Microtubules regulate pulmonary vascular smooth muscle contraction

Two patients with cluster-tic syndrome are reported. The first, a 43-years-old man, complaining of trigeminal pain in the right side of the face, accompanied by homolateral autonomic signs, such as ocular injection, sweating and drooped eyelid. The cluster attack was triggered by chewing, shaving and washing the face. The periodicity of bouts was six months. The pain was relieved by carbamazepine (800 mg/day). The second patient, a 43-year-old man, with an excruciant, neuralgic pain in the left side of the face, accompanied by tearing, conjuntival injection, drooped eyelid, rhinorrhea, photophobia and phonophobia. The neurologic examination showed triggered points in the first and second division of the trigeminal nerve. The patient was treated with verapamil (160 mg/day) and prednisone (60 mg/day), with relief of his symptoms. The periodicity of bouts was once a year. The literature was reviewed and 37 cases previosly reported are considered. We conclude that there are two different groups of patients. In the first group, the patients had cluster and trigeminal bouts in different time. In the second group, with only nine cases, the patients presented both cluster and trigeminal type of pain at the same time, as in the two cases reported here.     

Adrenomedullin is a potent inhibitor of angiotensin II-induced migration of human coronary artery smooth muscle cells

The migration of coronary artery medial smooth muscle cells (SMCs) into the intima is proposed to be an important process of intimal thickening in coronary atherosclerotic lesions. In the current study, we examined the possible interaction of adrenomedullin, a novel vasorelaxant peptide, and angiotensin II (Ang II) on human coronary artery SMC migration using Boyden's chamber method. Ang II stimulated SMC migration in a concentration-dependent manner between 10(6) and 10(8) mol/L. This stimulation was clearly blocked by the Ang II type 1 receptor antagonist losartan but not by the type 2 receptor antagonist PD 123319. The migration stimulatory effect of Ang II was chemotactic in nature for cultured human coronary artery SMCs but was not chemokinetic. Human adrenomedullin clearly inhibited Ang II-induced migration in a concentration-dependent manner. Human adrenomedullin stimulated cAMP formation in these cells. Inhibition by adrenomedullin of Ang II-induced SMC migration was paralleled by an increase in the cellular level of cAMP. 8-Bromo-cAMP, a cAMP analogue, and forskolin, an activator of adenylate cyclase, inhibited the Ang II-induced SMC migration. These results suggest that Ang II stimulates SMC migration via type 1 receptors in human coronary artery and adrenomedullin inhibits Ang II-induced migration at least partly through a cAMP-dependent mechanism. Taken together with the finding that adrenomedullin is synthesized in and secreted from vascular endothelial cells, this peptide may play a role as a local antimigration factor in certain pathological conditions.     

Angiotensin II-induced tyrosine phosphorylation in mesangial and vascular smooth muscle cells

It has previously been suggested that paradigms investigating schema theory should include tests of the effects of variability of practice on tasks experienced during the acquisition of learning. The present aim was to test this effect using a sports-related task. Two groups of children (n = 13 each) aged between 3 and 5 yr. old were pretested on an aiming task. They then received ten practice trials per day for ten days. One group (designated the specific group) practised only at the specified target distance, while another group (designated the specific plus variable group) practised at the target distance and at distances around the target. At the end of the practice period both groups were again tested. The data were analysed for distance and lateral errors, using two-way (group x time) analyses of covariance with one repeated measure on the latter factor. The interaction of groups x time was significant for the distance errors, indicating that at the end of the practice period the specific plus variable group had improved more than the specific group. This finding extends previous work on a similar variability paradigm to a sports-related setting.     

