Bovine Coronary Artery Smooth Muscle Research Articles

Pegylated Cholesterol and Methyl-Beta-Cyclodextrin are Modulators of L-Type Calcium Channel Current and Decrease Membrane Capacitance in Vascular Smooth Muscle Cells

Loading of pegylated cholesterol (PEG-cholesterol) by pretreatment of several hours decreases current density and augments voltage-dependent inactivation of L-type Ca channel current (ICa,L) and depletion of cholesterol by similar long pretreatment with methyl-beta-cyclodextrin (MbCD) increases the ICa,L density in A7r5 aortic smooth muscle cells. On the other hand, dehydroepiandrosterone (DHEA), a cholesterol-derived steroid hormone, rapidly induces voltage-dependent inhibition of ICa,L. If endocytosis and exocytosis of CaV1.2-containing membrane vesicles are involved in these modulations, they should induce a change in membrane capacitance (Cm). Cm could be affected also by a change of thickness and dielectric constant of lipid bilayer. Here we studied acute effects of PEG-cholesterol, MbCD and DHEA on Cm of isolated bovine coronary artery smooth muscle cells by whole-cell patch clamp technique. Ramp steps of 5 or 10 ms were applied repetitively before, during and after application of the modulators. Control Cm was 18.3±4.5 pF (mean±SD.). PEG-cholesterol and MbCD induced gradual and small decrease of Cm and their washout decelerated the rate of the decrease. Cm after 10 min of wash-in normalized by the initial value was: control without modulator, 1.01±0.02 (mean±SD.); 1 mM PEG-cholesterol, 0.96±0.02 (p<0.01, compared with control); 10 mM MbCD, 0.90±0.05 (p<0.0001); 0.1 mM DHEA, 1.00±0.03. Since the change of Cm is small, it little affects qualitative conclusion of the modulation of ICa,L density obtained by the pretreatment experiments with PEG-cholesterol and MbCD. The small but significant decrease of Cm reflects structural changes of the lipid bilayer which may be involved in the modulation of ICa,L by PEG-cholesterol and MbCD.

Does Dehydroepiandrosterone Directly Inhibit L-Type Ca Channel Current in Vascular Smooth Muscle?

Dehydroepiandrosterone (DHEA) is an abundantly released pro-hormone from adrenal gland and a neuro-modulator released locally in the brain. DHEA inhibits L-type Ca2+ channel current (ICa,L) at relatively high concentration. However, since serum concentration of DHEA-sulfate, an inactive form of DHEA, is ∼10−5 M and it is locally converted to DHEA by sulfatase, DHEA may regulate vascular contraction in situ. The mechanism of how DHEA inhibits ICa,L has remained unresolved. In the present study, we explored whether DHEA inhibits ICa,L by direct interaction with CaV1.2 in cultured A7r5 cell line from fetal rat aorta and isolated bovine coronary artery smooth muscle cells. We performed whole cell and cell-attached recording of ICa,L and applied DHEA either globally by bath application or locally to patch membrane via pipette solution. With whole recording ICa,L amplitude was inhibited by bath applied DHEA (0.1 mM) in a few minutes associated with an acceleration of the current decay. The washout of DHEA caused a rapid reversal. Single and multichannel ICa,Ls were recorded with 90 mM Ba2+ as a charge carrier in the presence of Bay K 8644 in the pipette solution. They exhibited characteristic long open time and constant unitary current amplitude from the start of recording. However, DHEA (0.1 mM) included in the pipette solution did not accelerate the transition to the long shut state in the single channel recording nor accelerate the time course of inactivation of the multichannel current for 5-10 min. On the other hand, mean currents from the cell-attached recording were reversibly inhibited by bath-applied DHEA. Therefore, if DHEA interacts directly with CaV1.2 in the membrane, it does not play major role in the steroid-induced inhibition of ICa,L.

