Biomedical subjects

K Takeda

Publications and source records attributed to K Takeda.

At least 289 records · Page 16Linked to original sources

PubMed2000

Potentiality of interleukin-18 as a useful reagent for treatment and prevention of Leishmania major infection.

Interleukin-18 (IL-18) is a proinflammatory cytokine that plays an important role in natural killer cell activation and the T helper 1 (Th1) cell response, particularly in collaboration with IL-12. Since Th1 cells play a pivotal role in the host defense against infection with intracellular microbes, such as Leishmania major, we investigated whether IL-18 is critically involved in protection against L. major infection by activation of Th1 cells. We administered IL-12 and/or IL-18 daily to L. major-susceptible BALB/c mice. Neither IL-12 (10 ng/mouse) nor IL-18 (1,000 ng/mouse) induced wound healing, while daily injection of IL-12 and IL-18 during the first week after infection strongly protected the mice from footpad swelling by induction and activation of Th1 cells. Furthermore, these mice acquired protective immunity. We also investigated a protective role of endogenous IL-18 by using anti-IL-18 antibody-treated C3H/HeN mice (an L. major-resistant strain) or IL-18 deficient (IL-18(-/-)) mice with a resistant background (C57BL/6). We found that in the absence of endogenous IL-18, these mice showed prolonged footpad swelling as well as diminished nitric oxide production. However, daily injection of IL-18 into IL-18(-/-) mice corrected their deficiencies, suggesting that these mice have Th1 cells that produce gamma interferon (IFN-gamma) in response to IL-18. Indeed, these mice had normal levels of Th1 cells. Thus, IL-18 is not responsible for inducing Th1 cells but participates in host resistance by its action in stimulating Th1 cells to produce IFN-gamma. Our results also indicate the high potentiality of IL-18 as a useful reagent for treatment as well as prevention against reinfection.

Animals↗

PubMed2000

Role of apoptosis signal-regulating kinase in regulation of the c-Jun N-terminal kinase pathway and apoptosis in sympathetic neurons.

We have previously shown that nerve growth factor (NGF) withdrawal-induced death requires the activity of the small GTP-binding protein Cdc42 and that overexpression of an active form of Cdc42 is sufficient to mediate neuronal apoptosis via activation of the c-Jun pathway. Recently, a new mitogen-activated protein (MAP) kinase kinase kinase, apoptosis signal-regulating kinase 1 (ASK1) which activates both the c-Jun N-terminal kinase (JNK) and p38 MAP kinase pathways and plays pivotal roles in tumor necrosis factor- and Fas-induced apoptosis, has been identified. Therefore, we investigated the role of ASK1 in neuronal apoptosis by using rat pheochromocytoma (PC12) neuronal cells and primary rat sympathetic neurons (SCGs). Overexpression of ASK1-DeltaN, a constitutively active mutant of ASK1, activated JNK and induced apoptosis in differentiated PC12 cells and SCG neurons. Moreover, in differentiated PC12 cells, NGF withdrawal induced a four- to fivefold increase in the activity of endogenous ASK1. Finally, expression of a kinase-inactive ASK1 significantly blocked both NGF withdrawal- and Cdc42-induced death and activation of c-jun. Taken together, these results demonstrate that ASK1 is a crucial element of NGF withdrawal-induced activation of the Cdc42-c-Jun pathway and neuronal apoptosis.

Animals↗

PubMed2000

Biological characteristics of the leukemia-associated transcriptional factor AML1 disclosed by hematopoietic rescue of AML1-deficient embryonic stem cells by using a knock-in strategy.

AML1 is one of the most frequently mutated genes associated with human acute leukemia and encodes the DNA-binding subunit of the heterodimering transcriptional factor complex, core-binding factor (CBF) (or polyoma enhancer binding protein 2 [PEBP2]). A null mutation in either AML1 or its dimerizing partner, CBFbeta, results in embryonic lethality secondary to a complete block in fetal liver hematopoiesis, indicating an essential role of this transcription complex in the development of definitive hematopoiesis. The hematopoietic phenotype that results from the loss of AML1 can be replicated in vitro with a two-step culture system of murine embryonic stem (ES) cells. Using this experimental system, we now demonstrate that this hematopoietic defect can be rescued by expressing the PEBP2alphaB1 (AML1b) isoform under the endogenous AML1-regulatory sequences through a knock-in (targeted insertion) approach. Moreover, we demonstrate that the rescued AML1(-/-) ES cell clones contribute to lymphohematopoiesis within the context of chimeric animals. Rescue requires the transcription activation domain of AML1 but does not require the C-terminal VWRPY motif, which is conserved in all AML1 family members and has been shown to interact with the transcriptional corepressor, Groucho/transducin-like Enhancer of split. Taken together, these data provide compelling evidence that the phenotype seen in AML1-deficient mice is due solely to the loss of transcriptionally active AML1.

