Regulation of asthmatic airway relaxation by estrogen and heat shock protein 90.

We tested the hypothesis that asthmatic mouse airways exhibit impaired relaxation to NO donors. Mouse tracheal rings were incubated overnight in serum from asthmatic human subjects or from nonasthmatic controls. The next day, cumulative concentration-response curves (CCRC) to sodium nitroprusside (SNP) and nitroglycerine (NTG) were obtained. Both SNP and NTG relaxed the pre-constricted normal tracheal rings. Tracheal rings exposed to serum from asthmatic patients exhibited a more than a threefold increase in the EC50 of SNP and NTG. Pre-incubation of tracheal rings with heat shock protein 90 inhibitors decreased the relaxation of both normal and asthmatic tracheal rings to SNP and NTG. Pre-incubation with estradiol did not affect normal tracheal ring relaxation but exhibited an increase in asthmatic tracheal ring relaxation, which was abolished by an estrogen receptor (ER) antagonist. ER subtype-selective agonists, but not GPR30 agonists, mimicked the action of estradiol on tracheal ring relaxation. Co-incubation of rings with radicicol and estradiol produced an ER-dependent increase in the relaxation response to SNP of both normal and asthmatic ASM. Estrogen-induced relaxation of ASM was abolished by overnight incubation with radicicol and this was associated with reduced expression of ERß. These data suggest that asthmatic ASM is considerably less responsive to NO-donors and that both estrogen and hsp90 play important roles in ASM relaxation.

Regulation of taurine transport by Escherichia coli heat-stable enterotoxin and guanylin in human intestinal cell lines.

The human colon carcinoma cell lines Caco-2 and HT-29 take up taurine actively. Treatment of Caco-2 cells with Escherichia coli heat-stable enterotoxin (STa) or with guanylin inhibited taurine uptake by approximately 40%. In contrast, neither STa nor guanylin changed the uptake of taurine in HT-29 cells. The inhibition in Caco-2 cells was associated with a decrease in the maximal velocity as well as in the affinity of the transporter. STa caused a 21-fold increase in guanosine 3',5'-cyclic monophosphate (cGMP) levels in Caco-2 cells with no change in cAMP levels. Neither cGMP nor cAMP levels were affected by STa treatment in HT-29 cells. Experiments with protein kinase inhibitors suggested that protein kinase A may mediate the observed effects of STa on taurine uptake. In accordance with this suggestion, treatment of Caco-2 cells with cholera toxin, which elevated intracellular cAMP levels, was found to inhibit taurine uptake. The steady state levels of the taurine transporter mRNA transcripts were not altered as a result of STa treatment. Studies with Caco-2 cells grown on permeable filters revealed that STa acts from the apical side. The taurine uptake from the apical side was inhibited by STa, but the taurine uptake from the basolateral side remained unaffected. It is suggested that the activity of the intestinal taurine transporter may be regulated by protein kinase A at a posttranslational level and that the intestinal absorption of taurine may be impaired during infection with enterotoxigenic strains of E. coli.

Functional significance of hsp90 complexes with NOS and sGC in endothelial cells.

Although the existence of hsp90-NOS and hsp90-sGC complexes is now firmly established, their role in many pathophysiological processes remain unclear. These complexes may represent physiological mechanisms aimed at maximizing intracellular cGMP production in response to endogenous or drug-derived NO in endothelial cells and thus affecting permeability, proliferation, migration and apoptosis. Along with minimizing NO scavenging by superoxide and reducing the formation of peroxynitrite, these complexes may also prolong sGC stability by retarding its degradation. Our work and that of others have demonstrated that, depending on the environment, sGC interaction with hsp90 can optimize sGC enzyme activity or modulate sGC survival. This review addresses the functional significance of hsp90 complexes with NOS (eNOS, iNOS) and sGC in endothelial cells relevant for maintaining endothelial barrier integrity and angiogenesis. Structural and functional characteristics of sGC, its expression, transcriptional and post-translational regulation, as they relate to sGC-hsp90 interactions, will also be examined.

RhoA S-nitrosylation as a regulatory mechanism influencing endothelial barrier function in response to G+-bacterial toxins.

