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T D Warner

Publications and source records attributed to T D Warner.

At least 109 records · Page 6Linked to original sources
PubMed1993

The metabolism of endothelin-1 and big endothelin-1 by the isolated perfused kidney of the rabbit.

The activity of human big endothelin-1 (bET-1) or endothelin-1 (ET-1) was investigated in the isolated perfused kidney of the rabbit. In some experiments the effluent superfused a rabbit jugular vein (RbJV) or rat colon (RC), and bolus doses of bET-1 or ET-1 were administered either directly over the tissue (OT) or through the kidney (TK). When injected OT, the contractile responses of the RbJV to ET-1 were > or = 100-200 times more than those to bET-1. However, at least 10 times the ET-1 dose given OT was needed TK to produce an equivalent contraction of the RbJV, showing that ET-1 was being inactivated or removed by the kidney. Injections of bET-1 TK produced increases in perfusion pressure of a magnitude 1/25th of those of ET-1. In some experiments administration of bET-1 TK was associated with the release into the perfusate of an ET-1-like factor that contracted the RbJV, but not the RC (used to detect any angiotensin II release), at doses that had little effect when administered OT, suggesting that bET-1 was being activated by the kidney. Phosphoramidon (10 microM) infused TK blocked (92 +/- 1% inhibition) the renal responses to bET-1 and reduced the overflow of ET-1-like material onto the tissues without affecting ET-1-induced renal vasoconstriction. Incubation of bET-1 with rabbit renal cortical microsomes (100 micrograms protein) resulted in the generation of ET-1-like activity, as assessed by bioassay, which was inhibited by phosphoramidon (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Vasoconstriction in the rat kidney induced by endothelin-1 is blocked by PD 145065.

We have previously shown that the receptors mediating the renal and systemic vasoconstrictor effects of endothelin-1 (ET-1) are of two distinct endothelin receptor subtypes. Here, we evaluate the effect of PD 145065, a nonselective endothelin receptor antagonist, on the renal vasoconstrictor effects of ET-1 in the rat kidney. ET-1 induced concentration-dependent increases in perfusion pressure in the isolated perfused kidney of the rat. The ETA receptor-selective antagonists BQ-123 (10 microM) and FR 139317 (10 microM) lowered the ET-1-induced rise in perfusion pressure by 57% and 61%, respectively, at 3 x 10(-10) M ET-1. By comparison, the ET-1-induced vasoconstriction was fully antagonized (96% inhibition) by PD 145065 (10 microM). In the anesthetized rat, ET-1 produced a dose-dependent increase in mean arterial pressure, which was attenuated by PD 145065. The initial depressor response to ET-1 was completely blocked by PD 145065, as were the reduction in renal blood flow and increase in renal vascular resistance induced by ET-1. These results suggest that both the ETA and a non-ETA receptor subtype play an important role in mediating the vasoconstrictor effects of ET-1 in the rat kidney.

Amino Acid Sequence↗
PubMed1993

Comparative studies with the endothelin receptor antagonists BQ-123 and PD 142893 indicate at least three endothelin receptors.

Endothelin-1 (ET-1), endothelin-3 (ET-3), sarafotoxin 6b (SX6b), and sarafotoxin 6C (SX6c) were used as agonists, and BQ-123 (ETA-selective) and PD 142893 (ET receptor-nonselective) were used as antagonists to characterize the receptors mediating the effects of the ET/SX peptides on a variety of isolated smooth-muscle preparations. Contractions of the rat thoracic aorta, rat isolated perfused mesentery, and guinea pig ileum and potentiation of twitch of the rat vas deferens were mediated by ETA receptors in that they showed the order of potency ET-1 = SX6b > ET-3 >> SX6C. These effects were antagonized by BQ-123 or PD 142893. Contractions of the rabbit pulmonary artery and rat stomach strip, inhibition of twitches in the guinea pig ileum, and vasodilatations of the rat isolated perfused mesentery showed the order of potency ET-1 = SX6b = ET-3 = SX6c and were unaffected by BQ-123, suggesting involvement of ETB receptors. However, in these tissues, PD 142893 antagonized only dilatations of the rat mesentery to ET-1 but not any of the other effects of ET-1. Thus, we suggest that there are three types of endothelin receptors: ETA and two subtypes of ETB.