Animals↗

PubMed2000

Role for mitochondrial oxidants as regulators of cellular metabolism.

Leakage of mitochondrial oxidants contributes to a variety of harmful conditions ranging from neurodegenerative diseases to cellular senescence. We describe here, however, a physiological and heretofore unrecognized role for mitochondrial oxidant release. Mitochondrial metabolism of pyruvate is demonstrated to activate the c-Jun N-terminal kinase (JNK). This metabolite-induced rise in cytosolic JNK1 activity is shown to be triggered by increased release of mitochondrial H(2)O(2). We further demonstrate that in turn, the redox-dependent activation of JNK1 feeds back and inhibits the activity of the metabolic enzymes glycogen synthase kinase 3beta and glycogen synthase. As such, these results demonstrate a novel metabolic regulatory pathway activated by mitochondrial oxidants. In addition, they suggest that although chronic oxidant production may have deleterious effects, mitochondrial oxidants can also function acutely as signaling molecules to provide communication between the mitochondria and the cytosol.

3T3 Cells↗

PubMed2000

Desmin splice variants causing cardiac and skeletal myopathy.

Desmin myopathy is a hereditary or sporadic cardiac and skeletal myopathy characterised by intracytoplasmic accumulation of desmin reactive deposits in muscle cells. We have characterised novel splice site mutations in the gene desmin resulting in deletion of the entire exon 3 during the pre-mRNA splicing. Sequencing of cDNA and genomic DNA identified a heterozygous de novo A to G change at the +3 position of the splice donor site of intron 3 (IVS3+3A-->G) in a patient with sporadic skeletal and cardiac myopathy. A G to A transition at the highly conserved -1 nucleotide position of intron 2 affecting the splice acceptor site (IVS2-1G-->A) was found in an unrelated patient with a similar phenotype. Expression of genomic DNA fragments carrying the IVS3+3A-->G and IVS2-1G-->A mutations confirmed that these mutations cause exon 3 deletion. Aberrant splicing leads to an in frame deletion of 32 complete codons and is predicted to result in mutant desmin lacking 32 amino acids from the 1B segment of the alpha helical rod. Functional analysis of the mutant desmin in SW13 (vim-) cells showed aggregation of abnormal coarse clumps of desmin positive material dispersed throughout the cytoplasm. This is the first report on the pathogenic potentials of splice site mutations in the desmin gene.

Adult↗

PubMed2000

Mice that overexpress Cu/Zn superoxide dismutase are resistant to allergen-induced changes in airway control.

Within the respiratory epithelium of asthmatic patients, copper/zinc-containing superoxide dismutase (Cu/Zn SOD) is decreased. To address the hypothesis that lung Cu/Zn SOD protects against allergen-induced injury, wild-type and transgenic mice that overexpress human Cu/Zn SOD were either passively sensitized to ovalbumin (OVA) or actively sensitized by repeated airway exposure to OVA. Controls included nonsensitized wild-type and transgenic mice given intravenous saline or airway exposure to saline. After aerosol challenge to saline or OVA, segments of tracheal smooth muscle were obtained for in vitro analysis of neural control. In response to electrical field stimulation, wild-type sensitized mice challenged with OVA had significant increases in cholinergic reactivity. Conversely, sensitized transgenic mice challenged with OVA were resistant to changes in neural control. Stimulation of tracheal smooth muscle to elicit acetylcholine release showed that passively sensitized wild-type but not transgenic mice released more acetylcholine after OVA challenge. Function of the M(2) muscarinic autoreceptor was preserved in transgenic mice. These results demonstrate that murine airways with elevated Cu/Zn SOD were resistant to allergen-induced changes in neural control.

Acetylcholine↗

PubMed2000

Mobilization of NK cells by exercise: downmodulation of adhesion molecules on NK cells by catecholamines.