Apocynin inhibits and its removal augments L‐type Ca2+ currents in coronary artery smooth muscle cells and ventricular myocytes

Exogenously applied H2O2 augments L‐type Ca2+ current (ICa,L). NADPH oxidase (NOX) initiates sequential reactions to generate reactive oxygen species (ROS) including H2O2. We studied the regulatory role of NOX‐generated ROS on ICa,L by applying apocynin (APO), a NOX inhibitor, and diphenyleneiodonium (DPI), a flavoprotein inhibitor. Whole cell ICa,L was recorded from bovine coronary arterial smooth muscle (BCASMC), rat ventricular myocytes (RVMC) and HEK 293 cells transiently expressing human cardiac CaV1.2. APO (0.1–10 mM) rapidly and dose‐dependently inhibited ICa,L: 10‐mM APO decreased ICa,L to 27, 34 and 16% in BCASMC, RVMC and 293 cells, respectively, while DPI (10 μM) inhibited more slowly and less intensively (to 64% in RVMC). The inhibitory effects of both inhibitors were mainly additive. Surprisingly, washout of high concentrations of APO rapidly recovered ICa,L to slightly above control in BCASMC and increased to 150% of control with prolonged current decay in RVMC. ROS levels measured simultaneously with ICa,L by increase rate of CM‐H2DCF fluorescence were decreased by APO and markedly increased by its removal in BCAVSM and RVMC. Possible accumulation of NADPH during NOX inhibition by APO explains the increased generation of ROS. We conclude that decrease of ROS inhibits and its increase augments cardiovascular ICa,L and NOX is involved in the regulation of ICa,L. This study is supported by NIH grant #R01HL85352

Apocynin Reversibly Inhibits L-type Ca2+ Channel Current Involvement of Reactive Oxygen Species

L-type Ca2+ channel current (ICa,L) is a major Ca2+ entry mechanism and contraction of arterial smooth muscle (ASM) is regulated by reactive oxygen species (ROS). We utilized apocynin (APO) as a tool to clarify the contribution of ROS in the regulation of ICa,L. APO, a natural organic compound contained in a variety of plants, is widely used as inhibitor of NADPH oxidase (NOX) that reduces oxygen to superoxide in the presence of NADPH to generate ROS including H2O2. We recorded whole cell ICa,L with Ba2+ as charge carrier from isolated bovine coronary ASM (BCASM) and HEK293 cells transiently expressing human cardiac CaV1.2. APO was introduced and washed out during continuous application of depolarization step to 0 mV from HP −80 mV at 1/min. We found that APO inhibited ICa,L in a dose-dependent manner between 0.1 and 10 mM decreasing its amplitude to ∼20% of control. APO also accelerated ICa,L decay during depolarization. Surprisingly, washout of the high concentration of APO caused rapid recovery of ICa,L. It could even produce a rebound increase of ICa,L with its peak at ∼3 min in BCASM. We performed fluorometric analysis of APO-induced change of cellular ROS by loading cells with 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA). Oxidation of CM-H2DCF to CM-DCF by ROS induces an increase of fluorescence. APO markedly inhibited the fluorescence increase, while washout of APO caused more intensive increase of fluorescence than control. The results are explained by the inhibition of NOX by APO which results in decrease of ROS and overproduction of ROS during washout utilizing accumulated NADPH produced by prior NOX inhibition. ROS which changes dynamically in situ, e.g. hypoxia and reoxygenation, seems to be vital to sustain ICa,L.

Mitochondrial-derived hydrogen peroxide inhibits relaxation of bovine coronary arterial smooth muscle to hypoxia through stimulation of ERK MAP kinase