Amino Acid Sequence↗
PubMed1993

Mediation via different receptors of the vasoconstrictor effects of endothelins and sarafotoxins in the systemic circulation and renal vasculature of the anaesthetized rat.

1. Using endothelin-1 (ET-1), endothelin-3 (ET-3), sarafotoxin 6b (SX6b) and sarafotoxin 6c (SX6c) as agonists and BQ-123 as a selective ETA receptor antagonist, we have examined the endothelin receptor subtypes mediating the systemic pressor and renal vasoconstrictor effects of the ET/SX family of peptides. 2. In anaesthetized rats, bolus intravenous injections of ET-1, ET-3, SX6b or SX6c (0.1, 0.25 and 0.50 nmol kg-1) produced initial transient depressor responses followed by sustained and dose-dependent increases in mean arterial pressure (MAP) with the following rank order of potency: SX6b > ET-1 >> SX6c > ET-3. In contrast, in the renal vasculature these peptides caused equipotent dose-dependent falls in renal blood flow (RBF) (ET-1 = ET-3 = SX6b = SX6c). 3. BQ-123 (1 mg kg-1, i.v. bolus) significantly reduced the systemic pressor effects of all the peptides but was largely ineffective against the renal vasoconstrictions. 4. These results indicate that although the systemic pressor effects of the ET/SX peptides are mediated via ETA receptors, the vasoconstriction in the kidney in vivo may be mediated predominantly via ETB-like receptors. This may be of therapeutic relevance, for an ETA-receptor-selective antagonist could offer only poor protection of the renal circulation from the deleterious effects of endogenously produced members of this peptide family.

Anesthesia↗
PubMed1993

Induction by endotoxin of nitric oxide synthase in the rat mesentery: lack of effect on action of vasoconstrictors.

1. Male Sprague-Dawley or Wistar rats were injected with bacterial lipopolysaccharide (LPS; 5 mg kg-1, i.p.) and killed after 1, 3, 6, 15, and 24 h. The brains, mesenteries, spleens, lungs, livers, kidneys, hearts, aortae and diaphragms were removed and frozen immediately. Control rats were injected with sterile saline and killed after 6 h. 2. The organs were homogenized in a semi-frozen state and NO synthase (NOS) activity measured in tissues from both LPS-treated and saline-treated groups by the ability of homogenates to convert [3H]-L-arginine to [3H]-L-citrulline in a NADPH-dependent manner. 3. The NOS activity in all organs taken from control animals was found to be calcium-dependent, with the highest activity being in the brain. After LPS-treatment an induced calcium-independent NOS was detected in all tissues tested, with the exception of the brain. The spleen, lung, mesentery and liver had the highest amounts of LPS-induced NOS activity. No induction of calcium-dependent NOS was detected. 4. Induction of NOS was maximum 6 h after administration of LPS and had returned to control levels in 24 h. 5. The constitutive NOS in brain and mesentery and the LPS-induced activities in the spleen, lung, liver and mesentery were inhibited by NG-monomethyl-L-arginine (L-NMMA) or NG-nitro-L-arginine methyl ester (L-NAME) according to concentration. The IC50 for L-NAME was 2.5 microM against the constitutive NOS from brain, and 20-25 microM against the inducible NOS. For L-NMMA the IC50 was 20-25 microM against either NOS isoform. 7. The vascular responses to endothelin-I (ET-1), the thromboxane A2-mimetic 11 alpha,9 alpha-epoxymethanoprostaglandin F2alpha (U46619), phenylephrine (PE) or 5-hydroxytryptamine (5-HT) were measured in the simultaneously perfused arterial and venous mesenteric vascular beds from both control and LPS-treated(6 h) rats. Vasoconstrictor responses to all agonists tested were unaffected by LPS treatment. In the presence of L-NAME (100 microM) vasoconstrictor responses were potentiated in both the arterial and venous portion of the mesenteric beds from both control and LPS-treated rats. The potentiation of responses to U46619 was significantly greater in beds from LPS-treated rats.8. Injection of LPS i.p. is associated with induction of NOS in all organs tested, except for the brain. In the mesentery this is not accompanied by a hyporesponsiveness to constrictor agents suggesting an increased sensitivity, particularly to U46619. This may explain the poor perfusion and tissue damage in the splanchnic circulation associated with sepsis.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1993

Characterization of receptors for endothelins in the perfused arterial and venous mesenteric vasculatures of the rat.

