Iron homeostasis in osteoporosis and its clinical implications.

Osteoporosis has until now been considered to be a disease associated with abnormal calcium metabolism. However, an increasing number of clinical observations strongly suggest the association of iron overload with bone diseases, particularly in osteoporosis in menopausal women. The recent identification of hepcidin sheds new light into the crucial role of iron homeostasis in bone metabolism. Decreasing iron overload in cell studies as well as in animal experiments has been shown to improve bone cell metabolism and growth in vitro and in vivo. In view of the significant iron overload found in the aging population, especially in females, the therapeutic potential of lowering iron overload for the treatment of osteoporosis is suggested.

The hemodynamic and metabolic profiles of Zucker diabetic fatty rats treated with a single molecule triple vasopeptidase inhibitor, CGS 35601.

CGS 35601 is a triple vasopeptidase inhibitor (VPI) of angiotensin converting enzyme (ACE), neutral endopeptidase (NEP), and endothelin (ET) converting enzyme-1 (ECE-1), with respective IC(50) values of 22, 2, and 55 nM. The aim of the present study was to establish the hemodynamic profile of Zucker diabetic fatty (Zdf)-Fatty rats, a high-fat diet gene-prone model developing spontaneous Type 2 diabetes (T2D) and the effects of CGS 35601. Male Zdf-Fatty (14 weeks, n = 17-23), Zdf-Lean (14 weeks, n = 8-10), and Wistar (14 weeks, n = 9-10) rats on distinct diets were implanted with a catheter in the left carotid and placed individually in a metabolic cage for 30 days. The hemodynamic profile and some metabolic biomarkers were assessed daily. After a 7-day stabilization period, the Zdf-Fatty rats were divided into two groups: Group 1, controls (n = 7-10) receiving vehicle-saline (250 microl/hr) and Group 2, (n = 10-13) receiving increasing doses of CGS 35601 (0.1, 1, and 5 mg/kg/day x 6 days each, intra-arterially) followed by a 5-day washout period. Mean arterial blood pressure (MABP) of young Zdf-Fatty rats was compared with age-matched Zdf-Lean and Wistar rats, which were found similar. MABP decreased by 5.9% (from baseline at 102 +/- 5 to 96 +/- 4 mmHg), 12.7% (to 89 +/- 6 mmHg) and 21.6% (to 80 +/- 4 mmHg), at 0.1, 1, and 5 mg/kg/day, respectively, in CGS 35601-treated Zdf-Fatty rats. Systolic and diastolic blood pressures were similarly reduced. The heart rate was not affected. Hyperglycemic status and insulin-resistance were not modulated by short-term treatment. CGS 35601 presented an excellent short-term safety profile. This novel molecule and class of VPI may be of interest for lowering vascular tone. Further long-term studies, once cardiovascular and renal complications have developed in this T2D rat model are warranted to define the efficacy of this class of VPI.

Triple VPI CGS 35601 reduces high blood pressure in low-renin, high-salt Dahl salt-sensitive rats.

We previously reported that CGS 35601, a potent triple inhibitor of angiotensin-converting enzyme, neutral endopeptidase, and endothelin-converting enzyme 1, completely normalized mean arterial blood pressure (MABP) in 36-week-old spontaneously hypertensive rats, a normal renin model. The aim of the present study was to determine the effects of this triple vasopeptidase inhibitor (VPI) on the hemodynamic profile of instrumented, conscious, and unrestrained Dahl salt-sensitive (DSS) rats, a gene-prone, high-salt diet-induced low-renin hypertension model. Male DSS rats (mean weight [+/-SEM], 385 +/- 10 g) were fed a normal diet (Group 1) or a high-salt diet (Groups 2 and 3; 8% NaCl in food) for 6 weeks and then instrumented with a carotid catheter and placed individually in metabolic cages for 30 days. The hemodynamic, hematological, and biochemical profiles were assessed daily. Dose-dependent treatment started after a 7-day stabilization period in Groups 1 and 2 (vehicle dosage, 250 microl/hr) and Group 3 (CGS 35601 dosages of 0.1, 1, and 5 mg/kg/day for 6 days per dose by means of constant intra-arterial infusion), followed by a 5-day washout period. Two additional groups included normotensive Wistar rats (Group 4) and DSS rats that received a double high-salt solid (8% NaCl) and liquid (1% NaCl) diet (Group 5). The MABP in rats receiving CGS 35601 decreased in a dose-dependent fashion toward the baseline level observed in DSS rats receiving a normal diet. The heart rate was unaffected. The hemodynamic profile returned to normal during the washout period. This novel triple VPI is a potent and effective antihypertensive agent with a safe short-term profile that may be of interest for treating hypertension and other cardiovascular diseases. Other hypertensive rat models are being tested.