Mitochondrial reactive oxygen species (ROS) are potentially important in vascular oxygen-sensing mechanisms because hypoxia appears to be a stimulus for mitochondrial ROS generation; however, scavenging of endogenous ROS does not alter relaxation of endothelium-denuded bovine coronary arteries (BCA) to hypoxia. The purpose of this study was to investigate the influence of increasing mitochondrial ROS on the relaxation of BCA to hypoxia. Increasing mitochondrial superoxide with inhibitors of electron transport (10 microM rotenone and antimycin) and by opening mitochondrial ATP-dependent K+ channels with 100 microM diazoxide were observed in this study to attenuate relaxation of BCA precontracted with 30 mM KCl to hypoxia by 68-76% and 38%, respectively. This effect of rotenone is not prevented by inhibiting NADPH oxidase (Nox) activation or scavenging superoxide with Peg-SOD; however, it is reversed 85% and 26% by increasing the consumption of intracellular peroxide by 0.1 mM ebselen and 32.5 U/ml Peg-catalase. Because inhibition of extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase (10 microM PD-98059), but not src kinase or rho kinase, also reverses the effects of rotenone by 69%, the peroxide-elicited force-enhancing effects of ERK appear to be attenuating the response to hypoxia. Rotenone increased the phosphorylation of ERK (by 163%). Activation of ERK in BCA with 0.1 mM peroxide or endogenous peroxide generated by stimulating Nox2 with a stretch treatment or contraction with 100 nM U-46619 also attenuated relaxation to hypoxia. Thus coronary arterial relaxation to hypoxia may be attenuated by pathophysiological conditions associated with increased peroxide generation by mitochondria or other sources that stimulate ERK.

Loss of function of CaV1.2 in cultured bovine coronary artery smooth muscle cells

L‐type Ca2+ current (ICa,L) through Cav1.2 α subunit pore controls contraction and phenotype change between contractile and proliferative type in vascular smooth muscle cell (VSMC). ICa,L enhances the expression of proteins that are characteristic for contractile phenotype. ICa,L is small in proliferative VSMC. The phenotype change to proliferative type is involved in atherogenesis. To explore underlying mechanism of the reduction of ICa,L reduction in proliferative VSMC, we have compared (1) the density of ICa,L between freshly‐isolated and proliferative cultured bovine coronary artery smooth muscle cell (BCASMC) and (2) the effect of CaV1.2 α subunit transfection to cultured BCASMC and HEK293 cells. Whole cell ICa,L was recorded with 10 mM Ba2+ as the charge carrier. ICa,L was 3.0 ± 0.4 pA/pF (n=28) in freshly isolated BCASMC but was not detectable in cultured BCASMC (N=15). Significant expression of CaV1.2 α subunit was confirmed by western blot in cultured BCASMC. ICa,L density was 4.2±0.7 pA/PF (n=15) in HEK293 cells transfected with human cardiac CaV1.2α‐GFP. However, ICa,L was not detectable in cultured BCASMC with the same transfection (n=15). We conclude that loss of function of expressed CaV1.2 is responsible for the reduction of ICa,L in the proliferatative vascular smooth muscle cells. This study is supported by NIH grant#R01HL85352

Inhibition of L-type Ca2+ Channel Window Current By Steroid Hormones in Coronary Artery Smooth Muscle Cells

Small and sustained depolarization induces window current (IWC) through L-type Ca2+ channel that supplies Ca2+ for arterial contraction. Existence of IWC is expected by voltage dependence of steady-state activation and inactivation. However, IWC of arterial smooth muscle has little been characterized by actual current recording. Dehydroepiandrosterone (DHEA) and DHEA-sulphate (DHEA-S) are most abundant adrenal steroid hormones and epiandrosterone (EPI) is a metabolite of DHEA. We recorded ICa,L and IWC with 10 mM Ba2+ as the charge carrier, performed noise analysis and examined effects of the steroids on IWC and its unitary current in bovine coronary artery smooth muscle cells. EPI and DHEA, but not DHEA-S, slightly inhibited ICa,L elicited by depolarization from large negative holding potentials. EPI and DHEA but not DHEA-S shifted steady-state inactivation curve slightly to a negative direction and strongly reduced residual availability at depolarized potentials. IWC elicited by staircase depolarizarion consisted of stair-duration of 20 s appeared at -40 mV and increased up to -10 mV. It decreased with further increase of depolarization. Maximal IWC was 23±2% (n=12) of maximal ICa,L. IWC was associated with marked current noise. IWC and the noise were strongly inhibited by EPI and DHEA, not affected by DHEA-S, and increased by Bay K 8644. Unitary current (iCa,L) and NPo were obtained from mean and variance of IWC. Uunitary conductance (γ) from iCa,L was 17.2 pS. EPI and DHEA decreased NPo and Bay K increased it both without affecting γ. In conclusion, EPI and DHEA inhibit L-type Ca2+ channel window current by accelerating inactivation and thus decreasing the number of opened channels.