HR 720, a novel angiotensin receptor antagonist inhibits the angiotensin II-induced trophic effects, fibronectin release and fibronectin-EIIIA+ expression in rat aortic vascular smooth muscle cells in vitro

The aim of this study was to evaluate the direct trophic effects of angiotensin II (AII) on rat vascular smooth muscle cells obtained from a single cellular isolate. Cell volume, protein synthesis, fibronectin (FN) release and FN-EIIIA+ mRNA isoform expression were analyzed in parallel. The effects of HR 720, a novel AT1 angiotensin receptor antagonist with some AT2 receptor affinity, were compared with those of selective AT1 antagonist EXP 3174. Both HR 720 and EXP 3174 inhibited in a concentration-dependent manner the maximum increase in cell volume induced by 10(-9) M Sar1-All (IC50 = 0.49 x 10(-9) M and 0.79 x 10(-9) M, respectively). Maximum [3H]leucine incorporation was also achieved at 10(-9) M All. HR 720 blocked the increase in protein synthesis with potency similar to EXP 3174; the respective IC50 values were 1.04 x 10(-9) M and 1.36 x 10(-9) M. All dose-dependently increased FN release, which was also equally inhibited by about 50% with both compounds at 10(-6) M. Furthermore, All enhanced FN-EIIIA+ mRNA in rat vascular smooth muscle cells (VSMC), which indicated a modulation of FN isoform expression which was inhibited by angiotensin II antagonists. In conclusion, All induced parallel and concentration-dependent increases in cell volume, protein synthesis, FN release and FN-EIIIA+ mRNA expression in vascular smooth muscle cells. These effects appeared to be essentially mediated by AT1 receptor stimulation as indicated by the equal inhibitory effects of HR 720 and EXP 3174.     

Integrins inhibit angiotensin II-induced contraction in rat aortic rings

Many extracellular matrix proteins contain the tripeptide sequence arginine-glycine-aspartate (RGD). This RGD motif is recognized by integrins, a family of adhesion receptors present on vascular smooth muscle cells. In the present study, we examined the ability of different RGD-containing peptides to affect the contraction of rat aortic rings in response to different agonists. We found that the peptide RGDS inhibited angiotensin-induced contraction in a dose dependent manner. In contrast, the peptides RGDW and RGES had no effect on angiotensin-induced contractility. We show that function-blocking antibodies to the integrins alphavbeta3 and alpha5beta1 also inhibit angiotensin-induced contraction. These effects were observed in the absence of an intact endothelium. In contrast, neither an antibody directed against the beta1 subunit nor the peptide RGDS had an effect on phenylephrine or 5-hydroxytryptamine-induced contraction. These data suggest that interactions of vascular smooth muscle with components of the surrounding extracellular matrix may influence the response of smooth muscle to agonists.     

Effects of genistein and staurosporine on angiotensin II-induced DNA synthesis, protein synthesis and mitogen-activated protein kinase activation in vascular smooth muscle cells

This study examines the effects of protein kinase inhibitors and activator on angiotensin II-induced DNA synthesis and protein synthesis of rat aortic smooth muscle cells. In quiescent confluent cells, angiotensin II induced a concentration-dependent increase in thymidine incorporation and leucine incorporation. The tyrosine kinase inhibitor genistein caused an inhibition of the angiotensin II-induced DNA synthesis but not of the agent-induced protein synthesis. The protein kinase C inhibitors staurosporine and calphostin C caused an inhibition of the angiotensin II-induced protein synthesis but not of the agent-induced DNA synthesis. The protein kinase C activator phorbol 12-myristate 13-acetate stimulated protein synthesis. Angiotensin II stimulated mitogen-activated protein (MAP) kinases and the angiotensin II-induced MAP kinase activation was inhibited by genistein but not by staurosporine. These findings suggest that angiotensin II-induced DNA synthesis is at least partly mediated via protein-tyrosine phosphorylation and angiotensin II-induced protein synthesis is at least partly mediated by activation of protein kinase C. It seems likely that MAP kinase activation is involved in DNA synthesis but not in protein synthesis induced by angiotensin II.     

