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Objective: Despite the increasing incidence of right ventricular (RV) failure in adult patients with congenital heart disease, current therapeutic options are still limited. By contrast to left-heart diseases, cell-based myocardial regeneration applied to the right ventricle is poorly studied, even though it may be a therapeutic solution. As human embryonic stem cell-derived cardiac progenitors seem to be good candidates owing to their proliferation capacity, our aim was to assess, in a large animal model of overloaded RV dysfunction, the feasibility and effects of such a cell therapy., Methods: Human MesP1(+)/SSEA-1(+) cardiogenic mesodermal cells were administered using multiple intramyocardial injections 4 months after a surgical procedure mimicking the repaired tetralogy of Fallot, and their effects were observed 3 months later on hemodynamic, rhythmic, and histologic parameters., Results: All pigs (sham n = 6, treated n = 6) survived without complication, and cell therapy was clinically well tolerated. Although functional, contractility, and energetics parameters evolved similarly in both groups, benefits regarding arrhythmic susceptibility were observed in the treated group, associated with a significant decrease of peri-myocyte fibrosis (5.71% ± 2.49% vs 12.12% ± 1.85%; P < .01) without interstitial fibrosis change (5.18% ± 0.81% vs 5.49% ± 1.01%). Such a decrease could be related to paracrine effects, as no human cells could be detected within the myocardium., Conclusions: Cell therapy using intramyocardial injections of human MesP1(+)/SSEA-1(+) cardiogenic mesodermal cells seems to have benefits regarding overloaded RV tissue remodeling and arrhythmic susceptibility, but this mode of administration is not sufficient to obtain a significant improvement in RV function., (Copyright © 2015 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

Cardiac dysfunction is often associated with an increase in the activity of the renin-angiotensin II-aldosterone system (RAAS). Here, we highlight the cross-talk between the Ca(2+) signalling generated by cardiac T-type current (I(CaT)) and RAAS signalling. Neonatal rat cardiomyocytes exposed to aldosterone, angiotensin II or aldosterone plus angiotensin II co-treatment (AA) show an increase in I(CaT) density, with no cumulative effect of the AA co-treatment. AA increases the amount of T-type channel Ca(v)3.1 mRNA in a time-dependent manner. Angiotensin II increases Ca(v)3.1 mRNA stability, whereas aldosterone increases the transcriptional activity of the Ca(v)3.1 gene promoter. However, in AA-treated cells, angiotensin II decreases aldosterone-induced promoter activity, and aldosterone decreases angiotensin II-induced mRNA stability. The mitogen-activated protein kinase kinase (MEK1/2), which is synergically phosphorylated in AA-treated cells, alters the translocation of glucocorticoid receptors (GR) into the nucleus and attenuates aldosterone-induced promoter activity. In contrast, MEK1/2 has no effect on the NFkB-induced increase in Ca(v)3.1 mRNA and MEK1/2 promoted CREB-target gene transcription. Aldosterone and AA-induced I(CaT) signalling result in a time-dependent activation of the phosphatase PP2A, which dephosphorylates MEK1/2 and CREB. Finally, angiotensin II alone also activates PP2A, which targets MEK1/2, but this activation is independent of I(CaT) calcium signalling and has no effect on CREB phosphorylation. In conclusion, our data demonstrate the cross-talk between a GR-mediated aldosterone response, angiotensin II and the I(CaT) signalling pathways and identify MEK1/2 as a point of connection. This cross-talk results in the fine control of GR- and/or CREB-target gene expression., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Haematopoietic c-kit+ progenitor cells may contribute to pulmonary vascular remodelling and pulmonary hypertension (PH). Stromal derived factor-1 (SDF-1/CXCL12) and its receptors CXCR4 and CXCR7 have been shown to be critical for homing and mobilisation of haematopoietic c-kit+ progenitor cells in the perivascular niche. We administered AMD3100, a CXCR4 antagonist, and CCX771, a CXCR7 antagonist, to chronic hypoxia exposed mice in order to study the role of c-kit+ progenitor cells in PH. CXCL12, CXCR4 and CXCR7 protein expression, haemodynamic parameters, right ventricular mass, extent of vascular remodelling and perivascular progenitor cell accumulation were studied. Chronic hypoxia-exposed mice showed increased total lung tissue expression of CXCR4, CXCR7 and CXCL12 after development of PH. This was associated with significantly increased right ventricular systolic pressure and evidence of right ventricular hypertrophy, vascular remodelling and perivascular c-kit+/sca-1+ progenitor cell accumulation. CCX771 administration did not abrogate these effects. In contrast, administration of AMD3100, whether alone or combined with CCX771, prevented vascular remodelling, PH and perivascular accumulation of c-kit+/sca-1+ progenitor cells, with a synergistic effect of these agents. This study offers important pathophysiological insights into the role of haematopoietic c-kit+ progenitors in hypoxia-induced vascular remodelling and may have therapeutic implications for PH.

Aims: We have investigated Ca²(+) signalling generated by aldosterone-induced T-type current (I(CaT)), the effects of I(CaT) in neonatal cardiomyocytes, and a putative role for I(CaT) in cardiomyocytes during cardiac pathology induced by stenosis in an adult rat., Methods and Results: Neonatal rat cardiomyocytes treated with aldosterone showed an increase in I(CaT) density, principally due to the upregulation of the T-type channel Ca(v)3.1 (by 80%). Aldosterone activated cAMP-response element-binding protein (CREB), and this activation was enhanced by blocking I(CaT) or by inhibiting protein phosphatase 2A (PP2A) activity. Aldosterone induced PP2A activity, an induction that was prevented upon I(CaT) blockade. I(CaT) exerted a negative feedback regulation on the transcription of the Ca(v)3.1 gene, and the activation of PP2A by I(CaT) led to increased levels of the pro-apoptotic markers caspase 9 and Bcl-x(S) and decreased levels of the anti-apoptotic marker Bcl-2. These findings were corroborated by flow cytometry analysis for apoptosis and necrosis. Similarly, in a rat model of cardiac disease, I(CaT) re-emergence was associated with a decrease in CREB activation and was correlated with increases in caspase 9 and Bcl-x(S) and a decrease in Bcl-2 levels., Conclusion: Our findings establish PP2A/CREB as targets of I(CaT)-generated Ca²(+) signalling and identify an important role for I(CaT) in cardiomyocyte cell death.

Objective: Mortality and morbidity related to right ventricular failure remain a problem for the long-term outcome of congenital heart diseases. Therapeutic innovation requires establishing an animal model reproducing right ventricular dysfunction secondary to chronic pressure-volume overload., Methods: Right ventricular tract enlargement by transvalvular patch and pulmonary artery banding were created in 2-month-old piglets (n = 6) to mimic repaired tetralogy of Fallot. Age-matched piglets were used as controls (n = 5). Right ventricular function was evaluated at baseline and 3 and 4 months of follow-up by hemodynamic parameters and electrocardiography. Right ventricular tissue remodeling was characterized using cellular electrophysiologic and histologic analyses., Results: Four months after surgery, right ventricular peak pressure increased to 75% of systemic pressure and pulmonary regurgitation significantly progressed, end-systolic and end-diastolic volumes significantly increased, and efficient ejection fraction significantly decreased compared with controls. At 3 months, the slope of the end-systolic pressure-volume relationship was significantly elevated compared with baseline and controls; a significant rightward shift of the slope, returning to the baseline value, was observed at 4 months, whereas stroke work progressed at each step and was significantly higher than in controls. Four months after surgery, QRS duration was significantly prolonged as action potential duration. Significant fibrosis and myocyte hypertrophy without myolysis and inflammation were observed in the operated group at 4 months., Conclusion: Various aspects of early right ventricular remodeling were analyzed in this model. This model reproduced evolving right ventricular alterations secondary to chronic volumetric and barometric overload, as observed in repaired tetralogy of Fallot with usual sequelae, and can be used for therapeutic innovation., (2010 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.)

