Porcine endothelial cells cocultured with smooth muscle cells became procoagulant in vitro.

Endothelial cell (EC) seeding represents a promising approach to provide a nonthrombogenic surface on vascular grafts. In this study, we used a porcine EC/smooth muscle cell (SMC) coculture model that was previously developed to examine the efficacy of EC seeding. Expression of tissue factor (TF), a primary initiator in the coagulation cascade, and TF activity were used as indicators of thrombogenicity. Using immunostaining, primary cultures of porcine EC showed a low level of TF expression, but a highly heterogeneous distribution pattern with 14% of ECs expressing TF. Quiescent primary cultures of porcine SMCs displayed a high level of TF expression and a uniform pattern of staining. When we used a two-stage amidolytic assay, TF activity of ECs cultured alone was very low, whereas that of SMCs was high. ECs cocultured with SMCs initially showed low TF activity, but TF activity of cocultures increased significantly 7-8 days after EC seeding. The increased TF activity was not due to the activation of nuclear factor kappa-B on ECs and SMCs, as immunostaining for p65 indicated that nuclear factor kappa-B was localized in the cytoplasm in an inactive form in both ECs and SMCs. Rather, increased TF activity appeared to be due to the elevated reactive oxygen species levels and contraction of the coculture, thereby compromising the integrity of EC monolayer and exposing TF on SMCs. The incubation of cocultures with N-acetyl-cysteine (2 mM), an antioxidant, inhibited contraction, suggesting involvement of reactive oxygen species in regulating the contraction. The results obtained from this study provide useful information for understanding thrombosis in tissue-engineered vascular grafts.

Beta-arrestins regulate atherosclerosis and neointimal hyperplasia by controlling smooth muscle cell proliferation and migration.

Atherosclerosis and arterial injury-induced neointimal hyperplasia involve medial smooth muscle cell (SMC) proliferation and migration into the arterial intima. Because many 7-transmembrane and growth factor receptors promote atherosclerosis, we hypothesized that the multifunctional adaptor proteins beta-arrestin1 and -2 might regulate this pathological process. Deficiency of beta-arrestin2 in ldlr(-/-) mice reduced aortic atherosclerosis by 40% and decreased the prevalence of atheroma SMCs by 35%, suggesting that beta-arrestin2 promotes atherosclerosis through effects on SMCs. To test this potential atherogenic mechanism more specifically, we performed carotid endothelial denudation in congenic wild-type, beta-arrestin1(-/-), and beta-arrestin2(-/-) mice. Neointimal hyperplasia was enhanced in beta-arrestin1(-/-) mice, and diminished in beta-arrestin2(-/-) mice. Neointimal cells expressed SMC markers and did not derive from bone marrow progenitors, as demonstrated by bone marrow transplantation with green fluorescent protein-transgenic cells. Moreover, the reduction in neointimal hyperplasia seen in beta-arrestin2(-/-) mice was not altered by transplantation with either wild-type or beta-arrestin2(-/-) bone marrow cells. After carotid injury, medial SMC extracellular signal-regulated kinase activation and proliferation were increased in beta-arrestin1(-/-) and decreased in beta-arrestin2(-/-) mice. Concordantly, thymidine incorporation and extracellular signal-regulated kinase activation and migration evoked by 7-transmembrane receptors were greater than wild type in beta-arrestin1(-/-) SMCs and less in beta-arrestin2(-/-) SMCs. Proliferation was less than wild type in beta-arrestin2(-/-) SMCs but not in beta-arrestin2(-/-) endothelial cells. We conclude that beta-arrestin2 aggravates atherosclerosis through mechanisms involving SMC proliferation and migration and that these SMC activities are regulated reciprocally by beta-arrestin2 and beta-arrestin1. These findings identify inhibition of beta-arrestin2 as a novel therapeutic strategy for combating atherosclerosis and arterial restenosis after angioplasty.