Disruption of the endothelial barrier in response to Gram positive (G+) bacterial toxins is a major complication of acute lung injury (ALI) and can be further aggravated by antibiotics which stimulate toxin release. The integrity of the pulmonary endothelial barrier is mediated by the balance of disruptive forces such as the small GTPase RhoA, and protective forces including endothelium-derived nitric oxide (NO). How NO protects against the barrier dysfunction is incompletely understood and our goal was to determine whether NO and S-nitrosylation can modulate RhoA activity and whether this mechanism is important for G+ toxin-induced microvascular permeability. We found that the G+ toxin listeriolysin-O (LLO) increased RhoA activity and that NO and S-NO donors inhibit RhoA activity. RhoA was robustly S-nitrosylated as determined by biotin-switch and mercury column analysis. MS revealed that three primary cysteine residues are S-nitrosylated including cys16, cys20 and cys159. Mutation of these residues to serine diminished S-nitrosylation to endogenous NO and mutant RhoA was less sensitive to inhibition by S-NO. G+-toxins stimulated the denitrosylation of RhoA which was not mediated by S-nitrosoglutathione reductase (GSNOR), thioredoxin (TRX) or thiol-dependent enzyme activity but was instead stimulated directly by elevated calcium levels. Calcium-promoted the direct denitrosylation of WT but not mutant RhoA and mutant RhoA adenovirus was more effective than WT in disrupting the barrier integrity of human lung microvascular endothelial cells. In conclusion, we reveal a novel mechanism by which NO and S-nitrosylation reduces RhoA activity which may be of significance in the management of pulmonary endothelial permeability induced by G+-toxins.

Angiotensin, bradykinin and the endothelium.

Angiotensins and kinins are endogenous peptides with diverse biological actions; as such, they represent current and future targets of therapeutic intervention. The field of angiotensin biology has changed significantly over the last 50 years. Our original understanding of the crucial role of angiotensin II in the regulation of vascular tone and electrolyte homeostasis has been expanded to include the discovery of new angiotensins, their important role in cardiovascular inflammation and the development of clinically useful synthesis inhibitors and receptor antagonists. While less applied progress has been achieved in the kinin field, there are continuous discoveries in bradykinin physiology and in the complexity of kinin interactions with other proteins. The present review focuses on mechanisms and interactions of angiotensins and kinins that deal specifically with vascular endothelium.

Pulmonary capillary endothelium-bound angiotensin-converting enzyme activity in acute lung injury.

BACKGROUND: Pulmonary capillary endothelium-bound (PCEB) angiotensin-converting ectoenzyme (ACE) activity alteration is an early, sensitive, and quantifiable lung injury index in animal models. We hypothesized that (1) PCEB-ACE alterations can be found in patients with acute lung injury (ALI) and (2) PCEB-ACE activity correlates with the severity of lung injury and may be used as a quantifiable marker of the underlying pulmonary capillary endothelial dysfunction. METHODS AND RESULTS: Applying indicator-dilution techniques, we measured single-pass transpulmonary hydrolysis of the synthetic ACE substrate (3)H-benzoyl-Phe-Ala-Pro (BPAP) in 33 mechanically ventilated, critically ill patients with a lung injury score (LIS) ranging from 0 (no lung injury) to 3.7 (severe lung injury) and calculated the kinetic parameter A(max)/K(m). Both parameters decreased early during the ALI continuum and were inversely related to APACHE II score and LIS. Hydrolysis decreased with increasing cardiac output (CO), whereas 2 different patterns were observed between CO and A(max)/K(m). CONCLUSIONS: PCEB-ACE activity decreases early during ALI, correlates with the clinical severity of both the lung injury and the underlying disease, and may be used as a quantifiable marker of underlying pulmonary capillary endothelial dysfunction.

Nitric oxide differentially regulates induction of type II nitric oxide synthase in rat vascular smooth muscle cells versus macrophages.

