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1.
Jpn J Pharmacol ; 85(4): 443-52, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11388649

ABSTRACT

A new sea urchin lectin from Toxopneustes pileolus, is D(+)galactose (Gal)-, D(+)fucose (Fuc)-specific. Incubation of rat peritoneal mast cells with the lectin in the presence of 0.3 mM CaCl2 for 10 min significantly and dose-dependently inhibited the histamine release induced by N-acetyl glucosamine (GlcNAc)-specific Datura stramonium agglutinin (DSA), an activator of the Gi-protein-dependent pathway in mast cells. This inhibition by the sea urchin lectin was sugar-specifically reversed in the presence of D(+)Gal or D(+)Fuc but not L(-)Fuc. The sea urchin lectin had no effect on the histamine release induced by compound 48/80, slightly inhibited the histamine release induced by substance P and mastoparan, and slightly enhanced the histamine release induced by melittin, but these effects were not dose-dependent. Compound 48/80, substance P, mastoparan and melittin are mast cell activators without sugar residues. It is suggested that the lectin binds to D(+)Gal residues of DSA to interfere with mast cell activation induced by DSA, a glycoprotein with arabinose and Gal residues. The effects of plant lectins with affinity to D(+)Gal, N-acetyl galactosamine and/or sialic acid and L(-)Fuc on the histamine release induced by DSA, compound 48/80 and substance P were also examined.


Subject(s)
Agglutinins/pharmacology , Galactose/pharmacology , Histamine Release/drug effects , Lectins/pharmacology , Plant Lectins , Sea Urchins/metabolism , Animals , Histamine Release/physiology , Lectins/isolation & purification , Male , Mast Cells/drug effects , Mast Cells/metabolism , Rats , Rats, Sprague-Dawley , Substance P/pharmacology , p-Methoxy-N-methylphenethylamine/pharmacology
2.
Am J Physiol Cell Physiol ; 279(5): C1432-42, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11029291

ABSTRACT

String-shaped reconstituted smooth muscle (SM) fibers were prepared in rectangular wells by thermal gelation of a mixed solution of collagen and cultured SM cells derived from guinea pig stomach. The cells in the fiber exhibited an elongated spindle shape and were aligned along the long axis. The fiber contracted in response to KCl (140 mM), norepinephrine (NE; 10(-7) M), epinephrine (10(-7) M), phenylephrine (10(-6) M), serotonin (10(-6) M), and histamine (10(-5) M), but not acetylcholine (10(-5) M). Phentolamine (10(-7) M) produced a parallel rightward shift of the NE dose-response curve. Moreover, NE-induced contraction was partially inhibited by nifedipine and completely abolished by the intracellular Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, the myosin light chain kinase inhibitor ML-9, the Rho kinase inhibitor Y-27632, and papaverine. A [(3)H]quinuclidinyl benzilate binding study revealed that the loss of response to acetylcholine was due to the loss of muscarinic receptor expression during culture. The expression of contractile proteins in the fibers was similar to that in cultured SM cells. These results suggest that, although the fiber is not a model for fully differentiated SM, contractile mechanisms are maintained.


Subject(s)
Isometric Contraction , Muscle Fibers, Skeletal/physiology , Muscle, Smooth/physiology , Acetylcholine/pharmacology , Adrenergic Agonists/pharmacology , Animals , Cells, Cultured , Collagen , Dose-Response Relationship, Drug , Gels , Genetic Markers , Guinea Pigs , Histamine/pharmacology , Isometric Contraction/physiology , Muscarinic Agonists/pharmacology , Muscle Fibers, Skeletal/drug effects , Muscle Proteins/metabolism , Muscle, Smooth/cytology , Muscle, Smooth/drug effects , Phenotype , Potassium Chloride/pharmacology , Serotonin/pharmacology
3.
Life Sci ; 64(22): 1975-87, 1999.
Article in English | MEDLINE | ID: mdl-10374923