1. Endothelin-1 and -3 induced marked arterial and venous constrictions in the perfused mesenteric vasculature of the rat with endothelin-3 being at least 20 times less active than endothelin-1, on both arterial and venous sides of the vasculature. 2. Two ETB selective agonists, BQ-3020 and IRL 1620 (500 pmol), induced weak constrictions of the venous mesenteric vasculature and were inactive in the arterial side at doses up to 1000 pmol. 3. In mesenteric vasculatures precontracted with either methoxamine (arterial side) or the thromboxane A2-mimetic, U46619 (venous side), acetylcholine or bradykinin produced vasodilations of both arterial and venous vessels, whereas endothelin-3 induced vasodilations only on the arterial side. 4. A selective ETA receptor antagonist, BQ-123, blocked, in a concentration-dependent and reversible fashion, the vasoconstrictions induced by endothelin-1 on both sides of the mesenteric circulation (IC50; arterial side: 0.013 microM; venous side: 0.032 microM). 5. In contrast, the vasodilator responses induced by endothelin-3 on the arterial side of the precontracted mesenteric vasculature were not affected by BQ-123. 6. The present study illustrates the presence of ETA receptors which are responsible for vasoconstriction by endothelins in the arterial and venous mesenteric vasculatures. Furthermore, we suggest that the vasodilations induced by endothelin-3 in the arterial vasculature uniquely, are ETB receptor-mediated.

Amino Acid Sequence↗
PubMed1993

Use of the endothelin antagonists BQ-123 and PD 142893 to reveal three endothelin receptors mediating smooth muscle contraction and the release of EDRF.

1. We have compared the receptors mediating the contractions of rings of rat thoracic aorta or rabbit pulmonary artery and rat stomach strips in response to the endothelin/sarafotoxin (ET/SX) family of peptides and to those mediating endothelium-dependent vasodilations within the isolated perfused mesentery of the rat. To discriminate ETA receptors from ETB receptors we have used the criteria that ET-1 is more active than SX6c on ETA receptors, and that the ET/SX peptides are equiactive on ETB receptors. We have also assessed the effects of the ETA receptor-selective antagonist BQ-123, and the non-selective ET receptor antagonist PD 142893 on the responses of each preparation to the ET/SX peptides. 2. ET-1-induced constrictions of the rat thoracic aorta (EC50 3 x 10(-10) M), a prototypic ETA receptor-mediated response, or isolated perfused mesentery of the rat were antagonized by BQ-123 (10(-5) M) or PD 142893 (10(-5) M). SX6c did not constrict either the rat isolated perfused mesentery or the rat thoracic aorta. Thus, ETA receptors mediate these constrictions. 3. ET-1 and SX6c were approximately equipotent in constricting rabbit pulmonary artery rings (EC50S 3-6 x 10(-10) M). Neither BQ-123 (10(-5) M) nor PD 142893 antagonized the contractions induced by ET-1. These effects suggest mediation by ETB receptors but PD 142893 (10(-5) M) did give a 3 fold antagonism of constrictions induced by SX6c. 4. SX6c was more potent than ET-1 in contracting the rat stomach strip (threshold concentrations 10(-10) and 3 x 10(-10) M). Contractions to ET-1 or SX6c were unaffected by BQ-123 (10-5 M), again indicative of ETB receptor-mediated events. PD 142893 (10-5 M) was ineffective against ET-1 but produced a 3 fold antagonism of SX6c.5. In the rat isolated perfused mesentery ET-1 or SX6c (0.3-300pmol) were equipotent in producing dose-related vasodilatations that were unaffected by BQ-123 (10-6 M), indicative of an ETB receptor mediated response. In contrast to the other ETB-mediated responses, PD 142893 (10-6 M) strongly antagonized these vasodilatations.6. Thus, ETA receptors mediate constrictions of the rat thoracic aorta and rat isolated perfused mesentery whereas ETB receptors mediate constrictions of the rabbit pulmonary artery and rat stomach strip and endothelium-dependent dilatations within the mesentery. However, within the group of ETB receptor-mediated responses, endothelium-dependent vasodilatations are sensitive to PD 142893, whereas contractions of the isolated smooth muscle preparations are not. Thus, the receptor present on the endothelium responsible for the release of nitric oxide in response to the ET/SX peptides is most probably different from that present on smooth muscle that mediates BQ-123-insensitive contractions.