Role of extracellular signal-regulated kinases in angiotensin II-stimulated contraction of smooth muscle cells from human resistance arteries

BACKGROUND: We assessed the role of extracellular signal-regulated kinases (ERKs) in Ang II-stimulated contraction and associated signaling pathways in vascular smooth muscle cells (VSMCs) from human small arteries. METHODS AND RESULTS: VSMCs derived from resistance arteries (<300 microm in diameter) from subcutaneous gluteal biopsies of healthy subjects (n=8) were used to assess Ang II-stimulated [Ca2+]i, pHi, and contractile responses. [Ca2+]i and pHi were measured with fura 2-AM and BCECF-AM, respectively, and contraction was measured photomicroscopically in cells grown on Matrigel matrix. To determine whether tyrosine kinases and ERKs influence Ang II-stimulated responses, cells were pretreated with 10(-5) mol/L tyrphostin A-23 (tyrosine kinase inhibitor) and PD98059 (MEK inhibitor). Ang II-stimulated MEK activity was determined by tyrosine phosphorylation of ERKs. The angiotensin receptor subtypes (AT1 and AT2) were assessed with [Sar1,Ile8]Ang II (a nonselective subtype antagonist), losartan (a selective AT1 antagonist), and PD123319 (a selective AT2 antagonist). Ang II dose-dependently increased [Ca2+]i (pD2=8.4+/-0.36, Emax=541+/-55 nmol/L), pHi (pD2=9. 4+/-0.29, Emax=7.19+/-0.01), and contraction (pD2=9.2+/-0.21, Emax=36+/-2.2%). Ang II induced rapid tyrosine phosphorylation of ERKs, which was inhibited by PD98059. Tyrphostin A-23 and PD98059 attenuated (P<0.05) Ang II-stimulated second messengers, and PD98059 reduced Ang II-induced contraction by >50%. [Sar1,Ile8]Ang II and losartan, but not PD123319, blocked Ang II-stimulated responses. CONCLUSIONS: These data demonstrate that in VSMCs from human peripheral resistance arteries, functional Ang II receptors of the AT1 subtype are coupled to signaling cascades involving Ca2+ and pHi pathways that are partially dependent on tyrosine kinases and ERKs. ERKs, the signaling cascades characteristically associated with cell growth, may play an important role in Ang II-stimulated contraction of human VSMCs.     

Dipyridamole inhibits PDGF- and bFGF-induced vascular smooth muscle cell proliferation

Dipyridamole is the only pharmacologic agent demonstrated to reduce polytetrafluoroethylene (PTFE) graft occlusion in hemodialysis patients. However, the mechanism of action of dipyridamole in preventing graft occlusion is unknown. The purpose of this study was to examine the direct effects of dipyridamole on both platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF)-induced vascular smooth muscle cell (VSMC) proliferation. Human aortic smooth muscle cells were grown to confluence in 96 well plates. A total of 5 x 10(-6) molar dipyridamole, PDGF 10 ng/ml, or bFGF 10 ng/ml were added to appropriate wells at the start of each experiment. Cell proliferation at 48 hours was determined using tritiated thymidine uptake. Intracellular cyclic AMP (cAMP) was measured using a competitive enzyme immunoassay. Treatment of VSMC with 5 microM dipyridamole dramatically reduced basal proliferation rates compared to controls [5229 +/- 1131 counts per minute (CPM) versus 387 +/- 68 CPM, P < 0.001]. Treatment with dipyridamole also reduced PDGF-stimulated VSMC proliferation (7311 +/- 1655 CPM vs. 593 +/- 110 CPM, P < 0.001) as well as the response to bFGF (5632 +/- 1270 CPM vs. 310 +/- 31 CPM, P < 0.001). Treatment of VSMC with either 5 or 20 microM dipyridamole did not change intracellular cAMP levels. Furthermore, the addition of dibutyryl cAMP to VSMC demonstrated only a modest inhibitory effect on proliferation. We conclude that dipyridamole inhibits both PDGF- and bFGF-stimulated VSMC proliferation. The effects of dipyridamole on VSMC proliferation do not appear to be entirely mediated by changes in intracellular cAMP concentrations. The direct effect of dipyridamole on VSMC proliferation may account for its efficacy in reducing PTFE graft thrombosis in hemodialysis patients.     

