Characterization of the alpha 1-adrenergic receptor subtype in a smooth muscle cell line

We have identified in the DDT1 smooth muscle cell line a [3H]dihydroergocryptine-binding site having the characteristics of an alpha 1-adrenergic receptor. Specific binding of [3H]dihydroergocryptine to DDT1 cells grown either in monolayer or suspension culture was reversible, saturable, and of high affinity, and the binding site demonstrated stereoselectivity. [3H]Dihydroergocryptine dissociation constants of 1.4 +/- 0.2 nM and 1.4 +/- 0.3 nM were observed for suspension and monolayer cells, respectively. However, the concentration of binding sites in suspension-cultured cells (65,100 +/- 8,300 sites/cell) was significantly greater (p less than 0.001) than that found in monolayer cells (27,900 +/- 4,300 sites/cell). The order of agonist competition for the binding site was epinephrine (Ki = 0.92 +/- 0.32 microM) greater than or equal to norepinephrine (Ki = 2.2 +/- 1.0 microM) greater than isoproterenol (Ki = 137 +/- 17 microM), consistent with an alpha-adrenergic interaction. Results of competition experiments with specific antagonists prazosin (alpha 1-selective) or yohimbine (alpha 2-selective) and a computer modeling technique indicated that the alpha-adrenergic receptor of the DDT1 cell was predominantly (greater than 95%) the alpha 1-subtype.     

Characterization of functional histamine H1 receptors on a cultured smooth muscle cell line

Cultured cells of the smooth muscle line DDT1MF-2, which was derived from a hamster vas deferens tumor, expressed histamine H1-type receptors and responded biochemically and functionally to H1-specific stimulation. The H1-receptor antagonist [3H]-pyrilamine bound specifically to 9.7 x 10(6) sites/DDT1MF-2 cell with a dissociation constant (Kd) of 219 nM. The addition of histamine to suspensions of fura-2-loaded DDT1MF-2 cells elicited a rapid, transient, and stimulus concentration-dependent increase in the intracellular concentration of Ca2+ with an EC50 of 3 x 10(-5) M, which demonstrated H1 receptor specificity. Moreover, in order to evaluate in vitro contractile response of individual DDT1MF-2 cells, the degree of intracellular actin polymerization was quantified by a DNase inhibition assay. The percentage of nonpolymerized or G-actin in DDT1MF-2 cells was reduced in a histamine concentration-dependent manner with an EC50 of 1 x 10(-5) M and H1 receptor specificity. Histamine-induced actin polymerization was accompanied by changes in cell shape that were consistent with cellular contraction, as assessed by flow cytometry. The H1-type receptors of cultured DDT1MF-2 cells thus couple histamine stimulation to a variety of functional responses of smooth muscle cells.     

Dihydropyridine sensitive calcium channels in a smooth muscle cell line

The pharmacological properties of voltage sensitive calcium channels (VSCC) were examined in a rat aortic smooth muscle cell line (A10). The inorganic VSCC blockers Co2+ and Cd2+ blocked 45Ca2+ uptake into these cells in both 5 mM K+ and 50 mM K+ (depolarizing) conditions. The organic VSCC antagonists nitrendipine, nimodipine, D-600 and diltiazem also blocked 45Ca2+ uptake at low concentrations. The relative potencies of blockade were similar to those found in intact vascular smooth muscle. The VSCC "agonist" BAY K8644 enhanced 45Ca2+ uptake and this effect could be reversed by nitrendipine. These results indicate that A10 cells possess VSCC and that these VSCC behave similarly to those in authentic smooth muscle.     

Adrenergic stimulation of inositol-phosphate production in a genital tract smooth muscle cell line

Catecholamines are important in the modulation of smooth muscle contractile activity; this study was undertaken to evaluate adrenoceptor stimulation of intracellular inositol-phosphate production in a genital tract smooth muscle myocyte. DDT1 MF-2 smooth muscle myocytes, derived from a hamster ductus deferens leiomyosarcoma, were loaded with 3H-inositol, incubated in 10 mM LiCl, then stimulated with adrenergic agonists with and without antagonists. Subsequently, the inositol phosphates were isolated by anion-exchange chromatography. In the presence of norepinephrine (NE), inositol trisphosphate (IP3) was produced by 30 s and peaked at 2 min; inositol 1-phosphate was also apparent by 30 s, and continued to increase over 15 min. Clonidine (an alpha-2 agonist), isoproterenol, and NE in the presence of phentolamine or prazosin (an alpha-1 antagonist) failed to increase IP3. In contrast, NE in the presence of yohimbine (an alpha-2 antagonist) or propranolol stimulated IP3 production to levels comparable to that stimulated by NE alone. These studies provide evidence that inositol phosphate production is involved in alpha-1 adrenergic signal transduction in DDT1 MF-2 myocyte.     

