Inhibition of platelet aggregation ex vivo is repressed in apolipoprotein E deficient mice.

In the present study, we assessed whether the endogenous platelet inhibitory mechanisms are altered in the early to moderate stages of the atherosclerotic process. Apolipoprotein E deficient mice (ApoE-/-), a mouse model of atherosclerosis, and their wild-type (WT) counterparts were used to assess agonist-stimulated synthesis of prostacyclin (PGI2), inhibition of platelet aggregation ex vivo, and intra-platelet cAMP levels. Basal U46619 and ADP -induced platelet aggregation in vitro were increased in ApoE-/- mice at 18-20 weeks in comparison with 8-10 weeks of age. Systemically administered endothelin-1 (ET-1) or bradykinin (BK) inhibited platelet aggregation in a similar fashion in 8- to 10-week-old ApoE-/- and WT mice, but not in the ApoE-/- mice at 18-20 weeks of age, although both peptides maintained their capacity to increase plasma levels of the PGI2. Intravenous infusion of PGI2 also failed to inhibit platelet aggregation ex vivo in 18- to 20-week-old ApoE-/- mice. Interestingly, both BK and PGI2 retained their ability to increase intraplatelet cAMP in WT and ApoE-/- mice. Our results suggest that a loss of activity of endogenous inhibitorymechanisms could contribute to the increased platelet reactivity in ApoE-/- mice, and that this phenomenon occurs early in the intermediate stage of the atherosclerotic process.

Endothelin-1: Biosynthesis, Signaling and Vasoreactivity.

Endothelin-1 (ET-1) is an extremely potent vasoconstrictor peptide originally isolated from endothelial cells. Its synthesis, mainly regulated at the gene transcription level, involves processing of a precursor by a furin-type proprotein convertase to an inactive intermediate, big ET-1. The latter peptide can then be cleaved directly by an endothelin-converting enzyme (ECE) into ET-1 or reach the active metabolite through a two-step process involving chymase hydrolyzing big ET-1 to ET-1 (1-31), itself needing conversion to ET-1 by neprilysin (NEP) to exert physiological activity. ET-1 signals through two G protein-coupled receptors, endothelin receptor A (ETA) and endothelin receptor B (ETB). Both receptors induce an increase in intracellular Ca(2+), mainly from the extracellular space through voltage-independent mechanisms, the receptor-operated channels and store-operated channels. ET-1 also induces signaling through epidermal growth factor receptor transactivation, oxidative stress induction, rho-kinase, and the activation (ETA) or inhibition (ETB) of the adenylate cyclase/cyclic adenosine monophosphate pathway. Arterial vasoconstriction is mediated mainly by the ETA receptor. ET-1, via endothelium-located ETB, relaxes arteries or constricts vessels following activation of the same receptor type on the smooth muscle, where it can interact with ETA. In addition, ETB-dependent vasoconstriction seems more prominent in the venous vasculature. A better understanding of how ET-1 is synthesized and how ETA and ETB receptors interact could help design better pharmacological agents in the treatment of cardiovascular diseases where targeting the ET-1 system is indicated.

High salt-induced hypertension in B2 knockout mice is corrected by the ETA antagonist, A127722.

BACKGROUND AND PURPOSE: The contribution of endothelin-1 (ET-1) in a B2KO mouse model of a high salt-induced arterial hypertension was investigated. EXPERIMENTAL APPROACH: Wild-type (WT) or B2KO mice receiving a normal diet (ND) or a high-salt diet (HSD) were monitored by radiotelemetry up to a maximum of 18 weeks. At the 12th week of diet, subgroups under ND or HSD received by gavage the ETA antagonist A127722 during 5 days. In addition, blood samples were collected and, following euthanasia, the lungs, heart and kidneys were extracted, homogenized and assayed for ET-1 by RIA. In a separate series of experiments, the ETA antagonist, BQ123 was tested against the pressor responses to a NOS inhibitor L-N(G)-nitroarginine methyl ester (L-NAME) in anaesthetized WT and B2KO mice. KEY RESULTS: In B2KO, but not WT mice, 12 weeks of HSD triggered a maximal increase of the mean arterial pressure (MAP) of 19.1 ± 2.8 mmHg, which was corrected by A127722 to MAP levels found in B2KO mice under ND. Significant increases in immunoreactive ET-1 were detected only in the lungs of B2KO mice under HSD. On the other hand, metabolic studies showed that sodium urinary excretion was markedly reduced in B2KO compared with WT mice under ND. Finally, BQ123 (2 mg·kg(-1)) reduced by 50% the pressor response to L-NAME (2 mg·kg(-1)) in B2KO, but not WT mice under anaesthesia. CONCLUSIONS AND IMPLICATIONS: Our results support the concept that functional B2 receptors oppose high salt-induced increments in MAP, which are corrected by an ETA receptor antagonist in this mouse model of experimental hypertension.

