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Background and Purpose: Cancer cells grow without the restraints of feedback control mechanisms, leading to increased cancer cell survival. The treatment of cancer is often complicated by the lack of response to chemotherapy leading to chemoresistance and persistent survival of tumour cells. In this work we studied the role of platelets in chemotherapy-induced cancer cell death and survival., Experimental Approach: Human adenocarcinoma cells, colonic (Caco-2) and ovarian (59 M) cells, were incubated with 5-fluorouracil (1-300 µg·mL(-1) ) or paclitaxel (1-200 µg·mL(-1) ) in the presence or absence of platelets (1.5 × 10(8) mL(-1) ) for 1, 24 or 72 h. Following incubation, cancer cells were harvested and cell survival/death was assayed using flow cytometry, Western blotting, real-time PCR, TaqMan® Gene Expression Assays and proteomics., Key Results: Human platelets increased the survival of colonic and ovarian adenocarcinoma cells treated with two standard anticancer drugs, 5-fluorouracil and paclitaxel. In the presence of platelets, cancer cells up-regulated anti-apoptotic and down-regulated pro-apoptotic genes, increased the number of cells in the synthesis of DNA and decreased the number in the quiescent phase, increased expression of cyclins, DNA repair proteins and MAPKs. The analysis of platelet-Caco-2 secretome demonstrated the release of the chemokine RANTES, thrombospondin-1, TGF-β and clusterin. Finally, human recombinant RANTES and thrombospondin-1 improved survival of Caco-2 cells challenged with paclitaxel., Conclusions and Implications: These data demonstrate that platelets increase adenocarcinoma cells survival, proliferation and chemoresistance to standard anticancer drugs. Modulating cancer cell-platelet interactions may offer a new strategy to improve the efficacy of chemotherapy., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

Background and Purpose: Tumour cells activate and aggregate platelets [tumour cell-induced platelet aggregation (TCIPA)] and this process plays an important role in the successful metastasis of cancer cells. To date, most studies on TCIPA have been conducted under no-flow conditions. In this study, we have investigated TCIPA in real time under flow conditions, using an ultrasound standing wave trap that allows formation and levitation of cancer cell clusters in suspension, thus mimicking the conditions generated by flowing blood., Experimental Approach: Using 59M adenocarcinoma and HT1080 fibrosarcoma cells and human platelets, cancer cell cluster-platelet aggregates were imaged in real time using epi-fluorescence microscopy (F-actin) and investigated in detail using confocal microscopy (matrix metalloproteinase-2-GPIIb/IIIa co-localization) and scanning electron and helium-ion microscopy (<1 nm resolution). The release of gelatinases from aggregates was studied using zymography., Key Results: We found that platelet activation and aggregation takes place on the surface of cancer cells (TCIPA), leading to time-dependent disruption of cancer cell clusters. Pharmacological modulation of TCIPA revealed that EDTA, prostacyclin, o-phenanthroline and apyrase significantly down-regulated TCIPA and, in turn, delayed cell cluster disruption, However, EGTA and aspirin were ineffective. Pharmacological inhibition of TCIPA correlated with the down-regulation of platelet activation as shown by flow-cytometry assay of platelet P-selectin., Conclusion and Implications: Our results show for the first time, that during TCIPA, platelet activation disrupts cancer cell clusters and this can contribute to metastasis. Thus, selective targeting of platelet aggregate-cancer cell clusters may be an important strategy to control metastasis., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Nanoparticles are tiny materials (<1000 nm in size) that have specific physicochemical properties different to bulk materials of the same composition and such properties make them very attractive for commercial and medical development. However, nanoparticles can act on living cells at the nanolevel resulting not only in biologically desirable, but also in undesirable effects. In contrast to many efforts aimed at exploiting desirable properties of nanoparticles for medicine, there are limited attempts to evaluate potentially undesirable effects of these particles when administered intentionally for medical purposes. Therefore, there is a pressing need for careful consideration of benefits and side effects of the use of nanoparticles in medicine. This review article aims at providing a balanced update of these exciting pharmacological and potentially toxicological developments. The classes of nanoparticles, the current status of nanoparticle use in pharmacology and therapeutics, the demonstrated and potential toxicity of nanoparticles will be discussed.

Background: Angiogenesis, the growth of new capillaries from pre-existing blood vessels, is regulated by a balance between its promoters and inhibitors. Platelets are an important circulating store of angiogenesis regulators. We have previously identified the angiogenesis inhibitor angiostatin in human platelets., Aim: To identify the mechanism of platelet angiostatin generation and its pharmacological regulation., Methods: Platelet aggregometry, flow cytometry, Western blot, zymography, immunofluorescence microscopy, matrigel-induced angiogenesis of human umbilical vein endothelial cells (HUVECs), and a panel of selective proteinase inhibitors were used to study the mechanism of angiostatin generation by platelets, its pharmacological regulation, and effects on angiogenesis. Release of pro-MMP-2 by HUVECs was also used to quantify angiogenesis., Results: Platelet membranes were identified as the site of angiostatin generation from plasminogen. Generation of angiostatin by platelet membranes was not affected by a matrix metalloproteinase (MMP) inhibitor, phenanthroline, but was inhibited by serine proteinase inhibitors aprotinin, leupeptin, plasminogen activator inhibitor-1, and selective inhibitor of urokinase plasminogen activator (uPA), uPA-STOP(TM). Angiostatin generation by intact platelets was inhibited by aprotinin, and the resulting incubate promoted angiogenesis to a greater extent than incubate where angiostatin generation occurred. Furthermore, HUVECs incubated with reaction mixture, where angiostatin generation was inhibited, released more pro-MMP-2 than HUVECs incubated with supernatants, where angiostatin generation occurred., Conclusions: We conclude that; (i) platelets constitutively generate angiostatin on their membranes; (ii) this mechanism is dependent on uPA, but not, MMPs; and (iii) inhibition of platelet angiostatin generation can further promote angiogenesis.

The immunomodulatory effects of physiological temperature change remain poorly understood and inter-relationships between changes in core temperature, stress hormones and cytokines during exertional hyperthermia are not well established. This experimental study was designed to examine how cytokine (tumour necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-12 and IL-1ra (receptor antagonist)) and hormone (epinephrine (Epi), norepinephrine (NE), growth hormone (GH) and cortisol (CORT)) responses are modified when the exercise-induced rise in core temperature is attenuated or exacerbated by immersion in a water bath. Ten men ((mean +/- SD) age: 26.9 +/- 5.7 years; height 1.75 +/- 0.07 m; body mass 76.0 +/- 10.9 kg; O(2 peak): 48.0 +/- 12.4 mL kg(-1) min(-1)) completed two 40-min cycle ergometer exercise trials at 65% O(2 peak) while immersed to mid-chest. Rectal temperature (T(re)) peaked at 39.1 +/- 0.03 and 37.5 +/- 0.13 degrees C during the hot (39 degrees C) and cold (18 degrees C) conditions, respectively. Blood samples were collected before, during (20- and 40-min) and after (30- and 120-min) exercise. Increases in circulating NE (>350%), Epi (>500%), GH (>900%), IL-12 (>150%) and TNF-alpha (>90%) were greatest after 40-min exercise in the heat. Substantial elevations of CORT (80%), IL-1ra (150%) and IL-6 (>400%) did not occur until after exercise was complete. Core temperature clamping decreased the rise in circulating stress hormone concentrations and abolished increases in plasma cytokine concentrations. These findings suggest that exercise-associated elevations of T(re) mediate increases of circulating stress hormones, which subsequently contribute to induction of circulating cytokine release.