Characterization of vascular smooth muscle cell phenotype in long-term culture

Summary Studies of bovine carotid artery smooth muscle cells, during long-term in vitro subcultivation (up to 100 population doublings), have revealed phenotypic heterogeneity among cells, as characterized by differences in proliferative behavoir, cell morphology, and contractile-cytoskeletal protein profiles. In vivo, smooth muscle cells were spindle-shaped and expressed desmin and alpha-smooth muscle actin (50% of total actin) as their predominant cytoskeletal and contractile proteins. Within 24 h of culture, vimentin rather than desmin was the predominant intermediate filament protein, with little change in alpha-actin content. Upon initial subcultivation, all cells were flattened and fibroblastic in appearance with a concommitant fivefold reduction in alpha-actin content, whereas the beta and gamma nonmuscle actins predominated. In three out of four cell lines studied, fluctuations in proliferative activity were observed during the life span of the culture. These spontaneous fluctuations in proliferation were accompanied by coordinated changes in morphology and contractile-cytoskeletal protein profiles. During periods of enhanced proliferation a significant proportion of cells reverted to their original spindle-shaped morphology with a simultaneous increase in alpha-actin content (20 to 30% of total actin). These results suggest that in long-term culture smooth muscle cells undergo spontaneous modulations in cell phenotype and may serve as a useful model for studying the regulation of intracellular protein expression. This work was supported by grants from from National Institutes of Health, Bethesda, MD, to DMW (HL35684), JW (HL36412), and JM and RL (SCOR HL 14212).     

Characterization of the calcium-dependent proteolytic system in a mouse muscle cell line

Many studies have demonstrated that the calcium-dependent proteolytic system (calpains and calpastatin) is involved in myoblast differentiation. It is also known that myogenic differentiation can be studied in vitro. In the present experiments, using a mouse muscle cell line (C₂C₁₂) we have analyzed both the sequences of appearance and the expression profiles of calpains 1, 2, 3 and calpastatin during the course of myoblast differentiation. Our results mainly show that the expression of ubiquitous calpains (calpain 1 and 2) and muscle-specific calpain (calpain 3) at the mRNAs level as well as at the protein level do not change significantly all along this biological process. In the same time, the specific inhibitor of ubiquitous calpains, calpastatin, presents a stable expression at mRNAs level as well as protein level, all along myoblast to myotube transition. A comparison with other myogenic cells is presented.     

Measurement of intracellular magnesium in a vascular smooth muscle cell line using a fluorescent probe

Until recently, direct measurement of intracellular free magnesium has been complex and difficult. However, fluorescent probes are now available, based on the same principle as well-established probes for free calcium. Using one such probe, mag-fura-2, we have estimated basal intracellular magnesium concentrations in the A7r5 rat vascular smooth muscle cell line. This level was unaffected by numerous pharmacological manipulations, including agonist stimulation and depolarisation. The possible implications of these findings are discussed.     

Characterization of sulfidopeptide leukotriene responses in sheep tracheal smooth muscle in vitro.