Acute or chronic exercise alters angiotensin II-induced contraction of rat aorta

Chronic exercise substantially shifted the dose-response curve for angiotensin II (ANG II)-induced contractions to the right with increases in the EC50 value in both endothelium-intact and endothelium-denuded aortae. An acute endurance exercise also caused a 3-fold increase in the EC50 value in both endothelium-intact and endothelium-denuded aortae from sedentary control rats. Whereas, no substantial increase in the EC50 value was observed in trained rats after an acute endurance exercise. Thus, chronic exercise attenuates ANG II receptor-mediated contraction of rat aortae. An acute exercise also caused the reduced responses to ANG II in sedentary control rats but not in chronically exercise-trained rats.     

Herbimycin A inhibits protein kinase C in vascular smooth muscle cells

A sensitive enantioselective high performance liquid chromatographic assay for determination of the dihydropyridine-type calcium antagonist amlodipine in human plasma samples is described. Chiral chromatography is performed on an alpha 1-acid glycoprotein column (i.e. Chiral-AGP) and the eluted enantiomers are trapped and compressed on two short columns before final achiral chromatography on a narrow bore column (i.e. Zorbax SB-Ph) using electrochemical detection. Both stereoselective quantitative analysis and enantiomeric ratio analysis, for samples with a known total concentration of amlodipine are described. The quantitative assay shows linearity over the range 0.5-10 ng ml-1 for the two enantiomers and the limit of detection is about 0.2 ng ml-1. The method has been applied to a pharmacokinetic study of the enantiomers of amlodipine in human subjects.     

Heparin inhibits human coronary artery smooth muscle cell migration

Heparin, an anticoagulant, has been shown to reduce neointimal proliferation and restenosis following vascular injury in experimental studies, but the clinical trials of heparin in coronary balloon angioplasty have been negative. The current study, therefore, examined the effect of heparin on basal or stimulated migration by serum and platelet-derived growth factor (PDGF)-BB in cultured human coronary artery smooth muscle cells (SMCs) by Boyden's chamber method. In addition, the reversibility of the heparin effect on human coronary artery SMC migration was examined. Fetal calf serum (FCS) and PDGF-BB stimulated SMC migration in a concentration-dependent manner. Heparin in moderate to high concentration (10 to 100 U/mL) exhibited concentration-related inhibition of FCS- and PDGF-BB-stimulated SMC migration; however, a low concentration (1 U/mL) of heparin had no inhibitory effects. Heparin also had weak inhibitory effects on nonstimulated SMC migration. The SMCs that were exposed to a high concentration (100 U/mL) of heparin for 6 hours were capable of migrating after a short lag period of removal of heparin from the culture medium. These SMCs also showed recovery of responses to FCS and PDGF-BB by migrating significantly greater than the nonstimulated level. Furthermore, heparin-containing medium did not contain detached cells. These results indicate that heparin inhibits human coronary artery SMC migration, especially when stimulated by FCS or PDGF-BB, and that this inhibitory effect of heparin is reversible and not simply a function of killing cells.     

Farnesol inhibits L-type Ca2+ channels in vascular smooth muscle cells

Earlier experiments with animal and human arteries have shown that farnesol, a natural 15-carbon (C15) isoprenoid, is an inhibitor of vasoconstriction (Roullet, J.-B., Xue, H., Chapman, J., McDougal, P., Roullet, C. M., and McCarron, D. A. (1996) J. Clin. Invest. 97, 2384-2390). We report here that farnesol reduced KCl- and norepinephrine-dependent cytosolic Ca2+ transients in fura-2-loaded intact arteries. An effect on Ca2+ signaling was also observed in cultured aortic smooth muscle cells (A10 cells). In these cells, farnesol reduced KCl-induced [Ca2+]i transients and mimicked the inhibitory effect of Ca2+-free medium on the [Ca2+]i response to both 12,13-phorbol myristate acetate, a protein kinase C activator, and thapsigargin, a specific endoplasmic reticulum ATPase inhibitor. Perforated patch-clamp experiments further showed in two vascular smooth muscle cell lines (A10 and A7r5), a reversible, dose-dependent inhibitory effect of farnesol on L-type Ca2+ currents (IC50 = 2.2 microM). Shorter (C10, geraniol) and longer (C20, geranylgeraniol) isoprenols were inactive. L-type Ca2+ channel blockade also occurred under tight (gigaohm) seal configuration using cell-attached, single-channel analysis, thus suggesting a possible action of farnesol from within the intracellular space. We finally demonstrated that farnesol did not affect Ca2+-sensitive pathways implicated in smooth muscle contraction, as tested with alpha-toxin permeabilized arteries. Altogether, our results indicate that farnesol is an inhibitor of vascular smooth muscle Ca2+ signaling with plasma membrane Ca2+ channel blocker properties. The data have implications for the endogenous and pharmacological regulation of vascular tone by farnesol or farnesol analogues.     

