Matrix metalloproteinase expression and activity following prostaglandin F(2 alpha)-induced luteolysis

Luteal tissue contains matrix metalloproteinases (MMPs) that cleave specific components of the extracellular matrix (ECM) and are inhibited by tissue inhibitors of metalloproteinases (TIMPs). We previously reported a decrease in luteal TIMP-1 within 15 min of prostaglandin F(2 alpha) (PGF(2 alpha))-induced luteolysis. An increase in the MMP:TIMP ratio may promote ECM degradation and apoptosis, as observed in other tissues that undergo involution. The objectives of these experiments were to determine whether 1) PGF(2 alpha) affects expression of mRNA encoding fibrillar collagenases (MMP-1 and -13), gelatinases A and B (MMP-2 and -9), membrane type (mt)-1 MMP (MMP-14), stromelysin (MMP-3), and matrilysin (MMP-7), and 2) PGF(2 alpha) increases MMP activity during PGF(2 alpha)-induced luteolysis in sheep. Corpora lutea (n = 3-10/time point) were collected at 0, 15, and 30 min and 1, 2, 4, 6, 12, 24, and 48 h after PGF(2 alpha) administration. Northern blot analysis confirmed the presence of all MMPs except MMP-9. Expression of mRNA for the above MMPs (except MMP-2) increased significantly (P < 0.05) by 30 min, and all MMPs increased significantly (P < 0.05) by 6 h after PGF(2 alpha) administration. Expression of MMP-14 mRNA increased significantly (P < 0.05) by 15 min post-PGF(2 alpha) and remained elevated through 48 h. MMP activity in luteal homogenates (following proenzyme activation and inactivation of inhibitors) was increased significantly (P < 0.05) by 15 min and remained elevated through 48 h post-PGF(2 alpha). MMP activity was localized (in situ zymography) to the pericellular area of various cell types in the 0-h group and was markedly increased by 30 min post-PGF(2 alpha). MMP mRNA expression and activity were significantly increased following PGF(2 alpha) treatment. Increased MMP activity may promote ECM degradation during luteolysis.     

Analysis of luteal tissue inhibitor of metalloproteinase-1, -2, and -3 during prostaglandin F(2alpha)-induced luteolysis

Increased matrix metalloproteinase (MMP) expression and activities help to mediate tissue involution through increasing extracellular matrix remodeling and promoting dedifferentiation and, ultimately, apoptosis. Therefore, we hypothesized that prostaglandin (PG) F(2alpha) administration would decrease expression of the tissue inhibitor of metalloproteinase (TIMP)-1, -2, and -3 and effectively increase the MMP:TIMP ratio, leading to glandular involution. In experiment 1, we tested the effects of PGF(2alpha) administration (Day 10 postestrus; Day 0 = estrus) on luteal TIMP-1, -2, and -3 mRNA and protein expression. Corpora lutea were collected at 0, 15, or 30 min or at 1, 2, 4, 6, 12, 24, and 48 h following PGF(2alpha) administration (n = 3-9 animals/time point). Following PGF(2alpha) administration, TIMP-1 mRNA levels decreased (P < 0.05) at 1 and 2 h relative to 0 h (controls), then increased to levels greater than controls at 4 and 6 h. In contrast, TIMP-2 and -3 mRNA levels did not decrease following PGF(2alpha) administration. The TIMP-1, -2, and -3 proteins were localized to large luteal cells (LLCs) within control (untreated) tissues. However, histodepletion of TIMP-1 within LLCs was evident within 30 min (earliest time point collected) following PGF(2alpha) injection and continued through 48 h. Luteal concentration of TIMP-1, as determined by RIA, was decreased (P < 0.05) by 15 min (earliest time point collected) following PGF(2alpha) administration and remained low through 48 h. In contrast, TIMP-2 and -3 immunolocalization was not altered by PGF(2alpha) administration. Experiment 2 was conducted to determine if PGF(2alpha) could initiate the preceding changes in TIMP-1 in early (Day 3) corpora lutea that can bind PGF(2alpha) but are refractory to its luteolytic effects. Serum concentrations of progesterone and luteal concentrations of TIMP-1 mRNA and protein were similar at 0 and 6 h after PGF(2alpha) injection on Day 3 postestrus. These data suggest that an early and sustained effect of PGF(2alpha) is the specific depletion of TIMP-1 within LLCs that are capable of responding to the luteolytic action of PGF(2alpha). This action may increase the MMP:TIMP-1 ratio, creating an environment that favors extracellular matrix degradation and, thereby, facilitates both functional and structural regression.     