The change of plasma catecholamine concentration correlates with the change of natural killer (NK) activity and NK cell number in peripheral blood mononuclear cells (PBMC) during and after moderate exercise. We studied the causal relation between exercise-induced catecholamine and expression of adhesion molecules on NK cells during and after exercise. The expression of CD44 and CD18 on CD3(-)CD56(+) NK cells was significantly reduced during exercise (P < 0.01). When PBMC were stimulated with 10(-8)M norepinephrine in vitro, the expression of these adhesion molecules on CD3(-)CD56(+) NK cells was downmodulated within 30 min. The binding capacity of NK cells to a CD44 ligand, hyaluronate, was reduced by the stimulation with norepinephrine (P < 0.01). The intravenous injection of norepinephrine in mice decreased the expression of CD44 and CD18 on CD3(-)NK1.1(+) cells (P < 0.01) and increased the number of CD3(-)NK1.1(+) cells in PBMC (P < 0.01). These findings suggest that exercise-induced catecholamines modulate the expression of adhesion molecules on NK cells, resulting in the mobilization of NK cells into the circulation.

Adult↗

PubMed2000

Two-step selective clamping of IVC for removal of hepatocellular carcinoma with intracaval extension.

Hepatocellular carcinoma (HCC) with retrohepatic intracaval extensions are difficult to treat. HCC may sometimes extend into the inferior vena cava (IVC) through two routes: via the right hepatic vein and via the inferior right hepatic vein. In such cases, in which tumor emboli are located both above and below the confluence of the hepatic vein with the IVC, we first remove the upper embolus during THVE, and then remove the lower one while the IVC is clamped obliquely in order to preserve the residual liver circulation.

Carcinoma, Hepatocellular↗

PubMed2000

Effect of increased intrareservoir pressure on upper urinary tract function in continent urinary diversion patients assessed by radioisotope renography.

To assess upper urinary tract function and determine an ideal continent reservoir capacity, we investigated the relationship between intrareservoir pressure and functional parameters using radioisotope renography. Forty-three patients for whom ileal reservoir construction was performed by Kock pouch (n = 33) or Hautmann's ileal neobladder (n = 14) approaches between November 1984 and September 1996 were studied. After the reservoir contents were catheterized, (99m)Tc-MAG 3 renoscintigraphy was carried out and time activity curves in the kidney (renogram) and reservoir (reservoirgram) were recorded, while saline solution was poured into the reservoir for monitoring of intrareservoir pressure. Three basic abnormal curves were noted in the excretory phase on renograms: a normal downward curve followed by an upward curve, a continuous upward curve associated with or without a sharp decline after the catheterization of the reservoir contents, and episodes of spike waves. Based on these curves, renograms were classified into five types: normal, high pressure, retention, obstruction, and reflux. Abnormal types were noted with 51 of 65 renal units (78.5%) in the Kock pouch group and 15 of 25 renal units (60.0%) in the neobladder group. This examination method may be useful for evaluating urodynamics in the upper urinary tract of patients with a continent ileal reservoir and provide data on adequate voiding volumes for the individual patient.

Adult↗

PubMed2000

Inhibition of type 4 phosphodiesterase by rolipram and Ginkgo biloba extract (EGb 761) decreases agonist-induced rises in internal calcium in human endothelial cells.

The effects of Gingko biloba extract EGb 761 on 5 isolated, vascular, cyclic nucleotide phosphodiesterase (PDE) isoforms were evaluated. EGb 761 preferentially inhibited PDE4 (IC(50)=25.1 mg/L), the isoform that is mainly present in endothelial cells, in a competitive manner (K:(i)=12.5 mg/L). Because changes in cyclic nucleotide levels may affect intracellular calcium ([Ca(2+)](i)) levels in endothelial cells, we examined the effects of EGb 761 on both resting [Ca(2+)](i) levels and agonist-induced rises in [Ca(2+)](i) in single human umbilical vein endothelial cells (HUVECs) in culture. The effects of EGb 761 were compared with those of rolipram, a selective PDE4 inhibitor that increases cellular cAMP levels, and the cAMP analogue dibutyryl cAMP (db-cAMP). EGb 761 (20 and 100 mg/L), rolipram (50 micromol/L), and db-cAMP (100 micromol/L) significantly inhibited histamine-, ATP-, and thrombin-induced [Ca(2+)](i) increases in HUVECs without modifying resting [Ca(2+)](i) levels. Similar results were obtained by using a Ca(2+)-free bath solution. EGb 761 (100 mg/L), but not rolipram (50 micromol/L) or db-cAMP (100 micromol/L), also inhibited Ca(2+) influx into cells having thapsigargin-depleted internal Ca(2+) stores and bathed in a Ca(2+)-free external solution. Our results are consistent with an inhibition of PDE activity that causes a reduction of agonist-induced increases in [Ca(2+)](i) in HUVECs, mainly by inhibition of Ca(2+) mobilization from internal stores. It thus may be that the cardiovascular effects of EGb 761 involve inhibition of PDE4 activity and subsequent modification of Ca(2+) signaling in endothelial cells.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2000

Downregulation of angiotensin II type 1 receptor by all-trans retinoic acid in vascular smooth muscle cells.