The effect of radiographic contrast media on human vascular smooth muscle cells

The relation between intravascular radiographic contrast media (RCM) and myointimal hyperplasia after percutaneous transluminal angioplasty is not known. We have investigated the cytotoxic effects of RCM on human vascular smooth muscle cells (VSMCs) and their effect on the growth of these cells. The cytotoxic effects of RCM were studied using human VSMCs. The cells after being grown to confluency were exposed for 60 min to 250 mgI ml-1 of diatrizoate, ioxaglate, iopromide, iotrolan and saturated mannitol solutions. The control group was treated with only 15% fetal calf serum (FCS) containing medium. The viability of the cells was examined using the trypan blue exclusion test. The effect of RCM on growth was assessed by exposing the VSMCs after growth arrest, for either 15 or 60 min to 250 mgI ml-1 of diatrozoate, ioxaglate, iopromide, iotrolan and saturated mannitol solution. There was no significant change in the viability of the VSMCs after 60 min exposure to iopromide, iotrolan, saturated mannitol solution, and after 15 min exposure to diatrizoate or ioxaglate. After exposure to diatrizoate or ioxaglate for 60 min, 16.5 +/- 2.2% or 9.2 +/- 2.6% dead cells were found, respectively (p < 0.05 versus control). In the growth assay of VSMCs, diatrizoate, ioxaglate and saturated mannitol solutions reduced the growth rate (p < 0.05 versus control). No significant change was observed with iopromide and iotrolan. In conclusion, ionic RCM have cytotoxic and cytostatic effects on VSMCs while non-ionic media have no effects. There is no direct stimulatory effect of contrast media on the growth of VSMCs. The cytotoxic and cytostatic effects of contrast media seems to be both osmolality and chemotoxicity dependent. Low osmolar non-ionic RCM are not likely to contribute to the mechanisms responsible for myointimal hyperplasia after angioplasty.     

Oleic acid and angiotensin II induce a synergistic mitogenic response in vascular smooth muscle cells

Oleic acid and angiotensin II (Ang II) are elevated and may interact to accelerate vascular disease in obese hypertensive patients. We studied the effects of oleic acid and Ang II on growth responses of rat aortic smooth muscle cells (VSMCs). Oleic acid (50 micromol/L) raised thymidine incorporation by 50% at 24 hours and cell number by 55% at 6 days (P<.05). Ang II (10(-11) to 10(-6) mol/L) did not significantly increase thymidine incorporation or VSMC number. Combining Ang II and 50 micromol/L oleic acid doubled thymidine incorporation and VSMC number. Losartan, an angiotensin type 1 (AT1) receptor antagonist, blocked the synergistic interaction between Ang II and oleic acid, whereas the AT2 receptor antagonist PD 123319 did not. Protein kinase C inhibition and downregulation, as well as inhibition of extracellular signal-regulated kinase (ERK) activation by PD 98059, eliminated the rise of thymidine incorporation in response to oleic acid and the synergistic interaction with Ang II. However, the response to 10% fetal bovine serum was unaffected. An antisense oligodeoxynucleotide to ERK-1 and ERK-2 reduced ERK protein expression and activation by 83% and 75%, respectively. Antisense prevented the rise of thymidine incorporation in response to oleic acid and the synergy with Ang II. Antisense reduced but did not prevent increased thymidine incorporation in response to serum. The data indicate that oleic acid and Ang II exert a synergistic mitogenic effect in VSMCs and suggest an important role for the AT1 receptor, PKC, and ERK in this synergy. The observations raise the possibility that a synergistic mitogenic interaction between oleic acid and Ang II accelerates vascular remodeling in obese hypertensive patients.     