ABSTRACT

Isolated single smooth muscle cells from the fundus of a guinea-pig stomach were permeabilized by use of streptolysin-O (0.5 U/ml). Most of the permeabilized cells responded to 0.6 microM Ca2+, but not to 0.2 microM Ca2+, with a resulting maximal cell shortening to approximately 71% of the resting cell length. These cells were relaxed again by washing with the Ca2+-free solution (2.5 nM free Ca2+) for 3-5 min. Addition of 10 microM acetylcholine (ACh) resulted in both a marked decrease in the concentration of Ca2+ required to trigger a threshold response and an increase in the maximal cell shortening, indicating that the cells retained the muscarinic receptor function. When the cell treated with a protein kinase C (PKC) inhibitor, K-252b (1 microM), for 3 min was exposed to 10 microM ACh in the presence of K-252b, the cell shortened within 2 min with a maximal cell shortening. When the cell shortening was induced by 10 microM ACh plus 1 microM Ca2+ in the presence of K-252b (1 microM) or more selective PKC inhibitors, such as calphostin C (1 microM) or PKC pseudosubstrate peptide (100 microM), the extension of the shortened cells, by washing with the Ca2+-free solution, was significantly inhibited. In contrast, K-252b (1 microM) did not inhibit the relaxation of Ca2+-induced shortened cells. These results suggest that the receptor-mediated activation of PKC in the process of ACh-induced cell shortening plays a role in the subsequent relaxation of the shortened cells.


Subject(s)
Muscle, Smooth/physiology , Protein Kinase C/physiology , Stomach/physiology , Acetylcholine/pharmacology , Animals , Bacterial Proteins , Calcium/pharmacology , Carbazoles/pharmacology , Cell Membrane Permeability/drug effects , Enzyme Inhibitors/pharmacology , Guinea Pigs , In Vitro Techniques , Indole Alkaloids , Muscle Relaxation/physiology , Muscle, Smooth/drug effects , Naphthalenes/pharmacology , Protein Kinase C/antagonists & inhibitors , Stomach/drug effects , Stomach/enzymology , Streptolysins/pharmacology
4.
Gen Pharmacol ; 29(5): 753-61, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9347321

ABSTRACT

1. Isolated single smooth muscle cells from the guinea pig stomach were permeabilized with Staphylococcus aureus alpha-toxin. 2. The permeabilized single cells showed a shortening in response to Ca2+ in an all-or-none manner. Moreover, the addition of acetylcholine (ACh) or guanosine 5'-triphosphate (GTP) resulted in a decrease in concentration of Ca2+ required to trigger a threshold response, suggesting that Ca2+ sensitization is induced by the stimulation of muscarinic acetylcholine receptors (mAChRs) or GTP-binding protein(s). 3. Short-term desensitization was induced by incubating the permeabilized cells with 100 microM ACh for 10 min. 4. In desensitized cells, the concentration of Ca2+ required to trigger a threshold response in the presence of ACh was increased, however, the cell shortening in response to Ca2+ in the absence of ACh and GTP-induced Ca2+ sensitization was not affected by short-term desensitization. 5. These results suggest that the receptor-operated augmentation of Ca2+ sensitivity is inhibited by short-term desensitization and that the development of short-term desensitization is due to an uncoupling of mAChR/GTP-binding protein(s).


Subject(s)
Calcium/pharmacology , Muscarinic Antagonists/pharmacology , Muscle, Smooth/drug effects , Receptors, Muscarinic/metabolism , Stomach/drug effects , Type C Phospholipases/pharmacology , Acetylcholine/pharmacology , Animals , Cell Membrane Permeability/drug effects , Gastric Fundus/cytology , Gastric Fundus/drug effects , Guanosine Triphosphate/pharmacology , Guinea Pigs , In Vitro Techniques , Muscle Contraction/drug effects , Muscle, Smooth/cytology , Stomach/cytology
5.
J Immunol ; 158(1): 393-404, 1997 Jan 01.
Article in English | MEDLINE | ID: mdl-8977215

ABSTRACT

Polycationic mast cell activators, such as compound 48/80 and substance P, have been reported to activate connective tissue-type mast cells specifically by interacting directly with the Gi family of trimeric GTP-binding protein. We now demonstrate that mouse bone marrow-derived mast cells (BMMC) developed in IL-3, an immature mast cell population lacking responsiveness to the Gi-coupled polycationic mast cell activators, underwent maturation toward a connective tissue-type mast cells-like phenotype that responded to polycationic compounds after only 4 to 6 days of coculture with Swiss 3T3 fibroblasts in concert with recombinant soluble c-kit ligand (KL), whereas 3T3 or KL alone was insufficient to mediate this process. Under optimal conditions, cocultured BMMC released approximately 30% beta-hexosaminidase and generated approximately 1 ng of PGD2/10(6) cells within a few minutes in response to compound 48/80 or substance P. Furthermore, these cells expressed cytokines, such as IL-1beta and IL-6, and PG endoperoxide synthase-2 1 to 4 h after stimulation with compound 48/80 or substance P. All these responses were suppressed effectively by pertussis toxin, implicating functional Gi coupling. Regardless of the remarkable change in polycationic compound sensitivity, there was only a minimal change in the constitutive expression of Gi3 alpha after coculture. These results together with the observation that before coculture BMMC responded to thrombin through its Gi-coupled receptor suggest that the alteration in a certain step(s) distinct from the level of Gi3 alpha protein expression is important for the acquisition of responsiveness to the polycationic compounds by the synergistic action of KL and 3T3 fibroblast-derived factor. Several lines of evidence have revealed that 3T3-derived factor appears to differ from the known cytokines, prostanoids, and adhesion molecules and is a labile soluble substance.