The first preclinical pharmacotoxicological safety assessment of CGS 35601, a triple vasopeptidase inhibitor, in chronically instrumented, conscious, and unrestrained spontaneously hypertensive rats.

CGS 35601 is a triple vasopeptidase inhibitor (VPI) of angiotensin-converting enzyme, neutral endopeptidase, and endothelin-converting enzyme-1 with respective IC50 values of 22, 2, and 55 nM. We characterized the safety profile and toxicity of escalating doses of CGS 35601 over a 20-day period in chronically instrumented, unrestrained, conscious, male, spontaneously hypertensive rats (SHR). Once instrumented with an arterial catheter, the SHR were placed in metabolic cages allowing daily assessment of hemodynamics and blood sampling for biochemical and hematological measurements. After a 7-day stabilization period, the SHR were divided into 2 groups: Gr. 1, (n = 13 to 18) receiving CGS 35601 at 0.01, 0.1, 1 and 5 mg kg(-1) day(-1) (continuous i.a. infusion) for 5 consecutive days/dose, followed by a 5-day washout; and Gr. 2, (n = 10) receiving vehicle (saline). The highest dose of CGS 35601 dose-dependently reduced MABP from 156 +/- 4 up to 94 +/- 5 mm Hg, whereas heart rate, metabolic, electrolytic, and hematological profiles, growth, diuresis, and renal activity were unaffected, and no hepatic or liver toxicities were observed. These results suggest that this novel triple VPI presents no safety concerns at this stage and may become of interest for the treatment of hypertension and other cardiovascular disorders. Long-term chronic experiments are needed to assess possible angioedema and increases in vascular permeability.

Antigen-induced bronchial hyperreactivity and bronchopulmonary inflammation in rats: effect of TNF receptor binding protein.

The effect of a receptor binding protein for tumor necrosis factor (TNFrbp) on cell infiltration, bronchial hyperreactivity, and release of inflammatory mediators were studied following antigen challenge in sensitized rats. A 3-fold increase in total cell number, mainly neutrophils and eosinophils, was noted in bronchoalveolar lavage (BAL) fluid 8 hours after antigen challenge. Antigen challenge also induced a significant hyperreactivity of the lower bronchus to carbachol and serotonin, but did not affect the reactivity of the trachea and upper bronchus. This increased responsiveness of the lower bronchus was transient, being detected 8 hours but not 24 hours after antigen challenge. Thromboxane B2 (TxB2), prostaglandin E2 (PGF2), and nitric oxide (NO) levels increased in the BAL fluid of sensitized rats 8 hours after antigen challenge by 197%, 172%, and 173%, respectively. TNFrbp treatment reduced by 83% the antigen-induced cell infiltration, with neutrophils being the cells most affected. The bronchial hyperreactivity induced by antigen challenge was also significantly inhibited by TNFrbp, whereas TxB2, PGE2, and NO levels in the BAL fluid were not affected. In our animal model, the cell infiltration and bronchial hyperreactivity appear to be mediated to some extent by TNF, but not by prostanoids nor NO.

Permeability of endothelial monolayers to albumin is increased by bradykinin and inhibited by prostaglandins.