Cell therapy holds promise for tissue regeneration, including in individuals with advanced heart failure. However, treatment of heart disease with bone marrow cells and skeletal muscle progenitors has had only marginal positive benefits in clinical trials, perhaps because adult stem cells have limited plasticity. The identification, among human pluripotent stem cells, of early cardiovascular cell progenitors required for the development of the first cardiac lineage would shed light on human cardiogenesis and might pave the way for cell therapy for cardiac degenerative diseases. Here, we report the isolation of an early population of cardiovascular progenitors, characterized by expression of OCT4, stage-specific embryonic antigen 1 (SSEA-1), and mesoderm posterior 1 (MESP1), derived from human pluripotent stem cells treated with the cardiogenic morphogen BMP2. This progenitor population was multipotential and able to generate cardiomyocytes as well as smooth muscle and endothelial cells. When transplanted into the infarcted myocardium of immunosuppressed nonhuman primates, an SSEA-1+ progenitor population derived from Rhesus embryonic stem cells differentiated into ventricular myocytes and reconstituted 20% of the scar tissue. Notably, primates transplanted with an unpurified population of cardiac-committed cells, which included SSEA-1- cells, developed teratomas in the scar tissue, whereas those transplanted with purified SSEA-1+ cells did not. We therefore believe that the SSEA-1+ progenitors that we have described here have the potential to be used in cardiac regenerative medicine.

We recently reported that corticosteroids increase the expression of the T-type channel Ca(v)3.1 through a transcriptional up-regulation of the Ca(v)3.1 encoding gene cacna1g. The nucleotide sequence analysis of cacna1g promoter revealed putative glucocorticoid response elements (GREs). However, the functional GREs involved in the regulation of cacna1g expression in neonatal cardiac myocytes are unknown. In the present study we have investigated the nuclear targets responsible for the transcriptional regulation of cacna1g. We identified five GREs from the nucleotide sequence of cacna1g promoter. Additionally, using punctual mutagenesis approach, three functional categories of GREs have been identified: (i) GRE-1 involved in promoter activity induced by aldosterone (Aldo, 1 microM); (ii) GRE-4 and GRE-5 involved in promoter activity induced by dexamethasone (Dex, 1 microM); and (iii) GRE-2 and GRE-3 involved in the basal level of neonatal promoter activity. The data presented here lead to better understanding of the molecular mechanisms underlying the regulation of Ca(v)3.1 channel expression by corticosteroids. These new findings have attractive physiological features during cardiac development and pathology such as arrhythmias.

The effect of the dexamethasone (Dex) on the regulation of the T-type Ca(2+) channel expressions was investigated in primary cultures of neonatal rat ventricular myocytes. We found that Dex (1 microM) increases the T-type Ca(2+) current (I(CaT)) associated with an increase in Ca(v)3.1 mRNA amount. We isolated the upstream region from Ca(v)3.1 encoding gene and tested the activity of the promoter in transfected ventricular myocytes. We found a minimal Dex-responsive region that displayed putative glucocorticoid receptor (GR) and nuclear factor kappa-B (NFkappaB) targets. The GR selective antagonist, RU38486 (10 microM), nearly turned off the transcriptional activity of Ca(v)3.1 encoding gene, and an NFkappaB inhibitor, pyrrolodine dithiocarbonate (10 microM), completely abolished the Dex-induced mRNA increase. However, Dex-induced GR and NFkappaB synthesis and nuclear translocation were not timely related to Ca(v)3.1 mRNA increase. These results indicate that both GR and NFkappaB were necessary, but not sufficient, to trigger the increase in Ca(v)3.1 mRNA amount. This study showed the relationship between glucocorticoid and T-type channels up-regulation that may be involved in cardiac development and pathology.

CXCL12 (SDF-1), which binds CXCR4, is involved in several physiological and pathophysiological processes. In heart, this axis seems to play a key role in cardiogenesis and is involved in the neovascularization of ischemic tissues. Rats have three known CXCL12 mRNA isoforms, of which only alpha and gamma are present in the normal heart. However, little is known about CXCL12 protein expression and localization. We investigated the pattern of protein expression and the localization of both CXCR4 and CXCL12 in the heart, using isolated cardiomyocytes and a rat myocardial infarction model. Western blots showed that cardiomyocytes contained a specific 67-kDa CXCR4 isoform and a 12-kDa CXCL12 isoform. Confocal and electron microscopy clearly showed that CXCR4 was present at the plasmalemma and CXCL12 in continuity of the Z-line, in the proximal part of T-tubules. In conclusion, we provide the first description of the expression and fine localization of CXCR4 and CXCL12 proteins in normal rat heart and cardiomyocytes. These results suggest that the CXCL12/CXCR4 axis may be involved in cardiomyocyte calcium homeostasis regulation. Our work and the well-known chemoattraction properties of the CXCL12/CXCR4 axis highlight the importance of deciphering the function of this axis in both normal and pathological hearts.

Objectives: The expression and distribution of connexins is abnormal in a number of cardiac diseases, including atrial fibrillation, and is believed to favor conduction slowing and arrhythmia. Here, we studied the role of atrial structural remodeling in the disorganization of gap junctions and whether redistributed connexins can form new functional junction channels., Methods: Expression of connexin-43 (Cx43) was characterized by immunoblotting and immunohistochemistry in human right atrial specimens and in rat atria after myocardial infarction (MI). Gap junctions were studied by electron and 3-D microscopy, and myocyte-myocyte coupling was determined by Lucifer yellow dye transfer., Results: In both chronically hemodynamically overloaded human atria in sinus rhythm and in dilated atria from MI-rats, Cx43 were dephosphorylated and redistributed from the intercalated disc to the lateral cell membranes as observed during atrial fibrillation. In MI-rats, the gap junctions at the intercalated disc were smaller (20% decrease) and contained very little Cx43 (0 or 1 gold particle vs. 42 to 98 in sham-operated rats). In the lateral membranes of myocytes, numerous connexon aggregates comprising non-phosphorylated Cx43 were observed. These connexon aggregates were in no case assembled into gap junction plaque-like structures. However, N-cadherin was well organized in the intercalated disc. There was very little myocyte-myocyte coupling in MI-rat atria and no myocyte-fibroblast coupling. Regression of the atrial remodeling was associated with the normalization of Cx43 localization., Conclusion: Structural alteration of the atrial myocardium is an important factor in the disorganization of connexins and gap junction. Moreover, redistributed Cx43 do not form junction channels.