Subject(s)
3T3 Cells/metabolism , Cations/pharmacology , Cytokines/biosynthesis , Exocytosis/immunology , GTP-Binding Proteins/biosynthesis , GTP-Binding Proteins/drug effects , Mast Cells/drug effects , Mast Cells/metabolism , Prostaglandins/biosynthesis , Stem Cell Factor/pharmacology , Animals , Bone Marrow Cells , Coculture Techniques , Cytokines/drug effects , Exocytosis/drug effects , GTP-Binding Proteins/metabolism , Male , Mice , Mice, Inbred BALB C , Pertussis Toxin , Prostaglandin D2/biosynthesis , Substance P/pharmacology , Virulence Factors, Bordetella/pharmacology , p-Methoxy-N-methylphenethylamine/pharmacology
6.
Gen Pharmacol ; 27(1): 123-8, 1996 Jan.
Article in English | MEDLINE | ID: mdl-8742508

ABSTRACT

1. Benzalkonium chloride (BAC) inhibited the histamine release from rat peritoneal mast cells induced by synthetic cationic polymers (compound 48/80 and PEI6), bradykinin, des-Arg1-bradykinin and des-Arg9-bradykinin and Datura stramonium agglutinin (DSA). 2. The anionic polymers heparin, de-N-sulfated heparin, poly-aspartic acid and poly-glutamic acid dose dependently inhibited the histamine release induced by cationic polymers, suggesting counteraction between anions and cations. 3. Inhibition by heparin was diminished but that of BAC remained after removal of extracellular heparin and BAC. 4. Mast cell activation by bradykinin and DSA was not inhibited by anionic polymers, suggesting that both bradykinin and DSA recognize membrane sites as receptors.


Subject(s)
Anticoagulants/pharmacology , Benzalkonium Compounds/pharmacology , Heparin/pharmacology , Histamine Release/drug effects , Mast Cells/drug effects , Preservatives, Pharmaceutical/pharmacology , Animals , Bradykinin/pharmacology , Glucuronates/pharmacology , Glucuronic Acid , Lectins/pharmacology , Male , Mast Cells/metabolism , Peritoneal Cavity/cytology , Rats , Rats, Sprague-Dawley
7.
Gen Pharmacol ; 26(7): 1583-9, 1995 Nov.
Article in English | MEDLINE | ID: mdl-8690250

ABSTRACT

1. We investigated the effect of Clostridium botulinum C3 ADP-ribosyltransferase upon beta-hexosaminidase release induced by various stimuli from streptolysin-O (0.5-1 U/ml)-permeabilized rat basophilic leukemia (RBL-2H3) cells. 2. The C3 transferase inhibited beta-hexosaminidase release induced by Ca2+ or by guanosine-5'-(3-thiotriphosphate) (GTP gamma S) plus Ca2+. 3. The C3 transferase also inhibited beta-hexosaminidase release induced by stimulating high affinity IgE and m3 muscarinic acetylcholine receptors. 4. The substrate for the C3 transferase was present in cytosol of RBL-2H3 cells, indicating the presence of rho p21. About 60% of the total cellular substrate protein remained within the cells permeabilized by 1 U/ml of streptolysin-O. 5. The protein rho p21 appears to be regulated by several pathways and it may function as an integration point for exocytosis.


Subject(s)
ADP Ribose Transferases/metabolism , Botulinum Toxins , Exocytosis/drug effects , GTP-Binding Proteins/metabolism , beta-N-Acetylhexosaminidases/metabolism , Animals , Cell Membrane Permeability , Clostridium botulinum/enzymology , GTP-Binding Proteins/analysis , Leukemia, Basophilic, Acute , Rats , Tumor Cells, Cultured , rho GTP-Binding Proteins
8.
Gen Pharmacol ; 26(6): 1171-8, 1995 Oct.
Article in English | MEDLINE | ID: mdl-7590104

