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.

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.

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.

Cytosolic free Ca2+ concentration in canine aortic endothelial cells lining the polyester arterial prosthesis.

A rise in baseline cytosolic free Ca2+ in canine vascular endothelial-like cells (VEC) lining the luminal surface of the polyester arterial prosthesis is described. In one, three and six month implantation experiments we employed six adult mongrel dogs, polyester arterial prostheses Arteknit Ra K, fluorescent Ca2+ indicator Fura-2 and digital imaging microscopy to study cytosolic free Ca2+ in cultured VEC. The electron microscopy scanning of the luminal surface in different regions of the graft were also performed. A rise in cytosolic free Ca2+ in the VEC lining the luminal surface of the prosthesis is probably the result of the immunologic reaction and mechanical stress which stimulate the proliferation activity of the endothelial cells. It seems that the baseline cytosolic free Ca2+ reflects the course of the endothelization process on the polyester arterial prosthesis.

Characterization of the prostaglandin receptors in human osteoblasts in culture.

Prostaglandins have complex actions on bone metabolism that depend on interactions with different types and subtypes of receptors. Our objective was to characterize the prostaglandins receptors present in primary cultures of human osteoblasts. RT-PCR analysis revealed the presence of DP, EP(4), IP, FP and TP receptor mRNA in primary cultures of human osteoblasts. FP receptor mRNA was detected only after 3 weeks of confluency, all the others were detected at every culture time tested. To verify the functionality of these receptors we challenged the cells with the prostanoids and synthetic analogues and determined the intracellular levels of cAMP. All receptors found by RT-PCR were coupled to second messengers except for the DP subtype. These results clearly show the presence of functional EP(4), IP, FP and TP receptors in human osteoblasts in culture.

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.

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.

Contribution of B(2) receptors for bradykinin in arthus reaction-induced plasma extravasation in wild-type or B(2) transgenic knockout mice.

The aim of the present study was to investigate the contribution of bradykinin (BK) B(1) and B(2) receptors in a model of type III hypersensitivity, the reverse passive Arthus reaction (RPA), in wild-type mice and transgenic B(2) knockout littermates. BK (10 microg mouse(-1)) or bovine serum albumin (0.5 mg mouse(-1)) induced a sustained Evans blue extravasation for more than 80 min in naive or rabbit anti-bovine serum albumin-treated mice (RPA model), respectively. The response to the two stimuli was prevented by the B(2) receptor antagonist, HOE-140, but not by [Leu(8)]desArg(9)-BK (B(1) receptor antagonist). In contrast to the wild-type littermates, RPA and bradykinin were unable to trigger an increase in plasma extravasation in B(2) knockout mice. Furthermore, endothelin-1 (5 microg mouse(-1)) and a selective NK-1 receptor agonist [Sar(9),Met (O(2))(11)]-SP (20 microg mouse(-1)), triggered a significant increase in peritoneal plasma extravasation in both wild-type and B(2) knockout animals. A pretreatment with indomethacin (200 microg mouse(-1)) significantly reduced the RPA-induced but not the BK-induced increase in Evans blue extravasation. Furthermore, RPA, but not BK, triggered a significant indomethacin-sensitive increase in peritoneal prostaglandin E(2) content. Our results suggest a pivotal role for B(2) receptors in the mechanism of plasma extravasation which occurs during the reverse passive Arthus reaction in the mouse. Moreover, our results suggest an important contribution of prostanoids in the plasma leakage mechanisms triggered by RPA but not by bradykinin.

ET(B) receptor blockade potentiates the pressor response to big endothelin-1 but not big endothelin-2 in the anesthetized rabbit.

The precursor of endothelin-1, big endothelin-1, is considered to be a more reliable marker of systemic production of vasoactive peptide. However, it is largely unclear whether ET(B) receptor-dependent clearance and endothelium-derived relaxing factors affect the precursor in a similar manner to mature ET-1. These ET(B)-dependent modulations of big ET-1 and big ET-2 pressor properties were therefore studied in the anesthetized rabbit. When injected into the left cardiac ventricle, ET-1 and ET-2 (0.01 to 1 nmol/kg) each induced biphasic responses (a depressor followed by a pressor response), whereas big ET-1 and big ET-2 (0.1 to 3 nmol/kg) caused only protracted pressor responses. The highest dose of big ET-1 caused significantly greater responses than ET-1, ET-2, or big ET-2. A selective ET(A) receptor antagonist, BQ-123 (1 mg/kg), markedly reduced pressor responses to all 4 peptides, whereas blockade of ET(B) receptors with BQ-788 (0.25 mg/kg) sharply potentiated the responses to ET-1, ET-2, and big ET-1, but not to big ET-2. Indomethacin (10 mg/kg) sharply potentiated the pressor response to ET-1 (1 nmol/kg), but not big ET-1, at all time points. In control animals, ET-1, but not big ET-1, also triggered an indomethacin-sensitive increase in circulating prostacyclin. Finally, systemically administered big ET-1, but not big ET-2, induced a phosphoramidon-sensitive increase in plasma IR-ET. Our results suggest a significant limiting role of ET(B) receptors on pressor responses to big ET-1. In contrast, the same receptor entities do not modulate the hemodynamic properties of the ET-2 precursor, given that, unlike big ET-1, it is poorly converted in the pulmonary or systemic circulation in anesthetized rabbits.

