Smooth Muscle Cell Ca Research Articles

Tmem16a‐induced ClCa currents are regulated via phosphotransferase activity

Ca2+‐activated Cl− currents (ICl(Ca)) represent a major excitatory mechanism in vascular smooth muscle cells (SMCs). In arterial SMCs, ICl(Ca) are down‐regulated by calmodulin‐dependent protein kinase II (CaMKII) and up‐regulated by calcineurin (CaN) and PP1. Recently, Tmem16a has emerged as a gene candidate for the native ICl(Ca) in pulmonary artery SMCs. HEK‐293 cells over‐expressing Tmem16a displayed time‐ and voltage‐dependent Cl− currents when dialyzed with 500 nM free Ca2+ that resembled the properties of ICl(Ca) from vascular SMCs. Similar to ICl(Ca) from VSMCs, Tmem16a‐induced ICl(Ca) also displayed enhanced rundown when cells were dialyzed with 5 mM ATP and this process was attenuated in the absence of ATP. Similar to ICl(Ca) in rabbit VSMCs (Ayon et al., J Biol Chem 284:32507–32521, 2009), dialysis of 30 nM okadaic acid, a nonselective PP1/PP2A inhibitor, impeded the recovery of Tmem16a‐dependent Cl− currents in the absence of ATP. Analysis of mouse Tmem16a protein sequence revealed 5 consensus sites for phosphorylation by CaMKII, which are believed to lie on protein segments facing the cytoplasm. A single point mutation of one of these putative CaMKII phosphorylation sites (Threonine 610 to Alanine) did not alter rundown of the channels. Taken together, these data suggest that much like native ICl(Ca), Tmem16a‐dependent Cl− currents also appear to be regulated by at least one phosphorylation step.

Calcium Homeostasis in Vascular Smooth Muscle Cells is altered in Type 2 Diabetes by Bcl‐2 Protein Modulation of InsP3R Calcium Release Channels

The current study examines the extent to which antiapoptotic Bcl‐2 proteins Bcl‐2 and Bcl‐XL contribute to aortic vascular smooth muscle cell (VSMC) Ca2+ dysregulation and contractility in the db/db mouse model of type 2 diabetes. Expression levels of Bcl‐XL were found to be higher in tissues isolated from diabetic (db/db) mice compared to non‐diabetic (db/m) controls. In single cells, inositol 1,4,5‐trisphosphate receptor (InsP3R)‐dependent [Ca2+]i signaling was enhanced in db/db VSMCs. This was attributed to alterations in the intrinsic properties of the InsP3R because there were no differences between db/db and db/m in the steady state [Ca2+]SR or InsP3R expression levels. Moreover, in permeabilized cells the rate of InsP3R‐dependent SR Ca2+ release was increased in db/db compared to db/m, and attenuated only in db/db cells by the Bcl‐2 protein inhibitor ABT‐737. Experiments in which [Ca2+]i and constriction were recorded simultaneously in cannulated intact arteries, application of ABT‐737 similarly attenuated enhanced agonist‐induced [Ca2+]i signaling only in intact db/db vessels. In contrast, ABT‐737 had no effect on agonist‐evoked contractility in either db/db or db/m vessels. In conclusion, elevated Bcl‐XL in diabetic VSMCs increases InsP3R excitability, the resulting dysregulated [Ca2+]i signaling, however, does not correlate with increased vessel reactivity.

CaV1.2 Channel N-terminal Splice Variants Modulate Functional Surface Expression in Resistance Size Artery Smooth Muscle Cells