Endothelin-1 (1-31): from chymase-dependent synthesis to cardiovascular pathologies.

The mast cell-derived serine protease chymase is importantly involved not only in degradation, but in synthesis of bioactive peptides as well. Several studies suggest that chymase is the predominant enzyme in the production of angiotensin II (Ang II) from angiotensin-I in interstitial tissues. Interestingly, chymase has also been suggested to mature endothelin-1 (ET-1) from its precursor, big-ET-1 in vitro. The lack of availability of specific chymase inhibitors, beyond the chymotrypsin-like inhibitor chymostatin, currently hampers the investigation of the chymase/ET-1/Ang II paradigm in physiology and cardiovascular diseases. Nonetheless, the recent advent of highly selective chymase inhibitors is shedding new light on the role of this enzymatic pathway in the several inflammatory prone vascular diseases as summarized in the present review. Considering increasing evidence towards significant interactions between Ang II and ET-1 in cardiovascular diseases, the present review will address the role of chymase in the production of those two peptides. Whether chymase-dependent production of ET-1 plays an important role in cardiovascular pathologies will also be discussed.

Chronic ethanol intake modulates vascular levels of endothelin-1 receptor and enhances the pressor response to endothelin-1 in anaesthetized rats.

BACKGROUND AND PURPOSE: The contribution of endothelin-1 (ET-1) to vascular hyper-reactivity associated with chronic ethanol intake, a major risk factor in several cardiovascular diseases, remains to be investigated. EXPERIMENTAL APPROACH: The biphasic haemodynamic responses to ET-1 (0.01-0.1 nmol kg(-1), i.v.) or to the selective ETB agonist, IRL1620 (0.001-1.0 nmol kg(-1), i.v.), with or without ETA or ETB antagonists (BQ123 (c(DTrp-Dasp-Pro-Dval-Leu)) at 1 and 2.5 mg kg(-1) and BQ788 (N-cis-2,6-dimethyl-piperidinocarbonyl-L-gamma-methylleucyl1-D-1methoxycarbonyltryptophanyl-D-norleucine) at 0.25 mg kg(-1), respectively) were tested in anaesthetized rats, after 2 weeks' chronic ethanol treatment. Hepatic parameters and ET receptor protein levels were also determined. KEY RESULTS: The initial hypotensive responses to ET-1 or IRL1620 were unaffected by chronic ethanol intake, whereas the subsequent pressor effects induced by ET-1, but not by IRL1620, were potentiated. BQ123 at 2.5 but not 1 mg kg(-1) reduced the pressor responses to ET-1 in ethanol-treated rats. Conversely, BQ788 (0.25 mg kg(-1)) potentiated ET-1-induced increases in mean arterial blood pressure in control as well as in ethanol-treated rats. Interestingly, in the latter group, increases in heart rate, induced by ET-1 at a dose of 0.025 mg kg(-1) were enhanced following ETB receptor blockade. Finally, we observed higher levels of ETA receptor in the heart and mesenteric artery and a reduction of ETB receptor protein levels in the aorta and kidney from rats chronically treated with ethanol. CONCLUSIONS AND IMPLICATIONS: Increased vascular reactivity to ET-1 and altered protein levels of ETA and ETB receptors could play a role in the pathogenesis of cardiovascular complications associated with chronic ethanol consumption.

