Expression Of Caldesmon Research Articles

Smooth Muscle Hypertrophy Following Partial Bladder Outlet Obstruction Is Associated with Overexpression of Non-Muscle Caldesmon

Partial bladder outlet obstruction (PBOO) induces remodeling of urinary bladder smooth muscle (detrusor). We demonstrate an increase in bladder wall mass, muscle bundle size, and a threefold increase in the cross-sectional area of detrusor myocytes following PBOO in male New Zealand White rabbits compared to that of controls. Some bladders with detrusor hypertrophy function close to normal (compensated), whereas others were dysfunctional (decompensated), showing high intravesical pressure, large residual urine volume, and voiding difficulty. We analyzed the expression of smooth muscle-specific caldesmon (h-CaD) and non-muscle (l-CaD) by Western blotting, RT-PCR, and real-time PCR. The expression of l-CaD is increased significantly at the mRNA and protein levels in the decompensated bladders compared to that of normal and compensated bladders. The CaD was also co-localized with myosin containing cytoplasmic fibrils in cells dissociated from obstructed bladders and cultured overnight. Our data show that the inability of decompensated bladders to empty, despite detrusor hypertrophy, is associated with an overexpression of l-CaD. The level of l-CaD overexpression might be a useful marker to estimate the degree of detrusor remodeling and contractile dysfunction in PBOO.

Multilineage potential of cells from the artery wall.

In diabetes or atherosclerosis, ectopic bone, fat, cartilage, and marrow often develop in arteries. However the mechanism is unknown. We have previously identified a subpopulation of vascular cells (calcifying vascular cells, CVC), derived by dilutional cloning of bovine aortic medial cells, and showed that they undergo osteoblastic differentiation and mineralization. We now show that CVC have the potential to differentiate along other mesenchymal lineages. To determine the multilineage potential of CVC, molecular and functional markers of multiple mesenchymal lineages were assessed. Chondrogenic potential of CVC was evidenced by expression of types II and IX collagen and cytochemical staining for Alcian blue. Leiomyogenic potential of CVC was evidenced by the expression of smooth muscle-alpha actin, calponin, caldesmon, and myosin heavy chain. Stromogenic potential of CVC was evidenced by the ability to support growth of colony-forming units of hematopoietic progenitor cells from human CD34+ umbilical cord blood cells for a period of 5 weeks. Adipogenic potential was not observed. CVC were immunopositive to antigens to CD29 and CD44 but not to CD14 or CD45, consistent with other mesenchymal stem cells. CVC retained multipotentiality despite passaging and expansion through more than 20 to 25 population triplings, indicating a capacity for self-renewal. These results suggest that the artery wall contains cells that have the potential for multiple lineages similar to mesenchymal stem cells but with a unique differentiation repertoire.

Postnatal changes in caldesmon expression and localization in cardiac myocytes.

Caldesmon is a heat-stable protein found in both muscle and non-muscle tissue. It binds to a number of contractile and cytoskeletal proteins and may be involved in regulating acto-myosin interaction in smooth muscle cells and/or the assembly of microfilaments in muscle and non-muscle cells. We have shown previously that caldesmon is localized at the Z-lines in adult cardiac myocytes and that both the low- and high-molecular-weight forms (/-caldesmon and h-caldesmon, respectively) are present in atrial and ventricular myocytes. Here we examined the expression of caldesmon and its localization in freshly isolated cardiac myocytes during postnatal development and when these myocytes were grown in culture. We found that /-caldesmon is expressed in both neonatal and adult rat ventricular myocytes. The expression of h-caldesmon, however, was not detected in myocytes from newborn animals but increased during the first 2 weeks of postnatal development. Caldesmon was generally not co-localized with alpha-actinin at the Z-lines in neonatal myocytes but became increasingly more so during the first 2 weeks of postnatal development. When myocytes from 5- and 10-day-old rats were grown in primary culture, h-caldesmon expression decreased and caldesmon could not be detected at the Z-lines in the cultured cells. These results indicate that caldesmon plays a role at the Z-lines in adult cardiac myocytes; however, its localization at the Z-lines is not necessary for the prenatal development that occurs at these sites or for the establishment of a contractile phenotype in cultured cardiac myocytes.