We have investigated the effects of leukotrienes (LTs) on isolated tracheal smooth muscle from sheep sensitive to Ascaris suum antigen. LTC4 and LTD4 produced dose-dependent contractions of sheep trachea, but LTE4 was virtually inactive. YM-17690, a non-analogous LT agonist, produced no contractile response up to 100 microM. Indomethacin (5 microM) had no effect on LTC4- and LTD4-induced contractions. L-Serine borate (45 mM), an inhibitor of gamma-glutamyl transpeptidase, shifted the dose-response curve of LTC4 to the left by 161-fold, and L-cysteine (6 mM), an inhibitor of aminopeptidase, shifted the dose-response curves of LTC4 and LTD4 to the left by 67- and 23-fold, respectively. YM-16638 (1 microM), an LT antagonist, shifted the dose-response curves of LTC4 and LTD4 to the right with pKB values of 6.57 and 7.13, respectively. YM-16638 did not affect LTC4-induced contractions of L-serine borate-treated tissues, indicating that the compound acts only on LTD4 receptors in sheep trachea, LTE4 (1 microM) shifted the dose-response curves of LTC4 and LTD4 to the right with pKB values of 6.87 and 7.31, respectively. YM-17690 (10 microM) showed effects similar to LTE4, suggesting that the compound acts as an LTE4 agonist in sheep trachea. These results suggest that in sheep tracheal smooth muscle (a) LTC4 and LTD4 produce contractions, (b) these LT-induced contractions are not mediated by cyclooxygenase products, (c) LTC4 is converted to LTD4 and then to LTE4, and (d) the potency of the LTC4- and LTD4-induced contractions is increased when their conversion to LTE4 is inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a G protein-activated phosphoinositide 3-kinase in vascular smooth muscle cell nuclei

Recent studies highlight the existence of an autonomous nuclear polyphosphoinositide metabolism related to cellular proliferation and differentiation. However, only few data document the nuclear production of the putative second messengers, the 3-phosphorylated phosphoinositides, by the phosphoinositide 3-kinase (PI3K). In the present paper, we examine whether GTP-binding proteins can directly modulate 3-phosphorylated phosphoinositide metabolism in membrane-free nuclei isolated from pig aorta smooth muscle cells (VSMCs). In vitro PI3K assays performed without the addition of any exogenous substrates revealed that guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) specifically stimulated the nuclear synthesis of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)), whereas guanosine 5'-(beta-thio)diphosphate was ineffective. PI3K inhibitors wortmannin and LY294002 prevented GTPgammaS-induced PtdIns(3,4,5)P(3) synthesis. Moreover, pertussis toxin inhibited partially PtdIns(3,4,5)P(3) accumulation, suggesting that nuclear G(i)/G(0) proteins are involved in the activation of PI3K. Immunoblot experiments showed the presence of Galpha(0) proteins in VSMC nuclei. In contrast with previous reports, immunoblots and indirect immunofluorescence failed to detect the p85alpha subunit of the heterodimeric PI3K within VSMC nuclei. By contrast, we have detected the presence of a 117-kDa protein immunologically related to the PI3Kgamma. These results indicate the existence of a G protein-activated PI3K inside VSMC nucleus that might be involved in the control of VSMC proliferation and in the pathogenesis of vascular proliferative disorders.     

Characterization of intestinal smooth muscle responses and binding sites for endothelin.

The contractile activity of and binding sites for endothelin-1 (ET-1) were investigated in isolated guinea-pig ileal longitudinal smooth muscle (GPILM). ET-1 produced concentration-dependent contractions of GPILM that either slowly subsided in the continued presence of ET-1 or rapidly subsided following washing of the tissue. The ED50 value for ET-1 contractions was 4.2 +/- 1.3 x 10(-9) M. The removal of extracellular calcium or pretreatment with nifedipine produced a complete inhibition of the contractions to ET-1. The IC50 value of nifedipine for inhibition of ET-1 mediated contractions was 3.0 +/- 0.8 x 10(-8) M. ET-1 produced a marked prolonged homologous desensitization of its contractile response but did not affect the responses mediated by carbachol, histamine, serotonin, substance P, and PLA2. High-affinity binding sites for 125I-labelled ET-1 were identified on microsomal membranes prepared from GPILM with Kd and Bmax values obtained by Scatchard analysis of 3.5 +/- 0.6 x 10(-10) M and 2138 +/- 159 fmol/mg protein, respectively. The binding of 125I-labelled ET-1 to GPILM microsomes was characterized by a rapid association (kob value of 0.077 min-1 at a radioligand concentration of 0.45 nM and an extremely slow dissociation (k1 value of 0.011 min-1; t1/2 value of 793 min). The binding was unaffected by the calcium channel antagonists nifedipine, verapamil, and diltiazem (10(-6) M); the receptor antagonists phenoxybenzamine, atropine, and naloxone (10(-6) M) and propranolol; and the peripheral benzodiazepine receptor antagonists Ro 5-4864 and PK 11195 and psychotomimetic drug phencyclidine (10(-5) M).(ABSTRACT TRUNCATED AT 250 WORDS)     