Characterization of the non-adrenergic/non-cholinergic response to perivascular nerve stimulation in the double-perfused mesenteric bed of the mouse.

BACKGROUND AND PURPOSE: Calcitonin gene-related peptide (CGRP), a capsaicin-sensitive neuromodulator of splanchnic vascular tone in several animal species, remains poorly investigated in mouse models. We therefore assessed whether endogenous CGRP is a non-adrenergic/non-cholinergic (NANC) neuromodulator in the mesenteric vascular bed of the mouse. EXPERIMENTAL APPROACH: Arterial and venous changes in perfusion pressure in response to perivascular nerve stimulation (PNS) were monitored in the mouse mesenteric bed under basal conditions or precontracted with KCl (artery) or U46619 (vein) in circuits pretreated with guanethidine, atropine, indomethacin and prazosin. Arterial responses to NANC were also characterized with a CGRP1 antagonist, halphaCGRP8-37. Finally, the PNS-induced release of arterial CGRP was measured by enzyme immunoassay. KEY RESULTS: HalphaCGRP8-37 enhanced PNS-induced arterial increases in perfusion pressure under basal tone. PNS-induced stimulation of NANC triggered an halphaCGRP8-37 or capsaicin- sensitive reduction in perfusion pressure of the pre-contracted arterial bed only. Chemical removal of the endothelium inhibited PNS- and halphaCGRP- induced reduction in perfusion pressure in the arterial mesenteric bed. Responses to NANC nerves were reduced by guanylate and adenylate cyclase inhibitors (1H-[1,2,4]oxadiazole[4,3-a] quinoxalin-1-one (ODQ)) and [9-(tetrahydro-2-furanyl)-9H-purin-6-amine] (SQ 22,536), respectively. A neuronal NOS inhibitor (7-nitroindazole; 7-NI) also enhanced the response to NANC in vessels from wild-type, eNOS KO but not iNOS KO mice. Finally, PNS enhanced the release of immunoreactive CGRP from the perfused arterial mesenteric bed. CONCLUSIONS AND IMPLICATIONS: Our study demonstrates a role for CGRP in the NANC-dependent reduction in perfusion pressure of the arterial but not venous mesenteric bed of the mouse.

Association of endothelin with lung hemorrhage induced by immune complexes in rats.

The participation of endothelins (ETs) in a model of neutrophil-dependent lung injury induced by intrabronchial instillation of rabbit antibodies to ovalbumin followed by i.v. injection of the antigens (Arthus reaction) was investigated. Hemorrhagic lesions were evaluated by measuring the extravasations of hemoglobin in lung parenchyma. From 5 min to 24 h after the Arthus reaction (AR), endothelin (ir-ET) levels in bronchoalveolar lavage fluid (BALF) and in plasma were measured by radioimmunoassay. BALF levels of ir-ET were not different between control and AR animals for the first 90 min after the antigen challenge but increased from 2 to 24 h after induction of AR. ET levels in the plasma did not change from the respective controls over the same 24 h period. Increased ir-ET in BALF was not affected by pretreatment with L-NAME (30 mg/kg, i.v.). A PAF antagonist (BN52021; 5 and 10 mg/kg, i.v.) increased ET content in BALF and decreased the intensity of the AR. Thiorphan (2 mg/kg, i.v.) inhibited the AR-induced hemorrhagic lesions in lungs. An ET(A) receptor antagonist, BQ-123 (1 mg/kg, i.v.) potentiated, whereas the ET(B) antagonist, BQ-788 (1 mg/kg, i.v.) inhibited the lung hemorrhage. It is concluded that ETs are released during and play a role in the lung AR.

Synthesis and degradation of endothelin-1.

