Activation of cPLA2 in vascular smooth muscle.
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Animals↗
Biomedical subjects
E F LaBelle
Publications and source records attributed to E F LaBelle.
Norepinephrine stimulates arachidonic acid release from vascular smooth muscle via activation of cPLA2.
The mechanism of agonist-activated arachidonate release was studied in segments of rat tail artery. Tail artery segments were prelabeled with [3H]arachidonate and then stimulated with norepinephrine (NE), and the radioactivity of the extracellular medium was determined. NE stimulated arachidonate release from the tissue without increasing arachidonic acid levels within cellular cytosol or crude membranes. About 90% of the extracellular radioactivity was shown to be unmetabolized arachidonate by TLC. Arachidonic acid release was not inhibited by the removal of the endothelium from the artery. NE exerted a half-maximal effect at a concentration of 0.2 microM. NE-stimulated arachidonate release was not inhibited by blockers of phospholipase C (U-73122), diacylglycerol lipase (RHC-80267), secretory phospholipase A2 (manoalide), calcium-insensitive phospholipase A2 (HELSS), or beta-adrenergic receptors (propranolol). NE-stimulated arachidonic acid release was inhibited by blockers of cytosolic phospholipase A2 (cPLA2) (AACOCF3), alpha 1-adrenergic receptors (prazosin), and specific G proteins (pertussis toxin). This indicated that NE stimulated arachidonate release from vascular smooth muscle via activation of alpha-adrenergic receptors, either Gi or Go, and cPLA2. NE-activated arachidonic acid release from vascular smooth muscle may play a role in force generation by the tissue. Perhaps arachidonic acid extends the effect of NE on one specific smooth muscle cell to its nearby neighbor cells.
Adrenergic alpha-Agonists↗
Phospholipase C beta 2 in vascular smooth muscle.
Receptor-mediated inositol 1,4,5-trisphosphate formation in most tissue is dependent on a variety of phospholipase C isoforms. To determine which phospholipase C isoforms were present in vascular smooth muscle compared to brain, liver, and spleen, we extracted proteins from these tissues and separated and identified the phospholipase C isoforms by immunoblotting. Aliquots of rat tail artery were examined by this procedure, together with aliquots of rat liver, spleen, cerebral cortex, hippocampus, cerebellum, aorta, and mesenteric artery. Phospholipase C gamma 1 was shown to be present in all of these tissues, while phospholipase C beta 1 was shown to be limited to fractions from brain. Phospholipase C delta 1 was detected in rat tail artery, mesenteric artery, aorta, and brain. Phospholipase C beta 2 was found in rat tail artery, liver, and brain. This is the first report of phospholipase C beta 2 in tissues other than HL60 cells. Since G proteins activate IP3 production via stimulation of phospholipase C beta isoforms in many tissues, and agonist-stimulated IP3 production in smooth muscle requires G protein activation, phospholipase C beta 2 may be required for agonist-stimulated force production in vascular smooth muscle.
Animals↗
Phospholipase D is activated by G protein and not by calcium ions in vascular smooth muscle.
We assessed the sensitivity of phospholipase D (PLD) activity in vascular smooth muscle to cytosolic Ca2+ by increasing cytosolic Ca2+ levels independently of agonist stimulation. When rat tail artery was preloaded with the Ca2+ indicator fluo 3 pentaacetoxymethyl ester, the addition of high extracellular K+, caffeine, or norepinephrine rapidly enhanced cytosolic Ca2+ levels. Neither increased extracellular K+ nor caffeine addition increased phosphatidylethanol production, indicating that cytosolic Ca2+ elevation alone did not stimulate PLD. In contrast, norepinephrine stimulated phosphatidylethanol production in this tissue. In strips of tail artery permeabilized with alpha-toxin and incubated in solutions containing free Ca2+ concentrations observed during physiological stimulation (pCa 6.4), PLD was not stimulated, whereas incubation with guanosine 5'-O-(3-thiotriphosphate) at pCa 7.0 activated this enzyme. Aluminum fluoride (AlF4-) stimulated PLD, and this activity was insensitive to pertussis toxin after stimulation by either norepinephrine or AlF4-. These results indicate that PLD in vascular smooth muscle is activated by norepinephrine via stimulation of a pertussis toxin-insensitive G protein and not via an increase in intracellular Ca2+ levels.