Vasopressin induced production of inositol trisphosphate and calcium efflux in a smooth muscle cell line

Phosphatidylinositol metabolism and ⁴⁵Ca²⁺ efflux were examined in a vascular smooth muscle cell line (A₇r₅). [Arg⁸]Vasopressin stimulated the rapid formation (measurable at 1 sec) of inositol phosphates in a concentration-dependent manner. The time course for formation of inositol phosphates was similar to that for ⁴⁵Ca²⁺ efflux from preloaded cells. The efflux of ⁴⁵Ca²⁺ in response to [Arg⁸]vasopressin could be inhibited by a vasopressin antagonist. This supports the hypothesis that inositol 1,4,5-trisphosphate plays a role in vasopressin stimulated calcium mobilisation from an intracellular source in cultured vascular smooth muscle cells.     

Vascular circadian rhythms in a mouse vascular smooth muscle cell line (Movas-1)

The circadian system in mammals is a hierarchy of oscillators throughout the organism that are coordinated by the circadian clock in the hypothalamic suprachiasmatic nucleus. Peripheral clocks act to integrate time-of-day information from neural or hormonal signals, regulating gene expression, and, subsequently, organ physiology. However, the mechanisms by which the central clock communicates with peripheral oscillators are not understood and are likely tissue specific. In this study, we establish a mouse vascular cell model suitable for investigations of these mechanisms at a molecular level. Using the immortalized vascular smooth muscle cell line Movas-1, we determined that these cells express the circadian clock machinery with robust rhythms in mRNA expression over a 36-h period after serum shock synchronization. Furthermore, norepinephrine and forskolin were able to synchronize circadian rhythms in bmal1. With synchronization, we observed cycling of specific genes, including the tissue inhibitor of metalloproteinase 1 and 3 (timp1, timp3), collagen 3a1 (col3a1), transgelin 1 (sm22alpha), and calponin 1 (cnn1). Diurnal expression of these genes was also found in vivo in mouse aortic tissue, using microarray and real-time RT-PCR analysis. Both of these revealed ultradian rhythms in genes similar to the cycling observed in Movas-1 in vitro. These findings highlight the cyclical nature of structurally important genes in the vasculature that is similar both in vivo and in vitro. This study establishes the Movas-1 cells as a novel cell model from which to further investigate the molecular mechanisms of clock regulation in the vasculature.     

Suppression of rat mammary tumor cell growth in vitro by glucocorticoids requires serum proteins. Characterization of wild type and glucocorticoid-resistant epithelial tumor cells

CON8 is a single-cell derived subclone of the 13762NF transplantable, hormone-responsive rat mammary tumor that proliferates rapidly in serum-free medium. Addition of either glucocorticoids or calf serum alone caused a slight stimulation of CON8 proliferation. However, glucocorticoids required the presence of specific serum proteins to strongly suppress CON8 cell growth. Furthermore, the anchorage-independent growth of CON8 cells was significantly reduced in the presence of glucocorticoids and serum. We have designated this serum activity GMGSF, for glucocorticoid modulating growth suppression factor. Inhibition of cell growth was limited to steroids with strong glucocorticoid biological activity, while exposure to the glucocorticoid antagonist RU38486 prevented this response. Half-maximal growth inhibition and half-maximal expression of a glucocorticoid-inducible gene product (2 nM) occurred slightly below the half-maximal receptor binding of [3H]dexamethasone (10nM). We have also selected a variant mammary epithelial tumor cell line, derived from CON8, denoted 8RUV7, whose proliferation and soft agar colony formation failed to be suppressed by glucocorticoids in the presence of serum. These glucocorticoid-resistant variant cells possess functional glucocorticoid receptors, competently produce the glucocorticoid-responsive gene product plasminogen activator inhibitor, and along with CON8 cells express milk fat globule protein antigens on their cell surface, indicative of their mammary epithelial cell character. We are using this variant line to genetically dissect the molecular mechanism of the glucocorticoid/GMGSF growth suppression pathway in mammary epithelial tumor cells.