Involvement of calcium in cyclic nucleotide metabolism in human vascular smooth muscle.

When cyclic nucleotide phosphodiesterase was purified from isolated smooth muscle layer of human aorta by DEAE-cellulose column chromatography, separated cyclic GMP phosphodiesterase activity was markedly stimulated in the presence of 10-20 micrometer of Ca2+ by a protein modulator which has similar physicochemical properties to troponin C. Synthetic compound, N-(6-aminohexl)-5-chloro-1-naphthalensulfonamide, which produced relaxations of arteries contracted by prostaglandin F2alpha or KCl was found to inhibit selectively this Ca2+-dependent cyclic GMP phosphodiesterase. This compound produced inhibition of superprecipitation of myosin B system obtained from mouse skeletal muscle and also inhibited adenosine triphosphatase activity of myosin B. Our data suggest that calcium is involved through a protein modulator in cyclic nucleotide metabolism of vascular smooth muscle and that the calcium-dependent protein modulator probably participates in the regulation of contractile response of vascular smooth muscle by affecting actomyosin ATPase activity.     

Role of calcium and cyclic adenosine 3':5' monophosphate in regulating smooth muscle contraction. Mechanisms of excitation-contraction coupling in smooth muscle.

Caclium initiates smooth muscle contraction by activating an enzyme, myosin light chain kinase. This enzyme catalyzes the transfer of phosphate from adenosine triphosphate to the 20,000 dalton light chain of myosin. In its phosphorylated form myosin interacts with actin to produce muscle contraction. The mechanism by which calcium activates myosin kinase requires (1) the binding of calcium to a 16,500 dalton calcium-binding protein (calmodulin), and (2) the binding of calmodulin-calcium to a 125,000 dalton catalytic subunit. This two protein complex is the active form of myosin light chain kinase. Smooth muscle relaxation is mediated by cyclic adenosine 3':5' monophosphate (cyclic AMP). One nechanism by which the latter may exert a direct effect on actin-myosin interaction is through the activation of a cyclic AMP-dependent protein kinase that can phosphorylate the 125,000 dalton component of myosin light chain kinase. Phosphorylation of myosin light chain kinase decreases the activity of the enzyme, thus favoring the unphosphorylated form of myosin, which cannot interact with actin to produce smooth muscle contraction.     

Differential skinning of smooth muscle: a new approach to excitation-contraction coupling.

In intact smooth muscle, the calcium sensitivity of force may increase as well as decrease. Using differently skinned smooth muscle preparations, two pathways involved in the change of calcium sensitivity of force have been identified: in microarteries skinned with beta-escin, GTP (possibly via a G protein) is involved in sensitization to Ca2+, while in porcine coronary arteries skinned with Triton X-100 cyclic AMP and possibly cyclic GMP are involved in desensitization. In both cases, there is a corresponding change in the phosphorylation of the regulatory light chain of myosin, suggesting that the balance of phosphorylating to dephosphorylating reactions is altered at constant [Ca2+].     

Regulation of vascular smooth muscle cell apoptosis. Modulation of bad by a phosphatidylinositol 3-kinase-dependent pathway.

Our objective was to define the signaling mechanisms by which mitogens such as insulin-like growth factor-I (IGF-I) regulate vascular smooth muscle cell (VSMC) apoptosis. We confirmed that IGF-I inhibits serum withdrawal-induced apoptosis of cultured VSMCs in a dose-dependent and time-dependent fashion. To test the hypothesis that the phosphatidylinositol (PI) 3-kinase signaling pathway regulates VSMC survival, we examined the relationship between PI 3-kinase activity and cell fate. PI 3-kinase was elevated in viable VSMCs maintained in serum-containing medium, declined significantly in response to serum withdrawal, and increased in response to IGF-I-induced survival. Moreover, blockade of PI 3-kinase with 2 structurally dissimilar inhibitors (wortmannin or LY294002) abolished the capacity of IGF-I to maintain VSMC viability. Similarly, transient transfection of a dominant-negative Deltap85 PI 3-kinase mutant construct abrogated the capacity of IGF-I to prevent VSMC death. Thus, PI 3-kinase is a critical antiapoptotic signal in VSMCs. To define the distal element of the antiapoptotic cascade, we tested the hypothesis that IGF-I inhibits the influence of the proapoptotic gene Bad. Indeed, IGF-I stimulates increased expression of the inactive, phosphorylated form of Bad by a PI 3-kinase-dependent pathway. Moreover, the proapoptotic effect of Bad was attenuated by the stimulation of IGF-I. Thus, growth factors appear to prevent VSMC death by activating signal transduction pathways linked to apoptotic regulatory genes.     