All-trans retinoic acid (atRA) is a biologically active metabolite of vitamin A that plays an important role in cell differentiation and proliferation. Although neointimal formation after balloon injury of rat carotid artery is inhibited by atRA, the mechanisms are not clearly understood. Because the renin-angiotensin system is one of the crucial components of atherosclerosis, we examined the effects of atRA on the expression of angiotensin II type 1 receptor (AT(1)-R) in vascular smooth muscle cells. atRA (1 micromol/L) decreased the AT(1)-R mRNA level by 50% after 24 hours; AT(1)-R number was also reduced to the same extent after 48 hours. atRA markedly suppressed promoter activity of the AT(1)-R promoter-luciferase construct, but AT(1)-R mRNA stability was not affected. Cycloheximide blocked the atRA-induced decrease in AT(1)-R mRNA expression, suggesting that this process requires de novo protein synthesis. Simultaneous treatment with an agonist (Ro40-6055) specific for retinoic acid receptor (RAR) and an agonist (Ro25-7836) specific for retinoid X receptor (RXR) suppressed the AT(1)-R mRNA expression comparable to that with treatment with atRA, suggesting that the RAR/RXR heterodimer mediates the effect of atRA in AT(1)-R downregulation. These results suggest that atRA suppressed AT(1)-R mRNA transcription through new protein synthesis induced by RAR/RXR-dependent transcription. This study provides novel insight into a role of atRA as an important molecule that regulates AT(1)-R gene expression and provides possible mechanisms for the suppression of neointimal formation by atRA.

Animals↗

PubMed2000

Downregulation of estrogen and progesterone receptors in the abnormal smooth muscle cells in pulmonary lymphangioleiomyomatosis following therapy. An immunohistochemical study.

Immunohistochemical and confocal microscopic studies were made on lung tissue from 10 women with lymphangioleiomyomatosis (LAM) to evaluate the distribution of estrogen receptors (ER) and progesterone receptors (PR) in the abnormal smooth muscle cells (LAM cells) that characterize this disorder. PR and ER were localized mainly in the nuclei of large, epithelioid LAM cells, in five patients in whom tissues were obtained before treatment. However, the reaction for PR and ER was essentially negative in similarly processed tissues from five patients studied after receiving hormonal therapy (progesterone and tamoxifen). In the untreated group, staining for ER and PR colocalized with that for HMB-45, but not with that for membrane type-1 matrix metalloproteinase (MT-1-MMP), which we have shown to be localized in proliferating LAM cells. These observations demonstrate that PR and ER are selectively expressed in a subpopulation of LAM cells that are larger in size, have a limited ability to proliferate, and do not produce MT-1-MMP, the enzyme that activates MMP-2 (which is secreted by LAM cells and is capable of lysing elastin and collagens). ER and PR in LAM cells appear to be downregulated by hormonal therapy.

Adult↗

PubMed2000

CD23 exhibits negative regulatory effects on allergic sensitization and airway hyperresponsiveness.

The effects of an anti-CD23 monoclonal antibody (B3B4) in CD23-deficient and CD23-overexpressing mice were compared in a murine model of allergic sensitization. After sensitization and challenge with OA, mice developed increased serum levels of OA-specific IgE and IgG(1) with airway eosinophilia and AHR when compared with nonsensitized animals. Anti-CD23 treatment was studied under two protocols: 10-d OA aerosol exposure and intraperitoneal sensitization followed by aerosol challenge. In both protocols anti-CD23 significantly reduced IgE and IgG(1) levels, abolished eosinophilia, and normalized AHR in BALB/c and wild-type CD23+/+ mice but not in CD23-/- mice. These changes were associated with increases in IFN-gamma and decreases in IL-4 production, suggesting that CD23 binding may affect not only IgE production but also the Th1/Th2 imbalance during the development of allergic AHR. Absence of CD23 in gene-deficient mice significantly enhanced OA-specific IgE and IgG(1) levels, airway eosinophilia, and AHR when compared with CD23+/+ wild-type littermates after sensitization and airway challenge. Sensitized and challenged CD23 transgenic mice also developed eosinophilic airway inflammation and methacholine hyperresponsiveness. However, the extent of AHR, BAL, and tissue eosinophilia in these animals showed a significant negative correlation with levels of CD23 expression on splenic T and B cells, demonstrating a limiting role of CD23 in the development of allergic AHR.