ABSTRACT

1. Polyethylenimine with a molecular weight of 600 (PEI6) was the simplest and the most useful to investigate mast cell-activating mechanisms via pertussis toxin (IAP)-sensitive G protein pathway. 2. IAP, lidocaine, or dibutyryl cyclic AMP were inhibitors of the histamine release induced by PEI6, but anti-allergic drug DSCG, the calcium antagonist, D-600, kinase inhibitors, H-7 and K252a, or the calmodulin inhibitor, W-7 were not. 3. The additive effects of compound 48/80 and PEI6 suggested that the action sites for PEI6 overlapped the binding sites of compound 48/80. 4. Mast cell activation induced by PEI6 was sugar-specifically inhibited by N-acetylglucosamine(Glc-NAc)-specific lectins and/or by sialic acid (Sia)-specific lectins, suggesting that the action sites for PEI6 were glycoproteins having GlcNAc and/or Sia residues. 5. Four glycoproteins seemed to be involved in histamine release, including the IAP-sensitive G-protein pathway.


Subject(s)
Mast Cells/physiology , Polyethyleneimine/pharmacology , Animals , Histamine Release , Pertussis Toxin , Rats , Virulence Factors, Bordetella , p-Methoxy-N-methylphenethylamine/pharmacology
9.
Gen Pharmacol ; 26(5): 1125-31, 1995 Sep.
Article in English | MEDLINE | ID: mdl-7557261

ABSTRACT

1. Carbachol-induced homologous desensitization of the secretory response was investigated by transfecting RBL-2H3 cells with cDNA encoding the human m3 muscarinic acetylcholine receptor (RBL-m3). 2. Exposure of RBL-m3 cells to 100 microM carbachol for 30 min in Ca2+-free medium inhibited the secretion induced by the subsequent addition of 10 microM carbachol plus Ca2+. 3. Desensitized cells bound [3H]quinuclidinyl benzilate with a similar Bmax and Kd to those of control cells. 4. The carbachol-induced transient increase in levels of inositol 1,4,5-trisphosphate was not changed by desensitization. 5. Homologous desensitization persisted when desensitized cells were permeabilized with Staphylococcal alpha-toxin.


Subject(s)
Carbachol/pharmacology , Leukemia, Basophilic, Acute/metabolism , Receptors, Muscarinic/drug effects , Animals , Atropine/pharmacology , Calcium/metabolism , Humans , Inositol 1,4,5-Trisphosphate/metabolism , Leukemia, Basophilic, Acute/enzymology , Leukemia, Basophilic, Acute/genetics , Plasmids , Quinuclidinyl Benzilate , Rats , Receptors, Muscarinic/genetics , Transfection , Tumor Cells, Cultured , Type C Phospholipases/pharmacology , beta-N-Acetylhexosaminidases/metabolism
10.
Gen Pharmacol ; 25(8): 1673-7, 1994 Dec.
Article in English | MEDLINE | ID: mdl-7721045

ABSTRACT

1. The effects of a protein-tyrosine kinase inhibitor, genistein, and a protein-tyrosine phosphatase inhibitor, orthovanadate, were tested on the Ca(2+)-free contraction of the estrogen-dominated rat, which has been proved to be induced mainly via protein kinase C entirely independently of Ca2+. 2. Genistein (30 microM) significantly inhibited the contraction indicating participation of tyrosine kinase activity in the contraction. 3. Orthovanadate caused contraction concentration-dependently and augmented the Ca(2+)-free contraction at concentrations of more than 1 microM. The contraction by orthovanadate was not inhibited so significantly by genistein (30 microM). 4. Possible participation of tyrosine kinase activity in Ca(2+)-free contraction is discussed in addition to the formerly reported participation of protein kinase C.


Subject(s)
Oxytocin/pharmacology , Protein Tyrosine Phosphatases/physiology , Protein-Tyrosine Kinases/physiology , Uterine Contraction/drug effects , Vanadates/pharmacology , Animals , Calcium/physiology , Drug Interactions , Female , Genistein , In Vitro Techniques , Isoflavones/pharmacology , Protein Tyrosine Phosphatases/antagonists & inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Rats
11.
Jpn J Pharmacol ; 66(2): 195-204, 1994 Oct.
Article in English | MEDLINE | ID: mdl-7532733