Using monolayers of bovine aortic endothelial cells (BAEC) in modified Boyden chambers, we examined the role of prostaglandins (PGs) in the bradykinin (BK)-induced increase of albumin permeability. BK induced a concentration-dependent increase of the permeability of BAEC, which reached 49.9 +/- 1% at the concentration of 10(-8) M. Two inhibitors of the prostaglandin G/H synthase, indomethacin (2.88 microM) and ibuprofen (10 microM), potentiated BK-induced permeability 1.8- and 3.9-fold, respectively. Exogenously administered PGE2 and iloprost, a stable analog of prostacyclin, attenuated the effect of BK in a concentration-dependent manner. Butaprost equally reduced the effect of BK, suggesting the participation of the EP2 receptor in this phenomenon. However, the EP4-selective antagonist AH-23848 did not significantly inhibit the protective effect of PGE2. The inhibitory effect of PGE2 was reversed by the adenylate cyclase inhibitor MDL-12330A (10 microM). These results suggest that BK-induced increase of permeability of BAEC monolayer to (125)I-labeled albumin is negatively regulated by PGs. This postulated autocrine activity of PGs may involve an increase in the intracellular level of cAMP.

Prostaglandin E(2) increases cyclic AMP and inhibits endothelin-1 production/secretion by guinea-pig tracheal epithelial cells through EP(4) receptors.

Prostaglandin E(2) (PGE(2)) increased adenosine 3' : 5'-cyclic monophosphate (cyclic AMP) formation in tracheal epithelial cells and concomitantly decreased the production/secretion of immunoreactive endothelin (irET). Naturally occurring prostanoids and selective and non-selective EP receptor agonists showed the following rank order of potency in stimulating cyclic AMP generation by epithelial cells: PGE(2) (EP-selective)>16,16-dimethyl PGE(2) (EP-selective)>11-deoxy PGE(2) (EP-selective)>>>iloprost (IP/EP(1)/EP(3)-selective), butaprost (EP(2)-selective), PGD(2) (DP-selective), PGF(2alpha) (FP-selective). The lack of responsiveness of the latter prostanoids indicated that the prostanoid receptor present in these cells is not of the DP, FP, IP, EP(1), EP(2) or EP(3) subtype. Pre-incubating the cells with the selective TP/EP(4)-receptor antagonists AH23848B and AH22921X antagonized the PGE(2)-evoked cyclic AMP generation. This suggested that EP(4) receptors mediate PGE(2) effects. However, in addition to any antagonistic effects at EP(4)-receptors, both compounds, to a different extent, modified cyclic AMP metabolism. The selective EP(1), DP and EP(2) receptor antagonist (AH6809) failed to inhibit PGE(2)-evoked cyclic AMP generation which confirmed that the EP(2) receptor subtype did not contribute to the change in cyclic AMP formation in these cells. The PGE(2)-induced inhibition of irET production by guinea-pig tracheal epithelial cells was due to cyclic AMP generation and activation of the cyclic AMP-dependent protein kinase since this effect was reverted by the cyclic AMP antagonist Rp-cAMPS. These results provide the first evidence supporting the existence of a functional prostaglandin E(2) receptor that shares the pharmacological features of the EP(4)-receptor subtype in guinea-pig tracheal epithelial cells. These receptors modulate cyclic AMP formation as well as ET-1 production/secretion in these cells.

Effects of a selective neutral endopeptidase and a nonselective neutral endopeptidase/endothelin-converting enzyme inhibitor on lipopolysaccharide-induced endotoxaemia in anaesthetized Sprague-Dawley rats.