Background: Roughly 400.000 cases of sepsis occur every year in the United States only and this is associated with a very high mortality. Bacterial lipopolysaccharide (LPS) triggers systemic inflammatory reactions in sepsis. However, down-stream cellular cascade initiated by LPS is still being elucidated. Nitric oxide (NO) and matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9) are known to be induced by LPS. We have investigated the release of NO, MMP-2 and MMP-9 following infusion of LPS to volunteers., Methods: IPS (2 ng kg-1) was infused to 10 healthy volunteers. Before the experiments were started the subjects had an intravenous catheters placed. An electrocardiogram was also placed and monitored constantly. Body temperature was measured by ear thermometer every 10 min Venous blood was collected and cell-free plasma assayed for the presence of MMP-2 and MMP-9 using zymography and NO using HPLC assay for NO metabolites, nitrite and nitrate., Results: The administration of LPS resulted in increased body temperature and tachycardia. Time-dependent release of MMP-9(30 fold increase from the baseline) peaking at 2 h following infusion of LPS was observed. LPS did not significantly modify the activity of MMP-2 (P > 0.05). Infusion of LPS did not significantly change the levels of nitrite and nitrate (from 60 +/- 11 to 67 +/- 10 micro m, P > 0.05)., Conclusion: The release of MMP-9, but not MMP-2 or NO, is a sensitive index of endotoxaemia in humans. MMP-9 release may contribute to the pathogenesis of sepsis via its pro-inflammatory effects on the vasculature.

Background/purpose: In neonates receiving extracorporeal membrane oxygenation (ECMO), platelet activation and dysfunction occur with the release of matrix metalloproteinase (MMP)-2, which stimulates platelet aggregation. Because inhaled nitric oxide (NO) reduces pulmonary hypertension and inhibits platelet aggregation, the authors examined the effects of inhaled NO on platelet activation induced by ECMO., Methods: Ten adult white New Zealand rabbits were instrumented for ECMO and assigned randomly to receive either inhaled NO at 40 ppm or 30% oxygen for 1 hour before ECMO and continued for 4 hours after starting ECMO. Platelet counts, collagen-induced platelet aggregation ex vivo, plasma MMP-2, and MMP-9 activities were measured., Results: (1) ECMO caused thrombocytopenia, decreased platelet aggregation, and increased plasma MMP-2 and MMP-9 activities in controls. (2) Inhaled NO inhibited platelet aggregation before ECMO but did not affect the ECMO-induced thrombocytopenia and platelet activation. (3) Inhaled NO significantly abolished the ECMO-induced increase in plasma MMP-2 but not MMP-9 activities., Conclusions: Although inhaled NO did not inhibit the platelet activation during ECMO in adult rabbits, it attenuated the increase in plasma MMP-2 activity that may be important for neonates treated with ECMO., (Copyright 2003, Elsevier Science (USA). All rights reserved.)

Has research on sleeping sickness, i.e., human African trypanosomiasis (HAT), been forgotten? To get an idea on funding, we consulted the Medline bibliographic database for the last 14 years. The number of publications on HAT was stagnant over the study period. By comparison there was a steady increase in the number of publications dealing with malaria. These findings suggest that interest in HAT research waned in favor of other endemics even though government or other funding agencies continued to finance research networks. To illustrate this situation, we present the funding and findings of our multidisciplinary working group in a wide range of domains including sleep, endocrine rhythms, identification of biological markers, research on physiopathologic mechanisms of the host-pathogen relationship, and development on new medications. Over the last 14 years, a total of 1 million Euros was spent to produce 68 publications on Medline, i.e., roughly 15000 [symbol: see text] per publication.

1. We have studied the effects of a novel agonist, solid-phase von Willebrand Factor (sVWF), on tumour cell-induced platelet aggregation (TCIPA). 2. Washed platelet suspensions were obtained from human blood and the effects of HT-1080 human fibrosarcoma cells and sVWF on platelets were studied using aggregometry, phase-contrast microscopy, and flow cytometry. 3. Incubation of platelets with sVWF (1.2 microg ml(-1)) and HT-1080 cells (5 x 10(3) ml(-1)) resulted in a two-phased reaction characterized first by the adhesion of platelets to sVWF, then by aggregation. 4. TCIPA in the presence of sVWF was inhibited by S-nitroso-glutathione (GSNO, 100 microM) and prostacyclin (PGI(2), 30 nM). 5. Platelet activation in the presence of tumour cells and sVWF resulted in the decreased surface expression of platelet glycoprotein (GP)Ib and up-regulation of GPIIb/IIIa receptors. 6. Pre-incubation of platelets with PGI(2) (30 nM) resulted in inhibition of sVWF-tumour cell-stimulated platelet surface expression of GPIIb/IIIa as measured by flow cytometry using antibodies directed against both non-activated and activated receptor. In contrast, GSNO (100 microM) did not affect sVWF-tumour cell-stimulated platelet surface expression of GPIIb/IIIa. 7. Flow cytometry performed with PAC-1 antibodies that bind only to the activated GPIIb/IIIa revealed that GSNO (100 microM) caused inhibition of activation of GPIIb/IIIa. 8. The inhibitors exerted no significant effects on TCIPA-mediated changes in GPIb. 9. Thus, sVWF potentiates the platelet-aggregatory activity of HT-1080 cells and these effects appear to be mediated via up-regulation of platelet GPIIb/IIIa. 10. Prostacyclin and NO inhibit TCIPA-sVWF-mediated platelet aggregation. The mechanisms of inhibition of this aggregation by PGI(2) differ from those of NO.

1. The pharmacological characteristics of solid-phase von Willebrand factor (svWF), a novel platelet agonist, were studied. 2. Washed platelet suspensions were obtained from human blood and the effects of svWF on platelets were measured using aggregometry, phase-contrast microscopy, flow cytometry and zymography. 3. Incubation of platelets with svWF (0.2 - 1.2 microg ml(-1)) resulted in their adhesion to the ligand, while co-incubations of svWF with subthreshold concentrations of ADP, collagen and thrombin resulted in aggregation. 4. 6B4 inhibitory anti-glycoprotein (GP)Ib antibodies abolished platelet adhesion stimulated by svWF, while aggregation was reduced in the presence of 6B4 and N-Acetyl-Pen-Arg-Gly-Asp-Cys, an antagonist of GPIIb/IIIa. 5. Platelet adhesion stimulated with svWF was associated with a concentration-dependent increase in expression of GPIb, but not of GPIIb/IIIa. 6. In contrast, collagen (0.5 - 10.0 microg ml(-1)) caused down-regulation of GPIb and up-regulation of GPIIb/IIIa in platelets. 7. Solid-phase vWF (1.2 microg ml(-1)) resulted in the release of MMP-2 from platelets. 8. Inhibition of MMP-2 with phenanthroline (10 microM), but not with aspirin or apyrase, inhibited platelet adhesion stimulated with svWF. 9. In contrast, human recombinant MMP-2 potentiated both the effects of svWF on adhesion and up-regulation of GPIb. 10. Platelet adhesion and aggregation stimulated with svWF were reduced by S-nitroso-n-acetyl-penicillamine, an NO donor, and prostacyclin. 11. Thus, stimulation of human platelets with svWF leads to adhesion and aggregation that are mediated via activation of GPIb and GPIIb/IIIa, respectively. 12. Mechanisms of activation of GPIb by svWF involve the release of MMP-2, and are regulated by NO and prostacyclin.

Objectives: Heart function is variably impaired after cardiopulmonary bypass. We hypothesized that, similar to other myocardial injury states, cardiopulmonary bypass leads to enhanced activity of nitric oxide synthase and matrix metalloproteinases., Methods: We obtained right atrial biopsy specimens and plasma samples at the onset and termination of cardiopulmonary bypass in 10 patients. Biopsy specimens were analyzed for nitric oxide synthase activity by using a citrulline assay, whereas plasma and tissue were analyzed for matrix metalloproteinase-9 and matrix metalloproteinase-2 activity by using zymography. Tissue inhibitor of metalloproteinase-4 was analyzed by means of Western blotting. The cellular expression of inducible nitric oxide, endothelial nitric oxide synthase, matrix metalloproteinase-2, and matrix metalloproteinase-9 was determined in right atrial biopsy samples from 3 additional patients by using the appropriate conjugated antibodies., Results: Nitric oxide synthase activity increased from the beginning to the end of bypass (4.46 +/- 1.07 vs 16.77 +/- 4.86 pmol citrulline/mg of protein per minute, respectively; P =.018). Pro-matrix metalloproteinase-9 activity increased in hearts (199 +/- 41 vs 660 +/- 177 density units/mg protein; P =.008) and plasma (14.1 +/- 4.6 vs 52.2 +/- 5.9 density units/mg protein; P =.008). Pro-matrix metalloproteinase-2 activity increased in the heart (201 +/- 23 vs 310 +/- 35 density units/mg protein, P <.05) but not in plasma. Tissue inhibitor of metalloproteinase-4 expression in the heart decreased (1574 +/- 280 vs 864 +/- 153 density units, P =.014)., Conclusions: Cardiopulmonary bypass activates enzymes mediating acute inflammation and organ injury (ie, nitric oxide synthase, matrix metalloproteinase-9, and matrix metalloproteinase-2). Decreased tissue inhibitor of metalloproteinase-4 expression allows relatively unopposed increases in matrix metalloproteinase tissue activity. We postulate that these changes play a role in the pathogenesis of heart dysfunction after bypass surgery.