Animals↗
Inositol 1,4,5-trisphosphate binding to porcine tracheal smooth muscle aldolase.
A cytoskeletal fraction of porcine tracheal smooth muscle (PTSM) was found to contain > 90% of total cellular aldolase (fructose 1,6-bisphosphate aldolase, EC 4.1.2.13) activity. PTSM aldolase was purified by DEAE and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) affinity chromatography and found to react with an antibody directed against human aldolase C, but not anti-aldolase A and B. The molecular mass of native aldolase was about 138 kDa (on Sephacryl S-300); SDS-denatured enzyme was 35 kDa (comigrated with rabbit skeletal muscle aldolase). Total cellular aldolase tetramer (aldolase4) content was 34.5 pmol/100 nmol lipid P(i). Ins(1,4,5)P3) binding activity coeluted with aldolase during Sephacryl 300, DEAE, and Ins(1,4,5)P3 affinity chromatography. Ins(1,4,5)P3 bound to purified aldolase (at 0 degree C) in a dose-dependent manner over the range [Ins(1,4,5)P3] 20 nM to 20 microM, with maximal binding of 1 mol of Ins(1,4,5)P3/mol aldolase4 and a Kd of 12-14 microM. Fru(1,6)P2 and Fru(2,6)P2 displaced bound Ins(1,4,5)P3) with a 50% inhibition at 30 and 170 microM, respectively. Ins(1,3,4)P3 (20 microM) and glyceraldehyde 3-phosphate (2 mM) were also potent inhibitors of Ins(1,4,5)P3 binding, but not inositol 4-phosphate or inositol 1,4-bisphosphate (20 microM each). Aldolase-bound Ins(1,4,5)P3 may play a role in phospholipase C-independent increases in free [Ins(1,4,5)P3].
Animals↗
Norepinephrine stimulates the direct breakdown of phosphatidyl inositol in rat tail artery.
When segments of rat tail artery were labeled with [3H]inositol and then stimulated with norepinephrine (NE), the inositol phosphates produced were primarily IP and IP2, together with a small but significant amount of Ins(1,4,5)P3 and a very small amount of Ins(1,3,4,5)P4. It has been unclear in many studies whether or not the relatively large levels of IP and IP2 produced in [3H]inositol-labeled tissue represent indirect products of phosphatidyl inositol(4,5)bis phosphate breakdown (through Ins(1,4,5)P3) or direct products of phosphatidyl inositol 4 monophosphate and phosphatidyl inositol breakdown. In order to answer this question tail artery segments were prelabeled with [3H]inositol and then permeabilized with beta escin and stimulated with norepinephrine and GTP gamma S, so that increases in IP, IP2, and Ins(1,4,5)P3 were still observed. If these permeable segments were stimulated with agonist in the presence of compounds known to inhibit Ins(1,4,5)P3 5-phosphatase, such as glucose 6P, (2,3)diphosphoglycerate, or Ins(1,4,5)P3, the levels of labeled Ins(1,4,5)P3 and labeled IP2 were increased, while the level of stimulated labeled IP was unchanged. This indicated that some of the IP2 and IP formed in these cells was produced from PIP2 but that some of these compounds might be formed from PIP or PI. When the isomers of inositol monophosphate, Ins 1P and Ins 4P, were separated by HPLC, it was shown that after prelabeled tail artery was stimulated by norepinephrine for periods of 1-2 min, the predominant isomer formed was Ins 4P, indicating either PIP2 or PIP as the source. However, after 5-20 min stimulation, both Ins 1P and Ins 4P were formed in equal amounts, suggesting that during sustained stimulation of smooth muscle PI itself was broken down directly. Therefore it appears that within 1-2 min of norepinephrine addition to vascular smooth muscle the bulk of the IP and IP2 produced are derived from PIP2 via IP3, while after 20 min of norepinephrine treatment much of the IP comes directly from PI. This suggests that the regulation of PLC in this tissue is more complicated than has been previously believed.
Animals↗
Norepinephrine-induced phosphatidylcholine hydrolysis by phospholipases D and C in rat tail artery.
