OBJECTIVETo explore the relationship between α-actinin content and cardiac function in rats during myocardial ischemia-reperfusion.

METHODSThirty-two rats were randomized equally into sham-operated group, 30 min ischemia group, 1 h ischemia group, and 1 h ischemia with 2 h reperfusion group. Acute myocardial ischemia was induced in the 3 ischemia groups by ligation of the left anterior descending coronary artery, and the cardiac functions were evaluated. The myocardial contents of α-actinin was measured by immunohistochemistry, and phospholipase C (PLC) and phosphatidylinositol-3-kinase (PI3K) contents were determined by ELISA after the operations.

RESULTSThe left ventricular systolic pressure (LVSP), +dp/dt max, and -dp/dt max tended to decrease during myocardial ischemia, and increased after reperfusion, and the left ventricular end-diastolic pressure (LVEDP) showed reverse changes. The levels of α-actinin decreased with prolonged ischemia, showing a significant difference in 1 h ischemia group from those in the other 3 groups. PI3K and PLC contents were significantly increased with prolonged myocardial ischemia. Stimulation by LY-294002 and U-73122 caused enhanced contraction of single cardiomyocytes, and also increased the fluorescence intensity of α-actinin in the cardiomyocytes compared with that in 1 h ischemia group.

CONCLUSIONSThe cardiac dysfunction during acute ischemia-reperfusion in rats may be related with the changes of myocardial α-actinin content, which are probably a result of increased PI3K and PLC contents in the ischemic myocardium.

Actinin ; metabolism ; Animals ; Myocardial Ischemia ; metabolism ; physiopathology ; Myocardial Reperfusion Injury ; metabolism ; physiopathology ; Myocardium ; metabolism ; Phosphatidylinositol 3-Kinase ; metabolism ; Rats ; Rats, Wistar ; Type C Phospholipases ; metabolism

OBJECTIVETo investigate the role of phospholipase C(PLC) in cytoskeleton rearrangement of mouse dendritic cells invaded by Mycobacterium tuberculosis.

METHODSMouse dendritic DC2.4 cells were co-cultured with Mycobacterium tuberculosis H37Rv. F-actin of DC2.4 cells were strained with phalloidin-TRITC, the microtubule was stained with anti-β-tubulin monoclonal antibody and FITC-conjugated AffiniPure anti-mouse IgG. The changes of cytoskeleton in DC2.4 cells induced by Mycobacterium tuberculosis H37Rv were determined by fluorescence microscopy and the rates of F-actin rearrangements were calculated. The expressions of PLC in cytoplasm and cytomembrane of DC2.4 cells were measured by ELISA. DC2.4 cells were pretreated with PLC inhibitor U73122, then F-actin rearrangements induced by invasion of Mycobacterium tuberculosis were observed.

RESULTSBacterial invasion was observed while DC2.4 cells were co-incubated with Mycobacterium tuberculosis H37Rv for 2 h. The rates of invasion were (26.1 ± 4.5)%, (39.9 ± 5.6)%, (51.2 ±5.9)%, (57.9 ± 6.1)% and (63.9 ± 6.8)% at 4, 6, 8, 10 and 12 h of co-culture, respectively; while those were (13.6 ± 3.1)%, (14.2 ± 3.9)%, (15.1 ± 4.3)%, (16.8 ± 4.0)% and (18.3 ± 5.2)% after blocked by PLC, respectively. The rates of the F-actin rearrangements at 2, 4, 6, 8, 10 and 12 h after DC2.4 cells were invaded by H37Rv were (26.9 ± 1.5)%, (59.3 ± 2.8)%, (72.7 ± 4.8)%, (78.2 ± 5.9)%, (63.3 ± 2.9)% and (43.2 ± 2.6)%, respectively; while those were (18.5 ± 1.2)%, (22.3 ± 1.7)%, (3.6 ± 2.5)%, (24.8 ± 2.3)%, (22.3 ± 1.3)% and (23.8 ± 1.8)% after blocked by PLC, respectively. There were no changes of the microtubule observed in DC2.4 cells at the same time points. The rates of the F-actin rearrangements before blocked by PLC were higher than those after PLC blockade at 4, 6, 8 and 10 h (P <0.05). The expressions of PLC in cytomembrane in DC2.4 cells increased after 2 h and reached its highest level at 8 h. The PLC inhibitor U73122 inhibited the expressions of PCL in cytomembrane of DC2.4 cells, but not in cytoplasm.