ABSTRACT

The N-acetyl glucosamine (GlcNAc)-specific lectin Datura stramonium agglutinin (DSA) rapidly and sugar-specifically released histamine from rat peritoneal mast cells, and pertussis toxin (IAP) inhibited it, suggesting that DSA activated mast cells via an IAP-sensitive G protein pathway. The additive effects of DSA and basic secretagogues such as compound 48/80 that activate IAP-sensitive G protein directly suggest that they shared the same mechanism of action including involvement of the IAP-sensitive G protein. Using lectin-blotting, blots of the corresponding glycoproteins detected by DSA diminished by haptenic sugar or pretreatment of the cells with N-glycosidase F, suggesting that the binding of DSA was responsible for the mast cell activation. The other GlcNAc-specific lectins such as Phytolacca americana mitogen, Solanum tuberosum agglutinin and wheat germ agglutinin (WGA) inhibited the histamine release induced by DSA, suggesting that these lectins were antagonists, but DSA was an agonist. Sialic acid-specific Macckia amurensis mitogen (MAM) inhibited the histamine release, and neuraminidase-treatment decreased mast cell activation induced by DSA. At least four mast cell glycoproteins that have affinity to DSA, WGA and MAM and are sensitive to neuraminidase-treatment were detected by lectin-blotting. Some of them may be binding sites coupled to histamine release including the IAP-sensitive G protein pathway. DSA is a useful tool for studying signal transduction of mast cells including the involvement of the IAP-sensitive G protein.


Subject(s)
Acetylglucosamine/metabolism , Histamine Release/drug effects , Lectins/pharmacology , Mast Cells/metabolism , Agglutinins/pharmacology , Animals , Bradykinin/pharmacology , Datura stramonium/metabolism , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , GTP-Binding Proteins/metabolism , Glycoproteins/metabolism , Haptens/pharmacology , Immunoblotting , Male , Mast Cells/drug effects , Neuraminidase/pharmacology , Oligosaccharides/pharmacology , Peritoneal Cavity/cytology , Pertussis Toxin , Plant Lectins , Plants, Medicinal , Plants, Toxic , Polyethyleneimine/pharmacology , Rats , Rats, Sprague-Dawley , Virulence Factors, Bordetella/pharmacology , p-Methoxy-N-methylphenethylamine/pharmacology
12.
Jpn J Pharmacol ; 66(2): 205-11, 1994 Oct.
Article in English | MEDLINE | ID: mdl-7532734

ABSTRACT

A confocal fluorescence microscope using fluo-3 and 9-(dicyanovinyl)- julolidine (DCVJ) was used to study the mast cell activation by the N-acetyl glucosamine oligomer specific lectin Datura stramonium agglutinin (DSA) and inhibition by antagonist lectins having affinity to N-acetyl glucosamine (GlcNAc). DSA induced a transient increase in intracellular free calcium concentration ([Ca2+]i) followed by cytoskeletal disassembly and reassembly in rat peritoneal mast cells. These changes induced by DSA resulted in histamine release. The time course of fluorescence intensity in mast cells loaded with fluo-3- or DCVJ and activated by DSA resembled those activated by the basic polymer compound 48/80. Inhibition of [Ca2+]i rise by antagonist lectins was responsible for the inhibition of cytoskeletal assembly and the consequent histamine release induced by DSA. At the level of the individual cell, a mast cell stimulated by DSA responds in an all-or-none fashion. DSA possible induced intracellular calcium mobilization and cytoskeletal change by recognizing the GlcNAc-oligomer residues of specific glycoproteins of mast cells.


Subject(s)
Datura stramonium/metabolism , Histamine Release/drug effects , Lectins/pharmacology , Mast Cells/drug effects , Plants, Medicinal , Plants, Toxic , Agglutinins/pharmacology , Animals , Calcium/metabolism , Cytoskeleton/drug effects , Male , Mast Cells/metabolism , Mast Cells/ultrastructure , Microscopy, Confocal , Microscopy, Fluorescence , Peritoneal Cavity/cytology , Plant Lectins , Rats , Rats, Sprague-Dawley , p-Methoxy-N-methylphenethylamine/pharmacology
13.
Eur J Pharmacol ; 263(1-2): 75-80, 1994 Sep 22.
Article in English | MEDLINE | ID: mdl-7821364

ABSTRACT

Ca2+ has been reported to exert an inhibitory effect on various kinds of smooth muscle. The physiological role of this inhibition is unclear. We investigated the inhibitory action of Ca2+ on the uterine smooth muscle of the rat in estrus, which shows a prominent Ca(2+)-induced inhibition. At concentrations of 0.1-30 microM Ca2+ inhibited the Ca(2+)-independent contraction of this muscle induced by oxytocin in Ca(2+)-free medium. We then investigated the inhibitory action of Ca2+ at various concentrations of Ca2+ in the bathing medium and found that Ca2+ at 1.0-10 microM also inhibited Ca(2+)-dependent contractions, which appeared phasically upon the onset of contractions. The magnitude of these phasic contractions was inversely proportional to the concentration of Ca2+ (between 1-10 microM). At 30 microM Ca2+, however, this inhibition was overcome and large pendular contractions began. Thus, the inhibition may regulate the initiation of smooth muscle contractions. The mechanism of this Ca(2+)-induced inhibition is also discussed with regard to an effect on actin.


