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.     

The development of insulin receptors and responses in the differentiating nonfusing muscle cell line BC3H-1

We have studied the development of high affinity insulin receptors and insulin-stimulated responses in the differentiating nonfusing muscle cell line BC3H-1. In the logarithmic growth phase, these myoblasts exhibit very low levels of insulin binding and no detectable insulin-stimulated glucose or amino acid uptake. Following the cessation of cell division and subsequent spontaneous differentiation, the resulting myocytes develop a 5-fold increase in specific 125I-insulin binding and demonstrate physiologic insulin-stimulated glucose and amino acid uptake (100% increase above baseline) with half-maximum stimulation at 1-3 nM in agreement with the known in vivo and in vitro insulin sensitivity of muscle tissue. Insulin stimulation of 2-deoxyglucose uptake is detectable within 3 min, becomes maximal within 15 min, and is mediated by a rapid increase of plasma membrane transport units, as determined by D-glucose-inhibitable cytochalasin B binding, resulting in a 2-fold increase in the Vmax for 2-deoxyglucose transport with no change in Km. Myocyte insulin binding is specific, reversible, and saturable, yielding equilibrium within 18 h at 4 degrees C. Scatchard analysis identified the high affinity insulin receptor with a Kd of 0.5 nM at 4 degrees C. The myocytes also demonstrate sensitive down-regulation of cell surface insulin receptors, with a maximum decrease of 50% in cell surface insulin binding following exposure to 20 nM insulin for 18 h at 37 degrees C. Since the differentiation of this muscle cell line from myoblasts to nonfusing myocytes is accompanied by the development of high affinity insulin receptors and physiologic insulin-stimulated glucose and alpha-methylaminoisobutyric acid uptake, this continuously cultured system provides an excellent model for the study of differentiation and mechanism of insulin action in muscle, its quantitatively most significant target tissue.     

Phorbol ester-mediated desensitization of histamine H1 receptors on a cultured smooth muscle cell line

The present study was undertaken in order to examine the effect of protein kinase C (PKC) on histamine H1 receptors (H1R) present on the smooth muscle cell line, DDT1MF-2. [3H]-pyrilamine binding revealed that specific [3H]-pyrilamine binding sites were reduced by pretreatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of PKC, but not the Kd. The TPA analogue, 4 alpha phorbol 12,13-didecanoate, which does not activate PKC, failed to induce down-regulation of H1R. TPA-induced down-regulation of H1R was inhibited by pretreatment with 1-(5-Isoquinilinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), a PKC inhibitor, in a dose dependent manner. The H-7 analogue, H-8, which is a less potent inhibitor of PKC, but a potent inhibitor of cyclic nucleotide dependent protein kinase, had no effect on H1R. Moreover, treatment with TPA inhibited histamine-induced increases in [Ca2+]i in cells loaded with the fluorescent indicator, indo-1. These data suggest that H1R in DDT1MF-2 cells are functionally regulated by PKC.     

Identification and characterization of functional D1 dopamine receptors in a human neuroblastoma cell line

Dopamine stimulated human neuroblastoma SK-N-MC cells to accumulated cyclic AMP. The D1 agonist SKF (R)-38393 also stimulated cyclic AMP production whereas the response to dopamine was inhibited by the D1 antagonist SCH (R)-23390. Membranes from SK-N-MC cells bound the D1 ligand [125I]SCH 23982 with a Kd of 2.1 nM and a Bmax of 102 fmol/mg protein. Binding was displaced by dopamine, SKF 38393, and SCH 23390. Up to 40% of the receptors were in an agonist high affinity, guanine nucleotide-sensitive state, compared to only 6% in rat striatum. A D1 photoaffinity probe labeled a 72 kDa protein in both SK-N-MC and rat striatal membranes. Thus, SK-N-MC human neuroblastoma cells contain D1 dopamine receptors which are similar to those found in mammalian striatum, but which are more tightly coupled to adenylate cyclase. SK-N-MC cells may be a useful model to investigate the properties and regulation of D1 dopamine receptors.     