Identification and characterization of the unique guanine nucleotide exchange factor, SmgGDS, in vascular smooth muscle cells

The guanine nucleotide exchange factor (GEF), SmgGDS, promotes nucleotide exchange by several GTPases in both the Ras and Rho families, especially by RhoA. Because RhoA plays an important role in regulating the contraction of vascular smooth muscle cells (VSMC), we examined the expression and function of SmgGDS in VSMC. SmgGDS is expressed in primary rat aortic smooth muscle (ASM) cells, primary bovine coronary artery smooth muscle (BCASM) cells, and the immortalized A7r5 line of rat ASM cells. Down regulation of SmgGDS expression by siRNA transfection resulted in a decrease of RhoA-GTP levels, enhanced cell spreading, and loss of the characteristic elongated morphology of VSMC. A similar morphology was also observed following treatment with the Rho-kinase inhibitor, Y27632. In contrast, cells with reduced RhoA expression exhibit an elongated shape. Subsequent immunofluorescent staining revealed a disruption of the myosin filament organization in the cells with reduced SmgGDS expression. Further studies analyzed the effect of SmgGDS siRNA transfection on the contraction of A7r5 cells and BCASM cells, which is also a Rho-regulated pathway. Transfection of SmgGDS siRNA or RhoA siRNA resulted in an impaired ability of the A7r5 and BCASM cells to undergo contraction in a collagen gel matrix. However, phosphorylation of the myosin-binding subunit of myosin phosphatase (MYPT1) or the light chain of myosin II (MLC) was not altered by downregulating expression of either SmgGDS or RhoA GTPase. Taken together these results identify SmgGDS as a novel regulator of myosin organization and contraction in VSMC.

Epiandrosterone activates BKCa channel in bovine coronary artery smooth muscle cells

Epiandrosterone (EPI), a metabolite of testosterone precursor dehydroepiandrosterone, causes relaxation of vascular smooth muscle (VSM) associated with inhibition of glucose‐6‐phosphate dehydrogenase (G6PD), which increases cellular reducing potential by converting NADP+ to NADPH. BKCa channel is a primary K+ channels in VSM. We studied whether EPI regulates BKCa channel and whether G6PD is involved in cultured bovine coronary artery smooth muscle cells (CASMC). Whole‐cell BKCa current (IBK) was recorded in the presence of 5.4 mM [K+]o/30 mM [K+]i and 0.1 or 1 μM [Ca2+]i. IBK was predominant K+ current in CASMC. IBK increased with moderate increase of depolarization, but large depolarizations caused inactivation presenting bell‐shaped I‐V relationship. The increase of [Ca2+]i caused negative shift of maximal BKCa conductance (GBKCa). EPI (&gt;3 μM) increased GBKCa and shifted voltage‐dependence of activation further to a negative direction; GBKCa increased by ∼50% at 10 μM and by ∼100% at 100 μM. EPI caused hyperpolarization that was reversed by TEA. Suppression of G6PD by siRNA did not affect GBKCa and EPI caused similar modulation of IBK in the G6PD KO myocytes. EPI is a novel activator of BKCa channel and the activation is independent of its inhibitory action on G6PD. EPI‐induced increase in IBK may cause relaxation of CASMC via hyperpolarization.

Subcellular Changes in NAD(P)H Redox Caused by Hypoxia in Bovine Coronary Artery Smooth Muscle Cells.

