Oclacitinib, a Janus Kinase Inhibitor, Reduces the Frequency of IL-4- and IL-10-, but Not IFN-γ-, Producing Murine CD4+ and CD8+ T Cells and Counteracts the Induction of Type 1 Regulatory T Cells.

The purpose of the present study was to broaden the knowledge and understanding of the effects of oclacitinib (OCL), a Janus kinase inhibitor, on T cells in the context of both the immune mechanisms underlying anti-inflammatory and anti-allergic properties of the drug and its safety. The results indicate that beneficial effects of OCL in the treatment of skin allergic diseases may be partially mediated by the inhibition of IL-4 production in CD4+ and CD8+ T cells. To a certain extent, the antiproliferative effect of OCL on CD8+ T cells may also contribute to its therapeutic effect. The study found that OCL does not affect the proliferation of CD4+ T cells or the number of IFN-γ- and IL-17-producing CD4+ and CD8+ T cells. Moreover, OCL was found to counteract the induction of type 1 regulatory T (Tr1) cells and to act as a strong inhibitor of IL-10 production in both CD4+ and CD8+ T cells. Thus, these results indicate that beneficial effects of OCL in the treatment of skin allergic diseases are not mediated through: (a) the abolishment of IFN-γ and IL-17-production in CD4+ and CD8+ T cells; (b) generation of Tr1 cells; (c) inhibition of CD4+ T cell proliferation; (d) induction of IL-10 production in CD4+ T cells. The results of this study strongly suggest that, with respect to the evaluated parameters, OCL exerts a suppressive effect on Th2- but not Th1-mediated immunity.

The pharmacokinetics and antiparasitic activity of ivermectin in Hutsul and Toric horses.

The aim of this study was to compare the pharmacokinetics of ivermectin and its antiparasitic activity in two horse breeds. Eight Hutsul and 14 Toric horses were administered ivermectin orally at a dose of 0.2 mg/kg body weight. Blood samples were collected for 96 hr, and faecal samples were collected one day before and on days 14 and 21 after drug administration. Ivermectin concentrations in plasma samples were determined by high-performance liquid chromatography. Ivermectin concentration was significantly higher in Toric than in Hutsul horses 90 min after ivermectin administration and was maintained at higher level for up to 96 hr. The area under the concentration versus the time curve from 0 to the last sampling point (AUC0→t ) and the maximum plasma concentration (Cmax ) were significantly higher in Toric than in Hutsul horses (1792.09 ± 246.22 µg × hr/L vs. 716.99 ± 255.81 µg × hr/L and 62.72 ± 17.97 ng/ml vs. 35.34 ± 13.61 ng/ml, respectively). No parasitic eggs were found in the faecal samples collected from both groups of horses on days 14 and 21 after drug administration. The obtained results indicate that although the pharmacokinetics of ivermectin may differ significantly between horse breeds, these differences do not affect the effectiveness of therapy.

Oclacitinib depletes canine CD4+ and CD8+ T cells in vitro.

Oclacitinib (OCL) is a novel immunosuppressive agent approved for dogs that controls itch and inflammation in allergic disease via the inhibition of the JAK/STAT pathway. This paper investigates the in vitro effect of OCL, a novel Janus kinase inhibitor, on selected canine regulatory (Treg) and effector (Teff) CD4+ and CD8+ T cells. Exposure of peripheral blood lymphocytes to OCL did not affect the transcription factor Foxp3 (Forkhead Box P3 protein) expression in CD25+CD4+ and CD25+CD8+ T cells. Moreover, OCL did not influence constitutive CD25 expression on these cells although it reduced the activation-induced CD25 expression on CD4+ T cells. Unexpectedly, the research demonstrated the cytoreductive and proapoptotic effects of OCL on the cells examined. Exposure to OCL caused a dramatic loss of both CD4+ and CD8+ T cells and this effect was observed in both Treg and Teff cell subsets. On the one hand, cytoreductive and proapoptotic effects of OCL toward CD4+ and CD8+ Teff cells, as well as the drug-induced inhibition of CD4+ T cell activation, may be considered as additional mechanisms involved in producing anti-inflammatory and anti-allergic properties of the drug. On the other hand, these effects also represent the immunosuppressive action in the sense of an unwanted effect because CD4+ and CD8+ Teff cells play a crucial role in the production of cellular immunity. Further studies are needed to determine whether the use of OCL actually creates the risk of such action.