The gene expression and levels of endothelins (ETs) are increased in various animal models of lipopolysaccharide-(LPS) induced septic shock as well as in patients with endotoxaemia (ENDO). A positive correlation was reported between the expression and production of ETs, and the severity of haemodynamic and haematological disturbances, organ injury and circulatory failure in ENDO. Previous studies using ET(A)- and/or ET(B)-receptor antagonists exacerbated the effects of LPS in anaesthetized and conscious rats. We investigated the effect of a selective neutral endopeptidase (NEP) (CGS 24592) or a mixed NEP/endothelin-converting enzyme (ECE) (CGS 26303) inhibitor in LPS-induced ENDO in anaesthetized Sprague-Dawley rats. Four hours post-LPS injection, blood pressure was 39% lower in the presence of CGS 26303, compared to control-saline or LPS-injected rats. In rats treated with CGS 26303, white blood cells and platelet counts decreased, whereas lymphocytes increased. In addition, progressive liver dysfunction, characterized by increases in plasma bilirubin and alanine transferase, became even more apparent (higher than in those injected with LPS). Plasma creatinine and blood urea were similar to those of the LPS-injected group. Similar results were observed with CGS 24592. Thus, these inhibitors enhanced some, but not all, of the LPS-induced deleterious effects.

Effects of tranexamic acid and aprotinin, two antifibrinolytic drugs, on PAF-induced plasma extravasation in unanesthetized rats.

Two antifibrinolytic drugs, tranexamic acid (TXA), and aprotinin (APR), are currently used to improve the recovery of patients following major surgery while reducing blood loss. Their mechanisms of action have yet to be fully understood. Here, we examined (1) the effects of TXA or APR on basal vascular permeability (VP) and (2) the effects of TXA or APR on platelet-activating factor (PAF)-induced increase of VP in normal unanesthetized rats. Evans blue dye (EB) bound to albumin was used as the marker of extravasation in selected tissues. In normal rats, PAF (1 microg/kg i.v.) increased VP in most selected tissues including bronchi, aorta, duodenum and pancreas without affecting blood pressure. TXA (up to 300 mg/kg i.v.) had no significant effect on basal VP in any tissues, while APR (30000 KIU/kg i.v.) decreased basal VP in 5 out of 8 tissues. Pre-treatment with TXA decreased PAF-induced increases of VP in the microcirculation of the thoracic and abdominal aorta, the duodenum and the pancreas, from 35% to 41%. TXA was mostly effective at an i.v. dose of 100 mg/kg with a 2 h of pre-treatment period. Pre-treatment with APR also reduced PAF-induced increases of VP in selected tissues by 35 to 61%. The i.v. dose of 30000 KIU/mg was optimal when injected at least 30 min before the administration of PAF + Evans blue. These results suggest that the beneficial effect of APR and TXA, following cardiopulmonary bypass (CPB) and other type of surgeries, may be attributed to the inhibition of plasma exudation mediated, at least in part, by PAF. Thus, TXA and APR may improve patients recovery by reducing the capillary leakage of albumin, associated with interstitial edema formation, and maintaining intravascular fluid volume.

Involvement of LTD(4)in allergic pulmonary inflammation in mice: modulation by cysLT(1)antagonist MK-571.

Cysteinyl leukotrienes are potent inflammatory molecules playing a major role in asthma. The involvement of these mediators in hypersensitivity in mice is not well known. This study aimed at elucidating their implication by using MK-571, a cysLT(1)receptor antagonist. Mice were sensitized with a suspension of ovalbumin (8 microg) adsorbed to alum (2 mg) and were challenged with an aerosolized ovalbumin solution (0.5%). Inflammatory cell infiltration in the bronchoalveolar lavage (mostly eosinophils) following antigen challenge was inhibited by dexamethasone (0.1, 1 and 5 mg kg(-1)s.c.) and MK-571 (1, 10, 100 mg kg(-1)i.v.) in a dose-dependent manner. Maximal inhibition was 95% with 5 mg kg(-1)dexamethasone and 90% with 100 mg kg(-1)MK-571. When injected together they showed an additive inhibitory effect on eosinophil infiltration. Bronchial hyperreactivity, measured by the increased pulmonary insufflation pressure to carbachol injections, was also inhibited dose-dependently by MK-571. The EC(50)values for carbachol were of 22.39+/-1.12 microg kg(-1)in sensitized and challenged animals that did not receive MK-571 and increased to 43.65+/-1.10, 50.12+/-1.15 and 83.18+/-1.16 microg kg(-1)in animals treated with 1, 10 and 100 mg kg(-1)MK-571 respectively. Lung microvascular leakage (as measured by Evans blue extravasation) induced by antigen bronchoprovocation was reduced by 22% after treatment with 10 mg kg(-1)MK-571. All these inhibitory effects of MK-571 suggest a role for leukotriene D(4)in this animal model of allergic asthma.