The endothelin-converting enzyme (ECE) is the main enzyme responsible for the genesis of the potent pressor peptide endothelin-1 (ET-1). It is suggested that the ECE is pivotal in the genesis of ET-1, considering that the knockout of both genes generates the same lethal developments during the embryonic stage. Several isoforms of the ECE have been disclosed, namely ECE-1, ECE-2, and ECE-3. Within each of the first two groups, several sub-isoforms derived through splicing of single genes have also been identified. In this review, the characteristics of each sub-isoform for ECE-1 and 2 will be discussed. It is important to mention that the ECE is, however, not the sole enzyme involved in the genesis of endothelins. Indeed, other moieties, such as chymase and matrix metalloproteinase II, have been suggested to be involved in the production of ET intermediates, such as ET-1 (1-31) and ET-1 (1-32), respectively. Other enzymes, such as the neutral endopeptidase 24-11, is curiously not only involved in the degradation and inactivation of ET-1, but is also responsible for the final production of the peptide via the hydrolysis of ET-1 (1-31). In this review, we will attempt to summarize, through the above-mentioned characteristics, the current wisdom on the role of these different enzymes in the genesis and termination of effect of the most potent pressor peptide reported to date.

Characterization of the NTPDase activities in the mesentery pre- and post-capillary circuits of the guinea pig.

NTPDase is one of the principal enzymes involved in the sequential hydrolysis of ATP. In the present study, the presence and functionality of NTPDase in the mesenteric vein and artery were examined. Adenosine triphosphate (ATP) (0.01-1000 pmol) induces a dose-dependent vasodilation in the isolated arterial and venous mesenteric vasculatures of the guinea pig. Adenosine diphosphate (ADP) (0.01-1000 pmol) but not adenosine monophosphate (AMP) (0.01-1000 pmol) induces a similar response in the mesenteric vascular circuit. L-NAME, a nitric oxide synthase inhibitor (200 microM, 30 min), significantly reduces the arterial dilatory effect of ATP and abolishes the responses to ADP and AMP. Complete removal of the endothelium with 3-[(3-cholamidopropyl) dimethylammonio]-1-propansulfonate (CHAPS) (20 mM, 2 x 45 s) abolishes ATP-induced responses. Infusion of ATP in the vascular circuit generated detectable amounts of ADP and AMP, as measured by HPLC. CHAPS treatment significantly reduced the level of ATP and the production of AMP in the arterial mesenteric circuit. In contrast to the arterial mesenteric vasculature, endothelium removal in the venous circuit triggered a marked potentiation of ADP release and, interestingly, a marked reduction in the release of AMP. Moreover, a specific inhibitor of NTP diphosphohydrolase, 1-hydroxynaphthlene-3,6-disulfonic acid BGO 136 (10 mM for 20 min), significatively reduced AMP production in both vascular preparations. These results confirm that the endothelium contributes to the vasoactive properties of ATP, ADP, and AMP. Our data also demonstrated a significant role of endothelium in NTPDase activity on ADP and AMP production prior to exogenous administration of ATP. The activity of this particular enzyme appears to be different from the reaction products viewpoint (i.e., the production of ADP) in the pre- and post-mesenteric circuits, suggesting two different isoforms with different substrate specificities.

Angiotensin II AT1 receptor internalization, translocation and de novo synthesis modulate cytosolic and nuclear calcium in human vascular smooth muscle cells.

The present study was designed to verify if human (h) Angiotensin II (Ang II) type-1 receptor (hAT1R) undergoes internalization, nuclear translocation, and de novo synthesis in primary culture of human aortic vascular smooth muscle cells (hVSMCs) and if overexpression of this receptor modulates sustained free cytosolic ([Ca]c) and nuclear ([Ca]n) calcium. 3-dimensional (3-D) confocal microscopy was used to monitor free intracellular Ca2+ and hAT1R-green fluorescence protein (GFP) fusion protein in cultured hVSMCs. Immunofluorescence studies showed the presence of hAT1R and the absence of hAT2R in normal hVSMCs. Using 3-D imaging technique, hAT1 receptors were localized at the sarcolemma and in the cytosolic and nuclear compartments. In native as well as in normal hAT1R or hAT1R-GFP overexpressing hVSMCs, Ang II (10(-9) and 10(-4) M) induced internalization and nuclear translocation of this type of receptor. The internalization of hAT1Rs is mediated via clathrin-coated pits and vesicles pathway. This phenomenon of trancellular trafficking of receptors was associated with an increase of hAT1R. The Ang II induced increase of hAT1R density was prevented by the protein synthesis inhibitor cycloheximide. Overexpression of hAT1R and hAT1R-GFP decreased both basal cytosolic and nuclear Ca2+. In normal hVSMCs and low hAT1R-GFP overexpressing hVSMCs, Ang II (10(-15) to 10(-4) M) induced a dose-dependent sustained increase of [Ca]c and [Ca]n with an EC50 near 5 x 10(-11) and 5 x 10(-9) M, respectively. Our results suggest that hAT1Rs are the predominant type of Ang II receptors in aortic hVSMCs and are present in the sarcolemma, the cytosolic and the nuclear compartments. Ang II rapidly induces hAT1R internalization, nuclear translocation, as well as nuclear de novo synthesis of this receptor. The hAT1R overexpression in hVSMCs modulates sustained [Ca]c and [Ca]n.

