oxadiazoles and Sepsis

Since 1999, human parechoviruses (HPeVs) have been classified as a separate group in the large and expanding family of Picornaviridae. In contrast to the well-established group of the human enteroviruses (HEVs), HPeVs have long been considered as irrelevant and have only been associated with mild disease manifestations in children. The identification of HPeV-3 in 2004 and its association with neonatal sepsis, refocused attention on this neglected group of viruses. Clinically HPeV infections may mimic HEV infections and are increasingly recognized as viral causes of sepsis-like illness and CNS infections in young children. Therapy is not available against HPeVs or HEVs. In this article, we will demonstrate that therapy against this group of picornaviruses is urgently needed and we will review the current knowledge of treatment options as well as the current developments in antiviral therapy against picornaviruses in the scope of treatment possibilities against HPeVs.

Topics: Animals; Antiviral Agents; Central Nervous System Infections; Disease Models, Animal; Humans; Infant, Newborn; Oxadiazoles; Oxazoles; Parechovirus; Picornaviridae Infections; Sepsis

Enterovirus infections are common in both children and adults and range from benign short-lived febrile illnesses to life-threatening infections. Recent developments in nucleic acid amplification techniques now allow the rapid and sensitive diagnosis of enterovirus infections, which in turn can lead to improvements in patient management that shorten hospitalizations and reduce costs. New antiviral drugs have been developed that inhibit enterovirus replication, and early clinical trials of these compounds suggest that effective therapy for enterovirus infections is now possible.

Topics: Adolescent; Adult; Antiviral Agents; Child; Child, Preschool; Enterovirus Infections; Female; Humans; Infant; Infant, Newborn; Meningitis, Viral; Oxadiazoles; Oxazoles; Polymerase Chain Reaction; Pregnancy; Pregnancy Complications, Infectious; Sepsis

The integrity of the intestinal barrier is critical for protecting the host against the pathogen. The role of hypoxia-inducible factor-1α (HIF-1α) in the intestinal barrier disfunction related to sepsis remained unclear. The purpose of the present study is to investigate the role of HIF-1α on oxidative damage, the intestinal mucosal permeability, structural and morphological changes during sepsis. Twenty-four Sprague Dawley (SD) rats were randomly divided into four groups of 6 rats each: the sham group (sham), sepsis group (subjected to cecal ligation and perforation, CLP), sepsis + DMOG group (40 mg/kg of DMOG by intraperitoneal injection for 7 consecutive days before CLP), and sepsis + BAY 87-2243 group (9 mg/kg of BAY 87-2243 orally administered for 3 consecutive days before CLP). Sepsis increased plasma levels of inflammatory mediators, oxidative stress markers and HIF-1α expression; caused pathological damage; increased permeability (P < 0.05); and decreased TJ protein expression in the intestinal mucosa of rats with sepsis (P < 0.05). The addition of DMOG up-regulated HIF-1α, then decreased the plasma levels of inflammatory mediators, oxidative stress markers, alleviated pathological damage to the intestinal mucosa and decreased intestinal permeability (P < 0.05); while BAY 87-2243 treatment had the opposite effects. Our findings showed that HIF-1α protects the intestinal barrier function of septic rats by inhibiting intestinal inflammation and oxidative damage, our results provide a novel insight for developing sepsis treatment.

Topics: Amino Acids, Dicarboxylic; Animals; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammation Mediators; Intestinal Mucosa; Models, Animal; Oxadiazoles; Pyrazoles; Rats; Rats, Sprague-Dawley; Sepsis; Wound Infection