1. The aim of the present study was to investigate the responses to acetylcholine (ACh; 3 nmol/L-30 micromol/L) and sodium nitroprusside (SNP; 3 nmol/L-30 micromol/L) of precontracted aortic rings from diabetic rats supplemented with docosahexaenoic acid (DHA). 2. Diabetes was induced by streptozotocin (STZ; 55 mg/kg). Diabetic and sham rats were fed, over a period of 8 weeks, either control diet or a DHA-supplemented diet. Aortic endothelial fatty acid composition was analysed by gas chromatography. The involvement of endothelial-derived nitric oxide (NO) and cyclo-oxygenase (COX) metabolites in response to ACh was assessed using the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (100 micromol/L) and the COX inhibitor indomethacin (1 micromol/L), respectively. 3. The DHA-supplemented diet induced a small increase in n-3 polyunsaturated fatty acids (PUFA; P < 0.001) owing to the incorporation of DHA in the endothelial cells of sham animals (1.6 +/- 0.2% in the DHA group compared with traces in the control group; P < 0.001) and diabetic animals (1.3 +/- 0.2% in the DHA group compared with traces in control group; P < 0.001), without a decrease in n-6 PUFA, despite a small decrease in arachidonic acid content (P < 0.05). Diabetes did not modify the incorporation of DHA in endothelial cells, but did significantly increase the arachidonic acid content (0.6 +/- 0.0 vs 0.4 +/- 0.1% in control group in the STZ and sham groups, respectively; P < 0.001). Acetylcholine-induced relaxation was significantly reduced in STZ groups compared with the sham groups (P < 0.001) and the DHA-supplemented diet did not modify these effects. In contrast, neither the DHA-supplemented diet nor diabetes affected the aortic relaxation induced by SNP. N(G)-Nitro-L-arginine methyl ester strongly inhibited the relaxant effects of ACh in the sham groups (P < 0.001) and abolished ACh-induced relaxation in the STZ groups (P < 0.001). The diet did not modify these effects. In the presence of indomethacin, the relaxation induced by ACh was decreased in the sham groups (P < 0.01), but not in the STZ groups. The DHA-supplemented diet did not have any effect on these responses. 4. In conclusion, these results suggest that, in the present study, the endothelial dysfunction occurring in the rat model of STZ-induced diabetes is associated with modifications of both the synthesis of COX derivatives and NO metabolism and is not affected by dietary supplementation with DHA.

The expression of cytochrome P450 (CYP) enzymes and cyclo-oxygenases (COX) was investigated in human saphenous veins by reverse transcription-polymerase chain reaction analysis. Non-varicose veins were obtained from patients undergoing aortocoronary bypass grafting, whereas varicose veins were obtained from patients undergoing stripping removal of varicose saphenous veins. In non-varicose veins, CYP1B1, CYP2C, CYP2E1 and CYP4A11 were detected, whereas CYP2J2, CYP3A5, COX-1 and COX-2 were detected almost exclusively in varicose veins. CYP4F2 was not detectable. Except for CYP4A11, the levels of individual CYP mRNA were higher in varicose veins than in control veins. Smooth muscle cell volume, determined by a colour image-analysis system, was increased approximately 1.5-fold in varicose veins. Because CYPs and COXs produce various vasoactive compounds, increased expression of these enzymes could be involved in the impairment of vascular tone and may contribute to varicose pathology. Then, CYP or COX modulators may be potentially active in the treatment of chronic venous insufficiency.

Etomidate is widely used for induction of anesthesia in the hemodynamically compromised patient, because of its moderate direct effect on arterial vasomotoricity and cardiac function, but its effect on blood pressure regulatory systems is not known. We studied the effect of etomidate (10(-8) to 10(-4) mol.L) on Ca++ mobilization elicited by angiotensin II (Ang II) in cultured aortic smooth muscle cells (VSMC) from 6-week-old Wistar Kyoto rats. Intracellular Ca++ (Cai++) variation was assessed in Fura 2-loaded VSMC, using fluorescent imaging microscopy. Ang II (10(-6) mol.L(-1))-induced transient Cai++ mobilization from internal stores was assessed in the absence of external Ca++. Ca++ influx was assessed upon reintroduction of external Ca++ (10(-3) mol.L(-1)). Etomidate moderately decreased both the amplitude (etomidate 10(-4) mol.L(-1): 68% of control value, P < 0.001) and the slope of Cai++ increase (56% of control, P < 0.001) from internal stores induced by Ang II. PD2 values (PD2 = -log(EC50)) for amplitude and slope were 6.4 +/- 0.7 and 6.0 +/- 0.3, respectively. Ang II-elicited Ca++ influx was also significantly decreased (45% of control, P < 0.001; PD2 = 5.5 +/- 0.3). Etomidate alters the Ca++ mobilization elicited by Ang II in rat aortic VSMC, suggesting that the vascular response to Ang II may be altered during etomidate anesthesia. However, this effect was observed at high concentration of etomidate, and may be limited when low doses of etomidate are used.

1. Angiotensin (Ang) II is a potent vasopressor agent, involved in the short-term control of arterial blood pressure during anaesthesia. The aim of the present study was to test the hypothesis that propofol, a widely used intravenous anaesthetic agent, could alter the arterial response to AngII and to evaluate its effect in genetic hypertension. 2. We studied the effect of increasing concentrations of propofol (5.6 x 10-7 to 5.6 x 10-4 mol/L) on aortic ring maximal isometric tension elicited by AngII and on AngII-induced Ca2+ mobilization in aortic smooth muscle cells from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). 3. Maximal tension developed by aortic rings from WKY rats was greater than that developed by rings from SHR. In both WKY rats and SHR, propofol at concentrations from 5.6 x 10-6 mol/L decreased maximal tension induced by AngII in a concentration-dependent manner. The magnitude of inhibition was higher in SHR than in WKY rats, whereas pD2 values were not different. In addition, Ca2+ mobilization induced by AngII was inhibited by propofol in a concentration-dependent manner, with the same magnitude and pD2 values. 4. These results suggest that the arterial response to AngII may be altered during propofol anaesthesia, particularly in hypertension.

Recent studies indicate that cardiac T-type Ca2+ current (ICaT) reappears in hypertrophied ventricular cells. The aim of this study was to investigate the role of angiotensin II (Ang II), a major inducer of cardiac hypertrophy, in the reexpression of T-type channel in left ventricular hypertrophied myocytes. We induced cardiac hypertrophy in rats by abdominal aorta stenosis for 12 weeks and thereafter animals were treated for 2 weeks with losartan (12 mg/kg per day), an antagonist of type 1 Ang II receptors (AT1). In hypertrophied myocytes, we showed that the reexpressed ICaT is generated by the CaV3.1 and CaV3.2 subunits. After losartan treatment, ICaT density decreased from 0.40+/-0.05 pA/pF (n=26) to 0.20+/-0.03 pA/pF (n=27, P<0.01), affecting CaV3.1- and CaV3.2-related currents. The amount of CaV3.1 mRNA increased during hypertrophy and retrieved its nonhypertrophic level after losartan treatment, whereas the amount of CaV3.2 mRNA was unaffected by stenosis. In cultured newborn ventricular cells, chronic Ang II application (0.1 micromol/L) also increased ICaT density and CaV3.1 mRNA amount. UO126, a mitogen-activated protein kinase kinase-1/2 (MEK1/2) inhibitor, reduced Ang II-increased ICaT density and CaV3.1 mRNA amount. Bosentan, an endothelin (ET) receptor antagonist, reduced Ang II-increased ICaT density without affecting the amount of CaV3.1 mRNA. Finally, cotreatment with bosentan and UO126 abolished the Ang II-increased ICaT density. Our results show that AT1-activated MEK pathway and autocrine ET-activated independent MEK pathway upregulate T-type channel expression. Ang II-increased of ICaT density observed in hypertrophied myocytes may play a role in the pathogenesis of Ca2+ overload and arrhythmias seen in cardiac pathology.