2-nor-leukotriene analogs: antagonists of the airway and vascular smooth muscle effects of leukotriene C4, D4 and E4

A structural analog of LTD4, 4R-hydroxy-5S-cysteinylglycyl-6Z-nonadecenoic acid (4R, 5S, 6Z-2-nor-LTD1) has been synthesized and pharmacologically characterized. It significantly antagonized the contractile action of LTD4, LTC4 and LTE4 in guinea pig airways. In addition, this compound antagonized the in vitro vasoconstrictive effects of LTD4 in the guinea pig pulmonary artery. The study of a series of structural analogs of 4R, 5S, 6Z-2-nor-LTD1 suggests that the spatial separation of the C-1 (eicosanoid) carboxyl relative to the hydroxyl is a critical determinant in LTD4 agonist/antagonist activity.     

Identification and characterization of leukotriene D4 receptors and signal transduction processes in rat basophilic leukemia cells

The leukotriene D4 (LTD4) receptor on rat basophilic leukemia (RBL-1) cell membranes was characterized using a radioligand binding assay. [3H]LTD4 binding to RBL-1 membrane receptors was stereoselective, specific, and saturable. The binding affinity and maximum binding density of [3H]LTD4 to RBL-1 membrane receptors were 0.9 +/- 0.2 nM and 800 +/- 125 fmol/mg protein, respectively. Binding of [3H]LTD4 to the receptors was enhanced by divalent cations (Ca2+, Mg2+, and Mn2+) and inhibited by guanine nucleotides and sodium ions, specifically, indicating that a guanine nucleotide-binding protein may regulate the agonist-receptor interaction. LTD4, LTE4 agonist and antagonist analogs competed with the radioligand in binding to the RBL-1 LTD4 receptors. The binding affinities of these analogs correlated with (a) those determined from the guinea pig lung LTD4 receptors and (b) the pharmacological activities in smooth muscle contraction. LTD4 and related agonists also induced time- and concentration-dependent phosphatidylinositol hydrolysis in RBL-1 cells. The LTD4 induction of inositol 1-phosphate was potent, stereoselective, specific, and was blocked by LTD4 receptor antagonists. The rank order potency of agonist-induced inositol 1-phosphate formation in RBL-1 cells was equivalent to the receptor binding affinity determined using either RBL-1 cell or guinea pig lung membranes. These studies have demonstrated the G protein coupled LTD4 receptors on RBL-1 cell membranes. Binding of agonists to the receptor may activate the G protein-regulated phospholipase C to induce hydrolysis of phosphatidylinositol. The hydrolytic products of phosphatidylinositol, possibly inositol trisphosphate and diacylglycerol, may be the intracellular messengers for LTD4 receptors in RBL-1 cells.     

Visualization and comparison of molecular dynamics simulations of leukotriene C4, leukotriene D4, and leukotriene E4

Molecular dynamics simulations of leukotriene C₄ (LTC₄), leukotriene D₄ (LTD₄), and leukotriene E₄ (LTE₄) were carried out, and the data were visualized in an animated video format. Three-dimensional ghost images show the positions of the heavy atoms of all three molecules throughout the simulations. The ghost images can be superimposed to give a single three-dimensional image in which the shapes of the most populated conformers of each molecule are apparent and can be compared. Leukotriene D₄ was found to occupy mostly T-shaped conformations, while LTC₄ occupied mostly cup-shaped conformations, and LTE₄ occupied a wide range of conformations spanning the LTD₄ and LTC₄ types. Digital filtering and graphing of the internal geometries of the molecules as a function of time revealed differences in dynamic behavior. The results are discussed in light of current knowledge about leukotriene receptors.     

Lipopolysaccharide down-regulates the leukotriene C4 synthase gene in the monocyte-like cell line,THP-1

We studied the effects of LPS on cysteinyl leukotriene (LT) synthesis and LTC(4) synthase expression in mononuclear phagocytes. Conditioning of the monocyte-like cell line, THP-1, with LPS for 7 days resulted in significantly decreased ionophore-stimulated LTC(4) release. The putative LPS receptor, Toll-like receptor 4, was expressed in THP-1 cells. LPS down-regulated LTC(4) synthase mRNA in THP-1 cells in a dose- and time-dependent manner, with down-regulation observed as early as 4 h. Conditioning of actinomycin D-treated cells with LPS resulted in no change in the rate of LTC(4) synthase mRNA decay. LPS treatment of THP-1 cells, transiently transfected with a LTC(4) synthase promoter (1.35 kb)-reporter construct, decreased promoter activity. Neutralization of TNF-alpha and inhibition of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase did not inhibit the effect of LPS. Treatment of cells with a Toll-like receptor 4-blocking Ab and an inhibitor of NF-kappaB activation resulted in inhibition of the LPS effect, while activation of NF-kappaB and p50/p65 overexpression down-regulated the LTC(4) synthase gene. LPS down-regulates cysteinyl LT release and LTC(4) synthase gene expression in mononuclear phagocytes by an NF-kappaB-mediated mechanism.     