Prostaglandin E2 exerts the proapoptotic and antiproliferative effects on bovine NK cells.

The aim of this research was to determine whether prostaglandin E2 (PGE2) affects bovine NK cells in respect of their counts, apoptosis and proliferation, and if it does, then which of the PGE2 receptor (EP) subtype(s) mediate(s) these effects. We here report that long-term, but not short-term, exposure of bovine peripheral blood mononuclear cells to PGE2 at 10(-5)M, 10(-6)M and 10(-7)M, but not at 10(-8)M, caused a significant increase in the percentage of early apoptotic cells among NK cell subset. Moreover, PGE2 at 10(-5)M and 10(-6)M, but not at 10(-7)M and 10(-8)M, induced a considerable decrease in the absolute count of NK cells. The magnitude of these effects increased with an increasing concentration of PGE2. The blockade of EP1, EP2, EP3 and EP4 receptors did not prevent the PGE2-induced apoptosis and depletion of NK cells. The results suggest that the proapoptotic effect of PGE2 is secondary in character and the induction of this effect is not mediated through EP receptors. Furthermore, the studies demonstrated that PGE2 at 10(-5)M and 10(-6)M, but not at 10(-7)M and 10(-8)M, highly significantly reduced the percentage of proliferating NK cells. The EP1, EP1/2 and EP3 receptor antagonists were unable to block this effect significantly, whereas the selective blockade of EP4 receptors prevented the PGE2-induced inhibition of NK cells proliferation. These results indicate that PGE2 at certain concentrations may impair the proliferation of NK cells and this effect is mediated via the EP4 receptor.

Involvement of regulatory T cells and selected cytokines in the pathogenesis of bronchial asthma.

Asthma pathogenesis is complex and involves the interplay of many factors and actions. Airway inflammation in allergic asthma is characterized by an exaggerated activation of T helper type 2 cells, IgE production and infiltration and activation of eosinophils. The results of studies conducted in recent years indicate that the deficit of naturally occurring Foxp3+CD25+CD4+ and Foxp3+CD25+CD8+ regulatory T cells and type 1 regulatory T cells plays a pivotal role in the development of this disease. Moreover, numerous studies have provided convincing evidence that a decrease in IL-10 production and an increase in IL-17 production have an important place in the pathophysiology of asthma. TGF-ß is another important cytokine involved in this disease. TGF-ß has a paradoxical status in relation to asthma pathogenesis because it seems to play a role in both suppressing and promoting asthma development. This review discusses briefly clinical and experimental data concerning the involvement of T regulatory cells and IL-10, IL-17 and TGF-ß in the pathogenesis of asthma.

The influence of prostaglandin E2 on the production of IFN-γ by bovine CD4(+), CD8(+) and WC1(+) T cells.