Rat tail arterial segments were incubated with [3H]choline to selectively label endogenous phosphatidylcholine. Norepinephrine (NE; 10(-5) M) addition for periods of 10 s to 30 min significantly increased the concentration of extracellular phosphatidylcholine metabolites, [3H]choline, and [3H]phosphocholine. The release of [3H]choline and [3H]phosphocholine from the segments was NE dose dependent (10(-6)-10(-3) M). NE also increased the formation of [3H]phosphatidylethanol in [3H]myristate-labeled tail artery in the presence of ethanol, characteristic of phospholipase D activity. NE-induced phosphatidylcholine hydrolysis was blocked by pretreatment with prazosin (10(-5) M) and was unchanged by pretreatment with propranolol (10(-5) M). 4 beta-phorbol 12,13-dibutyrate (PDBu, 10(-6) M) stimulated the release of [3H]choline, which was inhibited by pretreatment with staurosporine (10(-5) M). The stimulatory effect of NE on phosphatidylcholine metabolism was not altered by either pretreatment with staurosporine (10(-5) M) or calcium-free buffer. In summary, we have demonstrated NE-stimulated phosphatidylcholine hydrolysis by phospholipase D and C in intact vascular smooth muscle. This effect of NE was dose dependent and was mediated through the alpha 1-adrenergic receptor. Norepinephrine and PDBu stimulated phosphatidylcholine hydrolysis through different mechanism(s), and the stimulatory effect of NE did not seem to require protein kinase C and calcium influx.
Animals↗
Rapid increase in inositol phosphate levels in norepinephrine-stimulated vascular smooth muscle.
We examined the correlation between agonist-stimulated increases in inositol phosphates and force development in vascular smooth muscle. Segments of rat tail artery were preincubated with [3H]inositol and treated with norepinephrine (10(-5) M) for 3-10 s. Tissue levels of inositol monophosphate (IP), inositol bisphosphate (IP2), and inositol trisphosphate (IP3) were measured. IP and IP2 increased significantly after 3 s of norepinephrine treatment. IP3 increased significantly after 5 s of norepinephrine treatment. Analysis of tissue extracts by high-pressure liquid chromatography demonstrated that the only isomer of IP3 present in any tissue extract was the 1,4,5-isomer [Ins(1,4,5)P3]. Contractile response to norepinephrine stimulation showed that the increase in inositol phosphates coincides well with the time course of force development. This is the first report demonstrating such an early increase in Ins(1,4,5)P3 in agonist-stimulated vascular smooth muscle. These results are consistent with the hypothetical role of Ins(1,4,5)P3 as a mediator linking agonist-receptor activation to increased intracellular calcium and force development in norepinephrine-stimulated vascular smooth muscle.
Animals↗
Purification and characterization of dinitrophenylglutathione ATPase of human erythrocytes and its expression in other tissues.
S-(2,4-dinitrophenyl)glutathione (Dnp-SG) ATPase of human erythrocytes has been purified to apparent homogeneity by affinity chromatography. In reduced denaturing gels, the subunit Mr value of Dnp-SG ATPase was found to be 38,000. Dinitrophenyl glutathione (Dnp-SG) stimulated the hydrolysis of ATP by the purified enzyme whereas oxidized glutathione (GSSG) did not, indicating that Dnp-SG and GSSG are transported from the erythrocytes by different transporters. Results of Western blot analysis using the antibodies against Dnp-SG ATPase subunits indicated that the enzyme was expressed in human liver, lung, placenta and pancreas.
Adenosine Triphosphatases↗
G protein control of inositol lipids in intact vascular smooth muscle.
Agonist-induced PIP2 breakdown has been demonstrated in permeabilized vascular smooth muscle and shown to depend on a G protein. Segments of rat tail artery were permeabilized with ATP and EGTA after prelabeling with [3H]inositol. Norepinephrine and GTP gamma S were both able to increase levels of IP, IP2 and IP3 in the segments. The effects of both norepinephrine and GTP gamma S on the segments was non-additive. Aluminum fluoride also increased inositol phosphates in intact segments and norepinephrine-stimulated increases in IP, IP2 and IP3 were insensitive to pertussis toxin.
Aluminum↗
Ligand binding and internalization by the rat hepatic asialoglycoprotein receptor does not generate polyphosphoinositide derived second messengers.