Function of the endothelin(B) receptor in cardiovascular physiology and pathophysiology.

One of the two receptors by which the potent vasoactive effects of endothelin (ET)-1 are mediated is the ET(B) receptor (ET(BR)), which is found in several tissues, but, more importantly from a cardiovascular point of view, on the endothelial cell. The endothelial cell also has the unique capability of releasing ET-1, as well as other factors, such as the endothelial-derived relaxing factors and prostacyclin, which counteract the myotropic effects of the peptide. The secretory and contractile responses to ET-1 rely on G-protein-coupled ET(BR)s, as well as ET(A)-G-protein-coupled receptor-like proteins. The mitogenic properties of ET-1 via ET(A) receptors (ET(AR)s) coupled to mitogen-activated protein kinases and tyrosine kinases on the vascular smooth muscle may occur in conjunction with the anti-apoptotic characteristics of the endothelial ET(BR)s. Interestingly, most of the relevant antagonists and agonists for both ET(AR)s and ET(BR)s have been developed by the pharmaceutical industry. This highlights the therapeutical potential of compounds that act on ET receptors. In normal as well as in physiopathological conditions, the ET(BR) plays an important role in the control of vascular tone, and must be taken into account when using ET receptor antagonists for the treatment of cardiovascular diseases. For the management of congestive heart failure, renal failure and primary pulmonary hypertension, the most recent literature supports the use of selective ET(AR) antagonists rather than mixed antagonists of ET(AR)s and ET(BR)s. Nonetheless, validation of this view will have to await the first clinical trials comparing the actions of ET(A) to mixed ET(A)/ET(B) receptor antagonists.

Role of ETB and B2 receptors in the ex vivo platelet inhibitory properties of endothelin and bradykinin in the mouse.

1. We have developed a model to study the inhibitory properties of endogenous autacoids triggered by systemically-administered vasoactive peptides, on platelet aggregation ex vivo in the mouse. 2. Adenosine diphosphate (ADP) (0.5-10 microM) induces a concentration-dependent aggregation of platelet-rich plasma derived from C57BL/6 mice. Intravenously-administered endothelin-1 (0.01-1 nmolx kg(-1)), the selective ETB agonist, IRL-1620 (0.0 -1 nmol x kg(-1)) or bradykinin ( 1-100 nmol x kg(-1)) significantly reduced in a dose-dependent fashion the ADP-induced platelet aggregation. 3. The non-selective cyclo-oxygenase (COX) inhibitor, indomethacin, a selective COX-2 inhibitor NS-398 or the prostacyclin synthase inhibitor, tranylcypromine (10 mg x kg(-1)), markedly reduced the inhibitory properties of endothelin-1, whereas only a combination of both indomethacin, NS-398 or tranylcypromine and L-NAME (10 mg x kg(-1)) were required to abolish the response to bradykinin. 4. An ETB-selective antagonist (BQ-788) or knockout of the B2 receptor gene (in B2 knockout mice) abolishes the platelet inhibitory properties of endothelin-1 and bradykinin, respectively. 5. Our results suggest that intravenously-administered endothelin-1 and bradykinin, through ETB and B2 receptor activation, respectively, inhibit platelet aggregation ex vivo in the mouse. The inhibitory properties of endothelin-1 require the activation of COX-2 and the subsequent generation of prostacyclin. In addition to the two previously mentioned factors, nitric oxide is required for the anti-aggregatory effects of bradykinin.