Taurine indirectly increases [Ca]i by inducing Ca2+ influx through the Na(+)-Ca2+ exchanger.

Recent studies in heart cells have shown taurine to induce a sustained increase of both intracellular Ca2+ and Na+. These results led us to believe that the increase in Na+ by taurine could be due to Na+ entry through the taurine-Na+ cotransporter which in turn favours transarcolemmal Ca2+ influx through Na(+)-Ca2+ exchange. Therefore, we investigated the effect of beta-alanine, a blocker of the taurine-Na+ cotransporter and low concentrations of CBDMB (a pyrazine derivative, 5-(N-4chlorobenzyl)-2',4'-dimethylbenzamil), a Na(+)-Ca2+ exchanger blocker on taurine-induced [Ca]i increase in embryonic chick heart cells. Using Fura-2 Ca2+ imaging and Fluo-3Ca2+ confocal microscopy techniques, taurine (20 mM) as expected, induced a sustained increase in [Ca]i at both the cytosolic and the nuclear levels. Preexposure to 500 microM of the blocker of the taurine-Na+ cotransporter, beta-alanine, prevented the amino acid-induced increase of total [Ca]i. On the other hand, application of beta-alanine did not reverse the action of taurine on total [Ca]i. However, low concentrations of the Na(+)-Ca2+ exchanger blocker, CBDMB, reversed the taurine-induced sustained increase of cytosolic and nuclear free calcium (in presence or absence of beta-alanine). Thus, the effect of taurine on [Ca]i in heart cells appears to be due to Na+ entry through the taurine-Na+ cotransporter which in turn favours transarcolemmal Ca2+ influx through the Na(+)-Ca2+ exchanger.

Endothelin-1 and insulin activate the steady-state voltage dependent R-type Ca2+ channel in aortic smooth muscle cells via a pertussis toxin and cholera toxin sensitive G-protein.

In single rabbit aortic smooth muscle cells, and at a concentration known to induce a maximum sustained increase of intracellular Ca2+ via activation of the steady-state voltage dependent R-type Ca2+ channels, endothelin-1 (10(-7) M) and insulin (80 microU/ml) were found to induce a sustained increase in cytosolic free Ca2+ ([Ca]i) levels that was significantly attenuated by pre-treatment with either pertussis toxin (PTX), cholera toxin (CTX) or removal of extracellular Ca2+. However, both PTX and CTX failed to inhibit the sustained depolarization-evoked sustained Ca2+ influx and [Ca]i elevation via activation of the R-type Ca2+ channels. Moreover, ET-1 and insulin-evoked sustained increases in Ca2+ influx were not attenuated by the selective PKC inhibitor, bisindolylmaleimide (BIS), or the specific L-type Ca2+ channel blocker, nifedipine, but were completely reversed by the R-type Ca2+ channel blocker, (-) PN 200-110 (isradipine). These data suggest that both insulin and ET-1 activate the nifedipine-insensitive but isradipine-sensitive steady state voltage dependent R-type Ca2+ channels present on rabbit VSMCs and these channels are directly coupled to PTX and CTX sensitive G protein(s).

Increases of T-type Ca2+ current in heart cells of the cardiomyopathic hamster.

In the present study, the whole-cell voltage clamp technique was used in order to record the T- and L-type Ca2+ currents in single heart cells of newborn and young normal and hereditary cardiomyopathic hamsters. Our results showed that the I/V relationship curve as well as the kinetics of the L-type Ca2+ currents (ICa(L)) in both normal and cardiomyopathic heart cells were the same. However, the proportion of myocytes from normal heart hamster that showed L-type ICa was less than that of heart cells from cardiomyopathic hamster. The I/V relationship curve of the T-type ICa (ICa(T)) was the same in myocytes of both normal and cardiomyopathic hamsters. The main differences between ICa(T) of cardiomyopathic and normal hamster are a larger window current and the proportion of ventricular myocytes that showed this type of current in cardiomyopathic hamster. The high density of ICa(T) as well as the large window current and proportion of myocytes showing ICa(T) may explain in part Ca2+ overload observed in cardiomyopathic heart cells of the hamster.

Early fetal like slow Na+ current in heart cells of cardiomyopathic hamster.

Using the whole-cell voltage-clamp technique, early embryonic tetrodotoxin (TTX) and Mn(2+)-insensitive slow Na+ current was detected in 10-22 week old fetal human heart cells as well as in 1 day old and young cardiomyopathic hamster myocytes. This slow Na+ current in both heart cell preparations has the same kinetics and pharmacology. This type of slow Na+ current was absent in heart cells of newborn and young normal hamsters and became less present in myocytes of 19 and 22 week old human heart myocytes. Our results demonstrate that the slow Na+ channel does exist in early fetal human life and this type of channel continues to be functional after birth in myocytes of the hereditary cardiomyopathic hamster.