Nitric oxide inhibits Oct-1 DNA binding activity in cultured vascular smooth muscle cells

In this paper, the authors model the nonmonotonic relation between body mass index (BMI) (weight (kg)/height2 (m2)) and mortality in 13,242 black and white participants in the NHANES I Epidemiologic Follow-up Study in order to estimate the BMI at which minimum mortality occurs. The BMI of minimum mortality was 27.1 for black men (95% confidence interval (CI) 24.8-29.4), 26.8 for black women (95% CI 24.7-28.9), 24.8 for white men (95% CI 23.8-25.9), and 24.3 for white women (95% CI 23.3-25.4). Each confidence interval included the group average. Analyses conducted by smoking status and after exclusion of persons with baseline illness and persons who died during the first 4 years of follow-up led to virtually identical estimates. The authors determined the range of values over which risk of all-cause mortality would increase no more than 20% in comparison with the minimum. This interval was nine BMI units wide, and it included 70% of the population. These results were confirmed by parallel analyses using quantiles. The model used allowed the estimation of parameters in the BMI-mortality relation. The resulting empirical findings from each of four race/sex groups, which are representative of the US population, demonstrate a wide range of BMIs consistent with minimum mortality and do not suggest that the optimal BMI is at the lower end of the distribution for any subgroup.     

The actions of ketamine on vascular smooth muscle

The responses of rabbit aortic strips superfused with noradrenaline, adrenaline and 5-HT were studied alone and in combination with ketamine (50 mug/ml). Ketamine caused a slight depression of the isolated aorta but potentiated responses to adrenaline but not to noradrenaline or 5-hydroxytryptamine. Ketamine did not potentiate aortic strips contracted to a stable level by pyrogallol and adrenaline. Experiments carried out with COMT from homogenates of rat liver showed that, in contrast to pyrogallol (10(-5) M), ketamine (10(-3) M) did not inhibit the enzyme. Other experiments with rabbits given 6-hydroxydopamine showed that aortas of these rabbits responded in a similar manner to controls when treated with ketamine and catecholamines. Results obtained with aortas contracted by adrenaline and noradrenaline with ketamine present, followed by oil immersion, showed that ketamine prolonged greatly the relaxation induced by adrenaline and to a lesser extent the relaxation induced by noradrenaline. The results of these studies indicate that ketamine prevented catecholamines from reaching the intracellular site of COMT. In this respect, ketamine can be termed an inhibitor of uptake site 2. If this hypothesis is valid then the action of ketamine on vascular tissue might explain the cardiovascular effects of the drug in man and experimental animals.     

Effects of visnagin on cyclic nucleotide phosphodiesterases and their role in its inhibitory effects on vascular smooth muscle contraction

Several reports have been published which describe the technique of using an Endo GIA to resect submucosal tumors on the anterior wall of the stomach. Lesions on the posterior wall, however, especially near the esophagocardiac junction (ECJ), are difficult to resect using these reported techniques. This is because the surgeon must divide the omentum and enter the omental bursa in order to use a similar extraluminal technique. Furthermore, special care must be taken to ensure that resections do not involve the ECJ and narrow the esophagus. In order to overcome these difficulties, we have proposed a new technique for the laparoscopic excision of a submucosal tumor located on the posterior wall of the gastric fundus. The principle of this procedure involves the intraluminal resection of the submucosal tumor, including the surrounding stomach wall, using the Endo GIA. This technique is safe, simple, and effective. We believe that we are the first to address the excision of a submucosal lesion by resecting the full thickness of the posterior gastric wall lesion intraluminally.