Subject(s)
Calcium/pharmacology , Carbazoles , Indoles , Muscle, Smooth/drug effects , Uterine Contraction/drug effects , Actins/drug effects , Alkaloids/pharmacology , Animals , Calmodulin/antagonists & inhibitors , Cytochalasin D/pharmacology , Drug Interactions , Female , Myosin-Light-Chain Kinase/antagonists & inhibitors , Oxytocin/physiology , Rats , Sulfonamides/pharmacology , Vasodilator Agents/pharmacology
14.
Nihon Yakurigaku Zasshi ; 104(3): 251-62, 1994 Sep.
Article in Japanese | MEDLINE | ID: mdl-7959417

ABSTRACT

Desensitization of the m3-muscarinic acetylcholine-receptor in the smooth muscle of the digestive tract is discussed together with the changes in intracellular signal transduction. Isolated single cells that show an all-or-none contractile response to acetylcholine were desensitized by treatment with 0.1 mM acetylcholine for 10 min, resulting in an increase in the threshold concentration of acetylcholine for contraction, but without changing any of the binding characteristics. Permeabilized cells showed that the desensitization is via uncoupling between the receptor and G-protein. Secretory cells (rat basophil leukemia-2H3 cells) transfected with human m3-receptor showed desensitization when treated with 0.1 mM carbachol for 30 min. The coupling between the receptor and G-protein was not impaired, but some unknown Ca(2+)-independent mechanism may be involved. Smooth muscle tissue was tested for its time-course of desensitization, and a novel transient resensitization was found at 1 min of incubation with 0.1 mM carbachol. This resensitization, and the desensitization prior to it, were accompanied with changes in binding affinity. However, the affinity was not changed, in parallel with desensitization afterwards, but the positive feedback loop of Ca(2+)-influx caused by alkalization via receptor-stimulation was suppressed. After a 30-min treatment, a Ca(2+)-independent mechanism caused the uncoupling and affinity decrease. Treatment for 3 hr increased the number of binding sites without recovery of the response. The desensitizing process is very diverse to achieve selectivity, but its purpose is in unity.


Subject(s)
Muscle, Smooth/metabolism , Receptors, Muscarinic/metabolism , Signal Transduction , Animals , Calcium/metabolism , Cell Communication , Digestive System/metabolism , In Vitro Techniques , Muscle Contraction , Muscle, Smooth/cytology
15.
Gen Pharmacol ; 25(4): 691-5, 1994 Jul.
Article in English | MEDLINE | ID: mdl-7958730

ABSTRACT

1. Intracellular actin filament organization of gastric smooth muscle cells of the guinea pig in primary culture was examined with rhodamine-labelled phalloidin using a confocal laser fluorescence microscope. 2. The resting cells, both in the presence and absence of Ca2+, showed an even distribution of microfilamentous actin fibers. 3. The characteristic image of the stimulated cells with 10 microM acetylcholine in the presence of 1.8 mM Ca2+ was that the actin filaments were located only on the periphery of the cell. 4. The characteristic image of the cells stimulated as above, but in the absence of Ca2+, was that the actin filaments were unevenly distributed in the cell. 5. The characteristic image of the cells stimulated in the presence of 1 microM Ca2+, which inhibits the above contraction, was pultaceous with the actin filaments absent, indicating severing of actin filaments by a Ca(2+)-activated system, such as gelsolin.


Subject(s)
Actins/metabolism , Calcium/physiology , Muscle Contraction , Muscle, Smooth/physiology , Animals , Gastric Fundus/physiology , Guinea Pigs , In Vitro Techniques
16.
Gen Pharmacol ; 25(4): 729-37, 1994 Jul.
Article in English | MEDLINE | ID: mdl-7958735

ABSTRACT

1. RBL-2H3 cells permeabilized with alpha-toxin responded to dinitrophenol (30-40 mol/mol)-conjugated human serum albumin, as antigen, to secrete [14C]serotonin in the micromolar range of free Ca2+. 2. Calcium ion alone did not cause substantial secretion. 3. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) (100 microM) in combination with Ca2+ produced only negligible [14C]serotonin secretion. 4. GTP gamma S, in the presence of cytochalasin D, caused optimal secretion of [14C]serotonin in a Ca(2+)-dependent manner.