Voltage-dependent Ca(2+) (Ca(V)1.2) channels are the primary Ca(2+) influx pathway in arterial smooth muscle cells and are essential for contractility regulation by a variety of stimuli, including intravascular pressure. Arterial smooth muscle cell Ca(V)1.2 mRNA is alternatively spliced at exon 1 (e1), generating e1b or e1c variants, with e1c exhibiting relatively smooth muscle-specific expression in the cardiovascular system. Here, we examined physiological functions of Ca(V)1.2e1 variants and tested the hypothesis that targeting Ca(V)1.2e1 modulates resistance size cerebral artery contractility. Custom antibodies that selectively recognize Ca(V)1.2 channel proteins containing sequences encoded by either e1b (Ca(V)1.2e1b) or e1c (Ca(V)1.2e1c) both detected Ca(V)1.2 in rat and human cerebral arteries. shRNA targeting e1b or e1c reduced expression of that Ca(V)1.2 variant, induced compensatory up-regulation of the other variant, decreased total Ca(V)1.2, and reduced intravascular pressure- and depolarization-induced vasoconstriction. Ca(V)1.2e1b and Ca(V)1.2e1c knockdown reduced whole cell Ca(V)1.2 currents, with Ca(V)1.2e1c knockdown most effectively reducing total Ca(V)1.2 and inducing the largest vasodilation. Knockdown of α(2)δ-1, a Ca(V)1.2 auxiliary subunit, reduced surface expression of both Ca(V)1.2e1 variants, inhibiting Ca(V)1.2e1c more than Ca(V)1.2e1b. e1b or e1c overexpression reduced Ca(V)1.2 surface expression and whole cell currents, leading to vasodilation, with e1c overexpression inducing the largest effect. In summary, data indicate that arterial smooth muscle cells express Ca(V)1.2 channels containing e1b or e1c-encoded N termini that contribute to Ca(V)1.2 surface expression, α(2)δ-1 preferentially traffics the Ca(V)1.2e1c variant to the plasma membrane, and targeting of Ca(V)1.2e1 message or the Ca(V)1.2 channel proximal N terminus induces vasodilation.

Amplification of EDHF‐type vasodilatations in TRPC1‐deficient mice

TRPC1 channels are expressed in the vasculature and are putative candidates for intracellular Ca(2+) handling. However, little is known about their role in endothelium-dependent vasodilatations including endothelium-derived hyperpolarizing factor (EDHF) vasodilatations, which require activation of Ca(2+) -activated K(+) channels (K(Ca)). To provide molecular information on the role of TRPC1 for K(Ca) function and the EDHF signalling complex, we examined endothelium-dependent and independent vasodilatations, K(Ca) currents and smooth muscle contractility in TRPC1-deficient mice (TRPC1-/-). Vascular responses were studied using pressure/wire myography and intravital microscopy. We performed electrophysiological measurements, and confocal Ca(2+) imaging for studying K(Ca) channel functions and Ca(2+) sparks. TRPC1 deficiency in carotid arteries produced a twofold augmentation of TRAM-34- and UCL1684-sensitive EDHF-type vasodilatations and of endothelial hyperpolarization to acetylcholine. NO-mediated vasodilatations were unchanged. TRPC1-/- exhibited enhanced EDHF-type vasodilatations in resistance-sized arterioles in vivo associated with reduced spontaneous tone. Endothelial IK(Ca) /SK(Ca)-type K(Ca) currents, smooth muscle cell Ca(2+) sparks and associated BK(Ca)-mediated spontaneous transient outward currents were unchanged in TRPC1-/-. Smooth muscle contractility induced by receptor-operated Ca(2+) influx or Ca(2+) release and endothelium-independent vasodilatations were unaltered in TRPC1-/-. TRPC1-/- exhibited lower systolic blood pressure as determined by tail-cuff blood pressure measurements. Our data demonstrate that TRPC1 acts as a negative regulator of endothelial K(Ca) channel-dependent EDHF-type vasodilatations and thereby contributes to blood pressure regulation. Thus, we propose a specific role of TRPC1 in the EDHF-K(Ca) signalling complex and suggest that pharmacological inhibition of TRPC1, by enhancing EDHF vasodilatations, may be a novel strategy for lowering blood pressure.