Recent studies suggest that protein kinase C and, thus, possibly the rate of inositol phospholipid hydrolysis may regulate the function and distribution of the asialoglycoprotein (or galactosyl) receptor on isolated rat hepatocytes (Takahashi et al., Biochem. Biophys. Res. Commun., 1985, 126, 1054; Fallon and Schwartz, J. Biol. Chem., 1986, 261, 15081). We have studied the effects of asialoorosomucoid (ASOR) on the hydrolysis of [32P]-inositol phospholipids in isolated rat hepatocytes. When internalization of ASOR is maximal at 310 molecules/cell/sec, there is neither a decrease in the amount of [32P]-phosphatidylinositol-4,5-bisphosphate (PIP2) nor an increase in [32P]-phosphatidic acid (PA) up to 30 min after stimulation. On the other hand, 10(-6)M vasopressin, which was used as a positive control, caused a 35-40% decrease in the level of [32P]-PIP2 and a 70-80% increase in [32P]-PA within 30 sec. Addition of orosomucoid or ASOR, even at concentrations 1000-times the Kd, did not change the levels of any of the six phospholipids tested. Similarly, addition of ASOR did not increase the levels of soluble [3H]-inositol phosphates, whereas vasopressin caused a 6-fold increase in [3H]-inositol-1,4-diphosphate (IP2) and a 4-fold increase in [3H]-inositol-1,4,5-triphosphate (IP3) in isolated rat hepatocytes prelabeled with [3H]-inositol. We conclude that the receptor mediated endocytosis of asialoglycoproteins by rat hepatocytes does not stimulate hydrolysis of the inositol phospholipids.
Animals↗
A novel dinitrophenylglutathione-stimulated ATPase is present in human erythrocyte membranes.
Vesicles prepared from human erythrocyte membranes were found to catalyze ATP hydrolysis that was stimulated by dinitrophenylglutathione (Dnp-SG). This activity was dependent on temperature and Mg2+ and independent of ion pump ATPases present in erythrocyte membranes. The activity was a linear function of protein and time up to 60 min. The Km values of ATPase for Dnp-SG and ATP were found to be 49 microM and 1.67 mM, respectively. This suggests that in erythrocytes, the transport of Dnp-SG requires direct enzymatic hydrolysis of ATP and both Dnp-SG-stimulated ATPase activity and the ATP-dependent efflux of Dnp-SG from erythrocytes represent different activities of the same protein.
Adenosine Triphosphatases↗
Evidence for different transport systems for oxidized glutathione and S-dinitrophenyl glutathione in human erythrocytes.
The effect of oxidized glutathione (GSSG) on the ATP-dependent transport of S-dinitrophenyl glutathione (Dnp-SG) by inside-out vesicles prepared from human erythrocytes and by intact erythrocytes has been studied. It is demonstrated that the transport of Dnp-SG is not inhibited by GSSG in either intact erythrocytes or in inside-out vesicles. These results suggest that Dnp-SG and GSSG are transported out of human erythrocytes by separate systems.
Adenosine Triphosphate↗
Dinitrophenyl glutathione efflux from human erythrocytes is primary active ATP-dependent transport.
Dinitrophenyl S-glutathione is accumulated by inside-out vesicles made from human erythrocytes in a process totally dependent on ATP and Mg2+. The vesicles were shown to accumulate dinitrophenyl S-glutathione against a concentration gradient. The vesicles were able to concentrate this glutathione derivative even in the absence of membrane potential. This indicated that the ATP-dependent uptake of dinitrophenyl S-glutathione by inside-out vesicles represented an active transport process. Neither extravesicular EGTA nor intravesicular ouabain inhibited the transport process, indicating that neither the Ca2+-ATPase nor the Na+, K+-ATPase were involved. These results indicated that dinitrophenyl S-glutathione uptake by inside-out vesicles probably represented primary active transport. The uptake of dinitrophenyl S-glutathione was a linear function of time (up to 5 h) and vesicle protein. The rate of uptake was optimal between pH 7.0 and 8.0 and at 37 degrees C. The Km values determined for dinitrophenyl S-glutathione and ATP were 0.29 mM and 1 mM, respectively. The transport process was completely inhibited by vanadate and by p-hydroxymercuribenzene sulphonate and inhibited to a lesser extent by N-ethylmaleimide. GTP could efficiently substitute for ATP as an energy source for the transport process, but CTP and UTP were comparatively much less effective.
Adenosine Triphosphate↗
Amiloride-inhibited Na+-H+ exchange in human kidney medulla microsomes.