The aim of these studies was to assess the influence of prostaglandin E2 (PGE2) on the production of interferon-gamma (IFN-γ) by bovine CD4(+), CD8(+), and WC1(+) T cells and furthermore, should this effect exist, to identify the E-prostanoid (EP) receptor subtype(s) responsible for this influence. We here report that exposure of bovine peripheral blood mononuclear cells (PBMCs) to PGE2 significantly and dose-dependently decreased the percentage of IFN-γ-producing CD4(+) and CD8(+) T cells. It was also shown that PGE2 reduced IFN-γ production by WC1(+) T cells, but this effect was not dose dependent. The impairment of IFN-γ production should be recognized as an anti-inflammatory and immunosuppressive action, thus the obtained results confirm the paradoxical status of PGE2 as a proinflammatory factor with immunosuppressive activity. The blockade of EP1, EP2, EP3, and EP4 receptors did not prevent PGE2-induced reduction of IFN-γ production by CD4(+) and CD8(+) T cells, indicating that this effect of PGE2 is not mediated through EP receptors. On the contrary, the blockade of either EP2 or EP4 receptors, but not EP1 or EP3 receptors, prevented the PGE2-induced reduction of percentage of IFN-γ-producing WC1(+) T cells. These findings indicate that the ability of PGE2 to impair IFN-γ production by WC1(+) T cells is mediated via EP2 and EP4 receptors. These results suggest the possibility of pharmacological manipulation of IFN-γ production by WC1(+) T cells via selective antagonists and agonists of EP2 and EP4 receptors.

IκB kinase ß inhibitor, IMD-0354, prevents allergic asthma in a mouse model through inhibition of CD4(+) effector T cell responses in the lung-draining mediastinal lymph nodes.

IκB kinase (IKK) is important for nuclear factor (NF)-κB activation under inflammatory conditions. It has been demonstrated that IMD-0354, i.e. a selective inhibitor of IKKß, inhibited allergic inflammation in a mouse model of ovalbumin (OVA)-induced asthma. The present study attempts to shed light on the involvement of CD4(+) effector (Teff) and regulatory (Treg) T cells in the anti-asthmatic action of IMD-0354. The animals were divided into three groups: vehicle treated, PBS-sensitized/challenged mice (PBS group); vehicle treated, OVA-sensitized/challenged mice (OVA group); and IMD-0354-treated, OVA-sensitized/challenged mice. The analyzed parameters included the absolute counts of Treg cells (Foxp3(+)CD25(+)CD4(+)), activated Teff cells (Foxp3(-)CD25(+)CD4(+)) and resting T cells (CD25(-)CD4(+)) in the mediastinal lymph nodes (MLNs), lungs and peripheral blood. Moreover, lung histopathology was performed to evaluate lung inflammation. It was found that the absolute number of cells in all studied subsets was considerably increased in the MLNs and lungs of mice from OVA group as compared to PBS group. All of these effects were fully prevented by treatment with IMD-0354. Histopathological examination showed that treatment with IMD-0354 protected the lungs from OVA-induced allergic airway inflammation. Our results indicate that IMD-0354 exerts anti-asthmatic action, at least partially, by blocking the activation and clonal expansion of CD4(+) Teff cells in the MLNs, which, consequently, prevents infiltration of the lungs with activated CD4(+) Teff cells. The beneficial effects of IMD-0354 in a mouse model of asthma are not mediated through increased recruitment of Treg cells into the MLNs and lungs and/or local generation of inducible Treg cells.

Pharmacokinetics of oxytetracycline in broiler chickens following different routes of administration.

The aim of this study was to determine the pharmacokinetics of oxytetracycline (OTC) in broiler chickens following intravenous (IV), intramuscular (IM), subcutaneous (SC) and oral (PO) administrations at a dose of 15 mg/kg bodyweight. Plasma concentrations of OTC were determined using liquid chromatography-tandem mass spectrometry and non-compartmental pharmacokinetic analysis was then conducted. The absorption half-life time was 1.23 ± 0.36 h, 1.19 ± 0.52 h, and 0.49 ± 0.38 h after IM, SC and PO administration, respectively. The elimination half-life time was 27.41 ± 6.06 h, 10.23 ± 4.20 h, 7.83 ± 0.56 h, and 14.86 ± 9.23 h, and the mean residence time was 9.67 ± 1.7 h, 11.45 ± 1.76 h, 11.38 ± 0.59 h, and 10.37 ± 3.91 h after IV, IM, SC and PO administration, respectively. Bioavailability was 76.88 ± 12.90%, 92.20 ± 10.53% and 12.13 ± 4.56% after IM, SC and PO administration, respectively, which indicated that OTC is poorly absorbed from the gastrointestinal tract in broiler chickens.

