Photophysical properties and in vitro photocytotoxicity of disodium salt 2.4-di(alpha-methoxyethyl)-deuteroporphyrin-IX (Dimegine).

Photophysical and in vitro photocytotoxicity studies were performed for the photosensitizer Dimegine, a disodium salt 2.4-di(alpha-methoxyethyl)-deuteroporphyrin-IX with very low systemic toxicity. The singlet oxygen and luminescence quantum yield were ΦΔ = 0,65 ± 0,06, and Φƒ=0,11 ± 0,01 respectively, and were independent of the excitation wavelength. The photobleaching coefficients for Dimegine dissolved in phosphate buffer (pH 7.4), and DMEM medium at concentration 2 µM/l and in phosphate buffer (pH 7.0) at concentration 10 µM/l were 16·10-5, 9·10-5 and 2·10-5 respectively. In vitro cellular distribution and photocytotoxicity was studied in two human (U87 - primary glioblastoma and HT1376 - bladder cancer) and two rat cell lines (RG2 - glioma, and AY27 - bladder carcinoma). Fluorescence microscopy analysis shows primary Dimegine accumulation as small fluorescent inclusion bodies around the nuclei, suggesting an apoptotic over a necrotic cell death mechanism. The PDT efficacy was slightly higher for the rat cell lines over the human-derived cell lines, with LD50 values of 2,5 µM/l, 2.8 µM/l, 4.5 µM/l, 2.8 µM/l using 530 nm excitation wavelength for AY27, RG2, HT1376 and U87 respectively, and 1.8 µM/l, 2 µM/l, 5 µM/l, 2.4 µM/l using 625 nm excitation wavelength for AY27, RG2, HT1376 and U87 respectively. Comparison to literature data showed that Dimegine demonstrated improved phototherapeutic characteristics comparing to PpIX-mediated PDT.

The synergistic effect of PDT and oxacillin on clinical isolates of Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is a major pathogen in clinical microbiology. It is known to cause infections at various body sites and can be life-threatening. The development of resistance to many well-established antibiotic treatments and the prevalence of methicillin-resistant S. aureus (MRAS) among hospital patients and the general community pose challenges in treating the pathogen. The antimicrobial effect of photodynamic therapy (PDT) has been a subject of study for a long time and can offer new strategies for dealing with resistant strains. OBJECTIVE: In our study, we searched for a positive synergistic relationship between PDT and the standard antibiotics used to treat S. aureus and MRSA infections. MATERIALS AND METHODS: The phototoxic profile of deuteroporphyrin (DP) in both resistant and susceptible clinical strains of S. aureus was determined by plating of treated and untreated broth cultures. Electron microscopy imaging was done to explore possible sites of damage and free-radical accumulation in the cells during DP-PDT. Minimal inhibitory concentration (MIC) of oxacillin, gentamicin, vancomycin, rifampin, and fusidic acid was determined using the broth dilution method, and the checkerboard method was used to detect and evaluate the synergistic potential of DP-PDT and antibiotic combinations. A synergistic combination was further characterized using broth cultures and plating. RESULTS: DP-PDT using a light dose of 15 J/cm2 showed a bactericidal effect even with a small concentration of 17 µM DP. Transmission electron microscopy indicated profound damage in the cell wall and cell membrane, and the appearance of mesosome-like structures. Free radicals tend to localize in the cell membrane and inside the mesosome. No synergistic effect was detected by combining PDT with gentamicin, vancomycin, rifampin, and fusidic acid treatments. A positive synergistic effect was observed only in DP-PDT-oxacillin combined treatment using the checkerboard method. The effect was observed in clinical antibiotic-resistant isolates after DP-PDT using a light dose of 46 J/cm2 and small concentrations of DP. Oxacillin MIC decreased below 2 µg/ml in resistant strains under such conditions. Cultures which did not undergo new cycles of DP-PDT recovered their original oxacillin resistance after a few generations. CONCLUSIONS: PDT with porphyrins shows possible new therapeutic options in treating drug-resistant S. aureus at body sites suitable for irradiation. The synergistic effect of DP-PDT with oxacillin on clinical strains illustrates the potential of PDT to augment traditional antibiotic treatment based on cell wall inhibitors. Lasers Surg. Med. 50:535-551, 2018. © 2018 Wiley Periodicals, Inc.

[Perspectives of disodium salt 2.4-di(1-metoxyethyl)-deuteroporphyrin - IX ("Dimegin") application for photodynamic therapy in non-oncologic cases].

