Cholecystokinin-stimulated tyrosine phosphorylation of p125FAK and paxillin is mediated by phospholipase C-dependent and -independent mechanisms and requires the integrity of the actin cytoskeleton and participation of p21rho

Recent studies show that the effects of some oncogenes, integrins, growth factors and neuropeptides are mediated by tyrosine phosphorylation of the cytosolic kinase p125 focal adhesion kinase (p125FAK) and the cytoskeletal protein paxillin. Recently we demonstrated that cholecystokinin (CCK) C-terminal octapeptide (CCK-8) causes tyrosine phosphorylation of p125FAK and paxillin in rat pancreatic acini. The present study was aimed at examining whether protein kinase C (PKC) activation, calcium mobilization, cytoskeletal organization and small G-protein p21rho activation play a role in mediating the stimulation of tyrosine phosphorylation by CCK-8 in acini. CCK-8-stimulated phosphorylation of p125FAK and paxillin reached a maximum within 2.5 min. The CCK-8 dose response for causing changes in the cytosolic calcium concentration ([Ca2+]i) was similar to that for p125FAK and paxillin phosphorylation, and both were to the left of that for receptor occupation and inositol phosphate production. PMA increased tyrosine phosphorylation of both proteins. The calcium ionophore A23187 caused only 25% of the maximal stimulation caused by CCK-8. GF109203X, a PKC inhibitor, completely inhibited phosphorylation with PMA but had no effect on the response to CCK-8. Depletion of [Ca2+]i by thapsigargin had no effect on CCK-8-stimulated phosphorylation. Pretreatment with both GF109203X and thapsigargin decreased CCK-8-stimulated phosphorylation of both proteins by 50%. Cytochalasin D, but not colchicine, completely inhibited CCK-8- and PMA-induced p125FAK and paxillin phosphorylation. Treatment with Clostridium botulinum C3 transferase, which inactivates p21rho, caused significant inhibition of CCK-8-stimulated p125FAK and paxillin phosphorylation. These results demonstrate that, in pancreatic acini, CCK-8 causes rapid p125FAK and paxillin phosphorylation that is mediated by both phospholipase C-dependent and -independent mechanisms. For this tyrosine phosphorylation to occur, the integrity of the actin, but not the microtubule, cytoskeleton is essential as well as the activation of p21rho.

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Cell cycle progression of parthenogenetically activated mouse oocytes to interphase is dependent on the level of internal calcium

Journal of Cell Science ◽

10.1242/jcs.103.2.389 ◽

1992 ◽

Vol 103 (2) ◽

pp. 389-396 ◽

Cited By ~ 7

Author(s):

C. Vincent ◽

T.R. Cheek ◽

M.H. Johnson

Keyword(s):

Cell Cycle Progression ◽

Polar Body ◽

Calcium Ionophore ◽

Cytosolic Calcium ◽

Calcium Ionophore A23187 ◽

Mouse Oocyte ◽

Free Calcium ◽

Ionophore A23187 ◽

Parthenogenetic Activation ◽

Mouse Oocytes

Nuclear maturation of the mouse oocyte becomes arrested in metaphase of the second meiotic division (MII). Fertilization or parthenogenetic activation induces meiotic completion, chromosomal decondensation and formation of a pronucleus. This completion of meiosis is probably triggered by a transient increase in cytosolic calcium ions. When activated just after ovulation by a low concentration of the calcium ionophore A23187, the majority of the mouse oocytes go through a metaphase to anaphase transition and extrude their second polar body but they do not proceed into interphase; instead their chromatids remain condensed and a microtubular metaphase spindle reforms (metaphase III). However, a high percentage of these oocytes will undergo a true parthenogenetic activation assessed by the formation of a pronucleus, when exposed to a higher concentration of the calcium ionophore. The capacity of the mouse oocyte to pass into metaphase III is lost with increasing time post-ovulation. Direct measurement of intracellular calcium with Fura-2 reveals higher levels of cytosolic calcium in aged oocytes and/or using higher concentrations of calcium ionophore for activation. It is concluded that the internal free calcium level determines the transition to interphase.