Calponin and h-caldesmon expression in synovial sarcoma; the use of calponin in diagnosis.

To assess the frequency of expression and potential diagnostic utility of calponin and caldesmon in synovial sarcomas. Immunohistochemistry with antibodies to calponin and h-caldesmon was undertaken in paraffin sections from 50 synovial sarcomas (19 biphasic and 31 monophasic, of which one biphasic and eight monophasic tumours had poorly differentiated areas), 10 each of malignant peripheral nerve sheath tumour (MPNST), solitary fibrous tumour (SFT), dermatofibrosarcoma (DFSP), Ewing's sarcoma (ES/PNET), and neuroblastoma, eight alveolar rhabdomyosarcomas, five adult fibrosarcomas and five carcinosarcomas. Nine of 19 biphasic synovial sarcomas were positive for calponin in spindle and glandular areas, nine in spindle cells only, and one in glands only. All monophasic synovial sarcomas and poorly differentiated areas expressed calponin; in monophasic tumours this was focally (29% of cases), moderately (39%), or diffusely (32%) positive and the poorly differentiated areas were usually moderately or diffusely positive. Four synovial sarcomas showed focal reactivity for h-caldesmon. Calponin was found in 4/10 MPNST, 7/10 SFT, 4/10 DFSP, 3/5 fibrosarcomas and the spindle component of the carcinosarcomas. H-caldesmon was weakly positive in 1/10 MPNST, 4/10 SFT, 0/10 DFSP, 0/5 fibrosarcomas and 1/5 carcinosarcomas (glands only). Both markers were negative in the other small round cell tumours. Calponin can be used as an additional marker for synovial sarcoma. Its absence argues against the diagnosis. The presence of calponin might be useful in distinguishing poorly differentiated synovial sarcoma from other small round cell tumours. H-caldesmon is not helpful in diagnosis of synovial sarcoma.

Smooth muscle myosin heavy chain and caldesmon expression in the anterior vaginal wall of women with and without pelvic organ prolapse

Objective: The aim of this study was to quantify the expression of smooth muscle myosin heavy chain (SM-MHC) and caldesmon in the anterior vaginal wall of women with and without pelvic organ prolapse. Study Design: Immunoblot analysis was conducted on protein extracts from the vaginal muscularis of 15 women with and 11 women without pelvic organ prolapse by using specific antibodies for SM-MHC, nonmuscle MHC-B, and caldesmon. The fraction of muscularis containing smooth muscle was determined by morphometric analysis of histologic cross sections. Reverse transcriptase–polymerase chain reaction was used to amplify SM-MHC isoforms produced by alternative splicing in the myosin head. Results: Whereas the expression of SM-MHC was increased modestly (2-fold), expression of smooth muscle caldesmon was increased 6- to 7-fold in vaginal muscularis from women with prolapse. The relative distribution of SM-MHC isoforms was similar in both groups. Conclusions: Caldesmon is increased substantially in vaginal smooth muscle of women with pelvic organ prolapse. Caldesmon inhibits actin-activated myosin magnesium adenosine triphosphatase activity and inhibits the maintenance of contractile force. Thus, this disproportionate increase in caldesmon, relative to myosin, may result in inhibition of vaginal smooth muscle contractility and force maintenance. (Am J Obstet Gynecol 2001;185: 944-52.)

Cytoskeletal organization in tropomyosin-mediated reversion of ras-transformation: Evidence for Rho kinase pathway.