Vascular dysfunction plays a key role in sepsis but the role of perivascular adipose tissue (PVAT) in this condition is relatively unknown.. Sepsis was induced by cecal ligation and puncture (CLP). The responses of the aorta and superior mesenteric artery to norepinephrine in the presence or absence of PVAT were evaluated. Fluorescent probes measured the production of nitric oxide (NO) and reactive oxygen species (ROS). NO synthases (NOS) and β3-adrenoceptor expression were detected by immunofluorescence and S-nitrosylation by the biotin switch assay.. Aorta and superior mesenteric arteries from septic animals with intact PVAT showed a worsened response to the vasoconstrictor compared to vessels without PVAT. PVAT from the aorta (APVAT) produced NO and ROS whereas PVAT from the superior mesenteric artery (MPVAT) produced only ROS. Septic APVAT exhibited a higher density of NOS-1 and NOS-3. S-nitrosylation was found in APVAT. Donor (PVAT obtained from normal or septic rats):Host (normal vessel without PVAT) experiments showed that L-NAME, ODQ and β3-adrenergic receptor antagonist blocked the septic APVAT anti-contractile effect. None of these compounds affected MPVAT; tempol, but not apocynin, blocked its anti-contractile effect.. PVAT contributes to the anti-contractile effect in the aorta and mesenteric artery of septic rats through different pathways. β3-Adrenergic receptor and NO appear to be key mediators of this effect in APVAT, but not in MPVAT where ROS seem to be a relevant mediator. Therefore, PVAT is a relevant player of sepsis vascular dysfunction.

Topics: Acetophenones; Adipose Tissue; Adrenergic beta-3 Receptor Agonists; Animals; Aorta; Cyclic N-Oxides; Female; Mesenteric Arteries; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Norepinephrine; Oxadiazoles; Phenotype; Quinoxalines; Rats; Reactive Oxygen Species; Receptors, Adrenergic, beta-3; Sepsis; Spin Labels; Vasoconstriction

Endothelial barrier dysfunction is a hallmark in the pathogenesis of sepsis. Sphingosine-1-phosphate (S1P) has been proposed to be critically involved in the maintenance of endothelial barrier function predominately by activating S1P receptor-1 (S1P1). Previous studies have shown that the specific S1P1 agonist SEW2871 improves endothelial barrier function under inflammatory conditions. However, the effectiveness of SEW2871 and potential side effects remained largely unexplored in a clinically relevant model of sepsis. Therefore, this study aimed to evaluate the effects of SEW2871 in the Colon ascendens stent peritonitis (CASP) model.. Polymicrobial sepsis was induced in Sprague-Dawley rats using CASP model that enabled the monitoring of macro-hemodynamic parameters. Twelve hours after surgery, animals received either SEW2871 or sodium chloride. Mesenteric endothelial barrier function was evaluated 24 h after sepsis induction by intravital microscopy. Organ pathology was assessed in lungs. S1P levels, blood gas analyses, and blood values were measured at different time points. In parallel the effect of SEW2871 was evaluated in human dermal microvascular endothelial cells.. In vitro SEW2871 partially stabilized TNF-α-induced endothelial barrier breakdown. However, in vivo SEW2871 caused severe cardiac side effects in septic animals leading to an increased lethality. Sepsis-induced endothelial barrier dysfunction was not attenuated by SEW2871 as revealed by increased FITC-albumin extra-vasation, requirement of intravasal fluid replacement, and pulmonary edema. Interestingly, Sham-operated animals did not present any side effects after SEW2871 treatment.. Our study demonstrates that the application of SEW2871 causes severe cardiac side effects and cannot attenuate the inflammation-induced endothelial barrier breakdown in a clinically relevant sepsis model, suggesting that the time point of administration and the pro-inflammatory milieu play a pivotal role in the therapeutic benefit of SEW2871.

