Jerzy Jan Jaroszewski

Administration of a Nitric Oxide Synthase Inhibitor Counteracts Prostaglandin F2-Induced Luteolysis in Cattle

Abstract 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 F2α (PGF2α)-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 PGF2α-induced regression of the CL, saline (10 ml/h; n = 10) or a nonselective NOS inhibitor (Nω-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 PGF2α (aPGF2α; 100 μg) 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 aPGF2α 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 aPGF2α were higher (P < 0.05) than in animals injected only with aPGF2α. The PGF2α 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 aPGF2α (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 PGF2α.

Intraluteal administration of a nitric oxide synthase blocker stimulates progesterone and oxytocin secretion and prolongs the life span of the bovine corpus luteum.

To test the role of nitric oxide (NO) in secretory functions of bovine corpora lutea (CL), two groups of four Holstein heifers each were treated as follows: Group 1, Nomega-Nitro-L-Arginine Methyl Ester (L-NAME), an inhibitor of nitric oxide synthase (NOS), on Day 11 or 12 of the cycle and Group 2, L-NAME on Days 17 and 18 of the cycle. All treatments were administered by an intraluteal microdialysis system (MDS). Drugs were infused for 4-hr periods on the designated days, and the treatment periods were preceded and followed by 4-hr control periods. Perfusate and jugular blood samples were collected at half-hour intervals. Perfusate samples were analyzed for progesterone (P4), oxytocin (OT), prostaglandin F2alpha (PGF2alpha), and leukotriene C4 (LTC4); jugular plasma samples were analyzed for P4, OT, and LH. Perfusion of L-NAME on Day 11 or 12 consistently increased P4 concentration in the perfusate, but had no effect on the life span of the CL. Perfusion of L-NAME on Days 17-18 also elevated P4 levels in the perfusate, and in addition, maintained P4 levels in the plasma of three of the four treated animals through Day 25 of the cycle. L-NAME perfusion also increased OT release concomitant with P4 into the perfusate at both the mid- and late-luteal phase treatments. For the most part, concentrations of LH, OT, and P4 in the jugular plasma samples collected during the perfusions were unaffected by treatments. L-NAME perfusion caused small, but significant (P < 0.05) increases in perfusate PGF2alpha and LTC4 at Days 17 and 18 and in LTC4 on Day 11 or 12. These data indicate that NO plays a direct luteolytic role in regression of the bovine CL.

Nitric oxide as a local mediator of prostaglandin F2α-Induced regression in bovine corpus luteum: An in vivo study

To test whether nitric oxide (NO) is involved in prostaglandin (PG) F2α-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 F2α (aPGF2α; 5 mg/0.5 h); Group III, Nω-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 aPGF2α (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, aPGF2α 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 aPGF2α caused a further increase of P4 concentration (P < 0.05) induced by L-NAME or aPGF2α treatment and increased PGE2 and LTC4 (P < 0.001) concentrations to the level observed after aPGF2α treatment. Perfusion of CL with aPGF2α 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 aPGF2α treatment partially counteracted (P < 0.05) the luteal regression caused by aPGF2α administration. These results show that NO is involved in the process of luteolysis in the bovine CL and suggest that the luteolytic effect of aPGF2α may be mediated by NO as an important component of an autocrine/paracrine luteolytic cascade.

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 μM spermineNONOate, a NO donor, or with 100 μM Nω-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 (P4) or oxytocin (OT). L-NAME perfusion increased (P < 0.05) leukotriene C4 (LTC4) 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 P4 and OT and increased (P < 0.001) production of prostaglandin F2α (PGF2α) and LTC4. L-NAME stimulated (P < 0.001) P4 secretion, but did not influence (P > 0.05) OT, PGF2α or LTC4 production. Intraluteal administration (Experiment 3) of spermineNONOate increased (P < 0.001) LTC4 and PGF2α, decreased OT, but did not change P4 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.