Effects of nitric oxide and nitric oxide-derived species on prostaglandin endoperoxide synthase and prostaglandin biosynthesis.

Prostaglandins and NO. are important mediators of inflammation and other physiological and pathophysiological processes. Continuous production of these molecules in chronic inflammatory conditions has been linked to development of autoimmune disorders, coronary artery disease, and cancer. There is mounting evidence for a biological relationship between prostanoid biosynthesis and NO. biosynthesis. Upon stimulation, many cells express high levels of nitric oxide synthase (NOS) and prostaglandin endoperoxide synthase (PGHS). There are reports of stimulation of prostaglandin biosynthesis in these cells by direct interaction between NO. and PGHS, but this is not universally observed. Clarification of the role of NO. in PGHS catalysis has been attempted by examining NO. interactions with purified PGHS, including binding to its heme prosthetic group, cysteines, and tyrosyl radicals. However, a clear picture of the mechanism of PGHS stimulation by NO. has not yet emerged. Available studies suggest that NO. may only be a precursor to the molecule that interacts with PGHS. Peroxynitrite (from O2.-+NO.) reacts directly with PGHS to activate prostaglandin synthesis. Furthermore, removal of O2.- from RAW 267.4 cells that produce NO. and PGHS inhibits prostaglandin biosynthesis to the same extent as NOS inhibitors. This interaction between reactive nitrogen species and PGHS may provide new approaches to the control of inflammation in acute and chronic settings.     

Photo-control of nitric oxide synthase activity using a caged isoform specific inhibitor

Nitric oxide (NO) plays a critical role in a number of physiological processes and is produced in mammalian cells by nitric oxide synthase (NOS) isozymes. Because of the diverse functions of NO, pharmaceutical interventions which seek to abrogate adverse effects of excess NOS activity must not interfere with the normal regulation of NO levels in the body. A method has been developed for the control of NOS enzyme activity using the localized photochemical release of a caged isoform-specific NOS inhibitor. The caged form of an iNOS inhibitor has been synthesized and tested for photosensitivity and potency. UV and multiphoton uncaging were verified using a hemoglobin-based assay. IC(50) values were determined for the inhibitor (70+/-11 nM), the caged inhibitor (1098+/-172 nM), the UV uncaged inhibitor (67+/-26 nM) and the multiphoton uncaged inhibitor (73+/-11 nM). UV irradiation of the caged inhibitor resulted in a 86% reduction in iNOS activity after 5 min. Multiphoton uncaging had an apparent first order time constant of 0.007+/-0.001 min(-1). A therapeutic range exists, with molar excess of inhibitor to enzyme from 3- to 7-fold, over which the full dynamic range of the inhibition can be exploited.     

Role of endogenous nitric oxide synthase inhibitor in gastric mucosal injury

To explore the role of the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) in gastric mucosal injury, 3 models of gastric mucosal injury induced by ethanol, indomethacin, or cold stress were used in rats. The cultured human gastric mucosal epithelial cell line GES-1 infected by Helicobacter pylori (Hp) was selected to mimic human gastric mucosal injury. Gastric mucosal ulcer index (UI), levels of ADMA and NO, and activity of dimethylarginine dimethylaminohydrolase (DDAH) were determined in the mucosal injury models; in Hp-infected or ADMA-treated GES-1 cells, levels of ADMA, NO, and TNF-alpha and activity of DDAH were measured. The results showed that UI and levels of ADMA were markedly increased and accompanied by significantly decreased DDAH activity in the mucosal injury models. Incubation of GES-1 cells with Hp increased levels of TNF-alpha and ADMA and decreased activity of DDAH. Administration of ADMA also increased levels of TNF-alpha. The results suggest that ADMA plays an important role in facilitating gastric mucosal injury, an effect which is associated with inhibiting NO synthesis and inducing inflammatory reaction.     