Influence of oral co-administration of a preparation containing calcium and magnesium and food on enrofloxacin pharmacokinetics.

The objective of this study has been to determine the influence of food and ions on the pharmacokinetics of enrofloxacin (ENRO) in turkeys, administered per os at a dose of 10 mg/kg of body weight (b.w.). Co-administration of ENRO with ions or with food significantly retarded its absorption, and the interaction was more pronounced when the drug was given together with food. The bioavailability of ENRO was 65.78 ± 7.81% and 47.99 ± 9.48% with ions and food, respectively. The maximum concentration (Cmax) in plasma of animals exposed to ions reached 0.87 ± 0.26 µg/ml in a tmax of 2.07 ± 0.76 h; in animals which were fed while medicated, the analogous parameters were 0.36 ± 0.13 µg/ml and 8.06 ± 3.08 h. The PK/PD analysis demonstrated that a decrease in the concentration of ENRO in turkeys' blood due to the interaction with ions or food might impair the drug's clinical efficacy toward some pathogenic microorganisms in turkeys if a routine dose of 10 mg ENRO/kg b.w. is administered.

Effects of dexamethasone and meloxicam on bovine CD25+ CD8+ and CD25- CD8+ T cells--in vitro study.

The aim of undertaken research was an in vitro evaluation of the effects of dexamethasone and meloxicam on selected bovine CD8(+) T lymphocyte subpopulations. Dexamethasone induced a fast-occurring and lasting depletion of CD25(-)CD8(+) cells. This was primarily the result of the proapoptotic effect of dexamethasone, but the antiproliferative effect of the drug was clearly responsible for the deepening of this disturbance. Dexamethasone transiently increased the relative and absolute CD25(high)CD8(+) and CD25(low)CD8(+) cell numbers. This effect was not a consequence of increased proliferation, but at least partly resulted from the antiapoptotic effect of the drug on these cells. The obtained results indicate that induction of CD8(+) lymphocyte depletion and impairment of IFN-γ production by these cells participate in the production of the anti-inflammatory and immunosuppressive effect of dexamethasone in cattle. An increase in Foxp3, IL-10 and TGF-ß production by CD8(+) lymphocytes is not involved in the production of these effects because the drug did not affect the percentage of TGF-ß(+)CD8(+) cells, while paradoxically reducing the percentage of cells with the suppressive phenotype, i.e. IL-10(+)CD25(low)CD8(+) and Foxp3(+)CD25(low)CD8(+) cells. Meloxicam did not substantially affect CD8(+) lymphocytes as to their percentage, absolute number, apoptosis, proliferation, Foxp3 expression and IFN-γ, IL-10 and TGF-ß production. Thus, in the context of the parameters being estimated, meloxicam seems a relatively safe anti-inflammatory drug to be used in infectious diseases in cattle.

C (max) and t (max) verification using Fibonacci sequence and absorption rate.

The aim of this study was to verify the values of maximal observed concentration (C max,obs) and the time, at which maximum concentration is observed (t max,obs) using the analysis of the absorption rate constant (k ab). It focused on the changes in concentration over time (C-T) for drugs, for which several peaks of concentration occur. In addition, the attempt was made to use Fibonacci sequence to facilitate the visual analysis of the dynamics in changes of concentration on C-T graphs. The analyses were conducted with the use of three hypothetical data groups (groups I, II and III), which had distinct C-T profiles, and with the in vivo data form healthy subjects (n = 10) taking part in a bioequivalence study, who was given a single oral dose of topiramate (100 mg). The comparison of hypothetical and real in vivo data demonstrated that for the C-T curves, in which there are several peaks of concentration C max,obs and t max,obs values can easily be miscalculated when the increase in concentration is not properly related to the appropriate absorption phase (63.2, 87.50, 96.88 %). It was also demonstrated that the data transformation with the use of Fibonacci sequence exposes slight differences in the observed concentration values in a semi-logarithmic scale. The results of this study show that in case of C-T curves with several peaks of concentration, the verification of C max and t max data obtained taking into account different absorption phases enables more precise evaluation of these parameters.

