAP, and lasts longer than the blood insulin plateau. The RBF increase induced by insulin was only partially prevented by L-NAME. Ketoprofen failed to prevent the insulin-dependent RBF increase. Both facts suggested that complementary vasodilatatory agents accounted for the insulin effect on sheep renal hemodynamics.     

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.     

Effects of nitric oxide donor and nitric oxide synthase inhibitor on ruminal contractions in conscious sheep

The present study was planned to evaluate a role of nitric oxide (NO) in the regulation of regular ruminal contractions in conscious sheep. Intravenous infusion of S-nitroso-acetyl-DL-penicillamine (SNAP) at doses of 3-30 nmol kg(-1) min(-1)for 30 minutes inhibited both the amplitude and frequency of ruminal contractions in a dose-dependent manner. However, intravenous infusion of Nomega-nitro-L-arginine-methyl ester (L-NAME) at doses of 0.3-3.0 micromol kg(-1) min(-1)did not alter the basal tone of intraruminal pressure and the amplitude of ruminal contractions. The frequency of contractions was slightly inhibited by L-NAME infusion at 1.0 micromol kg(-1)min(-1). The effects of L-NAME were abolished by simultaneous infusion of L -arginine at 30 micromol kg(-1) min(-1). These results suggest that exogenous NO can diminish the ruminal contractions, while endogenous NO is not involved in the regulatory mechanism of basal tone and regular phasic contractions of the rumen in healthy sheep.     

Characterization of porcine peripheral blood leukocytes by light-scattering flow cytometry.

As a basis for other experiments using flow cytometry of porcine peripheral blood leukocytes, cell fractions were isolated by various methods and analyzed by forward angle light scatter and 90 degree light scatter. Cytospin smears of cell samples were also studied by leukocyte differential counts and nonspecific esterase staining. Three main populations of peripheral blood leukocytes [lymphocytes, monocytes, and granulocytes (primarily neutrophils)], were defined in the log 90 degree light scatter by forward angle light scatter histogram. Partial overlap was observed between lymphocyte and monocyte, and between monocyte and granulocyte domains. Correlation between leukocyte differential counts and flow cytometric quantification based on bitmap statistics of appropriate domains was between r = 0.872-0.892 for lymphocyte and granulocyte. Percoll density gradients were used for subfractionation of leukocyte populations, especially for the enrichment of granulocytes. The specific densities were calculated for lymphocytes (1.0585-1.0819 g/cc), monocytes (1.0585-1.0702 g/cc), granulocyte (1.0819-1.0936 g/cc), and erythrocytes (greater than 1.0952 g/cc). We suggest that light scatter characterization is a basis for future studies of porcine blood by flow cytometry.