We studied effects of nitric oxide (NO) released by different NO donors on induction of inducible NO synthase (iNOS) in rat aortic smooth muscle cells (RASMC) and rat macrophage cell line NR8383. iNOS protein expression induced by a CM (interleukin-1beta 250 U/mL, interferon-gamma 150 U/mL, and tumor necrosis factor-alpha 150 U/mL) was not affected by the NO donor SNAP (0.2 to 1 mmol/L) in RASMC at 24 hours of incubation but was dose-dependently decreased by SNAP in macrophages (maximal 60% inhibition). A fully functional -3.2-kb rat iNOS promoter was transfected into RASMC and macrophages. The CM-induced promoter activity in transfected macrophages was inhibited by SNAP (maximal 67% inhibition), but this inhibitory effect by SNAP was not observed in transfected RASMC. Electrophoretic mobility-shift assays demonstrated that nuclear factor-kappaB (NF-kappaB) binding patterns were different in 2 cell types and that the ratio of p50:p65 subunits was significantly lower in macrophages than in RASMC. Furthermore, NF-kappaB activity was not affected by SNAP in RASMC but was reduced by SNAP in macrophages. Another putative NO donor, NOR3 (1 mmol/L), completely inhibited iNOS induction by CM in RASMC, but this was accompanied by severe cytotoxicity, which resulted in cell death. Similar concentrations of SNAP did not exhibit cytotoxicity in RASMC, whereas macrophages demonstrated 88% viability compared with cells without SNAP. NO synthase inhibitor N(g)-monomethyl-L-arginine significantly inhibited CM-induced nitrite production in both cell types and stimulated iNOS protein expression in macrophages but did not affect iNOS expression in RASMC. These data strongly suggest that NO may affect transcriptional regulation of iNOS differently in RASMC versus macrophages, possibly by means of regulation of NF-kappaB activation.

Smooth muscle cell responsiveness to nitrovasodilators in hypertensive and normotensive rats.

Endothelium-derived relaxing factor and exogenous nitrovasodilators are thought to produce smooth muscle relaxation by activation of soluble guanylate cyclase. To investigate whether diminished cyclic GMP (cGMP) accumulation underlies the differences in vascular reactivity to nitrovasodilators between Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR), we determined cGMP formation in aortic smooth muscle cells from the two strains. Both cultured cells and aortic rings from 12- to 14-week-old SHR accumulated greater amounts of cGMP on stimulation with exogenous nitrovasodilators (ie, sodium nitroprusside) than those from WKY rats, whereas there was no difference observed in cells from prehypertensive animals (5- to 6-week old) between the two strains. Responsiveness of smooth muscle cells to endothelium-derived relaxing factor was investigated in cocultures of bovine aortic endothelial cells (BAE) and smooth muscle cells from SHR and WKY rats. cGMP accumulation elicited by endothelium-derived relaxing factor released either basally or in response to bradykinin and the calcium ionophore A23187 was greater in smooth muscle from 12- to 14-week-old SHR than from age-matched WKY rats (80 +/- 17 versus 11 +/- 2 for basal; 152 +/- 12 versus 80 +/- 26 for A23187; 163 +/- 21 versus 40 +/- 12 pmol/mg protein per 15 minutes for bradykinin) in SHR/BAE and WKY/BAE cocultures, respectively. Northern blot analysis of steady-state messenger RNA levels for the beta 1 subunit of soluble guanylate cyclase revealed higher levels of the message in SHR.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of vascular smooth muscle soluble guanylate cyclase activity, mRNA, and protein levels by cAMP-elevating agents.

Although the biochemical properties of soluble guanylate cyclase (sGC) have been extensively studied, little is known about the regulation of gene expression of sGC subunits by second messengers. cAMP analogues and elevating agents have been previously shown to alter gene expression in vascular cells. The aim of the present study was to investigate the effects of cAMP-elevating agents on sodium nitroprusside-stimulated sGC activity and to correlate activity changes with mRNA and protein levels in cultured rat aortic smooth muscle cells. Pretreatment of cells with 50 to 1000 mumol/L isobutylmethyl-xanthine or 0.01 to 10 mumol/L forskolin led to a time- and concentration-dependent decrease in sodium nitroprusside-induced cGMP accumulation, first evident after 3 hours of pretreatment with forskolin and 6 hours of pretreatment with isobutylmethylxanthine. Incubation of cells with a protein kinase A-selective inhibitor (H89 or KT 5720) partially or fully prevented the downregulation in sodium nitroprusside-induced cGMP accumulation caused by cAMP-elevating agents. Quantification of reverse transcriptase-polymerase chain reaction products by high-performance liquid chromatography revealed that mRNA for both alpha1- and beta1-subunits of sGC were decreased in cells pretreated with isobutylmethylxanthine and forskolin but not with dideoxyforskolin (inactive analogue). Moreover, protein levels for the sGC alpha1 subunit of cells pretreated with isobutylmethylxanthine and forskolin but not with dideoxyforskolin were decreased as indicated by Western blot analysis. These data indicate that cAMP-elevating agents decrease sGC activity, possibly by decreasing mRNA or protein levels or both.

