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Impedance of renal vascular smooth muscle cells (VSMCs) cultured on microelectrodes was measured by electric cell-substrate impedance sensing. Changes in measured impedance as a function of frequency were compared with the calculated values obtained from an extended cell-electrode model to estimate the junctional resistance, distance between the ventral cell surface and the substratum, and apical and basolateral membrane capacitances of renal VSMCs. This cell-electrode model was derived to accommodate the slender and rectangular shape of VSMCs. The calculated changes in impedance ([Z.sub.cal]) based on the model agreed well with the experimental measurement ([Z.sub.exp]), and the percentage error defined as |([Z.sub.cal] - [Z.sub.exp])/[Z.sub.exp]| was 1.0%. To test the sensitivity of the new model for capturing changes in cell-cell and cell-substrate interactions induced by changes in cellular environment, we then applied this model to analyze timpedance changes induced by an integrin binding peptide in renal VSMCs. Our result demonstrates that integrin binding peptide decreases junctional resistance between cells, increases the distance between the basolateral cell surface and substratum, and increases the apical membrane capacitance, whereas the basolateral membrane capacitance stays relatively stable. This model provides a generic approach for impedance analysis of cell layers composed of slender, rectangular cells. electric cell-substrate impedance sensing; cell attachment; cell adhesion; extracellular matrix; integrin

Blebbistatin is a myosin II-specific inhibitor. However, the mechanism and tissue specificity of the drug are not well understood. Blebbistatin blocked the chemotaxis of vascular smooth muscle cells (VSMCs) toward sphingosylphosphorylcholine (I[C.sub.50] = 26.1 [+ or -] 0.2 and 27.5 [+ or -] 0.5 [micro]M for GbaSM-4 and A7r5 cells, respectively) and platelet-derived growth factor BB (I[C.sub.50] = 32.3 [+ or -] 0.9 and 31.6 [+ or -] 1.3 [micro]M for GbaSM-4 and A7r5 cells, respectively) at similar concentrations. Immunofluorescence and fluorescent resonance energy transfer analysis indicated a blebbistatin-induced disruption of the actin-myosin interaction in VSMCs. Subsequent experiments indicated that blebbistatin inhibited the [Mg.sup.2+]-ATPase activity of the unphosphorylated (I[C.sub.50] = 12.6 [+ or -] 1.6 and 4.3 [+ or -] 0.5 [micro]M for gizzard and bovine stomach, respectively) and phosphorylated (I[C.sub.50] = 15.0 [+ or -] 0.6 [micro]M for gizzard) forms of purified smooth muscle myosin II, suggesting a direct effect on myosin II motor activity. It was further observed that the [Mg.sup.2+]-ATPase activities of gizzard myosin II fragments, heavy meromyosin (I[C.sub.50] = 14.4 [+ or -] 1.6 [micro]M) and subfragment 1 (I[C.sub.50] = 5.5 [+ or -] 0.4 [micro]M), were also inhibited by blebbistatin. Assay by in vitro motility indicated that the inhibitory effect of blebbistatin was reversible. Electron-microscopic evaluation showed that blebbistatin induced a distinct conformational change (i.e., swelling) of the myosin II head. The results suggest that the site of blebbistatin action is within the S1 portion of smooth muscle myosin II. Boyden chamber; fluorescence resonance energy transfer; ATPase; in vitro motility assay; electron microscopy

Surfactant protein D (SP-D) is a constituent of the innate immune system that plays a role in the host defense against lung pathogens and in modulating inflammatory responses. While SP-D has been detected in extrapulmonary tissues, little is known about its expression and function in the vasculature. Immunostaining of human coronary artery tissue sections demonstrated immunoreactive SP-D protein in smooth muscle cells (SMCs) and endothelial cells. SP-D was also detected in isolated human coronary artery SMCs (HCASMCs) by PCR and immunoblot analysis. Treatment of HCASMCs with endotoxin (LPS) stimulated the release of IL-8, a proinflammatory cytokine. This release was inhibited >70% by recombinant SP-D. Overexpression of SP-D by adenoviral-mediated gene transfer in HCASMCs inhibited both LPS-and TNF-[alpha]-induced IL-8 release. Overexpression of SP-D also enhanced uptake of Chlamydia pneumoniae elementary bodies into HCASMCs while attenuating IL-8 production induced by bacterial exposure. Both LPS and TNF-[alpha] increased SP-D mRNA levels by five-to eightfold in HCASMCs, suggesting that inflammatory mediators upregulate the expression of SP-D. In conclusion, SP-D is expressed in human coronary arteries and functions as an anti-inflammatory protein in HCASMCs. SP-D may also participate in the host defense against pathogens that invade the vascular wall. inflammation; Chlamydia pneumoniae; innate immunity; interleukin-8