Activation of calcium mobilization and calcium influx by alpha 1-adrenergic receptors in a smooth muscle cell line

Alpha 1-adrenergic receptor (alpha 1R) mediated increases in the cytosolic levels of free Ca+2 and the inositol phosphates were measured in a smooth muscle cell line, DDT1. Norepinephrine (NE) stimulated a rapid increase in cytosolic Ca+2 by two distinct components: 1) release of Ca+2 from intracellular sites (mobilization), and 2) influx of extracellular Ca+2. The mobilization component was not affected by removal of extracellular Ca+2 or addition of La+3 or Co+2 to the buffer. The influx component was abolished by EGTA, La+3, or Co+2, but was not affected by the voltage-operated Ca+2 channel blockers diltiazem or nifedipine. Depolarization of DDT1 cells with 100 mM KCl or with gramicidin did not induce Ca+2 influx. NE also increased inositol trisphosphate to 78% over basal levels within 1 minute. These results suggest that alpha 1R on DDT1 cells are coupled to both the mobilization of intracellular Ca+2 and to receptor-operated Ca+2 channels in the plasma membrane, and that polyphosphoinositide hydrolysis may play a role in these phenomena.     

Glucose regulates the expression of Gi-proteins in cultured BC3H-1 myocytes.

To examine whether glucose has regulatory effects on the expression of Gi-proteins, BC3H-1 myocytes were incubated for 24 hr in the presence of various concentrations of glucose (0-25 mM) and the amount of Gi-proteins was detected by pertussis toxin ADP-ribosylation and immunoblot analysis. Both detection methods showed a progressive decrease in the amount of Gi proteins in cells treated with increasing concentrations of glucose. A maximal reduction of 40% was observed after a 24 hr exposure to 25 mM glucose. The reduction in Gi-proteins correlated with a decrease in insulin-stimulated glucose transport.     

Electromechanical coupling of ferret airway smooth muscle in response to leukotriene C4

We investigated the possible electrophysiological basis for the slow, prolonged force generation by airway smooth muscle (ASM) produced by leukotriene C4 (LTC4). Preparations of ASM were made from ferret trachea and placed in tissue microchambers for study. Some of these preparations were arranged so that force transducers and intracellular microelectrodes (with tip resistances of 30-80 M omega) could be used to measure isometric force and cell membrane potential (Em) simultaneously from ASM cells stimulated by LTC4. We found that ferret tracheal muscle was relatively sensitive to LTC4 and that this sensitivity was not significantly affected by atropine (1 microM), phentolamine (1 microM), propranolol (3 microM), and pyrilamine (1 microM). In a 1 nM solution of LTC4, Em was -54.0 +/- 1.2 mV from 18 impalements (n) from 6 animals (N) compared with a base-line value of -61.6 +/- 0.8 mV (n/N = 29/8, P less than 0.0005). This change did not lead to force generation, however. Higher concentrations of LTC4 led to progressive decreases in Em to which force generation was closely coupled. Concentrations greater than or equal to 70 nM led to phasic oscillations in Em of 0.6-0.8 Hz and 1.7 mV in amplitude, which were abolished by 10 microM verapamil, although the base-line Em was unaffected by this concentration. Although 300 nM LTE4 by itself caused only a small depolarization of ferret trachealis, it substantially antagonized the electromechanical responsiveness of this smooth muscle to LTC4. We conclude that ferret ASM is relatively sensitive to LTC4 and that there is an electrical basis for the slow, prolonged force generation caused by this mediator.     