Non-NF-kappaB elements are required for full induction of the rat type II nitric oxide synthase in vascular smooth muscle cells.

We have investigated the role of the NF-kappaB binding sites and other promoter elements beyond NF-kappaB in iNOS induction in rat vascular smooth muscle cells (SMC). Rat aortic SMC transfected with iNOS promoter constructs with either mutation or deletion of the downstream NF-kappaB site exhibited about 50% reduction in promoter activity in response to a cytokine mixture, whereas either mutation or deletion of the upstream NF-kappaB site reduced promoter activity by 90%, suggesting that the latter site is the most important, and that co-existence of two NF-kappaB sites is necessary for iNOS induction. Nuclear NF-kappaB activity was robustly induced by TNF-alpha. However, TNF-alpha alone did not induce iNOS promoter activity, protein expression, or nitrite production, indicating that NF-kappaB activation alone is not sufficient for iNOS induction. The construct up to -890 bp, containing the downstream NF-kappaB site, exhibited little response to cytokines. The construct up to -1.0 kb, containing the two NF-kappaB sites exhibited only 22% of full promoter activity. The regions -1001 to -1368 bp and -2 to -2.5 kb contributed an additional 43 and 22% promoter activity, respectively. Internal deletion or reversal of the orientation of -1001 to -1368 bp in the full promoter resulted in 40% reduction in promoter activity. These data suggest that the co-existence of two NF-kappaB sites is essential for core promoter activity, but that full induction of the rat SMC iNOS gene requires other elements located between -1.0 to -1.37 and -2.0 to -2.5 kb of the promoter.

Mechanisms of tolerance to sodium nitroprusside in rat cultured aortic smooth muscle cells.

1. While exposure of smooth muscle cells to sodium nitroprusside (SNP) leads to the development of tolerance to soluble guanylate cyclase (sGC) activation, the mechanisms responsible for this phenomenon in intact cells remain unclear. In the present study, possible mechanisms of tolerance were investigated in a cell culture model where sGC activity was estimated from the accumulation of cyclic GMP in response to 10 microM SNP over a 15 min period in the presence of a phosphodiesterase (PDE) inhibitor. 2. Pretreatment of rat aortic smooth muscle cells with 10-500 microM SNP led to a dose-dependent downregulation of cyclic GMP accumulation upon subsequent SNP stimulation. This effect was evident as early as 2 h following incubation with 10 microM SNP, reached a plateau at 4 h and was blocked by co-incubation with 30 microM oxyhaemoglobin. 3. Pretreatment of smooth muscle cells with the PDE inhibitor, zaprinast, resulted in downregulation of the SNP-induced cyclic GMP accumulation in a time- and concentration-dependent manner, that was first evident after 12 h. Moreover, while the zaprinast-induced downregulation of cyclic GMP accumulation was completely inhibited by the protein kinase A (PKA) inhibitor, H89, tolerance to SNP was partially reversed by H89. 4. beta 1 sGC steady state mRNA levels of S-nitroso N-acetylpenicillamine (SNAP)- or 8Br-cyclic GMP-pretreated cells were unchanged, as indicated by Northern blot analysis. However, Western blot analysis revealed that alpha 1 protein levels were decreased in zaprinast, but not in SNP, SNAP or 8Br-cyclic GMP pretreated cells. 5. While thiol depletion did not prevent the development of tolerance, pretreatment of cells with SNP in the presence of reducing agents partially or completely restored the ability of cells to respond to SNP. 6. We conclude that tolerance to SNP results from two distinct mechanisms: an early onset, NO-mediated event that is reversed by reducing agents and a more delayed, PKA-sensitive process that is mediated through increases in cyclic GMP and a decrease in sGC protein levels.