Intercellular calcium waves in primary cultured rat mesenteric smooth muscle cells are mediated by connexin43.

Intercellular Ca(2+) wave propagation between vascular smooth muscle cells (SMCs) is associated with the propagation of contraction along the vessel. Here, we characterize the involvement of gap junctions (GJs) in Ca(2+) wave propagation between SMCs at the cellular level. Gap junctional communication was assessed by the propagation of intercellular Ca(2+) waves and the transfer of Lucifer Yellow in A7r5 cells, primary rat mesenteric SMCs (pSMCs), and 6B5N cells, a clone of A7r5 cells expressing higher connexin43 (Cx43) to Cx40 ratio. Mechanical stimulation induced an intracellular Ca(2+) wave in pSMC and 6B5N cells that propagated to neighboring cells, whereas Ca(2+) waves in A7r5 cells failed to progress to neighboring cells. We demonstrate that Cx43 forms the functional GJs that are involved in mediating intercellular Ca(2+) waves and that co-expression of Cx40 with Cx43, depending on their expression ratio, may interfere with Cx43 GJ formation, thus altering junctional communication.

γ-Radiation induces apoptosis via sarcoplasmatic reticulum in guinea pig ileum smooth muscle cells

We investigated the effects of γ-radiation on cells isolated from the longitudinal smooth muscle layer of the guinea pig ileum, a relatively radioresistant tissue. Single doses (up to 50 Gy) reduced the amount of sarcoplasmatic reticulum and condensed the myofibrils, as shown by electron microscopy 3 days post-irradiation. After that, contractility of smooth muscle strips was reduced. Ca2+ handling was altered after irradiation, as shown in fura-2 loaded cells, with elevated basal intracellular Ca2+, reduced amount of intrareticular Ca2+, and reduced capacitive Ca2+ entry. Radiation also induced apoptosis, judged from flow cytometry of cells loaded with proprium iodide. Electron microscopy showed that radiation caused condensation of chromatin in dense masses around the nuclear envelope, the presence of apoptotic bodies, fragmentation of the nucleus, detachment of cells from their neighbors, and reductions in cell volume. Radiation also caused activation of caspase 12. Apoptosis was reduced by the administration of the caspase inhibitor Z-Val-Ala-Asp-fluoromethyl-ketone methyl ester (Z-VAD-FMK) during the 3 day period after irradiation, and by the chelator of intracellular Ca2+, 1,2-bis(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA), from 1 h before until 2 h after irradiation. BAPTA also reduced the effects of radiation on contractility, basal intracellular Ca2+, amount of intrareticular Ca2+, capacitative Ca2+ entry, and apoptosis. In conclusion, the effects of gamma radiation on contractility, Ca2+ handling, and apoptosis appear due to a toxic action of intracellular Ca2+. Ca2+-induced damage to the sarcoplasmatic reticulum seems a key event in impaired Ca2+ handling and apoptosis induced by γ-radiation. © 2008 Elsevier B.V. All rights reserved.

Characterization of outward K(+) currents in isolated smooth muscle cells from sheep urethra.

The perforated-patch technique was used to measure membrane currents in smooth muscle cells from sheep urethra. Depolarizing pulses evoked large transient outward currents and several components of sustained current. The transient current and a component of sustained current were blocked by iberiotoxin, penitrem A, and nifedipine but were unaffected by apamin or 4-aminopyridine, suggesting that they were mediated by large-conductance Ca(2+)-activated K(+) (BK) channels. When the BK current was blocked by exposure to penitrem A (100 nM) and Ca(2+)-free bath solution, there remained a voltage-sensitive K(+) current that was moderately sensitive to blockade with tetraethylammonium (TEA; half-maximal effective dose = 3.0 +/- 0.8 mM) but not 4-aminopyridine. Penitrem A (100 nM) increased the spike amplitude and plateau potential in slow waves evoked in single cells, whereas addition of TEA (10 mM) further increased the plateau potential and duration. In conclusion, both Ca(2+)-activated and voltage-dependent K(+) currents were found in urethral myocytes. Both of these currents are capable of contributing to the slow wave in these cells, suggesting that they are likely to influence urethral tone under certain conditions.