Background: Matrix metalloproteinase-2 (MMP-2) has been shown to activate a non-thromboxane, non-ADP pathway of platelet aggregation. In contrast, nitric oxide (NO) is known to inhibit platelet adhesion and aggregation. Therefore, we have studied the release of MMP-2 during platelet adhesion to fibrinogen, the effects of phenanthrolione, an MMP-2 inhibitor, on adhesion and the interactions of inhibitor with a NO donor, S-nitroso-N-acetyl-D, L-penicillamine (SNAP)., Material and Methods: Human platelets were isolated from blood of healthy volunteers and platelet adhesion to fibrinogen-coated plates was studied by measuring thrombin-stimulated release of platelet a-granule constituent, platelet factor 4. In addition, the mode of action of phenanthroline and NO on platelets was investigated by assaying the levels of intraplatelet cyclic GMP., Results: Thrombin-stimulated platelet adhesion to fibrinogen was associated with increased release of MMP-2 from platelets. Phenanthroline (0.1-100 KM) reduced platelet adhesion to fibrinogen. The adhesion was also inhibited by SNAP (0.1-100 KM), an effect abolished by 1H-[1,2,4] oxadiazolol [4,3,-a] quinoxalin-1-one (ODQ), a selective inhibitor of the soluble guanylate cyclase. Co-administration of phenanthroline and SNAP resulted in a synergistic inhibition of platelet adhesion, an effect that was not associated with enhanced cyclic GMP generation by platelets. Furthermore, ODQ did not reverse the synergistic effect of these compounds on adhesion., Conclusions: 1. MMP-2 promotes platelet adhesion to fibrinogen. 2. Phenanthroline and NO synergize to inhibit platelet adhesion to fibrinogen acting through a cyclic GMP-independent mechanism(s).

Sleeping sickness, once under control, is a re-emergent endemic parasitic disease in intertropical Africa. Its originality resides in its duality. Two trypanosome groups (Trypanososma brucei gambiense vs.rhodesiense ) are transmitted to humans by tsetse flies from two geographical areas (Western and Central Africa humid forest vs. Eastern Africa arboreous savannah), provoking a slowly or a rapidly evolutive disease. The two stage (haemolymphatic vs. neurological invasion) pathogenic evolution leads to the duality of the immune response, depending on the host-parasite inter-relation differences in the blood and the brain. In the blood, the immune processes involved are both specific (anti-variant surface glycoprotein (VSG) antibodies) and non-specific (complement-mediated lysis, opsonification-facilitated phagocytosis and antibody dependent cell-mediated cytotoxicity). Although macrophages are activated in the blood and infiltrate the brain, nitric oxide decreases in the blood and increases in the brain, with a breakage in the blood-brain barrier, leading to brain lesions through the production of deleterious molecules. Prophylactic means are affected by the duality of pathogenic processes. This finally leads to a two stage disease (haemolymphatic vs. neurological) with two different therapeutic strategies. The sleep-wake cycle and other biological rhythms are also marked by the disappearance of circadian rhythmicity demasking basic ultradian activities and relationships, such as the interdependence of endocrine profiles and the sleep-wake alternation. 2001 Harcourt Publishers Ltd

The JCR:LA-cp rat is obese and insulin resistant and develops a major vasculopathy, with associated ischemic damage to the heart. Male rats were treated with 17alpha-ethinylestradiol (EE), LY117018, and/or the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME). LY117018 decreased plasma cholesterol esters, with a 40% reduction in total cholesterol. EE increased triglyceride levels and modestly decreased cholesterol esters. L-NAME increased blood pressure and aortic contractile sensitivity to phenylephrine and inhibited acetylcholine-induced relaxation. LY117018 decreased the force of contraction. The L-NAME-mediated increase in force of contraction and decrease in response to acetylcholine was inhibited by LY117018. L-NAME-induced hypertension was prevented by LY117018. Platelet aggregation was not different between obese and lean rats and was unaffected by L-NAME. LY117018, both in the absence and presence of L-NAME, inhibited platelet aggregation. The effects of LY117018 are apparently mediated through both NO-dependent and -independent mechanisms. The changes induced by EE and LY117018 may reflect the activation of multiple mechanisms, both estrogen receptor-dependent and -independent. The changes induced by LY117018 are significant and may prove to be cardioprotective in the presence of the insulin resistance syndrome.

At the meningoencephalitis stage, human African trypanosomiasis (HAT), sleeping sickness, causes dysregulation of the circadian rhythm of the sleep/wake cycle, rather than hypersomnia. In bedridden patients, total sleep time does not exceed 9 hours. The change in the 24-hour distribution of sleep and wakefulness is proportional to severity of clinical symptoms and laboratory abnormalities. The internal structure of sleep is also altered. All patients present sleep onset rapid eye movement periods (SOREMP), i.e., several sleep episodes beginning with rapid eye movement (REM) sleep. In mild cases, treatment with melarsoprol reverses circadian dysregulation, and SOREMP either decrease in number or disappear. Other circadian disturbances may be observed in HAT. These may include circadian dysrhythmia of hormonal secretions, but the relationship between hormonal pulses and sleep/wake states is preserved. The circadian rhythm of secretion of prolactin, renin, growth hormone and cortisol disappears in severe cases, but persists in mild ones. The amplitude and mean 24-hour value of plasma melatonin are normal with nocturnal peaks and no diurnal secretion. However, peak melatonin secretion occurs 2 hours earlier than in healthy African controls. In conclusion, HAT-induced dysregulation of circadian rhythm is proportional to disease severity. Presence of SOREMP and precocity of peak melatonin secretion support disturbance of the serotoninergic network rather than direct action on the biological clock.

A correlation exists between the ability of tumor cells to aggregate platelets and their tendency to metastasize. Tumor cell-induced platelet aggregation (TCIPA) facilitates the embolization of the vasculature with tumor cells and the formation of metastatic foci. It is well documented that matrix metalloproteinases (MMPs) play an integral part in tumor spread and the metastatic cascade. Therefore, we have examined the role of MMPs during TCIPA and its regulation by nitric oxide (NO) in vitro. Human HT-1080 fibrosarcoma and A549 lung epithelial cancer cells induced TCIPA in a concentration-dependent manner that was monitored by aggregometry. This aggregation resulted in the release of MMIP-2 from platelets and cancer cells, as measured by zymography. HT-1080 cells released significantly more MMP-2 than A549 cells and were more efficacious in inducing TCIPA. Inhibition of MMP-2 with phenanthroline (1-1000 microM), a synthetic inhibitor of MMPs, and by neutralizing anti-MMIP-2 antibody (10 microg/ml) reduced TCIPA induced by HT-1080 cells. TCIPA was abolished by simultaneous inhibition of platelet function with acetylsalicylic acid (100 microM; thromboxane pathway inhibitor), apyrase (250 microg/ml; ADP pathway inhibitor), and phenanthroline. NO donors such as S-nitroso-n-acetylpenicillamine and S-nitrosoglutathione (both at 0.01-100 microM) inhibited TCIPA and MMP-2 release from platelets and tumor cells. The inhibitory actions of S-nitroso-n-acetylpenicillamine and S-nitrosoglutathione were reversed by 1H-[1,2,4]oxadiazole[4,3]quinoxalin-1-one (0.01-30 microM), a selective inhibitor of the soluble guanylyl cyclase. We conclude that (a) human fibrosarcoma cells aggregate platelets via mechanism(s) that are mediated, in part, by MMP-2; (b) NO inhibits TCIPA, in part, by attenuating the release of MMP-2; and (c) these effects of NO are cGMP-dependent.