Augmented EDHF signaling in rat uteroplacental vasculature during late pregnancy

A successful pregnancy outcome relies on extensive maternal cardiovascular adaptation, including enhanced uteroplacental vasodilator mechanisms. The objective of the present study was to determine the contribution of the endothelium-derived hyperpolarizing factor (EDHF) signaling in pregnancy-enhanced uterine vasodilation, to define the role of Ca(2+)-activated K(+) channels in mediating EDHF effects, and to explore the impact of endothelial Ca(2+) signaling in pregnancy-specific upregulation of EDHF. Fura 2-based measurements of smooth muscle cell (SMC) and endothelial cell cytosolic Ca(2+) concentration ([Ca(2+)](i)) were performed simultaneously with measurements of the diameter of uterine radial arteries from nonpregnant (NP) and late pregnant (LP) rats. Changes in SMC membrane potential of pressurized arteries from LP rats were assessed using glass microelectrodes. After blockade of nitric oxide and prostacyclin production, a cumulative application of ACh induced rapid and effective dilatation of uterine vessels from both NP and LP rats. This vasodilation was associated with SMC hyperpolarization and SMC [Ca(2+)](i) reduction and was abolished by a high-K(+) solution, demonstrating that N(G)-nitro-L-arginine (L-NNA)- and indomethacin-resistant responses are attributable to EDHF. Pregnancy significantly potentiates EDHF-mediated vasodilation in part due to enhanced endothelial Ca(2+) signaling. L-NNA- and indomethacin-resistant responses were insensitive to iberiotoxin but abolished by a combined treatment with apamin and charybdotoxin, supporting the key role of small- and intermediate-conductance K(+) channels in mediating EDHF signaling in the maternal uterine resistance vasculature.

A mathematical model of vasoreactivity in rat mesenteric arterioles. II. Conducted vasoreactivity

This study presents a multicellular computational model of a rat mesenteric arteriole to investigate the signal transduction mechanisms involved in the generation of conducted vasoreactivity. The model comprises detailed descriptions of endothelial (ECs) and smooth muscle (SM) cells (SMCs), coupled by nonselective gap junctions. With strong myoendothelial coupling, local agonist stimulation of the EC or SM layer causes local changes in membrane potential (V(m)) that are conducted electrotonically, primarily through the endothelium. When myoendothelial coupling is weak, signals initiated in the SM conduct poorly, but the sensitivity of the SMCs to current injection and agonist stimulation increases. Thus physiological transmembrane currents can induce different levels of local V(m) change, depending on cell's gap junction connectivity. The physiological relevance of current and voltage clamp stimulations in intact vessels is discussed. Focal agonist stimulation of the endothelium reduces cytosolic calcium (intracellular Ca(2+) concentration) in the prestimulated SM layer. This SMC Ca(2+) reduction is attributed to a spread of EC hyperpolarization via gap junctions. Inositol (1,4,5)-trisphosphate, but not Ca(2+), diffusion through homocellular gap junctions can increase intracellular Ca(2+) concentration in neighboring ECs. The small endothelial Ca(2+) spread can amplify the total current generated at the local site by the ECs and through the nitric oxide pathway, by the SMCs, and thus reduces the number of stimulated cells required to induce distant responses. The distance of the electrotonic and Ca(2+) spread depends on the magnitude of SM prestimulation and the number of SM layers. Model results are consistent with experimental data for vasoreactivity in rat mesenteric resistance arteries.

Rotenone partially reverses decreased BKCa currents in cerebral artery smooth muscle cells from streptozotocin‐induced diabetic mice

1. Reactive oxygen species (ROS) cause vascular complications and impair vasodilation in diabetes mellitus. Large-conductance Ca(2+)-activated potassium channels (BK(Ca)) modulate vascular tone and play an important negative feedback role in vasoconstriction. In the present study, we tested the hypothesis that ROS regulate the function of BK(Ca) in diabetic cerebral artery smooth muscle cells. 2. Diabetes was induced in male BALB/c mice by injection of streptozotocin (STZ; 180 mg/kg, i.p., dissolved in sterile saline). Control and diabetic mice were treated with 12.7 micromol/L rotenone, an inhibitor of the mitochondrial electron transport chain complex I, or placebo every other day for 5 weeks. The whole-cell patch clamp-technique and functional vasomotor methods were used to record BK(Ca) currents and myogenic tone of cerebral artery smooth muscle cells. 3. In the diabetic group, there was a significant decrease in spontaneous transient outward currents in cerebral artery smooth muscle cells compared with control. Although the currents were only moderately increased in rotenone-treated diabetic mice, they remained significantly lower than in the control group. Furthermore, the macroscopic BK(Ca) currents that were decreased in diabetic mice were partially recovered in rotenone-treated diabetic mice (P < 0.05 vs untreated diabetic group). 4. The posterior cerebral artery from diabetic mice had a significantly higher myogenic tone than the control group, but this impaired contraction was partially reversed in the rotenone-treated diabetic group (P < 0.05 vs untreated diabetic group). 5. The H(2)O(2) concentration was significantly increased in cerebral arteries from diabetic mice compared with control. This increase in H(2)O(2) was significantly blunted by rotenone treatment. 6. In conclusion, rotenone partially reverses the decreased macroscopic BK(Ca) currents in STZ-induced Type 1 diabetic mice and this reversal of BK(Ca) currents may be related to the inhibitory effects of rotenone on H(2)O(2) production. Reactive oxygen species, particularly H(2)O(2), are important regulators of BK(Ca) channels and myogenic tone in diabetic cerebral artery.