Cytoskeleton-dependent activation of the inducible nitric oxide synthase in cultured aortic smooth muscle cells.

1. Vascular endothelial and smooth muscle cells generate nitric oxide (NO) via different nitric oxide synthase (NOS) isozymes. Activation of the endothelial constitutive NOS (ecNOS) contributes to the maintenance of cardiovascular homeostasis, whereas expression of the endotoxin- and cytokine-inducible pathway (iNOS) within the vascular smooth muscle is thought to be responsible for the cardiovascular collapse which occurs during septic shock and antitumour therapy with cytokines. Since the cytoskeleton is involved in the activation of certain genes and in some effects of endotoxin in macrophages, we investigated the role of microtubules and microfilaments in the activation of the NO pathway in cultured vascular cells. 2. Depolymerization of microtubules by either nocodazole or colchicine prevented lipopolysaccharide (LPS)- and interleukin-1 beta-induction of NO-dependent cyclic GMP accumulation. Steady state levels of iNOS mRNA, assessed by Northern blot and RT-PCR, and iNOS protein, assessed by Western blotting, were also decreased by either colchicine or nocodazole treatment. 3. Taxol enhanced microtubule polymerization alone, and prevented microtubule depolymerization elicited by nocodazole and colchicine. Associated with its effect on microtubule assembly, taxol prevented the inhibitory effects of nocodazole and colchicine on cyclic GMP accumulation and iNOS mRNA levels. 4. Disruption of microfilaments by cytochalasins had no inhibitory effect on the activation of the inducible NO pathway. 5. In contrast to cytokine-stimulated smooth muscle cells, modulation of either microtubule or microfilament assembly did not affect the constitutive NO pathway in endothelial cells, as endothelial cell- and NO-dependent cyclic GMP accumulation in endothelial-smooth muscle co-cultures remained unchanged. 6. Our findings demonstrate that microtubules play a prominent role in the activation of the inducible NO pathway in response to inflammatory mediators in smooth muscle cells but not of the constitutive synthesis of NO in endothelial cells.

Induction of nitric oxide synthase by protein synthesis inhibition in aortic smooth muscle cells.

1. The role of de novo protein synthesis in inducible NO synthase (iNOS) activation was investigated in vitro by evaluating the effects of protein synthesis inhibitors cycloheximide (CH) and anisomycin (ANI) on iNOS activity, protein and mRNA levels in rat aortic smooth muscle cells (RASMC). 2. As determined by cyclic GMP accumulation, substrate (L-arginine)- and inhibitor (N(G)-monomethyl-L-arginine, NMMA)-sensitive iNOS activity was significantly elevated in CH- or ANI-treated RASMC after 24 h. 3. Lipopolysaccharide (LPS) produced a time-dependent increase in cyclic GMP levels with maximal stimulation at 6 h and a decline to near baseline at 24 h. CH attenuated LPS-induced cyclic GMP accumulation at 3 and 6 h. However, cyclic GMP levels were superinduced at later times by CH. The concentration-dependence of cyclic GMP stimulation by cycloheximide was biphasic both in the absence and presence of LPS, with maximal stimulation at 10 microM and inhibition at higher concentrations. 4. Increased iNOS activity by CH was associated with elevated levels of immunoreactive iNOS protein as judged by Western blotting in LPS- and CH-treated cells. 5. CH-induced iNOS activity and superinduction of iNOS by CH in cells treated with LPS were both significantly inhibited by actinomycin D, a transcription inhibitor. 6. RT-PCR revealed elevated iNOS mRNA levels after 12 h of exposure to CH. The combination of LPS and CH caused a significant increase in iNOS gene expression relative to LPS- or CH stimulation alone. 7. These results show that partial protein synthesis inhibition by CH alone upregulates iNOS mRNA and superinduces iNOS mRNA in cytokine-treated RASMC, which is translated to the functional enzyme generating biologically active NO. Thus iNOS activation in these cells not only requires new protein synthesis but it also appears to be negatively regulated by newly synthesized proteins.

