Increased vascular smooth muscle contractility in TRPC6-/- mice

Among the TRPC subfamily of TRP (classical transient receptor potential) channels, TRPC3, -6, and -7 are gated by signal transduction pathways that activate C-type phospholipases as well as by direct exposure to diacylglycerols. Since TRPC6 is highly expressed in pulmonary and vascular smooth muscle cells, it represents a likely molecular candidate for receptor-operated cation entry. To define the physiological role of TRPC6, we have developed a TRPC6-deficient mouse model. These mice showed an elevated blood pressure and enhanced agonist-induced contractility of isolated aortic rings as well as cerebral arteries. Smooth muscle cells of TRPC6-deficient mice have higher basal cation entry, increased TRPC-carried cation currents, and more depolarized membrane potentials. This higher basal cation entry, however, was completely abolished by the expression of a TRPC3-specific small interference RNA in primary TRPC6(-)(/)(-) smooth muscle cells. Along these lines, the expression of TRPC3 in wild-type cells resulted in increased basal activity, while TRPC6 expression in TRPC6(-/-) smooth muscle cells reduced basal cation influx. These findings imply that constitutively active TRPC3-type channels, which are up-regulated in TRPC6-deficient smooth muscle cells, are not able to functionally replace TRPC6. Thus, TRPC6 has distinct nonredundant roles in the control of vascular smooth muscle tone.     

Caveolin-1 regulates contractility in differentiated vascular smooth muscle

Caveolin is a principal component of caveolar membranes. In the present study, we utilized a decoy peptide approach to define the degree of involvement of caveolin in PKC-dependent regulation of contractility of differentiated vascular smooth muscle. The primary isoform of caveolin in ferret aorta vascular smooth muscle is caveolin-1. Chemical loading of contractile vascular smooth muscle tissue with a synthetic caveolin-1 scaffolding domain peptide inhibited PKC-dependent increases in contractility induced by a phorbol ester or an alpha agonist. Peptide loading also resulted in a significant inhibition of phorbol ester-induced adducin Ser662 phosphorylation, an intracellular monitor of PKC kinase activity, ERK1/2 activation, and Ser789 phosphorylation of the actin binding protein caldesmon. alpha-Agonist-induced ERK1-1/2 activation was also inhibited by the caveolin-1 peptide. Scrambled peptide-loaded tissues or sham-loaded tissues were unaffected with respect to both contractility and signaling. Depolarization-induced activation of contraction was not affected by caveolin peptide loading. Similar results with respect to contractility and ERK1/2 activation during exposure to the phorbol ester or the alpha-agonist were obtained with the cholesterol-depleting agent methyl-beta-cyclodextrin. These results are consistent with a role for caveolin-1 in the coordination of signaling leading to the regulation of contractility of smooth muscle.     

The effects of EGTA on vascular smooth muscle contractility in calcium-free medium

The effect of EGTA, commonly present in Ca2+-free physiological saline solution, on the contractile responses induced by Ca2+ and phenylephrine was studied in dog mesenteric arteries and aortas of rats and rabbits. EGTA substantially enhanced the contractile responses of these vascular strips or rings to added Ca2+ after a prolonged preincubation period in the Ca2+-free medium. The maximal level of the enhanced contractile responses was independent of EGTA concentration, but the rate of the maximal responses was faster at higher EGTA concentration, presumably as a result of faster removal of intracellular Ca2+. Such a Ca2+-induced response was sensitive to the Ca2+ antagonist, nifedipine. EGTA present at low concentrations (50 and 400 microM) in Ca2+-free medium also inhibited the phenylephrine-induced contractile response more prominently for the longer preincubation periods of the vascular tissues in Ca2+-free medium. Our results suggest that EGTA, even when added at low concentrations to the vascular smooth muscle for a sufficiently long period in Ca2+-free medium, may cause destabilization of the cell membranes leading to increased permeability to subsequently added Ca2+. EGTA may also remove the superficially bound Ca2+ and subsequently reduce the intracellular Ca2+ pool via extraction of the intracellular Ca2+ at the cell membrane surfaces.     

Increased calcium influx in dystrophic muscle

We examined pathways which might result in the elevated resting free calcium [( Ca2+]i) levels observed in dystrophic mouse (mdx) skeletal muscle fibers and myotubes and human Duchenne muscular dystrophy myotubes. We found that mdx fibers, loaded with the calcium indicator fura-2, were less able to regulate [Ca2+]i levels in the region near the sarcolemma. Increased calcium influx or decreased efflux could lead to elevated [Ca2+]i levels. Calcium transient decay times were identical in normal and mdx fibers if resting [Ca2+]i levels were similar, suggesting that calcium-sequestering mechanisms are not altered in dystrophic muscle, but are slowed by the higher resting [Ca2+]i. The defect appears to be specific for calcium since resting free sodium levels and sodium influx rates in the absence of Na+/K(+)-ATPase activity were identical in normal and dystrophic cells when measured with sodium-binding benzofuran isophthalate. Calcium leak channels, whose opening probabilities (Po) were voltage independent, could be the major calcium influx pathway at rest. We have shown previously that calcium leak channel Po is significantly higher in dystrophic myotubes. These leak channels were selective for calcium over sodium under physiological conditions. Agents that increased leak channel activity also increased [Ca2+]i in fibers and myotubes. These results suggest that increased calcium influx, as a result of increased leak channel activity, could result in the elevated [Ca2+]i in dystrophic muscle.     