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Calcium ionophore, A23187, induces commitment to differentiation but inhibits the subsequent expression of erythroid genes in murine erythroleukemia cells

Blood ◽

10.1182/blood.v77.6.1362.1362 ◽

1991 ◽

Vol 77 (6) ◽

pp. 1362-1370 ◽

Cited By ~ 25

Author(s):

JO Hensold ◽

G Dubyak ◽

DE Housman

Keyword(s):

Transcriptional Activation ◽

Calcium Ionophore ◽

Erythroid Differentiation ◽

Cytosolic Calcium ◽

Calcium Ionophore A23187 ◽

Ionophore A23187 ◽

Cellular Processes ◽

Cellular Events ◽

Erythroleukemia Cells ◽

Murine Erythroleukemia

Abstract Murine erythroleukemia (MEL) cells are a useful model for studying the processes that regulate erythroid differentiation because exposure of these cells to a variety of chemical inducing agents results in expression of erythroid-specific genes and the resultant loss of cellular immortality. Previously it has been suggested that the calcium ionophore, A23187, has effects on the early cellular events that lead to the commitment of these cells to differentiation, but was not in itself sufficient to induce differentiation. We demonstrate here that A23187, as well as another calcium ionophore, ionomycin, are capable of inducing commitment to differentiation. Unlike other inducing agents, continual exposure to A23187 inhibits transcription of the erythroid- specific genes, beta-globin and Band 3. This effect is not attributable to an increase in cytosolic calcium concentration, because cells induced by ionomycin produce normal amounts of hemoglobin. These effects of A23187 on MEL cells confirm that commitment to differentiation is a distinct event from the subsequent transcriptional activation of erythroid genes. The ability of both ionophores to induce commitment to differentiation suggests that an increase in cytosolic calcium can trigger commitment to differentiation. These agents should prove useful in investigating the cellular processes that are responsible for commitment to differentiation.

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Rapid ultrastructural changes in the dense tubular system following platelet activation

Blood ◽

10.1182/blood.v80.3.718.718 ◽

1992 ◽

Vol 80 (3) ◽

pp. 718-723 ◽

Cited By ~ 29

Author(s):

L Ebbeling ◽

C Robertson ◽

A McNicol ◽

JM Gerrard

Keyword(s):

Intracellular Calcium ◽

Platelet Activation ◽

Calcium Ionophore ◽

Cytosolic Calcium ◽

Calcium Ionophore A23187 ◽

Ultrastructural Changes ◽

Ionophore A23187 ◽

Tubular System ◽

Protein Kinase C Activators ◽

Morphologic Changes

Abstract The dense tubular system (DTS) functions to regulate platelet activation by sequestering or releasing calcium, similar to the sarcotubules of skeletal muscle. In resting platelets, the DTS exists as thin elongated membranes. Within 10 seconds of the addition of thrombin, platelets show a major ultrastructural change in their DTS: from the thin elongated form to a rounded vesicular form. These morphologic changes were demonstrated with two different stains and two different fixation methods. Platelets exposed to the calcium ionophore A23187 showed the same ultrastructural changes in the DTS. In contrast, the DTS remains in a thin elongated form when platelets are stimulated by the protein kinase C activators phorbol 12-myristate 13-acetate (PMA) and oleoylacetylglycerol (OAG). These morphologic changes may be related to the discharge of calcium from the DTS because this is stimulated by thrombin and A23187, but not by PMA. Preincubation of the platelets with the intracellular calcium chelator 5,5′-dimethyl-bis-(0- aminophenoxy)-ethane-N,N,N′,N tetra acetic acid (BAPTA) largely prevented both the thrombin-induced rise in intracellular calcium and the changes in DTS morphology, suggesting that the changes in DTS morphology are secondary to the increase in cytosolic calcium. The results provide a morphologic correlate to existing biochemical evidence showing that the DTS is involved early during paltelet activation.

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Cell-membrane phospholipase C is involved in inducing the antiviral effect of interferon

Bioscience Reports ◽

10.1007/bf01119795 ◽

1989 ◽

Vol 9 (5) ◽

pp. 531-539 ◽

Cited By ~ 2

Author(s):

L. M. Popescu ◽

C. Cernescu ◽

I. I. Moraru ◽

St. N. Constantinescu ◽

F. Baltã ◽

...

Keyword(s):

Phospholipase C ◽

Vesicular Stomatitis Virus ◽

Second Messengers ◽

Calcium Ionophore ◽

Antiviral Effect ◽

Vero Cells ◽

Calcium Ionophore A23187 ◽

Ionophore A23187 ◽

Antibody Molecule ◽

Vesicular Stomatitis

A monospecific inhibitory antibody directed to phospholipase C (phosphoinositidase C) blocked the antiviral effect of human interferons alpha and beta when tested on human quiescent fibroblasts challenged with the vesicular stomatitis virus. This action was due to specific inhibition of polyphosphoinositide hydrolysis because (a) the F(ab′)2 fragment of the antibody molecule was also inhibitory; (b) excess antibodies directed to phospholipase A2 and to a phosphatidylcholine-preferring phospholipase C did not have any inhibitory effect, and (c) the combination of 12-O-tetradecanoylphorbol-acetate and calcium ionophore A23187 had an interferon-like antiviral effect which was not influenced by the inhibitory anti-phospholipase C antibodies. To avoid an interferon-like effect due to induction of interferon by second messengers, Vero cells, which lack interferon biosynthesis, were also used. Liposomes containing inositol 1,4,5-triphosphate and 1-oleoyl-2-acetyl-rac-glycerol protected Vero cells against the infection with the vesicular stomatitis virus. These results taken together show that phosphoinositide-derived second messengers are involved in triggering the antiviral effect of interferons alpha and beta.

