N-Terminomic Identification of Intracellular MMP-2 Substrates in Cardiac Tissue.

Proteases are enzymes that induce irreversible post-translational modifications by hydrolyzing amide bonds in proteins. One of these proteases is matrix metalloproteinase-2 (MMP-2), which has been shown to modulate extracellular matrix remodeling and intracellular proteolysis during myocardial injury. However, the substrates of MMP-2 in heart tissue are limited, and lesser known are the cleavage sites. Here, we used degradomics to investigate the substrates of intracellular MMP-2 in rat ventricular extracts. First, we designed a novel, constitutively active MMP-2 fusion protein (MMP-2-Fc) that we expressed and purified from mammalian cells. Using this protease, we proteolyzed ventricular extracts and used subtiligase-mediated N-terminomic labeling which identified 95 putative MMP-2-Fc proteolytic cleavage sites using mass spectrometry. The intracellular MMP-2 cleavage sites identified in heart tissue extracts were enriched for proteins primarily involved in metabolism, as well as the breakdown of fatty acids and amino acids. We further characterized the cleavage of three of these MMP-2-Fc substrates based on the gene ontology analysis. We first characterized the cleavage of sarco/endoplasmic reticulum calcium ATPase (SERCA2a), a known MMP-2 substrate in myocardial injury. We then characterized the cleavage of malate dehydrogenase (MDHM) and phosphoglycerate kinase 1 (PGK1), representing new cardiac tissue substrates. Our findings provide insights into the intracellular substrates of MMP-2 in cardiac cells, suggesting that MMP-2 activation plays a role in cardiac metabolism.

Cardioprotective Action of a Novel Synthetic 19,20-EDP Analog Is Sirt Dependent.

ABSTRACT: Mounting evidence suggests that cytochrome P450 epoxygenase-derived metabolites of docosahexaenoic acid, called epoxydocosapentaenoic acids (EDPs), limit mitochondrial damage after cardiac injury. In particular, the 19,20-EDP regioisomer has demonstrated potent cardioprotective action. Thus, we investigated our novel synthetic 19,20-EDP analog SA-22 for protection against cardiac ischemia-reperfusion (IR) injury. Isolated C57BL/6J mouse hearts were perfused through Langendorff apparatus for 20 minutes to obtain baseline function, followed by 30 minutes of global ischemia. Hearts were then treated with vehicle, 19,20-EDP, SA-22, or SA-22 with the pan-sirtuin inhibitor nicotinamide or the SIRT3-selective inhibitor 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP) at the start of 40 minutes reperfusion (N = 5-8). We assessed IR injury-induced changes in recovery of myocardial function, using left ventricular developed pressure and systolic and diastolic pressure change. Tissues were assessed for electron transport chain function, SIRT1 and SIRT3, optic atrophy type 1, and caspase-1. We also used H9c2 cells in an in vitro model of hypoxia/reoxygenation injury (N = 3-6). Hearts perfused with SA-22 had significantly improved postischemic left ventricular developed pressure, systolic and diastolic recovery (64% of baseline), compared with vehicle control (15% of baseline). In addition, treatment with SA-22 led to better catalytic function observed in electron transport chain and SIRT enzymes. The protective action of SA-22 resulted in reduced activation of pyroptosis in both hearts and cells after injury. Interestingly, although nicotinamide cotreatment worsened functional outcomes, cell survival, and attenuated sirtuin activity, it failed to completely attenuate SA-22-induced protection against pyroptosis, possibly indicating EDPs exert cytoprotection through pleiotropic mechanisms. In short, these data demonstrate the potential of our novel synthetic 19,20-EDP analog, SA-22, against IR/hypoxia-reoxygenation injury and justify further development of therapeutic agents based on 19,20-EDP.

Data-Independent Acquisition Proteomics and N-Terminomics Methods Reveal Alterations in Mitochondrial Function and Metabolism in Ischemic-Reperfused Hearts.

