Effect of chloroquine on mast cell membranes

The effect of chloroquine (CQ) on phospholipid turnover andde novo synthesis in isolated rat mast cells (IRMC) was studied by determining the incorporation of³²P and¹⁴C-glycerol into IRMC phospholipids. Incubation of mast cells with chloroquine increased³²P incorporation into PI and PS whilst it decreased³²P incorporation into PC, PE and PA. In mast cells pretreated with CQ and subsequently stimulated with compound 48/80,³²P incorporation into PI, PS and PA fractions was enhanced, while it was decreased into PC and PE, in comparison to 48/80 stimulated IRMC. ¹⁴C-glycerol incorporation into total IRMC phospholipids was not significantly changed by CQ and compound 48/80 treatment and neither was any dose-dependent effect of CQ on individual phospholipids detected. Our results indicate that chloroquine, similarly to other cationic amphiphilic drugs, may alter membrane PL turnover without changingde novo synthesis of phospholipids.     

Effect of chloroquine on mast cell membranes

The effect of chloroquine (CQ) on phospholipid turnover andde novo synthesis in isolated rat mast cells (IRMC) was studied by determining the incorporation of³²P and¹⁴C-glycerol into IRMC phospholipids. Incubation of mast cells with chloroquine increased³²P incorporation into PI and PS whilst it decreased³²P incorporation into PC, PE and PA. In mast cells pretreated with CQ and subsequently stimulated with compound 48/80,³²P incorporation into PI, PS and PA fractions was enhanced, while it was decreased into PC and PE, in comparison to 48/80 stimulated IRMC.¹⁴C-glycerol incorporation into total IRMC phospholipids was not significantly changed by CQ and compound 48/80 treatment and neither was any dose-dependent effect of CQ on individual phospholipids detected. Our results indicate that chloroquine, similarly to other cationic amphiphilic drugs, may alter membrane PL turnover without changingde novo synthesis of phospholipids.     

Effect of beta-adrenoceptor blocking drugs on32P incorporation into and arachidonic acid liberation from phospholipids in stimulated rat mast cells

The lipophilic beta-adrenoceptor blocking (BAB) drugs metipranolol, propranolol and exaprolol significantly decreased 48/80-and A23187-induced³²P incorporation into rat mast cell phospholipids. Exaprolol was the most active, followed by propranolol and metipranolol. Atenolol and metipranolol significantly decreased the 48/80-stimulated, and metipranolol and exaprolol the A23187-stimulated³H-arachidonic acid liberation from isolated mast cells.     

Metipranolol-induced changes in membrane phospholipids of isolated mast cells

Metipranolol significantly decreased³²P incorporation into membrane phosphatidylcholine but increased the radioactivity in phosphatidylinositol of nonstimulated and 48/80-stimulated rat mast cells. In stimulated cells metipranolol significantly decreased³H-arachidonic acid liberation from total membrane phospholipids as well as thromboxane B₂ formation. The effect of metipranolol on stimulated rat mast cells is most probably a result of its direct effect on membrane phospholipid and arachidonic acid turnover.     

The effect of betaadrenoceptor blocking drugs on histamine liberation,prostaglandin synthesis and phospholipid turnover in isolated mast cells stimulated with concanavalin A

Exaprolol, metipranolol and propranolol decreased significantly histamine liberation, degranulation,⁴⁵Ca uptake and thromboxane B₂ formation in isolated rat mast cells stimulated with concanavalin A and phosphatidylserine. Moreover, exaprolol and metipranolol decreased³²P incorporation into membrane phospholipids and metipranolol and propranolol reduced the liberation of arachidonic acid from membrane phospholipids of stimulated mast cells. Exaprolol significantly increased the arachidonic acid liberation from these cells. Possible mechanisms of interaction of betaadrenoceptor blocking drugs with isolated mast cells are discussed.     

Alterations in membrane phospholipid turnover,arachidonic acid liberation and thromboxane formation in isolated mast cells treated with propranolol and atenolol

Propranolol substantially changed³²P redistribution in isolated mast cell membrane phospholipids by increasing the activity of phosphatidylinositol and phosphatidic acid and decreasing the activity of phosphatidylcholine. Atenolol failed to affect³²P distribution. Both atenolol and propranolol decreased arachidonic acid liberation from membrane phospholipids of mast cells stimulated with compound 48/80 and concanavalin A plus phosphatidylserine. These findings confirmed the interaction of amphiphilic drugs with membrane phospholipids.