The large increase in placental surface area and fetal villous vascular development in the third trimester of pregnancy requires degradation and reformation of the placental basal lamina. Degradation is carried out by matrix metalloproteinases (MMPs) secreted by adjacent cells. Although the gelatinases, MMP-2 and MMP-9, which are released by extravillous cytotrophoblasts (CTs) are believed to play crucial roles in early placental expansion, neither has been reported in third trimester villous trophoblasts nor has appropriate (basolateral) release of any MMP by the highly polarized syncytiotrophoblast (ST) been demonstrated. We demonstrated villous trophoblast expression of both MMP-2 and MMP-9 by in situ immunohistochemistry and by Western blot analysis and zymography of lysates and culture supernatants of highly purified villous CTs. We also found that epidermal growth factor (EGF)-stimulated CT differentiation into ST and stimulation by the phorbol diester, PMA, both increase MMP-9 secretion. The direction of MMP release was determined with confluent cultures of ST on porous membranes. We found that >90% of MMP-2 and MMP-9 were released from the basolateral surface. We conclude that villous STs express and release gelatinases from their basolateral surfaces in a regulated manner and suggest that such polarized release may be important to villous tissue remodeling.

The human respiratory tract is covered with airway surface liquid (ASL) that is essential for lung defense and normal airway function. The quantity and composition of ASL is regulated by active ion transport across the airway epithelium. Abnormal electrolyte transport produces changes in ASL volume and composition, inhibits mucociliary clearance and leads to chronic infection of airway surfaces, as is evident in cystic fibrosis. Agonists that induce intracellular increases in cAMP or Ca2+ are generally associated with electrolyte secretion. While these mechanisms have been studied in detail for many years, modulation of ion channels by nitric oxide (NO) has emerged only recently as a significant determinant of ion channel function. NO is a physiological regulator of transepithelial ion movement and alterations of its generation and action may play an important role in the pathogenesis of lung disorders characterized by hypersecretion of ASL. This review presents the current understanding of regulation of airway epithelial ion channels by NO and attempts to highlight the importance of this regulation for lung defense.

Lung inflammation is associated with enhanced expression of proinflammatory cytokines and increased production of nitric oxide (NO) by inducible NO synthase (iNOS). To investigate the possible relationship between cytokine-induced expression of iNOS and epithelial ion channel function, we measured whole-cell current in A549 cells treated with a mixture of cytokines: tumor necrosis factor, interleukin-1 beta, and interferon-gamma for 12 h. Cytokines significantly increased the expression and activity of iNOS, and reduced generation of cGMP in response to stimulation with NO donor S-nitroso-glutathione (GSNO). Patch-clamp studies showed that 100 microM GSNO increased the whole-cell current from 11.2 +/- 1.8 to 19.6 +/- 2.7 pA/pF (n = 16) in control cells, but had no effect in cytokine-treated cells (n = 9). N-(3-(Aminomethyl)benzyl)acetamidine (1400W), a selective inhibitor of iNOS, restored activation of the current by GSNO in cytokine-treated cells, indicating a crucial role for iNOS in this process. Cells treated with cytokines showed increased levels of peroxynitrite (ONOO(-)), compared with the control, or cells that were treated with the cytokines and 1400W or superoxide dismutase/catalase. Treatment of cells with 100 microM ONOO(-) had no effect on the whole-cell current, but in contrast to untreated cells, subsequent application of GSNO did not activate the current. In conclusion, cytokine-induced expression of iNOS affects activation of the whole-cell current via NO/cGMP pathway, likely by increasing the generation of ONOO(-).

Matrix metalloproteinase (MMP)-2 has been historically associated with the process of vascular remodeling through the cleavage of extracellular matrix proteins. However, we recently found that MMP-2 also cleaves the endothelium-derived peptide big endothelin-1, ET-1[1-38] and yields the novel vasoconstrictor ET-1[1-32]. We therefore investigated the effects of MMP-2 inhibitors as potential vasodilators. MMP inhibition with ortho-phenanthroline (0.3 to 30 micromol/L) induced vasorelaxation of isolated rat mesenteric arteries (maximum of relaxation=74.5+/-27.6% at 30 micromol/L). However, phosphoramidon (0.3 to 30 micromol/L), which inhibits some metalloenzymes, but not MMP-2, did not dilate the arteries. Selective inhibition of endogenous MMP-2 with the novel tissue-permeable cyclic peptide CTTHWGFTLC (CTT, 10 micromol/L) also caused vasorelaxation (by 85+/-6%), whereas STTHWGFTLS (10 micromol/L), an inactive CTT analogue, did not dilate the arteries. Interestingly, the vasorelaxation that results from MMP-2 inhibition was endothelium-independent. Thus, we examined whether MMP-2 acted on peptides derived from the smooth muscle or the perivascular nerves. Recombinant human MMP-2 cleaved calcitonin gene-related peptide (CGRP) specifically at the Gly(14)-Leu(15) peptide bond and reduced the vasodilatory potency of CGRP by 20-fold. Inhibition of MMP-2 increased the amount of intact CGRP in arteries and enhanced vasorelaxation induced by anandamide, which stimulates CGRP release. Vasorelaxation in response to MMP-2 inhibition was abolished by CGRP[8-37], a selective CGRP receptor antagonist, and by capsaicin, which depletes arterial perivascular nerves of CGRP. We conclude that vascular MMP-2 cleaves endogenous CGRP and promotes vasoconstriction. These data suggest a novel mechanism of regulating the vasoactive and, possibly, the neurohormonal actions of CGRP and establish MMP-2 as a modulator of vascular function.

Objective: Although bleeding associated with thrombocytopenia often complicates extracorporeal membrane oxygenation (ECMO), the mechanisms of platelet dysfunction during ECMO remain poorly understood. We investigated the role of matrix metalloproteinase (MMP)-2, which recently has been shown to mediate a novel pathway of platelet aggregation, in the platelet dysfunction induced by ECMO., Design: Prospective longitudinal case study., Setting: Level III neonatal intensive care unit., Patients: Ten neonates treated with ECMO., Intervention: ECMO procedure., Measurements: Platelet counts and collagen-induced platelet aggregation ex vivo; plasma markers of platelet (soluble P-selectin) and endothelial (soluble E-selectin and total nitrite/nitrate) activation; plasma MMP-2 and MMP-9 activities; and concentrations of tissue inhibitors of MMPs., Main Results: During ECMO, time-dependent platelet activation, as evidenced by thrombocytopenia, decreased platelet aggregation, and increased plasma soluble P-selectin concentrations were found in the absence of endothelial activation, as shown by normal plasma concentrations of soluble E-selectin and nitric oxide metabolites (nitrite/nitrate). There was a time-dependent increase in plasma MMP-2 but not MMP-9 activity; tissue inhibitors of MMPs were not detected. Plasma soluble P-selectin concentrations significantly correlated with simultaneous plasma MMP-2 (r2 = .37, p < .0001) but not with MMP-9 activities. Platelet dysfunction persisted despite repeated platelet transfusions to maintain platelet counts >100 x 10(9)/L., Conclusions: ECMO resulted in the activation of platelets but not endothelial cells. During ECMO, platelet dysfunction persisted despite platelet transfusions. MMP-2 may play a role in the development of platelet dysfunction caused by ECMO.