Increased retinoid signaling in vascular smooth muscle cells by proinflammatory cytokines

Retinoids have been shown to modulate inflammation and the immune response in many cell types including macrophages, endothelial cells, and vascular smooth muscle cells. However, present knowledge of whether inflammatory mediators modulate vitamin A status in these cells is limited. To identify the role of inflammation on retinoid metabolism in vascular smooth muscle cells, the cells were exposed to a combination of proinflammatory cytokines: interleukin-1beta, interferon-gamma, and lipopolysaccharides. Without stimulation with proinflammatory cytokines, vascular smooth muscle cells expressed retinol dehydrogenases-2 and 5 mRNA detected by RT-PCR. Stimulation with the combination of cytokines induced a substantial increase of retinol dehydrogenase-5 mRNA. This was associated with increased production of ligands for retinoic acid receptors, when assayed in a retinoic acid receptor-dependent luciferase reporter system. Our results demonstrate that inflammatory mediators activate the retinoid metabolic pathway in vascular smooth muscle cells, which potentially may modulate the inflammatory response in the vascular wall.     

Cholinesterase in muscle of dystrophic hamsters (Bio-40.54)

Isozyme patterns of cholinesterase (ChE) from heart, tongue, and skeletal muscle of normal and dystrophic hamsters are presented. Two principal bands, bands 1 and 2, were evaluated. Band 1 migrates faster towards the anode than does band 2. While bands 1 and 2 stain for AChE and were found in control muscles, only band 2 was stained by a pseudocholinesterase (BuChE) and was decreased in samples from dystrophic hamsters. The decrease in BuChE was most pronounced in dystrophic heart muscle. The low level of BuChE measured for dystrophic animal tissue was similar to isozyme patterns found in embryonic tissue and in denervated muscle. BuChE obtained by acrylamide gel electrophoresis along with 16S AchE appears to be a useful biochemical marker of nerve-muscle interactions.     

Kaurane and pimarane-type diterpenes from the Viguiera species inhibit vascular smooth muscle contractility

The research, development and use of natural products as therapeutic agents, especially those derived from plants, have been increasing in recent years. Despite the fact that plants provide a rich source of novel biologically active compounds, only a small percentage have been phytochemically investigated and studied for their medical potential. Viguiera is a genus that belongs to the family Asteraceae and to the sunflower tribe Heliantheae, which is widespread mostly in Mexico and in other areas of the Andes and upland areas of Brazil. A review on the secondary metabolites pointed out that sesquiterpene lactones and diterpenes, of the kaurane and pimarane-type, are the main compounds produced by these plants. Some reports have shown that kaurane- and pimarane-type diterpenes exert several biological activities such as anti-inflammatory action, antimicrobial and antispasmodic activities. Kaurenoic and pimaradienoic acids, which are the main secondary metabolites isolated by our research group from the roots of Viguiera robusta and V. arenaria, respectively, have been evaluated on vascular smooth muscle contractility. We showed that these diterpenoids are able to inhibit the vascular contractility mainly by blocking extracellular Ca(2+) influx. Additionally, in this review we discuss the structure-activity relationship of the diterpenes regarding their inhibitory activity on vascular contractility.     

The effect of euthanasia technique on vascular arachidonic acid metabolism and vascular and intestinal smooth muscle contractility

This study was designed to determine the effects that specific euthanasia methods have on vascular arachidonic acid metabolism and vascular and intestinal smooth muscle contractility. Rats were euthanatized by decapitation (DC), pentobarbital overdose (PB), or anesthesia with CO2, methoxyflurane or ether followed by DC (CO2-DC, Met-DC, Ether-DC, respectively). Rabbits were killed by a similar protocol, but CO2 overexposure replaced Ether-DC. The rat and rabbit aortas produced mainly 6-keto PGF1 alpha, the prostacyclin metabolite, and lesser amounts of PGE2. No qualitative differences were seen in arachidonate metabolites. However, aortic tissue from rabbits and rats killed by Met-DC produced more prostacyclin. In contrast, aorta from rabbits euthanatized by CO2-DC produced less prostacyclin than controls, whereas aorta from rats killed in the same way yielded greater amounts of prostacyclin. Aortic tissue from rabbits killed by Met-DC and CO2-OD was less responsive to acetylcholine (ACH). Intestinal contractility to ACH was increased in rabbits when Met-DC was used as the method of euthanasia, while colon from rats sacrificed by Met-DC showed decreased responsiveness to ACH. Colon from rats killed by intraperitoneal PB exhibited altered contractility to ACH and norepinephrine. The results of this study show that methoxyflurane, carbon dioxide (rabbit) and pentobarbital (rat) alter the vascular synthesis of prostacyclin and smooth muscle contractility. We conclude that the method of euthanasia affects certain physiologic parameters and careful consideration should be given to the selection of a particular euthanasia technique.     