Here, we report the design of unprecedented, non-FRET based cGMP-biosensors, named FlincGs, to assess the dynamics of nitric oxide (NO) and atrial natriuretic peptide (ANP) induced synthesis of intracellular cGMP, [[cGMP].sub.i]. Regulatory fragments of PKG I [alpha], PKG I [beta], and an N-terminal deletion mutant of PKG | [alpha] were fused to circular permutated EGFP to generate [alpha]-, [beta]-, and [delta]-FlincG, with high dynamic ranges and apparent [K.sub.D],cGMP values of 35 nM, 1.1 [micro]M, and 170 nM, respectively. All indicators displayed significant selectivity for cGMP over cAMP, and 1.5- to 2.1-fold increases in fluorescence intensity at 510 nm when excited at 480 nm. Surprisingly, FlincGs displayed an additional excitation peak at 410 nm. [delta]FlincG permitted ratiometric (480/410 nm) measurements, with a cGMPspecific 3.5-fold ratio change. In addition, [delta]-FlincG presented cGMP association and dissociation kinetics sufficiently fast to monitor rapid changes of [[cGMP].sub.i] in intact cells. In unpassaged, adenoviral transfected vascular smooth muscle (VSM) cells, [delta]-FlincG had an E[C.sub.50], cGMP of 150 nM, and revealed transient global cGMP elevations to sustained physiological NO ([EC.sub.50,DEA/NO] = 4 nM), and the decay phase depended on the activity of PDE-5. In contrast, ANP elicited sustained submembrane elevations in [[cGMP].sub.i], which were converted to global cGMP elevations by inhibition of PDE-5 by sildenafil. These results indicate that FlincG is an innovative tool to elucidate the dynamics of a central biological signal, cGMP, and that NO and natriuretic peptides induce distinct cGMP patterning under the regulation of PDE-5, and therefore likely differentially engage cGMP targets. fluorescent biosensors | natriuretic peptides | nitrix oxide | confocal microscopy | cGMP-dependent protein kinase

Because inflammatory processes may promote the development of atherosclerosis, we examined the activation of cytokine genes in rat vascular smooth muscle cells in vitro after treatment with bacterial lipopolysaccharide (LPS). Interleukin-1 (IL-l), IL-6 and tumor necrosis factor-[Alpha] (TNF-[Alpha]) mRNA increased in response to LPS. Activation of nuclear factor-[Kappa]B (NF-[Kappa]B) presumably results in NF-[Kappa]B binding to regulatory regions of target genes and activating transcription. We therefore compared the kinetics of NF-[Kappa]B activation, cytokine message production, and TNF-[Alpha] secretion. Maximum active NF-[Kappa]B was found at 30 min after the addition of LPS and decreased thereafter. Increased IL-6 mRNA was detected at 30 min, increased TNF-[Alpha] mRNA at 60 min, and increased IL-1 mRNA at 120 min. Secretion of TNF-[Alpha] was dependent on LPS concentration and was first detected 120 min after LPS addition. Aspirin, which has been shown to inhibit NF-[Kappa]B activation and cytokine secretion in other cell types, did not inhibit NF-[Kappa]B activation or TNF-[Alpha] secretion. However, aspirin reduced the amount of both TNF-[Alpha] and IL-6 mRNA present 30 min after LPS addition by half (P [is less than] 0.05). atherosclerosis; nuclear factor-[Kappa]B; tumor necrosis factor-[Alpha]; aspirin; cytokine gene transcription