PubMed Health⌕ Search

Biomedical subjects

J D Catravas

Publications and source records attributed to J D Catravas.

At least 37 records · Page 2Linked to original sources
PubMed1998

Zn2+ inhibits nitric oxide formation in response to lipopolysaccharides: implication in its anti-inflammatory activity.

There is compelling evidence to indicate an anti-inflammatory action of Zn2+. Most inflammatory diseases are associated with an increase of the inducible form of nitric oxide (NO) synthase. Additionally, inflammatory mediators such as histamine or bradykinin stimulate the constitutive NO synthase. Thus, the present study was undertaken to investigate whether Zn2+ inhibits production of inducible NO synthase and/or constitutive NO synthase activity to produce NO. Lipopolysaccharide, 5 mg/kg i.v., administered to Zn2+-deficient (ZD) rats, rats supplemented with Zn2+ sulfate (ZG), 10 mg/kg s.c., or controls resulted in a significant reduction of their serum Zn2+. The levels of N(G)-nitro-L-arginine methylester (L-NAME)-sensitive cyclic GMP (cGMP) in aortas isolated from ZD or ZG were significantly lower than those obtained from control animals. Zinc (100-150 microM) produced a dose-dependent inhibition of lipopolysaccharide or interleukin-1beta-induced NO formation in isolated rat aortic smooth muscle cells. Compared to cyclohexamide or actinomycin-D, the time course of inhibition of NO formation by 150 microM Zn2+ did not suggest an effect of Zn2+ on inducible NO synthase protein synthesis. Moreover, Zn2+ (150 microM) significantly reduced the rate of conversion of [3H]arginine to [3H]citrulline in lung homogenates from lipopolysaccharide-treated rats. Incubation of rat aortic smooth muscle cells and bovine pulmonary artery endothelial cell co-cultures with Zn2+ (150 microM) caused a significant reduction in basal and bradykinin- or A-23187-induced formation of cGMP. Thus, our results indicate that Zn2+ is capable of inhibiting lipopolysaccharide- or interleukin-1beta-induced NO formation as well as NO formation by constitutive NO synthase basally or in response to bradykinin or A-23187, and may explain the reported anti-inflammatory activity of Zn2+.

Animals↗
PubMed1998

Effect of acute coronary occlusion on the size of the dynamically perfused coronary capillary bed in the dog.

The aim of the present study was to investigate the influence of reduced left anterior descending (LAD) coronary arterial blood flow on the size of the perfused coronary capillary surface area (CCSA) in dogs. The transcoronary hydrolysis (v) of the specific ACE substrate, [3H]benzoyl-Phe-Ala-Pro, was estimated and the parameter Amax/Km (proportional to the size of the perfused CCSA) was calculated. By means of a ligature placed around the LAD, LAD blood flow was transiently reduced to 36.0 +/- 4.1 (E1) and 17.4 +/- 4.3% (E2) of control; in a separate maneuver the first diagonal branch of the LAD was ligated to achieve 40.0 +/- 6.7% (E3) of control flow. The v values remained unchanged at around 0.7 for E1, E2, and E3 determinations, suggesting unaltered substrate transit time through the coronary capillary bed. Amax/Km values decreased to 36 +/- 5, 17 +/- 4, and 47 +/- 10% of control for E1, E2, and E3 determinations, respectively, reflecting a flow-proportional decrease in CCSA. Values of the transpulmonary measures of v and Amax/Km performed at the beginning and end of the protocol were unchanged. These results support the hypothesis that reduction in coronary blood flow will produce proportional decreases in the size of the CCSA. This new procedure can thus serve as a useful tool for investigating alterations in the size of the CCSA in different species and under various pathophysiologic challenges.

Animals↗
PubMed1998

Differential action of angiotensin II and activity of angiotensin-converting enzyme in human bypass grafts.

OBJECTIVE: The activity of the renin-angiotensin system may be important in determining the performance of coronary artery bypass grafts. We have examined the activity of tissue angiotensin-converting enzyme and the effects of angiotensin II in vessels used as bypass grafts. METHODS: Organ bath studies were used to determine the vasoactive effect of angiotensin II. The activity of the angiotensin-converting enzyme was assessed by metabolism of a specific synthetic substrate. RESULTS: The saphenous vein produced greater maximum responses to angiotensin II than did the internal thoracic artery. This response was not modified by inhibition of nitric oxide synthase, cyclooxygenase, or by an endothelin receptor antagonist in either vessel. Losartan, an AT1 receptor antagonist, inhibited the vasoconstrictor response in both blood vessels. Homogenates of saphenous vein and internal thoracic artery displayed tissue angiotensin-converting enzyme activity, which was inhibited by captopril. Enzyme activity was threefold greater in the vein. Both the contractile response to angiotensin II and the enzyme activity were retained in venous grafts removed up to 20 years after coronary bypass surgery. CONCLUSIONS: These data demonstrate that marked differences exist in angiotensin-converting enzyme activity and AT1 receptor responses in the saphenous vein compared with the internal thoracic artery. These findings may have important implications for the performance of the vein when used as a coronary artery bypass graft and may have clinical implications for the use of angiotensin-converting inhibitors and AT1 receptor antagonists in the prevention and treatment of vein graft disease.