Amino Acid Sequence↗
PubMed1993

Characterization of endothelin receptors mediating the effects of the endothelin/sarafotoxin peptides on autonomic neurotransmission in the rat vas deferens and guinea-pig ileum.

1. To characterize the receptors mediating the effects of the endothelin/sarafotoxin family of peptides on the responses to electrical stimulation of the rat vas deferens (RVD) and guinea-pig ileum (GPI) we have used endothelin-1 (ET-1), ET-3, sarafotoxin 6b (SX6b) and SX6c as agonists and the endothelin-receptor antagonists BQ-123 (ETA receptor selective) and PD 142893 (non-selective). 2. In the RVD, ET-1 and SX6b increased the twitches induced by field stimulation starting at a threshold concentration of 10(-10) M while the threshold concentration for ET-3 was 3 x 10(-9) M. SX6c (up to 3 x 10(-8) M) did not potentiate the twitches. SX6b produced significantly (P < 0.05) greater potentiations than ET-1 at concentrations of 3 x 10(-9) M and higher, and 10(-7) M ET-3 also produced a significantly greater effect than ET-1 at the same concentration. Thus, at threshold the rank order of peptides was ET-1 = SX6b > ET-3 >>> SX6c, and at concentrations of 3 x 10(-8) M and higher, SX6b > ET-3 > ET-1 >>> SX6c. 3. In the presence of BQ-123 or PD 142893 (10(-5) M) the threshold concentrations for ET-1 to augment the twitches were increased 30 fold. In the same conditions neither SX6b nor ET-3 potentiated the responses. The relative activities of the endothelin/sarafotoxin peptides and the effectiveness of the endothelin receptor antagonists are consistent with postjunctional ETA receptors mediating these effects. 4. In the transmurally stimulated GPI the endothelin/sarafotoxin peptides produced two effects; an increase in the basal tension of the tissues and an inhibition of the twitch responses. To increase the basal tension the peptides had the order of potency ET-1 > SX6b>> ET-3 = SX6c. These direct effects of ET-1 or SX6b were strongly antagonized (100 fold) by either BQ-123 (10-5M) or PD 142893(10-5 M). Thus, ETA receptors mediate contractions of the GPI induced by these peptides.5. The endothelin/sarafotoxin peptides were approximately equipotent at depressing twitches of the GPI in response to transmural stimulation (EC50s, 4 x 10-11 to 1.5 x 10-10 M). The depressions induced byET-1 were unaffected by either BQ-123 (10-5 M) or PD 142893 (10-5 M). BQ-123 produced a small(three fold) antagonism of the inhibitory effects of ET-3 or SX6c. These results indicate that a receptor of the ETB type mediates the inhibitory effects of the endothelin/sarafotoxin peptides on neurotransmission in the GPI.6. Thus, both ETA receptors and ETB receptors mediate the effects of the endothelin/sarafotoxinpeptides on neurotransmission.

Amino Acid Sequence↗
PubMed1993

Characterization of endothelin synthetic pathways and receptor subtypes: physiological and pathophysiological implications.