Animals↗

PubMed2000

Timing of administration of anti-VLA-4 differentiates airway hyperresponsiveness in the central and peripheral airways in mice.

The development of airway hyperresponsiveness (AHR) is correlated with the infiltration into the lungs of activated eosinophils and T lymphocytes. In large part, influx of eosinophils into the lung is dependent on very late activating antigen-4 (VLA-4) expression. However, the kinetics of eosinophil recruitment and the development of AHR are not fully delineated. Airway function was monitored by changes in lung resistance (RL) and dynamic compliance (Cdyn) to methacholine (MCh) inhalation after anti-VLA-4. After ovalbumin (OVA) sensitization and airway challenge of BALB/c mice, AHR increased as did the number of lung inflammatory cells. Administration of anti-VLA-4 to sensitized mice 2 h before the first (of three) OVA airway challenges significantly prevented changes in RL. Moreover, injection of the antibody from 2 h before the first challenge to 42 h after the last challenge significantly prevented the increases in RL, as well as eosinophil and lymphocyte numbers in the bronchoalveolar lavage fluid (BALF); interleukin-5 (IL-5) and leukotriene concentrations in BALF were also significantly inhibited. Interestingly, treatment with anti-VLA-4 only prevented changes in Cdyn and goblet cell hyperplasia when administered 2 h before the first challenge. These studies demonstrate that the timing of anti-VLA-4 administration can selectively affect pathologic processes that contribute to altered airway function in the central and peripheral airways after allergen challenge.

Airway Resistance↗

PubMed2000

Interleukin (IL)-5 but not immunoglobulin E reconstitutes airway inflammation and airway hyperresponsiveness in IL-4-deficient mice.

We studied the role of interleukin (IL)-4, IL-5, and allergen-specific immunoglobulin (Ig) E in the development of allergen-induced sensitization, airway inflammation, and airway hy-perresponsiveness (AHR). Normal, IL-4-, and IL-5-deficient C57BL/6 mice were sensitized intraperitoneally to ovalbumin (OVA) and repeatedly challenged with OVA via the airways. After allergen sensitization and airway challenge, normal and IL-5-deficient, but not IL-4-deficient, mice developed increased serum levels of total and antigen-specific IgE levels and increased IL-4 production in the lung tissue compared with nonsensitized control mice. Only normal mice showed significantly increased IL-5 production in the lung tissue and an eosinophilic infiltration of the peribronchial regions of the airways, whereas both IL-4- and IL-5-deficient mice had little or no IL-5 production and no significant eosinophilic airway inflammation. Associated with the inflammatory responses in the lung, only normal mice developed increased airway responsiveness to methacholine after sensitization and airway challenge; in both IL-4- and IL-5-deficient mice, airway responsiveness was similar to that in nonsensitized control mice. Reconstitution of sensitized, IL-4-deficient mice before allergen airway challenge with IL-5, but not with allergen-specific IgE, restored eosinophilic airway inflammation and the development of AHR. These data demonstrate the importance of IL-4 for allergen-driven airway sensitization and that IL-5, but not allergen-specific IgE, is required for development of eosinophilic airway inflammation and AHR after this mode of sensitization and challenge.

Animals↗

PubMed2000

Gene dosage affects the cardiac and brain phenotype in nonmuscle myosin II-B-depleted mice.