Effects of disodium salt 2,4-di(1-metoxyethyl)-deuteroporphyrine-IX (Dimegin) and the light from Soret band (¼395-405 nm) at the viability of microbial cells and at their potential to form microbial biofilms have been compared with traditional antiseptics. Irradiation of microbial cells of S. aureus, E. coli, C. albicans and others with diode light (power density 0.05 Wt/cm2) caused a bactericidial effect similar to that obtained with standard anticeptics (chlorhexidine and dioxidine). A comparative study of the effectiveness of Dimegin and Photoditazine (a soluble salt of chlorine e6) as photosensitizers have been performed using the test system of erythrocyte hemolysis in vitro under irradiation with light from the Sore band. Results have shown insignificant difference in the photodynamic effect with similar doses of absorbed light and preparation concentration.

Heme oxygenase inhibition enhances neutrophil migration into the bronchoalveolar spaces and improves the outcome of murine pneumonia-induced sepsis.

A reduction of the neutrophil migration into the site of infection during cecal ligation and puncture-induced sepsis increases host mortality. Inhibition of heme oxygenase (HO) prevents this neutrophil paralysis and improves host survival in the cecal ligation and puncture model. Taking into account that almost 50% of all sepsis cases are a consequence of pneumonia, we designed the present study to determine the role of HO in an experimental model of pneumonia-induced sepsis. The objective of this study was to evaluate whether the inhibition of HO improves the outcome and pathophysiologic changes of sepsis induced by an intratracheal instillation of Klebsiella pneumoniae. The pretreatment of mice subjected to pneumonia-induced sepsis with ZnDPBG (zinc deuteroporphyrin 2,4-bis glycol), a nonspecific HO inhibitor, increased the number of neutrophils in the bronchoalveolar spaces, reduced the bacterial load at the site of infection, and prevented the upregulation of CD11b and the downregulation of CXCR2 on blood neutrophils. Moreover, the pretreatment with ZnDPBG decreased alveolar collapse, attenuating the deleterious changes in pulmonary mechanics and gas exchanges and, as a consequence, improved the survival rate of mice from 0% to ∼20%. These results show that heme oxygenase is involved in the pathophysiology of pneumonia-induced sepsis and suggest that HO inhibitors could be helpful for the management of this disease.

Effect of light exposure on metalloporphyrin-treated newborn mice.

BACKGROUND: Neonatal hyperbilirubinemia arises from increased bilirubin production and decreased bilirubin elimination. Although phototherapy safely and effectively reduces bilirubin levels, recent evidence shows that it has adverse effects. Therefore, alternative treatments are warranted. Metalloporphyrins, competitive inhibitors of heme oxygenase (HO), the rate-limiting enzyme in bilirubin production, effectively reduce bilirubin formation; however, many are photoreactive. Here, we investigated possible photosensitizing effects of chromium mesoporphyrin (CrMP) and zinc deuteroporphyrin bis-glycol (ZnBG). METHODS AND RESULTS: Administration of CrMP or ZnBG to 3-d-old mouse pups (3.75-30.0 µmol/kg intraperitoneally) and exposure to cool white (F20T12CW) and blue (TL20W/52) fluorescent lights (+L) for 3 h, resulted in a dose-dependent mortality (50% lethal dose (LD50) = 21.5 and 19.5 µmol/kg, respectively). In contrast to ZnBG, there was no significant difference in survival between the CrMP+L and CrMP groups. Following 30 µmol/kg ZnBG+L, we found significant weight loss, decreased liver antioxidant capacities, and increased aspartate aminotransaminase levels. At 6-d post-light exposure, ZnBG+L-treated pups showed gross and histologic skin changes at doses >7.5 µmol/kg. No lethality was observed following treatment with 30 µmol ZnBG/kg plus exposure to blue light-emitting diodes. Phototoxicity of ZnBG was dependent on light source, emission spectrum, and irradiance. CONCLUSION: Low doses of ZnBG (<3.75 µmol/kg) retained maximal HO inhibitory potency without photosensitizing effects, and therefore are potentially useful in treating neonatal hyperbilirubinemia.

Effects of zinc deuteroporphyrin bis glycol on newborn mice after heme loading.