Tropomyosin (TM) family of cytoskeletal proteins is implicated in stabilizing actin microfilaments. Many TM isoforms, including tropomyosin-1 (TM1), are down-regulated in transformed cells. Previously we demonstrated that TM1 is a suppressor of the malignant transformation, and that TM1 reorganizes microfilaments in the transformed cells. To investigate how TM1 induces microfilament organization in transformed cells, we utilized ras-transformed NIH3T3 (DT) cells, and those transduced to express TM1, and/or TM2. Enhanced expression of TM1 alone, but not TM2, results in re-emergence of microfilaments; TM1, together with TM2 remarkably improves microfilament architecture. TM1 induced cytoskeletal reorganization involves an enhanced expression of caldesmon, but not vinculin, alpha-actinin, or gelsolin. In addition, TM1-induced cytoskeletal reorganization and the revertant phenotype appears to involve re-activation of RhoA controlled pathways in DT cells. RhoA expression, which is suppressed in DT cells, is significantly increased in TM1-expressing cells, without detectable changes in the expression of Rac or Cdc42. Furthermore, expression of a dominant negative Rho kinase, or treatment with Y-27632 disassembled microfilaments in normal NIH3T3 and in TM1 expressing cells. These data suggest that reactivation of Rho kinase directed pathways are critical for TM1-mediated microfilament assemblies.

Differential expression of high molecular weight caldesmon in colorectal pericryptal fibroblasts and tumour stroma.

AIM: To investigate an extent of smooth muscle differentiation of pericryptal fibroblasts. METHODS: Expression of high molecular weight caldesmon (h-CD) and alpha smooth muscle actin was investigated in 123 invasive...

Caldesmon inhibits nonmuscle cell contractility and interferes with the formation of focal adhesions.

Caldesmon is known to inhibit the ATPase activity of actomyosin in a Ca(2+)-calmodulin-regulated manner. Although a nonmuscle isoform of caldesmon is widely expressed, its functional role has not yet been elucidated. We studied the effects of nonmuscle caldesmon on cellular contractility, actin cytoskeletal organization, and the formation of focal adhesions in fibroblasts. Transient transfection of nonmuscle caldesmon prevents myosin II-dependent cell contractility and induces a decrease in the number and size of tyrosine-phosphorylated focal adhesions. Expression of caldesmon interferes with Rho A-V14-mediated formation of focal adhesions and stress fibers as well as with formation of focal adhesions induced by microtubule disruption. This inhibitory effect depends on the actin- and myosin-binding regions of caldesmon, because a truncated variant lacking both of these regions is inactive. The effects of caldesmon are blocked by the ionophore A23187, thapsigargin, and membrane depolarization, presumably because of the ability of Ca(2+)-calmodulin or Ca(2+)-S100 proteins to antagonize the inhibitory function of caldesmon on actomyosin contraction. These results indicate a role for nonmuscle caldesmon in the physiological regulation of actomyosin contractility and adhesion-dependent signaling and further demonstrate the involvement of contractility in focal adhesion formation.

Enhanced interstitial expression of caldesmon in IgA nephropathy and its suppression by glucocorticoid-heparin therapy.