Pharmacological and functional characterization of bradykinin receptors in rat cultured vascular smooth muscle cells

The pharmacological properties of bradykinin receptors were characterized in rat cultured vascular smooth muscle cells (VSMCs) using [3H]-bradykinin as a ligand. Analysis of binding isotherms gave an apparent equilibrium dissociation constant (K(D)) of 1.2 +/- 0.2 nM and a maximum receptor density (Bmax) of 47.3 +/- 4.4 fmol/mg protein. The specific binding of [3H]-bradykinin to VSMCs was inhibited by the B2 receptor-selective agonists (bradykinin and kallidin) and antagonists ([D-Arg0, Hyp3, Thi5, D-Tic7, Oic8]-bradykinin (Hoe 140) and [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin) with an order of potency as kallidin = bradykinin = Hoe 140 > [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin, but not by a B1 receptor-selective agonist (des-Arg9-bradykinin) and antagonist ([Leu8, des-Arg9]-bradykinin). Stimulation of VSMCs by bradykinin produced a concentration-dependent inositol phosphate (IP) accumulation, and initial transient peak of [Ca2+]i with half-maximal responses (pEC50) were 7.53 and 7.69, respectively. B2 receptor-selective antagonists (Hoe 140 and [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin) significantly antagonized the bradykinin-induced responses with pK(B) values of 8.3-8.7 and 7.2-7.9, respectively. Pretreatment of VSMCs with pertussis toxin (100 ng/ml, 24 h) did not alter the bradykinin-induced inositol phosphate accumulation and [Ca2+]i changes in VSMCs. Removal of external Ca2+ led to a significant attenuation of responses induced by bradykinin. Influx of external Ca2+ was required for the bradykinin-induced responses, since Ca2+-channel blockers, nifedipine, verapamil, and Ni2+, partially inhibited the bradykinin-induced IP accumulation and Ca2+ mobilization. These results demonstrate that bradykinin stimulates phosphoinositide hydrolysis and Ca2+ mobilization via a pertussis toxin-insensitive G-protein in rat VSMCs. Bradykinin B2 receptors may be predominantly mediating IP accumulation and subsequently induction of Ca2+ mobilization may function as the transducing mechanism for bradykinin-stimulated contraction of vascular smooth muscle.     

Molecular and cellular analysis of histamine H1 receptors on cultured smooth muscle cells

Histamine is an important mediator of immediate hypersensitivity for both animals and humans. The action of histamine on target tissues is believed to be mediated by specific cell surface receptors, especially H1 and H2 receptors for hypersensitivity and inflammatory reactions, which involve stimulation of smooth muscle contractility, alterations in vascular permeability, and modifications in the activities of macrophages and lymphocytes. Although the nature of histamine receptors in the brain and peripheral tissues has been studied extensively by many laboratories, the molecular mechanism of histamine receptor-mediated reactions is not fully understood, mainly because histamine receptors are incompletely characterized from the biochemical point of view. In previous studies, we have found that the cultured smooth muscle cell line DDT1MF-2, derived from hamster vas deferens, expresses low-affinity histamine H1 receptors and responds biochemically and functionally to H1-specific stimulation (Mitsuhashi and Payan, J Cell Physiol 134:367, 1988). This cell line provides a model for analyzing the biochemical responses of H1 receptor-mediated reactions in peripheral tissues. In this review, we summarized our recent progress in the study of low-affinity H1 receptors on DDT1MF-2 cells.     

Regulation of differentiation of the BC3H1 muscle cell line through cAMP-dependent and -independent pathways

Serum mitogens, fibroblast growth factor (FGF), and type beta transforming growth factor (TGF-beta) suppress differentiation of the mouse muscle cell line BC3H1; however, the signal transduction pathways whereby these growth factors exert their effects on this system are unknown. The goal of this study was to determine whether the program for differentiation of BC3H1 cells was susceptible to negative regulation by signaling pathways involving cAMP or protein kinase C and whether these intracellular effectors participate in the mechanism by which growth factors prevent establishment of the myogenic phenotype. Exposure of BC3H1 cells to dibutyryl cAMP, 8-bromo-cAMP, or compounds that stimulate adenylate cyclase, i.e. forskolin, prostaglandin E1, and cholera toxin, prevented up-regulation of muscle-specific gene products following growth arrest in mitogen-deficient medium. Conversely, addition of cAMP to differentiated BC3H1 myocytes caused down-regulation of muscle-specific mRNAs. In contrast to the ability of cAMP to block differentiation, chronic exposure to O-tetradecanoylphorbol-13-acetate, the potent activator of protein kinase C, exhibited no apparent effects on expression of muscle-specific gene products. The proto-oncogenes c-myc and c-fos were up-regulated rapidly by cAMP in a manner similar to that observed previously by serum, FGF, and TGF-beta. However, these growth factors failed to increase intracellular cAMP levels, and they did not induce ornithine decarboxylase, which was subject to positive regulation by cAMP and O-tetradecanoyl-13-acetate. Together, these data indicate that differentiation of BC3H1 cells is subject to negative regulation through a cAMP-dependent pathway and that serum mitogens, FGF, and TGF-beta inhibit differentiation through a mechanism independent of cAMP or protein kinase C.     