We studied cardiac outward K currents (transient and sustained) by the whole-cell patch-clamp technique and the Kv4.2, Kv4.3, Kv1.4, Kv1.5, Kv1.2 and Kv2.1 expression of voltage-gated K channel by RT-PCR, in ventricular myocytes from two models of catecholamine-depleted adult rats. We induced endogenous catecholamine depletion by reserpine treatment and used adrenalectomized rats as a model of plasma catecholamine depletion. In reserpine-treated rats (97% decrease in endogenous norepinephrine content of the heart), the amplitude of the transient outward current was decreased by 48% and Kv4.2 and Kv4.3 mRNA levels were decreased by 57% and 34%, respectively. The amount of Kv1.5 mRNA tripled, with no change in sustained current density. This increase was not confirmed by immunostaining for the Kv1.5 protein. The amplitude of K currents and their corresponding mRNA levels returned to control values following recovery from reserpine treatment. In contrast, in adrenalectomized rats (98% decrease in plasma epinephrine concentration), we observed no change in the amplitude of outward K currents or in Kv mRNA levels. These results suggested a role for sympathetic innervation and endogenous norepinephrine in the regulation of transcription of cardiac outward K currents in physiological and pathological situations.

1. The effect of propofol on arterial tone in hypertension is poorly understood. We examined the effect of increasing concentrations of propofol (5.6 x 10-8 to 2.8 x 10-3 mol/L) on isometric tension developed by noradrenaline (10-7 mol/L)-contracted aortic rings from 12-week-old Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). 2. In both WKY rats and SHR, propofol induced a dose-dependent inhibition of contraction induced by noradrenaline, but the amplitude of relaxation was larger in the SHR than in WKY rats. 3. The effects of propofol was endothelium independent in WKY rats, whereas in SHR relaxation induced by propofol was greater in endothelium-intact than in endothelium-denuded rings. 4. In conclusion, we found significant differences in the effect of propofol in hypertensive rats, which may be related to differences in structural and functional properties of the arterial wall observed in hypertension.

Background: Angiotensin II (AngII) is a potent vasoconstrictor involved in the short-term control of arterial blood pressure. Isoflurane was reported to decrease vascular tone through an alteration of vascular smooth muscle cell vasomotor response to several agonists, but its effect on AngII signaling is not known. On the other hand, vascular response to AngII is altered in hypertension. In this study, the authors tested the hypothesis that (1) isoflurane alters AngII-induced intracellular Ca mobilization in aortic vascular smooth muscle cell from Wistar Kyoto and spontaneously hypertensive rats, and (2) this effect could be associated with an alteration of the organization of microtubular network, reported to be involved in AngII signaling., Methods: The effect of 0.5-3% isoflurane was studied (1) on AngII (10 m)-induced intracellular Ca mobilization, intracellular Ca release from internal stores, and Ca influx in Fura-2 loaded cultured aortic vascular smooth muscle cell isolated from 6-week-old Wistar Kyoto and spontaneously hypertensive rats, using fluorescent imaging microscopy; and (2) on the organization of cytoskeletal elements, using immunofluorescence labeling., Results: In both stains, isoflurane decreased in a concentration-dependent manner AngII-induced intracellular Ca mobilization, Ca release from internal stores, and Ca influx through nifedipine-insensitive Ca channels. This effect occurred at a lower concentrations of isoflurane in Wistar Kyoto rats than in spontaneously hypertensive rats. In both strains, the effect of isoflurane on AngII- Ca mobilization was abolished by impairment with nocodazole, vinblastine, or paclitaxel of microtubules polymerization. Isoflurane directly altered tubular network organization in a concentration-dependent and reversible manner., Conclusions: Isoflurane decreased AngII-induced Ca mobilization at clinically relevant concentrations, suggesting that vascular response to AngII could be altered during isoflurane anesthesia. The hypertensive strain was found less sensitive than the normotensive one. In both strains, the isoflurane effect was associated with a microtubular network interaction.

Rats treated with DOCA salts and subjected to abdominal aortic stenosis display left ventricle hypertrophy associated with a decrease in cardiac I(to) current density and prolongation of the action potential duration. We investigated the molecular basis of these electrophysiological defects by analyzing the amount of mRNA corresponding to the genes encoding the a subunits of the left ventricle K+ channel at the steady state. The mRNAs corresponding to the a subunits of the K+ channel (Kv1.2, Kv1.4, Kv1.5, Kv2. 1, Kv4.2 and Kv4.3) were measured by quantitative RT-PCR using a specific Kv internal standard. In control rats, the Kvl.5 gene was only expressed at a low level, whereas the Kv4.2 and Kv4.3 genes were expressed at a high level. Regardless of the etiology of the hypertrophy, the amounts of Kv1.4 and Kv1.5 mRNAwere similar in treated, sham and control rats. The amounts of Kv1.2 and Kv2.1 mRNA were markedly lower in DOCA-salt treated rats (66%) than in sham-DOCA rats, but no effect was observed after stenosis. The very conservative Kv4.2 and Kv4.3 genes were found to be downregulated simultaneously in both type of hypertrophy. However, the steady-state amount of Kv4 mRNA was even lower in rats with DOCA-salt-induced hypertrophy than in those with stenosis-induced ventricular hypertrophy. Therefore, the decrease in I(to) density, consecutively to pressure- and volume-overload, is due to a large decrease in the amount of Kv4.2 and Kv4.3 mRNA. In addition, DOCA-salt treatment alters the amounts of Kv transcripts independently to cardiac hypertrophy, suggesting that the mineralocorticoid may be involved in Kv gene expression.

Angiotensin II (Ang II) is involved in hypertension-related arterial wall hypertrophy [1]. Regulation of AT II transduction pathway in vascular smooth muscle cells (VSMC) may involve cytoskeleton and extracellular matrix (ECM) [2]. We assessed the role of components of ECM on Cai2+ increase induced by Ang II in Wistar-Kyoto (WKY) and Spontaneously Hypertensive Rats (SHR) aortic VSMC. The effect of Ang II (1 mumol) on Ca2+ mobilization was studied in cultured VSMC isolated from the aorta of 6-wk old WKY (MAP (m +/- SE) = 98 +/- 4 mmHg) and SHR (136 +/- 5 mmHg; p < 0.05), using fluorescent imaging microscopy (Fura-2 AM). Cai2+ release from internal stores and Ca2+ influx were assessed in the absence and upon reintroduction of external Ca2+ respectively. Cells were cultured on uncoated glass coverslips (control) or coated with either collagen I (10 micrograms/mL), collagen IV (7 micrograms/mL), vitronectin (0.1 microgram/mL), fibronectin (3 micrograms/mL) and extracellular matrix extract (matrigel, 1/10) and studied at confluence. Paxillin was located in cells by indirect immunofluorescence micrography. Results are expressed in % of Control. Statistical significance (p < 0.05) was assessed with Student's t-test for unpaired data. The effects on Ang II-induced Ca2+ mobilization of growing cells on ECM are in Table. Paxillin in Control cells appeared as dots at the cell boundaries. Density increased in cells grown on collagen I with a diffuse distribution in the WKY cells. On matrigel, paxillin was located in a belt-like fashion at the periphery of the cell. These effects were not linked to differences in cell cycle (flux cytometry).