Characterization and immunohistochemical localization of nucleoside triphosphate diphosphohydrolase (NTPDase) in pig adrenal glands (presence of a non-sedimentable isoform).

Considering that adrenal glands possess a variety of purinoceptors associated with various cell types and that some of these cells (chromaffin cells) secrete large amounts of adenine nucleotides, it was of interest to localize nucleoside triphosphate diphosphohydrolase (NTPDase) in these glands and to define the biochemical characteristics of this ectonucleotidase. Immunolocalization produced a moderate reaction in capsula and medulla, with no signal in zona glomerulosa and zona reticularis. In contrast, a very strong reaction was found in zona fasciculata. Biochemical analysis of particulate fractions isolated from whole glands revealed high levels of ATPase and ADPase activities. This appeared to be attributable to the NTPDase since the level of ADPase was as high as ATPase. Both ATPase and ADPase activities were similarly inhibited by sodium azide. Additionally electrophoretograms with these two substrates showed comparable patterns. Western blots with 'Ringo', an antibody that recognizes the different isoforms of mammalian NTPDases, showed the presence of isoforms of NTPDases at 54 and 78 kDa, respectively. Interestingly, the 54 kDa isoform remains in the supernatant of a chromaffin granule lysate after ultracentrifugation. Up until now little interest has been given to the relationship between adrenal medulla and cortex. Presence of purinoceptors and ectonucleotidases in both these regions together with the effects of ATP in vivo and in vitro in different species indicate that purines play a significant role in adrenal glands.

Contribution of endogenous endothelin-1 and endothelin-A-receptors to the hypertensive state of endothelin-B heterozygous (+/-) knockout mice.

We observed that heterozygous knockout (+/-, KO) of either endothelin-A- (ET(A)) or -B- (ET(B)) receptors significantly reduced the pressor responses to systemically administered endothelin-1 (ET-1) in ET(A) or ET(B) (+/-) KO mice when compared to wild-type (WT) mice (data not shown). Also, we observed that basal mean arterial pressure (MAP) is significantly higher in ET(B) (+/-) (92.7 +/- 1.2 mmHg) (n = 53, p < 0.05) but not ET(A) (+/-) KO mice (70.6 +/- 1.8 mmHg) (n = 23) when compared to their anaesthetized WT littermates (70.1 +/- 0.7 mmHg) (n = 118). A 90 min treatment with either BQ-123 (10 mg/kg), an ET(A)-selective antagonist, or BQ-928 (10 mg/kg), a mixed ET(A)/ET(B) antagonist, administered intraperitoneally, significantly reduced basal MAP of ET(B) (+/-) KO mice almost to the level of their WT treated counterparts (94.9 +/- 4.9 mmHg) (n = 6) vs (+ BQ-123: 59.7 +/- 0.3 mmHg, n = 8); (+ BQ-928: 72.4 +/- 2.6 mmHg, n = 5). It is worthy of note that BQ-123 significantly reduced basal MAP in WT mice but to a lesser extent than in ET(B) (+/-) KO mice (69.6 +/- 2.3 mmHg, n = 8) vs (+ BQ-123: 57.3 +/- 1.4 mmHg, n = 8). In contrast, the ET(B)-selective antagonist, BQ-788 (10 mg/kg i.p.), had no significant effect on MAP even after 90 min of treatment (ET(B) (+/-) KO: (92.3 +/- 2.3 mmHg, n = 6) vs (+ BQ-788: 89.7 +/- 3.1 mmHg, n = 6); WT: (70.5 +/- 3.7 mmHg, n = 7) vs (+ BQ-788: 71.2 +/- 2.0 mmHg, n = 6). Therefore heterozygous KO of either ET(A)- or ET(B)-receptors significantly alters the phenotypic pressor properties of ET-1. We also suggest that there is less ET clearance in ET(B) (+/-) KO mice than in WT mice, which can explain the ET(A)-dependent hypertensive state of the former strain.

Nitric oxide relaxes the vascular smooth muscle independently of endothelin-1- and U46619-induced intracellular increase of calcium.