Subcellular Changes in NAD(P)H Redox Caused by Hypoxia in Bovine Coronary Artery Smooth Muscle Cells.

Ultrasound Microbubble Delivery of Ca 2+ Signaling Second Messengers into Bovine Coronary Arterial Smooth Muscle Cells

Ultrasound microbubble technique has been widely used for gene transfer and drug delivery into cells. So far little is known whether this delivery strategy can be used for studies of intracellular signaling molecules in the regulation of cell function. The present study was designed to test the applicability of this ultrasound microbubble mediated delivery method to the studies of Ca2+ signaling second messengers. Freshly isolated bovine coronary arterial smooth muscle cells (CASMCs) were used to monitor Ca2+ release response and Ca2+-dependent superoxide production. These cells were loaded with Fura-2 Am and dihydroethidium (DHE), and Ca2+ transient and conversion of DHE to ethidium (Eth) were monitored simultaneously. Ca2+ mobilizing second messengers inositol-1,4,5-triphosphate (IP3), cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) were mixed with 40 μl microbubble solution, respectively before added into the reaction bath solution at 37°C. Ultrasound with a frequency of 1MHZ was then applied to drive microbubbles into cells. We found that IP3 (5 μM), cADPR (10 μM) or NAADP (40 nM) induced a rapid increase in fura 2 F340/F380 fluorescent ratio accompanied by a relatively slow increase in DHE/Eth ratio within CASMCs. Maximal Δ[Ca2+]i reached 729 ± 31, 691 ± 24, 592 ± 47 nM when we introduced IP3, cADPR or NAADP, respectively. These increases in intracellular Ca2+ are accompanied by increases in superoxide production by 60–80%. These results suggest that this ultrasound microbubble delivery method is suitable for effective introduction of intracellular second messenger compounds to activate Ca2+ release and Ca2+-dependent NAD(P)H oxidase. (Supported by NIH Grants HL57244, HL70726, and HL075316)

NADPH OXIDASE‐MEDIATED O 2 −· PRODUCTION AMPLIFIES VASOCONSTRICTOR RESPONSE OF SMALL CORONARY ARTERIES TO M 1 ‐MUSCARINIC RECEPTOR ACTIVATION

The present study test the hypothesis that NADPH oxidase (Nox) mediated O2−· production amplifies coronary arterial constrictor response to M1-receptor (M1R) agonists. Fluorescence imaging analysis showed that M1R agonist, acetylcholine (Ach), simultaneously increased intracellular Ca2+ and O2−· levels in bovine coronary arterial smooth muscle cells (CASMCs) with a maximal increase of 821 + 67 nM in Ca2+ levels accompanied by 75% in O2−· production. However, when cells were transfected with siRNA of p47phox(a key subunit for Nox activation), Ach-induced increases in Ca2+ and O2−· were significantly attenuated. Similarly, p47phoxsiRNA significantly reduced oxotremorine (OXO)-induced O2−· production in CASMCs by 67%. Functionally, OXO-induced constrictor response of endothelium-denuded small bovine coronary arteries was significantly reduced by Nox inhibitor, DPI or O2−· scavenger cocktail (tempol + catalase). Moreover, introduction of siRNA for p47phox, NOX1 and NOX4 into arterial smooth muscle by ultrasound microbubble technology significantly blocked this OXO-induced contraction. We concluded that Nox-derived O2−· is involved in M1R-mediated intracellular Ca2+ release in CASMCs and thereby importantly contributes to amplification of vasoconstrictor response of coronary arteries to M1R activation (Supported by NIH grants HL057244, HL075316 and AHA predoctoral fellowship 0410061Z).