Myocardial ischemia-reperfusion (IR) (stunning) injury triggers changes in the proteome and degradome of the heart. Here, we utilize quantitative proteomics and comprehensive degradomics to investigate the molecular mechanisms of IR injury in isolated rat hearts. The control group underwent aerobic perfusion, while the IR injury group underwent 20 min of ischemia and 30 min of reperfusion to induce a stunning injury. As MMP-2 activation has been shown to contribute to myocardial injury, hearts also underwent IR injury with ARP-100, an MMP-2-preferring inhibitor, to dissect the contribution of MMP-2 to IR injury. Using data-independent acquisition (DIA) and mass spectroscopy, we quantified 4468 proteins in ventricular extracts, whereby 447 proteins showed significant alterations among the three groups. We then used subtiligase-mediated N-terminomic labeling to identify more than a hundred specific cleavage sites. Among these protease substrates, 15 were identified following IR injury. We identified alterations in numerous proteins involved in mitochondrial function and metabolism following IR injury. Our findings provide valuable insights into the biochemical mechanisms of myocardial IR injury, suggesting alterations in reactive oxygen/nitrogen species handling and generation, fatty acid metabolism, mitochondrial function and metabolism, and cardiomyocyte contraction.

The roles of intracellular proteolysis in cardiac ischemia-reperfusion injury.

Ischemic heart disease remains a leading cause of human mortality worldwide. One form of ischemic heart disease is ischemia-reperfusion injury caused by the reintroduction of blood supply to ischemic cardiac muscle. The short and long-term damage that occurs due to ischemia-reperfusion injury is partly due to the proteolysis of diverse protein substrates inside and outside of cardiomyocytes. Ischemia-reperfusion activates several diverse intracellular proteases, including, but not limited to, matrix metalloproteinases, calpains, cathepsins, and caspases. This review will focus on the biological roles, intracellular localization, proteolytic targets, and inhibitors of these proteases in cardiomyocytes following ischemia-reperfusion injury. Recognition of the intracellular function of each of these proteases includes defining their activation, proteolytic targets, and their inhibitors during myocardial ischemia-reperfusion injury. This review is a step toward a better understanding of protease activation and involvement in ischemic heart disease and developing new therapeutic strategies for its treatment.

Matrix metalloproteinase-2 proteolyzes mitofusin-2 and impairs mitochondrial function during myocardial ischemia-reperfusion injury.

During myocardial ischemia and reperfusion (IR) injury matrix metalloproteinase-2 (MMP-2) is rapidly activated in response to oxidative stress. MMP-2 is a multifunctional protease that cleaves both extracellular and intracellular proteins. Oxidative stress also impairs mitochondrial function which is regulated by different proteins, including mitofusin-2 (Mfn-2), which is lost in IR injury. Oxidative stress and mitochondrial dysfunction trigger the NLRP3 inflammasome and the innate immune response which invokes the de novo expression of an N-terminal truncated isoform of MMP-2 (NTT-MMP-2) at or near mitochondria. We hypothesized that MMP-2 proteolyzes Mfn-2 during myocardial IR injury, impairing mitochondrial function and enhancing the inflammasome response. Isolated hearts from mice subjected to IR injury (30 min ischemia/40 min reperfusion) showed a significant reduction in left ventricular developed pressure (LVDP) compared to aerobically perfused hearts. IR injury increased MMP-2 activity as observed by gelatin zymography and increased degradation of troponin I, an intracellular MMP-2 target. MMP-2 preferring inhibitors, ARP-100 or ONO-4817, improved post-ischemic recovery of LVDP compared to vehicle perfused IR hearts. In muscle fibers isolated from IR hearts the rates of mitochondrial oxygen consumption and ATP production were impaired compared to those from aerobic hearts, whereas ARP-100 or ONO-4817 attenuated these reductions. IR hearts showed higher levels of NLRP3, cleaved caspase-1 and interleukin-1ß in the cytosolic fraction, while the mitochondria-enriched fraction showed reduced levels of Mfn-2, compared to aerobic hearts. ARP-100 or ONO-4817 attenuated these changes. Co-immunoprecipitation showed that MMP-2 is associated with Mfn-2 in aerobic and IR hearts. ARP-100 or ONO-4817 also reduced infarct size and cell death in hearts subjected to 45 min ischemia/120 min reperfusion. Following myocardial IR injury, impaired contractile function and mitochondrial respiration and elevated inflammasome response could be attributed, at least in part, to MMP-2 activation, which targets and cleaves mitochondrial Mfn-2. Inhibition of MMP-2 activity protects against cardiac contractile dysfunction in IR injury in part by preserving Mfn-2 and suppressing inflammation.

Loss of PI3Kα Mediates Protection From Myocardial Ischemia-Reperfusion Injury Linked to Preserved Mitochondrial Function.