With progressive renal disease, structural derangement increasingly encompasses the tubulointerstitial compartment. Tubulointerstitial injury is a critical determinant of renal functional reserve and prognosis in renal disease. Interstitial cells acquiring characteristic of myofibroblasts are an important contributor to interstitial fibrosis. Caldesmon, a calmodulin or actin binding protein, is a molecular marker of differentiation in smooth muscle cells and has recently been shown by us to be a good marker of mesangial cell activation in IgA nephropathy patients. METHODS. We studied whether the expression of caldesmon in interstitium of the kidney was enhanced in the process of glomerular disease and whether it would be a marker of interstitial activation in specific disease states. We performed immunohistochemical staining with anti-caldesmon antibodies in 38 biopsy specimens from IgA nephropathy patients and analysed them quantitatively with a computer-aided manipulator. Interstitial caldesmon expression were compared with histological changes and clinical parameters. Caldesmon expression was enhanced where interstitial cell infiltration and fibrosis were found. Immunoelectron microscopy revealed that caldesmon staining in the renal interstitium was cytoplasmic, and in the processes of myofibroblast-like cells. Caldesmon expression was more prominent in the intense CD68 infiltrated group than in the low positive cells infiltrated group. Patients showing high intensity of interstitial caldesmon expression had significantly higher urinary protein excretion than those showing low intensity of caldesmon expression. Next, 15 patients were treated with glucocorticoid and heparin for 4-8 weeks and re-biopsies were performed. Caldesmon expression was reduced in concomitant with decreased interstitial cell infiltration. Follow-up of these patients (average 24 months) revealed a significant suppression of urinary protein excretion and significant improvement of creatinine clearance. These results suggest that the interstitial caldesmon expression is associated with the progression of IgA nephropathy, and glucocorticoid--heparin therapy may reverse the phenotypic change of interstitial cells during the disease process of glomerulonephritis.

Structural interactions between actin, tropomyosin, caldesmon and calcium binding protein and the regulation of smooth muscle thin filaments.

The basic structure and functional properties of smooth muscle thin filaments were established about 10 years ago. Since then we and others have been working on the details of how tropomyosin, caldesmon and the Ca(2+)-binding protein regulate actin interaction with myosin. Our work has tended to emphasize the similarities between caldesmon and troponin function whilst others have been more concerned with the differences. The need to resolve the resulting differences has stimulated us to find new and more direct ways of investigating the mechanism of thin filament regulation. In recent years an apparent divergence has opened up between functional measurements, which indicate an allosteric-cooperative regulatory mechanism in which caldesmon and Ca(2+)-binding protein control actin-tropomyosin state in the same way as troponin, and structural measurements which show thin filament structures unlike striated muscle thin filaments. The challenge is to interpret function in terms of structure. We have combined functional studies with expression and mutagenesis of caldesmon and with structural methods including X-ray crystalography of tropomyosin-caldesmon crystals, electron microscopy and helical reconstruction of actin-tropomyosin-caldesmon complexes and high resolution nuclear magnetic resonance spectroscopy of the C-terminus of caldesmon in interaction with actin and calmodulin. We have used this information to propose a structural mechanism for caldesmon regulation of the smooth muscle thin filament.

Enhanced interstitial expression of caldesmon and its suppression by glucocorticoid and heparin therapy in IgA nephropathy patients

Enhanced interstitial expression of caldesmon and its suppression by glucocorticoid and heparin therapy in IgA nephropathy patients

Enhanced glomerular expression of caldesmon in IgA nephropathy and its suppression by glucocorticoid-heparin therapy.

Activation and consequent phenotypic modulation of mesangial cells is considered to play a crucial role in the process of glomerular disease progression. Caldesmon, a calmodulin and actin-binding protein, is a molecular marker of the phenotypic change in smooth-muscle cells. We studied whether the expression of caldesmon in mesangial cells was enhanced in the process of IgA nephropathy and whether it would be a marker of mesangial activation indicating prognostic significance in specific disease states. We performed immunohistochemical staining with anticaldesmon and alpha-smooth-muscle actin (alpha-SMA) antibodies in 32 biopsy specimens from IgA nephropathy patients and analysed them quantitatively with a computer-aided manipulator. The glomerular expression of caldesmon was enhanced in IgA nephropathy patients. We compared caldesmon expression with composite histological scores (cell score and matrix score), clinical parameters and expressions of alpha-SMA. There was a statistically significant correlation between the caldesmon score and the histological scores (cell score and matrix score, P<0.0001, P<0.01 respectively). Patients showing a high intensity of caldesmon expression (defined as caldesmon score > or = 35; H-group) had significantly higher urinary protein excretion than those showing a low intensity of caldesmon expression (defined as caldesmon score < 35; L-group) (1.2 +/- 1.2 g/24 h vs 0.41 +/- 0.53 g/24 h, P<0.05). Caldesmon and alpha-SMA expression had a statistically significant correlation (P<0.000). Next, 13 patients were treated with glucocorticoid-heparin for 4-8 weeks and re-biopsies were performed. After the therapy, the caldesmon and alpha-SMA scores were significantly lower than those before the therapy (P<0.01). These results suggest that the expression of caldesmon in glomeruli is associated with the progression of IgA nephropathy, and that glucocorticoid heparin therapy may reverse the phenotype of mesangial cells during the disease process of glomerulonephritis.

