scholarly journals Massive calcium–activated endocytosis without involvement of classical endocytic proteins

2010 ◽  
Vol 137 (1) ◽  
pp. 111-132 ◽  
Author(s):  
Vincenzo Lariccia ◽  
Michael Fine ◽  
Simona Magi ◽  
Mei-Jung Lin ◽  
Alp Yaradanakul ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Cardiac Myocytes ◽  
Adenosine Triphosphate ◽  
Hek293 Cells ◽  
Membrane Domains ◽  
Baby Hamster Kidney ◽  
Bromoenol Lactone ◽  
Cytoplasmic Side

We describe rapid massive endocytosis (MEND) of >50% of the plasmalemma in baby hamster kidney (BHK) and HEK293 cells in response to large Ca transients. Constitutively expressed Na/Ca exchangers (NCX1) are used to generate Ca transients, whereas capacitance recording and a membrane tracer dye, FM 4–64, are used to monitor endocytosis. With high cytoplasmic adenosine triphosphate (ATP; >5 mM), Ca influx causes exocytosis followed by MEND. Without ATP, Ca transients cause only exocytosis. MEND can then be initiated by pipette perfusion of ATP, and multiple results indicate that ATP acts via phosphatidylinositol-bis 4,5-phosphate (PIP2) synthesis: PIP2 substitutes for ATP to induce MEND. ATP-activated MEND is blocked by an inositol 5-phosphatase and by guanosine 5′-[γ-thio]triphosphate (GTPγS). Block by GTPγS is overcome by the phospholipase C inhibitor, U73122, and PIP2 induces MEND in the presence of GTPγS. MEND can occur in the absence of ATP and PIP2 when cytoplasmic free Ca is clamped to 10 µM or more by Ca-buffered solutions. ATP-independent MEND occurs within seconds during Ca transients when cytoplasmic solutions contain polyamines (e.g., spermidine) or the membrane is enriched in cholesterol. Although PIP2 and cholesterol can induce MEND minutes after Ca transients have subsided, polyamines must be present during Ca transients. MEND can reverse over minutes in an ATP-dependent fashion. It is blocked by brief β-methylcyclodextrin treatments, and tests for involvement of clathrin, dynamins, calcineurin, and actin cytoskeleton were negative. Therefore, we turned to the roles of lipids. Bacterial sphingomyelinases (SMases) cause similar MEND responses within seconds, suggesting that ceramide may be important. However, Ca-activated MEND is not blocked by reagents that inhibit SMases. MEND is abolished by the alkylating phospholipase A2 inhibitor, bromoenol lactone, whereas exocytosis remains robust, and Ca influx causes MEND in cardiac myocytes without preceding exocytosis. Thus, exocytosis is not prerequisite for MEND. From these results and two companion studies, we suggest that Ca promotes the formation of membrane domains that spontaneously vesiculate to the cytoplasmic side.

Stimulation of Ca(2+)-dependent exocytosis of the sperm acrosome by cAMP acting downstream of phospholipase A2

Reproduction ◽  
2000 ◽  
pp. 57-68 ◽  
Author(s):  
J Garde ◽  
ER Roldan
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Phosphodiesterase Inhibitors ◽  
Dibutyryl Camp ◽  
Camp Phosphodiesterase ◽  
Ram Sperm ◽  
Camp Formation ◽  
Stimulation Of

Spermatozoa undergo exocytosis in response to agonists that induce Ca2+ influx and, in turn, activation of phosphoinositidase C, phospholipase C, phospholipase A2, and cAMP formation. Since the role of cAMP downstream of Ca2+ influx is unknown, this study investigated whether cAMP modulates phospholipase C or phospholipase A2 using a ram sperm model stimulated with A23187 and Ca2+. Exposure to dibutyryl-cAMP, phosphodiesterase inhibitors or forskolin resulted in enhancement of exocytosis. However, the effect was not due to stimulation of phospholipase C or phospholipase A2: in spermatozoa prelabelled with [3H]palmitic acid or [14C]arachidonic acid, these reagents did not enhance [3H]diacylglycerol formation or [14C]arachidonic acid release. Spermatozoa were treated with the phospholipase A2 inhibitor aristolochic acid, and dibutyryl-cAMP to test whether cAMP acts downstream of phospholipase A2. Under these conditions, exocytosis did not occur in response to A23187 and Ca2+. However, inclusion of dibutyryl-cAMP and the phospholipase A2 metabolite lysophosphatidylcholine did result in exocytosis (at an extent similar to that seen when cells were treated with A23187/Ca2+ and without the inhibitor). Inclusion of lysophosphatidylcholine alone, without dibutyryl-cAMP, enhanced exocytosis to a lesser extent, demonstrating that cAMP requires a phospholipase A2 metabolite to stimulate the final stages of exocytosis. These results indicate that cAMP may act downstream of phospholipase A2, exerting a regulatory role in the exocytosis triggered by physiological agonists.