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Synergistic Activation of Tyrosine Phosphorylation by Orthovanadate plus Calcium Ionophore A23187 or Aromatic 1,2-Diols

Biochemistry ◽

10.1021/bi00172a031 ◽

1994 ◽

Vol 33 (6) ◽

pp. 1518-1525 ◽

Cited By ~ 5

Author(s):

William R. Huckle ◽

H. Shelton Earp

Keyword(s):

Tyrosine Phosphorylation ◽

Calcium Ionophore ◽

Calcium Ionophore A23187 ◽

Ionophore A23187 ◽

Synergistic Activation

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Melittin inhibits epidermal growth factor-induced protein tyrosine phosphorylation: comparison with phorbol myristate acetate and calcium ionophore A23187

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research ◽

10.1016/0167-4889(94)90056-6 ◽

1994 ◽

Vol 1222 (3) ◽

pp. 471-476 ◽

Cited By ~ 2

Author(s):

Mourad Errasfa ◽

Arnold Stern

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Tyrosine Phosphorylation ◽

Phorbol Myristate Acetate ◽

Calcium Ionophore ◽

Calcium Ionophore A23187 ◽

Ionophore A23187 ◽

Protein Tyrosine Phosphorylation ◽

Myristate Acetate ◽

Epidermal Growth

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Calcium ionophore, A23187, induces commitment to differentiation but inhibits the subsequent expression of erythroid genes in murine erythroleukemia cells

Blood ◽

10.1182/blood.v77.6.1362.bloodjournal7761362 ◽

1991 ◽

Vol 77 (6) ◽

pp. 1362-1370 ◽

Cited By ~ 1

Author(s):

JO Hensold ◽

G Dubyak ◽

DE Housman

Keyword(s):

Transcriptional Activation ◽

Calcium Ionophore ◽

Erythroid Differentiation ◽

Cytosolic Calcium ◽

Calcium Ionophore A23187 ◽

Ionophore A23187 ◽

Cellular Processes ◽

Cellular Events ◽

Erythroleukemia Cells ◽

Murine Erythroleukemia

Murine erythroleukemia (MEL) cells are a useful model for studying the processes that regulate erythroid differentiation because exposure of these cells to a variety of chemical inducing agents results in expression of erythroid-specific genes and the resultant loss of cellular immortality. Previously it has been suggested that the calcium ionophore, A23187, has effects on the early cellular events that lead to the commitment of these cells to differentiation, but was not in itself sufficient to induce differentiation. We demonstrate here that A23187, as well as another calcium ionophore, ionomycin, are capable of inducing commitment to differentiation. Unlike other inducing agents, continual exposure to A23187 inhibits transcription of the erythroid- specific genes, beta-globin and Band 3. This effect is not attributable to an increase in cytosolic calcium concentration, because cells induced by ionomycin produce normal amounts of hemoglobin. These effects of A23187 on MEL cells confirm that commitment to differentiation is a distinct event from the subsequent transcriptional activation of erythroid genes. The ability of both ionophores to induce commitment to differentiation suggests that an increase in cytosolic calcium can trigger commitment to differentiation. These agents should prove useful in investigating the cellular processes that are responsible for commitment to differentiation.

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Distinct activation of Na+-H+ exchange by gastrin and CCK peptide in acini from guinea pig

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1988.254.1.g25 ◽

1988 ◽

Vol 254 (1) ◽

pp. G25-G32

Author(s):

M. J. Bastie ◽

M. Delvaux ◽

M. Dufresne ◽

J. S. Saunier-Blache ◽

N. Vaysse ◽

...