Angiotensin converting enzyme activity is present in the endothelium-denuded aorta.

Angiotensin converting enzyme activity was measured in intact aortic rings utilizing the synthetic tripeptide [3H]-benzoyl-Phe-Ala-Pro as the substrate. Intact aortic rings possessed angiotensin converting enzyme activity which was blocked by captopril. Enzyme activity was reduced by approximately 30% after removal of the endothelium by chemical or mechanical methods. The remaining activity was also captopril-sensitive. These results suggest that in addition to endothelium, angiotensin converting enzyme activity is present in other vascular cells and may contribute to the metabolism of angiotensin I generated within the vessel wall.

Prevention of nitric oxide synthase induction in vascular smooth muscle cells by microtubule depolymerizing agents.

We investigated the role of microtubules in the induction of nitric oxide synthase in cultured vascular smooth muscle cells. We found that like interleukin-1 alpha, lipopolysaccharide elicited a time and concentration-dependent accumulation of cyclic GMP via induction of nitric oxide synthase. Nocodazole and colchicine, two chemically distinct microtubule depolymerizing agents, completely prevented lipopolysaccharide- and interleukin-induced (and nitric oxide-mediated) cyclic GMP generation. In contrast to lipopolysaccharide and interleukin-1 alpha, cyclic GMP accumulation in response to sodium nitroprusside, an exogenous nitrovasodilator, was not altered by either nocodazole or colchicine. Our findings demonstrate that microtubule depolymerizing agents inhibit nitric oxide synthase induction and suggest a prominent role for microtubules in mediating the activation of the inducible nitric oxide pathway in smooth muscle cells.

Downregulation of nitrovasodilator-induced cyclic GMP accumulation in cells exposed to endotoxin or interleukin-1 beta.

1. Induction of nitric oxide synthase (iNOS) results in overproduction of nitric oxide (NO), which may be a principal cause of the massive vasodilatation and hypotension observed in septic shock. Since NO-induced vasorelaxation is mediated via the soluble isoform of guanylate cyclase (sGC), the regulation of sGC activity during shock is of obvious importance, but yet poorly understood. The aim of the present study was to investigate the activation of sGC by sodium nitroprusside (SNP) before and after exposure of rat aortic smooth muscle cells to endotoxin (LPS) or interleukin-1 beta (IL-1 beta). 2. Exposure of rat aortic smooth muscle cells to SNP (10 microM) elicited up to 200 fold increases in cyclic GMP. This effect was attenuated by 30-70% in IL-1 beta- or LPS-pretreated cells, in a pretreatment time-and IL-1 beta- or LPS-concentration-dependent manner. When, however, cells were exposed to IL-1 beta or LPS and then stimulated with the particulate guanylate cyclase activator, atriopeptin II, no reduction in cyclic GMP accumulation was observed. 3. Pretreatment of rats with LPS (5 mg kg-1, i.v.) for 6 h led to a decrease in aortic ring SNP-induced cyclic GMP accumulation. 4. The IL-1 beta-induced reduction in SNP-stimulated cyclic GMP accumulation in cultured cells was dependent on NO production, as arginine depletion abolished the downregulation of cyclic GMP accumulation in response to SNP. 5. Reverse-transcriptase-polymerase chain reaction analysis revealed that the ratio of steady state mRNA for the alpha, subunit of sGC to glyceraldehyde phosphate dehydrogenase was decreased in LPS- or IL-1 beta-treated cells, as compared to vehicle-treated cells. 6. Protein levels of the alpha 1 sGC subunit remained unaltered upon exposure to LPS or IL-1 beta, suggesting that the early decreased cyclic GMP accumulation in IL-1 beta- or LPS-pretreated cells was probably due to reduced sGC activation. Thus, the observed decreased responsiveness of sGC to NO stimulation following cytokine or LPS challenge may represent an important homeostatic mechanism to offset the extensive vasodilatation seen in sepsis.

Inhibition of endothelial-bound angiotensin converting enzyme, in vivo.