A balance between circulating and locally released vasoconstrictors, such as endothelin-1 (ET-1), and vasodilators, such as nitric oxide, controls vascular smooth muscle tone. In the study reported here, using the technique of simultaneous measurements of intracellular free calcium ([Ca2+]i) and tension, we investigated the effects of a nitric oxide donor, sodium nitroprusside (NaNP) on endothelin-1- and U46619- [a thromboxane angiotensin-II (TXA-II) mimetic] induced sustained increases in tension and [Ca2+]i in intact and endothelium-denuded rabbit thoracic aortas. Our results showed that, in both intact and endothelium-denuded preparations, the nitric oxide donor NaNP (10(-6) M) reverses the ET-1- (10(-7) M) and U46619- (10(-7) M) induced sustained increase in tension but not in [Ca2+]i. However, it did not reduce the ET-1- and U46619-induced responses. Our data suggest that nitric oxide production modulates vascular smooth muscle tension via a mechanism that is independent of that generated by vasoconstrictors such as ET-1 and TXA-II.

Endothelin-B-receptors-dependent-inhibition of platelet aggregation in the CD-1 mouse.

An experimental protocol of adenosine diphosphate-(ADP) induced platelet aggregation in the mouse was designed in order to study the roles played by endothelin-A (ET(A)) and endothelin-B (ET(B)) receptors in the ET-1-induced inhibition of ex vivo platelet aggregation. The pressor effects of ET-1 or IRL-1620 were firstly determined in vivo in anesthetized (ketamine/xylazine) CD-1 mice (males and females; 25-30 g). All agents were administered intravenously (via the jugular vein) and blood samples were collected from the carotid artery into heparinized Eppendorfs (15 U/ml). To obtain platelet-rich plasma (PRP) the blood was immediately centrifuged for 12 min at low speed (1100 g). Platelet-poor plasma (PPP) was then prepared by centrifugation of the whole blood sample at high speed (3700 g) for 30 min. PPP was used to calibrate the aggregometer at 100% transmission. Platelet aggregation was monitored ex vivo as a change in light transmission through PRP following the injection of ADP (5 microM). ET-1 (0.01-2.0 nmol/kg) induced a significant and dose-dependent inhibition of platelet aggregation ex vivo (12-84%). The selective ET(B) agonist, IRL-1620 (0.05-2.0 nmol/kg), also triggered a marked inhibition of platelet aggregation. Indomethacin (10 mg/kg), a nonselective cyclooxygenase inhibitor, abolished the inhibitory effect of ET-1. The selective ET(A) antagonist, BQ-123 (1 nmol/kg), abolished the in vivo pressor effect of exogenous ET-1, without affecting its anti-aggregatory activity. The selective ET(B) antagonist, BQ-788 (0.5 nmol/kg), did not modify the elevation of blood pressure produced by the ET-1; however, it did abrogate dose-dependently the inhibitory effect of ET-1 on platelet aggregation. These results suggest that the anti-aggregatory effect of ET-1, in anesthetized CD-1 mice, relies mainly upon the activation of ET(B) receptors. The mechanism whereby ET-1 exerts this effect, is partially indirect and requires at least the production and the release of prostanoids (possibly PGI2) into the blood stream.

Endothelin-1 induces an endothelin-A-receptor-dependent and L-NAME-sensitive vasoconstriction in the isolated perfused guinea-pig kidney.