The vasodilator effects of thrombin depend on activation of proteinase-activated receptor (PAR)-1 and the subsequent release of endothelin (ET)-1, which stimulates the generation of nitric oxide and PGs. We recently showed that thrombin released matrix metalloproteinase-2 (MMP-2) from rat arteries. We have now studied the significance of this release for the vasodilator effects of thrombin. Thrombin (>/=100 pmol), but not a PAR-1-activating peptide (TFLLR-NH(2)), produced a long-lasting (>10 min) vasorelaxation of rat mesenteric arteries, as detected by a microperfusion bioassay. Thrombin induced a simultaneous release of vascular MMP-2 into arterial perfusates, as revealed by zymography. Interestingly, the vasodilator effects of thrombin were inhibited by a tissue inhibitor of MMP-2 (TIMP-2, 10 pmol). Moreover, infusion of exogenous MMP-2 (5 pmol) resulted in vasorelaxation. These vasodilatory effects of thrombin and MMP-2 were significantly (P < 0.05) inhibited by endothelium denudation and by PD-142893 (2 nmol), an antagonist of ET receptors. Furthermore, both thrombin and MMP-2 constricted endothelium-denuded arteries. These results show that the vasodilator effects of thrombin may depend, in part, on a release of vascular MMP-2 and downstream activation of ETs. Thus MMP-2-dependent signaling may complement the PAR-1-dependent pathway of vasodilator action of thrombin.

Background: Matrix metalloproteinases (MMPs) contribute to collagen degradation and remodeling of the extracellular matrix after myocardial infarction; however, their role in myocardial dysfunction immediately after ischemia and reperfusion is unknown., Methods and Results: We measured the release of MMPs into the coronary effluent of isolated, perfused rat hearts during aerobic perfusion and reperfusion after ischemia. Aerobically perfused control hearts expressed pro-MMP-2 and MMP-2, as well as an unidentified 75-kDa gelatinase. These enzymes were also detected in the coronary effluent. After 20 minutes of global no-flow ischemia, there was a marked increase in pro-MMP-2 in the coronary effluent that peaked within the first minute of reperfusion. The release of pro-MMP-2 into the coronary effluent during reperfusion was enhanced with increasing duration of ischemia and correlated negatively with the recovery of mechanical function during reperfusion (r(2)=0.99). MMP-2 antibody (1.5 to 15 microg/mL) and the inhibitors of MMPs doxycycline (10 to 100 micromol/L) and o-phenanthroline (3 to 100 micromol/L) improved whereas MMP-2 worsened the recovery of mechanical function during reperfusion., Conclusions: These results show that acute release of MMP-2 during reperfusion after ischemia contributes to cardiac mechanical dysfunction. The inhibition of MMPs may be a novel pharmacological strategy for the treatment of ischemia-reperfusion injury.

We have recently found matrix metalloproteinase-2 (MMP-2) in human platelets and reported that the release of this enzyme during platelet activation stimulates aggregation. We have now identified matrix metalloproteinase-9 (MMP-9) in human platelets and resistance-sized (approximately 200 microm) arteries. Resting platelets released small quantities of pro-MMP-9. Maximal release of MMP-9 was detected during partial (appr. 30% maximum) aggregation with thrombin. However, maximal release of MMP-2 was associated with maximal aggregation. MMP-9 antibodies induced aggregation of resting platelets and potentiated aggregation of platelets induced by thrombin and collagen. Moreover, MMP-9 microisolated from arteries as well as recombinant human MMP-9 (0.1-30 ng/ml) inhibited thrombin and collagen-induced aggregation. We conclude that MMP-9 is an inhibitor of aggregation and in this action opposes the effects of MMP-2. The MMP-2/MMP-9 system may play an important role in the regulation of platelet-platelet and platelet-vessel wall interactions.

Matrix metalloproteinase-2 (MMP-2, gelatinase A) and its tissue inhibitor (TIMP-2) are mainly known for their roles in the (patho)physiological remodeling of the vasculature, angiogenesis, tissue repair, tumor invasion, inflammation, and atherosclerotic plaque rupture. A mechanism of action of MMP-2 is the proteolytic breakdown of specific extracellular matrix proteins. The amino acid sequences in interstitial collagen (Gly-Leu/Ile) and laminin-5 (Ala-Leu) that are cleaved by MMP-2 are homologous to a region (Gly(32)-Leu(33)) within human big endothelin-1[1 to 38] (big ET-1). Big ET-1 requires cleavage to an active form to produce vasoconstriction. We tested the hypothesis that vascular MMP-2 can cleave big ET-1, thus generating a vasoconstrictor peptide. In perfused rat mesenteric arteries with an intact endothelium, inhibition of vascular MMP-2 with TIMP-2 reduced (by 16.2+/-4.2%) the vasoconstrictor effects of big ET-1 (50 pmol). However, when the endothelium was mechanically removed, TIMP-2 abolished (>90%) the vasoconstriction of big ET-1, and this effect was mimicked by an anti-MMP-2 antibody. Incubation of big ET-1 with recombinant human MMP-2 resulted in the specific cleavage of the Gly(32)-Leu(33) bond of big ET-1. Moreover, the resultant peptide ET-1[1 to 32] exerted greater vasoconstrictor effects than big ET-1. We conclude that vascular MMP-2 contributes to the vasoconstrictor effects of big ET-1 by cleaving big ET-1 to yield a novel and potent vasoconstrictor, ET-1[1 to 32]. These data implicate, for the first time, the endogenous MMP-2/TIMP-2 system in the regulation of vascular reactivity.

Matrix metalloproteinase-2 (MMP-2, gelatinase A) and thrombin contribute to many long-term (patho)physiological processes requiring the proteolytic breakdown of the vascular extracellular matrix (e.g., normal tissue repair, remodeling, tumor invasion, atherosclerosis plaque rupture). Thrombin (10 to 1000 nM, 0.5 to 50 U/ml) induced a rapid secretion of MMP-2 from freshly isolated rat aortic tissue (detectable after 1 min of thrombin exposure). This secretion was mediated by an unidentified thrombin receptor, distinct from the proteinase activated receptors (PAR)-1 and -2. Protein tyrosine kinase/phosphatase activity differentially modulated the basal and the thrombin-induced release of MMP-2. The inhibitors of protein tyrosine kinase, herbymicin A, genistein, and tyrphostin 1288 (1 to 100 microM), enhanced the basal release of MMP-2 but did not affect the thrombin-induced secretion of MMP-2. The inhibitor of phosphotyrosine phosphatases, vanadate (100 microM), selectively inhibited the thrombin-induced, but not the basal, release of MMP-2. Rapid release of vascular MMP-2 by thrombin could contribute to short-term processes where thrombin is involved such as the regulation of platelet aggregation and vascular reactivity. Vascular tyrosine kinase/phosphatase likely modulates this action of thrombin to prevent exaggerated platelet aggregation, thrombosis, and vasospasm.

The contribution of hyperthermia to the differential leukocytosis of exercise remains obscure. This study examined changes in circulating sympathoadrenal hormone concentrations and patterns of leukocyte and lymphocyte subset (CD3(+), CD4(+), CD8(+), CD19(+), CD3(-)16(+)/56(+)) redistribution during exercise, with and without a significant rise of rectal temperature (T(re)). Ten healthy men [age 26.9 +/- 5.7 (SD) yr, body mass 76.0 +/- 10.9 kg, body fat 13.9 +/- 4.6%, peak O(2) consumption: 48.0 +/- 12.4 ml x kg(-1) x min(-1)] exercised for 40 min (65% peak O(2) consumption) during water immersion at 39 or 18 degrees C. T(re) increased from 37.2 to 39.3 degrees C (P < 0.0001) after 40 min of exercise in 39 degrees C water but was held constant to an increment of 0.5 degrees C during exercise in 18 degrees C water. Application of this thermal clamp reduced exercise-associated increments of plasma epinephrine (Epi) and norepinephrine (NE) by >50% (P < 0.05) and abolished the postexercise increase in cortisol. Thermal clamping also reduced the exercise-induced leukocytosis and lymphocytosis. Multiple regression demonstrated that T(re) had no direct association with lymphocyte subset mobilization but was significantly (P < 0.0001) correlated with hormone levels. Epi was an important determinant of total leukocytes, lymphocytes, and CD3(+), CD4(+), CD8(+), and CD3(-)CD16(+)/56(+) subset redistribution. The relationship between NE and lymphocyte subsets was weaker than that with Epi, with the exception of CD3(-)CD16(+)/56(+) counts, which were positively (P < 0.0001) related to NE. Cortisol was negatively associated with leukocytes, CD14(+) monocytes, and CD19(+) B- and CD4(+) T-cell subsets but was positively related to granulocytes. We conclude that hyperthermia mediates exercise-induced immune cell redistribution to the extent that it causes sympathoadrenal activation, with alterations in circulating Epi, NE, and cortisol.