Background Identifying new therapeutic targets for preventing the myocardial ischemia-reperfusion injury would have profound implications in cardiovascular medicine. Myocardial ischemia-reperfusion injury remains a major clinical burden in patients with coronary artery disease. Methods and Results We studied several key mechanistic pathways known to mediate cardioprotection in myocardial ischemia-reperfusion in 2 independent genetic models with reduced cardiac phosphoinositide 3-kinase-α (PI3Kα) activity. P3Kα-deficient genetic models (PI3KαDN and PI3Kα-Mer-Cre-Mer) showed profound resistance to myocardial ischemia-reperfusion injury. In an ex vivo reperfusion protocol, PI3Kα-deficient hearts had an 80% recovery of function compared with ≈10% recovery in the wild-type. Using an in vivo reperfusion protocol, PI3Kα-deficient hearts showed a 40% reduction in infarct size compared with wild-type hearts. Lack of PI3Kα increased late Na+ current, generating an influx of Na+, facilitating the lowering of mitochondrial Ca2+, thereby maintaining mitochondrial membrane potential and oxidative phosphorylation. Consistent with these functional differences, mitochondrial structure in PI3Kα-deficient hearts was preserved following ischemia-reperfusion injury. Computer modeling predicted that PIP3, the product of PI3Kα action, can interact with the murine and human NaV1.5 channels binding to the hydrophobic pocket below the selectivity filter and occluding the channel. Conclusions Loss of PI3Kα protects from global ischemic-reperfusion injury linked to improved mitochondrial structure and function associated with increased late Na+ current. Our results strongly support enhancement of mitochondrial function as a therapeutic strategy to minimize ischemia-reperfusion injury.

Cardiomyocyte-specific CYP2J2 and its therapeutic implications.

INTRODUCTION: Cytochrome P450s (CYPs) are a superfamily of monooxygenases with diverse biological roles. CYP2J2 is an isozyme highly expressed in the heart where it metabolizes endogenous substrates such as N-3/N-6 polyunsaturated fatty acids (PUFA) to produce lipid mediators involved in homeostasis and cardioprotective responses. Expanding our knowledge of the role CYP2J2 has within the heart is important for understanding its impact on cardiac health and disease. AREAS COVERED: The objective of this review was to assess the state of knowledge regarding cardiac CYP2J2. A literature search was conducted using PubMed-MEDLINE (from 2022 and earlier) to evaluate relevant studies regarding CYP2J2-mediated cardioprotection, small molecule modulators, effects of CYP2J2 substrates toward biologically relevant effects and implications of CYP2J2 polymorphisms and sexual dimorphism in the heart. EXPERT OPINION: Cardiac CYP2J2-mediated metabolism of endogenous and exogenous substrates have been shown to impact cardiac function. Identifying individual factors, like sex and age, that affect CYP2J2 require further elucidation to better understand CYP2J2's clinical relevance. Resolving the biological targets and activities of CYP2J2-derived PUFA metabolites will be necessary to safely target CYP2J2 and design novel analogues. Targeting CYP2J2 for therapeutic aims offers a potential novel approach to regulating cardiac homeostasis, drug metabolism and cardioprotection.

Staurosporine-induced cleavage of apoptosis-inducing factor in human fibrosarcoma cells is independent of matrix metalloproteinase-2.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein which mediates staurosporine (STS) - induced cell death. AIF cleavage and translocation to the cytosol is thought to be calpain-1-dependent as calpain inhibitors reduce AIF proteolysis; however, many calpain inhibitors also inhibit matrix metalloproteinase-2 (MMP-2) activity, an intracellular and extracellular protease implicated in apoptosis. Here we investigated whether MMP-2 activity is affected in response to STS and if it contributes to AIF cleavage. Human fibrosarcoma HT1080 cells were treated with STS (0.1 µM, 0.25-24 h). A significant increase in cellular MMP-2 activity was seen by gelatin zymography after a 6 h STS treatment, prior to induction of cell necrosis. Western blot showed the time-dependent appearance of two forms of AIF (∼60 and 45 kDa) in the cytosol which were significantly increased at 6 h. Surprisingly, knocking down MMP-2 or inhibiting its activity with MMP-2 preferring inhibitors ARP-100 or ONO-4817, or inhibiting calpain activity with ALLM or PD150606, did not prevent the STS-induced increase in cytosolic AIF. These results show that although STS rapidly increases MMP-2 activity, the cytosolic release of AIF may be independent of the proteolytic activities of MMP-2 or calpain.