CONCLUSIONMycobacterium tuberculosis can provoke to F-actin rearrangements through PLC molecule, which would further lead to Mycobacterium tuberculosis invasion of DC2.4 cells.

Actins ; metabolism ; Animals ; Cell Line ; Coculture Techniques ; Cytoskeleton ; metabolism ; Dendritic Cells ; cytology ; microbiology ; Mice ; Microtubules ; metabolism ; Mycobacterium tuberculosis ; pathogenicity ; Type C Phospholipases ; metabolism

OBJECTIVETo estimate the effect of aluminum on hippocampal intracellular Ca²+ concentration and expression of phospholipase C (PLC) and NMDA receptor α (NMDARα) genes in hippocampus as well as the neural behaviors in weaning rats through subchronic exposure in order to explore the mechanism which aluminum impaired the ability of learning and memory of central nervous system development.

METHODSWeaning Wistar rats were randomly divided into four groups based on their body weight. Aluminium chloride was administered by water at the doses of 0.2%, 0.4% and 0.6% (m/v) for 90 days. Platform experiment was used to detect the activity of learning and memory. Fura-2/AM calcium ions fluorescence indicator was used to measure Ca²+ concentration in hippocampal neurons. Western blot method was used to detect the expressions of PLC and NMDARα genes.

RESULTSThe incubation of rats in platform experiment [(232.20 ± 57.45), (35.00 ± 9.37), (16.10 ± 5.57) s] shortened while increase of mistake times (1.10 ± 0.74, 2.20 ± 0.92, 3.40 ± 1.51) was significantly associated with the dose of aluminum (P < 0.01). The Ca(2+) concentration decreased significantly in the rats of aluminum exposed groups (P < 0.01). The expression of PLC and NMDARα in aluminum exposed groups (0.30 ± 0.06, 0.18 ± 0.04, 0.16 ± 0.03; 0.38 ± 0.03, 0.32 ± 0.02, 0.25 ± 0.02) decreased significantly compared with that in the control group (0.47 ± 0.07, 0.48 ± 0.04) (P < 0.01) and there was a dose-effect relationship in the NMDARα expression.

CONCLUSIONSubchronic exposure of aluminium could impair the ability of learning and memory in rats during development, inhibit the expression of NMDARα and PLC and reduce Ca²+ concentration, suggesting that the disorder of Ca²+ signaling system might be one of mechanisms of aluminium damaging the ability of learning and memory.

Aluminum ; toxicity ; Animals ; Calcium ; metabolism ; Hippocampus ; metabolism ; Learning ; drug effects ; Male ; Memory ; drug effects ; Rats ; Rats, Wistar ; Receptors, N-Methyl-D-Aspartate ; metabolism ; Type C Phospholipases ; metabolism

OBJECTIVETo determine the effects of leptospiral strains with different virulence on intracellular free Ca(2+)level and its relation with phospholipase C (PLC) activity of L.interrogans.

METHODSL.interrogans-j infection cell modals were established with Vero and J774A.1 cell lines. Vero and J774A.1 cells were co-incubated with L.interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 (strong virulence) and serogroup Pomona serovar pomona strain 56608 (weak virulence) and L.biflexa serogroup Samaranga serovar patoc strain Patoc I (non-virulence). Intracellular free Ca(2+)levels were detected by laser scanning confocal microscopy with specific fluorescence labeling of fluoj3/AM. Using [(3)H] PIP2 as the substrate, the PLC activities in the culture supernatant, and cytoplasma and cytomembrane of the three strains of Leptospira were measured by isotope assay.