Infants with hemolytic diseases frequently develop hyperbilirubinemia and are treated with phototherapy, which only eliminates bilirubin after its production. A better strategy might be to directly inhibit heme oxygenase (HO), the rate-limiting enzyme in bilirubin production. Metalloporphyrins (Mps) are heme analogs that competitively inhibit HO activity in vitro and in vivo and suppress plasma bilirubin levels in vivo. A promising Mp, zinc deuteroporphyrin bis glycol (ZnBG), is orally absorbed and effectively inhibits HO activity at relatively low doses. We determined the I(50) (the dose needed to inhibit HO activity by 50%) of orally administered ZnBG in vivo and then evaluated ZnBG's effects on in vivo bilirubin production, HO activity, HO protein levels, and HO-1 gene expression in newborn mice after heme loading, a model analogous to a hemolytic infant. The I(50) of ZnBG was found to be 4.0 µmol/kg body weight (BW). At a dose of 15 µmol/kg BW, ZnBG reduced in vivo bilirubin production, inhibited heme-induced liver HO activity and spleen HO activity to and below baseline, respectively, transiently induced liver and spleen HO-1 gene transcription, and induced liver and spleen HO-1 protein levels. We conclude that ZnBG may be an attractive compound for treating severe neonatal hyperbilirubinemia caused by hemolytic disease.

Bystander effects induced by diffusing mediators after photodynamic stress.

The bystander effect, whereby cells that are not traversed by ionizing radiation exhibit various responses when in proximity to irradiated cells, is well documented in the field of radiation biology, Here we demonstrate that considerable bystander responses are also observed after photodynamic stress using the membrane-localizing dye deuteroporphyrin (DP). Using cells of a WTK1 human lymphoblastoid cell line in suspension and a transwell insert system that precludes contact between targeted and bystander cells, we have shown that the bystander signaling is mediated by diffusing species. The extranuclear localization of the photosensitizer used suggests that primary DNA damage is not the trigger for initiating these bystander responses, which include elevated oxidative stress, DNA damage (micronucleus formation), mutagenesis and decreased clonogenic survival. In addition, oxidative stress in the bystander population was reduced by the presence of the membrane antioxidant vitamin E in the targeted cells, suggesting that lipid peroxidation may play a key role in mediating these bystander effects. The fluence responses for these bystander effects are non-linear, with larger effects seen at lower fluences and toxicity to the target cell population. Hence, when considering outcomes of photodynamic action in cells and tissue, bystander effects may be significant, especially at sublethal fluences.

Cardioprotection by hypoxia-inducible factor 1 alpha transfection in skeletal muscle is dependent on haem oxygenase activity in mice.

AIMS: The present study investigates whether the cardioprotection achieved by gene delivery of hypoxia-inducible factor-1 alpha (HIF-1 alpha) depends on the downstream factor haem oxygenase (HMOX)-1. METHODS AND RESULTS: Immortalized cardiomyocytes (HL-1 cells) were transfected with HIF-1 alpha or HMOX-1 and injured with hydrogen peroxide (H(2)O(2)), and death was evaluated by trypan blue staining. Quadriceps muscles of mice were treated with DNA for HIF-1 alpha and HMOX-1, or sham-treated and electroporated, and 3 days later, hearts were isolated and subjected to global ischaemia and reperfusion. Some HIF-1 alpha- and sham-treated mice received the HMOX blocker zinc deuteroporphyrin 2,4-bis-glycol (ZnBG) (n = 6-8 in each group). HL-1 cells were stimulated with bilirubin or the carbon monoxide donor CORM-2 before injury with H(2)O(2). HL-1 cells which were transfected with HIF-1 alpha or HMOX-1 had an increased survival to H(2)O(2)-induced injury compared with empty vector (n = 10-12 per group; P < 0.01 for both). When HMOX-1-luciferase reporter mice were treated with HIF-1 alpha in the quadriceps muscle, increased luciferase activity was found locally, but nowhere else. Mice pre-treated with HIF-1 alpha or HMOX-1 had a reduced infarct size, improved post-ischaemic function, and increased serum bilirubin (P < 0.05). ZnBG inhibited all these effects afforded by HIF-1 alpha. Stimulation of HL-1 cells with bilirubin and CORM-2 reduced cell death evoked by H(2)O(2) (P < 0.05 for both, n = 11-15 in each group). CONCLUSION: HIF-1 alpha and HMOX-1 provided protection against H(2)O(2)-induced damage in HL-1 cells. Remote gene delivery of HIF-1 alpha afforded cardioprotective effects. These were dependent on HMOX activity, as an HMOX blocker abolished the effects, and they were mimicked by pre-treatment with HMOX-1. Downstream to HMOX-1, bilirubin as well as carbon monoxide may be organ effectors.