Subject(s)
Bacterial Toxins/pharmacology , Calcium/physiology , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , Hemolysin Proteins/pharmacology , Leukemia, Basophilic, Acute/metabolism , Receptors, IgE/physiology , Adenine/metabolism , Animals , Cell Membrane Permeability/drug effects , Rats , Serotonin/metabolism , Tumor Cells, Cultured , beta-N-Acetylhexosaminidases/metabolism
17.
Jpn J Pharmacol ; 64(1): 1-8, 1994 Jan.
Article in English | MEDLINE | ID: mdl-7513032

ABSTRACT

The effects of seven lectins with various sugar-specificities on histamine release from rat peritoneal mast cells induced by non-immunologic stimuli were studied. The non-immunologic stimuli used were three basic secretagogues, compound 48/80, bradykinin and PEI6 (polyethylenimine with a molecular weight of 600). In this study, we observed inhibition of the histamine release by Macckia amurensis mitogen and Solanum tuberosum agglutinin (100 micrograms/ml at 37 degrees C for 10 min), which are specific for sialic acid-alpha 2,3-N-acetyl galactosamine (Sia alpha 2,3GalNAc) and N-acetyl glucosamine (GlcNAc) oligomers, respectively. The effects of Phytolacca americana mitogen and Sambucus sieboldiana agglutinin were different. Three lectins specific for mucin type oligosaccharides inhibited the histamine release induced by compound 48/80 but not that induced by bradykinin or PEI6. Since bradykinin and PEI6 additively enhanced the histamine release induced by compound 48/80, they partially shared the same signalling pathways. Glycoproteins with bisecting GlcNAc and Sia residues, as described previously (Jpn. J. Pharmacol. 57, 79-90, 1991), seemed to be one of the action sites for compound 48/80, bradykinin and PEI6. In addition to the direct activation of the pertussis toxin-sensitive G proteins, we propose another mechanism of non-immunologic stimuli via specific glycoproteins on rat peritoneal mast cells. The apparent sugar residues involved were asparagine-linked oligosaccharides with Sia (especially Sia alpha 2,3Gal), GlcNAc oligomers and/or bisecting GlcNAc.


Subject(s)
Acetylglucosamine/metabolism , Histamine Release/drug effects , Lectins/pharmacology , Mast Cells/drug effects , Sialic Acids/metabolism , Agglutinins/pharmacology , Animals , Bradykinin/pharmacology , Electrophoresis, Polyacrylamide Gel , Glycoproteins/analysis , Histamine Antagonists/pharmacology , In Vitro Techniques , Mast Cells/chemistry , Mast Cells/metabolism , Mitogens/pharmacology , N-Acetylneuraminic Acid , Neuraminidase/pharmacology , Oligosaccharides , Peritoneal Cavity/cytology , Polyethyleneimine/pharmacology , Rats , p-Methoxy-N-methylphenethylamine/pharmacology
18.
Gen Pharmacol ; 24(5): 1085-90, 1993 Sep.
Article in English | MEDLINE | ID: mdl-8270165

ABSTRACT

1. Single smooth muscle cells from the guinea-pig taenia caecum and the fundus of guinea-pig stomach were prepared by collagenase digestion under different, mild conditions. 2. Most of the cells either from taenia caecum or from the fundus of stomach responded repeatedly, showing an all-or-none response to acetylcholine (ACh). 3. The threshold concentrations of ACh were different for the cells of the two tissues. Although individual cells showed an all-or-none response to ACh, the average responses of all the cells were graded, like that of whole tissues. 4. Thus, isolated single smooth muscle cells from different tissues and under different conditions responded to ACh in an all-or-none manner such as the twitch observed in skeletal muscle. 5. These results suggest that the isolation of cells reveals the fundamental characteristics of smooth muscle cells as excitable.


Subject(s)
Acetylcholine/pharmacology , Muscle, Smooth/drug effects , Animals , Cecum/cytology , Cecum/drug effects , Gastric Fundus/cytology , Gastric Fundus/drug effects , Guinea Pigs , In Vitro Techniques , Muscle Contraction/drug effects , Muscle, Smooth/cytology
19.
J Pharmacol Exp Ther ; 266(1): 367-73, 1993 Jul.
Article in English | MEDLINE | ID: mdl-8101221