Adult↗
PubMed1998

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.

Animals↗
PubMed1998

Effects of antisense oligonucleotide to iNOS on hemodynamic and vascular changes induced by LPS.

Lipopolysaccharide (LPS) causes impaired vascular contractility proposed to be mediated by induction of nitric oxide synthase (iNOS). Antisense (AS) oligonucleotide inhibits the translation of target mRNA into functional proteins. We hypothesize that in vivo pretreatment with AS oligonucleotide targeted to iNOS mRNA can prevent LPS-induced hyporeactivity to norepinephrine (NE). Three groups of conscious male Wistar rats received one of the following: saline, AS, or mismatch (MM) oligonucleotide at 0.4 mg/kg iv at 12 and 24 h before LPS (5 mg/kg iv). The fourth group received saline only. Mean arterial pressure (MAP) and heart rate (HR) were continuously recorded before and 6 h after LPS or saline administration. Aorta, lung lavage, and lung tissue were collected for determination of iNOS protein expression and NOS activity. Small mesenteric arteries ( approximately 250 micron) were isolated, denuded of endothelium, and maintained at a constant intraluminal pressure of 40 mmHg for study in vitro. LPS produced significant tachycardia that was not altered by AS or MM oligonucleotide. AS, but not MM oligonucleotide, reduced the accumulation of cGMP, the increase in conversion of L-[3H]arginine to L-[3H]citrulline, and iNOS protein expression in tissue from LPS-treated rats. Small mesenteric arterial contraction to NE was significantly impaired in vessels from LPS-treated rats and was restored by AS, but not MM, oligonucleotide. In a rat model of septic shock, AS oligonucleotide to iNOS mRNA inhibits NOS activity and iNOS protein expression and prevents the vascular hyporeactivity to NE, which may contribute to hypotension in shock.

Animals↗
PubMed1998

Unaltered pulmonary capillary surface area in the presence of changing arterial resistance.

We hypothesized that capillary recruitment may not be solely dependent on extracapillary factors. To test this hypothesis, rabbits were anesthetized and placed on total cardiac bypass at a constant, physiological pulmonary blood flow. Vascular occlusion techniques were combined with measurement of the transpulmonary metabolism of an angiotensin-converting enzyme substrate, allowing the concomitant assessment of changes in segmental resistances and dynamically perfused capillary surface area. Intra-arterial serotonin infusion increased upstream pulmonary vascular resistances without affecting dynamically perfused capillary surface area. Intra-arterial isoproterenol infusion diminished serotonin-induced increased upstream resistances, also without affecting capillary surface area. These findings support the hypothesis that pulmonary capillary recruitment may not be solely dependent on extracapillary factors.

Animals↗
PubMed1998

Release of a leukocyte activation inhibitor by staurosporine-treated pulmonary artery endothelial cells.

Bovine pulmonary arterial endothelial cells (BPAE) treated with the protein kinase C (PKC) inhibitor staurosporine inhibited O-2. generation by neutrophils exposed to phorbol myristate acetate (PMA) but did not affect O-2. generated enzymatically by xanthine/xanthine oxidase (X/XO). Similar results were obtained with conditioned medium from staurosporine-pretreated BPAE. The inhibitory effects of staurosporine-treated BPAE on O-2. generation were not altered by the superoxide dismutase inhibitor diethylcarbamazine. This BPAE-derived inhibitor was continuously released from staurosporine-pretreated BPAE for at least 5 h. The exact nature of the inhibitor remains unknown, but it appears to be a positively charged molecule with molecular weight <10,000. Treatment of either BPAE or neutrophils with staurosporine or conditioned medium from staurosporine-treated BPAE prevented the neutrophil-mediated decrease in endothelium-bound angiotensin-converting enzyme activity and cytotoxicity in BPAE. In contrast, staurosporine potentiated the H2O2- and X/XO-mediated endothelial cytotoxicity. These data suggest that staurosporine-treated endothelial cells release a soluble factor that inhibits neutrophil activation and protects endothelial cells from neutrophil-mediated injury.

Animals↗
PubMed1998

Differential distribution of angiotensin AT2 receptors in the normal and failing human heart.