Carbon monoxide-prostaglandin E2 interaction in the hypothalamic circulation.

The heme oxygenase (HO)-carbon monoxide pathway was earlier shown to increase hypothalamic blood flow after inhibition of nitric oxide synthesis in rats. We hypothesized that this effect is mediated by prostaglandin E2 (PGE2). Inhibition of constitutive HO activity decreased cerebral PGE2 production and simultaneously increased hypothalamic nitric oxide synthase (NOS) activity without changing hypothalamic blood flow. Furthermore, HO blockade induced cyclooxygenase-dependent decrease and NOS-mediated increase of the hypothalamic blood flow after inhibition of NOS and cyclooxygenase, respectively. Therefore, constitutive carbon monoxide release seems to have two indirect effects on the hypothalamic circulation: vasodilation mediated by PGE2 and vasoconstriction as a result of NOS inhibition.

Role of locus coeruleus heme oxygenase-carbon monoxide-cGMP pathway during hypothermic response to restraint.

Central heme oxigenase-carbon monoxide (HO-CO) pathway has been shown to play a pyretic role in the thermoregulatory response to restraint. However, the specific site in the central nervous system where CO may act modulating this response remains unclear. LC is rich not only in sGC but also in heme oxygenase (HO; the enzyme that catalyses the metabolism of heme to CO, along with biliverdin and free iron). Therefore, the possible role of the HO-CO-cGMP pathway in the restraint-induced-hypothermia by LC neurons was investigated. Body temperature dropped about 0.7 degrees C during restraint. ZnDPBG (a HO inhibitor; 5 nmol, intra-LC) prevented the hypothermic response during restraint. Conversely, induction of the HO pathway in the LC with heme-lysinate (7.6 nmol, intra-LC) intensified the hypothermic response to restraint, and this effect was prevented by pretreatment with ODQ (a sGC inhibitor; given intracerebroventricularly, 1.3 nmol). Taken together, these data suggest that CO in the LC produced by the HO pathway and acting via cGMP is implicated in thermal responses to restraint.

Pharmacokinetics, tissue distribution and excretion of a new photodynamic drug deuxemether.

Deuxemether was a new photodynamic drug effective for many kinds of solid tumor therapy, which was mainly composed of 3-(or 8-)-(1-methoxyethyl)-8-(or 3-)-(1-hydroxyethyl)-deutero-porphyrin IX (MHD) and 3,8-di(1-methoxyethyl)-deuteroporphyrin IX (DMD). The aims of this study were to elucidate its pharmacokinetic characteristics, tissue distribution, plasma protein binding and excretion properties and underlying mechanisms of deuxemether in rats based on the simultaneous determination of MHD and DMD. The pharmacokinetic profiles of both MHD and DMD in rats after intravenous doses were linear and best fitted to a two compartment model, characterized with a rapid distribution phase (MHD: t(1/2)alpha, 0.09-0.14 h; DMD: t 1/2 alpha, 0.07-0.11h) and a relatively slow elimination phase (MHD: t 1/2 beta, 2.03-3.20 h; DMD: t 1/2 beta, 2.51-3.20 h). The tissue distributions of MHD and DMD in rats were rather limited as evidenced from their low distribution volume (0.75-1.70 L/kg) and the results of tissue distribution study. Protein binding of MHD and DMD were moderate (65.36-89.99% for MHD; 45.43-76.23% for DMD), independent of drug concentrations and similar between human and rat plasma over a concentration range of 0.50-50.0 microg/mL. Both MHD and DMD were predominantly (>74.1%) eliminated from rats as the parent drugs through the hepatobiliary systems and finally excreted into the feces. The multidrug resistance-associated proteins 2 (MRP2) inhibitors, bromosulfophthalein and probenecid, substantially inhibited the hepatobiliary elimination of MHD and DMD while the P-gp inhibitor digoxin had little effect, suggesting that MRP2 may contribute to the rapid and extensive hepatobiliary excretion of deuxemether. There were no significant differences between MHD and DMD for all pharmacokinetic characteristics studied. In conclusion, this study provided firstly the full pharmacokinetic characteristics and mechanisms of deuxemether, which would be helpful for its clinical regiment design.