Kv1.5 is a major component underlying the A-type potassium current in retinal arteriolar smooth muscle.

Little is known about the molecular characteristics of the voltage-activated K(+) (K(v)) channels that underlie the A-type K(+) current in vascular smooth muscle cells of the systemic circulation. We investigated the molecular identity of the A-type K(+) current in retinal arteriolar myocytes using patch-clamp techniques, RT-PCR, immunohistochemistry, and neutralizing antibody studies. The A-type K(+) current was resistant to the actions of specific inhibitors for K(v)3 and K(v)4 channels but was blocked by the K(v)1 antagonist correolide. No effects were observed with pharmacological agents against K(v)1.1/2/3/6 and 7 channels, but the current was partially blocked by riluzole, a K(v)1.4 and K(v)1.5 inhibitor. The current was not altered by the removal of extracellular K(+) but was abolished by flecainide, indicative of K(v)1.5 rather than K(v)1.4 channels. Transcripts encoding K(v)1.5 and not K(v)1.4 were identified in freshly isolated retinal arterioles. Immunofluorescence labeling confirmed a lack of K(v)1.4 expression and revealed K(v)1.5 to be localized to the plasma membrane of the arteriolar smooth muscle cells. Anti-K(v)1.5 antibody applied intracellularly inhibited the A-type K(+) current, whereas anti-K(v)1.4 antibody had no effect. Co-expression of K(v)1.5 with K(v)beta1 or K(v)beta3 accessory subunits is known to transform K(v)1.5 currents from delayed rectifers into A-type currents. K(v)beta1 mRNA expression was detected in retinal arterioles, but K(v)beta3 was not observed. K(v)beta1 immunofluorescence was detected on the plasma membrane of retinal arteriolar myocytes. The findings of this study suggest that K(v)1.5, most likely co-assembled with K(v)beta1 subunits, comprises a major component underlying the A-type K(+) current in retinal arteriolar smooth muscle cells

Modification of smooth muscle Ca2+-sparks by tetracaine: evidence for sequential RyR activation.

Spontaneous Ca(2+)-sparks were imaged using confocal line scans of fluo-4 loaded myocytes in retinal arterioles. Tetracaine produced concentration-dependent decreases in spark frequency, and modified the spatiotemporal characteristics of residual sparks. Tetracaine (10 microM) reduced the rate of rise but prolonged the average rise time so that average spark amplitude was unaltered. The mean half-time of spark decay was also unaffected, suggesting that spark termination, although delayed, remained well synchronized. Sparks spread transversely across the myocytes in these vessels, and the speed of spread within individual sparks was slowed by approximately 60% in 10 microM tetracaine, as expected if the spark was propagated across the cell but the average P(o) for RyRs was reduced. Staining of isolated vessels with BODIPY-ryanodine and di-4-ANEPPS showed that RyRs were located both peripherally, adjacent to the plasma membrane, and in transverse extensions of the SR from one side of the cell to the other. Immuno-labelling of retinal flat mounts demonstrated the presence RyR(2) in arteriole smooth muscle but not RyR(1). We conclude that Ca(2+)-sparks in smooth muscle can result from sequential activation of RyRs distributed over an area of several microm(2), rather than from tightly clustered channels as in striated muscle.

Ca2+-activated Cl- current in retinal arteriolar smooth muscle.