Route of prostaglandin F2alpha injection and luteolysis in mares (38519).

Nine groups of pony mares (3/group) were used in a 3 times 3 factorial experiment. The factors were dose of PGF-2 alpha (0, 0.25 of 1.25 mg and route of administration (im, iu or il). Mares were laparotomized and treated on day 7 postovulation. Jugular blood was collected for progesterone RIA at 0 (pretreatment) and 1,6,12,24,48, and 72 hr posttreatment. In mares given either 0.25 mg or 1.25 mg PGF-2alpha, progesterone concentrations were not significantly different among the three routes at any of the posttreatment times studied except at 6 hr posttreatment. In mares given 0.25 mg, progesterone concentrations at 6 hr was less (p less than 0.05) for mares injected im than for mares injected iu. Compared to pretreatment progesterone values, PGF2-alpha (0.25 mg and 1.25 mg groups combined) administration significantly decreased progesterone concentration by 12 hr posttreatment in mares injected im and 24 hr in mares injected iu or il. In the iu group, a significant increase in progesterone concentration occurred between 1 and 6 hr followed by a significant decrease at 12 hr posttreatment. There were no significant differences among the three routes for intervals from treatment to estrus or ovulation, length of posttreatment estrus or length of interovulatory interval. Injection of either 0.25 mg or 1.25 mg PGF-2alpha significantly shortened the interval from treatment to estrus. Although 0.25 mg tended to shorten the interval from treatment to ovulation and interovulatory interval, these two end points were significantly shortened only in mares given 1.25 mg PGF-2alpha. Results indicated that local administration (iu or il) did not improve the luteolytic efficacy of PGF-2alpha over systemic administration (im).     

Improved oxygenation with prostaglandin F2alpha with and without inhaled nitric oxide in dogs

Dogs of mixedbreed (n = 7) were anesthetized, rightlung atelectasis was established, and the cyclooxygenase pathway was blocked with ibuprofen. Measurements of pulmonary gas exchange wereperformed (fractional concentration of inspiredO₂ = 0.95) after infusions ofprostaglandin F₂(PGF₂; 2 µg · kg¹ · min¹),ventilation with nitric oxide (NO; 40 ppm), or both(PGF₂ + NO) in random order.The arterial PO₂(Pa_O2) under control conditions was 117 ± 16 Torr (shunt = 33 ± 2.5%), was unchanged with NO alone(Pa_O2 = 114 ± 17 Torr; shunt = 35.7 ± 3.1%), but was significantlyimproved with PGF₂ alone(Pa_O2 = 180 ± 28 Torr; shunt = 23.2 ± 2.8%) and with the combination ofPGF₂ + NO(Pa_O2 = 202 ± 30 Torr; shunt = 20.9 ± 2.5%). The addition of NO didnot significantly enhance the effectiveness of thePGF₂ onPa_O2.Simulation of these data in a computer model, combining pulmonary gasexchange and pulmonary blood flow, reproduced the results on the basisthat vasoconstriction with PGF₂was maximal under hypoxia in the atelectatic lung and reduced byhyperoxia in the ventilated lung, consistent with the hypothesis ofO₂ dependence ofPGF₂ vasoconstriction.