The endothelins are a family of three widely expressed 21 amino acid peptides. Apart from the ability to constrict vascular smooth muscle, they have numerous other potent effects, from enhancing mitogenesis to influencing nervous function. The pathway of their production involves the initial synthesis of a predominantly inactive 38 to 41 amino acid precursor, big endothelin, which is cleaved to the active form by an as yet unidentified, but widely distributed, endothelin-converting enzyme. This enzyme has been partially characterized and appears to be a metalloprotease, active at neutral pH, which is inhibited by phosphoramidon. However, other enzymes, including those contained in inflammatory cells, can also convert big endothelin to its active form. Research in the past 2 to 3 years has revealed at least two endothelin receptors. These are widely distributed throughout the body and are present, for instance, on vascular and non-vascular smooth muscle, nervous tissue, and endothelial and epithelial cells. There is also a fairly large degree of heterogeneity between species in the distribution of these endothelin receptors. The endothelins have been widely implicated in a number of disease states and the recent development of selective and non-selective antagonists of these receptors, such as BQ-123, PD 142893, and Ro 46-2005 is finally making available the tools required to elucidate their true roles.

Animals↗
PubMed1992

Insulin secretion from pancreatic B cells caused by L-arginine-derived nitrogen oxides.

L-arginine causes insulin release from pancreatic B cells. Data from three model systems support the hypothesis that L-arginine-derived nitrogen oxides (NOs) mediate insulin release stimulated by L-arginine in the presence of D-glucose and by the hypoglycemic drug tolbutamide. The formation of NO in pancreatic B cells was detected both chemically and by the NO-induced accumulation of guanosine 3',5'-monophosphate. NG-substituted L-arginine analogs inhibited the release of both insulin and NO. Protein immunoblot and histochemical analysis with antiserum to type I NO synthase suggest that the formation of NO in pancreatic B cells is catalyzed by an NADPH- (reduced form of nicotinamide adenine dinucleotide phosphate), Ca2+/calmodulin-dependent type I NO synthase of about 150 kilodaltons.

Amino Acid Oxidoreductases↗
PubMed1992

Detection by bioassay and specific enzyme-linked immunosorbent assay of phosphoramidon-inhibitable endothelin-converting activity in brain and endothelium.

The endothelin-converting enzyme (ECE) activity present in endothelial cells and rat and human brains was characterized using a selective and rapid bioassay for endothelin-1 (ET-1) or endothelin-3 (ET-3) together with a sensitive enzyme-linked immunosorbent assay. We found that ECE activity was predominantly in the membrane fraction of endothelial cells from which it could be extracted by treatment with detergent. In rat brain tissue, the ECE activity was in the membrane fraction and was not solubilized by detergent treatment. Further dissection of the brain revealed that there was a strong localization of ECE activity in the hypothalamus, midbrain, and medulla oblongata in agreement with earlier observations of ET-like immunoreactivity and binding sites. Experiments with human brain tissue also showed the presence of ECE activity. In conclusion, our studies confirmed the presence of ECE activity within endothelial cells, and showed ECE to be localized in brain tissue in sites consistent with the selective distribution of the ET-1 synthetic pathway. In all tissues studied, the ECE activity was significantly inhibited by phosphoramidon or ethylenediaminetetra-acetate.

Animals↗
PubMed1992

Characterization of serine protease-derived metabolites of big endothelin in the cytosolic fraction from human polymorphonuclear leukocytes.

We have characterized by bioassay and reversed-phase high-performance liquid chromatography analysis (rp-HPLC) the conversion of human big endothelin-1 (bET-1) to endothelin-1 (ET-1) by the cytosolic fraction from human polymorphonuclear leukocytes (PMNs). Either the general serine protease inhibitor 3,4-dichloroisocoumarin (DCI; 50 microM) or the selective elastase inhibitor ONO-5046 (100 microM) blocked the formation of ET-1 from bET-1. Interestingly, human leukocyte elastase formed some of the same products from bET-1 as the PMN cytosol, but generated negligible amounts of ET-1. However, coincubation of the elastase-derived fragments of bET-1 with the PMN cytosol in the presence of ONO-5046 resulted in a 17-fold increase in the formation of ET-1, indicating that an elastase-derived intermediate of bET-1 was subsequently cleaved by a soluble protease(s) to form mature ET-1. We have identified by electrospray-mass spectrometry (ESMS) analysis this intermediate as bET-1(1-22). Analysis of bET-1 digestion by human leukocyte cathepsin G revealed the formation of a biologically active metabolite chromatographically distinct from ET-1, identified as bET-1(1-31) by ESMS. These findings indicate the presence of complex enzyme systems in human PMNs capable of activating bET-1.