PURPOSE: To characterize the biophysical, pharmacologic, and functional properties of the Ca(2+)-activated Cl(-) current in retinal arteriolar myocytes. METHODS: Whole-cell perforated patch-clamp recordings were made from myocytes within intact isolated arteriolar segments. Arteriolar tone was assessed using pressure myography. RESULTS: Depolarizing of voltage steps to -40 mV and greater activated an L-type Ca(2+) current (I(Ca(L))) that was followed by a sustained current. Large tail currents (I(tail)) were observed on stepping back to -80 mV. The sustained current and I(tail) reversed close to 0 mV in symmetrical Cl(-) concentrations. The ion selectivity sequence for I(tail) was I(-)> Cl(-)> glucuronate. Outward I(tail) was sensitive to the Cl(-) channel blockers 9-anthracene-carboxylic acid (9-AC; 1 mM), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS; 1 mM), and disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS; 1 mM), but only DIDS produced a substantial (78%) block of inward tail currents at -100 mV. I(tail) was decreased in magnitude when the normal bathing medium was substituted with Ca(2+)-free solution or if I(Ca(L)) was inhibited by 1 microM nimodipine. Caffeine (10 mM) produced large transient currents that reversed close to the Cl(-) equilibrium potential and were blocked by 1 mM DIDS or 100 microM tetracaine. DIDS had no effect on basal vascular tone in pressurized arterioles but dramatically reduced the level of vasoconstriction observed in the presence of 10 nM endothelin-1. CONCLUSIONS: Retinal arteriolar myocytes have I(Cl(Ca)), which may be activated by Ca(2+) entry through L-type Ca(2+) channels or Ca(2+) release from intracellular stores. This current appears to contribute to agonist-induced retinal vasoconstriction.

Deletion of TRPC4 and TRPC6 in Mice Impairs Smooth Muscle Contraction and Intestinal Motility In Vivo.

BACKGROUND & AIMS: Downstream effects of muscarinic receptor stimulation in intestinal smooth muscle include contraction and intestinal transit. We thought to determine whether classic transient receptor potential (TRPC) channels integrate the intracellular signaling cascades evoked by the stimulated receptors and thereby contribute to the control of the membrane potential, Ca-influx, and cell responses. METHODS: We created trpc4-, trpc6-, and trpc4/trpc6-gene-deficient mice and analyzed them for intestinal smooth muscle function in vitro and in vivo. RESULTS: In intestinal smooth muscle cells TRPC4 forms a 55 pS cation channel and underlies more than 80% of the muscarinic receptor-induced cation current (mI(CAT)). The residual mI(CAT) depends on the expression of TRPC6, indicating that TRPC6 and TRPC4 determine mI(CAT) channel activity independent of other channel subunits. In TRPC4-deficient ileal myocytes the carbachol-induced membrane depolarizations are diminished greatly and the atropine-sensitive contraction elicited by acetylcholine release from excitatory motor neurons is reduced greatly. Additional deletion of TRPC6 aggravates these effects. Intestinal transit is slowed down in mice lacking TRPC4 and TRPC6. CONCLUSIONS: In intestinal smooth muscle cells TRPC4 and TRPC6 channels are gated by muscarinic receptors and are responsible for mI(CAT). They couple muscarinic receptors to depolarization of intestinal smooth muscle cells and voltage-activated Ca(2+)-influx and contraction, and thereby accelerate small intestinal motility in vivo.

Pharmacological profiling of store-operated Ca(2+) entry in retinal arteriolar smooth muscle