Effects of eicosanoids, neuromediators and bioactive peptides on murine airways.

The effects of several mediators including prostanoids, neuromediators, bioactive peptides and leukotrienes were investigated on the trachea, upper bronchi, lower bronchi and lung parenchyma of selected strains of mice mounted in a cascade superfusion system. The upper airways (trachea, upper bronchi) responded with greater maxima than lower airways (lower bronchi, lung parenchyma). Acetylcholine, carbachol, serotonin and 9, 11-dideoxy-9alpha,11alpha-epoxymethano-prostaglandin F(2alpha)serotonin>/=acetylcholine. Prostaglandins E(2), F(2alpha) and D(2)90% relaxation in some cases. The rank order of potency for the prostaglandins was E(2)>/=F(2alpha)D(2) with the exception of the lower bronchi on which prostaglandins had the following order of potency: F(2alpha)>/=E(2)D(2). The effects of prostaglandins were similar in four commonly used strains of mice (CD-1, BALB/c, C57BL/c6 and C3H) with some variations in efficacy. Iloprost was a weak mouse airway relaxant. It had the greatest effect on the trachea and bronchi of BALB/c and C57BL/c6 mice, whereas it had little or no effect on the airways of the CD-1 and C3H mouse strains. Vasoactive intestinal peptide potently relaxed the carbachol and precontracted the mouse trachea and bronchi. However, vasopressin, another bioactive peptide, potently and efficaciously contracted the mouse trachea and upper bronchi but had little effect on the lower bronchi. Vasopressin was the most potent and efficacious contractile agonist tested in this study. Contractions were observed with endothelins-1, -2 and -3 on mouse trachea and bronchi, but marked tachyphylaxis was present. Sarafotoxin s6c followed the same pattern suggesting the presence of endothelin ET(B) receptors on the mouse airways. Of all leukotrienes assayed (B(4), C(4), D(4) and E(4)) only leukotriene C(4) weakly contracted the mouse trachea and upper bronchi, but tachyphylaxis was most evident.

Adenosine induces cyclic-AMP formation and inhibits endothelin-1 production/secretion in guinea-pig tracheal epithelial cells through A(2B) adenosine receptors.

1. The adenosine receptor subtype mediating adenosine 3' : 5'-cyclic monophosphate (cyclic AMP) formation and the effect of its activation on endothelin-1 (ET-1) secretion were studied in primary cultures of tracheal epithelial cells. 2. Adenosine analogues showed the following rank order of potency (pD(2) value) and intrinsic activity on the generation of cyclic AMP by tracheal epithelial cells: 5'-N-ethylcarboxyamidoadenosine (NECA, A(1)/A(2A)/A(2B), pD(2): 5.44+/-0.16)>adenosine (ADO, non selective, pD(2): 4.99+/-0. 09; 71+/-9% of NECA response) >/=2-Cl-adenosine (2CADO, non selective, pD(2): 4.72+/-0.14; 65+/-9% of NECA response)>>>CGS21680 (A(2A); inactive at up to 100 microM). 3. Cyclic AMP formation stimulated by NECA in guinea-pig tracheal epithelial cells was inhibited by adenosine receptor antagonist with the following order of apparent affinity (pA(2) value): Xanthine amine congeners (XAC, A(2A)/A(2B), 7.89+/-0.22)>CGS15943 (A(2A)/A(2B), 7.24+/-0. 26)>ZM241385 (A(2A), 6.69+/-0.14)>DPCPX (A(1), 6.51+/-0. 14)>3n-propylxanthine (weak A(2B), 4.30+/-0.10). This rank order of potency is typical for A(2B)-adenosine receptor. 4. Adenosine decreased basal and LPS-stimulated irET production in a concentration-dependent manner. Moreover, NECA but not CGS21680 inhibited LPS-induced irET production. 5. The inhibitory effect of NECA on LPS-induced irET production was reversed by XAC (pA(2)=8.84+/-0. 12) and DPCPX (pA(2)=8.10+/-0.22). 6. These results suggested that adenosine increased cyclic AMP formation and inhibited irET production/secretion by guinea-pig tracheal epithelial cells through the activation of a functional adenosine receptor that is most likely the A(2B) subtype. This adenosine receptor may be involved in the regulation of the level of ET-1 production/secretion by guinea-pig tracheal epithelial cells in physiological as well as in pathophysiological conditions.