     

A nitric oxide synthesis inhibitor decreased prostaglandin production in rat mesenteric vasculature

Endothelial cells synthesize and release nitric oxide (NO) and prostacyclin (PGI₂) which are involved in the regulation o f vascular tone and blood pressure. Our objective was to evaluate the effects of inhibiting NO synthesis on vascular prostaglandin (PG) and cyclic nucleotide production, as well as the pressor response to norepinephrine (NE). Isolated mesenteric arterial beds were perfused with Krebs-Henseleit solution containing 100 μM N^G-monomethyl-L-arginine (L-NMMA), 100 μM L-arginine (LA), or vehicle. After a 30 min equilibration 0.1, 0.5, 1, or 5 μM NE was infused into the superior mesenteric artery and the perfusion pressure was monitored. The basal perfusion pressure did not differ significantly between groups. The pressure-response curve was shifted to the right in the L-NMMA group vs. the LA and control groups. Perfusion was similarly performed with a Krebs-Henseleit solution containing 100 μM L-NMMA, LA, D-arginine, or vehicle. Perfusates were collected before and after NE infusion for the measurement of PGE₂, 6-keto-PGF_1α, TxB₂, cAMP, and cGMP. In the L-NMMA group the release of PGE₂ and 6-keto-PGF1α was decreased, and the release of cGMP was prevented. Production of cAMP did not differ between the four groups before NE infusion, and NE increased cAMP release in the L-NMMA group and controls. The results indicate that inhibition of NO synthesis by L-NMMA enhanced the pressor response to NE, possibly mediated by the decreased cGMP and PGI₂ production in resistance vessels.     

Role of zinc in isoform-selective inhibitor binding to neuronal nitric oxide synthase

In previous studies [Delker, S. L., et al. (2010), J. Am. Chem. Soc. 132, 5437-5442], we determined the crystal structures of neuronal nitric oxide synthase (nNOS) in complex with nNOS-selective chiral pyrrolidine inhibitors, designed to have an aminopyridine group bound over the heme where it can electrostatically interact with the conserved active site Glu residue. However, in addition to the expected binding mode with the (S,S)-cis inhibitors, an unexpected "flipped" orientation was observed for the (R,R)-cis enantiomers. In the flipped mode, the aminopyridine extends out of the active site where it interacts with one heme propionate. This prompted us to design and synthesize symmetric "double-headed" inhibitors with an aminopyridine at each end of a bridging ring structure [Xue, F., Delker, S. L., Li, H., Fang, J., Jamal, J., Marta?sek, P., Roman, L. J., Poulos, T. L., and Silverman, R. B. Symmetric double-headed aminopyridines, a novel strategy for potent and membrane-permeable inhibitors of neuronal nitric oxide synthase. J. Med. Chem. (submitted for publication)]. One aminopyridine should interact with the active site Glu and the other with the heme propionate. Crystal structures of these double-headed aminopyridine inhibitors in complexes with nNOS show unexpected and significant protein and heme conformational changes induced by inhibitor binding that result in removal of the tetrahydrobiopterin (H(4)B) cofactor and creation of a new Zn(2+) site. These changes are due to binding of a second inhibitor molecule that results in the displacement of H(4)B and the placement of the inhibitor pyridine group in position to serve as a Zn(2+) ligand together with Asp, His, and a chloride ion. Binding of the second inhibitor molecule and generation of the Zn(2+) site do not occur in eNOS. Structural requirements for creation of the new Zn(2+) site in nNOS were analyzed in detail. These observations open the way for the potential design of novel inhibitors selective for nNOS.     

2-thiouracil is a selective inhibitor of neuronal nitric oxide synthase antagonising tetrahydrobiopterin-dependent enzyme activation and dimerisation

2-thiouracil (TU), an established antithyroid drug and melanoma-seeker, was found to selectively inhibit neuronal nitric oxide synthase (nNOS) in a competitive manner (K(i)=20 microM), being inactive on the other NOS isoforms. The drug apparently interfered with the substrate- and tetrahydrobiopterin (BH(4))-binding to the enzyme. It caused a 60% inhibition of H(2)O(2) production in the absence of L-arginine and BH(4), and antagonised BH(4)-induced dimerisation of nNOS, but did not affect cytochrome c reduction. These results open new perspectives in the understanding of the antithyroid action of TU and provide a new lead structure for the development of selective nNOS inhibitors to elucidate the interdependence of the substrate and pteridine sites and to modulate pathologically aberrant NO formation.     