T-type calcium current (I(CaT)) is distributed among a large variety of species and tissues. The main functions of I(CaT) are thought to be related to pacemaker activity and to the cell cycle. Using the whole-cell patch-clamp configuration, we showed that fetal rat ventricular cells exhibit an I(CaT) with electrophysiological and pharmacological characteristics similar to those already described for this current. We investigated I(CaT) density and found that this current was mainly expressed in fetal cells and remained stable until birth (3.1+/-0.3 pA/pF for 18-day-old fetus, n=9). I(CaT) density decreased soon after birth (2.0+/-0.3 pA/pF, n=6, 1.1+/-0.2 pA/pF, n=5, for 1- and 5-day-old rats, respectively) and was no longer detected in 21-day-old rats. The rat ventricular cells express an alpha 1H isoform in addition to a homologous alpha 1G variant. Interestingly, the Ni(2+) sensitivity of I(CaT) indicates that in newborn myocytes, I(CaT) is only generated by alpha 1G subunits, whereas both alpha 1G and alpha 1H subunits participate in the fetal I(CaT). Moreover, the relative contribution of each subunit varies during fetal developmental stages, with a major contribution of alpha 1H in 16-day-old fetuses. Through quantitative RT-PCR we showed that the amount of both alpha 1G and alpha 1H transcripts are developmentally regulated. In fetuses of less than 18 days and in newborn rats after 1 day old, the transcriptional levels of alpha 1G and alpha 1H subunits clearly mismatch the functional contribution of these subunits to I(CaT). However, in perinatal period, the amount of alpha 1G mRNA seems to be in accordance to alpha 1G-related I(CaT) density. In conclusion, we showed that I(CaT) is mainly expressed during fetal stages, that alpha 1G and alpha 1H differentially participate to I(CaT) and that alpha 1G and alpha 1H isoforms are regulated by both transcriptional and post-transcriptional mechanisms., (Copyright 2002 Academic Press.)

The effect of catecholamine depletion (induced by prior treatment with reserpine) was studied in Wistar rat ventricular myocytes using whole cell voltage-clamp methods. Two calcium-independent outward currents, the transient outward potassium current (I(to)) and the sustained outward potassium current (I(sus)), were measured. Reserpine treatment decreased tissue norepinephrine content by 97%. Action potential duration in the isolated perfused heart was significantly increased in reserpine-treated hearts. In isolated ventricular myocytes, I(to) density was decreased by 49% in reserpine-treated rats. This treatment had no effect on I(sus). The I(to) steady-state inactivation-voltage relationship and recovery from inactivation remained unchanged, whereas the conductance-voltage activation curve for reserpine-treated rats was significantly shifted (6.7 mV) toward negative potentials. The incubation of myocytes with 10 microM norepinephrine for 7-10 h restored I(to), an effect that was abolished by the presence of actinomycin D. Norepinephrine (0.5 microM) had no effect on I(to). However, in the presence of both 0.5 microM norepinephrine and neuropeptide Y (0.1 microM), I(to) density was restored to its control value. These results suggest that the sympathetic nervous system is involved in I(to) regulation. Sympathetic norepinephrine depletion decreased the number of functional channels via an effect on the alpha-adrenergic cascade and norepinephrine is able to restore expression of I(to) channels.

In rats, removal of the carotid arterial or abdominal aortic endothelium results in an acute increase of diameter and compliance. In humans, acute local administration of a specific NO synthase inhibitor increases radial artery compliance but not the diameter. The purpose of this review is to determine whether in spontaneously hypertensive rats (SHR), a cause-and-effect relationship may be observed between endothelial function and arterial stiffness with possible consequences on pulse pressure (PP) control. The study is based on a comparative time-dependent analysis of the following in young and old SHR: aortic blood pressure measurements and reactivity, ultrasonographic arterial stiffness assessment, aortic histomorphometry and staining, and molecular biology with evaluation of endothelium function. In young SHR, aortic mean blood pressure and PP increase proportionally, whereas isobaric arterial stiffness is unchanged or poorly modified. The endothelial NO response to norepinephrine is normal or upregulated as a response to predominant vasoconstrictive influences. In contrast, in old SHR, PP and mean blood pressure change disproportionately with age, together with an enhanced isobaric arterial stiffness. The endothelial NO response to norepinephrine is abolished, in association with endothelium-dependent heightened norepinephrine reactivity and enhanced accumulation of vessel extracellular matrix. In this latter case, exogenous NO acutely and selectively lowers the increased PP. Thus, during SHR aging, a negative feedback may be observed between NO bioactivity and PP through changes in arterial structure and function. Whether this alteration contributes to the development of systolic hypertension in old populations remains to be determined.

The interaction of the vascular smooth muscle cells (VSMCs) with the components of the matrix determines several functions of the cell, such as growth and differentiation. In contrast, an alteration in angiotensin (Ang) II-induced Ca(2+) mechanisms in VSMCs was reported in genetic hypertension. In this study, we wished to assess the effect of different components of the extracellular matrix on the increase of [Ca(2+)](i) induced by Ang II in VSMCs from spontaneously hypertensive rats (SHR) compared with those from normotensive Wistar-Kyoto rats (WKY). Results demonstrate for the first time that elements of the extracellular matrix modulate the Ang II-induced Ca(2+) transport mechanisms. This modulation is different in cells from WKY compared with those from SHR. Thus, growing cells from SHR on collagen I, collagen IV, fibronectin, vitronectin, or Matrigel induced a significant decrease in Ang II-induced Ca(2+) release from internal stores, whereas in cells from WKY, no effect could be observed except for those grown on collagen I, which increased Ca(2+) release. Fibronectin and vitronectin, however, induced a decrease in Ang II-induced Ca(2+) influx in WKY, whereas no effect could be observed in SHR. Conversely, collagen I and collagen IV induced an increase in this influx in SHR but not in WKY, whereas Matrigel increased the influx in both strains. These results suggest a modulation of the Ang II-associated signaling events by the matrix elements via the focal adhesion points. The understanding of these synergies should provide insight into issues such as development of hypertrophy of large vessels in hypertension.

1. We investigated the role of arachidonic acid metabolism and assessed the participation of mast cells and leukocytes in neurogenic inflammation in rat paw skin. We compared the effect of lipoxygenase (LOX) and cyclo-oxygenase (COX) inhibitors on oedema induced by saphenous nerve stimulation, substance P (SP), and compound 48/80. 2. Intravenous (i.v.) pre-treatment with a dual COX/LOX inhibitor (RWJ 63556), a dual LOX inhibitor/cysteinyl-leukotriene (CysLt) receptor antagonist (Rev 5901), a LOX inhibitor (AA 861), a five-lipoxygenase activating factor (FLAP) inhibitor (MK 886), or a glutathione S-transferase inhibitor (ethacrynic acid) significantly inhibited (40 to 60%) the development of neurogenic oedema, but did not affect cutaneous blood flow. Intradermal (i.d.) injection of LOX inhibitors reduced SP-induced oedema (up to 50% for RWJ 63556 and MK 886), whereas ethacrynic acid had a potentiating effect. 3. Indomethacin and rofecoxib, a highly selective COX-2 inhibitor, did not affect neurogenic and SP-induced oedema. Surprisingly, the structurally related COX-2 inhibitors, NS 398 and nimesulide, significantly reduced both neurogenic and SP-induced oedema (70% and 42% for neurogenic oedema, respectively; 49% and 46% for SP-induced oedema, respectively). 4. COX-2 mRNA was undetectable in saphenous nerves and paw skin biopsy samples, before and after saphenous nerve stimulation. 5. A mast cell stabilizer, cromolyn, and a H(1) receptor antagonist, mepyramine, significantly inhibited neurogenic (51% and 43%, respectively) and SP-induced oedema (67% and 63%, respectively). 6. The co-injection of LOX inhibitors and compound 48/80 did not alter the effects of compound 48/80. Conversely, ethacrynic acid had a significant potentiating effect. The pharmacological profile of the effect of COX inhibitors on compound 48/80-induced oedema was similar to that of neurogenic and SP-induced oedema. 7. The polysaccharide, fucoidan (an inhibitor of leukocyte rolling) did not affect neurogenic or SP-induced oedema. 8. Thus, (i) SP-induced leukotriene synthesis is involved in the development of neurogenic oedema in rat paw skin; (ii) this leukotriene-mediated plasma extravasation might be independent of mast cell activation and/or of the adhesion of leukocytes to the endothelium; (iii) COX did not appear to play a significant role in this process.