Selective inflammatory response induced by intratracheal and intravenous administration of poly-L-arginine in guinea pig lungs.

Major basic protein (MBP) is a cationic protein found in eosinophil granules that was postulated to participate in the pathogenesis of bronchial asthma. Recently, it has been demonstrated that MBP level in serum or bronchoalveolar lavage (BAL) fluid was correlated with bronchial hyperresponsiveness (BHR) in asthmatics. A number a studies have established that MBP actions could be mimicked by synthetic polycations as poly-L-arginine. In this study, we investigated the effects of intratracheal and intravenous administration of poly-L-arginine on lung inflammatory response development. The intratracheal injection of poly-L-arginine at the doses of 1, 10, 100 nmol/animal increased the number of eosinophils (up to 3.2 fold) and neutrophils (up to 12 fold) in BAL fluid. Eosinophil and neutrophil infiltration was reversed by 88% and 67% respectively following low molecular weight heparin treatment (500 microg/animal). The intravenous injection of increasing doses of poly-L-arginine (1, 10, 100, 500 nmol/animal) increased the number of eosinophils (up to 2.7 fold) but not neutrophil infiltration in guinea pig lungs. Eosinophil infiltration was reversed by 87% following low molecular weight heparin treatment (1.5 mg/animal). Intratracheal treatment with poly-L-arginine (100 nmol/animal) produced an important increase of beta-glucuronidase, histamine, eosinophil peroxidase (EPO) and albumin levels in BAL fluid, whereas the intravenous treatment (500 nmol/animal) did not. These results show that the route of administration of poly-L-arginine greatly influences its effect on inflammatory cell recruitment since both administration routes elicited eosinophil migration but only the intratracheal route stimulated the migration of neutrophils. Moreover, poly-L-arginine appeared to induce other inflammatory responses since it increased beta-glucuronidase, histamine, EPO and albumin levels in BAL fluid following intratracheal treatment. These results also showed that low molecular weight heparin significantly blocks the inflammatory responses elicited by poly-L-arginine.

Technology evaluation: ISIS-3521.

It is well known that the PKC family of enzymes is involved in the propagation of intracellular signals and is implicated in cancers, inflammatory processes, cardiovascular and endocrinological diseases. Relatively low isozyme specificity has largely limited the clinical use of PKC antagonists. The members of the PKC family differ from each other at the mRNA level and the selectivity of antisense compounds is distinguished by this feature. According to ISIS Pharmaceuticals Inc antisense compounds are highly selective inhibitors even within a family of closely-related genes [321211]. The use of these compounds could be invaluable as tools to discover the mechanisms and roles of specific PKC isozyme in normal and diseased tissues and could provide the information for better cancer treatments [226799]. The isozyme of PKC-alpha is believed to play an important role in the proliferation of several types of cancer cells [234471-323703]. Recently, ISIS Pharmaceuticals received a patent US-05885970, covering the antisense technique targeting human PKC-alpha for cancer therapy (US-0588970). In the past few years, several effective antisense oligonucleotides (AS ONs) targeting murine and human PKC-alpha isozymes have been developed and a series of positive results have been obtained in cell culture and in nude mice cancer transplantation [327453]. Phase I clinical trials have shown that relatively high doses were well tolerated with no obvious side-effects [226799]. Whether these AS ONs are beneficial to patients suffering from cancer, either alone or in combination with other chemotherapy drugs is still under evaluation in a clinical setting.