Human Airway Contraction and Formoterol-Induced Relaxation Is Determined by Ca2+ Oscillations and Ca2+ Sensitivity

The etiology of airway hyperresponsiveness associated with asthma requires an understanding of the regulatory mechanisms mediating human airway smooth muscle cell (SMC) contraction. The objective of this study was to determine how human airway SMC contraction (induced by histamine) and relaxation (induced by formoterol) are regulated by Ca(2+) oscillations and Ca(2+) sensitivity. The responses of human small airways and their associated SMCs were studied in human lung slices cut from agarose-inflated lungs. Airway contraction was measured with phase-contrast video microscopy. Ca(2+) signaling and Ca(2+) sensitivity of airway SMCs were measured with two-photon fluorescence microscopy and Ca(2+)-permeabilized lung slices. The agonist histamine induced contraction of human small airways by stimulating both an increase in intracellular Ca(2+) concentration in the SMCs in the form of oscillatory Ca(2+) waves and an increase in Ca(2+) sensitivity. The frequency of the Ca(2+) oscillations increased with histamine concentration, and correlated with increased contraction. Formoterol induced airway relaxation at low concentrations by initially decreasing SMC Ca(2+) sensitivity. At higher concentrations, formoterol additionally slowed or inhibited the Ca(2+) oscillations of the SMCs to relax the airways. The action of formoterol was only slowly reversed. Human lung slices provide a powerful experimental assay for the investigation of small airway physiology and pharmacology. Histamine induces contraction by simultaneously increasing SMC Ca(2+) signaling and Ca(2+) sensitivity. Formoterol induces long-lasting relaxation by initially reducing the Ca(2+) sensitivity and, subsequently, the frequency of the Ca(2+) oscillations of the airway SMCs.

The contribution of inositol 1,4,5-trisphosphate and ryanodine receptors to agonist-induced Ca2+ signaling of airway smooth muscle cells

The relative contribution of inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) and ryanodine receptors (RyRs) to agonist-induced Ca(2+) signaling in mouse airway smooth muscle cells (SMCs) was investigated in lung slices with phase-contrast or laser scanning microscopy. At room temperature (RT), methacholine (MCh) or 5-hydroxytryptamine (5-HT) induced Ca(2+) oscillations and an associated contraction in small airway SMCs. The subsequent exposure to an IP(3)R antagonist, 2-aminoethoxydiphenyl borate (2-APB), inhibited the Ca(2+) oscillations and induced airway relaxation in a concentration-dependent manner. 2-APB also inhibited Ca(2+) waves generated by the photolytic release of IP(3). However, the RyR antagonist ryanodine had no significant effect, at any concentration, on airway contraction or agonist- or IP(3)-induced Ca(2+) oscillations or Ca(2+) wave propagation. By contrast, a second RyR antagonist, tetracaine, relaxed agonist-contracted airways and inhibited agonist-induced Ca(2+) oscillations in a concentration-dependent manner. However, tetracaine did not affect IP(3)-induced Ca(2+) release or wave propagation nor the Ca(2+) content of SMC Ca(2+) stores as evaluated by Ca(2+)-release induced by caffeine. Conversely, both ryanodine and tetracaine completely blocked agonist-independent slow Ca(2+) oscillations induced by KCl. The inhibitory effects of 2-APB and absence of an effect of ryanodine on MCh-induced airway contraction or Ca(2+) oscillations of SMCs were also observed at 37 degrees C. In Ca(2+)-permeable SMCs, tetracaine inhibited agonist-induced contraction without affecting intracellular Ca(2+) levels indicating that relaxation also resulted from a reduction in Ca(2+) sensitivity. These results indicate that agonist-induced Ca(2+) oscillations in mouse small airway SMCs are primary mediated via IP(3)Rs and that tetracaine induces relaxation by both decreasing Ca(2+) sensitivity and inhibiting agonist-induced Ca(2+) oscillations via an IP(3)-dependent mechanism.