1. We determined apparent Ki constants of two inhibitors, captopril and CL242,817, for pulmonary endothelial-bound angiotensin converting enzyme (ACE) in anaesthetized rabbits. [3H]-benzoyl-Phe-Ala-Pro was used as the substrate. The apparent kinetic parameters Km and Amax (product of Vmax and microvascular plasma volume) were measured, as was the ratio (Amax/Km) (measured under first order reaction conditions) before and 30s after the i.v. administration of captopril 10 nmol kg-1 or CL242,817, 35 nmol kg-1. 2. Under mixed order reaction conditions, ([S] greater than or equal to Km), apparent Km values increased from 12.2 +/- 1.9 microM to 32.9 +/- 3.3 microM (P less than 0.05) in the captopril-treated rabbits and from 9.3 +/- 2.3 microM to 45.8 +/- 9.8 microM (P less than 0.05) in the CL242,817-treated rabbits, indicative of competitive inhibition. However, apparent Amax values decreased from 10.3 +/- 2.1 to 4.5 +/- 0.8 mumol min-1 (P less than 0.05) and 8.9 +/- 1.7 to 4.8 +/- 0.5 mumol min-1 (P less than 0.05), respectively. 3. Under first order reaction conditions ([S] much less than Km), the Amax/Km ratio decreased from 763 +/- 100 to 125 +/- 38 ml min-1 (P less than 0.05) and 1009 +/- 149 to 126 +/- 44 ml min-1 (P less than 0.05) in the captopril- and CL242,817-treated groups respectively. 4. When the single pass transpulmonary binding of 80pmol [3H]-RAC-X-65 (an ACE inhibitor) was measured in additional rabbits, a significant (P < 0.05) decrease in RAC-X-65 binding was observed 30s after captopril (80% decrease) or CL242,817 (85% decrease), a result expected for a loss of catalytically active enzyme mass due to tightly bound captopril or CL242,817. 5. These results indicate that, in vivo, both captopril and CL242,817 are competitive, tight binding inhibitors of lung ACE. Furthermore, they suggest means for evaluating the interaction of other potential ACE inhibitors with the pulmonary endothelial membrane-bound enzyme, in vivo, possibly in phase I clinical trials.

Sequential development of angiotensin receptors and angiotensin I converting enzyme during angiogenesis in the rat subcutaneous sponge granuloma.

1. The vasoconstrictor peptide antiotensin II (AII) can stimulate angiogenesis, an important process in wound healing, tumour growth and chronic inflammation. To elucidate mechanisms underlying AII-enhanced angiogenesis, we have studied a subcutaneous sponge granuloma model in the rat by use of 133Xe clearance, morphometry and quantitative in vitro autoradiography. 2. When injected directly into the sponge, AII (1 nmol day-1) increased 133Xe clearance from, and fibrovascular growth in sponge granulomas, indicating enhanced angiogenesis 6 to 12 days after implantation. This AII-enhanced angiogenesis was inhibited by daily doses (100 nmol/sponge) of the specific but subtype non-selective AII receptor antagonist (Sar1, Ile8)AII, and by the selective non-peptide AT1 receptor antagonists losartan and DuP 532. In contrast, AII-enhanced neovascularization was not inhibited by the AT2 receptor antagonist PD123319, nor was it mimicked by the AT2 receptor agonist CGP42112A (each at 100 nmol/sponge day-1). 3. AI (1 nmol/sponge day-1), the angiotensin converting enzyme (ACE) inhibitors captopril (up to 100 micrograms/sponge day-1) and lisinopril (40 micrograms/sponge day-1), or AII receptor antagonists did not affect angiogenesis in the absence of exogenous AII. 4. [125I]-(Sar1, Ile8)AII binding sites with characteristics of AT1 receptors were localized to microvessels and to non-vascular cells within the sponge stroma from 4 days after implantation, and were at higher density than in skin throughout the study. 5. [125I]-(Sar1, Ile8)AII binding sites with characteristics of AT2 receptors were localized to non-vascular stromal cells, were of lower density and appeared later than did AT1 sites. 6. The ACE inhibitor [125I]-351A bound to sites with characteristics of ACE, 14 days after sponge implantation. [125I]-351A bound less densely to sponge stroma than to skin. 7. We propose that AII can stimulate angiogenesis, acting via AT1 receptors within the sponge granuloma. AT1 and AT2 receptors and ACE develop sequentially during microvascular maturation, and the role of the endogenous angiotensin system in angiogenesis will depend on the balanced local expression of its various components. Pharmacological modulation of this balance may provide novel therapeutic approaches in angiogenesis-dependent diseases.