ABSTRACT

Isoproterenol (ISO)-induced relaxation of oxytocin-induced Ca(++)-independent contraction of the rat uterus was examined. Oxytocin induced Ca(++)-dependent phasic contraction in a solution containing Ca++ (normal contraction) and Ca(++)-independent sustained contraction in Ca(++)-free solution (Ca(++)-free contraction). Both contractions were completely suppressed by cyclic AMP-elevating relaxants such as ISO, dibutyryl cyclic AMP and forskolin. Moreover, the ISO concentration needed to inhibit the Ca(++)-free contraction was lower than that needed for normal contraction, although the relaxing effect of dibutyryl cyclic AMP and forskolin during Ca(++)-free contraction was not significantly different from that during Ca(++)-dependent contraction. The ISO-induced relaxation of the uterus in Ca(++)-free solution may involve three mechanisms. The first is cyclic AMP-dependent relaxation shown by high concentrations (more than 1 nM) of ISO. The second is stabilization via K+ channels by intermediate concentrations (10 pM to 1 nM) of ISO. These two actions appear to be mediated through beta-1 adrenoceptors. The third is, however, via an unknown subtype of adrenoceptor stimulated by extremely low concentrations (1 pM to 10 pM) of ISO. All of these relaxing mechanisms are independent of Ca++.


Subject(s)
Calcium/physiology , Muscle, Smooth/physiology , Receptors, Adrenergic, beta/physiology , Uterine Contraction/physiology , Adrenergic beta-Antagonists/pharmacology , Animals , Bucladesine/pharmacology , Calcium/metabolism , Colforsin/pharmacology , Cyclic AMP/metabolism , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Female , Intracellular Fluid/metabolism , Isoproterenol/pharmacology , Muscle Relaxation/drug effects , Muscle Relaxation/physiology , Muscle, Smooth/drug effects , Muscle, Smooth/metabolism , Potassium/pharmacology , Potassium Channels/drug effects , Rats , Rats, Wistar , Uterine Contraction/drug effects
20.
Br J Pharmacol ; 109(2): 330-5, 1993 Jun.
Article in English | MEDLINE | ID: mdl-8358538

ABSTRACT

1. Muscarinic stimulation of the smooth muscle of guinea-pig taenia caeci was produced with 10(-4) M carbachol for 15 s, 30 s, 1 min, 2 min and 30 min, and the time course of developing desensitization was studied by measuring the muscle contractility and the binding characteristics of muscarinic receptors. 2. The contractile response to carbachol was analyzed using dose-response curves. The response to 10(-7) M carbachol was reduced by treatment for 15 s with 10(-4) M carbachol (fast desensitization), but recovered partially after 30 s treatment and completely after 1 min treatment (resensitization). Contractility was reduced again after 2 min and 30 min treatment (re-desensitization). 3. The affinity of carbachol for muscarinic receptors was changed by the carbachol treatment in a manner similar to the contractility. Thus, the affinity was reduced at 15 s, restored slightly at 30 s and completely at 1 and 2 min, and was reduced again at 30 min. 4. 5'-Guanylylimidodiphosphate (GppNHp), a non-hydrolysable analogue of guanosine triphosphate (GTP) reduced the affinity of muscarinic receptors for carbachol via guanine nucleotide-binding regulatory proteins (G proteins). A similar effect was observed in tissues desensitized by 15 s carbachol treatment. This effect disappeared after 30 s, recovered completely after 1 and 2 min, and disappeared again after 30 min carbachol treatment. 5. Neither the dissociation constant (Kd value) nor the maximal binding (Bmax) of [3H]-quinuclidinyl benzilate ([3H]-QNB) to muscarinic receptors were changed by the carbachol treatment. 6. These results indicate that the whole process of desensitization, resensitization and re-desensitization are related to changes in the binding ability of muscarinic receptors, in their coupling with G proteins and in the post-receptor steps of the signal transduction. We emphasize that the desensitizing process involves an early transient phase of resensitization that could be caused by restoration of both the affinity of carbachol for muscarinic receptors and their coupling with G proteins. This novel resensitization mechanism may have some physiological significance for cellular homoeostasis by modulating cellular responsiveness transiently or even in an oscillatory manner during the process of desensitization.


Subject(s)
GTP-Binding Proteins/metabolism , Muscle, Smooth/metabolism , Receptors, Muscarinic/drug effects , Animals , Carbachol/metabolism , Cecum/drug effects , Female , Guanine Nucleotides/metabolism , Guanylyl Imidodiphosphate/pharmacology , Guinea Pigs , In Vitro Techniques , Kinetics , Male , Muscle Contraction/drug effects , Muscle, Smooth/drug effects , Muscle, Smooth, Vascular/drug effects , Quinuclidinyl Benzilate/pharmacology , Receptors, Muscarinic/metabolism
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