Cathepsin G↗
PubMed1992

Different patterns of release of endothelium-derived relaxing factor and prostacyclin.

1. Release of endothelium derived relaxing factor (EDRF) and prostacyclin (PGI2) from endothelial cells (EC) cultured from bovine aortae was measured by bioassay and radioimmunoassay, respectively, during infusions (10 min) of bradykinin (BK), adenosine diphosphate (ADP), arachidonic acid (AA), alkaline buffers and the free-bases (FB) of L-arginine or D-arginine. Release of EDRF from the luminally perfused rabbit aorta was also measured during infusions (10 min) of acetylcholine (ACh), substance P and ADP. 2. Bradykinin (10 or 30 nM) infused through the column of EC induced release of both EDRF and PGI2, neither of which was maintained for the duration of the infusion. 3. ADP (1.6 or 4 microM) infused through the column of EC induced release of a EDRF which was maintained for the duration of the infusion and a release of PGI2 which lasted for a much shorter period. 4. Arachidonic acid (30 or 90 microM) infused through the column of EC caused a sustained release of EDRF and PGI2, both of which outlasted the infusion of AA. 5. L-Arginine FB, D-arginine FB or alkaline buffer infused through the column of EC released EDRF, but only small amounts of PGI2. The release of EDRF outlasted the period of infusion and was due to an increase in the pH of the Krebs solution perfusing the EC. 6. Infusions of ACh (0.25-1 microM) or ADP (4-16 microM) caused a sustained release of EDRF from the luminally-perfused rabbit aorta, whereas infusion of substance P (3.3-10 microM) caused only a transient release of EDRF. 7. These results show that distinct patterns of EDRF release exist to different agonists in both cultured and in situ EC, and that EDRF and PGI2 do not necessarily follow the same time course of release. Furthermore, sustained release of EDRF does not require the constant infusion of the precursor, L-arginine, whereas sustained release of PGI2 only occurs when AA, the precursor of PGI2, is present in the extracellular medium.

Acetylcholine↗
PubMed1992

Human brain contains a metalloprotease that converts big endothelin-1 to endothelin-1 and is inhibited by phosphoramidon and EDTA.

Incubation of big endothelin-1 (bET-1) with protein derived from the detergent-extracted 100,000 g pellet prepared from human brain tissue resulted in the formation of endothelin-1 (ET-1) at a rate of 90 fmol mg-1 protein min-1. This formation was inhibited in a concentration-dependent manner by either phosphoramidon or EDTA, with half-maximal inhibitory concentrations of 2 and 20 microM, respectively. No conversion of big endothelin-3 (bET-3) to endothelin-3 (ET-3) was detected under the same conditions. These results show the presence in the human brain of a metalloprotease-like enzymatic activity which selectively converts bET-1 and ET-1. Together with earlier reports of mRNA for ET-1 this suggests the presence of the entire synthetic pathway for ET-1 in human brain.

Brain↗
PubMed1992

Regional differences in endothelin converting enzyme activity in rat brain: inhibition by phosphoramidon and EDTA.