Matrix metalloproteinases (MMPs) are involved in the remodeling and degradation of the extracellular matrix. Recently, it has been found that MMPs also contribute to processes not directly related to tissue remodeling, such as platelet aggregation or degranulation of airway gland cells. Since mucus secretion is closely related to ion channel function, we investigated whether MMPs could also be involved in the regulation of ion channels. We used human airway submucosal cell line Calu-3 to study the effects of MMPs on whole-cell current and transepithelial short-circuit current (I(sc)). Phenanthroline, a specific inhibitor of MMPs, increased whole-cell current with the half-maximally effective dose of 5.2 microM, and reversibly activated I(sc) in transepithelial measurements. Current stimulated by phenanthroline displayed linear current-voltage relationships and had inhibitor pharmacology and ion selectivity consistent with cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel activity. Zymography and Western blot showed significant expression of MMP-2 in Calu-3 cells. Moreover, anti-MMP-2 antibodies (1 microg/mL) increased whole-cell current and I(sc), whereas human recombinant MMP-2 (10 ng/mL) reduced it. We also studied the expression of MMPs and the effects of phenanthroline on whole-cell current in A549 cells, which are derived from airway surface epithelium and do not express CFTR Cl- channels. While these cells also showed significant expression of MMP-2, inhibition of this enzyme with phenanthroline exerted no significant effect on whole-cell current. It is concluded that MMP-2 is involved in the regulation of CFTR Cl- channels in human airways.

Objectives: Hemodynamic instability and generalized organ dysfunction are common after cardiopulmonary bypass in human beings. Previous studies have suggested that alterations of nitric oxide metabolism may be associated with this impaired function. Using a canine model we tested whether nitric oxide synthase activity is increased after cardiopulmonary bypass. We also tested whether administration of a nitric oxide donor can influence nitric oxide synthase activity after cardiopulmonary bypass., Methods: After induction of anesthesia, dogs were randomized to receive cardiopulmonary bypass (n = 12) or to serve as controls (n = 12). They were further randomized to receive a continuous infusion of a nitric oxide donor, S-nitrosoglutathione, or an equivalent volume of placebo. Cardiopulmonary bypass was maintained for 90 minutes, and then 4 hours later dogs were put to death. Cardiac and coronary artery sections were frozen in liquid nitrogen immediately after death for later determination of nitric oxide synthase activity using a citrulline assay., Results: After cardiopulmonary bypass, 4 of 6 placebo-treated but only 2 of 6 S-nitrosoglutathione treated animals required phenylephrine infusion (3.1 +/- 3.1 microgram/min and 0.2 +/- 0.4 microgram/min, respectively, P =.05) to maintain a predetermined blood pressure. Furthermore, after cardiopulmonary bypass, Ca2+-dependent nitric oxide synthase activity in the left ventricle, atrium, and coronary artery did not increase compared with activity in the control animals, but Ca2+-independent nitric oxide synthase activity did increase (P =.005): left ventricle (+28.0% +/- 9.0%), atrium (+45.0% +/- 12.0%) and coronary artery (+17.0% +/- 12.0%)., Conclusions: We have found that (1) cardiopulmonary bypass results in increased activity of Ca2+-independent nitric oxide synthase, (2) S-nitrosoglutathione can prevent the increase of Ca2+-independent nitric oxide synthase after cardiopulmonary bypass, and (3) Ca2+-independent nitric oxide synthase may contribute to hemodynamic dysfunction after cardiopulmonary bypass.

The effects of nitric oxide (NO) and metalloproteinases (MMP-2 and MMP-9) in the pathogenesis of hyperoxia-induced lung damage in newborn rats were examined. Three-day-old rat pups were subjected to hyperoxia (> or = 95% O2) or room air for 7 and 14 days. Some animals were treated with NG-L-nitro-L-arginine methyl ester (L-NAME, 10 mg kg(-1), s.c., daily). Histology, morphometry, oedema, Ca2+-dependent and -independent NO synthase (NOS) activities, expression of NOS isoforms and the activities of MMP-2 and MMP-9 were measured in lungs of hyperoxic and control animals. Exposure of rats to hyperoxia for 7 days resulted in alveolar sac injury characterized by the presence of cellular debris, red cell extravasation and inflammatory infiltration with mononuclear cells. Lung water content, epithelial, smooth muscle layers and total airway thickness was similar to controls. In contrast, exposure of rats to hyperoxia for 14 days resulted in lung oedema, inflammation and epithelial proliferation. Hyperoxia caused a decrease in Ca2+-dependent NOS activity, an effect that was associated with increased expression of eNOS protein. In control rats, Ca2+-dependent NOS activity and expression of eNOS were reduced at 14 days. Hyperoxia caused 10 fold increase in the activity of Ca2+-independent NOS that remained significantly elevated after 14 days of exposure to hyperoxia. The activity of this enzyme was unchanged in control rats. In lungs of hyperoxic rats, the immunoblot showed time-dependent, biphasic expression (peak at 7 days) of iNOS. The profile of expression of iNOS in control rats was similar. The activities of MMPs were increased in lungs of hyperoxic animals. The L-NAME treatment of hyperoxic animals reduced lung oedema and epithelial proliferation, but enhanced the activities of MMPs. L-NAME exerted no significant effects in control rats. It is concluded that increased generation of NO contributes to the pathogenesis of hyperoxia-induced lung damage in newborn rats.

In this study we investigated the effect of oral contraceptive (OC) use (OCU) and non-use (OCNU) on growth hormone (GH) responses to exercise in the same females (n = 7, age 22-31 years) during the normal course of OC therapy. Continuous (60% maximum oxygen consumption, VO2max for 20 min) and intermittent exercise (>80% VO2max) protocols of equal total duration, and similar external work were performed during phases of OCNU (days 3-5 of the menstrual cycle) and OCU (days 7-11). Levels of GH, lactate, 17 beta-estradiol, and progesterone were measured. Lactate responses were significantly greater (P<0.05) during intermittent than continuous exercise, with no effect of OC use. However, significantly greater GH responses were found during the OCU phase than the OCNU phase in both the continuous (+94%) and intermittent (+250%) exercise protocols. Estradiol and progesterone levels increased significantly during exercise in all four conditions. We suggest that the increased GH responses observed during the OCU-phase were potentiated by the elevated levels levels of total estrogens (endogenous 17 beta-estradiol and exogenous ethinyl estradiol). It is suggested that training programs for female athletes could be timed in accordance with the menstrual cycle to benefit from an increased GH response to exercise during phases of OC use or the luteal phase of women not on OC therapy.

Involvement of nitric oxide (NO) in nociceptive transmission is well documented. However, there is controversy concerning the exact role of NO in mediation of nociception at different levels of the nervous system. Most studies agree that NO promotes hyperalgesia at the level of the spinal cord. Conversely, at supraspinal sites exogenously applied NO has been found to be both pro- and antinociceptive. In light of this discrepancy, the aim of the present study was to compare the effects of NO donors on nociceptive transmission at spinal and supraspinal sites of the central nervous system using mechanical (paw pressure; PP) and thermal (tail-flick; TF) noxious stimulation. Four NO donors which release NO through different mechanisms were used: S-nitrosoglutathione (SNOG; 3-600 nmol), S-nitroso-N-acetylpenicillamine (SNAP; 0.18-4.5 nmol), hydroxylamine (HYD; 60-1200 nmol) and 3-morpholino-sydnonimine (SIN-1; 490-970 nmol). They were injected intrathecally (i.t.) or intracerebroventricularly (i.c.v.) to male Wistar rats and nociceptive thresholds were evaluated in TF and PP tests. It was found that NO donors administered i.t. or i.c.v. produced a dose-dependent hyperalgesia in the PP test. The hyperalgesia induced by mechanical stimuli was stronger after i.t. than after i.c.v. administration of NO donors. The SIN-1-induced hyperalgesia, as evaluated by teh PP test, was reversed by i.t. pretreatment with haemoglobin (1.5-4 nmol) a NO scavenger, and methylene blue (267-1070 nmol) a guanylate cyclase and NO synthase inhibitor, suggesting that NO exerts its action by facilitating cyclic guanosine 3',5'-monophosphate (GMP) formation. Unlike in the PP test, SNAP and SNOG had no effect on the nociceptive threshold in the TF test, and only SIN-1 administered i.t. produced a weak hyperalgesia in that test, while HYD caused a mild but significant prolongation of the TF reflex. The above data show that NO produces hyperalgesia principally in response to noxious mechanical stimuli. This effect seems to be predominantly mediated in the spinal cord, however, it occurs at both levels of the central nervous system.