RESULTSThe baseline intracellular free Ca(2+)levels in the normal Vero and J774A.1 cells were (102.3+/-8.2)% and (105.9+/-7.3)%,respectively. The fluorescence intensity in the two cell lines incubated with L.biflexa strain Patoc I were fluctuated in range of (102.3+/-8.2)%approximate, equals(102.2+/-8.3)% during the observation period. The intracellular free Ca(2+)levels in the two cell lines infected with L.interrogans strain 56601 showed elevation with double peak patterns, with first peaks of (430.5+/-35.7)%, (747.5+/-18.5)% and the second peak of (380.6+/-17.4)%, (804.6+/-22.4)%, respectively. When the cells were infected with L.interrogans strain 56608, the intracellular free Ca(2+)levels were rising slowly with a single slope-like pattern, with the maximal of (235.0+/-19.3)% in Vero cells and (402.4+/-17.4)% in J774A.1 cells, which were significantly lower than those in the cells infected with L.interrogans strain 56601 (P<0.01). The culture supernatants, and cytoplasma and cytomembrane proteins of all three strains displayed PLC activities (P<0.05).

CONCLUSIONThe cells infected with L.interrogans of different virulence show distinct intracellular free Ca(2+)levels and peak patterns. The different host cell lines can affect the intracellular free Ca(2+)levels, which is not related to the PLC activity in the leptospiral strains.

Animals ; Calcium ; metabolism ; Cells, Cultured ; Cercopithecus aethiops ; Endocytosis ; Humans ; Leptospira interrogans ; enzymology ; pathogenicity ; Macrophages ; metabolism ; microbiology ; Type C Phospholipases ; metabolism ; Vero Cells ; Virulence

The aim of the present study was to investigate the effects of inositol 1,4,5-trisphosphate (IP(3))-generating agonist UTP on spontaneous transient outward currents (STOCs), and explore the role of intracellular Ca(2+) release in the current response mediated by IP(3) in porcine coronary artery smooth muscle cells (CASMCs). The coronary artery was excised from the fresh porcine heart and cut into small segments (2 mm × 5 mm) and then transferred to enzymatic dissociation solution for incubation. Single CASMCs were obtained by two-step enzyme digestion at 37 °C. STOCs were recorded and characterized using the perforated whole-cell patch-clamp configuration in freshly isolated porcine CASMCs. The currents were amplified and filtered by patch-clamp amplifier (Axopatch 200B), and then the digitized data were recorded by pClamp 9.0 software and further analyzed by MiniAnalysis 6.0 program. The results were as follows: (1) UTP led to conspicuous increases in STOC amplitude by (57.54±5.34)% and in frequency by (77.46±8.42)% (P<0.01, n=38). (2) The specific blocker of phospholipase C (PLC) - U73122 (5 μmol/L) remarkably reduced STOC amplitude by (31.04±7.46)% and frequency by (41.65±16.59)%, respectively (P<0.05, n=10). In the presence of U73122, UTP failed to reactivate STOCs (n=7). (3) Verapamil (20 μmol/L) and CdCl2 (200 μmol/L), two blockers of L-type voltage-dependent Ca(2+) channels, had little effects on STOCs initiated by UTP (n=8). (4) 1 μmol/L bisindolylmaleimide I (BisI), a potent blocker of protein kinase C (PKC), significantly increased STOC amplitude by (65.44±24.66)% and frequency by (61.35±21.47)% (P<0.01, n=12); UTP (40 μmol/L), applied in the presence of 1 μmol/L BisI, could further increase STOC activity (P<0.05, P<0.01, n=12). Subsequent application of ryanodine (50 μmol/L) abolished STOC activity. (5) In the presence of UTP (40 μmol/L), inhibition of IP(3) receptors (IP(3)Rs) by 2-aminoethoxydiphenyl borate (2-APB, 40 μmol/L) reduced STOC amplitude by (24.08±3.97)% (P<0.05, n=8), but had little effect on STOC frequency (n=8). While application of 2-APB (80 μmol/L) significantly reduced STOC amplitude by (31.43±6.34)% and frequency by (40.59±19.01)%, respectively (P<0.05, P<0.01, n=6). Subsequent application of ryanodine (50 μmol/L) completely blocked STOC activity. Pretreatment of cells with 2-APB (40 μmol/L) or ryanodine (50 μmol/L), UTP (40 μmol/L) failed to reactivate STOCs. The results suggest that UTP activates STOCs mainly via PLC and IP(3)-dependent mechanisms. Complex Ca(2+)-mobilization pathways are involved in UTP-mediated STOC activation in porcine CASMCs.
Animals ; Boron Compounds ; pharmacology ; Calcium ; metabolism ; Coronary Vessels ; cytology ; Inositol 1,4,5-Trisphosphate ; metabolism ; Myocytes, Smooth Muscle ; metabolism ; Protein Kinase C ; metabolism ; Ryanodine ; pharmacology ; Signal Transduction ; Swine ; Type C Phospholipases ; metabolism ; Uridine Triphosphate ; metabolism