Luteolytic Effect of Prostaglandin F2α on Bovine Corpus Luteum Depends on Cell Composition and Contact

Prostaglandin F(2 alpha) (PGF(2 alpha)) is a main luteolytic factor in vivo; however, its direct luteolytic influence on steroidogenic cells of bovine corpus luteum (CL) is controversial and not fully understood. The aim of the study was to clarify PGF(2 alpha) action on bovine CL in different in vivo and in vitro conditions and to examine whether the contact among all main types of CL cells is necessary for luteolytic PGF(2 alpha) action. In experiment 1, the bovine CL (day 15 of the oestrous cycle) was perfused using in vivo microdialysis system with dinoprost (an analogue of PGF(2 alpha)) for 0.5 h. Dinoprost caused a short-time increase in progesterone (P4), whose concentration decreased thereafter (at 6-, 10-, 12- and 24-h after treatment). In experiment 2, the direct effect of PGF(2 alpha) on P4 accumulation in CL steroidogenic cells cultured in monolayer (day 15 of the cycle) was determined. PGF(2 alpha) after 24 h of incubation increased P4 accumulation in steroidogenic CL cells. In experiment 3 steroidogenic, endothelial CL and immune cells (day 15 of the cycle) were incubated with PGF(2 alpha) in cocultures for 24 h in glass tubes and the levels of P4, stable metabolites of nitric oxide (NO) and leukotriene (LT) C(4) were determined. Although PGF(2 alpha) treatment increased P4 secretion in homogeneous steroidogenic CL cell culture, the decrease in P4 secretion in cocultures of all types of CL cells was observed. The secretion of NO and LTC(4) increased after the treatment of PGF(2 alpha) both in pure cultures of CL cells and in cocultures. The interactions between endothelial and immune cells with steroidogenic CL cells are needed for luteolytic PGF(2 alpha) action within the bovine CL. Our results indicate that the cell coculture model, including the main types of CL cells, is the most approximate to study PGF(2 alpha) role in vitro.

Synthesis of prostacyclin and its effect on the contractile activity of the inflamed porcine uterus

The goal of the study was to estimate the content of prostacyclin (PGI(2)), the levels of PGI synthase (PTGIS) and receptor (PTGIR) protein expression, and the cellular localization of these factors in the inflammatory-changed porcine uterus. The effect of PGI(2) on the contractility of the inflamed uteri was also determined. On Day 3 of the estrous cycle (Day 0 of the study), 50 mL of either saline or Escherichia coli suspension (10(9) colony-forming units/mL) were injected into each uterine horn. Acute endometritis developed in all bacteria-inoculated gilts, however on Day 8 of the study a severe form of acute endometritis was noted more often than on Day 16. Bacteria injections increased the contents of 6-keto-prostaglandin F(1α) in endometrium, myometrium, washings, and the level of PTGIS in endometrium on Days 8 and 16, and the content of PTGIR in endometrium on Day 16. In the inflamed uteri on both study days, stronger immunoreactivity for PTGIS was observed in part of the luminal and glandular epithelial cells and in a portion of the endometrial arteries, and for PTGIR in part of the luminal epithelium and endothelial cells in a portion of the endometrial arteries. On Day 8, PGI(2) decreased contraction intensity in endometrium/myometrium and myometrium of the saline-treated uteri and increased the contraction intensity in both types of strips from the inflamed organs. Our study reveals that inflammation of the porcine uterus upregulates PGI(2) synthesis and that PGI(2) increases contractility, which suggests that PGI(2) might be essential for the course of uterine inflammation.

In vitro effects of dexamethasone on bovine CD25+CD4+ and CD25-CD4+ cells.

This paper investigates the in vitro effect of dexamethasone on bovine CD25highCD4+, CD25lowCD4+ and CD25-CD4+ T cells. Only a small percentage of bovine CD25highCD4+ (2-4%) and CD25lowCD4+ (1-2%) cells expressed Foxp3. Dexamethasone caused considerable loss of CD25-CD4+ cells, but it increased the relative and absolute numbers of CD25highCD4+ and CD25lowCD4+ lymphocytes, while at the same time reducing the percentage of Foxp3+ cells within the latter subpopulations. Considering all these, as well as the intrinsically poor Foxp3 expression in bovine CD25+CD4+, it can be concluded that the drug most probably increased the number of activated non-regulatory CD4+ lymphocytes. It has been found that changes in cell number were at least partly caused by proapoptotic effect of the drug on CD25-CD4+ cells and antiapoptotic effect on CD25highCD4+ and CD25lowCD4+ cells. The results obtained from this study indicate that the involvement of CD4+ lymphocytes in producing the anti-inflammatory and immunosuppressive effect of dexamethasone in cattle results from the fact that the drug had a depressive effect on the production of IFN-γ by CD25-CD4+ cells. Secretion of TGF-β and IL-10 by CD4+ lymphocytes was not involved in producing these pharmacological effects, because the drug did not affect production of TGF-β and, paradoxically, it reduced the percentage of IL-10+CD4+ cells.