Role of the spinal cord heme oxygenase-carbon monoxide-cGMP pathway in the nociceptive response of rats.

The aim of the present study was to investigate the role of the spinal cord heme oxygenase (HO)-carbon monoxide (CO)-soluble guanylate cyclase (sGC)-cGMP pathway in nociceptive response of rats to the formalin experimental nociceptive model. Animals were handled and adapted to the experimental environment for a few days before the formalin test was applied. For the formalin test 50 microl of a 1% formalin solution was injected subcutaneously in the dorsal surface of the right hind paw. Following injections, animals were observed for 1 h and flinching behavior was measured as the nociceptive response. Thirty min before the test, rats were pretreated with intrathecal injections with the HO inhibitor, zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG) or heme-lysinate, which is known to induce the HO pathway. Control animals were treated with vehicles. We observed a significant increase in nociceptive response of rats treated with ZnDPBG, and a drastic reduction of flinching nociceptive behavioral response in the heme-lysinate treated animals. Furthermore, the HO pathway seems to act via cGMP, since methylene blue (a sGC inhibitor) prevented the reduction of flinching nociceptive behavioral response caused by heme-lysinate. These findings strongly indicate that the HO pathway plays a spinal antinociceptive role during the formalin test, acting via cGMP.

Investigation of conditions involved in the susceptibility of the dermatophyte Trichophyton rubrum to photodynamic treatment.

BACKGROUND: Photodynamic treatment (PDT) refers to a treatment with light-activated agents (photosensitizers) in combination with visible light and molecular oxygen. Recently, we have demonstrated that the porphyrins, 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) and deuteroporphyrin monomethylester (DP mme) are excellent photosensitizers to be used against Trichophyton rubrum both in vitro and ex vivo. OBJECTIVES AND METHODS: The objective of this study was to investigate the key factors involved in PDT efficacy of both photosensitizers in an ex vivo situation during different fungal growth stages using a recently developed ex vivo model. The study focused on the influence of pH and ion strength of incubation media, photochemical properties of the photosensitizers (spectra and singlet oxygen production), and the effect of several scavengers of reactive oxygen species (sodium azide, histidine, mannitol) and phenylmethylsulphonylfluoride (keratinase inhibitor) on the PDT efficacy. RESULTS AND CONCLUSIONS: The results show that an optimal pH and low concentrations of calcium are crucial for a selective binding of Sylsens B to the fungus, leading to an increased PDT efficacy. This selective binding to T. rubrum cannot be accomplished for DP mme. It can be concluded that the prerequisite for successful treatment is a use of a low molarity solution of pH 5, supplemented with a chelating agent and a keratinase activity-repressing agent. Under these conditions, PDT with Sylsens B inactivates, initially via singlet oxygen, effectively the fungus in different fungal growth stages.

Influence of the heme-oxygenase pathway on cerebrocortical blood flow.

Heme-oxygenase (HO)-derived carbon monoxide (CO) is generated in the cardiovascular and in the central nervous systems. Endogenous CO exerts direct vascular effects and has also been shown to inhibit nitric oxide synthase (NOS). In the current study, the heme-oxygenase blockade [zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG), 45 micromol/kg intraperitoneally] decreased cerebral CO production and increased cerebrocortical blood flow (CBF) in anesthetized rats. This latter effect was abrogated by the NOS blockade (50 mg/kg L-NAME intravenously). Furthermore, inhibition of CO production had no effect on stepwise hypoxia/hypercapnia-stimulated increases in CBF. Our results indicate that endogenous CO reduces the resting CBF via inhibition of NOS but fails to influence the CBF response to hypoxia and hypercapnia in adult rats.

A novel ex vivo skin model to study the susceptibility of the dermatophyte Trichophyton rubrum to photodynamic treatment in different growth phases.