Effects of dual endothelin-converting enzyme/neutral endopeptidase inhibitors, CGS 26303 and CGS 26393, on lipopolysaccharide or interleukin-1 beta-stimulated release of endothelin from guinea pig tracheal epithelial cells.

A number of studies using endothelin (ET) receptor antagonists support the participation of ETs in a variety of cardiovascular, renal, and other disorders. It has also been established that a number of cytokines, which are released in such diseases, modulate the expression and production of ETs and thus activate the ET system. This effect may represent one pathway by which these inflammatory mediators operate. By regulating endothelin-converting enzyme (ECE) activities, and thus ET synthesis, one can potentiate or attenuate the production of ETs and the receptor affinity/density in such pathologic conditions. Here, the stimulated (lipopolysaccharide or interleukin-1 beta) production of ET-1 from guinea pig tracheal epithelial cells was abolished by CGS 26303 or CGS 26393, two ECE/neutral endopeptidase (NEP) inhibitors, but was unaffected by CGS 24592, a specific NEP inhibitor. Therefore, such dual, and eventually selective ECE inhibitors are effective agents to prevent the stimulated production of ETs.

Adenosine-induced inhibition of basal endothelin-1 production from guinea-pig tracheal epithelial cells: a mechanism involving the release of cAMP.

Guinea-pig tracheal epithelial cells, like other pulmonary cells in various species, have been reported to synthesize and release endothelins (ET). Their production is inducible by many agents and inhibited by ET-converting enzyme inhibitors, corticosteroids and beta agonists. Here, we studied the effect of adenosine on (1) the formation of adenosine 3', 5'-cyclic monophosphate (cAMP) and (2) concomitant effect on the basal release of ET from primary cultured tracheal epithelial cells isolated from guinea-pigs (GPTEpC). Adenosine (10(-7)-10(-3) M), the endogenous non-selective purinergic receptor agonist, induced the production of cAMP in a concentration-dependent manner (pD2=4.99+/-0.09). Concomitantly, adenosine decreased by a maximum of 39% ET-1-production over the same concentration range (with a 96% correlation). The inhibitory effect over ET-1-production was abolished by CGS15943 (10(-4) M), a non-selective A1/A2A receptor antagonist, and a xanthine amine congener (10(-4) M), a selective A1 receptor antagonist. Thus, these results suggest that adenosine attenuates the production of ET-1 from GPTEpC by stimulating the release of cAMP via the direct activation of adenosine receptors.

Developmental changes in synthesis of and responsiveness to prostaglandins I2 and E2 in hypoxic lamb lungs.

Previous studies have shown that the attenuated hypoxic pulmonary vasoconstriction (HPV) of young newborn lamb lungs was enhanced by cyclooxygenase inhibition. We sought to determine whether this reflected greater synthesis of and (or) responsiveness to dilator prostaglandins (PG). Protocol 1 measured responses to graded hypoxia and perfusate concentrations of 6-keto-PGF1alpha (the stable metabolite of PGI2) and PGE2 in isolated lungs from 1-day- and 1-month-old lambs. Protocol 2 compared dose responses and segmental vascular resistances during infusion of PGI2 and PGE2 in hypoxic, cyclooxygenase-inhibited, lungs from 1- to 2-day-old and 1- to 3-month-old lambs. Lungs of 1-day-old lambs with attenuated responses to 4% O2 had significantly higher perfusate concentrations of 6-keto-PGF1alpha and PGE2, but responses to both PGE2 and the more potent vasodilator, PGI2 did not differ with age. These data support the hypothesis that attenuated HPV in young newborn lamb lungs is due to increased synthesis of dilator PG, particularly PGI2.

Effects of dexamethasone on the basal and cytokine-stimulated release of endothelin-1 from guinea-pig cultured tracheal epithelial cells.