Topics: Animals; Disease Models, Animal; Humans; Lysophospholipids; Male; Oxadiazoles; Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; Sepsis; Sphingosine; Sphingosine-1-Phosphate Receptors; Thiophenes; Tumor Necrosis Factor-alpha

Microvascular failure is hallmark of sepsis in humans and is recognized as a strong predictor of mortality. In the mouse subjected to cecal ligation and puncture (CLP) to induce a clinically relevant sepsis, renal microvascular permeability increases and peritubular capillary perfusion declines rapidly in the kidney leading to acute kidney injury (AKI). Sphingosine-1-phosphate (S1P) is a key regulator of microvascular endothelial function. To investigate the role of S1P in the development of microvascular permeability and peritubular capillary hypoperfusion in the kidney during CLP-induced AKI, we used a pharmacologic approach and a clinically relevant delayed dosing paradigm. Evans blue dye was used to measure renal microvascular permeability and intravital video microscopy was used to quantitate renal cortical capillary perfusion. The S1P receptor 1 (S1P1) agonist SEW2871 [5-[4-phenyl-5-(trifluoromethyl)-2-thienyl]-3-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazole] and S1P2 antagonist JTE-013 [N-(2,6-dichloro-4-pyridinyl)-2-[1,3-dimethyl-4-(1-methylethyl)-1H-pyrazolo[3,4-b]pyridin-6-yl]-hydrazinecarboxamide] were administered at the time of CLP and produced a dose-dependent but partial reduction in renal microvascular permeability at 6 hours after CLP. However, neither agent improved capillary perfusion at 6 hours. With delayed administration at 6 hours after CLP, only SEW2871 reversed microvascular permeability when measured at 18 hours. Importantly, SEW2871 also restored capillary perfusion and improved renal function. These data suggest that S1P1 and S1P2 do not regulate the early decline in renal capillary perfusion. However, later in the course of sepsis, pharmacologic stimulation of S1P1, even when delaying therapy until after injury has occurred, improves capillary and renal function, suggesting this approach should be evaluated as an adjunct therapy during sepsis.

Topics: Animals; Capillaries; Dose-Response Relationship, Drug; Kidney; Ligation; Male; Mice; Microcirculation; Molecular Targeted Therapy; Oxadiazoles; Permeability; Punctures; Pyrazoles; Pyridines; Receptors, Lysosphingolipid; Sepsis; Thiophenes; Time Factors

Activated protein C (APC) exerts anticoagulant effects via inactivation of factors Va and VIIIa and cytoprotective effects via protease activated receptor (PAR)1. Inhibition of endogenous APC in endotoxemia and sepsis results in exacerbation of coagulation and inflammation, with consequent enhanced lethality.. We here sought to dissect the distinct roles of the anticoagulant and cytoprotective functions of endogenous APC in severe Gram-negative pneumonia-derived sepsis (melioidosis).. We infected wild-type (WT) mice with Burkholderia pseudomallei, a common sepsis pathogen in southeast Asia, and treated them with antibodies inhibiting both the anticoagulant and cytoprotective functions of APC (MPC1609) or the anticoagulant functions of APC (MAPC1591) only. Additionally, we administered SEW2871 (stimulating the S1P1-pathway downstream from PAR1) to control and MPC1609-treated mice.. MPC1609, but not MAPC1591, significantly worsened survival, increased coagulation activation, facilitated bacterial growth and dissemination and enhanced the inflammatory response. The effects of MPC1609 could not be reversed by SEW2871, suggesting that S1P1 does not play a major role in this model.. These results suggest that the mere inhibition of the anticoagulant function of APC does not interfere with its protective role during Gram-negative pneumosepsis, suggesting a more prominent role for cytoprotective effects of APC .

Topics: Animals; Antibodies, Monoclonal; Bacterial Load; Blood Coagulation; Burkholderia pseudomallei; Cytokines; Cytoprotection; Disease Models, Animal; Female; Inflammation; Inflammation Mediators; Liver; Lung; Lysophospholipids; Melioidosis; Mice; Mice, Inbred C57BL; Oxadiazoles; Protein C; Receptor, PAR-1; Sepsis; Signal Transduction; Sphingosine; Thiophenes; Time Factors