BACKGROUND: Dermatophytes are fungi that can cause infections of skin, hair and nails because of their ability to feed on keratin. Superficial mycoses are among the most prevalent infectious diseases worldwide. Two important restrictions of current therapeutic options are the recurrence of the infection and prolonged treatment. This is especially true for infections caused by Trichophyton rubrum, a widely distributed dermatophyte. The application of photosensitizers for treatment of fungal infections is, within the field of photodynamic treatment (PDT), relatively new. Recently, we demonstrated that the porphyrins 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) and deuteroporphyrin monomethylester (DP mme) were excellent photosensitizers towards T. rubrum when using red light. OBJECTIVES AND METHODS: To evaluate the photodynamic effectiveness of the porphyrins in a situation that mimics the clinical situation, we developed an ex vivo model using human stratum corneum. This model offers the possibility of applying PDT at different time points during the germination and subsequent development of T. rubrum microconidia. The model was used for two different incubation media, Dulbecco's modified Eagle medium (DMEM) and distilled water. RESULTS AND CONCLUSIONS: We demonstrated that the PDT susceptibility of T. rubrum depended on the time of PDT application after spore inoculation. A decrease in susceptibility was observed with increasing time of PDT application for both photosensitizers in DMEM. Changing the incubation medium to distilled water resulted in an increased fungicidal effect for Sylsens B and in a decreased effect for DP mme. We conclude that T. rubrum is susceptible to PDT in a situation that mimics the clinical situation. The fungicidal effect of PDT on fungal spores is of particular importance.

Role of the peripheral heme oxygenase-carbon monoxide pathway on the nociceptive response of rats to the formalin test: evidence for a cGMP signaling pathway.

The aim of the present study was to investigate the role of the peripheral heme oxygenase (HO)-carbon monoxide (CO) pathway on nociceptive response of rats to the formalin experimental model of pain. Animals were handled and adapted to the experimental environment for a few days before the formalin test was applied. For the formalin test, 50 microl of a 1% formalin solution was used and injected subcutaneously in the dorsal surface of the right hind paw. Following injections, animals were observed for 1 h, and flinching behavior was measured as the nociceptive response. Twenty minutes before the test rats were pretreated with podal injections with the HO inhibitor, zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG) or heme-lysinate, which is known to induce the HO pathway. Control animals were treated with vehicles. We observed a significant increase on nociceptive response of rats treated with ZnDPBG, and a drastic reduction of flinching nociceptive behavioral response in the heme-lysinate and CO treated animals. Among the three different HO products, CO seems to account for the heme-lysinate effect because the injection of the gas attenuated the flinching response whereas biliverdine and deferoxanine (an iron chelator) failed to cause any significant change. Furthermore, CO seems to act via cGMP, since methylene blue (a soluble guanylate cyclase inhibitor) prevented the reduction of the flinching nociceptive behavioral response caused by heme-lysinate. These findings strongly indicate that CO is the HO pathway product that plays an antinociceptive role during the formalin test, acting via cGMP.

Role of heme-oxygenase pathway on vasopressin deficiency during endotoxemic shock-like conditions.

The septic shock is characterized by decrease in median arterial pressure; many researchers have been related a deficiency in vasopressin release during the septic shock. Lipopolysaccharide administration is used to induce septic shock model in animals. We investigated the heme-oxygenase (HO) inhibition during the endotoxemic shock-like conditions. The LPS administration induced a significant decrease in MAP (-15.4 +/- 1.2 mmHg at second hour, -25.8 +/- 8.7 mmHg at fourth hour, and -22.3 +/- 8.6 mmHg at sixth hour) with a concomitant increase in heart rate (486.3 +/- 55.0, 531.8 +/- 53.8, and 510.0 +/- 55.3 bpm, respectively), a significant decrease in diuresis (from 1.1 +/- 0.7 to 0.4 +/- 0.3/100g body weight at fourth hour), and a transitory decrease in body temperature (from 37.0 +/- 0.5 to 35.4 +/- 0.8 degrees C at second hour). An increase in plasma arginine vasopressin (AVP) concentration (from 3.2 +/- 0.9 to 19.0 +/- 5.7 pg/mL at the first hour) occurred in these animals and was present for 2 h after LPS administration, returning close to basal levels thereafter and remaining unchanged until the end of the experiment. When LPS was combined with the i.c.v. administration of HO inhibitor, we observed a sustained increase in plasma AVP concentration, attenuation in the drop of MAP, and increase in antidiuresis induced by LPS treatment. These data suggest that central HO pathway may activate a control mechanism that attenuates AVP secretion during endotoxemia and may consequently regulate the MAP and diuretic output.

Autonomic cardiovascular responses to heme oxygenase inhibition in conscious rats.