The reactivity of old hypertensive rat aortas has not been investigated in relation to each phenotype of the blood pressure curve, mean arterial pressure (MAP), and pulse pressure (PP). Aortic reactivities from 3- to 78-week-old Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were studied with the use of organ chambers and invasive blood pressure, carotid diameter, and histomorphometry. MAP and PP were elevated in SHR, but at 78 weeks, a selective increase of PP without further MAP increase was observed for the same carotid diameter as WKY. Aortic relaxation in response to carbamylcholine decreased similarly with age in both strains. With (+) or without (-) endothelium (E), maximal developed tension (MDT) under KCl increased linearly with age in SHR, proportionally to wall thickness and MAP increase. Under norepinephrine (NE), MDT of E(-) aortas from SHR and controls increased with age and reached plateaus at 12 weeks, whereas MDT of E(+) aortas from SHR increased linearly with age. Because the NE-induced MDT was higher for E(-) than E(+), the difference estimated endothelial function. This difference reached plateaus from 12 to 78 weeks in WKY but was abolished beyond 12 weeks in SHR, a finding also observed under NO-synthase inhibition. In old hypertensive rats, (1) increased KCl reactivity is endothelium independent but influenced by the MAP-dependent aortic hypertrophy with resulting increased vascular smooth muscle reactivity, whereas (2) increased NE reactivity is endothelium dependent in association with increased PP, altered endothelial function, and extracellular matrix, with resulting enhanced intrinsic arterial stiffness.

Objectives: To investigate the role of transforming growth factor-beta1 (TGF-beta1) on Ca2+-dependent mechanisms elicited by angiotensin II in aortic vascular smooth muscle cells (VSMC) of Wistar- Kyoto (WKY) rats and spontaneously hypertensive rats (SHR)., Methods: Cai2+ release induced by angiotensin II (1 micromol/ l) was studied in cultured VSMC isolated from the aortas of 6-week-old WKY rats and SHR. Intracellular Ca2+ (Cai2+) was assessed in Fura-2 loaded cells using fluorescent imaging microscopy. Angiotensin II receptors were analysed by binding studies., Results: Pretreatment of VSMC for 24 h with TGF-beta1 significantly increased angiotensin II-induced Cai2+ mobilization from internal stores in SHR, while Ca2+ influx was not altered. This effect involves tyrosine kinase and is not due to an increase in angiotensin II binding sites, or a change in the affinity of the receptors. By contrast, TGF-beta1 did not modify the response of VSMC from WKY rats to angiotensin II., Conclusions: These results help our understanding of the interactions between the pathways activated by TGF-beta1 and the G protein-coupled receptor signalling pathway, and their role in genetic hypertension.

The vascular mechanism of action of cicletanine, an antihypertensive agent, was studied on isolated Wistar rat aortas (24-months-old) in presence and in absence of endothelium in two different stress conditions, normoxic and hypoxic, in presence of norepinephrine (NE). Under normoxic conditions, in presence of endothelium, cicletanine (10(-9)-10(-5)M) induced a concentration-dependent relaxation, whereas in absence of endothelium, cicletanine (10(-9)-10(-5)M) was ineffective although it relaxed the smooth muscle at higher concentrations (10(-4)M). At pharmacologic concentrations (below or equal 10(-5)M), relaxation induced by cicletanine, in presence of endothelium, was prevented by N(omega)-nitro-L-arginine (L-NNA) (P <.005) and relaxation induced by the highest concentration (10(-4)M) was reversed by BaCl2 (P <.005). Under hypoxic conditions, in presence of NE and endothelium, the aorta displayed an increased developed tension that was significantly (P <.05) attenuated by cicletanine (10(-5)M) and insensitive to indomethacine (10(-7)M). When the two compounds were added together, the relaxation induced by cicletanine was significantly improved (P <.005). These results indicated that cicletanine, under stress conditions, relaxes vascular smooth muscle through an endothelium-dependent action mediated by the nitric oxide (NO) synthase pathway. We proposed that the observed vascular effects could be associated with the counter-regulation mechanisms linked to the antihypertensive action of cicletanine.

We studied the effect of basic fibroblast growth factor (b-FGF) on different Ca(2+) mechanisms elicited by angiotensin II (Ang II) in normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Intracellular Ca(2+) (Ca(2+)(i)) variations were studied in cultured vascular smooth muscle cells (VSMCs) isolated from the aorta of 5- to 6-week-old WKY rats and SHR. Ca(2+)(i) was assessed in Fura-2-loaded cells with fluorescent imaging microscopy. Ang II subtype 1 receptor activation by Ang II (1 micromol/L) induced a transient increase in Ca(2+)(i) that was partially attenuated by genistein, a tyrosine kinase inhibitor. Pretreatment of VSMCs with b-FGF for 24 hours markedly stimulated the Ang II-induced Ca(2+)(i) release from the internal stores in WKY rats, whereas it was without effect in SHR. This was not consequent to a change in the affinity of Ang II subtype 1 receptors or an increase in their density. Inhibition of mitogen-activated protein kinase with PD 98059 reduced this stimulatory effect of the cytokine in the WKY rats. On the other hand, b-FGF stimulated the Ang II-induced Ca(2+) influx in both strains. Similar results were observed when Ca(2+) influx was induced with thapsigargin. Genistein and PD 98059 abolished the effect of b-FGF. These results show for the first time that b-FGF regulates Ca(2+) mechanisms induced by Ang II and that this regulation is different in SHR than in normotensive control animals. The extracellular signal-regulated kinase cascade is implicated in this cross-regulation with G protein-signaling pathway at 2 levels and possibly more: 1 at the tyrosine kinases and the other downstream of the extracellular signal-regulated kinase family. These results may prove useful in understanding the interaction between these 2 pathways and their implication in genetic hypertension.

1. Thoracic aortas of normotensive (Wistar-Kyoto (WKY) and Lyon normotensive (LN)) and hypertensive (spontaneously hypertensive rats (SHR) and Lyon hypertensive (LH)) rats from two groups (Japanese (WKY rats and SHR) and Lyon (LN and LH rats)) were compared using organ chambers. Changes in endothelium and smooth muscle reactivity to noradrenaline (NA), carbamylcholine and N omega-nitro-L-arginine (L-NNA) were analysed to distinguish between changes in reactivity that are associated with the presence of hypertension and those that are dependent on group (Japanese vs Lyon). 2. Aortas of hypertensive rats had lower pD2 values for NA than aortas from normotensive rats. These differences were associated with hypertension (P < 0.005 and P < 0.01) and group (P < 0.005 and P < 0.005) in presence or absence of endothelium, respectively, whereas no difference was seen in the maximal developed tension in response to NA. 3. Aortas also differed by a reduced ability to relax in response to carbamylcholine in hypertensive rats; this effect is hypertension (P < 0.05) and group (P < 0.005) dependent, without any change in carbamylcholine pD2 values. 4. Changes in maximum developed tension in the presence of L-NNA were found to be endothelium dependent and pressure and group independent. Furthermore, the change in tension induced by L-NNA appears significantly more pronounced in SHR than in LH rats (P < 0.05). 5. These results indicate that the common defect associated with hypertension appears to be linked to the endothelium through alpha-adrenoceptors and muscarinic receptors in both the Japanese and Lyon groups. However, SHR differs markedly from LH rats by having a higher developed tension in response to NA, this increased tension being counterbalanced by the release of nitric oxide, as observed in the presence of L-NNA.