Expression and Functions of Nox Isoforms in Bovine Coronary Arterial Smooth Muscle Cells ‐ Contribution to Extracellular O 2 ·− sLevels

Expression and Functions of Nox Isoforms in Bovine Coronary Arterial Smooth Muscle Cells ‐ Contribution to Extracellular O ₂ ^·− sLevels

Simultaneous Monitoring of Intra‐ and Extracellular O 2 ·− Production Associated with NAD(P)H Oxidase in Single Coronary Arterial Smooth Muscle Cells

The present study was designed to develop a novel method to simultaneously monitor extra- and intracellular O2·− production in single bovine coronary arterial smooth muscle cells (CASMCs). Based on the fluorescent oxidation of dihydroethidium (DHE) to ethidium (Eth) and Eth-DNA binding inside and outside cells, salmon DNA (5 mg/ml) was added on surface of isolated CASMCs and then Matrigel solution was loaded on the top as supportive matrix to trap DNA when it was polymerized at 37°C. Before monitoring O2·− production, CASMCs were loaded with 25 μM DHE for 60 min. Using a high speed wavelength switching fluorescence imaging system, the dynamic changes in Eth-DNA fluorescence (O2·− production) were simultaneously monitored inside and outside CASMCs. It was found that oxotremorine (OXO) induced increase in extracellular O2·− production was earlier and more rapid than intracellular one. A similar pattern was also observed when CASMCs were challenged by another M1-receptor agonist, acetylcholine. In the presence of superoxide dismutase (500 U/ml), NAD(P)H oxidase inhibitor, diphenylene iodonium (50 μM) or apocynin (30 μM), or in CASMCs transfected with siRNA of Nox1, OXO-induced O2·− production inside and outside CASMCs was substantially blocked. However, angiotensin II induced a different pattern showing a parallel increase in O2·− Eth-DNA signal both outside and inside cells. In conclusion, a simultaneous monitoring assay of intra- and extracellular O2·− production is established, which can be used for studies of dynamic changes in NAD(P)H oxidase activity and for clarification of the orientation of O2·− production from single CASMCs (Supported by NIH Grants HL57244, HL70726, and HL075316).

Enhanced production and action of cyclic ADP–ribose during oxidative stress in small bovine coronary arterial smooth muscle

Recent studies in our lab and by others have indicated that cyclic ADP–ribose (cADPR) as a novel second messenger is importantly involved in vasomotor response in various vascular beds. However, the mechanism regulating cADPR production and actions remains poorly understood. The present study determined whether changes in redox status influence the production and action of cADPR in coronary arterial smooth muscle cells (CASMCs) and thereby alters vascular tone in these arteries. HPLC analyses demonstrated that xanthine (X, 40 μM)/xanthine oxidase (XO, 0.1 U/ml), a superoxide-generating system, increased the ADP–ribosyl cyclase activity by 59% in freshly isolated bovine CASMCs. However, hydrogen peroxide (H 2O 2, 1–100 μM) had no significant effect on ADP–ribosyl cyclase activity. In these CASMCs, X/XO produced a rapid increase in [Ca 2+] i (Δ[Ca 2+] i = 201 nM), which was significantly attenuated by a cADPR antagonist, 8-Br-cADPR. Both inhibition of cADPR production by nicotinamide (Nicot) and blockade of Ca 2+-induced Ca 2+ release (CICR) by tetracaine (TC) and ryanodine (Rya) significantly reduced X/XO-induced rapid Ca 2+ responses. In isolated, perfused, and pressurized small bovine coronary arteries, X at 2.5–80 μM with a fixed XO level produced a concentration-dependent vasoconstriction with a maximal decrease in arterial diameter of 45%. This X/XO-induced vasoconstriction was significantly attenuated by 8-Br-cADPR, Nicot, TC, or Rya. We conclude that superoxide activates cADPR production, and thereby mobilizes intracellular Ca 2+ from the SR and produces vasoconstriction in coronary arteries.