Objective: Pharmacological profiling of store-operated Ca(2+) entry (SOCE) and molecular profiling of ORAI and TRPC expression in arterioles. 
Methods: Fura-2 based microfluorimetry was used to assess CPA-induced SOCE in rat retinal arteriolar myocytes. Arteriolar ORAI and TRP transcript expression were screened using RT-PCR. 
Results: SKF96365 and LOE908 blocked SOCE (IC(50) s of 1.2µM and 1.4µM, respectively). Gd(3+) and La(3+) potently inhibited SOCE (IC(50) s of 21nM and 42nM, respectively), but Ni(2+) showed lower potency (IC(50) = 11.6µM). 2-aminoethyldiphenyl borate (2APB) inhibited SOCE (IC(50) = 3.7µM) but enhanced basal influx (>100µM). Verapamil and nifedipine had no effect at concentrations that inhibit L-type Ca(2+) channels, but diltiazem inhibited SOCE by approximately 40% (=0.1µM). RT-PCR demonstrated transcript expression for ORAI 1, 2 and 3, and TRPC1, 3, 4 and 7. Transcripts for TRPV1 and 2, which are activated by 2APB, were also expressed. 
Conclusion: The pharmacological profile of SOCE in retinal arteriolar smooth muscle appears unique when compared to other vascular tissues. This suggests that the molecular mechanisms underlying SOCE can differ, even in closely related tissues. Taken together, the pharmacological and molecular data are most consistent with involvement of TRPC1 in SOCE, although involvement of ORAI or other TRPC channels cannot be excluded. © 2012 John Wiley & Sons Ltd.

T- and L-type Ca2+ currents in freshly dispersed smooth muscle cells from the human proximal urethra

The purpose of the present study was to characterise Ca2+ currents in smooth muscle cells isolated from biopsy samples taken from the proximal urethra of patients undergoing surgery for bladder or prostate cancer. Cells were studied at 37 degreesC using the amphotericin B perforated-patch configuration of the patch-clamp technique. Currents were recorded using Cs+-rich pipette solutions to block K+ currents. Two components of current, with electrophysiological and pharmacological properties typical of T- and L-type Ca2+ currents, were present in these cells. When steady-state inactivation curves for the L current were fitted with a Boltzmann equation, this yielded a V-1/2 of -45 +/- 5 mV. In contrast, the T current inactivated with a V-1/2 of -80 +/- 3 mV. The L currents were reduced in a concentration-dependent manner by nifedipine (ED50 = 159 +/- 54 nm) and Ni2+ (ED50 = 65 +/- 16 muM) but were enhanced when external Ca2+ was substituted with Ba2+. The T current was little affected by TTX, reduction in external Na+, application of nifedipine at concentrations below 300 nm or substitution of external Ca2+ with Ba2+, but was reduced by Ni2+ with an ED50 of 6 +/- 1 mum. When cells were stepped from -100 to -30 mV in Ca2+-free conditions, small inward currents could be detected. These were enhanced 40-fold in divalent-cation-free solution and blocked in a concentration-dependent manner by Mg2+ with an ED50 of 32 +/- 16 mum. These data support the idea that human urethral myocytes possess currents with electrophysiological and pharmacological properties typical of T- and L-type Ca2+ currents.

Partial urethral obstruction of rabbit urinary bladder: stereological evidence that the increase in muscle content is mostly driven by changes in number, rather than size, of smooth muscle cells

The effects of partial urethral obstruction on the detrusor muscle of rabbit urinary bladder were investigated using stereological sampling and estimation tools. Twelve female Norfolk rabbits (2.5-3.0 kg body weight) were divided into four groups: 3, 7 and 12 weeks after surgical intervention to produce a standard partial obstruction and unobstructed controls. Following removal, bladder axes (craniocaudal, dorsoventral and laterolateral) and organ weights were recorded. Bladders were prepared for light microscopy by multistage random sampling procedures. Stereological methods were used to estimate the volume of muscle and the packing density and total number of myocyte nuclei in each bladder. We also estimated mean myocyte volume and the mean cross-sectional area and length of myocytes. Group comparisons were made by one-way analysis of variance. Changes in bladder axes were mainly laterolateral and craniocaudal. Mean bladder weight increased roughly six-fold by 3 weeks and 17-fold by 12 weeks and was accompanied, on average, by 12- and 33-fold increases in total muscle volume. These variables did not differ at 3 and 7 weeks post-obstruction. Increases in muscle content were not accompanied by changes in packing densities but were associated with increases in the total numbers of myocyte nuclei (13-fold by 3 weeks, 28-fold by 12 weeks). Mean myocyte volume did not vary significantly between groups but cells in obstructed groups were shorter and wider. These findings support the notion that partial outflow obstruction leads to an increase in the number, but not mean volume, of myocytes. If due solely to myocyte mitosis, the total of 43 x 10(8) cells found at 12 weeks could be generated by the original complement of 15 x 10(7) cells if an average of only 2.1 x 10(6) new cells was produced every hour. In reality, even this modest proliferation rate is unlikely to be achieved because myocyte proliferation rates are very low and it is possible that new myocytes can arise by differentiation of mesenchymal or other precursor cells.