Hyporeactivity to vasoconstrictors is one of the clinical manifestations of sepsis in man and experimental animals. The objective of the investigation was to examine whether atorvastatin can prevent hyporeactivity to norepinephrine (NE) in mouse aorta in sepsis, and if so, what are the mechanisms involved. Sepsis in mice was induced by cecal ligation and puncture. The aorta was harvested for tension experiment, nitric oxide (NO) and cyclic guanosine monophosphate measurements, and inducible NO synthase (iNOS) and α(1D)-adrenoceptor mRNA expression studies. In comparison with sham-operated controls, sepsis significantly decreased the contractile response to NE in the mouse aorta. Pretreatment with atorvastatin of septic animals completely restored NE-induced contractions to levels similar to those of sham-operated controls and significantly increased survival time and mean arterial pressure. Atorvastatin also attenuated iNOS-induced overproduction of NO, as well as iNOS mRNA expression. Accordingly, hyporeactivity to NE was not evident in tissues pretreated with selective iNOS inhibitor 1400W in sepsis. Although basal cyclic guanosine monophosphate accumulation in the aorta was reduced in sepsis, pretreatment of the tissues with soluble guanylyl cyclase inhibitor 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ) partially restored the reactivity to NE. Interestingly, hyporeactivity to NE in sepsis was associated with a decreased α(1D)-adrenoceptor mRNA expression in the mouse aorta. Atorvastatin pretreatment, however, prevented the decrease in α(1D)-adrenoceptor mRNA expression in septic animals. In conclusion, atorvastatin seems to prevent hyporeactivity to vasoconstrictor NE in the aorta from septic mice through attenuation of overproduction of NO as well as improved α(1D)-adrenoceptor mRNA expression. The findings of the present study may explain the beneficial effects of atorvastatin on improved hemodynamic functions in sepsis.

Topics: Animals; Atorvastatin; Enzyme Inhibitors; Guanylate Cyclase; Heptanoic Acids; Mice; Nitric Oxide; Norepinephrine; Oxadiazoles; Polymerase Chain Reaction; Pyrroles; Quinoxalines; Receptors, Adrenergic, alpha-1; RNA, Messenger; Sepsis; Vasoconstrictor Agents

Sepsis induces massive production of inflammatory mediators, such as nitric oxide (NO), and causes neuroendocrine and cardiovascular alterations. This study investigates the involvement of the central NO-cGMP pathway in arginine vasopressin (AVP) and oxytocin (OXY) gene expression during sepsis induced by cecal ligation and puncture (CLP). Male Wistar rats received an i.c.v. injection of ODQ (0.25 μg/μL), a selective inhibitor of the heme site of soluble guanylate cyclase, or of 1% dymethilsulfoxide (DMSO), as vehicle. Thirty minutes after the injections, sepsis was induced by cecal ligation and puncture or the animals were sham operated. The ODQ pre-treatment did not alter the progressive NO increase observed after CLP. In the supraoptic nucleus (SON), this pretreatment increased the relative gene expression ratio of AVP and OXY in the initial phase of sepsis, but in the late phase, the gene expression of both hormones was reduced. In the paraventricular nucleus (PVN), soluble guanylate cyclase inhibition caused an even larger decrease in the relative gene expression ratio of AVP and OXY during sepsis. These results are indicative of a role of the NO-cGMP pathway in hormonal synthesis in the SON and PVN of the hypothalamus during polymicrobial sepsis.

Topics: Animals; Arginine Vasopressin; Cyclic GMP; Enzyme Inhibitors; Gene Expression Regulation; Male; Nitric Oxide; Oxadiazoles; Oxytocin; Paraventricular Hypothalamic Nucleus; Quinoxalines; Rats; Rats, Wistar; Sepsis; Signal Transduction; Supraoptic Nucleus