We have previously shown that human platelets express matrix metalloproteinase-2 (MMP-2) and that the release of this enzyme during platelet activation mediates the ADP- and thromboxane-independent part of aggregation. We have now used immunogold electron microscopy, flow cytometry. Western blot analysis and zymography methods to study the ultrastructural localization of MMP-2 in human washed platelets. Platelet aggregation was stimulated by collagen and the MMP-2 immunoreactivity of platelets was followed during various stages of aggregation. In resting platelets, MMP-2 was randomly distributed in the platelet cytosol without detectable association with platelet granules. Platelet aggregation caused the translocation of MMP-2 from the cytosol to the extracellular space. During the early stages of aggregation, MMP-2 remained in close association with the platelet plasma membrane. We conclude that the interactions of MMP-2 with platelet surface membranes mediate the aggregatory response induced by this enzyme.

We have investigated the effects of early phases of chronic stress on generation and actions of nitric oxide (NO) in JCR:LA-cp rats both lean (+/+) and obese (cp/cp). Restraint stress was carried out for a 15-min single exposure or for 1 hr every day during 4, 9 or 14 days. The stress reaction was evidenced by significant increase in plasma cortisol. The exposure to stress for 14 days led to a neuronal damage in lean rats as evidenced by a decrease in glutamate uptake and an increase in the release of lactate in synaptosomes. This effect was not observed in obese rats. Concomitantly, the levels of glutamate increased in the hippocampus at 14 days in lean, but not obese rats, that showed higher basal levels of glutamate than lean rats. The activity of NO synthase (NOS) and guanosine cyclic monophosphate levels increased in the hippocampus preceding the neuronal damage. The neuronal lesions were prevented by inhibition of NOS without affecting cortisol levels. In the cardiovascular system, chronic stress exerted no significant effect on blood pressure, aortic contractility or platelet aggregation. However, there were significant changes in plasma nitrite/nitrate that reached maximum at 4 to 9 days. It is concluded that the generation of NO contributes to the systemic response to the organism to stress. In the brain, NO appears to be detrimental as this molecule mediates glutamate-dependent hippocampal damage, this effect being cortisol-independent. In contrast, in the vascular system, increased generation of NO may attenuate the vasoconstrictor and platelet aggregatory effects of catecholamines and other mediators of stress.

1. The effects of nitric oxide (NO) on vascular reactivity and platelet function in the obese (cp/cp) and lean (+/?) JCR:LA-cp rats were investigated. 2. Phenylephrine (PE; 0.1 nM-10 microM) induced contraction of isolated aortic rings in both genotypes (cp/cp and +/?) of JCR:LA-cp rats. The sensitivity to contraction with PE was enhanced in cp/cp compared with +/? rings. Rings from both genotypes showed an increased contraction upon removal of the endothelium. 3. Acetylcholine (ACh; 0.1 nM-10 microM)-induced endothelium-dependent relaxation of rings was not significantly different in the two genotypes. Both were inhibited to a similar extent by NG-nitro-L-arginine methyl ester (L-NAME; 0.01-1 mM) when administered in vitro. 4. The nitric oxide synthase (NOS) inhibitor (L-NAME; 0.3, 1 or 3 mg ml(-1), p.o.) when administered in vivo increased blood pressure in cp/cp rats but not in +/? rats. 5. L-NAME resulted in greater inhibition of ACh-induced relaxation in cp/cp rings compared with +/? rings. 6. L-NAME treatment in vivo caused a decrease in cyclic GMP and NOS activity in rings from cp/cp but not +/? rats. 7. The NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP; 0.1 nM-10 microM)-induced relaxation of rings from +/? rats, an effect enhanced by the treatment with L-NAME in vivo. 8. Oral administration of L-NAME did not enhance the vasorelaxant effect of SNAP on rings of aorta from cp/cp animals. 9. Platelet aggregation and NOS activity were similar in both genotypes and were not modified by oral administration of L-NAME. 10. These results show that unimpaired generation of NO is crucial for maintenance of vascular tone particularly under conditions of vascular insult exemplified by insulin resistance, obesity and dyslipidemia detected in cp/cp rats.

Changes in plasma hormonal concentrations during exercise have been ascribed to the type, duration, and intensity of exercise, physical fitness of subjects, oxygen availability and debt, and acid-base balance. However, relatively few studies have examined the possible role of exercise-induced hyperthermia. This paper reviews previous studies on this subject and describes a series of experiments carried out in our laboratories to define the role of changes in body temperature in the release of hormones during exercise. In a first series of experiments, we studied the relationship between thermoregulatory and growth hormone responses to severe exercise at 23 degrees C for 2 h in fit euhydrated subjects, controlling the core temperature increase to a maximum of 40 degrees C by varying wind speed. Exponential relationships were found between increases in core temperature and plasma growth hormone, prolactin, and catecholamines during exercise, suggesting the existence of a thermal threshold for stimulation of hormonal release during exercise. The effect of endurance exercise with and without a thermal clamp (immersion in cold and warm water) on hormonal and leukocyte responses was examined. Again, a significant exponential relationship was found between increases in core temperature and hormonal responses. Thermal clamping significantly diminished the hormonal and the leukocytic responses to exercise, suggesting that an exercise-induced thermal threshold of approximately 38 degrees C exists where hormonal responses are observed. Therefore, core temperature increases may be integrated in the controlling system of hormonal and leukocytic responses to exercise.

In this paper, the effects of exercise on human sleep (in temperate, cold, and hot climates) are compared with those of exposure to extreme environments (tropical, polar climates). Exercise has two effect: (i) when the exercise load is too heavy or if the subject is not trained to the exercise conditions, the hypothalamo-pituitary-adrenocortical axis (HPA) is strongly activated (somatic stress reaction), and a diachronic (delayed) decrease in total sleep time and slow-wave sleep (SWS) occurs with a synchronic (concomitant) sleep disruption (such as a decrease in REM sleep); (ii) a diachronic enhancement of SWS and (or) REM sleep occurs during moderate training and in athletes, with a moderate HPA activation (neurogenic stress reaction). Heat acclimatization (neurogenic stress response) results in a diachronic increase in SWS, contrary to acute heat exposure (somatic stress) which leads to a diachronic decrease in SWS. Nocturnal cold exposure (somatic and (or) neurogenic stress) provokes a synchronic decrease in REM sleep with an activation of stress hormones, which are reduced by previous acclimation (neurogenic pathway); SWS remains undisturbed in the cold, as it occurs at the beginning of the night before body cooling. In conclusion, when the brain can deal with the stressor (neurogenic stress), diachronic increases in SWS and (or) REM sleep occur. When these "central" mechanisms are overloaded, the classical "somatic" stress reaction occurs with diachronic and synchronic disruptions of the sleep structure.

Insulin resistance is associated with an increased risk of cardiovascular disease that is probably related to abnormalities of vascular wall function. The JCR:LA-cp rat is a unique animal model of human vascular disease that exhibits a profound insulin resistance, vasculopathy, and cardiovascular disease, and allows study of the relationships between insulin resistance and vascular function. Conductance and resistance arteries serve different functions, thus vascular disease may affect these types of artery differently. Concentration-response curves to norepinephrine, phenylephrine, and acetylcholine were studied in conductance vessels (aortic rings) and resistance vessels (mesenteric arteries) from 12-week-old male obese and lean JCR:LA-cp rats. The aortic rings and mesenteric arteries from obese rats showed increased maximal response to phenylephrine compared with those from lean rats, whereas only the mesenteric arteries from obese rats showed increased maximal response to norepinephrine. In aortic rings, relaxation to acetylcholine was similar for both genotypes, but the mesenteric arteries of obese rats showed impaired relaxation to acetylcholine. We conclude that the sensitivity to vasoconstriction is enhanced in aortic rings and mesenteric arteries of obese male JCR:LA-cp rats, but endothelial function is impaired only in the mesenteric resistance arteries of these animals. Hence functional aberrations in the obese, insulin-resistant state are more pronounced in mesenteric resistance arteries than in a major conductance artery.