Insulin activates ATP-sensitive potassium channels via phosphatidylinositol 3-kinase in cultured vascular smooth muscle cells

The effects of insulin on the vasculature are significant because insulin resistance is associated with hypertension. To increase the understanding of the effects of insulin on the vasculature, we analyzed changes in potassium ion transport in cultured vascular smooth muscle cells (VSMCs). Using the potential-sensitive fluorescence dye bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC4(3)], we found that insulin induced membrane hyperpolarization after 2 min in A10 cells. Insulin-induced hyperpolarization was suppressed by glibenclamide, an ATP-sensitive potassium (K(ATP)) channel blocker. Using a cell-attached patch clamp experiment, the K(ATP) channel was activated by insulin in both A10 cells and isolated VSMCs from rat aortas, indicating that insulin causes membrane hyperpolarization via K(ATP) channel activation. These effects were not dependent on intracellular ATP concentration, but wortmannin, a phosphatidylinositol 3-kinase (PI3-K) inhibitor, significantly suppressed insulin-induced K(ATP) channel activation. In addition, insulin enhanced phosphorylation of insulin receptor, insulin receptor substrate (IRS)-1 and protein kinase B (Akt) after 2 min. These data suggest that K(ATP) channel activation by insulin is mediated by PI3-K. Furthermore, using a nitric oxide synthase (NOS) inhibitor, we found that NOS might play an important role downstream of PI3-K in insulin-induced K(ATP) channel activation. This study may contribute to our understanding of mechanisms of insulin resistance-associated hypertension.     

Stabilization of follicle-stimulating hormone-receptor complexes may involve calcium-dependent transglutaminase activation.

Calcium-dependent transglutaminase (TGase) activity, determined by incorporation of [1,4-14C]diaminobutane dihydrochloride (putrescine) into casein, was demonstrated in a light membrane fraction prepared from bovine calf testicular homogenates. Purification of these membranes by sucrose density gradient centrifugation produced a follicle-stimulating hormone (FSH) receptor-enriched fraction containing TGase activity which cosolubilized with the FSH receptor and could be incorporated with detergent-solubilized receptor into liposomes. In the present study, we show that calcium increases specific binding of FSH to receptor in a concentration-related manner, and is associated with an increase (13.2-fold at 20 mM) in the affinity (Ka) of the receptor with no significant (P greater than 0.05) change in receptor concentration. Treatment of the light membrane fraction with monodansylcadaverine (MDC, 1 mM), a specific inhibitor of TGase, did not affect specific binding of FSH, but resulted in only a 3.9-fold increase in Ka at 20 mM calcium with no change in receptor concentration. Specific binding of FSH to receptor at 4 degrees C was also enhanced by calcium. Scatchard analysis of competitive binding inhibition data showed a Ka at 20 mM calcium similar to that observed with MDC. Dissociation of [125I]hFSH-receptor complexes formed at 30 degrees C in the presence of calcium was significantly less than dissociation of complexes formed at 30 degrees C in the absence of calcium. When [125I]hFSH-receptor complexes were formed at 30 degrees C in the presence of calcium and dissociated in calcium-deficient buffer, dissociation increased 3-fold. Similar results were obtained in the presence of MDC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms by which smooth muscle sensitivity may be altered by calcium

6-Hydroxydopamine (6-OHDA) increased the sensitivity of rat, rabbit and guinea pig portal veins to norepinephrine (NE), methoxamine (ME), barium (Ba++) and calcium (Ca++) but not to potassium (K+). Reserpine potentiated the responses of NE, ME and Ca++ but not Ba++ or K+ in rabbit and guinea pig veins and did not alter the sensitivity of rat veins to all agonists tested. Cocaine only potentiated the NE responses of the veins and decreased the sensitivity of rabbit and guinea pig veins to K+. 6-OHDA increased 48Ca-influx in all veins whereas reserpine increased 45Ca-influx only in rabbit and guinea pig veins. Cocaine failed to increase 45Ca-influx in all veins tested. NE, ME and K+ increased 45Ca-influx in the veins from the different animals. The agonist-induced 45Ca-influx was greater in most of the supersensitive veins than in the control veins. In veins that failed to develop supersensitivity, agonist-induced 45Ca-influx did not differ from that of the control veins. It is concluded that the development of super- and subsensitivity exhibits species variation and that the alteration in Ca++ influx would be consistent with the changes in sensitivity of the venous smooth muscle.     