Carbon monoxide (CO) is produced in the course of heme degradation from biliverdin by heme oxygenase (HO) in various tissues, including the central nervous system. Recent studies suggest the inhibition of HO activity increases arterial pressure mediated by the autonomic nervous system. The present study was designed to investigate the autonomic regulation of cardiovascular responses to inhibition of endogenous CO production by the HO inhibitor Zinc deuteroporphyrin 2, 4-bis glycol (ZnDPBG) by using direct sympathetic nerve recordings in conscious, chronically instrumented rats. ZnDPBG induced increases in mean arterial pressure (MAP) (P<0.05) and renal sympathetic nerve activity (RSNA) (P<0.05) but no significant change in heart rate (P>0.05) in intact rats. In atropine-treated rats, ZnDPBG also induced increases in MAP (P<0.05) and RSNA (P<0.05) but no change in heart rate (P>0.05). In sinoaortic denervated rats, ZnDPBG induced increases in MAP (P<0.05), heart rate (P<0.05), and RSNA (P<0.05). ZnDPBG shifted the baroreflex curve for RSNA upward and to the right, which was characterized by increases in the maximum and minimum response and midpoint pressure without altering the maximum gain. These results indicate that inhibition of HO activity within the central nervous system causes sympathoexcitation, resulting in an increase in arterial pressure. We conclude that the CO/HO system plays an important role in cardiovascular regulation by modulating sympathetic tone.

Heme oxygenase/carbon monoxide-biliverdin pathway down regulates neutrophil rolling, adhesion and migration in acute inflammation.

BACKGROUND AND PURPOSE: Heme oxygenase (HO) activity is known to down-regulate inflammatory events. Here, we address the role of HO and its metabolites, carbon monoxide (CO) and biliverdin (BVD), in leukocyte rolling, adhesion and neutrophil migration during inflammatory processes. EXPERIMENTAL APPROACH: Intravital microscopy was used to evaluate leukocyte rolling and adhesion in the mesenteric microcirculation of mice. TNFalpha and IL-1beta were determined by ELISA and HO-1 protein expression by Western blot. KEY RESULTS: Intraperitoneal challenge with carrageenan enhanced HO-1 protein expression in mesentery and bilirubin concentration in peritoneal exudates. Pretreatment of mice with a non-specific inhibitor of HO (ZnDPBG) or with a HO-1 specific inhibitor (ZnPP IX) enhanced neutrophil migration, rolling and adhesion on endothelium induced by carrageenan. In contrast, HO substrate (hemin), CO donor (DMDC) or BVD reduced these parameters. The reduction of neutrophil recruitment promoted by HO metabolites was independent of the production of chemotactic cytokines. Inhibitory effects of CO, but not of BVD, were counteracted by treatment with a soluble guanylate cyclase (sGC) inhibitor, ODQ. Furthermore, inhibition of HO prevented the inhibitory effect of a nitric oxide (NO) donor (SNAP) upon neutrophil migration, while the blockade of NO synthase (NOS) activity by aminoguanidine did not affect the CO or BVD effects. CONCLUSIONS AND IMPLICATIONS: Metabolites of HO decreased leukocyte rolling, adhesion and neutrophil migration to the inflammatory site by a mechanism partially dependent on sGC. Moreover, inhibition by NO of neutrophil migration was dependent on HO activity.

Genetic overexpression of eNOS attenuates hepatic ischemia-reperfusion injury.

Previous studies have shown that endothelial nitric oxide (NO) synthase (eNOS)-derived NO is an important signaling molecule in ischemia-reperfusion (I-R) injury. Deficiency of eNOS-derived NO has been shown to exacerbate injury in hepatic and myocardial models of I-R. We hypothesized that transgenic overexpression of eNOS (eNOS-TG) would reduce hepatic I-R injury. We subjected two strains of eNOS-TG mice to 45 min of hepatic ischemia and 5 h of reperfusion. Both strains were protected from hepatic I-R injury compared with wild-type littermates. Because the mechanism for this protection is still unclear, additional studies were performed by using inhibitors and activators of both soluble guanylyl cyclase (sGC) and heme oxygenase-1 (HO-1) enzymes. Blocking sGC with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and HO-1 with zinc (III) deuteroporphyrin IX-2,4-bisethyleneglycol (ZnDPBG) in wild-type mice increased hepatic I-R injury, whereas pharmacologically activating these enzymes significantly attenuated I-R injury in wild-type mice. Interestingly, ODQ abolished the protective effects of eNOS overexpression, whereas ZnDPBG had no effect. These results suggest that hepatic protection in eNOS-TG mice may be mediated in part by NO signaling via the sGC-cGMP pathway and is independent of HO-1 signal transduction pathways.