Complete ablation of nonmuscle myosin heavy chain II-B (NMHC-B) in mice resulted in cardiac and brain defects that were lethal during embryonic development or on the day of birth. In this paper, we report on the generation of mice with decreased amounts of NMHC-B. First, we generated B(DeltaI)/B(DeltaI) mice by replacing a neural-specific alternative exon with the PGK-Neo cassette. This resulted in decreased amounts of NMHC-B in all tissues, including a decrease of 88% in the heart and 65% in the brain compared with B(+)/B(+) tissues. B(DeltaI)/B(DeltaI) mice developed cardiac myocyte hypertrophy between 7 months and 11 months of age, at which time they reexpressed the cardiac beta-MHC. Serial sections of B(DeltaI)/B(DeltaI) brains showed abnormalities in neural cell migration and adhesion in the ventricular wall. Crossing B(DeltaI)/B(DeltaI) with B(+)/B(-) mice generated B(DeltaI)/B(-) mice, which showed a further decrease of approximately 55% in NMHC-B in the heart and brain compared with B(DeltaI)/B(DeltaI) mice. Five of 8 B(DeltaI)/B(-) mice were born with a membranous ventricular septal defect. Moreover, 5 of 5 B(DeltaI)/B(-) mice developed myocyte hypertrophy by 1 month; B(DeltaI)/B(-) mice also reexpressed the cardiac beta-MHC. More than 60% of B(DeltaI)/B(-) mice developed overt hydrocephalus and showed more severe defects in neural cell migration and adhesion than did B(DeltaI)/B(DeltaI) mice. These data on B(DeltaI)/B(DeltaI) and B(DeltaI)/B(-) mice demonstrate a gene dosage effect of the amount of NMHC-B on the severity and time of onset of the defects in the heart and brain.

Animals↗

PubMed2000

Adenovirus-mediated tumor suppressor p53 gene therapy for anaplastic thyroid carcinoma in vitro and in vivo.

The present study was designed to evaluate the therapeutic efficacy of adenovirus-mediated wild-type (wt) tumor suppressor p53 expression in four human thyroid carcinoma cell lines harboring p53 mutations (ARO, FRO, NPA, and WRO) and normal human thyroid follicular cells with wt-p53 in vitro and in vivo. In vitro infection of replication-deficient recombinant adenovirus vector expressing wt-p53 led to a dose-dependent cell killing in both normal and carcinoma cells. In contrast, adenovirus expressing Escherichia coli beta-galactosidase showed little effect. The sensitivity to p53-mediated cell killing varied among the cells used. It was, at least partly, dependent on their adenovirus infectivity in carcinoma cells, whereas normal thyroid cells were relatively resistant to p53-mediated cell death despite its highest adenovirus infectivity. The mechanism of cell killing by wt-p53 was shown, by flow cytometric analysis, to be apoptosis. Furthermore, wt-p53 expression renders two out of four carcinoma cell lines (FRO and NPA) more sensitive to doxorubicin and one (FRO) to 5-fluorouracil, independent of treatment schedule. In vivo experiments, using FRO and NPA cells, showed that growth of sc tumors in nude mice was nearly completely inhibited by direct injection of adenovirus expressing wt-p53 [1 x 10(9) plaque-forming units/tumor]. This effect was augmented by its combination with doxorubicin treatment (4 mg/kg, thrice a week), which led to tumor regression. Our results therefore indicate that adenovirus-mediated wt-p53 gene introduction seems to be a potential clinical utility in gene therapy for anaplastic thyroid carcinomas, particularly when combined with chemotherapy.

Adenoviridae↗

PubMed2000

Migration of human vascular smooth muscle cells involves serum-dependent repeated cytosolic calcium transients.

Migration of vascular smooth muscle cells (VSMC) is a key event in the formation of neointima during atherosclerosis. Fura-2 loaded VSMCs were used to investigate calcium homeostasis during cell migration. Multiple spontaneous transient increases in cytosolic free calcium [Ca(2+)](i)were observed in single human VSMCs migrating on type I collagen. Such [Ca(2+)](i)transients were dependent on the presence of serum or PDGF-BB. Removal of serum, or loading cells with BAPTA, abolished the transients and decreased cell migration speed. The transients were not affected by disruption of cell polarization by dihydrocytochalasin B. Adhesion was used to investigate the specific role of cell-substrate interactions in the generation of transients. Transients are seen in VSMCs adhering either on collagen or on poly-L-lysine, suggesting that generation of transients is not strictly dependent on integrins. Buffering [Ca(2+)](i) with BAPTA led to accumulation of (beta)1 integrins at the cellular tail, and to increased release of integrin on the extracellular matrix. These results demonstrate a role for [Ca(2+)](i) transients in the rapid, serum-dependent migration of VSMCs. These [Ca(2+)](i)transients are present in migrating VSMCs only when two simultaneous events occur: (1) substrate independent spreading and (2) stimulation of cells by serum components such as PDGF-BB.

Antibodies, Monoclonal↗