1. It has been demonstrated previously that conversion of big endothelin-1 (bET-1) to endothelin-1 (ET-1) is inhibited in vitro and in vivo by phosphoramidon. In addition, ET-1 binding sites and mRNA have been shown within the brain. Here we expand upon our previous observation that rat brain contains phosphoramidon-inhibitable endothelin converting enzyme (ECE) and show that this activity is not uniformly distributed throughout the brain. 2. ECE activity was detected by a bioassay which depended upon the 10,000 fold difference in potency between bET-1 and ET-1 as stimulants of guanosine 3':5'-cyclic monophosphate (cyclic GMP) accumulation in kidney epithelial (PK1) cells of the pig. Data were confirmed by specific enzyme-linked immunosorbent assay (ELISA) employing antibody directed against ET-1/3(17-21). 3. Following homogenization of the whole brain and ultracentrifugation the 100,000 g pellet contained greater than 4 times more ECE activity than the cytosol. Washing of the pellet with KCl (1 M) and extraction with the detergent CHAPS (20 mM) revealed a phosphoramidon-inhibitable ECE within the residual particulate fraction (nominally classified as the cytoskeletal fraction). Phosphoramidon (IC50, approx. 5 microM) or EDTA inhibited the conversion of bET-1 to ET-1 by the cytoskeletal fraction of rat brain by more than 60%.2+ 4. Following dissection of rat brain into olfactory bulb, cerebral cortex, striatum, hippocampus, cerebellum, midbrain (including thalamus), hypothalamus and medulla oblongata (including pons) the greatest ECE was detected in the hypothalamus and medulla oblongata.After fractionation, the ECE-activities in the cytoskeletal fractions prepared from the hypothalamus or medulla oblongata were inhibited concentration-dependently by phosphoramidon or EDTA, with maximum inhibitions of>80% and >70%, respectively.5. These data show that rat brain contains a phosphoramidon- and EDTA-inhibitable ECE which maybe similar to that present in endothelial cells. The localization of this enzyme correlates with published reports of immunoreactive-ET-l, ET-1-binding sites, and messenger RNA for ET-1 in the rat brain, and suggests the presence of the entire synthetic pathway for ET-1.

Animals↗
PubMed1992

Nitric oxide synthase in ferret brain: localization and characterization.

1. In the present study, we have investigated the distribution of nitric oxide synthase in the ferret brain. Nitric oxide synthase was determined biochemically and immunochemically. 2. In the rat brain, the highest nitric oxide synthase activity has been detected in the cerebellum. However, in the ferret brain, the highest activity was found in the striatum and the lowest in the cerebellum and cerebral cortex. The enzymatic activity was localized predominantly in the cytosolic fractions, it was dependent on NADPH and Ca2+, and inhibited by NG-nitro-L-arginine or NG-methyl-L-arginine. 3. Western blot analysis revealed that all regions of the ferret brain contained a 160 kD protein crossreacting with an antibody to nitric oxide synthase purified from the rat cerebellum, and the levels of relative intensity of staining by the antibody correlated with the distribution of nitric oxide synthase activity. 4. These results indicate that the ferret brain contains a nitric oxide synthase similar to the rat brain, but the distribution of enzymatic activity in the ferret brain differs markedly from the rat brain.

Amino Acid Oxidoreductases↗
PubMed1992

Interactions of endothelins and EDRF in bovine native endothelial cells: selective effects of endothelin-3.

The tone of vascular smooth muscle is influenced by factors released from the endothelium, including endothelin (ET)-1 and endothelium-derived relaxing factor (EDRF). To better understand the interactions between these two mediators, we examined the release of both immunoreactive ET-1 (ir-ET-1) and EDRF from bovine aortic intact endothelium. Bovine aortas were opened longitudinally, washed, and clamped with the endothelium uppermost between two plates. The upper plate contained six openings forming identical and independent wells of endothelial cell monolayer. In experiments examining the release of EDRF, measured as accumulated NO2- and NO3- (NO chi -), we found that ET-3, calcium ionophore A23187 (A23187), acetylcholine (ACh), or ADP caused significant increase in NO chi- release, whereas ET-1 did not. These were significantly reduced in the presence of the EDRF/NO synthase inhibitor, NG-methyl-L-arginine (L-NMA). In a parallel series of experiments measuring EDRF release by stimulation of guanosine 3',5'-cyclic monophosphate (cGMP) accumulation in rat fetal lung (RFL)-6 cells, ET-3 but not ET-1 was also found to be active as a releaser of EDRF. A23187 caused an increase of ir-ET-1 release, whereas ACh, ADP, or the NO-containing compound sodium nitroprusside decreased the release of ir-ET-1. The depression in ir-ET-1 release in the presence of ACh or ADP was not seen when the endothelium was treated with L-NMA. When the cells were pretreated with 8-bromoguanosine 3',5'-cyclic monophosphate (8-bromo-cGMP), the release of ir-ET-1 in response to A23187 was significantly depressed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