Impaired vascular contractility and blood pressure homeostasis in the smooth muscle alpha-actin null mouse.

The smooth muscle (SM) alpha-actin gene activated during the early stages of embryonic cardiovascular development is switched off in late stage heart tissue and replaced by cardiac and skeletal alpha-actins. SM alpha-actin also appears during vascular development, but becomes the single most abundant protein in adult vascular smooth muscle cells. Tissue-specific expression of SM alpha-actin is thought to be required for the principal force-generating capacity of the vascular smooth muscle cell. We wanted to determine whether SM alpha-actin gene expression actually relates to an actin isoform's function. Analysis of SM alpha-actin null mice indicated that SM alpha-actin is not required for the formation of the cardiovascular system. Also, SM alpha-actin null mice appeared to have no difficulty feeding or reproducing. Survival in the absence of SM alpha-actin may result from other actin isoforms partially substituting for this isoform. In fact, skeletal alpha-actin gene, an actin isoform not usually expressed in vascular smooth muscle, was activated in the aortas of these SM alpha-actin null mice. However, even with a modest increase in skeletal alpha-actin activity, highly compromised vascular contractility, tone, and blood flow were detected in SM alpha-actin-defective mice. This study supports the concept that SM alpha-actin has a central role in regulating vascular contractility and blood pressure homeostasis, but is not required for the formation of the cardiovascular system.     

Electrophysiological observations on diaphragm muscle from normal and dystrophic hamsters

The cellular electrical activity of diaphragm from F1B normal and BIO 14.6 dystrophic hamsters has been investigated using microelectrodes. Resting membrane potentials and action potentials were recorded from control muscles and from muscles exposed to 2,4-dinitrophenol. The action potentials of normal and dystrophic diaphragms were similar in amplitude and configuration. Treatment with 2,4-dinitrophenol caused the action potential amplitude of both diaphragms to decline by similar amounts. The control resting membrane potential of diaphragm from dystrophic hamsters is not significantly different from that of normal hamsters. Treatment with 2,4-dinitrophenol caused a linear decrease in the resting membrane potentials of both groups of muscles. Dystrophic muscle, however, showed a more rapid decline in excitability when exposed to 2,4-dinitrophenol. This suggests that adenosine triphosphate production in dystrophic muscle is partially inhibited as has been suggested by other workers.     

Increased endothelial and vascular smooth muscle cell adhesion on nanostructured titanium and CoCrMo

In the body, vascular cells continuously interact with tissues that possess nanostructured surface features due to the presence of proteins (such as collagen and elastin) embedded in the vascular wall. Despite this fact, vascular stents intended to restore blood flow do not have nanoscale surface features but rather are smooth at the nanoscale. As the first step towards creating the next generation of vascular stent materials, the objective of this in vitro study was to investigate vascular cell (specifically, endothelial, and vascular smooth muscle cell) adhesion on nanostructured compared with conventional commercially pure (cp) Ti and CoCrMo. Nanostructured cp Ti and CoCrMo compacts were created by separately utilizing either constituent cp Ti or CoCrMo nanoparticles as opposed to conventional micron-sized particles. Results of this study showed for the first time increased endothelial and vascular smooth muscle cell adhesion on nanostructured compared with conventional cp Ti and CoCrMo after 4 hours' adhesion. Moreover, compared with their respective conventional counterparts, the ratio of endothelial to vascular smooth muscle cells increased on nanostructured cp Ti and CoCrMo. In addition, endothelial and vascular smooth muscle cells had a better spread morphology on the nanostructured metals compared with conventional metals. Overall, vascular cell adhesion was better on CoCrMo than on cp Ti. Results of surface characterization studies demonstrated similar chemistry but significantly greater root-mean-square (rms) surface roughness as measured by atomic force microscopy (AFM) for nanostructured compared with respective conventional metals. For these reasons, results from the present in vitro study provided evidence that vascular stents composed of nanometer compared with micron-sized metal particles (specifically, either cp Ti or CoCrMo) may invoke cellular responses promising for improved vascular stent applications.     

Thick filaments in vascular smooth muscle

Two sets of myofilaments were demonstrated after incubation of strips of rabbit portal-anterior mesenteric vein under moderate stretch in a physiological salt solution. Thick filaments had a mean diameter of 18 nm and reached a maximum length of 1.4 µm with a mean length of 0.61 µm. In transverse sections, 2.5–5 nm particles were resolved as subunits of the thick filaments. Thin filaments had an average diameter of 8.4 nm and generally conformed to the structure believed to represent actin filaments in smooth and striated muscles. In the areas of maximum concentration there were 160–328 thick filaments/µm² and the lowest ratio of thin to thick filaments was 12:1. Thick filaments were present in approximately equal numbers in vascular smooth muscle relaxed by theophylline, in Ca⁺⁺-free solution, or contracted by norepinephrine. The same preparatory procedures used with vascular smooth muscle also enabled us to visualize thick filaments in guinea pig and rabbit taenia coli and vas deferens.