BACKGROUNDAngiotensin II (Ang II) acting at angiotensin AT(1) receptor (AT(1)R) has well documented effects on cardiovascular structure such as the promotion of cardiovascular hypertrophy and fibrosis, which are believed to be opposed by angiotensin AT(2) receptor (AT(2)R) stimulation. The expressions of AT(1)R and AT(2)R are up-regulated in senescent hearts. The purpose of this study was to investigate the interaction of signal transduction between AT(1)R and AT(2)R, and to detect whether there is any difference in the interaction in rat hearts of different age.

METHODSIn 3.5-, 12-, 18- and 24-month-old rats, the heart cell membrane activities of protein kinase C (PKC) and tyrosine kinase were measured when AT(1)R and AT(2)R were both activated by Ang II or just the AT(1)R was activated by Ang II and PD123319. The activities of cytosolic phospholipase A(2) (cPLA(2)) and the levels of cGMP were investigated when AT(1)R and AT(2)R were both activated by Ang II or just the AT(2)R was activated by Ang II and losartan.

RESULTSWhen AT(1)R and AT(2)R were both activated compared to when the AT(1)R was activated, the activities of PKC were not different in hearts from 3.5- and 12-month-old rats, but decreased significantly in 18- and 24-month-old rats; the activities of tyrosine kinase were not different in 3.5-month-old rats but decreased significantly in 12-, 18- and 24-month-old rats. The activities of cPLA(2) were all decreased significantly in rats of different age when AT(1)R and AT(2)R were both activated compared to when the AT(2)R was activated. Treatment with Ang II alone compared to Ang II and losartan decreased the levels of cGMP (fmol/mg) in rats of different age (102.7 +/- 12.7 versus 86.0 +/- 8.0 in 3.5-month-old rats, P < 0.05; 81.0 +/- 9.4 versus 70.0 +/- 6.3 in 12-month-old rats, P < 0.05; 69.8 +/- 5.6 versus 54.2 +/- 5.3 in 18-month-old rats, P < 0.01; 57.7 +/- 8.0 versus 39.0 +/- 3.0 in 24-month-old rats, P < 0.01).

CONCLUSIONSThe activation of AT(1)R inhibited the signal transduction of AT(2)R during the aging variation, and the activation of AT(2)R inhibited the signal transduction of AT(1)R in rat heart of different age.

Aging ; metabolism ; Animals ; Cyclic GMP ; analysis ; Male ; Myocardium ; metabolism ; Phospholipases A2 ; metabolism ; Protein Kinase C ; metabolism ; Protein-Tyrosine Kinases ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor, Angiotensin, Type 1 ; physiology ; Receptor, Angiotensin, Type 2 ; physiology ; Signal Transduction ; physiology