Nitric Oxide (NO) Induces Airway Smooth Muscle Relaxation by Inhibiting Ca2+ Oscillations

To investigate how nitric oxide (NO) induces airway smooth muscle cell (SMC) relaxation, we examined airway contraction, Ca2+ signalling, and Ca2+ sensitivity in mouse lung slices in response to agonists and an NO donor, NOC‐5. In airways contracted with 5‐HT or ACh, NOC‐5 induced a concentration‐dependent relaxation consisting of an initial relaxation followed by partial re‐contraction. Inhibition of cGMP‐specific phosphodiesterases (PDE‐5) with zaprinast increased NOC‐5‐induced relaxation. 8‐Br‐cGMP, a PDE‐resistant analog of cGMP, induced sustained airway relaxation. Agonist‐induced airway contraction was mediated by SMC Ca2+ oscillations and airway relaxation correlated with a NOC‐5‐induced reduction in the frequency of these Ca2+ oscillations. NOC‐5 inhibited the Ca2+ oscillations and contraction triggered by the photolytic release of IP3 in SMCs. However, in Ca2+ permeabilized SMCs, NO only induced a small airway relaxation when the [Ca2+]i was clamped at a high level. We conclude that NO relaxes airway SMCs by decreasing the frequency of Ca2+ oscillations by blocking Ca2+ release via IP3 receptors and not by a major change in Ca2+ sensitivity.

Smooth Muscle Cell Arachidonic Acid Release, Migration, and Proliferation Are Markedly Attenuated in Mice Null for Calcium-independent Phospholipase A2β

Pharmacologic evidence suggests that the lipid products generated by one or more calcium-independent phospholipases A(2) (iPLA(2)s) participate in the regulation of vascular tone through smooth muscle cell (SMC) Ca(2+) signaling and the release of arachidonic acid. However, the recent identification of new members of the iPLA(2) family, each inhibitable by (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one, has rendered definitive identification of the specific enzyme(s) mediating these processes difficult. Accordingly, we used iPLA(2)beta(-/-) mice to demonstrate that iPLA(2)beta is responsible for the majority of thapsigargin and ionophore (A23187)-induced arachidonic acid release from SMCs. Both thapsigargin and A23187 stimulated robust [(3)H]arachidonate (AA) release from wild-type aortic SMCs that was dramatically attenuated in iPLA(2)beta(-/-) mice (>80% reduction at 5 min; p < 0.01). Moreover, iPLA(2)beta(-/-) mice displayed defects in SMC Ca(2+) homeostasis and decreased SMC migration and proliferation in a model of vascular injury. Ca(2+)-store depletion resulted in the rapid entry of external Ca(2+) into wild-type aortic SMCs that was significantly slower in iPLA(2)beta-null cells (p < 0.01). Furthermore, SMCs from iPLA(2)beta-null mesenteric arterial explants demonstrated decreased proliferation and migration. The defects in migration and proliferation in iPLA(2)beta-null SMCs were restored by 2 mum AA. Remarkably, the cyclooxygenase-2-specific inhibitor, NS-398, prevented AA-induced rescue of SMC migration and proliferation in iPLA(2)beta(-/-) mice. Moreover, PGE(2) alone rescued proliferation and migration in iPLA(2)beta(-/-) mice. We conclude that iPLA(2)beta is an important mediator of AA release and prostaglandin E(2) production in SMCs, modulating vascular tone, cellular signaling, proliferation, and migration.