Objective: The vascular mechanism of action of cicletanine, an antihypertensive agent, was studied on isolated Wistar rat aorta in presence and in absence of endothelium both in normoxic and hypoxic conditions., Design and Methods: Isolated aorta, from 24 month-old rats, were precontracted with noradrenaline (10(-7) M), in presence and in absence of endothelium and exposed to cumulative cicletanine concentrations in presence and absence of either L-NNA (10(-4) M) or indomethacin (Indo) (10(-7) M). Thereafter, aorta were precontracted by noradrenaline 10(-7) M, and hypoxia was induced by switching gas mixture from 95%O2/5%CO2 to 95%N2/5%CO2 during 10 minutes. Results are expressed as mean +/- sem and statistical analysis were done using one-way analysis of variance., Results: When aorta were precontracted with noradrenaline (10(-7) M), in presence of endothelium, cicletanine (10(-9)-10(-4) M), induced a biphasic concentration-dependent relaxation (EC50 approximately 10(-7) M and 3 x 10(-5) M). In absence of endothelium, the effect of cicletanine was abolished (10(-9) and 10(-5) M). Whereas, at higher concentration (10(-4) M), the magnitude of the relaxation reached 94 +/- 2% and 67 +/- 5% of the initial developed tension in presence and in absence of endothelium respectively. The endothelium-dependent relaxation induced by cicletanine was significantly reduced by Indo (10(-7) M) (p < 0.05) and L-NNA (10(-4) M) (p < 0.005). Addition of 10 mM of BaCl2 significantly reversed the relaxation induced by the higher concentration of cicletanine used (p < 0.005). Under hypoxic conditions, the aorta, in presence of endothelium, displayed an increased developed tension which was significantly attenuated by cicletanine., Conclusion: These results indicated that cicletanine relaxes vascular smooth muscle through both, an endothelium-dependent action which was mediated by cyclooxygenase and NOsynthase pathways and an endothelium-independent action that was mediated through K+ channels opening. Under hypoxic conditions, our findings indicate that the effects of cicletanine, appear related to an endothelium protective action associated to NO release.

Conduit arteries of hypertensive rats are thicker and stiffer than those of normotensive controls. The possible role played by collagen type I and II subtypes in the mechanism of arterial stiffness remains unknown. The carotid and aortic arterial wall of rats of Japanese (Wistar-Kyoto and spontaneously hypertensive rats) and Lyon (normotensive and hypertensive rats) origin were studied. The stiffness of the carotid wall material (ultrasound), the histomorphometry of the aortic wall with the content in collagen I and III subtypes and their corresponding mRNA were analyzed. Independently of hypertension, the Japanese group differed from the Lyon group by a stiffer carotid wall material at any given value of wall stress; a lesser degree of aortic hypertrophy with a higher percentage of elastin, and a higher density of collagen III but not of collagen I. All other hemodynamic and histomorphometric parameters were affected by both the origin of the rats (Japanese vs. Lyon) and the presence of hypertension. Large artery stiffness in genetically hypertensive rats was not only influenced by hypertension itself, but also by differences in the contents of collagen subtypes which are also found in their corresponding normotensive controls.

Na+-H+ exchanger is thought to play an important role in the regulation of the intracellular pH (pHi) and in the cardiac cell injury induced by ischemia and reperfusion. The aim of this study was to assess the effect of cicletanine, an antihypertensive compound in humans, which has been reported to have cardioprotective effect under an ischemia-reperfusion process, on Na+-H+ exchanger activity in ventricular cardiomyocytes isolated from chick embryo. Na+-H+ exchanger activity was assessed by the rate of pHi recovery after an acid load. A dual effect was observed: at low concentration of cicletanine (10(-7) M), Na+-H+ exchanger activity was significantly decreased, whereas at higher concentrations (10(-6)-10(-5) M), a significant stimulation of the exchanger was observed. These results suggest that cicletanine modulates pHi recovery in cardiac cells after cellular acid load.

Background: The human radial artery has been proposed as an alternative to coronary grafts in subjects with coronary ischemic disease. Because this muscular artery is known to be prone to spasm, changes in its mechanical properties in response to catecholamines are important to investigate., Methods: We describe a new echo-tracking technique of high resolution that allows investigation, in vitro, of the diameter and the wall thickness of arterial cylindrical segments. In addition, a classic tissue bath experiment is used to study the vasoreactivity of arterial rings. Mechanical properties of the radial artery are determined over a 0 to 200 mm Hg range of transmural pressure in the presence and absence of norepinephrine in the perfusion medium., Results: With tissue bath experiments, the norepinephrine dose-response curve was characterized by an EC50 value of 1.48+/-1.09 10(-6) mol/L and a maximal developed tension at 10(-5) mol/L. The results obtained with pressurized segments gave similar results with an EC50 value of 8.1+/-2.3 10(-7) mol/L and a maximal change in diameter at 10(-5) mol/L norepinephrine. Under the influence of 10(-5) mol/L norepinephrine, the radial artery constriction reached 22%, significantly affecting the unstressed diameter. Compliance did not show any significant change in the overall transmural pressure range, whereas distensibility significantly increased and elastic modulus significantly decreased., Conclusion: The study shows that the capacitive properties of the human muscular radial artery are maintained with norepinephrine not only through decreased stiffness of wall material but also through reduced unstressed diameter. Thus drugs inducing smooth muscle relaxation may be helpful in preventing radial artery spasm after coronary grafts.

Objective: The aim of this study was to analyse the arterial wall mechanics and the vasoreactive properties of the radial artery in comparison with those of the internal mammary artery and to discuss their implications for coronary bypass grafts., Methods: Measurements of pressure and diameter were obtained from cylindrical segments, whereas measurements of reactivity were obtained from ring segments from the same arteries. We used an echo-tracking technique of high resolution enabling to investigate, in vitro, the diameter and the wall thickness of arterial cylindrical segments. Furthermore, the compliance, distensibility and incremental elastic modulus of the radial and of the mammary arteries were determined for a wide range of transmural pressure (0-200 mmHg) in presence and absence of norepinephrine (NE)., Results: Our results show that NE caused vasoconstriction of the two arteries. Strain was found significantly higher for the radial artery than for the internal mammary artery at any given value of stress both in the presence and in the absence of NE. In presence of NE, compliance for radial artery, in the overall transmural pressure range, did not change, whereas, distensibility was significantly increased and the elastic modulus was significantly decreased. Under the same conditions, the distensibility of the mammary artery tended to decrease and its elastic modulus to increase. In parallel, the vasoreactive properties of the two arteries confirmed the previous results showing that radial artery developed a significant higher tension to vasoconstricting agents (KCl, NE and phenylephrine (PHE)) and higher relaxation to isradipine than internal mammary artery. Moreover, radial artery displayed a lesser sensitivity to sodium nitroprusside than internal mammary artery. Furthermore, sensitivity to NE was found to be 7-fold higher for radial artery than for internal mammary artery., Conclusion: Taken together, data on the mechanical and reactive properties of radial and internal mammary arteries show why the radial artery displayed a higher potential for spasm than the internal mammary artery and why the use of Ca2+ channel blocker can decrease the incidence of occlusion and spasm.