Contraction of smooth muscle is initiated by an increase in cytosolic Ca2+ leading to activation of Ca2+/calmodulin-dependnet myosin light chain (MLC) kinase and phosphorylation of MLC. The types of contraction and signaling mechanisms mediating contraction differ depending on the region. The involvement of these different mechanisms varies depending on the source of Ca2+ and the kinetic of Ca2+ mobilization. Ca2+ mobilizing agonists stimulate different phospholipases (PLC-beta, PLD and PLA2) to generate one or more Ca2+ mobilizing messengers (IP3 and AA), and diacylglycerol (DAG), an activator of protein kinase C (PKC). The relative contributions of PLC-beta, PLA2 and PLD to generate second messengers vary greatly between cells and types of contraction. In smooth muscle cell derived form the circular muscle layer of the intestine, preferential hydrolysis of PIP2 and generation of IP3 and IP3-dependent Ca2+ release initiate the contraction. In smooth muscle cells derived from longitudinal muscle layer of the intestine, preferential hydrolysis of PC by PLA2, generation of AA and AA-mediated Ca2+ influx, cADP ribose formation and Ca2+/-induced Ca2+ release initiate the contraction. Sustained contraction, however, in both cell types is mediated by Ca2+/-independent mechanism involving activation of PKC- epsilon by DAG derived form PLD. A functional linkage between G13, RhoA, ROCK, PKC- epsilon, CPI-17 and MLC phosphorylation in sustained contraction has been implicated. Contraction of normal esophageal circular muscle (ESO) in response to acetylcholine (ACh) is linked to M2 muscarinic receptors activating at least three intracellular phospholipases, i.e. phosphatidylcholine-specific phospholipase C (PC-PLC), phospholipase D (PLD) and the high molecular weight (85 kDa) cytosolic phospholipase A2 (cPLA2) to induce phosphatidylcholine (PC) metabolism, production of diacylglycerol (DAG) and arachidonic acid (AA), resulting in activation of a protein kinase C (PKC)-dependent pathway. In contrast, lower esophageal sphincter (LES) contraction induced by maximally effective doses of ACh is mediated by muscarinic M3 receptors, linked to pertussis toxin-insensitive GTP-binding proteins of the Gq/11 type. They activate phospholipase C, which hydrolyzes phosphatidylinositol bisphosphate (PIP2), producing inositol 1, 4, 5-trisphosphate (IP3) and DAG. IP3 causes release of intracellular Ca2+ and formation of a Ca2+/-calmodulin complex, resulting in activation of myosin light chain kinase and contraction through a calmodulin-dependent pathway.
Acetylcholine ; Arachidonic Acid ; Cyclic ADP-Ribose ; Cytosol ; Esophageal Sphincter, Lower ; GTP-Binding Proteins ; Hydrolysis ; Inositol ; Intestines ; Metabolism ; Molecular Weight ; Muscle, Smooth* ; Myocytes, Smooth Muscle ; Myosin Light Chains ; Myosin-Light-Chain Kinase ; Negotiating ; Phosphatidylcholines ; Phosphatidylinositols ; Phospholipase D ; Phospholipases ; Phospholipases A2 ; Phosphorylation ; Phosphotransferases ; Protein Kinase C ; Receptor, Muscarinic M3 ; Receptors, Muscarinic ; Second Messenger Systems ; Type C Phospholipases ; Whooping Cough

We report two cases of gingival plasma cell granuloma in a 34-yr-old and 40-yr-old two male renal transplant recipients with cyclosporine A (CsA)-induced gingival overgrowth (GO). Histologically, these lesions were composed of mature plasma cells, showing polyclonality for both lambda and kappa light chains and fibrovascular connective tissue stroma. In addition to the fact that CsA-induced plasma cell granuloma is rare, the salient features of our cases were the secretion of interleukin-6 and overexpression of phospholipase C-gamma1 of the tumor cells, which may explain the mechanisms of CsA- induced GO.
Cyclosporine/adverse effects ; Female ; Gingival Diseases/*chemically induced/enzymology/immunology/pathology ; Granuloma, Plasma Cell/*chemically induced/enzymology/immunology/pathology ; Humans ; Immunohistochemistry ; Immunosuppressive Agents/adverse effects ; Interleukin-6/metabolism ; Kidney Transplantation ; Male ; Middle Aged ; Phospholipase C gamma ; Type C Phospholipases/metabolism

OBJECTIVETo elucidate GPR40/FFA1 and its downstream signaling pathways in regulating insulin secretion.