Vasorelaxant and Hypotensive Effects of Allium cepa Peel Hydroalcoholic Extract in Rat

The aim of present study was to investigate the effect of onion (Allium cepa) peel hydroalcoholic extract (OPE) on rat hypertension induced by high-fructose diet and aorta contractility. The OPE was prepared by maceration method using 70% ethanol. The thoracic aorta from male adult rat (Wistar) was dissected and suspended in Krebs-Henseleit solution under 1 g resting tension. Tissue preparation was contracted by KCl (80 mM) or phenylephrine (Phe, 1 microM) and then the extract was applied cumulatively (0.0625-2 mg mL(-1)). Hypertension was induced in negative control and three groups of rats by adding fructose (10% WN/V) in drinking water for 6 weeks but control group received tap water. Hypertensive groups received saline or OPE at 200, 400 and 800 mg kg(-1) daily for last 3 weeks by gavage. Results showed that OPE reduces aorta contractions induced by KCl or Phe in a concentration-dependent manner (p < 0.001). Removing aorta endothelium did not attenuate the OPE activity. Inhibition of nitric oxide, cGMP and prostaglandin synthesis by L-NAME (100 microM), methylene blue (10 microM) and indomethacin (10 microM), respectively, did not attenuate OPE activity. Atropine abolished ACh-induced relaxation in Phe precontracted aorta but not the OPE-induced relaxation. Although the extract did not change heart rate but after 3 weeks reduced the hypertension induced by fructose (p < 0.001). Present results indicated that OPE reduces aortic contractions possibly via inhibition of calcium influx but without involving NO, cGMP, endothelium and prostaglandins. The OPE hypotensive effect could be due to extract quercetin content, antioxidant activity and inhibiting vascular smooth muscle cells Ca2+ influx.

Complex Regulation of Ca2+‐Activated Cl− Channels by Phosphatases in Pulmonary Arterial Smooth Muscle Cells

Ca2+‐activated Cl− currents (ICl(Ca)) represent a major excitatory mechanism in vascular smooth muscle cells (SMCs). Our group previously showed that CaMKII and Calcineurin (CaN) exert opposed regulatory effects on ICl(Ca) in arterial SMCs. The specific aim of this study was to determine whether other phosphatases modulate ICl(Ca) in rabbit pulmonary artery (PA) myocytes. RT‐PCR performed on rabbit brain, lung and PA cDNA revealed single bands of the expected product size for PP1, PP2A and CaN in all tissues studied. In a previous study, our group (Ayon et al., Faseb J. 21: 873.5, 2007) showed NIPP‐1, a peptide inhibitor of PP1, blocked recovery of ICl(Ca) in cells dialyzed with no ATP. In contrast, Fostriecin, a specific inhibitor of PP2A, had no effect on ICl(Ca) recovery. To further test the PP1 hypothesis, we examined the effects the catalytic subunits of PP1 and PP2A have on ICl(Ca) in cells dialyzed with 3 mM ATP. Surprisingly, PP1 failed to offset ICl(Ca) rundown, while filling the intracellular milieu with PP2A led to ICl(Ca) recovery. The results of our experiments revealed differential responses when manipulating endogenous phosphatases as opposed to supplying these phosphatases exogenously. These results highlight unsuspected complex mechanisms of regulation of ICl(Ca) by phosphatases in PASMCs. [NIH (HL075477 and NCRR 5P20 RR15581), British Heart Foundation (PG/05/038), The Wellcome Trust]

Paraoxonase 1 gene transfer lowers vascular oxidative stress and improves vasomotor function in apolipoprotein E‐deficient mice with pre‐existing atherosclerosis