Experimental pharmacology and studies on hypertension frequently use genetically hypertensive animal models like the SHR or the Lyon hypertensive rat LH. In order to further characterize these two models we measured the expression levels of three major extracellular matrix components in the aortic wall, tropoelastin (TE) and type I and type III collagen, during postnatal development. The type I collagen expression decreases progressively during the first twelve weeks of postnatal development without significant differences between SHR and LH, or their normotensive controls, WKY or LN respectively. No differences were detected either for the expression levels of TE and type III collagen between the hypertensive strains and their respective controls. However, direct comparison of the two hypertensive strains SHR and LH, revealed a specific, strong increase of TE and type III expression for the LH at 5 and 12 weeks (p < 0.001 and 0.005 respectively). The evolution of the ratios of expression levels between the two collagens (type III/type I) on one side and of TE and collagen type I (TE/type I) on the other side were similar for the hypertensive strains and their respective controls, but diverged significantly for LH and SHR animals (up to p < 0.001 depending on the age group). Both indicators, III/I and TE/I, are considerably higher in LH compared to SHR from 5 weeks of postnatal development onwards. Our results indicate that the genes for TE and type I and III collagen are regulated during postnatal development of LH and SHR. It is however not possible at this point to establish a link between mRNA levels and hypertension in these animals. Nevertheless, the ratios III/I and TE/I seem to be good phenotypic markers for the characterisation of LH and SHR strains.

1. The sensitivity of cromakalim-activated current (Icrom) to manipulations of extracellular cationic composition was examined in whole-cell voltage clamp recordings from freshly-dispersed, adult guinea-pig ventricular myocytes. In bathing media with different concentrations of K+ (1, 2.5, 5.4 and 12 mM) the Icrom reversal potential (Erev) varied in strict correspondance with the K+ equilibrium potential and inward Icrom amplitude was proportional to the external K+ concentration. 2. Replacement of 12mM K+ with 12mM Rb+ induced a slight positive shift of Erev indicating that PRb+/PK+ = 1.06. K+ replacement with 12mM Cs+ reduced or abolished inward Icrom and produced a negative shift of Erev by at least 50 mV; an upper limit of PCs+/PK+ was fixed at 0.18. 3. Addition of Rb+ (1-30 mM) to 2.5 mM K(+)-containing medium produced a concentration-dependent increase in inward Icrom and positive shift of Erev suggesting that K+ and Rb+ have similar permeabilities and conductivities and do not interfere with each other in the channel. 4. CS+ (0.01-30 mM), added to medium containing 12 mM Rb+, induced a potent, voltage-dependent inhibition of inwardly rectifying current (IK1; IC50 = 0.2-3 mM). Voltage-dependent inhibition of inward Icrom was observed only at considerably higher CS+ concentrations (IC50 = 4-30 mM). Extracellular Rb+ and CS+ did not substantially alter the amplitude of outward Icrom. 5. The results support the contention that the ATP-sensitive K+ channel is the primary target of cromakalim action in ventricular myocytes.

Several 1,2,3,4-tetrahydro-6-methyl-4-(substituted phenyl)-2-thioxo-5-pyrimidinecarboxylic acid methyl esters were synthesized by condensing thiourea with methyl acetoacetate and substituted benzaldehydes according to Biginelli reaction. The structures of the compounds were confirmed by spectral and elemental analysis and their calcium antagonistic activities were investigated. Furthermore, the compounds synthesized in previous and present studies were evaluated for their antiaggregating activities.

A series of 1,2,3,4-tetrahydro-6-methyl-4-(substituted phenyl)-2-thioxo-5-pyrimidinecarboxylic acid methyl esters were synthesized by condensing thiourea with methyl acetoacetate and nonsubstituted or differently substituted benzaldehydes in absol. ethanol using HCl as a catalyst according to the Biginelli reaction. The structures of the compounds were confirmed by elemental and spectroscopic analysis. -The compounds were evaluated for their calcium antagonistic activity on the basis of their potency in inhibiting [3H] PN 200-110 binding on microsomes obtained from rat skeletal muscle.

S11568, (+-)[((amino-2-ethoxy)-2-ethoxy]-methyl)-2-(dichloro-2', 3'-phenyl)-4-ethoxycarbonyl-3-methoxycarbonyl-5-methyl-6-dihydro-1,4-pyr idine HCl, is a new dihydropyridine derivative that is water soluble and relatively insensitive to light. The Ca2+ channel inhibitory activity of S11568 was tested in whole-cell patch clamp recordings from cultured embryonic chick cardiomyocytes in 40 mM Ba2(+)-containing medium that revealed T-type and L-type components of inward current through calcium channels. S11568 inhibited L-type Ca2+ current with an IC50 value near 1 microM but was without effect on the T-type barium current.

Biochemical and electrophysiological techniques were used to describe the Ca2+ channel blocking properties of a new dihydropyridine derivative, S11568 (+/-)- ([(amino-2-ethoxy)-2-ethoxy]methyl)-2-(dichloro-2',3'-phenyl)-4- ethoxy-carbonyl-3-methoxycarbonyl-5-methyl-6-dihydro-1,4-pyridine and its enantiomers S12967 ((+)-S11568) and S12968 ((-)-S11568). In binding studies, S11568 and S12968 displaced specifically bound [3H]PN 200-110 from cardiac and vascular smooth muscle preparations with potencies of 5.6-51 nM, respectively. S12967 was 6- to 18-fold less potent than S12968. A good correlation was found between the IC50 value for the inhibition of 45Ca2+ uptake by A7r5 aortic smooth muscle cells and binding data. Whole-cell patch clamp studies in both guinea-pig ventricular myocytes and A7r5 cells yielded similar results. At holding potential (VH) -50 mV, S12968 inhibited L-type Ca2+ current with an IC50 value near 70 nM, 2- to 3-fold more potently than S11568 and 30-fold more potently than S12967. With VH -100 mV, all three compounds were less potent, with IC50 values ranging from 500 nM to 3 microM. These results demonstrate conclusively that S12968 is the more active enantiomer. Furthermore, the pronounced voltage dependence of its actions in vitro suggests that in vivo it could exhibit good selectivity for vascular smooth muscle over cardiac muscle.

The biochemical and pharmacological properties of the (Na+,K+)-ATPase have been studied at different stages of chick embryonic heart development in ovo and under cell culture conditions. The results show the existence of two families of ouabain binding sites: a low affinity binding site with a dissociation constant (Kd) of 2-6 microM for the ouabain-receptor complex and a high affinity binding site with a Kd of 26-48 nM. Levels of high affinity sites gradually decrease during cardiac ontogenesis to reach a plateau near 14 days of development. Conversely the number of low affinity binding sites is essentially invariant between 5 days and hatching. Cultured cardiac cells display the same binding characteristics as those found in intact ventricles. Inhibition of 86Rb+ uptake in cultured cardiac cells and an increase in intracellular Na+ concentration, due to (Na+,K+)-ATPase blockade, occur in a ouabain concentration range corresponding to the saturation of the low affinity ouabain site. Ouabain-stimulated 45Ca2+ uptake increases in parallel with the increase in the intracellular Na+ concentration. It is suppressed in Na+-free medium or when Na+ is replaced by Li+ suggesting that the increase is due to the indirect activation of the Na+/Ca2+ exchange system in the plasma membrane. Dose-response curves for the inotropic effects of ouabain on papillary muscle and on ventricular cells in culture indicate that the development of the cardiotonic properties is parallel to the saturation of the low affinity binding site for ouabain. Therefore, inhibition of the cardiac (Na+,K+)-ATPase corresponding to low affinity ouabain binding sites seems to be responsible for both the cardiotonic and cardiotoxic effects of the drug.