We have previously reported a significant contribution of ET(A)- and ET(B)-receptors in the eicosanoid-releasing properties of endothelin-1 (ET-1) in the guinea-pig perfused lung. More recently, it was found that intravenously administered ET-1 in the guinea-pig triggers the release of hypotensive and bronchoconstrictive eicosanoids which is strongly modulated by endogenous nitric oxide. In this study we investigate the contribution of both ET receptors as well as of the eicosanoids and nitric oxide to the vasoconstrictor properties of ET-1 in the guinea-pig kidney. The kidney was isolated from stunned and spinal cord-sectioned guinea-pig (300-350 g) and rapidly suspended and perfused (3 ml/min) with oxygenated Krebs solution at 37 degrees C. ET-1 (1-1000 pmol) induced dose-dependent increases in perfusion pressure in the perfused guinea-pig kidney. Furthermore, the pressor effect of a dose of ET-1 (40 pmol) was markedly reduced after a 15-min infusion of the ET(A) antagonist, BQ-123 (1 microM). The ET-1-induced vasoconstriction was restored 50 min after the cessation of BQ-123 infusion. In contrast, the selective ET(B)-receptor agonist, IRL-1620 (1-1000 pmol) was found to be inactive. Indomethacin (5 microM) and the ET(B)-receptor antagonist, BQ-788 (10 nM) did not affect the constrictor effect of ET-1. The infusion of ET-1 (0.5 and 1 microM, 5 min) in the guinea-pig kidney did not induce the release of prostacyclin or thromboxane. Finally, a 60 min infusion with the nitric oxide synthase inhibitor, L-NAME (200 microM) markedly potentiated the constrictor effects of ET-1. Our results suggest that unlike the pulmonary vasculature, the guinea-pig kidney responds to ET-1 by a direct constriction, which is modulated by nitric oxide but not eicosanoids (supported by the MRCC).

Endothelin-1 regulates cytosolic and nuclear Ca2+ in human endocardial endothelium.

Endocardial endothelium is far less studied than its systemic and pulmonary counterparts and its role in cardiac function is not well known. In the study reported here, we succeeded in isolating and culturing endocardial endothelial cells (EECs) from 21-week-old human fetal heart and verified whether endothelin-1 (ET-1) receptors are present on these cells and whether their activation regulates cytosolic ([Ca]c) and nuclear calcium ([Ca]n). Using fluo-3 Ca2+ measurement and three-dimension confocal microscopy techniques, superfusion of fetal human EECs from right and left ventricles with increasing concentrations of ET-1 induced a dose-dependent sustained increase of free cytosolic and nuclear Ca2+ levels with an EC50 near 10(-10) M and 10(-11) M, respectively. The sustained increase of cytosolic and nuclear Ca2+ by ET-1 in both EEC preparations was completely blocked by the calcium chelator ethylene glycol-bis (beta-aminoethylether)-N,N,N',N'-tetra-acetic acid (EGTA) but was insensitive to the L-type Ca2+ channel blocker, nifedipine. The effect of ET-1 was prevented by the ET(A)-receptor antagonist BQ 123. However the ET(B)-receptor antagonist BQ 788 had no effect. Our results suggest that ET-1 receptors are present in human EECs and that their stimulation induces sustained Ca2+ influx through ET(A)-receptor stimulation of a nifedipine-insensitive Ca2+ channel, probably the R-type Ca2+ channel. The presence of Ca2+-dependent responses to ET-1 supports a possible modulatory role of the endocardial endothelium in myocardial functions.

Presence of functional endothelin-1 receptors in nuclear membranes of human aortic vascular smooth muscle cells.

Our previous work showed that the nucleus plays a role in excitation-contraction coupling and that the channels and receptors could be present at the nuclear membrane. In the study reported here, the objective was to test the hypothesis that endothelin-1 (ET-1) receptors are functional at the level of the nuclear membranes and that their stimulation importantly regulates free nucleoplasmic Ca2+ level. Using a Fluo-3 Ca2+ measurement technique in human vascular smooth muscle cells (HVSMC), superfusion with increasing concentrations of extracellular ET-1 induced a dose-dependent sustained increase of free cytosolic ([Ca]c), nuclear ([Ca]n) Ca2+ and contraction with an EC50 near 3 x 10(-10) M. Like the extracellular ET-1, the cytosolic application of ET-1 using the perforated sarcolemma membrane technique, induced a dose-dependent increase of nuclear free calcium of HVSMC with an EC50 of 2 x 10(-11) M. These results strongly suggest that ET-1 receptors are functional at the level of the nuclear membranes. Furthermore, the sensitivity of ET-1 receptors at the nuclear membrane level seems to be higher than that of the receptors at the sarcolemma membrane. Finally, our results suggest that cytosolic ET-1 may play a role in preventing HVSMC nuclear calcium overload, thus protecting the cells from apoptosis.