Transgenesis of human paraoxonase 1 (PON1), a HDL-associated enzyme that destroys lipid peroxides, has been reported to reduce early atherogenesis in mice. The present study explored the therapeutic potential of human PON1 gene transfer in old apolipoprotein E-deficient (apoE(-/-)) mice with advanced atherosclerosis. ApoE(-/-) mice (18 months, regular chow) were transfected with PON1 adenovirus (AdPON1, n=10) or control adenovirus (AdRR5, n=10). Non-transfected apoE(-/-) (n=9) and C57Bl/6J (WT, n=6) mice served as controls. Three weeks later, plaque size and composition, and endothelial cell (EC) and smooth muscle cell (SMC) function were assessed in the aorta. PON1 gene transfer raised total PON1 serum activity 13-15 fold during the 3-week study period, without affecting hypercholesterolaemia or lesion size. However, PON1 decreased the oxLDL content of the plaque. Plaque-free thoracic aorta rings from apoE(-/-) mice displayed, like rings from WT mice, complete relaxation to acetylcholine (ACh, 86+/-2%), ATP (90+/-2%) or UTP (83+/-3%). In contrast, in plaque-bearing segments amplitude (55+/-7%, 68+/-8%, 52+/-8% respectively) and sensitivity were decreased. EC function was completely (ATP, UTP) or largely (ACh) restored by AdPON1. Furthermore, apoE(-/-) SMCs released less intracellular calcium than WT upon sarco-endoplasmic reticulum calcium ATPase (SERCA) inhibition by cyclopiazonic acid. This defect was also restored by AdPON1 transfection. These data indicate that AdPON1 gene transfer improved vascular wall oxidative stress, EC function, and SMC Ca(2+) homeostasis in segments with pre-existing atherosclerosis, independently of an effect on plaque size.

Effects of Dendroaspis Natriuretic Peptide on Calcium-Activated Potassium Current and Its Mechanism

In this study, we sought to investigate the effect of dendroaspis natriuretic peptide (DNP) on calcium-activated potassium current (I K(Ca)) and its mechanism in gastric antral circular smooth muscle cells (SMCs) using the whole-cell patch-clamp technique. DNP concentration-dependently increased macroscopic I K(Ca) and spontaneous transient outward currents (STOCs) in freshly isolated guinea pig gastric antral circular SMCs. The effects of DNP on I K(Ca) and/or STOCs were not blocked by applying calcium-free bath solution or the ryanodine receptor (RyR) antagonist ryanodine (10 microM), but they were inhibited by the inositol triphosphate receptor (IP3R) inhibitor heparin or the guanylate cyclase inhibitor LY83583. Moreover, a DNP-induced increase in STOCs was potentiated by the cyclic guanosine monophosphate (cGMP)-sensitive phosphoesterase inhibitor zaprinast. In conclusion, our results suggest that DNP increases I K(Ca) in gastric antral circular SMCs by increasing cGMP production and activating IP3Rs.

Effects of Albuterol Isomers on the Contraction and Ca2+ Signaling of Small Airways in Mouse Lung Slices

The beta(2)-adrenergic agonist, albuterol, is used as a bronchodilator by patients with asthma and consists of a racemic mixture of (R)- and (S)-albuterol. However, the action of the individual enantiomers is poorly understood. Consequently, we investigated the effects of (R)-, (S)- and racemic-albuterol on airway smooth muscle cell (SMC) contraction and Ca(2+) signaling in mouse lung slices with phase-contrast and confocal microscopy. (R)-albuterol relaxed airways contracted with methacholine (MCh) in a dose-dependent manner. By contrast, (S)-albuterol had no effect on airways. (R)-albuterol had a greater relaxant effect than a double concentration of racemic albuterol. Because MCh-induced contraction of airway SMCs is mediated by Ca(2+) oscillations and an increase in Ca(2+) sensitivity, the effects of albuterol on these responses were examined. Both (R)- and racemic albuterol decreased the frequency of the MCh-induced Ca(2+) oscillations by a similar amount. However, (R)-albuterol was more effective than racemic albuterol in decreasing the Ca(2+) sensitivity of the airway SMCs in "model" lung slices with a clamped [Ca(2+)](i). In contrast, (S)-albuterol had no effect on the Ca(2+) oscillations or the Ca(2+) sensitivity. In conclusion, (R)-albuterol consistently induced a greater airway relaxation than racemic albuterol, and (S)-albuterol appears to be responsible for this reduced efficacy.