Keyword(s):

Guinea Pig ◽

Calcium Concentration ◽

Calcium Ionophore ◽

Calcium Ionophore A23187 ◽

Ionophore A23187 ◽

Pancreatic Acini ◽

Cck Receptors ◽

Kinase C ◽

22Na Uptake ◽

Cck Antagonists

The effect of cholecystokinin (CCK)-gastrin family peptides (caerulein, unsulfated gastrin-17, and pentagastrin) and secretin in activating amiloride-sensitive 22Na uptake were investigated in guinea pig pancreatic acini. Secretin had no effect, but CCK-gastrin peptides stimulated the amiloride-sensitive 22Na uptake. The effect of caerulein was inhibited by dibutyryl guanosine 3',5'-cyclic monophosphate (cGMP) and asperlicin, indicating that activation of the Na+-H+ antiport caused by caerulein is mediated by CCK receptors. The effect of gastrin was dibutyryl cGMP and asperlicin insensitive, whereas the effect of pentagastrin was inhibited by the CCK antagonists but with a low affinity, indicating that the effect of gastrin and that of pentagastrin was CCK receptor independent. The calcium ionophore A23187 caused an increase in amiloride-sensitive 22Na uptake. However, the effect of caerulein, which increased internal calcium concentration, was not modified after depletion of intracellular calcium, and that of CCK-gastrin family peptides was not dependent on external calcium concentration. Activation of amiloride-sensitive 22Na uptake was also induced by 12-O-tetradecanoylphorbol 13-acetate and 1-oleoyl-2-acetyl-glycerol. Activation of protein kinase c may be involved in the mechanism of caerulein or gastrin in activating the Na+-H+ exchange.

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Mechanisms of Nonimmunological Histamine and Tryptase Release from Human Cutaneous Mast Cells

Anesthesiology ◽

10.1097/00000542-200004000-00026 ◽

2000 ◽

Vol 92 (4) ◽

pp. 1074-1081 ◽

Cited By ~ 61

Author(s):

Mette Veien ◽

Fania Szlam ◽

Jeannine T. Holden ◽

Koji Yamaguchi ◽

Donald D. Denson ◽

...

Keyword(s):

Mast Cell ◽

Mast Cells ◽

Phospholipase A2 ◽

Histamine Release ◽

Phospholipase C ◽

Cell Activation ◽

Calcium Ionophore ◽

Calcium Ionophore A23187 ◽

Mast Cell Activation ◽

Ionophore A23187

Background If mast cells are stimulated they release multiple mediators that delineate markers for immunologic and nonimmunologic reactions; histamine and tryptase are the two best known. Although histamine can be assayed in plasma, it is a nonspecific marker with a very short half-life. Tryptase has a longer half-life, but its release has not been proven to be specific for anaphylaxis. The authors investigated the mechanisms of nonimmunologic histamine release from human cutaneous mast cells to understand the mechanisms of mediator release and to determine whether tryptase was specific for allergic mediated activation. Methods Dispersed mast cell suspensions isolated from neonatal foreskins underwent challenge with vancomycin, calcium ionophore A23187, morphine, and atracurium, and histamine tryptase release was measured. The effects of calcium and magnesium, along with phospholipase C and phospholipase A2 inhibitors, also were investigated. Results Tryptase and histamine both were released by the known nonimmunologic stimuli (pharmacologic agents used in the current study; r2 = 0.6). Furthermore, vancomycin- and atracurium-induced histamine release was calcium dependent. Phospholipase C and phospholipase A2 inhibitors decreased vancomycin-induced histamine release, but not calcium ionophore A23187-induced release. Conclusions Tryptase is not a specific marker of mast cell activation (ie., anaphylaxis), and signaling mechanisms for mast cell activation involve activation of phospholipase C and phospholipase A2 pathways that are also involved in other cellular activation mechanisms.

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Carbachol regulates cholecystokinin receptor on pancreatic acinar cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1987.252.1.g77 ◽

1987 ◽

Vol 252 (1) ◽

pp. G77-G83

Author(s):

T. Honda ◽

H. Adachi ◽

M. Noguchi ◽

S. Sato ◽

S. Onishi ◽

...

Keyword(s):

Binding Sites ◽

Calcium Ionophore ◽

Inhibitory Effect ◽

Calcium Ionophore A23187 ◽

Pancreatic Acinar Cells ◽

Amylase Secretion ◽

Ionophore A23187 ◽

Pancreatic Acini ◽

Cholecystokinin Receptor ◽

Apparent Loss

We have examined the effect of carbamylcholine on the binding of cholecystokinin (CCK) to dispersed acini from rat pancreas. The CCK receptor on pancreatic acini possesses two classes of binding sites. Simultaneous addition of carbamylcholine inhibited binding of CCK to acini due to an apparent loss of high affinity CCK binding sites. Atropine prevented the inhibitory effect of carbamylcholine, whereas calcium ionophore A23187 did not alter binding of CCK. 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited binding of CCK in the same manner as carbamylcholine. Inhibition by carbamylcholine was reversible and the recovery was time dependent. By contrast, inhibition of binding of CCK by TPA did not reverse after a 60-min incubation without the agent. These findings, at least in part, account for the inhibition of the CCK-induced stimulation of amylase secretion by carbamylcholine. The action of TPA on binding of CCK suggests the possible involvement of the activation of protein kinase C in the inhibition of binding.

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