endothelin-1 and tetrafluoroaluminate

We determined whether activation of G proteins can affect the force developed for a given intracellular Ca(2+) concentration ([Ca(2+)]; i.e., the Ca(2+) sensitivity) by mechanisms in addition to changes in regulatory myosin light chain (rMLC) phosphorylation. Responses in alpha-toxin-permeabilized canine tracheal smooth muscle were determined with Ca(2+) alone or in the presence of ACh, endothelin-1 (ET-1), or aluminum fluoride (AlF; acute or 1-h exposure). Acute exposure to each compound increased Ca(2+) sensitivity without changing the response to high [Ca(2+)] (maximal force). However, chronic exposure to AlF, but not to chronic ACh or ET-1, increased maximal force by increasing the force produced for a given rMLC phosphorylation. Studies employing thiophosphorylation of rMLC showed that the increase in force produced by chronic AlF exposure required Ca(2+) during activation to be manifest. Unlike the acute response to receptor agonists, which is mediated solely by increases in rMLC phosphorylation, chronic direct activation of G proteins further increases Ca(2+) sensitivity in airways by additional mechanisms that are independent of rMLC phosphorylation.

Topics: Acetylcholine; Adenosine Triphosphate; Aluminum Compounds; Animals; Calcium; Dogs; Drug Resistance; Endothelin-1; Female; Fluorides; GTP-Binding Proteins; In Vitro Techniques; Magnesium Compounds; Male; Muscle Contraction; Muscle, Smooth; Myosin Light Chains; Phosphorylation; Time Factors; Trachea

Endothelin-1 (ET-1) has been shown to induce DNA synthesis in primary astrocytes by stimulating the extracellular signal-regulated kinase (ERK) pathway. To clarify the mechanisms responsible for the anchorage-dependent growth of astrocytes, the relationships between cell adhesion and ERK activation were investigated. Here it is reported that ET-1 promotes the formation of stress fibers and focal adhesions and the tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, as well as Src activation and association of phosphorylated FAK with Grb2. Pretreatment of astrocytes with cytochalasin D or C3-transferase, which inhibits actin polymerization or Rho activity, respectively, prevented the activation/phosphorylation of Src, FAK, and paxillin after ET-1 stimulation; by contrast, the ERK pathway was not significantly affected. This differential activation of FAK/Src and ERK pathways was also observed with astrocytes 10 and 60 min after replating on poly-L-ornithine-precoated dishes. Collectively, these findings indicate that activation of FAK and Src is dependent on actin cytoskeleton integrity, Rho activation, and adhesion to extracellular matrix, whereas ERK activation is independent of these intracellular events and seems to correlate with activation of the newly identified protein tyrosine kinase PYK2. Induction of DNA synthesis by ET-1, however, was reduced dramatically in astrocytes pretreated with either cytochalasin D or C3-transferase. This study provides a demonstration of Rho- and adhesion-dependent activation of FAK/Src, which collaborates with adhesion-independent activation of PYK2/ERK for DNA synthesis in ET-1-stimulated astrocytes.

Topics: Actins; Adaptor Proteins, Signal Transducing; Aluminum Compounds; Animals; Astrocytes; Cell Adhesion Molecules; Cell Division; Cells, Cultured; Cerebral Cortex; Corpus Striatum; Cytoskeletal Proteins; Cytoskeleton; DNA; Endothelin-1; ErbB Receptors; Fetus; Fluorides; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; GRB2 Adaptor Protein; GTP-Binding Proteins; Mitogen-Activated Protein Kinases; Nerve Tissue Proteins; Paxillin; Phosphoproteins; Phosphorylation; Protein Binding; Protein Kinase C; Protein-Tyrosine Kinases; Proteins; Rabbits; Rats; rho GTP-Binding Proteins; Signal Transduction; src Homology Domains; src-Family Kinases; Stress, Mechanical; Tyrosine

Cultured astrocytes express bradykinin (BK) receptors, which are coupled to phospholipase C (PLC) through G-protein to mediate phosphoinositide (PI) hydrolysis. The regulation of this BK receptor-G protein-PLC pathway by cAMP and endothelin-1 (ET-1) was explored by short-term (20 min) and long-term (24 h) treatment with 100 mu M dibutyryl cyclic AMP (dBcAMP) or 10 nM ET-1. Short-term treatment of cells with dBcAMP had no effect on BK-induced PI hydrolysis; however, long-term treatment resulted in potentiation of the BK response. Similar effects were seen after 10 mu M forskolin pretreatment of the cells. We further explored the site of action of 24 h dBcAMP pretreatment and found that AlF(4)-, ionomycin- or A3187-induced PI hydrolysis was not affected but (3H)BK binding was increased. These results indicate that the site of action of dBcAMP is the BK receptor and Scatchard plot analysis showed that the Bmax was increased but the Kd decreased. Cycloheximide (0.5 mu M) blocked the increase in (3H)BK binding, indicating that new synthesis of receptor protein might occur during 24 h pretreatment with dBcAMP. Twenty minutes pretreatment of cells with ET-1 resulted in desensitization of the ET-1 induced P1 response, while the BK response was unaffected. After 24 h pretreatment with ET-1, desensitization to ET-1 still occurred, while BK-induced PI hydrolysis was markedly potentiated. (3H)BK binding and AlF(4)--induced but not A23187- or ionomycin-induced PI hydrolysis were increased, indicating that the site of action of long-term ET-1 treatment was the BK receptor and G protein; Scatchard analysis showed an increase in Bmax but no effect on Kd. These effects were blocked by cycloheximide, indicating that new synthesis of both receptor protein and G protein might occur during 24 h pretreatment with ET-1. (3H)Thymidine uptake was inhibited or potentiated by dBcAMP and ET-1, respectively. Possible dBcAMP-induced differentiation and ET-1-induced proliferation may contribute to the increased expression of receptor proteins.

Topics: Aluminum Compounds; Animals; Astrocytes; Bradykinin; Bucladesine; Calcium; Cells, Cultured; Colforsin; Cyclic AMP; Endothelin-1; Fluorides; Hydrolysis; Inositol Phosphates; Ionophores; Rats; Rats, Wistar; Thymidine