Residual fraction of the area under the curve as a qualitative criterion in pharmacokinetic studies.

The aim of the present study was to determine whether the residual area under the curve (AUC(res%); expressed as % of total value of AUC) could be used as a parameter for the qualitative evaluation of pharmacokinetic studies.We propose new criteria for the qualitative evaluation of pharmacokinetic analysis. Two sets of hypothetical data that illustrate the relationship between concentration and time were used for the analysis of drug pharmacokinetics. Non-compartmental analysis was applied for the calculations. The results obtained from the hypothetical data were compared with those obtained from an in vivo study in which 3-week-old broiler chickens were administered 10 mg/kg b.w. enrofloxacin intravenously (iv) or per os (po). In the first set of data (A-D), AUC(res%) values were as follows: A= 16.29% and B = 20.79% for iv administration and C = 29.61% and D = 27.90% for po administration. In the next set of data (E-G), AUC(res%) values after oral administration were 25.30% (E), 23.18% (F), and 20.79% (G). The AUC(res%) values after iv administration of enrofloxacin were similar to po administration; the range of iv and po administration values were 14.35% to 17.50% and 11.14% to 28.33% of the total AUC, respectively. The analysis of the hypothetical data indicates that AUC(res%) is not an optimal method for the evaluation of pharmacokinetic studies.

Role of tumor necrosis factor-alpha and nitric oxide in luteolysis in cattle.

Although prostaglandin (PG) F2alpha is known to be a principal luteolytic factor, its action on the bovine corpus luteum (CL) is mediated by other intra-ovarian factors. Tumor necrosis factor-alpha (TNFalpha) and its specific receptors are present in the bovine CL with the highest expressions at luteolysis. TNFalpha in combination with interferon-gamma reduced progesterone (P4) secretion, increased PGF2alpha and leukotriene C4 (LTC4) production, and induced apoptosis of the luteal cells in vitro. Low concentrations of TNFalpha caused luteolysis, which resulted in a decreased level of P4, and increased levels of PGF2alpha, LTC4 and nitrite/nitrate (stable metabolites of nitric oxide-NO) in the blood. Inhibition of local NO production counteracts spontaneous and PGF2alpha-induced luteolysis. Therefore, NO is a likely candidate for the molecule that mediates PGF2alpha and TNFalpha actions during luteolysis. Both PGF2alpha and TNFalpha increase NO concentrations in blood, and stimulate NO synthase expression on protein level in the bovine CL cells. NO stimulates PGF2alpha and LTC4 secretion, inhibits P4 production and reduces the number of viable luteal cells. TNFalpha and NO induce apoptotic death of the CL by modulating expression of bcl-2 family genes and by stimulating expression and activity of caspase-3. The above findings indicate that TNFalpha and NO play crucial roles in functional and structural luteolysis in cattle.

Effects of prostaglandin F(2alpha) and nitric oxide on the secretory function of bovine luteal cells.

The objective of the present study was to investigate the influence of prostaglandin F(2alpha) (PGF (2alpha)) and nitric oxide (NO) on production of steroids and PGs by culturing bovine luteal cells obtained from ovaries on days 8-12 of the estrous cycle with a nitric oxide (NO) donor (Spermine NONOate), and a NO synthase inhibitor (N(G)-nitro-L-arginine methyl ester dihydrochloride: L-NAME). When the cells were exposed for 24 h to PGF(2alpha) (10(-7)-10(-5) M), production of progesterone (P(4)) increased significantly at all doses used (P<0.05). Moreover, PGF(2alpha) stimulated PGF(2alpha) production (P<0.01), depressed testosterone (T) production (P<0.05), but did not affect synthesis of prostaglandin E(2) (PGE(2)). Spermine NONOate decreased P(4) production to 66%, 47% and 34% of the control concentration after treatment with 10(-5) M, 10(-4) M and 10(-3) M, respectively, but did not affect T production, and increased PGF(2alpha) synthesis (P<0.05) and PGE(2) (P<0.01) at all doses used. L-NAME increased production of P(4) (P<0.01) but did not affect (P>0.05) secretion of T, PGF(2alpha) and PGE(2). Estradiol-17beta (E(2)) was detectable on the level of sensitivity of assay and was not significantly altered by any treatments. The overall results suggest that PGF(2alpha) and NO produced locally in bovine CL play roles in the regulation of the secretory function of the bovine CL as auto/paracrine factors.