Expression of smooth muscle caldesmon in developing chicken gizzard

Caldesmon is an actin/calmodulin/tropomyosin binding protein located in the thin filaments of smooth muscle cells and microfilaments of nonmuscle cells. Two isoforms of caldesmon, h- and l-types, shown to exist in vertebrate smooth and nonmuscle cells respectively, are produced by alternative splicing of the caldesmon mRNA encoded by a single gene. To study the expression of smooth muscle specific h-caldesmon during the differentiation of mesenchymal cells into smooth muscle cells, soluble protein and total RNA from the gizzard primordium in the gut region of 5-day and gizzards of 7-, 9-, 13-, 17- and 21-day embryos and 2-day post-hatch chicks were extracted and analyzed for caldesmon expression at both protein and mRNA levels. Western blot analysis of proteins and immunofluorescence microscopy of tissue sections were carried out using an antibody specific for h-caldesmon. Total RNA was analyzed by Northern blotting using a caldesmon cDNA probe, and h- and l-caldesmon cDNAs were identified due to the difference in their molecular sizes (4.8 and 4.1 kb respectively). The mRNA was also analyzed by reverse transcribed-polymerase chain reaction (RT-PCR) and Southern blot analysis. Our results show that the l-caldesmon mRNA was expressed at higher levels in the gizzard primordium during the early stages of development, and decreased gradually during growth. The h-caldesmon protein and mRNA, not expressed at day 5, is minimally expressed at day 7 and is fully turned on by day 9. Additionally, sequence analyses of the RT-PCR products of l-caldesmon showed that it lacked the spacer region, as predicted. RT-PCR analysis of total RNA gave two h-caldesmon fragments. These two fragments were identified as two different isoforms of h-caldesmon since they both contained the spacer region. They also showed homology in the region of exon 4 and had differences in the region of exon 3b.

Protein kinase C activation and its role in the development of vascular complications in diabetes mellitus.

Hyperglycemia is the major causal factor in the development of diabetic vascular complications and can mediate their adverse effects through multiple pathways. One of those mechanisms is the activation of protein kinase C (PKC) by hyperglycemia-induced increases in diacylglycerol (DAG) level, partly due to de novo synthesis. The activation of PKC regulates various vascular functions by modulating enzymatic activities such as cytosolic phospholipase A2 and Na+,K+-ATPase, and gene expressions including extracellular matrix components and contractile proteins. Some of the resulting vascular abnormalities include changes in retinal and renal blood flow, contractility, permeability, proliferation, and basement membrane. Among the various isoforms of PKC predominantly the beta isoforms are activated in cultured vascular cells exposed to high glucose and vascular tissues isolated from animal models of diabetes mellitus. Administration of vitamin E, which decreases DAG level possibly through the activation of DAG kinase, prevents hemodynamic changes in retina and renal glomeruli of diabetic rats. In addition, the inhibition of PKC beta isoforms by a specific inhibitor (LY333531) can normalize the changes in gene expression of cytokines, caldesmon, and hemodynamics. These results provide supportive evidence that the activation of PKC, especially the beta isoforms, is involved in the development of diabetic vascular complications, and that PKCbeta inhibitors can be used in the treatment of diabetic vascular complications.

Transcriptional regulation of the chicken caldesmon gene. Activation of gizzard-type caldesmon promoter requires a CArG box-like motif.