Membrane vesicles from the medulla of the human kidney are capable of amiloride-inhibited sodium-proton exchange. Sodium uptake into the vesicles is stimulated by low intravesicular pH (6.0) and inhibited by both low extravesicular pH (6.0) and amiloride (2 mM). Sodium uptake is a linear function of the vesicular protein concentration and can be reversed by raising the osmotic strength of the solution to shrink the vesicles or by adding the ionophore nigericin. Acridine orange was accumulated by the vesicles in response to a pH gradient (pH 6.0, inside, 8.0 outside), and the accumulation of acridine orange could be reduced by increased extravesicular sodium. Thus sodium could stimulate proton movement just as protons could stimulate sodium movement across the vesicular membrane.
Acridine Orange↗
ATP-dependent proton transport in human renal medulla.
An electrogenic proton-translocating ATPase (H+-ATPase) has been described in turtle urinary bladder and bovine and rat renal medulla. In the present study, a membrane fraction with ATP-dependent H+ transport activity was isolated from human renal medulla. Intravesicular acidification was assessed by acridine orange absorbance changes. Proton transport was abolished by N-ethylmaleimide but not oligomycin or vanadate, differentiating this H+-ATPase from mitochondrial F0-F1 H+-ATPase and gastric H+-K+-ATPase. In addition, vesicular proton uptake was demonstrated to be independent of sodium and potassium cotransport. Proton translocation rate increased when transmembrane potential was clamped with valinomycin supporting an electrogenic mechanism. Hydrogen ion transport was dependent on the presence of chloride or bromide, since substitution by fluoride or nitrate markedly decreased intravesicular acidification. The transport characteristics of this proton-translocating ATPase are similar to those described for turtle urinary bladder and bovine and rat renal medulla, which have been assumed to play a role in urinary acidification by the medullary collecting duct.
Acridine Orange↗
Phosphoinositides in mitogenesis: neomycin inhibits thrombin-stimulated phosphoinositide turnover and initiation of cell proliferation.
Thrombin stimulates 32Pi incorporation into phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bis-phosphate (PIP2), and phosphatidylinositol (PI), and initiates DNA synthesis in hamster (NIL) fibroblasts at a half-maximal concentration of 125 ng/ml. Neomycin, which binds PIP2 and PIP, inhibits both thrombin-stimulated initiation of cell proliferation and 32P pI incorporation into at concentrations above 2 mM without affecting thrombin binding, thymidine uptake, or cellular protein synthesis. At lower concentrations, neomycin inhibits thrombin-stimulated release of inositol 1,4,5-trisphosphate (IP3), by selectively binding PIP2, but does not inhibit 32P incorporation into PI or initiation of DNA synthesis. Phosphoinositide recycling and diacylglycerol release therefore appear necessary for initiation of cell proliferation by thrombin. IP3-stimulated Ca++ mobilization may not be required for thrombin mitogenesis, however, since neomycin can block IP3 release without inhibiting initiation.
Animals↗
Reconstituted amiloride-inhibited sodium transporter from rabbit kidney medulla is responsible for Na+-H+ exchange.
Microsomes formed from rabbit kidney medulla and reconstituted proteoliposomes formed from these microsomes were capable of amiloride-inhibited Na+ transport that was insensitive to valinomycin either with or without K+. This indicated that the Na+ transport process was electroneutral. This Na+ transport process was insensitive to extravesicular Cl- or HCO-3 and not stimulated by high intravesicular gradients of K+, Ca2+ or Mg2+, which indicated that the process did not require NaCl or NaHCO3 co-transport or Na+/K+, Na+/Ca2+ or Na+/Mg2+ counter-transport. Na+ uptake into microsomes or proteoliposomes was inhibited by extravesicular K+, Ca2+, Mg2+ or La3+, which indicated that these ions interacted with the Na+-binding site on the transport protein. Na+ uptake into microsomes was stimulated by intravesicular protons and inhibited by extravesicular protons. This suggested that microsomes were capable of Na+-H+ exchange and this was confirmed when Na+ was shown to stimulate H+ efflux from microsomes. The amiloride-inhibited Na+ transporter from medulla microsomes which has been reconstituted into proteoliposomes is most likely a Na+-H+ exchanger. This Na+ transporter was totally insensitive to the uncoupler 1799, either in the presence or absence of valinomycin plus K+ and less sensitive to NH3 than to amiloride. This indicated that amiloride inhibited Na+ transport not merely by acting as a weak-base uncoupler but by directly interacting with the protein responsible for Na+-H+ exchange.
Amiloride↗