Endothelins (ETs) are potent bronchoconstrictor agents postulated to contribute to the pathophysiology of asthma and other respiratory disorders. An increase in both the expression and release of immunoreactive (ir) ETs was reported in bronchial epithelial cells and the bronchoalveolar lavage fluid of asthmatic patients. We investigated whether dexamethasone (DEX), a potent anti-inflammatory and anti-asthmatic drug, regulates the basal and stimulated release of ETs from guinea-pig cultured tracheal epithelial cells. These airway epithelial cells spontaneously release ET-1 over 24 h. When incubated in the presence of 10(-7) and 10(-6) M DEX for 24 h, basal production of ET-1 decreased by 32 and 29%. Lipopolysaccharide (LPS; 1, 5, 10 micrograms/mL), tumor necrosis factor-alpha (TNF alpha; 5, 10 ng/mL), and interleukin-1 beta (IL-1 beta; 1, 5 ng/mL) significantly increased the basal release of ET-1 after 24 h. When these cells were pretreated with DEX (10(-7) M) for a 24-h period, then incubated in the presence of LPS (10 micrograms/mL), TNF alpha (10 ng/mL), or IL-1 beta (1 ng/mL) for another 24 h, the stimulated release of ET-1 was inhibited by 48, 31, and 38%, respectively. At 10(-6) M, DEX decreased the stimulated release by 45, 37, and 46%, respectively. The present results show that DEX can regulate the basal release and inhibit the pro-inflammatory cytokine-stimulated production of ET-1 from guinea-pig cultured tracheal epithelial cells. They suggest that the beneficial effect of glucocorticoids in asthma may be related to the inhibition of ET synthesis.

Aprotinin, an antifibrinolytic drug, attenuates bradykinin-induced permeability in conscious rats via platelets and neutrophils.

Two antifibrinolytic drugs, tranexamic acid (TXA) and aprotinin (APR), are used to improve the recovery of patients following cardiac surgery while reducing blood loss. Their mechanisms of action have yet to be fully understood. To investigate their possible mechanisms of action during cardiopulmonary bypass, we examined (i) the effects of TXA and APR on bradykinin (BK) induced vascular permeability (VP) in conscious rats, (ii) the roles of platelets and neutrophils in this reaction, and (iii) the effects of TXA or APR on BK responses in platelet- or neutrophil-depleted rats. Evans blue dye (EB) was used as the marker of extravasation. The animals were treated with antiplatelet serum for platelet depletion or with methotrexate for neutrophil depletion. In normal rats, BK increased VP in most tissues. Thrombocytopenia and neutropenia also increased basal VP. TXA had no significant effect whereas APR decreased basal VP. In the second series of experiments, APR significantly attenuated BK-induced increases in VP, whereas TXA was completely ineffective. Platelet depletion did not affect BK-induced increases of VP, except for a massive plasma exudation in the lung parenchyma. Neutrophil depletion also had no effect on BK-induced increases of VP, except for an attenuation in the duodenum. In the third and last series of experiments, TXA potentiated the effect of BK in the upper and lower bronchi of platelet-depleted rats, compared with the effects of TXA on BK in normal animals, except in the lung parenchyma, where TXA blocked the increase of VP induced by BK. APR also potentiated the effect of BK in the lower bronchi of platelet-depleted rats. Overall, the inhibitory effect of APR on the VP induced by BK in normal rats was attenuated in platelet-depleted rats. Like TXA, APR blocked the increase of VP induced by BK in the lung parenchyma of platelet-depleted rats. In neutrophil-depleted rats, TXA did not affect the permeabilizing response to BK. In those rats, the inhibitory effect of APR against BK increases of VP was attenuated. These results show that the beneficial effect of APR, but not TXA, following cardiac surgery may be attributed to the inhibition of plasma exudation mediated, in part, by BK. In addition, platelets and neutrophils do not appear to be involved in BK-mediated plasma exudation. However, both cell types are essential for the regulation of basal VP. Finally, the mechanism underlying the protective inhibitory effect of APR on BK-induced increases of VP involves, at least in part, platelets and neutrophils, since the inhibitory effect of APR is attenuated in thrombocytopenic and neutropenic rats. Both cell types are not involved in the action of TXA on VP. Therefore, maintaining platelet and neutrophil counts following cardiopulmonary bypass could enhance the protective effect of APR.