Caldesmon, which plays a vital role in the actomyosin system, is distributed in smooth muscle and non-muscle cells, and its isoformal interconversion between a high M(r) form and low M(r) form is a favorable molecular event for studying phenotypic modulation of smooth muscle cells. Genomic analysis reveals two promoters, of which the gizzard-type promoter displays much higher activity than the brain-type promoter. Here, we have characterized transcriptional regulation of the gizzard-type promoter. Transient transfection assays in chick gizzard smooth muscle cells, chick embryo fibroblasts, mouse skeletal muscle cell line (C2C12), and HeLa cells revealed that the promoter activity was high in smooth muscle cells and fibroblasts, but was extremely low in other cells. Cell type-specific promoter activity depended on an element, CArG1, containing a unique CArG box-like motif (CCAAAAAAGG) at -315, while multiple E boxes were not directly involved in this event. Gel shift assays showed the specific interaction between the CArG1 and nuclear protein factors in smooth muscle cells and fibroblasts. These results suggest that the CArG1 is an essential cis-element for cell type-specific expression of caldesmon and that the function of CArG1 might be controlled under phenotypic modulation of smooth muscle cells.

Embryonic chicken gizzard: expression of the smooth muscle regulatory proteins caldesmon and myosin light chain kinase

The patterns of expression of the smooth muscle regulatory proteins caldesmon and myosin light chain kinase were investigated in the developing chicken gizzard. Immunofluorescent studies revealed that both proteins were expressed as early as E5 throughout the mesodermal gizzard anlage, together with actin, alpha-actin-in and a small amount of nonmuscle myosin. These proteins appear to form the scaffold for smooth muscle development, defined by the onset of smooth muscle myosin expression. During E6, a period of extensive cell division, smooth muscle myosin begins to appear in the musculi laterales close to the serosal border and, later, also in the musculi intermedii. Until about E10, myosin reactivity expands into the pre-existing thin filament scaffold. Later in development, the contractile and regulatory proteins co-localize and show a regular uniform staining pattern comparable to that seen in adult tissue. By using immunoblotting techniques, the low-molecular mass form of caldesmon and myosin light chain kinase were detected as early as E5. During further development, the expression of caldesmon switched from the low-molecular mass to the high-molecular mass form; in neonatal and adult tissue, high-molecular mass caldesmon was the only isoform expressed. The level of expression of myosin light chain kinase increased continously during embryonic development, but no embryo-specific isoform with a different molecular mass was detected.

Embryonic chicken gizzard: expression of the smooth muscle regulatory proteins caldesmon and myosin light chain kinase

Embryonic chicken gizzard: expression of the smooth muscle regulatory proteins caldesmon and myosin light chain kinase

Synthesis and Expression of Smooth Muscle Phenotype Markers in Primary Culture of Rabbit Aortic Smooth Muscle Cells: Influence of Seeding Density and Media and Relation to Cell Contractility

Rabbit aortic smooth muscle cells (SMC) were seeded at moderate or high densities and grown either in the presence of serum or in the serum-substitution formula Monomed. Expression and synthesis of marker proteins caldesmon, calponin, smooth muscle myosin, and vinculin were monitored during SMC cultivation. Contractility was tested by the ability of cultured SMC to deform silicone membranes following ionomycin treatment. The results show that cells of moderate density grown in Monomed, as opposed to those grown in 5% serum, have the smooth muscle isoform of caldesmon 1.6-fold higher, calponin 1.4-fold and smooth muscle myosin 1.4-fold higher on Day 14 of cultivation. Synthesis of these proteins corresponded to their expression in SMC. The metavinculin:vinculin ratio slightly decreased over the first days with a following reestablishment on Day 8. Contraction was observed until Day 13, compared with Day 7 for cells grown in the presence of serum. High seeding density also prevented a decrease in the expression of smooth muscle markers with the exception of smooth muscle caldesmon whose content in the high density SMC culture was not significantly different from that in the moderate density culture. The period of contractility of SMC in the high density culture was also similar to that in the moderate density culture in the presence of serum. We conclude that cultivation of primary SMC in Monomed allows the maintenance of cells in the contractile phenotype more effectively than high initial seeding density.