Cardiac expression of angiotensin II (Ang II) AT1 and AT2 receptor subtypes is species dependent, and changes in their relative proportion may influence myocardial hypertrophy and fibrosis. Regional differences in the distribution of Ang II receptors in the normal and failing human heart were assessed using 125I-(Sar1,Ile8)Ang II binding and quantitative autoradiography. Receptor subtypes were distinguished by their affinity for selective nonpeptide antagonists (losartan and PD123319) and sensitivity to dithiothreitol. Ventricular and atrial tissues displayed a heterogeneous distribution of ligand binding sites. AT2 receptors predominated, representing 70% to 77% of the sites in normal and noninfarcted myocardium. Endocardial, interstitial, perivascular and infarcted regions in the ventricles of patients with end-stage ischemic heart disease or dilated cardiomyopathy exhibited a significantly greater density (P < .001) of high affinity AT2 binding sites (Kd = 0.57 nmol/liter) compared with adjacent noninfarcted myocardium. Regions displaying the relative increase in AT2 binding sites corresponded to areas of fibroblast proliferation and collagen deposition, shown by picrosirius red staining. AT1 binding sites were localized to nerves, occurred at relatively low density in coronary vessels and represented only 23% to 29% of myocardial 125I-(Sar1,Ile8)Ang II binding sites. The border zone between infarcted and noninfarcted myocardium characteristically contained numerous microvessels, exhibiting perivascular AT2 receptors and endothelial angiotensin converting enzyme activity, as demonstrated by binding of 125I-351A. Specific myocardial AT2 receptor mRNA transcripts (approximately 3 kb) were identified and exhibited alternative splicing of untranslated 5' exons. The differential distribution of cardiac Ang II receptor subtypes and selective increase in binding to AT2 sites in the diseased heart suggest that cells bearing the AT2 receptor represent a significant target for Ang II, possibly contributing to its growth-related actions.

Adolescent↗
PubMed1997

Endothelial ectoenzyme assays estimate perfused capillary surface area in the dog lung.

Whether the pulmonary vascular bed accommodates flow-induced increases in blood volume mainly through recruitment of previously unperfused capillaries or distension of already perfused vessels remains controversial. The modified first order reaction parameter of an enzyme and substrate, Amax/K(m), is, under nontoxic conditions, proportional to enzyme mass. Thus for ACE, an endothelium-bound ectoenzyme uniformly distributed along the luminal surface of the pulmonary capillary bed, Amax/K(m) is proportional to the dynamically perfused capillary surface area (PCSA). We estimated single-pass translobar hydrolysis and calculated the corresponding Amax/K(m) values of the synthetic ACE substrate 3H-benzoyl-Phe-Ala-Pro (BPAP), under first-order reaction conditions, in isolated blood-perfused dog lung lobes. We additionally studied blood flow distribution using radioactive microsphere techniques. Experiments were performed under zone III conditions over a wide range of lobar blood flow rates (Qb). As Qb was increased, Amax/K(m) rose linearly, while lobar vascular resistance (LVR) decreased, suggesting capillary recruitment rather than distension. Single pass BPAP hydrolysis (v approximately 2.9 at resting Qb) was not altered over a wide range of Qb, indicative of unchanging capillary transit times. When full capillary recruitment was achieved (at Qb > 70 ml/min/g lung wet weight), further Qb elevations failed to increase Amax/K(m), but decreased BPAP hydrolysis, denoting shorter transit times through the fully recruited capillary bed. Our data indicate that, as previously shown for rabbit lung, in this canine model, increases in pulmonary blood volume are mainly accommodated through recruitment of previously unperfused capillaries throughout the entire lung.

Animals↗
PubMed1997

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.

1-Sarcosine-8-Isoleucine Angiotensin II↗
PubMed1996

cGMP accumulation and gene expression of soluble guanylate cyclase in human vascular tissue.

Gene expression of soluble guanylate cyclase (sGC) and cGMP accumulation in response to sodium nitroprusside (SNP) were studied in cultured human vascular cells and freshly harvested vascular tissue. As revealed by reverse transcriptase-polymerase chain reaction, cultured smooth muscle and endothelial cells, as well as freshly isolated human vascular tissue, express mRNA for the alpha 3 and beta 3 subunits but not for the alpha 2 and beta 3 subunits is evident even in the absence of increased cGMP accumulation in response to SNP. cGMP accumulation in human cells cultured from different vascular beds typically increased two- to five-fold (maximum of 11.4-fold) over baseline following stimulation with 100 microM SNP. Bovine, murine, canine, and avian vascular smooth muscle cells accumulated similar or lower amounts of cGMP than human cells, whereas porcine, rat, and rabbit smooth muscle cells accumulated greater amounts of cGMP. In freshly harvested human vessels, cGMP accumulation in response to SNP was found to increase fifteen-fold over baseline. In contrast to the SNP-induced cGMP accumulation, cGMP levels in response to particulate guanylate cyclase activator atriopeptin II were equal to or greater in cultured human cells than in fresh human vascular tissue. We conclude that human vascular cells (fresh and cultured) express the mRNA for both a large (alpha 3) and a small (beta 3) sGC subunit and that fresh human cells are more sensitive to SNP stimulation.