METHODSGPR40/FFA1 expression was detected by immunofluorescence imaging. We employed linoleic acid (LA), a free fatty acid that has a high affinity to the rat GPR40, and examined its effect on cytosolic free calcium concentration ([Ca2+]i) in primary rat beta-cells by Fluo-3 intensity under confocal microscopy recording. Downregulation of GPR40/FFA1 expression by antisense oligonucleotides was performed in pancreatic beta-cells, and insulin secretion was assessed by enzyme-linked immunosorbent assay.

RESULTSLA acutely stimulated insulin secretion from primary cultured rat pancreatic islets. LA induced significant increase of [Ca2+]i in the presence of 5.6 mmol/L and 11.1 mmol/L glucose, which was reflected by increased Fluo-3 intensity under confocal microscopy recording. LA-stimulated increase in [Ca2+]i and insulin secretion were blocked by inhibition of GPR40/FFA1 expression in beta-cells after GPR40/FFA1-specific antisense treatment. In addition, the inhibition of phospholipase C (PLC) activity by U73122, PLC inhibitor, also markedly inhibited the LA-induced [Ca2+]i increase.

CONCLUSIONLA activates GPR40/FFA1 and PLC to stimulate Ca2+ release, resulting in an increase in [Ca2+]i and insulin secretion in rat islet beta-cells.

Animals ; Calcium ; metabolism ; Enzyme Activation ; Insulin ; secretion ; Insulin-Secreting Cells ; drug effects ; metabolism ; secretion ; Linoleic Acid ; pharmacology ; Male ; Rats ; Rats, Sprague-Dawley ; Receptors, G-Protein-Coupled ; physiology ; Type C Phospholipases ; physiology

The one event on signaling mechanism is the cleavage by adenyl cyclase of ATP into second messenger, cyclic AMP. The other transfer system of inositol metabolism, it is widely recognized that hydrolysis of the minor membrane lipid phosphoinositide bisphosphate (PIP₂) initiated by occupation of certain receptors and catalyzed by phospholipase C, lead to toe generation of the two intracellular messengers, inositol triphosphate (IP₃) and diacylglycerol (DG). IP₃ is converted to inositol tetrakisphosphate (IP₄) by IP₃ kinase. In the present study, it is that purification of calmodulin is used by phenyl-Sepharose CL-4B chromatography, it's molecular weigh, 17,000 in SDS-polyacrylamide gel electrophoresis. In order to observe the affinity between calmodulin (CaM)-Affigel 15 and IP₃ kinase, and isolated IP₃ kinase, was applied in CaM-Affigel with Ca²⁺ equilibrium buffer and EGTA equilibrium buffer. We compared with binding and elution effect of IP₃ kinase in several condition of buffer. In affinity of binding, Ca²⁺ equilibrium buffer was in the most proper condition, and elution, CaM/Ca²⁺buffer (CE 1 10.36, CE2 12.76pM/min/mg of protein) was effected much more than EGTA buffer (E2 1.48, E 2.43pM/min/mg of protein), but CaM/Ca²⁺stimulate the activity of IP₃ kinase. And then, several detergents such as sodium deoxycholate, tween 20, cholic acid, polyethylene glycol, chaps were applied. The 0.2% chaps buffer (E2 23.19, E3 8.05pnM/min/mg of protein) was the most effective in elution of IP3 kinase.
Adenosine Triphosphate ; Adenylyl Cyclases ; Brain* ; Calmodulin ; Cholic Acid ; Chromatography ; Cyclic AMP ; Deoxycholic Acid ; Detergents ; Egtazic Acid ; Electrophoresis ; Hydrolysis ; Inositol* ; Membranes ; Metabolism ; Occupations ; Phosphotransferases* ; Polyethylene Glycols ; Polysorbates ; Second Messenger Systems ; Toes ; Type C Phospholipases