Inactivation of calcium-activated chloride channels in smooth muscle by calcium/calmodulin-dependent protein kinase

To determine the mechanisms responsible for the termination of Ca2+-activated Cl− currents (ICl(Ca)), simultaneous measurements of whole cell currents and intracellular Ca2+ concentration ([Ca2+]i) were made in equine tracheal myocytes. In nondialyzed cells, or cells dialyzed with 1 mM ATP, ICl(Ca) decayed before the [Ca2+]i decline, whereas the calcium-activated potassium current decayed at the same rate as [Ca2+]i. Substitution of AMP-PNP or ADP for ATP markedly prolonged the decay of ICl(Ca), resulting in a rate of current decay similar to that of the fall in [Ca2+]i. In the presence of ATP, dialysis of the calmodulin antagonist W7, the Ca2+/calmodulin-dependent kinase II (CaMKII) inhibitor KN93, or a CaMKII-specific peptide inhibitor the rate of ICl(Ca) decay was slowed and matched the [Ca2+]i decline, whereas H7, a nonspecific kinase inhibitor with low affinity for CaMKII, was without effect. When a sustained increase in [Ca2+]i was produced in ATP dialyzed cells, the current decayed completely, whereas in cells loaded with 5′-adenylylimidodiphosphate (AMP-PNP), KN93, or the CaMKII inhibitory peptide, ICl(Ca) did not decay. Slowly decaying currents were repeatedly evoked in ADP- or AMP-PNP-loaded cells, but dialysis of adenosine 5′-O-(3-thiotriphosphate) or okadaic acid resulted in a smaller initial ICl(Ca), and little or no current (despite a normal [Ca2+]i transient) with a second stimulation. These data indicate that CaMKII phosphorylation results in the inactivation of calcium-activated chloride channels, and that transition from the inactivated state to the closed state requires protein dephosphorylation.

Formation of heteromeric gap junction channels by connexins 40 and 43 in vascular smooth muscle cells

Connexin (Cx) 43 and Cx40 are coexpressed in several tissues, including cardiac atrial and ventricular myocytes and vascular smooth muscle. It has been shown that these Cxs form homomeric/homotypic channels with distinct permeability and gating properties but do not form functional homomeric/heterotypic channels. If these Cxs were to form heteromeric channels, they could display functional properties not well predicted by the homomeric forms. We assessed this possibility by using A7r5 cells, an embryonic rat aortic smooth muscle cell line that coexpresses Cxs 43 and 40. Connexons (hemichannels), which were isolated from these cells by density centrifugation and immunoprecipitated with antibody against Cx43, contained Cx40. Similarly, antibody against Cx40 coimmunoprecipitated Cx43 from the same connexon fraction but only Cx40 from Cx (monomer) fractions. These results indicate that heteromeric connexons are formed by these Cxs in the A7r5 cells. The gap junction channels formed in the A7r5 cells display many unitary conductances distinct from homomeric/homotypic Cx43 or Cx40 channels. Voltage-dependent gating parameters in the A7r5 cells are also quite variable compared with cells that express only Cx40 or Cx43. These data indicate that Cxs 43 and 40 form functional heteromeric channels with unique gating and conductance properties.