Multifunctional intracellular matrix metalloproteinases: implications in disease.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that were first discovered as proteases, which target and cleave extracellular proteins. During the past 20 years, however, intracellular roles of MMPs were uncovered and research on this new aspect of their biology expanded. MMP-2 is the first of this protease family to be reported to play a crucial intracellular role where it cleaves several sarcomeric proteins inside cardiac myocytes during oxidative stress-induced injury. Beyond MMP-2, currently at least eleven other MMPs are known to function intracellularly including MMP-1, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-14, MMP-23 and MMP-26. These intracellular MMPs are localized to different compartments inside the cell including the cytosol, sarcomere, mitochondria, and the nucleus. Intracellular MMPs contribute to the pathogenesis of various diseases. Cardiovascular renal disorders, inflammation, and malignancy are some examples. They also exert antiviral and bactericidal effects. Interestingly, MMPs can act intracellularly through both protease-dependent and protease-independent mechanisms. In this review, we will highlight the intracellular mechanisms of MMPs activation, their numerous subcellular locales, substrates, and roles in different pathological conditions. We will also discuss the future direction of MMP research and the necessity to exploit the knowledge of their intracellular targets and actions for the design of targeted inhibitors.

Can N-3 polyunsaturated fatty acids be considered a potential adjuvant therapy for COVID-19-associated cardiovascular complications?

Coronavirus disease 2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has currently led to a global pandemic with millions of confirmed and increasing cases around the world. The novel SARS-CoV-2 not only affects the lungs causing severe acute respiratory dysfunction but also leads to significant dysfunction in multiple organs and physiological systems including the cardiovascular system. A plethora of studies have shown the viral infection triggers an exaggerated immune response, hypercoagulation and oxidative stress, which contribute significantly to poor cardiovascular outcomes observed in COVID-19 patients. To date, there are no approved vaccines or therapies for COVID-19. Accordingly, cardiovascular protective and supportive therapies are urgent and necessary to the overall prognosis of COVID-19 patients. Accumulating literature has demonstrated the beneficial effects of n-3 polyunsaturated fatty acids (n-3 PUFA) toward the cardiovascular system, which include ameliorating uncontrolled inflammatory reactions, reduced oxidative stress and mitigating coagulopathy. Moreover, it has been demonstrated the n-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are precursors to a group of potent bioactive lipid mediators, generated endogenously, which mediate many of the beneficial effects attributed to their parent compounds. Considering the favorable safety profile for n-3 PUFAs and their metabolites, it is reasonable to consider n-3 PUFAs as potential adjuvant therapies for the clinical management of COVID-19 patients. In this article, we provide an overview of the pathogenesis of cardiovascular complications secondary to COVID-19 and focus on the mechanisms that may contribute to the likely benefits of n-3 PUFAs and their metabolites.

International Union of Basic and Clinical Pharmacology. CIX. Differences and Similarities between Human and Rodent Prostaglandin E2 Receptors (EP1-4) and Prostacyclin Receptor (IP): Specific Roles in Pathophysiologic Conditions.

Prostaglandins are derived from arachidonic acid metabolism through cyclooxygenase activities. Among prostaglandins (PGs), prostacyclin (PGI2) and PGE2 are strongly involved in the regulation of homeostasis and main physiologic functions. In addition, the synthesis of these two prostaglandins is significantly increased during inflammation. PGI2 and PGE2 exert their biologic actions by binding to their respective receptors, namely prostacyclin receptor (IP) and prostaglandin E2 receptor (EP) 1-4, which belong to the family of G-protein-coupled receptors. IP and EP1-4 receptors are widely distributed in the body and thus play various physiologic and pathophysiologic roles. In this review, we discuss the recent advances in studies using pharmacological approaches, genetically modified animals, and genome-wide association studies regarding the roles of IP and EP1-4 receptors in the immune, cardiovascular, nervous, gastrointestinal, respiratory, genitourinary, and musculoskeletal systems. In particular, we highlight similarities and differences between human and rodents in terms of the specific roles of IP and EP1-4 receptors and their downstream signaling pathways, functions, and activities for each biologic system. We also highlight the potential novel therapeutic benefit of targeting IP and EP1-4 receptors in several diseases based on the scientific advances, animal models, and human studies. SIGNIFICANCE STATEMENT: In this review, we present an update of the pathophysiologic role of the prostacyclin receptor, prostaglandin E2 receptor (EP) 1, EP2, EP3, and EP4 receptors when activated by the two main prostaglandins, namely prostacyclin and prostaglandin E2, produced during inflammatory conditions in human and rodents. In addition, this comparison of the published results in each tissue and/or pathology should facilitate the choice of the most appropriate model for the future studies.