Control by fibroblast growth factor of differentiation in the BC3H1 muscle cell line

The regulation of creatine phosphokinase (CPK) expression by polypeptide growth factors has been examined in the clonal mouse muscle BC3H1 cell line. After arrest of cell growth by exposure to low concentrations of serum, BC3H1 cells accumulate high levels of muscle-specific proteins including CPK. The induction of this enzyme is reversible in the presence of high concentrations of fetal calf serum, which cause quiescent, differentiated cells to reenter the cell cycle. Under these conditions, the rate of CPK synthesis is drastically reduced. We show in the present communication that either pituitary-derived fibroblast growth factor (FGF) or brain-derived FGF are as effective as serum in repressing the synthesis of CPK when added to quiescent, differentiated cells. The decrease in the rate of synthesis of CPK occurs within 22 h after the addition of pituitary FGF to the cells. Pituitary FGF had very little effect, if any, on the rate CPK degradation. The overall rate of protein synthesis and the pattern of synthesis of the major polypeptides made by these cells was not altered by the addition of FGF. Although pituitary FGF was mitogenic for BC3H1 cells, the rate of cell growth was not absolutely correlated with the extent of repression of CPK. Brain-derived FGF fully repressed CPK induction under conditions where it showed no significant mitogenic activity. These results show that the expression of a muscle-specific protein, CPK, can be controlled by a single defined polypeptide growth factor in fully differentiated cultures, and that initiation of cell division is not required for their regulation to take place.     

Membrane glycoproteins are involved in the differentiation of the BC3H1 muscle cell line

The nonfusing muscle cell line BC3H1 expresses a family of muscle-specific proteins when the fetal bovine serum (FBS) concentration is reduced from 20 to 1%. We have used a series of glycosylation inhibitors to assess the role played by glycoproteins in the initiation of differentiation in this cell line. Tunicamycin (TNM) and 2-deoxy-D-glucose, added to cells when the FBS concentration was reduced, blocked creatine phosphokinase (CPK) induction by 70-95%. These effects were dose dependent and reversible. TNM and 2-deoxy-D-glucose also reversed CPK induction in differentiated cells. Leupeptin and N-acetylglucosamine did not reverse these effects. 1-Deoxynojirimycin, 1-deoxymannojirimycin, and swainsonine have no effect on induced CPK expression, whereas castanospermine, a glucosidase I inhibitor, blocked its induction completely. As attempts to use conditioned medium from cells grown in 1 or 20% FBS have no effect on this differentiation process we conclude that high mannose structures, but not complex form glycoproteins, bound to the surface of BC3H1 cells play a role in transducing signals for differentiation and are probable mediators of cell/cell contact.     

Inhibitory effect of D1-like and D3 dopamine receptors on norepinephrine-induced proliferation in vascular smooth muscle cells

The sympathetic nervous system plays an important role in the regulation of blood pressure. There is increasing evidence for positive and negative interactions between dopamine and adrenergic receptors; the activation of the alpha-adrenergic receptor induces vasoconstriction, whereas the activation of dopamine receptor induces vasorelaxation. We hypothesize that the D1-like receptor and/or D3 receptor also inhibit alpha1-adrenergic receptor-mediated proliferation in vascular smooth muscle cells (VSMCs). In this study, VSMC proliferation was determined by measuring [3H]thymidine incorporation, cell number, and uptake of 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT). Norepinephrine increased VSMC number and MTT uptake, as well as [3H]thymidine incorporation via the alpha1-adrenergic receptor in aortic VSMCs from Sprague-Dawley rats. The proliferative effects of norepinephrine were attenuated by the activation of D1-like receptors or D3 receptors, although a D1-like receptor agonist, fenoldopam, and a D3 receptor agonist, PD-128907, by themselves, at low concentrations, had no effect on VSMC proliferation. Simultaneous stimulation of both D1-like and D3 receptors had an additive inhibitory effect. The inhibitory effect of D3 receptor was via protein kinase A, whereas the D1-like receptor effect was via protein kinase C-zeta. The interaction between alpha1-adrenergic and dopamine receptors, especially D1-like and D3 receptors in VSMCs, could be involved in the pathogenesis of hypertension.