Studies on the mechanism of action of prostaglandin F2 alpha induced luteolysis in rats

The effects of prostaglandin F2 alpha (PGF2 alpha) administration on the utilization of low density lipoprotein (LDL) and progesterone secretion were examined in dispersed luteal cells from rat ovaries. Immature rats were rendered pseudopregnant with administration of pregnant mare serum gonadotropin and human chorionic gonadotropin. Animals were sacrificed at different times after PGF2 alpha (5 mg/kg) or vehicle administration on day-5 of pseudopregnancy. Administration of PGF2 alpha in vivo decreased human chorionic gonadotropin (hCG) binding to luteal cell membranes in vitro but enhanced binding of LDL. Utilization of labelled cholesterol for steroid synthesis from reconstituted LDL [(3H)-CL-LDL] by dispersed luteal cells was enhanced following PGF2 alpha administration. This suggests that the LDL pathway is not suppressed during prostaglandin induced luteolysis. Progesterone and total progestin secretion in response to N6-2'-0-Dibutyryladenosine 3'5'-cyclic monophosphate (cAMP) was decreased at 2, 4 and 24 hours following PGF2 alpha administration demonstrating a post-cAMP defect in steroidogenesis. Addition of the hydroxylated sterols, 20 or 25-OH cholesterol as substrate stimulated progesterone secretion in vehicle treated rats in a dose dependent fashion with 20-OH cholesterol being more potent. Progesterone secretion in response to stimulation with luteinizing hormone (LH) and cAMP from vehicle treated rats was less than that observed with 20 or 25-OH cholesterol, indicating that endogenous substrate may be a limiting factor in steroid synthesis. The maximal capacity of luteal tissue to produce progestins following PGF2 alpha administration was determined with 20-OH cholesterol as the substrate. The results suggest that the post-cAMP defect at 4 hours following PGF2 alpha administration may be due to failure of the cells to mobilize endogenous cholesterol. However at 24 hours following PGF2 alpha administration the decreased ability of luteal cells to convert cholesterol to pregnenolone may contribute to decreased progesterone synthesis.     

Anchored plasticity opens doors for selective inhibitor design in nitric oxide synthase

Nitric oxide synthase (NOS) enzymes synthesize nitric oxide, a signal for vasodilatation and neurotransmission at low concentrations and a defensive cytotoxin at higher concentrations. The high active site conservation among all three NOS isozymes hinders the design of selective NOS inhibitors to treat inflammation, arthritis, stroke, septic shock and cancer. Our crystal structures and mutagenesis results identified an isozyme-specific induced-fit binding mode linking a cascade of conformational changes to a new specificity pocket. Plasticity of an isozyme-specific triad of distant second- and third-shell residues modulates conformational changes of invariant first-shell residues to determine inhibitor selectivity. To design potent and selective NOS inhibitors, we developed the anchored plasticity approach: anchor an inhibitor core in a conserved binding pocket, then extend rigid bulky substituents toward remote specificity pockets, which become accessible upon conformational changes of flexible residues. This approach exemplifies general principles for the design of selective enzyme inhibitors that overcome strong active site conservation.     

Zinc and nitric oxide synthase inhibitor l-NAME attenuate NPY-induced feeding in mice

The influences of zinc (Zn) and the nitric oxide synthase (NOS) inhibitor l-NAME on peripheral neuropeptide Y (NPY)-induced feeding in mice were investigated. Male mice received NPY (200 ng/d/mouse subcutaneously) and were separated into four groups based on cotreatments (with or without Zn [0.1 mg/mL]) and with or without l-NAME [0.2 mg/mL]) administered in drinking water for 10 d. A control group that received saline injection was also studied. The results showed that NPY, with or without any studied chemicals, did not affect body weight gain or body fat content. However, the mice that were administered NPY alone had increased energy intakes, higher serum triglyceride and free fatty acid, and lower serum glucose than saline-injected controls. NPY-treated mice that were given Zn and l-NAME cotreatments had compatible results of determined variables in comparison with control mice. This study showed that Zn and l-NAME attenuated NPY-mediated feeding and selected serum variables in mice. However, the mechanisms of the interactions among NPY, Zn and NOS, and their effects on appetite regulation, remain to be elucidated.     