Axl/phosphatidylinositol 3-kinase signaling inhibits mineral deposition by vascular smooth muscle cells

The calcification of blood vessels correlates with increased morbidity and mortality in patients with atherosclerosis, diabetes, and end-stage kidney disease. The receptor tyrosine kinase Axl is emerging as an important regulator of adult mammalian physiology and pathology. This study tests the hypothesis that Axl prevents the deposition of a calcified matrix by vascular smooth muscle cells (VSMCs) and that this occurs via the phosphatidylinositol 3-kinase (PI3K) signaling pathway. First, we demonstrate that Axl is expressed and phosphorylated in confluent VSMCs and that its expression is markedly downregulated as these cells calcify their matrix. Second, we demonstrate that overexpression of wild-type Axl, using recombinant adenoviruses, enhances Axl phosphorylation and downstream signaling via PI3K and Akt. Furthermore, overexpression of Axl significantly inhibits mineral deposition by VSMCs, as assessed by alizarin red staining and (45)Ca accumulation. Third, the addition of a PI3K inhibitor, wortmannin, negates the inhibition of mineralization by overexpression of wild-type Axl, suggesting that activation of downstream signaling via PI3K is crucial for its inhibitory activity. In contrast, Axl-mediated signaling is not enhanced by overexpression of kinase-dead Axl and mineralization is accelerated, although beta-glycerophosphate is still required for this effect. Finally, the caspase inhibitor zVAD.fmk attenuates the increased mineralization induced by kinase-dead Axl, suggesting that kinase-dead Axl stimulates mineralization by inhibiting the antiapoptotic effect of endogenous Axl. Together, these results demonstrate that signaling through Axl inhibits vascular calcification in vitro and suggest that therapeutics targeting this receptor may open up new avenues for the prevention of vascular calcification in vivo.     

Myoinositol and phosphatidylinositol metabolism in synaptosomes from galactose-fed rats.

The effects of experimental galactose toxicity on inositol and phosphatidylinositol (PtdIns) metabolism in synaptosomes from 0- to 30-day-old rats were investigated. Galactose toxicity was induced by feeding mothers a 40% galactose diet from the 12th day of pregnancy until 19 days postpartum when the offspring were weaned onto the maternal diet. There was no decrease in myoinositol concentrations and only a small decrease in PtdIns in synaptosomes from galactose-fed rats relative to glucose-fed controls. Synaptosomes from rats on the two diets converted equivalent amounts of [U-14C]glucose to inositol and PtdIns. Acetylcholine stimulated [2-3H]inositol incorporation into PtdIns while producing a net decrease in PtdIns concentration in synaptosomes from 22- to 30-day-old rats. However, the phospholipid response to acetylcholine in synaptosomes from galactose-fed rats was impaired. Thus, the acetylcholine-stimulated labeling of PtdIns was 40--50% lower in these synaptosomes while the effect on PtdIns concentration was reduced by a maximum of 55%. The data suggest that galactose-fed rats may have either a deficiency in the number of acetylcholine receptors or a defect in some step between receptor-neurotransmitter interaction and PtdIns breakdown.     

Similarities and differences in smooth muscle alpha-actin induction by TGF-beta in smooth muscle versus non-smooth muscle cells.

Transforming growth factor-beta (TGF-beta) has been shown to stimulate smooth muscle (SM) alpha-actin expression in smooth muscle cells (SMCs) and non-SMCs. We previously demonstrated that the 2 CArG boxes A and B and a novel TGF-beta control element (TCE) located within the first 125 bp of the SM alpha-actin promoter were required for TGF-beta inducibility of SM alpha-actin in SMCs. The aims of the present study were (1) to determine whether the TCE exhibits SMC specificity or contributes to TGF-beta induction of SM alpha-actin expression in non-SMCs (ie, endothelial cells and fibroblasts) and (2) to determine whether TGF-beta can induce expression of multiple TCE-containing SMC differentiation marker genes, such as SM22alpha, h(1) calponin, and SM myosin heavy chain (SM MHC) in non-SMCs. Results of transient transfection assays demonstrated that mutation of CArG A, CArG B, or the TCE within a 125-bp promoter context completely abolished TGF-beta inducibility of SM alpha-actin in endothelial cells and fibroblasts. However, in contrast to observations in SMCs, inclusion of regions upstream from (-155) completely repressed TGF-beta responsiveness in non-SMCs. Electrophoretic mobility shift assays showed that TGF-beta enhanced binding of a serum response factor to the CArG elements and the binding of an as-yet-unidentified factor to the TCE in endothelial cells and fibroblasts, but to a much lesser extent compared with SMCs. TGF-beta also stimulated expression of the SMC differentiation marker SM22alpha in non-SMCs. However, in contrast to SMCs, TGF-beta did not induce expression of h(1) calponin and SM MHC in non-SMCs. In summary, these results suggest a conserved role for CArG A, CArG B, and the TCE in TGF-beta-induced expression of SM alpha-actin in SMCs and non-SMCs that is modified by a complex interplay of positive- and negative-acting cis elements in a cell-specific manner. Furthermore, observations that TGF-beta stimulated expression of several early but not late differentiation markers in non-SMCs indicate that TGF-beta alone is not sufficient to induce transdifferentiation of non-SMCs into SMCs.     