Overproduction of nitric oxide and activation of soluble guanylate cyclase (sGC) are important in sepsis-induced hypotension and hyporesponsiveness to vasoconstrictors. A time course of the expression and activity of sGC in a sepsis model [cecal ligation and puncture (CLP)] was evaluated in rats. Soluble GC alpha-1 and beta-1 subunit mRNA levels increased in the lungs, but not in the aorta. However, in both tissues, the protein levels increased 24 h after sepsis and remained high for up to 48 h. Sodium nitroprusside-stimulated cGMP accumulation was higher 48 h after CLP in the lung and aorta. NOS-2 protein expression peaked 24 h after CLP, decreasing thereafter. The impact of inhibiting the expression of sGC early (8 h) or late (20 h) on vascular reactivity and the indexes of organ damage and mortality were also studied. Late administration of methylene blue (MB) or ODQ (1H-[1,2,4]-oxadiazole[4,3-a]quinoxalin-1-one) restored the blood pressure and vascular responsiveness to vasoconstrictors to normal levels but was ineffective in early sepsis. Late MB injection reduced the plasma levels of urea, creatinine, and lactate. MB improved the survival if administered late, but it increased the mortality when administrated early after sepsis onset. The increased sGC expression/activity may be relevant for the late hypotension and hyporesponsiveness to vasoconstrictors in sepsis. In accordance, MB increased survival if administered in late sepsis, but not in early sepsis. Therefore, differential responsiveness to sGC during the course of sepsis may determine the success or failure of treatment with sGC inhibitors.

Topics: Animals; Aorta, Thoracic; Blood Pressure; Cecum; Cyclic GMP; Disease Models, Animal; Drug Administration Schedule; Enzyme Inhibitors; Female; Guanylate Cyclase; Methylene Blue; Mortality; Oxadiazoles; Quinoxalines; Rats; Rats, Wistar; RNA, Messenger; Sepsis; Vasoconstriction

In septic shock, systemic vasodilation and myocardial depression contribute to the systemic hypotension observed. Both components can be attributed to the effects of mediators that are released as part of the inflammatory response. We previously found that lysozyme (Lzm-S), released from leukocytes, contributed to the myocardial depression that develops in a canine model of septic shock. Lzm-S binds to the endocardial endothelium, resulting in the production of nitric oxide (NO), which, in turn, activates the myocardial soluble guanylate cyclase (sGC) pathway. In the present study, we determined whether Lzm-S might also play a role in the systemic vasodilation that occurs in septic shock. In a phenylephrine-contracted canine carotid artery ring preparation, we found that both canine and human Lzm-S, at concentrations similar to those found in sepsis, produced vasorelaxation. This decrease in force could not be prevented by inhibitors of NO synthase, prostaglandin synthesis, or potassium channel inhibitors and was not dependent on the presence of the vascular endothelium. However, inhibitors of the sGC pathway prevented the vasodilatory activity of Lzm-S. In addition, Aspergillus niger catalase, which breaks down H(2)O(2), as well as hydroxyl radical scavengers, which included hydroquinone and mannitol, prevented the effect of Lzm-S. Electrochemical sensors corroborated that Lzm-S caused H(2)O(2) release from the carotid artery preparation. In conclusion, these results support the notion that when Lzm-S interacts with the arterial vasculature, this interaction results in the formation of H(2)O(2), which, in turn, activates the sGC pathway to cause relaxation. Lzm-S may contribute to the vasodilation that occurs in septic shock.

Topics: Aminoquinolines; Animals; Carotid Artery, Internal; Catalase; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclooxygenase Inhibitors; Dogs; Dose-Response Relationship, Drug; Enzyme Inhibitors; Ethanol; Free Radical Scavengers; Guanylate Cyclase; Humans; Hydrogen Peroxide; Hydroquinones; In Vitro Techniques; Indomethacin; Mannitol; Mesenteric Artery, Superior; Methylene Blue; Muramidase; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Oxadiazoles; Phenylephrine; Prostaglandins; Protein Kinase Inhibitors; Quinoxalines; Receptors, Cytoplasmic and Nuclear; Sepsis; Signal Transduction; Soluble Guanylyl Cyclase; Thionucleotides; Time Factors; Vasoconstrictor Agents; Vasodilation