4,5-Disubstituted-1,3-oxazolidin-2-imine derivatives: a new class of orally bioavailable nitric oxide synthase inhibitor

In our search for a novel class of inducible nitric oxide synthase (iNOS) inhibitors, 1,3-oxazolidin-2-imine was found to weakly inhibit iNOS. Further modifications of this compound resulted in a remarkable increase in both the in vivo and in vitro inhibitory activity and selectivity for iNOS.     

Hyperactivity caused by a nitric oxide synthase inhibitor is countered by ultra-wideband pulses.

Potential action of ultra-wideband (UWB) electromagnetic field pulses on effects of N(G)-nitro- L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS), on nociception and locomotor activity was investigated in CF-1 mice. Animals were injected IP with saline or 50 mg/kg L-NAME and exposed for 30 min to no pulses (sham exposure) or UWB pulses with electric field parameters of 102+/-1 kV/m peak amplitude, 0.90+/-0.05 ns duration, and 160+/-5 ps rise time (mean+/-S.D.) at 600/s. Animals were tested for thermal nociceptive responses on a 50 degrees C surface and for spontaneous locomotor activity for 5 min. L-NAME by itself increased mean first-response (paw lift, shake, or lick; jump) and back-paw-lick response latencies and mean locomotor activity. Exposure to UWB pulses reduced the L-NAME-induced increase in back-paw-lick latency by 22%, but this change was not statistically significant. The L-NAME-induced hyperactivity was not present after UWB exposure. Reduction and cancellation of effects of L-NAME suggest activation of opposing mechanism(s) by the UWB pulses, possibly including increase of nitric oxide production by NOS. The action, or actions, of UWB pulses appears to be more effective on locomotor activity than on thermal nociception in CF-1 mice.     

Regulation of prostaglandin generation in carrageenan-induced pleurisy by inducible nitric oxide synthase in knockout mice

In the present study, by comparing the responses in wild-type mice (iNOSWT) and mice lacking (iNOSKO) the inducible (or type 2) nitric oxide synthase (iNOS), we investigated the correlation between endogenous nitric oxide (NO) and prostaglandin (PG) generation in carrageenan-induced pleurisy. The inflammatory response in iNOSKO mice was significantly reduced in respect to iNOSWT animals, as demonstrated by the exudate volume (-63%) and numbers of infiltrating cells (-62%). The levels of NOx in the pleural exudate from carrageenan-treated mice were significantly (p < 0.01) decreased in iNOSKO mice (16 +/- 7.6 nmoles/mice) compared to iNOSWT animals (133 +/- 9 nmoles/mice). Similarly, the amounts of PGE2 in the pleural exudates of carrageenan-treated animals were significantly (p < 0.01) lower in iNOSKO compared to iNOSWT mice (120 +/- 20 pg/mice vs. 308 +/- 51 pg/mice). Also the amounts of 6-keto-PGF(1 alpha) produced by lungs from carrageenan-treated iNOSKO mice (1.01 +/- 0.10 ng/tissue mg) were significantly (p < 0.01) reduced compared to iNOSWT carrageenan-treated mice (2.1 +/- 0.09 ng/tissue mg). In conclusion our results confirm, by the use of iNOSKO mice that in carrageenan-induced pleurisy NO positively modulates PG biosynthesis.     

Influence of nitric oxide and noradrenaline on prostaglandin F(2)(alpha)-induced oxytocin secretion and intracellular calcium mobilization in cultured bovine luteal cells