14,15-Epoxyeicosa-5(Z)-Enoic-mSI

Endothelium-dependent hyperpolarizations and relaxation of vascular smooth muscle induced by acetylcholine and bradykinin are mediated by endothelium-derived hyperpolarizing factors (EDHFs). In bovine coronary arteries, arachidonic acid metabolites, epoxyeicosatrienoic acids (EETs), function as EDHFs. The 14,15-EET analog 14,15-epoxyeicosa-5(Z)-enoic-methylsulfonylimide (14,15-EEZE-mSI) was synthesized and tested for agonist and antagonist activity. In U46619-preconstricted bovine coronary arterial rings, 14,15-, 11,12-, 8,9-, and 5,6-EET induced maximal concentration-related relaxation averaging 75% to 87% at 10 micromol/L, whereas, 14,15-EEZE-mSI induced maximal relaxation averaging only 7%. 14,15-EEZE-mSI (10 micromol/L) preincubation inhibited relaxation to 14,15- and 5,6- EET but not 11,12- or 8,9- EET. 14,15-EEZE-mSI also inhibited indomethacin-resistant relaxation to arachidonic acid and indomethacin-resistant and l-nitroarginine-resistant relaxation to bradykinin and methacholine. It did not alter the relaxation to sodium nitroprusside, iloprost, or the K+ channel openers bimakalim or NS1619. In cell-attached patches of isolated bovine coronary arterial smooth muscle cells, 14,15-EEZE-mSI (100 nmol/L) blocked the 14,15-EET-induced (100 nmol/L) activation of large-conductance, calcium-activated K+ channels. Mass spectrometric analysis of rat renal cortical microsomes incubated with arachidonic acid showed that 14,15-EEZE-mSI (10 micromol/L) increased EET concentrations while decreasing the concentrations of the corresponding dihydroxyeicosatrienoic acids. Therefore, 14,15-EEZE-mSI inhibits relaxation to 5,6- and 14,15- EET and the K+ channel activation by 14,15-EET. It also inhibits the EDHF component of bradykinin-induced, methacholine-induced, and arachidonic acid-induced relaxation. These results suggest that 14,15- or 5,6 -EET act as an EDHF in bovine coronary arteries.

CD40 ligand (CD40L) does not stimulate proliferation of vascular smooth muscle cells

The present study investigates the effects of CD40 ligand (CD40L) on mitogenic signalling, proliferation, and migration of cultured bovine coronary artery smooth muscle cells (SMC). A time- and concentration-dependent phosphorylation of the extracellular signal-regulated kinases-1/2 (ERK-1/2) and the mitogen-activated protein kinase p38 (p38-MAPK) was observed upon stimulation with soluble CD40L (sCD40L). This phosphorylation was inhibited by neutralizing antibodies against the CD40 and CD40L, respectively. Activation of the phosphatidylinositol-3-phosphate (PI-3) kinase pathway by sCD40L, as determined by the measurement of Akt phosphorylation, was not detected. However, there was evidence for direct activation of the NFkappaB system (degradation of IkappaBalpha and nuclear translocation of the p65 NFkappaB subunit) by sCD40L. Accordingly, sCD40L caused a small but significant increase in DNA synthesis. However, sCD40L-induced DNA synthesis was not followed by proliferation (increase in cell number). Furthermore, sCD40L did not potentiate SMC mitogenesis induced by known mitogens such as platelet-derived growth factor-BB, thrombin or serum. The lack of cell proliferation was not caused by a concomitant induction of SMC apoptosis by sCD40L. The possible role of membrane-bound CD40L in SMC mitogenesis was also studied using different membrane preparations (platelets, lymphocytes). However, no mitogenic effects of membrane-bound CD40L were detected. Finally, sCD40L did not induce SMC migration. From these data it is concluded that CD40L activates mitogenic signalling and DNA synthesis but does not contribute to proliferation or migration of vascular SMC.