A Synthetic Epoxydocosapentaenoic Acid Analogue Ameliorates Cardiac Ischemia/Reperfusion Injury: The Involvement of the Sirtuin 3-NLRP3 Pathway.

While survival rates have markedly improved following cardiac ischemia-reperfusion (IR) injury, the resulting heart damage remains an important issue. Preserving mitochondrial quality and limiting NLRP3 inflammasome activation is an approach to limit IR injury, in which the mitochondrial deacetylase sirtuin 3 (SIRT3) has a role. Recent data demonstrate cytochrome P450 (CYP450)-derived epoxy metabolites, epoxydocosapentaenoic acids (EDPs), of docosahexaenoic acid (DHA), attenuate cardiac IR injury. EDPs undergo rapid removal and inactivation by enzymatic and non-enzymatic processes. The current study hypothesizes that the cardioprotective effects of the synthetic EDP surrogates AS-27, SA-26 and AA-4 against IR injury involve activation of SIRT3. Isolated hearts from wild type (WT) mice were perfused in the Langendorff mode with vehicle, AS-27, SA-26 or AA-4. Improved postischemic functional recovery, maintained cardiac ATP levels, reduced oxidative stress and attenuation of NLRP3 activation were observed in hearts perfused with the analogue SA-26. Assessment of cardiac mitochondria demonstrated SA-26 preserved SIRT3 activity and reduced acetylation of manganese superoxide dismutase (MnSOD) suggesting enhanced antioxidant capacity. Together, these data demonstrate that the cardioprotective effects of the EDP analogue SA-26 against IR injury involve preservation of mitochondrial SIRT3 activity, which attenuates a detrimental innate NLRP3 inflammasome response.

Sildenafil corrects the increased contractility of rat detrusor muscle induced by alprostadil in vitro.

BACKGROUND: Sildenafil (PDE5-inhibitor) and alprostadil (PGE1) are used in combination clinically for the management of some cases of erectile dysfunction. Despite the roles of prostaglandins (PG) and nitric oxide (NO) pathways in contractility of bladder smooth muscle are frequently studied, the effect of sildenafil/alprostadil combination and the crosstalk between NO/cGMP and PG pathways on bladder activity is not documented. METHODS: Organ-bath experiments were performed using isolated rat detrusor muscle. Direct and neurogenic contractions were induced using ACh and electric stimulation (EFS, 4Hz, 80V, 1ms), respectively. The contractile responses in absence and presence of the tested drugs at different concentrations were compared. Results are expressed as mean ± SEM (n = 5-7). RESULTS: Alprostadil (0.01-10 µM) concentration-dependently potentiated ACh (100µM)- and EFS (4 Hz)- induced contraction. Maximum potentiation of ACh-contraction in presence of alprostadil was 40 ± 5%. Sildenafil potentiated ACh-induced contraction at low concentrations (0.01-1 µM), but inhibited it at higher ones (10-100 µM). IBMX (non-selective PDE-inhibitor, 0.01-100µM) and SNP (NO-donor, 1nM-1 mM) produced the same biphasic pattern. The potentiatory phase of sildenafil was inhibited by atropine (0.1µM), L-NAME (non-selective NOS-inhibitor, 100µM), N-PLA (nNOS-inhibitor, 30µM) or MB (nonselective GC-inhibitor, 10µM). In presence of sildenafil (0.1µM), the concentration-response curve of alprostadil (0.01-10µM) on both ACh and EFS-induced contraction was clearly shifted downward. CONCLUSIONS: A crosstalk between PGE1 and NO/cGMP pathways may exist. At low concentrations only, the effect of sildenafil on bladder contractility is dependent on NO/cGMP. cGMP intracellularly-elevated by sildenafil, may inhibit the activity of PLC and hence the cascade of EP1-receptors, thus masking the hyperactivity of bladder caused by alprostadil, which adds to the advantages of this combination.

Evaluation of some prostaglandins modulators on rat corpus cavernosum in-vitro: Is relaxation negatively affected by COX-inhibitors?