Nitric oxide (NO) is a mediator that modulates vessel wall tone and hemostatic-thrombotic balance. Platelet function is regulated by NO generated from platelets, endothelial cells and leukocytes. Nitric oxide has been shown to inhibit platelet adhesion, aggregation, and stimulate disaggregation of preformed platelet aggregates. Many of the effects of NO are mediated by its stimulation of guanylate cyclase and the formation of cyclic GMP and its subsequent transduction mechanism. In vivo, NO is likely to interact with prostacyclin, metabolites of ecto-nucleotidase, and lipoxygenase to modulate platelet function in a synergistic manner. An imbalance of NO production (deficiency or overproduction) has been implicated in the pathogenesis of various vascular disorders including thrombosis, atherosclerosis, septicemia, and ischemia-reperfusion injury. It is likely that some of detrimental effects of NO are mediated through its reaction with superoxide anion to form the potent oxidant, peroxynitrite. Nitric oxide gas and NO donors are used for the pharmacological treatment of various vascular disorders. Because inhaled NO has been documented to improve systemic oxygenation and reduce the need for extracorporeal membrane oxygenation, it has been widely used in neonates with severe hypoxemia. An inhibition of platelet function, resulting in a prolonged bleeding time, has been shown in adults receiving inhaled NO. Because bleeding complications may occur in high-risk infants, it is important to evaluate the effect of inhaled NO on platelet function and its correlation with clinical consequences such as intracranial hemorrhage. For these reasons, hemostasis should be carefully monitored during the administration of inhaled NO to critically ill neonates.

Epithelial Cl- channels are regulated by various physiological factors, including guanosine 3',5'-cyclic monophosphate (cGMP). Because cGMP mediates many of the physiological actions of nitric oxide (NO), we have studied both the presence of endogenous NO and the effects of exogenous NO on Cl- currents in A549 human lung epithelial cells. We have detected Ca(2+)-dependent NO synthase activity in A549 cells. Using the perforated patch-clamp technique, we have shown that inhibition of this enzyme by NG-monomethyl-L-arginine decreased Cl- current, an effect that was reversed by the NO donor S-nitrosoglutathione (GSNO). In addition, the NO donors GSNO and S-nitroso-N-acetyl-D,L-penicillamine increased whole-cell Cl- currents in A549 cells. This stimulatory effect of the NO donors was sensitive to inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, suggesting that channels other than the cystic fibrosis transmembrane conductance regulator (CFTR) are involved in the action of NO on A549 cells. In addition, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a selective inhibitor of soluble guanylyl cyclase, decreased NO-mediated stimulation of Cl- currents. Our results suggest that, in lung epithelial cells, NO regulates a non-CFTR Cl- conductance acting via a cGMP-dependent mechanism.

The pathogenesis of human African trypanosomiasis (HAT) has been the object of considerable research interest but has remained incompletely understood. The importance of cytokines in the pathophysiology of this protozoan infection is now widely recognized, but the full spectrum of cytokines involved has yet to be determined. In the present investigation we compared the plasma concentrations of TNF-alpha and IL-10 in normal African controls and patients suffering from advanced meningocephalic (late-stage) Trypanosomiasis brucei (T.b.) gambiense infections, before and after treatment with the arsenical trypanocide melarsoprol. We found that patients with late-stage T. b. gambiense exhibit chronically elevated circulating levels of both of these cytokines, and that these levels quickly decline following melarsoprol treatment. These findings confirm that TNF-alpha is involved in the immunopathogenesis of late-stage African trypanosomiasis and suggest that IL-10 may also play an important regulatory role in this disease.

Blood platelets limit blood loss at sites of vascular injury by forming a mechanical plug. They are also involved in thrombosis, atherosclerosis, inflammation and metastasis. Platelet activation is essential for these physiological and pathological reactions and depends upon their adhesion to the vessel wall and attachment to each other in the aggregation process. The two known pathways of aggregation are mediated by the release of endoperoxides/thromboxane A2 and ADP which amplify platelet aggregation. Here we report the identification of a new pathway of aggregation which is mediated by the release of a metalloproteinase enzyme, gelatinase A.

To test how leukocyte responses to endurance exercise were modified by clamping body temperature, nine men (27.3 +/- 6.0 yr) completed four 80-min immersions to midchest at water temperatures of 23 or 39 degrees C; two tests included 40-min of cycle ergometer exercise at 65% of aerobic power. When the subjects were exercising, rectal temperature peaked at 39.1 +/- 0.4 degrees C in the warm water and 37.8 +/- 0.3 degrees C in the cool water. When the subjects were sitting in warm water, rectal temperature closely matched the core temperature during exercise in cool water, whereas when they were sitting in cool water, rectal temperatures decreased to 36.4 +/- 0.6 degrees C. Total and differential white cell counts were determined by using a Coulter counter, and cortisol and growth hormone concentrations were determined by radioimmunoassay; all data were adjusted for changes of blood and plasma volumes. Heat clamping during exercise substantially reduced the rise in white cell, lymphocyte, and granulocyte counts but not the increase in monocyte count. Clamping also abolished previously observed associations between cell counts and cortisol and weakened associations with growth hormone concentrations (D. A. McCarthy and M. M. Dale. Sports Med. 6: 333-363, 1988). We conclude that both exercise and a rise of core temperature contribute to the changes in white cell and subset counts during and immediately after moderate exercise. Both cortisol and growth hormone concentrations appear to be mediators of these responses.

Human African trypanosomiasis (sleeping sickness) is a unique disease model of disrupted circadian rhythms in the sleep-wake cycle and cortisol and prolactin secretion. This study examined the temporal relationship between growth hormone (GH) secretion and the sleep-wake cycle in 8 infected African patients and 6 healthy indigenous African subjects. Twenty-four-hour sleep patterns were recorded by polysomnography and hourly blood samples analyzed for plasma GH. No relationships between the mean normalized plasma GH levels (Z scores) and the sleep stages (wakefulness, sleep stages 1 and 2 ('light' sleep), slow-wave sleep (stages 3 and 4, SWS), and rapid eye movement (REM) sleep) were found in the patients or healthy subjects. However, when the time of sampling of the plasma GH concentrations was lagged by 16 min with respect to the occurrence of the various sleep stages, significant correlations were found between plasma GH concentrations and SWS in both healthy subjects and patients. Thus, the association between SWS and GH secretion persisted even in the presence of disrupted circadian rhythms, further supporting the concept that sleep and the stimulation of GH secretion are outputs of a common mechanism.

The L-arginine:nitric oxide (NO) pathway is believed to exert many of its physiological effects via stimulation of the soluble guanylyl cyclase (SGC); however, the lack of a selective inhibitor of this enzyme has prevented conclusive demonstration of this mechanism of action. We have found that the compound 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) inhibits the elevation of cGMP induced by the NO donor S-nitroso-DL-penicillamine in human platelets and rat vascular smooth muscle (IC50 = 10-60 nM and <10 nM, respectively) and that this is accompanied by prevention of the platelet inhibitory and vasodilator actions of NO donors. ODQ also inhibited the antiaggregatory action of NO generated by the platelets but did not affect the action of prostacyclin or that of a cGMP mimetic. In addition, ODQ inhibited the vasodilator actions of endogenously released NO and of NO generated after induction of NO synthase in vascular preparations. It did not, however, affect the increase in vascular smooth muscle cGMP or the dilatation induced by atrial natriuretic factor. ODQ had no effect on NO synthase activity, nor did it react with NO. It did, however, potently (IC50 approximately 10 nM) inhibit the activity of the SGC in cytosol obtained from crude extract of rat aortic smooth muscle. Thus ODQ prevents the actions of NO on platelets and vascular smooth muscle through its potent inhibitory effect on the SGC.