Role of FKBP12.6 in cADPR-induced activation of reconstituted ryanodine receptors from arterial smooth muscle.

cADP ribose (cADPR) serves as second messenger to activate the ryanodine receptors (RyRs) of the sarcoplasmic reticulum (SR) and mobilize intracellular Ca(2+) in vascular smooth muscle cells. However, the mechanisms mediating the effect of cADPR remain unknown. The present study was designed to determine whether FK-506 binding protein 12.6 (FKBP12.6), an accessory protein of the RyRs, plays a role in cADPR-induced activation of the RyRs. A 12.6-kDa protein was detected in bovine coronary arterial smooth muscle (BCASM) and cultured CASM cells by being immunoblotted with an antibody against FKBP12, which also reacted with FKBP12.6. With the use of planar lipid bilayer clamping techniques, FK-506 (0.01-10 microM) significantly increased the open probability (NP(O)) of reconstituted RyR/Ca(2+) release channels from the SR of CASM. This FK-506-induced activation of RyR/Ca(2+) release channels was abolished by pretreatment with anti-FKBP12 antibody. The RyRs activator cADPR (0.1-10 microM) markedly increased the activity of RyR/Ca(2+) release channels. In the presence of FK-506, cADPR did not further increase the NP(O) of RyR/Ca(2+) release channels. Addition of anti-FKBP12 antibody also completely blocked cADPR-induced activation of these channels, and removal of FKBP12.6 by preincubation with FK-506 and subsequent gradient centrifugation abolished cADPR-induced increase in the NP(O) of RyR/Ca(2+) release channels. We conclude that FKBP12.6 plays a critical role in mediating cADPR-induced activation of RyR/Ca(2+) release channels from the SR of BCASM.

An approach to constructing three-dimensional tissue.

The authors propose an approach to constructing three-dimensional tissue with capillaries using cellulose hollow fibers. Fibronectin (FN) was immobilized on hollow fibers to assure cell attachment. Bovine coronary artery smooth muscle cells (BCASMC) and L cells were seeded on FN-immobilized fibers and cultured for an extended period of time. The cells proliferated and formed multicellular layers on the fibers. The hollow fibers were removed by enzymatic digestion using cellulase. The cellulase treatment did not damage L cells, although some cells fell off from the fibers. On the other hand, no deterioration was observed in the BCASMC aggregate structure. The BCASMC aggregates maintained several lumens after removal of the hollow fibers by cellulase digestion. The authors believe that their approach offers a useful method to tissue engineering in preparation of three-dimensional tissue structure.

Nitric oxide inhibits Ca(2+) mobilization through cADP-ribose signaling in coronary arterial smooth muscle cells.

The present study was designed to determine whether the cADP-ribose-mediated Ca(2+) signaling is involved in the inhibitory effect of nitric oxide (NO) on intracellular Ca(2+) mobilization. With the use of fluorescent microscopic spectrometry, cADP-ribose-induced Ca(2+) release from sarcoplasmic reticulum (SR) of bovine coronary arterial smooth muscle cells (CASMCs) was determined. In the alpha-toxin-permeabilized primary cultures of CASMCs, cADP-ribose (5 microM) produced a rapid Ca(2+) release, which was completely blocked by pretreatment of cells with the cADP-ribose antagonist 8-bromo-cADP-ribose (8-Br-cADPR). In intact fura 2-loaded CASMCs, 80 mM KCl was added to depolarize the cells and increase intracellular Ca(2+) concentration ([Ca(2+)](i)). Sodium nitroprusside (SNP), an NO donor, produced a concentration-dependent inhibition of the KCl-induced increase in [Ca(2+)](i), but it had no effect on the U-46619-induced increase in [Ca(2+)](i). In the presence of 8-Br-cADPR (100 microM) and ryanodine (10 microM), the inhibitory effect of SNP was markedly attenuated. HPLC analyses showed that CASMCs expressed the ADP-ribosyl cyclase activity, and SNP (1-100 microM) significantly reduced the ADP-ribosyl cyclase activity in a concentration-dependent manner. The effect of SNP was completely blocked by addition of 10 microM oxygenated hemoglobin. We conclude that ADP-ribosyl cyclase is present in CASMCs, and NO may decrease [Ca(2+)](i) by inhibition of cADP-ribose-induced Ca(2+) mobilization.