The distribution and influence of calcitonin gene-related peptide (CGRP) on the perfusion pressure in the isolated ovarian artery in the pig.

A moderate number of delicate CGRP-immunoreactive (CGRP-IR) varicose nerve terminals were found at the adventitia-media border of the sexually mature porcine ovarian artery by means of a routine single immunolabelling technique. Additionally, a pharmacological analysis was performed of the function fulfilled by alpha-CGRP and its C-terminal fragment (Tyr27) alpha-CGRP(27-37) in the porcine isolated ovarian artery, collected on days 8-13 of the oestrous cycle. It was shown that alpha-CGRP (10(-8) M) caused a decrease in the perfusion pressure of the porcine isolated ovarian artery of 16% (p < 0.05), while (Tyr27) alpha-CGRP(27-37), added at the same concentration, reduced perfusion pressure by 13% (p > 0.05). Thus, we concluded that alpha-CGRP released from perivascular terminals may cause relaxation of the ovarian artery and, furthermore, that the potency of this action is dependent on the length of the chain of this peptide produced during the deactivation of the molecule by tissue proteases.

NPY: its occurrence and relevance in the female reproductive system.

Neuropeptide Y (NPY), an amidated peptide composed of 36 amino acid residues, is the most widely distributed neuropeptide that performs a broad spectrum of physiological functions in both the central and peripheral nervous systems. Among numerous other actions, this peptide is involved, at the periphery, in the neural regulation of blood pressure and blood flow through the organs, and also, acting via Y2 and/or Y5 receptors, in the regulation of angiogenesis. NPY influences blood vessels via its own Y receptors, predominantly of the Y1 subtype. As a sympathetic co-transmitter NPY causes vasoconstriction, stimulates vascular growth and potentiates the contractile activity of noradrenaline (NA), and as a parasympathetic neurotransmitter it is involved in the regulation of vasodilatation within e.g. the uterine artery. In the female reproductive system, NPY not only regulates the blood flow, but also the contractile activity of non-vascular smooth muscle cells of the uterus and oviduct, as well as the secretory function of the ovary. Both the concentration of NPY and its influence on the blood flow through the female reproductive organs are finely tuned by fluctuations in the concentration of ovarian steroid hormones. Thus, the present review was aimed at summarizing the current knowledge dealing with the physiological relevance of NPY in the regulation of female gonad and genital tract function, with a special regard to the pig as a model animal.

Nitric oxide as a local mediator of prostaglandin F2alpha-induced regression in bovine corpus luteum: an in vivo study.