Inflammatory mediators have been implicated as a cause of reversible myocardial depression in septic shock. We previously reported that the release of lysozyme-c (Lmz-S) from leukocytes from the spleen or other organs contributes to myocardial dysfunction in Escherichia coli septic shock in dogs by binding to a cardiac membrane glycoprotein. However, the mechanism by which Lzm-S causes this depression has not been elucidated. In the present study, we tested the hypothesis that the binding of Lzm-S to a membrane glycoprotein causes myocardial depression by the formation of nitric oxide (NO). NO generation then activates soluble guanylyl cyclase and increases cyclic guanosine monophosphate (cGMP), which in turn triggers contractile impairment via activation of cGMP-dependent protein kinase (PKG). We examined these possibilities in a right ventricular trabecular preparation in which isometric contraction was used to measure cardiac contractility. We found that Lzm-S's depressant effect could be prevented by the non-specific NO synthase (NOS) inhibitor N(G)-monomethyl-l-arginine (l-NMMA). A guanylyl cyclase inhibitor (ODQ) and a PKG inhibitor (Rp-8-Br-cGMP) also attenuated Lzm-S's depressant effect as did chemical denudation of the endocardial endothelium (EE) with Triton X-100 (0.5%). In EE tissue, we further showed that Lzm-S caused NO release with use of 4,5 diaminofluorescein, a fluorescent dye that binds to NO. The present study shows that the binding of Lzm-S to EE generates NO, and that NO then activates the myocardial guanosine 3',5' monophosphate pathway leading to cardiac depression in sepsis.

Topics: Animals; Cell Membrane; Cyclic GMP; Dogs; Endocardium; Endothelium; In Vitro Techniques; Muramidase; Myocardial Contraction; Myocardium; Nitric Oxide; omega-N-Methylarginine; Oxadiazoles; Quinoxalines; Sepsis

A rodent model of sepsis was used to establish the relationship between caspase inhibition and inhibition of apoptotic cell death in vivo. In this model, thymocyte cell death was blocked by Bcl-2 transgene, indicating that apoptosis was predominantly dependent on the mitochondrial pathway that culminates in caspase-3 activation. Caspase inhibitors, including the selective caspase-3 inhibitor M867, were able to block apoptotic manifestations both in vitro and in vivo but with strikingly different efficacy for different cell death markers. Inhibition of DNA fragmentation required substantially higher levels of caspase-3 attenuation than that required for blockade of other apoptotic events such as spectrin proteolysis and phosphatidylserine externalization. These data indicate a direct relationship between caspase inhibition and some apoptotic manifestations but that small quantities of uninhibited caspase-3 suffice to initiate genomic DNA breakdown, presumably through the escape of catalytic quantities of caspase-activated DNase. These findings suggest that putative caspase-independent apoptosis may be overestimated in some systems since blockade of spectrin proteolysis and other cell death markers does not accurately reflect the high degrees of caspase-3 inhibition needed to prevent DNA fragmentation. Furthermore, this requirement presents substantial therapeutic challenges owing to the need for persistent and complete caspase blockade.

Topics: Animals; Apoptosis; Biomarkers; Caspase 3; Caspase Inhibitors; Cysteine Proteinase Inhibitors; DNA Fragmentation; Female; Genes, bcl-2; Mice; Mice, Inbred C57BL; Mice, Transgenic; Oxadiazoles; Pyrazines; Rats; Rats, Sprague-Dawley; Sepsis; Spectrin; T-Lymphocytes

We investigated whether blockade of the smooth muscle cell (SMC) inducible nitric oxide synthase (iNOS)-soluble guanylyl cyclase (sGC) vasodilator pathway would restore the fall in vasoreactivity produced by sepsis following cecal ligation and perforation (CLP) in rats. Contraction of adjacent aortic rings paired for the presence or absence of endothelial cells (EC) was recorded following high [K(+)](e) (40 mm) or norepinephrine (NE, 10(-8) to 10(-5) m) in the presence of the nitric oxide synthase inhibitor (NOS), N(G)-nitro-l-arginine methyl ester (l-NAME, 0.3 mm) or the sGC inhibitor, 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ, 5 mum). In EC-denuded rings, sepsis halved SMC contraction induced by high [K(+)](e) or NE; neither l-NAME nor ODQ produced an increase in NE E(max) or high [K(+)](e)-evoked contraction. In conclusion, SMC contractility is globally reduced in CLP; this reduction does not appear to be explained by induction of SMC NOS in this CLP model.

