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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.

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.

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).

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.

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.

Unlabelled: We studied the effect of propofol (5.6-560 micromol/L; 1-100 microg/mL) on the mechanisms involved in Ca(2+) mobilization elicited by angiotensin II (AngII) in Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. We studied the variations in intracellular Ca(2+) ([Ca(2+)](i)) concentrations in cultured aortic vascular smooth muscle cells (VSMCs) isolated from 6-wk-old WKY and SHR rats loaded with the Ca(2+)-sensitive fluorescent dye, Fura-2, using fluorescent imaging microscopy. In the absence of external Ca(2+), AngII (1 micromol/L) induced a transient [Ca(2+)](i) mobilization from internal stores that was larger in SHR than in WKY rats. Ca(2+) influx was assessed after external Ca(2+) (1 mmol/L) reintroduction. Propofol (1-100 microg/mL) added 5 min before the experiments did not alter AngII-induced Ca(2+) release from internal stores in either strain. By contrast, Ca(2+) influx elicited by AngII was significantly decreased by propofol. This effect occurred at a smaller concentration of propofol in the SHR than in the WKY rats. When Ca(2+) stores were depleted by exposure of cells to thapsigargin, an inhibitor of the sarcoendoplasmic reticulum Ca(2+)-ATPase, reintroduction of Ca(2+) to the medium induced a capacitative Ca(2+) influx of similar magnitude than that elicited by AngII. This influx was also significantly decreased by propofol at 100 microg/mL ( WKY: 27 +/- 3% of control values, n = 107; SHR: 16 +/- 3%, n = 47; P < 0.001). In conclusion, propofol decreased AngII-induced Ca(2+) influx through voltage-independent channels, without altering Ca(2+) release from internal stores in aortic VSMCs. The hypertensive rats were found to be more sensitive to the effect of propofol than the normotensive rats. This suggests that the response of VSMCs to AngII may be altered by propofol., Implications: In rat aortic vascular smooth muscle cells, propofol reduced angiotensin II-elicited Ca(2+) entry through capacitative Ca(2+) channels without altering Ca(2+) release from intracellular stores. Spontaneously hypertensive rats were more sensitive to these effects of propofol than normotensive rats. The response of vascular smooth muscle cells to angiotensin II may be altered by propofol.

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.

This study was to assess the role of different components of the extracellular matrix (ECM) on the mobilization of Cai++ induced by angiotensin II in vascular smooth muscle cells (VSMC) from hypertensive (SHR) and normotensive (WKY) rats. The effect of AII (10-6 M) on Cai++ release was studied in VSMC isolated from the aorta of 5-week-old WKY and SHR using fluorescent imaging microscopy (fura-2). Cai++ mobilization was characterized by amplitude, slope of Cai++ increase and total amount of Cai++. Cells were cultured on glass coverslips (control) or coated with either collagen I, collagen IV, vitronectin, fibronectin and extracellular matrix (ECM) and studied at confluence between passage 3 and 9. A significant increase of Cai++ released by AII has been observed with cells from WKY cultured on collagen I (meam +/- SEM, amplitude: 192 +/- 12% of control values, slope: 194 +/- 13%, total amount Cai++: 173 +/- 12%, n = 270, p < or = 0.0001 for each, unpaired t-test). Conversely, response with SHR was not significatively modified. Cai++ mobilization was not significatively modified after culture of VSMC from SHR and WKY on collagen IV. A significative decrease of the slope (WKY: 66 +/- 6%, p < or = 0.0001; SHR: 83 +/- 5%, p < or = 0.03) and of the amount of Cai++ (WKY: 74 +/- 7%, p < or = 0.01; SHR: 74 +/- 5%, p < or = 0.01) has been observed after culture of VSMC from the 2 strains on vitronectin. A decrease in amplitude (53 +/- 3%, p < or = 0.0001), slope (38 +/- 4%, p < or = 0.0001) and Cai++ release (69 +/- 5%, p < or = 0.004, n = 106) has been observed in VSMC from SHR seeded on fibronectin. Conversely, in VSMC from WKY, Cai++ mobilisation has not been modified compared with control cells. Culture of VSMC from SHR on ECM induced a significative decrease of amplitude (49 +/- 2%), slope (54 +/- 4%) and Cai++ release (53 +/- 3%, p < or = 0.0001 for each, n = 122), while in WKY, ECM induced a significative stimulation of these parameters (amplitude: 157 +/- 11%, slope: 149 +/- 13% and Cai++ release: 130 +/- 9%, p < or = 0.0001 for each, n = 247). These results show that the Cai++ mobilization induced by AII is modified by the adhesion of cells to different ECM components. This suggests a modulation of the A II-associated signalling events via the focal adhesion points. Furthermore, a difference in this modulation is observed between SHR and WKY when cells are seeded on collagen I, fibronectin or ECM. These modulations of Cai++ mobilization could play a role in the regulation of growth and differentiation of cells during the development of hypertension.

We hypothesized that the cytoskeletal network in vascular smooth muscle cells (VSMC) is critical to the signaling pathways from angiotensin (ANG) II-receptor subtype 1 (AT(1)) activation to intracellular Ca(2+) (Ca(2+)(i)) release from internal stores and Ca(2+) influx. This was tested in spontaneously hypertensive rats (SHR), in which differences were reported in cultured aortic VSMC Ca(2+)(i) regulation and G protein function compared with those in normotensive Wistar-Kyoto (WKY) rats. In cultured aortic VSMC, disorganization of actin filaments with cytochalasin D (2 micromol/l) decreased the ANG II-induced Ca(2+)(i) release from internal stores and the ANG II-induced Ca(2+) influx in SHR in a reversible fashion, whereas it was without effect in WKY rats. On the other hand, blocking the dynamic state of the microtubule network significantly reduced ANG II-induced Ca(2+)(i) release from internal stores but was without effect on Ca(2+) influx in either SHR or WKY rats. This study demonstrates for the first time that, in the SHR, actin filaments play a major role in linking AT(1)-receptor activation to both Ca(2+)(i) release mechanisms and capacitative Ca(2+) influx. Furthermore, a functionally intact microtubule system is a necessary prerequisite for ANG II-induced Ca(2+)(i) release in both strains.