Highlights From The Literature

Highlights From The Literature

Smooth muscle sparklet Cav channels defined: 1.2 is the number

Systemic blood pressure and tissue blood flow are regulated by the contractile status of smooth muscle cells located within the wall of small arteries and arterioles. One important signal that regulates arterial smooth muscle cell contractility is the intracellular calcium ion (Ca2+) concentration. Although Ca2+ was once considered to act only as a global intracellular signaling element, research performed over the past decade has revealed Ca2+ to act in a far more dynamic and versatile manner than previously thought possible.

Protein kinase C regulates vascular myogenic tone through activation of TRPM4

Myogenic vasoconstriction results from pressure-induced vascular smooth muscle cell depolarization and Ca(2+) influx via voltage-dependent Ca(2+) channels, a process that is significantly attenuated by inhibition of protein kinase C (PKC). It was recently reported that the melastatin transient receptor potential (TRP) channel TRPM4 is a critical mediator of pressure-induced smooth muscle depolarization and constriction in cerebral arteries. Interestingly, PKC activity enhances the activation of cloned TRPM4 channels expressed in cultured cells by increasing sensitivity of the channel to intracellular Ca(2+). Thus we postulated that PKC-dependent activation of TRPM4 might be a critical mediator of vascular myogenic tone. We report here that PKC inhibition attenuated pressure-induced constriction of cerebral vessels and that stimulation of PKC activity with phorbol 12-myristate 13-acetate (PMA) enhanced the development of myogenic tone. In freshly isolated cerebral artery myocytes, we identified a Ca(2+)-dependent, rapidly inactivating, outwardly rectifying, iberiotoxin-insensitive cation current with properties similar to those of expressed TRPM4 channels. Stimulation of PKC activity with PMA increased the intracellular Ca(2+) sensitivity of this current in vascular smooth muscle cells. To validate TRPM4 as a target of PKC regulation, antisense technology was used to suppress TRPM4 expression in isolated cerebral arteries. Under these conditions, the magnitude of TRPM4-like currents was diminished in cells from arteries treated with antisense oligonucleotides compared with controls, identifying TRPM4 as the molecular entity responsible for the PKC-activated current. Furthermore, the extent of PKC-induced smooth muscle cell depolarization and vasoconstriction was significantly decreased in arteries treated with TRPM4 antisense oligonucleotides compared with controls. We conclude that PKC-dependent regulation of TRPM4 activity contributes to the control of cerebral artery myogenic tone.

Ca 2+ Regulation in Detrusor Smooth Muscle From Ovine Fetal Bladder After In Utero Bladder Outflow Obstruction

We characterized intracellular Ca(2+) regulation in fetal bladders following outflow obstruction by examining the Ca(2+) response to agonists in smooth muscle cells. Severe bladder outflow obstruction was induced in male fetal sheep by placing a urethral ring and urachal ligation midway through gestation at 75 days. Fetuses were examined 30 days after surgery. Intracellular Ca(2+) in single smooth muscle cells isolated from the bladder wall was measured with epifluorescence microscopy using fura-2(AM) during exposure to agonists, such as carbachol and adenosine triphosphate, and to other activators, such as caffeine and KCl. Detrusor smooth muscle cells from obstructed bladders had resting intracellular Ca(2+) similar to that in sham operated controls. The maximal response to carbachol was decreased following obstruction (p <0.05). Construction of dose-response curves also demonstrated higher EC(50) (p <0.05). However, these changes were not mirrored by caffeine evoked Ca(2+) release, which was not significantly different between the obstruction group and sham operated controls. Kinetic analysis of carbachol transients further revealed an attenuated maximal rate of increase in obstructed bladders (p <0.01). The magnitude of intracellular Ca(2+) to purinergic neurotransmitter adenosine triphosphate was also found to be smaller in cells from obstructed bladders (p <0.05), although transmembrane influx by high K depolarization was not significantly affected. Muscarinic and purinergic pathways were down-regulated in fetal detrusor muscle following outflow obstruction. These major functional receptors appeared to be more susceptible to obstruction than other Ca(2+) regulators. Their impairment may contribute to the compromised contractile function seen in in utero bladder outflow obstruction.