INTRODUCTION: Prostaglandins (PGs) play an important role in corpus cavernosum relaxation, as evidenced by alprostadil being used as a drug for erectile dysfunction. Reports about the effect of cyclooxygenase (COX) inhibitors on erectile function are highly contradictory. AIM: To compare the potential effects of some COX inhibitors with varying COX-1/COX-2 selectivities (indomethacin, ketoprofen and diclofenac) with that of the selective COX-2 inhibitor (DFU) on corpus cavernosal tone in-vitro. The role played by PGE1, PGI2-analogue and PGE4 receptor (EP4)-agonist in controlling corpus cavernosum function and the modulation of their action by sildenafil is also studied. METHODS: Organ bath experiments were performed using isolated rat corpus cavernosum. Direct relaxations and changes to electric field stimulation (EFS, 2-16 Hz, 60 V, 0.8 ms, 10 s train)-induced relaxation by the effect of the selected drugs were studied. Strips were precontracted using phenylephrine (PE, 10-5 M). Results are expressed as mean ± SEM of 5-9 rats. RESULTS: Alprostadil, iloprost and L902688 (selective EP4 agonist) induced direct relaxation where L902688 showed greater relaxant effect. Sildenafil potentiated the Emax of alprostadil and iloprost but not L902688. EFS and acetylcholine (ACh)-induced relaxations were significantly potentiated in presence of indomethacin, ketoprofen and diclofenac (20, 100 µM) but not in presence of selective COX-2 inhibitor (DFU, 1 µM). GR32191B (Thromboxane A2 receptor antagonist, 10-6 M) significantly reduced the potentiatory effect of indomethacin. Only diclofenac succeeded to potentiate sodium nitroprusside (SNP)-induced relaxation. CONCLUSIONS: EP4 receptors may play an important nitric oxide (NO)/cGMP-independent role in corpus cavernosal relaxation. Nonselective COX inhibitors seem of no harm concerning cavernosal tissue relaxation, possibly because they inhibit the synthesis of the highly contracting mediator thromboxane A2.

Hypoactivity of rat detrusor muscle in a model of cystitis: exacerbation by non-selective COX inhibitors and amelioration by a selective DP1 receptor antagonist.

Various studies have confirmed that prostaglandins (PG) alter the bladder motor activity and micturition reflex in both human and animals. However, no sufficient data is reported about the effect of cyclooxygenase (COX) inhibitors neither in normal bladder physiology nor in pathological conditions. This study aims to compare the potential effects of some COX inhibitors with varying COX-1/COX-2 selectivities (indomethacin, ketoprofen, and diclofenac) with that of the selective COX-2 inhibitor (DFU) on bladder function. The role played by some PGs and their receptors in controlling detrusor muscle function in normal condition and in cystitis is also studied. Organ bath experiments were performed using isolated rat detrusor muscle. Direct and neurogenic contractions were induced using ACh and electric stimulation (EFS), respectively. A model of hemorrhagic cystitis was induced by single injection of cyclophosphamide (300 mg/kg) in rats, and confirmed by histophathological examination. Results are expressed as mean ± SEM of 5-9 rats. Alprostadil and iloprost (1 nM- 10 µM) concentration-dependently potentiated ACh (100 µM)- and EFS (4 Hz)-induced contraction, with maximum potentiation of 40.01 ± 5.29 and 27.59 ± 6.64%, respectively, in case of ACh contractions. In contrast, ONO-AE1-259 (selective EP2 agonist, 1 nM-10 µM) inhibited muscle contraction. SC51322 (EP1-antagonist, 10 µM) and RO1138452 (IP antagonist, 10 µM) inhibited both direct and neurogenic responses. Hemorrhagic cystitis reduced both ACh and EFS responses as well as the potentiatory effect of iloprost and the inhibitory effect of RO1138452 on ACh contractions. ONO-AE3-237 (DP1 antagonist, 1 µM) significantly potentiated contractions in cystitis but showed no effect in normal bladder. A significant inhibition of contractile response was observed in presence of indomethacin, ketoprofen, and diclofenac at all tested concentrations (20, 50, and 100 µM). Highest effect was induced by diclofenac. The effect of these COX inhibitors on EFS contractions was intensified in case of cystitis, indomethacin being the most potent. Atropine (1 nM) significantly reduced indomethacin effect on ACh contraction only in normal rats. On the other hand, DFU (10-6 M) significantly potentiated the contractile effect of ACh in case of cystitis although it showed no effect in normal rats. EP1 receptors seem to play an important role in rat bladder contractility. DP1 receptors as COX-2, on the other hand, gain an important role only in case of cystitis. The use of non-selective COX inhibitors in cystitis may be associated with bladder hypoactivity; selective COX-2 inhibitors may be a safer option.