To test whether nitric oxide (NO) is involved in prostaglandin (PG) F2alpha-induced regression of the bovine corpus luteum (CL) in vivo, heifers were treated as follows: Group 1, saline (3 ml/h); Group 2, dinoprost, an analogue of prostaglandin F2alpha (aPGF2alpha; 5 mg/0.5 h); Group III, Nomega-nitro-L-arginine methyl ester (L-NAME; 200 mg/4 h), an inhibitor of nitric oxide synthase; and Group IV, L-NAME (400 mg/4 h) and aPGF2alpha (5 mg/0.5 h). All treatments were administered by an intraluteal microdialysis system (MDS) on day 15 of the cycle. Perfusate and jugular plasma samples were collected at half-hour intervals; additionally, jugular plasma samples were collected once daily from day 16 to day 21 of the cycle. In the perfusate samples, aPGF2alpha increased P4 (P < 0.05), PGE2 (P < 0.001), and LTC4 (P < 0.05) concentrations; L-NAME increased P4 (P < 0.05) but did not change PGE2 and LTC4 (P > 0.05) concentrations as compared with the period before treatment. Simultaneous perfusion of CL with L-NAME and aPGF2alpha caused a further increase of P4 concentration (P < 0.05) induced by L-NAME or aPGF2alpha treatment and increased PGE2 and LTC4 (P < 0.001) concentrations to the level observed after aPGF2alpha treatment. Perfusion of CL with aPGF2alpha caused luteal regression within 24 h, while perfusion with L-NAME prolonged the life span of CL to day 21 (P < 0.05). Concomitant L-NAME and aPGF2alpha treatment partially counteracted (P < 0.05) the luteal regression caused by aPGF2alpha administration. These results show that NO is involved in the process of luteolysis in the bovine CL and suggest that the luteolytic effect of aPGF2alpha may be mediated by NO as an important component of an autocrine/paracrine luteolytic cascade.

Localization of neuropeptide Y and norepinephrine in the porcine ovarian artery and their influence on the local blood pressure.

The aim of the present study was to determine: (i) the presence of dopamine-beta-hydroxylase (DbetaH)- and neuropeptide Y (NPY)-immunoreactive (IR) nerve fibres in the wall of the porcine ovarian artery, (ii) the influence of NPY and norepinephrine (NE) on the contractile activity of the pig ovarian arteries, and (iii) the pharmacological analysis of the interaction between NPY and NE in the isolated porcine ovarian arteries collected from immature pigs and from animals in different days of the estrous cycle. Ovarian arteries for immunohistochemistry and isolated arteries for pharmacological studies were excised from immature pigs and mature animals on days 1-5, 8-13 and 17-20 of the estrous cycle. The study showed that both DbetaH- and NPY-IR nerve fibres were present in the pig ovarian arteries in all periods examined, and, that in some fibres DbetaH and NPY were co-localized. Both NE (10(-6) M) and NPY (10(-7) M) increased blood pressure of examined preparations, however, NE caused stronger changes in the vessel wall tension (P<0.001), than did NPY. NE significantly increased (P<0.001) blood pressure of all isolated arteries, however, this response was stronger in vessels from days 1-5 of the cycle, when compared to days 8-13 (P<0.01), 17-20 and immature pigs (P<0.001). NPY increased significantly blood pressure in isolated arteries from days 8-13 and 17-20 of the cycle (P<0.001), while in preparations taken from immature pigs and animals on days 1-5 of the estrous cycle this response was somewhat weaker (P<0.01). A higher elevation (P<0.001) of blood pressure after NPY administration was observed in isolated arteries from days 17-20 of the cycle, when compared to vessels from days 1-5 and 8-13 and those from immature pigs. Moreover, NE significantly intensified (P<0.001) an increase in the blood pressure in isolated arteries pre-treated with NPY in all periods examined. NPY insignificantly (P>0.05) potentiated increase of blood pressure in NE pre-treated vessels of immature pigs and in isolated arteries from days 17-20 of the cycle and significantly (P<0.05) in vessels from days 1-5 and 8-13 of the estrous cycle. Our results indicate that DbetaH- and NPY-IR nerve fibres are present in the pig ovarian arteries. NE and NPY administered alone increased blood pressure in the pig isolated ovarian artery and simultaneous administration of both substances caused each other potentiation of vasocontractile effect, however, the strength of observed changes was dependent on the stage of the estrous cycle.

Influence of nitric oxide on the secretory function of the bovine corpus luteum: dependence on cell composition and cell-to-cell communication.