Molecular cloning and analysis of the rat inducible nitric oxide synthase gene promoter in aortic smooth muscle cells.

We have cloned five DNA fragments (-0.32, -0.48, -1.7, -3.2, and -5.1 kb) of the 5'-flanking region of the rat inducible nitric oxide synthase (iNOS) gene from rat genomic DNA. The functional importance of the 5'-flanking region was determined by transient expression of iNOS promoter-luciferase constructs in cultures of rat aortic smooth muscle cells. The -0.48 kb construct, containing one nuclear factor kappaB (NF-kappaB) binding site, expressed basal promoter activity but showed only a 1.5- and 1.7-fold increase in luciferase activity in response to lipopolysaccharide (LPS) or a cytokine mixture, respectively. However, the -3.2 kb construct (containing a second NF-kappaB binding site) showed full promoter activity with a 24-fold increase in response to LPS or cytokine mixture. The -5.1 kb construct showed no further increase in luciferase activity, suggesting that the 1.9 kb upstream of -3.2 kb may not be important in rat iNOS regulation. Rat iNOS promoter induction did not appear to be transcriptionally regulated by NO since NOS inhibitors did not affect induction. These data are in marked contrast to the mouse iNOS promoter in which a DNA sequence as short as a -85 bp, containing one NF-kappaB site, confers 10-fold inducibility by LPS. The present findings demonstrate that the rat iNOS gene is transcriptionally regulated by cytokines and LPS, but, unlike the mouse gene, the downstream NF-kappaB site does not appear to be a key region in responses to cytokines and LPS. These data suggest that the regulation of the rat gene may require the coexistence of at least two NF-kappaB sites or other elements upstream of -0.48 kb of the 5'-flanking region.

Effects of adenosine and analogs on adenylate cyclase activity in cultured bovine aortic endothelial cells.

We studied the effects of adenosine and analogs on adenylate cyclase (AC) activity in membranes from long-term cultured bovine aortic endothelial cells, using [alpha-32]ATP as substrate and chromatographic separation of [32P]cAMP. Compared to our previous findings in cultured bovine pulmonary arterial endothelial cells (Legrand et al., Biochem Pharmacol 38: 423-430, 1989), the present results were qualitatively and quantitatively comparable between the two cell types. In aortic cells, AC activity was stimulated in a concentration-dependent manner by isoproterenol, forskolin and 5'-guanylylimidodiphosphate (Gpp(NH)p), by 2.6-, 5.2- and 4.8-fold respectively. The A2 adenosine agonist 5'-(N-ethyl)-carboxamidoadenosine induced a smaller (60%) increase of AC activity. Adenosine (10(-3) M) partially inhibited (30%) the Gpp(NH)p-stimulated AC activity. Similarly, adenosine partially reversed, but 2',5'-dideoxyadenosine (DDA) totally blocked (IC50: 540 microM), the forskolin-induced stimulation of AC activity. DDA and 2'-deoxyadenosine-3'-monophosphate (2'-deoxy-3'-AMP) also inhibited the isoproterenol-induced stimulation of AC activity (IC50: 350 and 23 microM respectively). Adenosine-induced inhibition of stimulated AC activity does not appear to involve adenosine A1 receptors since the specific A1 agonist cyclohexyladenosine did not reverse forskolin stimulation of AC activity. Instead, it suggests a direct action of adenosine on the catalytic subunit of the adenylate cyclase (P site). We conclude that membranes from long-term cultured bovine aortic endothelial cells, express beta-adrenergic and adenosine A2 receptors coupled to adenylate cyclase activation. The two P site agonists, DDA and 2'-deoxy-3'-AMP, and, with a weaker effect, adenosine itself, inhibited the activated cyclase at the P site. The natural nucleotide 2'-deoxy-3'-AMP was a strong inhibitor in aortic cell types (as in pulmonary arterial endothelial cells) and may possibly act as a modulator of adenylate cyclase in these cells.