Phenotypic changes of human smooth muscle cells during development: Late expression of heavy caldesmon and calponin

Expression of the regulatory contractile proteins, heavy caldesmon (h-caldesmon) and calponin was studied in human aortic smooth muscle cells (SMCs) during development and compared with the expression of α-SM-actin and smooth muscle-myosin heavy chain (SM-MHCs). For this study, novel monoclonal antibodies specific to SM-MHCs, h-caldesmon, and calponin were developed and characterized. Aortic SMCs from fetuses of 8–10 and 20–22 weeks of gestation express α-SM-actin and SM-MHCs, but neither h-caldesmon nor calponin were expressed as demonstrated by immunoblotting and immunofluorescence techniques. In the adult aortic tunica media, SMCs contain all four markers. Thus, the expression of calponin, similar to the expression of α-SM-actin, SM-MHCs, and h-caldesmon, is developmentally regulated in aortic SMCs. In the adult aortic subendothelial (preluminal) part of tunica intima, numerous cells containing SM-MHCs, but lacking h-caldesmon and calponin, were found. These results illustrate the similarity of SMCs from intimal thickenings and immature (fetal) SMCs. Expression of contractile proteins in the developing SMCs is coordinately regulated; however, distinct groups of proteins appear to exist whose expression is regulated differently. Actin and myosin, being major contractile proteins, also play a structural role and appear rather early in development, whereas caldesmon and calponin, being involved in regulation of contraction, can serve as markers of higher SMC differentiation steps. In contrast, h-caldesmon and calponin were already present in visceral SMCs (trachea, esophagus) of the 10-week-old fetus. These results demonstrate that the time course of maturation of visceral SMCs is different from that of vascular SMCs.

Contractile rabbit aortic smooth muscle cells in culture. Preparation and characterization.

Investigations were conducted to determine whether rabbit aortic smooth muscle cells (SMC) reproduce their essential in situ features in culture. Enzymatically isolated cells in culture were compared with their in situ state in terms of myosin and caldesmon isoform expression, sensitivity to Ca(2+)-mobilizing agonists, and contractility. Protein marker expression was assessed by electrophoresis and quantitative immunoblotting, and intracellular free Ca2+ ([Ca2+]i) measurements were accomplished using indo-1, a Ca(2+)-sensitive fluorescent dye. Contraction of SMC grown on deformable silicone films was monitored optically. Before the onset of cell division (3 to 6 days in culture), SMC still contained significant although decreasing amounts of smooth muscle myosin (SM1 and SM2 isoforms) and they started to express nonmuscle-type myosin. The relative content of 150-kDa caldesmon decreased, whereas the expression of 77-kDa caldesmon increased during this period. In the confluent primary culture (11 days), SM1 was expressed, but 150-kDa caldesmon was hardly detectable. Histamine (10(-5) mol/L), serotonin (10(-6) mol/L), and thrombin (1.5 units/mL) contracted deendothelialized rings of rabbit aorta, but only histamine was able to elevate [Ca2+]i 2.5- to 3-fold and induce reversible contraction of primary nondividing cells. [Ca2+]i elevation in response to histamine was due both to Ca2+ mobilization from intracellular stores and Ca2+ flux across the plasma membrane. After the onset of proliferation, SMC regained the ability to elevate [Ca2+]i in response to serotonin and thrombin but lost the ability to contract. Thus, primary cultured quiescent rabbit aortic SMC (3 to 6 days in culture) retain the essential features of vascular SMC in situ (eg, smooth muscle specific contractile and regulatory proteins, vasoactive hormone sensitivity, and contractility).