Correlations between no observed effect level and selected parameters of the chemical structure for veterinary drugs.

No observed effect level (NOEL) represents the lowest value among numerous parameters used for the analysis of risk and the safety of a chemical substance and medicinal product. In the present study, the correlation between NOEL and selected topological parameters was determined for a group of 135 veterinary drugs. Due to the high values of the standard deviation for most of the parameters, the method of NOEL mean values analysis in quintile groups was used. The direct correlations between NOEL and the partition coefficient octanol/water (LogKOW), logarithm of water solubility value (LogSw), polar surface area (PSA), apolar surface area (aPSA), hydrogen bond acceptors and hydrogen bond donors were very low. In quintile groups a significant correlation (P<0.05) between NOEL and LogKOW, aPSA, PSA, as well as between NOEL and equations based on topological parameters were found. The results obtained indicate that, among the analysed parameters, LogSw and PSA play a crucial role in relation to a drug activity, at the NOEL level, in the tested group of compounds. Moreover, the calculation tools, based on the analysis of the molecular structure of topological parameters, can be useful for the prognosis of parameters describing drug safety.

Bioavailability of veterinary drugs in vivo and in silico

The physical and chemical properties of a drug determine the behaviour of its molecule in a living organism. In this paper, the relationship between selected physical and chemical parameters and drug bioavailability in vivo was investigated. Data sets from 75 compounds, which can be administered per os to 11 various animal species were analyzed. The selected parameters for the prediction of the in vivo bioavailability were the lipophilicity (LogP) and the polar surface area (PSA) or apolar surface area (aPSA) of a molecule. It was shown that the calculation of the hybrid parameters aPSA/PSA and LogP + (aPSA/PSA) allows to estimate the oral bioavailability of a drug and its allocation to clusters with either an oral bioavailability of <70% or >70%. The performed analysis also showed that an extreme low value of the PSA of a molecule (<40 square ångströms) combined with the extreme high value of LogP (>4) is associated with a lower oral bioavailability (<50%). The results obtained indicate the existence of a relationship between the mean LogP value and aPSA/PSA in silico, and the bioavailability of veterinary drugs as determined in vivo.

In vitro effects of meloxicam on the number, Foxp3 expression, production of selected cytokines, and apoptosis of bovine CD25+CD4+ and CD25-CD4+ cells

The purpose of this study was to evaluate the effect of meloxicam (MEL) on selected immune parameters of bovine CD25highCD4+, CD25lowCD4+, and CD25-CD4+ cells. Peripheral blood mononuclear cells (PBMCs) collected from 12-month-old heifers were treated with MEL at a concentration corresponding to the serum level of this medication following administration at the recommended dose (MEL 5 × 10-6 M) and at a concentration 10 times lower (MEL 5 × 10-7 M). After 12 and 24 h of incubation with the drug, the percentage of CD25highCD4+ cells decreased; however, this disturbance was quickly reversed. Furthermore, the absolute number of CD25highCD4+ cells in the PBMC populations treated with MEL 5 × 10-6 M for 48 and 168 h was increased. Prolonged (168 h) exposure to the drug increased the percentage of Foxp3+ cells in the CD25highCD4+ cell subpopulation. The higher dose of MEL was found to significantly increase the percentage of IFN-γ+ cells among the CD25-CD4+ cells. These results indicated that MEL does not exert an immunosuppressive effect by depleting CD4+ cells and suppression of IFN-γ+ production by these cells. Furthermore, IL-10 and TGF-β production was not changed following exposure to MEL.