Topics: Animals; Aorta; Disease Models, Animal; Endothelial Cells; Enzyme Inhibitors; Guanylate Cyclase; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Oxadiazoles; Quinoxalines; Rats; Rats, Wistar; Receptors, Cytoplasmic and Nuclear; Sepsis; Soluble Guanylyl Cyclase

Sepsis and posttraumatic inflammatory processes are accompanied by definite changes in microvascular permeability, particularly in the lung. These permeability changes may occur because of damaged regulatory mechanisms at the level of the capillary wall. Pericytes are adventitial cells located within the basement membrane of capillaries. These cells contain multiple cytoplasmic processes that envelope endothelial cells, and are consequently thought to stabilize capillary walls and participate in microcirculation and endothelial cell permeability. Data from this laboratory and other laboratories have confirmed that pericytes are contractile cells, adding to the evidence that pericytes may influence or help regulate capillary permeability. We have already determined that hydrogen peroxide (H2O2) causes dose-dependent relaxation in microvascular lung pericytes (MLPs) at 10 minutes and, conversely, dose-dependent contraction at 30 minutes. It is the aim of this study to determine the mechanism of this biphasic contractile response. Specifically, we will determine whether cyclic adenosine monophosphate (cAMP)- or cyclic guanosine monophosphate (cGMP)-dependent protein kinase intracellular pathways are responsible for the hydrogen peroxide-induced contractility of MLPs.. Rat MLPs were isolated by previously published protocol and cultured on collagen gel matrices. MLPs were pretreated with either ODQ, a soluble guanylate cyclase inhibitor (100 mumol/L), for 15 minutes; GKIP, a protein kinase G inhibitor (100 mumol/L), for 1 hour; SQ22536, an adenylate cyclase inhibitor (100 mumol/L), for 15 minutes; or H89, a protein kinase A inhibitor (10 mumol/L), for 1 hour. Hydrogen peroxide was then introduced to each MLP culture at 10 mumol/L, 100 mumol/L, and 1 mmol/L. After each of these treatments, the surface area of the collagen gels was digitally quantified at 10 and 30 minutes.. SQ22536 attenuated both relaxation at 10 minutes and the contraction seen at 30 minutes for all concentrations of H2O2. H89 caused a marked basal relaxation and prevented the cells from contracting at 30-minute exposures to all concentrations of H2O2. Both ODQ and GKIP attenuated the relaxation at 10 minutes but had no affect on the later contraction.. The cGMP-dependent protein kinase pathway is a mechanism for H2O2-induced relaxation of MLPs. Up-regulation of cAMP and cGMP is responsible for early H2O2-induced relaxation and late contraction. Protein kinase A (cAMP-dependent protein kinase pathway) may be an important intracellular signaling protein in the H2O2-induced contraction of MLPs or may be unable to down-regulate cAMP once inhibited. This evidence further supports the concept that there are separate intracellular pathways that regulate divergent cellular responses. This idea parallels the clinical concept of reversible and irreversible dysfunction of cellular processes in shock, and that the cellular dysfunction is initiated by separate intracellular pathways.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Analysis of Variance; Animals; Cell Survival; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Hydrogen Peroxide; Isoquinolines; Lung; Male; Muscle Contraction; Oxadiazoles; Pericytes; Quinoxalines; Rats; Rats, Sprague-Dawley; Respiratory Distress Syndrome; Sepsis; Sulfonamides