The objective of the present study was to investigate the role of cell-to-cell contact in the influence of nitric oxide (NO) on the secretory function of the bovine corpus luteum (CL). In Experiment 1, separate small luteal cells (SLC) or large (LLC) luteal cells were perfused with 100 micro M spermineNONOate, a NO donor, or with 100 micro M Nomega-nitro-L-arginine methyl ester (L-NAME), a NO synthase (NOS) inhibitor; in Experiment 2, a mixture of LLC and SLC and endothelial cells was cultured and incubated with spermineNONOate or L-NAME; in Experiment 3, spermineNONOate was perfused into the CL (100 mg/4 hr) by a microdialysis system in vivo. Perfusion of isolated SLC and LLC with the NO donor or NOS inhibitor (Experiment 1) did not affect (P > 0.05) secretion of progesterone (P(4)) or oxytocin (OT). L-NAME perfusion increased (P < 0.05) leukotriene C(4) (LTC(4)) secretion by both SLC and LLC cells. Treatment of mixtures of luteal cells with an NO donor (Experiment 2) significantly decreased (P < 0.001) secretion of P(4) and OT and increased (P < 0.001) production of prostaglandin F(2alpha) (PGF(2alpha)) and LTC(4). L-NAME stimulated (P < 0.001) P(4) secretion, but did not influence (P > 0.05) OT, PGF(2alpha) or LTC(4) production. Intraluteal administration (Experiment 3) of spermineNONOate increased (P < 0.001) LTC(4) and PGF(2alpha), decreased OT, but did not change P(4) levels in perfusate samples. These data indicate that cell-to-cell contact and cell composition play important roles in the response of bovine CL to treatment with NO donors or NOS inhibitors, and that paracrine mechanisms are required for the full secretory response of the CL in NO action. Endothelial cells appear to be required for the full secretory response of the CL to NO.

Administration of a nitric oxide synthase inhibitor counteracts prostaglandin F2-induced luteolysis in cattle.

The objective of this study was to determine whether nitric oxide (NO) is produced locally in the bovine corpus luteum (CL) and whether NO mediates prostaglandin F2alpha (PGF2alpha)-induced regression of the bovine CL in vivo. The local production of NO was determined in early I, early II, mid, late, and regressed stages of CL by determining NADPH-d activity and the presence of inducible and endothelial NO synthase immunolabeling. To determine whether inhibition of NO production counteracts the PGF2alpha-induced regression of the CL, saline (10 ml/h; n = 10) or a nonselective NOS inhibitor (Nomega-nitro-l-arginine methyl ester dihydrochloride [L-NAME]; 400 mg/h; n = 9) was infused for 2 h on Day 15 of the estrous cycle into the aorta abdominalis of Holstein/Polish Black and White heifers. After 30 min of infusion, saline or cloprostenol, an analogue of PGF2alpha (aPGF2alpha; 100 microg) was injected into the aorta abdominalis of animals infused with saline or L-NAME. NADPH-diaphorase activity was present in bovine CL, with the highest activity at mid and late luteal stages (P < 0.05). Inducible and endothelial NO synthases were observed with the strongest immunolabeling in the late CL (P < 0.05). Injection of aPGF2alpha increased nitrite/nitrate concentrations (P < 0.01) and inhibited P4 secretion (P < 0.05) in heifers that were infused with saline. Infusion of L-NAME stimulated P4 secretion (P < 0.05) and concomitantly inhibited plasma concentrations of nitrite/nitrate (P < 0.05). Concentrations of P4 in heifers infused with L-NAME and injected with aPGF2alpha were higher (P < 0.05) than in animals injected only with aPGF2alpha. The PGF2alpha analogue shortened the cycle length compared with that of saline (17.5 +/- 0.22 days vs. 21.5 +/- 0.65 days P < 0.05). L-NAME blocked the luteolytic action of the aPGF2alpha (22.6 +/- 1.07 days vs. 17.5 +/- 0.22 days, P < 0.05). These results suggest that NO is produced in the bovine CL. NO inhibits luteal steroidogenesis and it may be one of the components of an autocrine/paracrine luteolytic cascade induced by PGF2alpha.