Although prostaglandin (PG) F(2alpha) released from the uterus has been shown to cause regression of the bovine corpus luteum (CL), the neuroendocrine, paracrine, and autocrine mechanisms regulating luteolysis and PGF(2alpha) action in the CL are not fully understood. A number of substances produced locally in the CL may be involved in maintaining the equilibrium between luteal development and its regression. The present study was carried out to determine whether noradrenaline (NA) and nitric oxide (NO) regulate the sensitivity of the bovine CL to PGF(2alpha) in vitro and modulate a positive feedback cascade between PGF(2alpha) and luteal oxytocin (OT) in cows. Bovine luteal cells (Days 8-12 of the estrous cycle) cultured in glass tubes were pre-exposed to NA (10(-5) M) or an NO donor (S-nitroso-N:-acetylpenicillamine [S-NAP]; 10(-4) M) before stimulation with PGF(2alpha) (10(-6) M). Noradrenaline significantly stimulated the release of progesterone (P(4)), OT, PGF(2alpha), and PGE(2) (P: < 0.01); however, S-NAP inhibited P(4) and OT secretion (P: < 0.05). Oxytocin secretion and the intracellular level of free Ca(2+) ([Ca(2+)](i)) were measured as indicators of CL sensitivity to PGF(2alpha). Prostaglandin F(2alpha) increased both the amount of OT secretion and [Ca(2+)](i) by approximately two times the amount before (both P: < 0.05). The S-NAP amplified the effect of PGF(2alpha) on [Ca(2+)](i) and OT secretion (both P: < 0.001), whereas NA diminished the stimulatory effects of PGF(2alpha) on [Ca(2+)](i) (P: < 0.05). Moreover, PGF(2alpha) did not exert any additionally effects on OT secretion in NA-pretreated cells. The overall results suggest that adrenergic and nitrergic agents play opposite roles in the regulation of bovine CL function. While NA stimulates P(4) and OT secretion, NO may inhibit it in bovine CL. Both NA and NO are likely to stimulate the synthesis of luteal PGs and to modulate the action of PGF(2alpha). Noradrenaline may be the factor that is responsible for the limited action of PGF(2alpha) on CL and may be involved in the protection of the CL against premature luteolysis. In contrast, NO augments PGF(2alpha) action on CL and it may be involved in the course of luteolysis.     

Exercise conditioning attenuates the hypertensive effects of nitric oxide synthase inhibitor in rat

Many individuals with cardiovascular diseases undergo periodic exercise conditioning with or with out medication. Therefore, this study investigated the interaction of exercise training and chronic nitric oxide synthase (NOS) inhibitor (Nitro-L-Arginine Methyl Ester, L-NAME) treatment on blood pressure and its correlation with aortic nitric oxide (NO), antioxidant defense system and oxidative stress parameters in rats. Fisher 344 rats were divided into four groups: (1) sedentary control, (2) exercise training (ET) for 8 weeks, (3) L-NAME (10 mg/kg, subcutaneous for 8 weeks) and (4) ET + L-NAME. Blood pressure (BP) was monitored weekly for 8 weeks with tail-cuff method. The animals were sacrificed 24 h after last treatments and thoracic aortic rings were isolated and analyzed. Exercise conditioning resulted in a significant increase in respiratory exchange ratio (RER), aortic NO production, NO synthase activity and inducible iNOS protein expression. Training significantly enhanced aortic GSH levels, GSH/GSSG ratio and up-regulation of aortic CuZn-SOD, Mn-SOD, catalase (CAT) glutathione peroxidase (GSH-Px) activity and protein expression and significantly decreased aortic lipid peroxidation. Chronic L-NAME administration resulted in a significant depletion of aortic NO, NOS activity, endothelial (eNOS) and iNOS protein expression, GSH level, GSH/GSSG ratio, down-regulation of aortic antioxidant enzyme activities and protein expressions. Aortic xanthine oxidase (XO) activity significantly increased with increased lipid peroxidation and protein oxidation after L-NAME administration. The biochemical changes were accompanied by increased in BP. Interaction of training and chronic NOS inhibitor treatment resulted in normalization of BP and aortic antioxidant enzyme activity and protein expression, up-regulation of aortic GSH/GSSG ratio, NO levels, Mn-SOD protein expression, depletion of GSSG, protein oxidation and lipid peroxidation. The data suggest that training attenuated the oxidative injury caused by chronic NOS inhibitor treatment by up-regulating the NO and antioxidant systems and lowering the BP in rats.