Abstract 1918 P122 Protein, a Positive Regulator for Phospholipase C-σ1, Is Upregulated and Involved in Enhanced Calcium Signaling in Human Coronary Spasm

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Reiichi Murakami ◽  
Tomohiro Osanai ◽  
Hirofumi Tomita ◽  
Ken Okumura
Keyword(s):  
Protein Expression ◽  
Phospholipase C ◽  
Protein A ◽  
Coronary Spasm ◽  
Amino Acid Replacement ◽  
Hek293 Cells ◽  
Gene Expressions ◽  
Human Embryonic Kidney Cells ◽  
Control Subjects ◽  
Intracellular Ca

We previously showed that the activity of phospholipase C (PLC)-σ1, a key enzyme for Ca 2+ signaling in the coronary artery smooth muscle, was enhanced threefold in patients with coronary spastic angina (CSA) compared with control subjects. Structural mutation of PLC-σ1 (864G-A) variant with the amino acid replacement of arginine 257 by histidine is one mechanism for the enhanced PLC-σ1 activity, but this was observed in only 10% of CSA patients. PLC-σ1 was shown to be positively regulated by p122 protein. We examined the possible role of p122 protein in the mechanism for enhanced PLC-σ1 activity. In 11 patients with CSA and 9 control subjects without CSA, skin fibroblasts were obtained at the coronary angiography and were cultured. Protein and gene expressions of p122 were determined by Western blot analysis and real-time quantitative RT-PCR, respectively. The protein expression of p122 was enhanced in CSA threefold compared with control subjects (237±17 vs 85±13 units, p<0.0001). The gene expression of p122 was also enhanced in CSA by 36.1±8.7% compared with control (p<0.01). We further examined whether the upregulated p122 protein is associated with the enhancement of intracellular Ca 2+ signaling. Human embryonic kidney cells (HEK293) were cultured and transfected by muscarine M1 receptor. In the cells expressing normal PLC-σ1, acethylcholine (ACh) at 10 −6 and 10 −5 mol/L caused a dose-dependent, rapid transient increase in [Ca 2+ ] i followed by a lower but sustained phase of the increase. We further transfected the HEK293 cells by p122, which resulted in the increased expression of p122 protein two-to threefold. [Ca 2+ ] i at baseline was 23±1 nmol/L in the cells without p122 transfection and 39±2 nmol/L in those with p122 (P<0.01). The peak increase in [Ca 2+ ] i from the baseline after ACh was significantly greater in the cells transfected with p122 than in those without transfection (68±6 versus 33±4 nmol/L at 10 −6 mol/L Ach, and 128±11 versus 67±8 nmol/L at 10 −5 mol/L ACh, both P<0.01). The sustained phase of [Ca 2+ ] i increase was prolonged in the cells with p122 transfection compared with those without transfection. In conclusion, the enhanced p122 protein expression is involved in the pathogenesis of CSA by enhancing intracellular Ca 2+ signaling.

Effect of glucocorticoid on prostaglandin F2α-induced prostaglandin E2 synthesis in osteoblast-like cells: inhibition of phosphoinositide hydrolysis by phospholipase C as well as phospholipase A2

1994 ◽  
Vol 131 (5) ◽  
pp. 510-515 ◽  
Author(s):  
Osamu Kozawa ◽  
Haruhiko Tokuda ◽  
Atsushi Suzuki ◽  
Jun Kotoyori ◽  
Yoshiaki Ito ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Phospholipase A ◽  
Phosphoinositide Hydrolysis ◽  
Prostaglandin E ◽  
Dependent Manner ◽  
Kinase C ◽  
Dose Dependent

Kozawa O, Tokuda H, Suzuki A, Kotoyori J, Ito Y, Oiso Y. Effect of glucocorticoid on prostaglandin F2α-induced prostaglandin E2 synthesis in osteoblast-like cells: inhibition of phosphoinositide hydrolysis by phospholipase C as well as phospholipase A2. Eur J Endocrinol 1994;131:510–15. ISSN 0804–4643 It is well known that osteoporosis is a common complication of patients with glucocorticoid excess. We showed previously that prostaglandin (PG) F2α stimulates the synthesis of PGE2, a potent bone resorbing agent, and that the activation of protein kinase C amplifies the PGF2α-induced PGE2 synthesis through the potentiation of phospholipase A2 activity in osteoblast-like MC3T3-E1 cells. In the present study, we examined the effect of dexamethasone on PGE2 synthesis induced by PGF2α in MC3T3-E1 cells. The pretreatment with dexamethasone significantly inhibited the PGE2 synthesis in a dose-dependent manner in the range between 0.1 and 10 nmol/l in these cells. This effect of dexamethasone was dependent on the time of pretreatment up to 8 h. Dexamethasone also inhibited PGE2 synthesis induced by melittin, known as a phospholipase A2 activator. Furthermore, dexamethasone significantly inhibited the enhancement of PGF2α- or melittin-induced PGE2 synthesis by 12-O-tetradecanoylphorbol-13-acetate, known as a protein kinase C activator. In addition, dexamethasone significantly inhibited PGF2α-induced formation of inositol phosphates in a dose-dependent manner between 0.1 and 10 nmol/l in MC3T3-E1 cells. These results strongly suggest that glucocorticoid inhibits PGF2α-induced PGE2 synthesis through the inhibition of phosphoinositide hydrolysis by phospholipase C as well as phospholipase A2 in osteoblast-like cells. Osamu Kozawa, Department of Biochemistry, Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi 480-03, Japan

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