Contractile protein interactions in smooth muscle.

Smooth muscle tone and 'holding economy' depend on the rate constants governing the cross-bridge cycle. Thus, calcium activation via calmodulin-dependent myosin light chain phosphorylation may determine the apparent rate constant ('f') at which cross-bridges enter the force-generating state, forming actin-attached, strongly bound cross-bridges. This phosphorylation of the light chain may be inhibited in skinned fibers by a peptide mimic of the calmodulin recognition site of the myosin light chain kinase (RS 20) that relaxes smooth muscle. In smooth muscle, the apparent cross-bridge detachment rate constant ('g') also seems to be variable, a low constant allowing for a high holding economy and low shortening velocity in the 'latch state'. It may also account for force maintenance at low levels of myosin phosphorylation. Additionally, cross-bridge attachment may, however, be also controlled by other regulatory proteins such as calponin and caldesmon.     

An electron-microscopic study of smooth muscle cell dye coupling in the pig coronary arteries. Role of gap junctions.

Arterial smooth muscles behave like a syncytium, since they are electrically coupled. It is generally assumed that electrical coupling and dye coupling are mediated by gap junctions. No gap junctions could be detected by transmission electron microscopy in media of coronary arteries. We looked for the presence of gap junction protein in vascular smooth muscle by immunohistochemistry with light microscopy. Immunohistologically detectable connexin is expressed by smooth muscle cells of the media of pig coronary arteries, where staining occurs as a discrete punctation. We investigated the dye coupling in strips of pig coronary artery. The fluorescent dye lucifer yellow was microiontophoretically injected into a smooth muscle cell through an intracellular microelectrode. The dye was visualized on the entire strip, then on semithin sections with a fluorescence microscope, and at the ultrastructural level by using an anti-lucifer yellow antibody revealed by the protein A-gold technique. In all the tissues examined, the cells were dye-coupled. We conclude that in arterial media the smooth muscle cells are dye-coupled, despite the absence of detectable gap junctions by transmission electron microscopy, and suggest that dye coupling could occur via isolated gap junction channels.     

Intracoronary serum smooth muscle myosin heavy chain levels following PTCA may predict restenosis

Recently a novel biochemical method that uses an immunoassay to quantitate serum smooth muscle myosin heavy chain (SMMHC) levels was developed for diagnosis of aortic dissection.) The purpose of this study was to determine whether SMMHC released from the coronary arterial wall can be used to predict restenosis after percutaneous transluminal coronary angioplasty (PTCA). Fifty-two consecutive patients undergoing successful PTCA for single vessel disease were examined (40 men, 12 women, 63 +/- 8 years). Intracoronary blood samples were obtained distal to the lesion, and from the femoral artery after PTCA. In 10 patients, blood samples were taken immediately after the final balloon inflation, and 10 and 20 minutes after PTCA. SMMHC levels were measured by ELISA using SMMHC-specific monoclonal antibodies. Follow-up coronary angiography was performed 3 months after PTCA. Intracoronary serum SMMHC levels were significantly higher than those obtained from the femoral artery (10.6 +/- 1.5 vs 2.1 +/- 0.1 ng / ml, p < or = 0.001). Of 40 patients without apparent dissection, the 23 patients who did not develop restenosis in the follow-up study were found to have had higher levels of intracoronary SMMHC levels immediately after PTCA compared to the 17 patients with restenosis (15.2 +/- 2.9 vs 7.1 +/- 1.2 ng /ml, p < or = 0.05). We suggest that elevated intracoronary SMMHC levels after PTCA may reflect the extent of injury to the arterial wall. Intracoronary SMMHC may be a possible biochemical marker for the prediction of restenosis.     

Molecular biology of smooth muscle.

Several myosin isoforms are expressed in smooth muscle, based upon both heavy and light chain polymorphisms. These myosin isoenzymes reveal distinct detachment rate constants, thus regulating the responsiveness of the VSMC contractile apparatus to Ca2+, as well as its energetical and mechanical behavior.