Animals↗
PubMed1996

Anesthesia with sodium pentobarbital enhances lipopolysaccharide-induced cardiovascular dysfunction in rats.

Lipopolysaccharide (LPS)-induced hypotension and impaired aortic contraction to norepinephrine (NE) are thought to be consequent to induction of nitric oxide synthase (iNOS). Anesthesia is often employed in studies of the mechanisms mediating LPS-induced cardiovascular dysfunction in rats. Since sympathetic nervous system activity and compensatory mechanisms can be altered by anesthesia, this study was designed to determine a) if the cardiovascular dysfunction associated with LPS (5 mg/kg, i.v.)-induced endotoxin shock is enhanced in anesthetized compared with conscious male Wistar rats, and b) the potential role of iNOS in these responses to LPS. Arterial pressure and heart rate were continuously measured via a femoral arterial cannula. Six hours after LPS, conscious rats had a stable mean arterial pressure (MAP) and were tachycardic, while anesthetized rats showed a significant decrease in MAP without tachycardia. Small mesenteric arterioles (200-300 microns) were isolated, and the endothelium was removed six h after LPS. Intraluminal diameter was continuously recorded while vessels were maintained at a constant intraluminal pressure of 40 mmHg. Norepinephrine-induced contraction and oscillations/min were impaired to a greater extent in arterioles from LPS-treated anesthetized rats than in those from conscious rats. Calcium-dependent and -independent nitric oxide formation, reflected as cGMP accumulation, were also determined in aortic rings treated with a chelator of Ca2+, EGTA, or the inhibitor of nitric oxide synthase activity, L-NAME. In rings from saline-treated conscious and anesthetized rats, cGMP accumulation was significantly reduced by EGTA and L-NAME, indicating calcium-dependent constitutive (cNOS) activity. However, in aortic rings from LPS-treated conscious and anesthetized rats, cGMP accumulation was not affected by EGTA and was significantly greater in rings from anesthetized vs. conscious rats. These results suggest that cardiovascular dysfunction is more prominent in LPS-treated anesthetized vs. conscious rats. This effect may be related to increased induction of iNOS in the presence of anesthesia.

Adjuvants, Anesthesia↗
PubMed1996

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.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1996

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.

Animals↗
PubMed1996

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.

Animals↗
PubMed1996

Relationship between volume of bathing medium and ectoenzyme activity in monolayers of cultured BPAEC.

It is unclear whether all or a fraction of the capillary plasma volume (Vcp) serves as the reaction volume (Vr) for pulmonary capillary endothelial ectoenzymes, in vivo. Cultured endothelial cell (EC) monolayers provide a convenient model for studying EC-bound enzyme-Vr relationships. Because the Michaelis-Menten parameter [maximum velocity of enzyme reaction (Vmax) = E x kcat/Vr, where E is enzyme mass and kcat is the constant of product formation] is inversely proportional to Vr, we hypothesized that increasing the volume of medium (Vm) bathing EC monolayers would proportionally reduce the calculated Vmax (or Vmax/K(m), where K(m) is the Michaelis constant) values of an ectoenzyme reacting with a substrate only if, and as long as, Vm = Vr. To test this hypothesis, studies were performed in bovine pulmonary arterial EC grown to confluence. Activities of angiotensin-converting enzyme (ACE) and 5'-nucleotidase (NCT) were assayed in Earle's balanced salts solution utilizing [3H]benzoyl-Phe-Ala-Pro ([3H]BPAP) and 5'-[14C]AMP as substrates, respectively. Under first-order reaction conditions and at constant substrate concentrations ([BPAP] = 15 nM, [AMP] = 1 microM), Vmax/K(m) ratios of ACE and NCT declined to 20% of their original values, as Vm increased from 0.6 to 2 ml. ACE activity was also studied at constant substrate mass (BPAP = 7 pmol) under first-order reaction conditions. Again, enzyme activity (Vmax/K(m)) declined proportionally to increasing Vm. Under zero-order reaction conditions ([BPAP] = 250 microM), ACE activity (Vmax) was similarly related to Vm. Linear regression analyses revealed that ACE or NCT would recognize up to at least 3 ml Vm, a volume vastly exceeding that of Vcp in a section of the capillary bed composed of an equivalent number of ECs, thus suggesting that Vcp could serve as the reaction volume for pulmonary capillary EC ectoenzymes in vivo.

5'-Nucleotidase↗