     

Inhibitory effect of adenine nucleotides and anti-allergic drugs on phosphorylation of phosphatidylinositol in rat mast cell granules

Rat mast cell granules were obtained by sonication of highly purified rat mast cells and isolated in a Percoll gradient. Phosphorylation of endogenous phosphatidylinositol in rat mast cell granules, which is catalyzed by phosphatidylinositol kinase in the granules, was assayed by measuring the incorporation of 32P from [gamma 32P]ATP into phosphatidylinositol 4-phosphate. Lipids were isolated with methanol/chloroform/HCl and were separated by thin-layer chromatography on oxalic acid impregnated silica gel plates. Phosphatidylinositol 4-phosphate areas were identified by staining with iodine, scraped and measured for 32P radioactivity. The phosphorylation reaction was inhibited by 50-500 microM adenosine, ADP and 500 microM AMP in a concentration-dependent manner. Among several anti-allergic drugs investigated. 100-1000 microM theophylline and 10-100 microM azelastine inhibited the phosphorylation reaction, but disodium cromoglycate and ketotifen had little effect.     

Polyamines stimulate the phosphorylation of phosphatidylinositol in rat mast cell granules

Rat mast cell granules were obtained by homogenization of highly purified rat mast cells and isolated in a Percoll gradient. Diphosphoinositide (DPI) synthesis in rat mast cell granules was assayed by measuring the incorporation of 32P from [gamma 32] ATP into DPI in the absence of exogenous phosphatidylinositol (PI). Lipids were isolated with methanol/chloroform/HCl and were separated by thin-layer chromatography on oxalic acid impregnated silica gel plates. DPI areas were identified by staining with iodine, scraped and measured for 32P radioactivity. The addition of polyamines, spermine and spermidine to the granules caused an increase in DPI synthesis, which can be catalyzed by PI kinase. This effect of polyamines on DPI synthesis in the rat mast cell granules was dose-dependent and maximal effects were observed at 1 mM spermine and 10 mM spermidine respectively. When the effect of 1 mM spermine on 32P incorporation into DPI in rat mast cell granules was investigated serially, 32P incorporation into DPI in rat mast cell granules incubated with spermine for 15 min was enhanced significantly (p less than 0.05) compared with that in the granules in the absence of spermine.     

Human pancreatic GRF stimulates phosphatidylinositol labeling in cultured anterior pituitary cells

The effects of natural and synthetic human pancreatic growth hormone-releasing factor (hpGRF) on 32P incorporation into phospholipids were studied in cultured rat anterior pituitary cells. Natural hpGRF (1:30 dilution) significantly (P less than 0.01) stimulated phosphatidylinositol labeling at all times studied (15, 30, and 60 min). Synthetic hpGRF-(1-40)OH also significantly (P less than 0.05; P less than 0.01) increased 32P incorporation into phosphatidylinositol in a dose-related (1-30 nM) and time-dependent (5, 10, and 30 min) manner. In contrast, phosphatidylcholine and phosphatidylethanolamine labeling was not affected at any time studied. Somatostatin (30 nM) did not affect basal or hpGRF-stimulated phosphatidylinositol labeling but inhibited the hpGRF stimulation of cyclic AMP accumulation and growth hormone release. These results suggest that the phosphatidylinositol cycle may be involved in the mechanism of action of hpGRF in the anterior pituitary gland.     

Mast cell secretion: differences between immunologic and non-immunologic stimulation

Non-immunologic and immunologic stimulation of mast cells have been compared. Non-immunologic stimulation relys heavily on cellular Ca, is unaffected by neuraminidase treatment, shows a rapid inactivation, and elicits no increase in the incorporation of³H-methyl groups into the lipid fraction. In contrast, stimulation by immunologic agents relys primarily on extracellular Ca, is inhibited by neuraminidase treatment, shows a comparatively slow rate of inactivation, and causes a significant increase in the incorporation of³H-methyl groups into the lipid fraction. We found no evidence of crossinactivation or desensitization between immunologic and non-immunologic agents. However, pretreatment of mast cells with neurotensin desensitized them to subsequent stimulation by compound 48/80. Our results support the hypothesis that immunologic and non-immunologic agents activate exocytotic mast cell secretion via separate mechanisms.     

Phorbol myristate acetate stimulates formation of diphosphoinositide in rat mast cell granules

Rat mast cell granules were obtained by homogenization of highly purified rat mast cells and isolated in a Percoll gradient. Diphosphoinositide (DPI) synthesis in rat mast cell granules was assayed by measuring the incorporation of 32P from [gamma 32P]ATP into DPI in the absence of exogenous phosphatidylinositol (PI). Lipids were isolated with methanol/chloroform/HCl and were separated by thin-layer chromatography on oxalic acid impregnated silica gel plates. DPI areas were identified by staining with iodine, scraped and measured for 32P radioactivity. The addition of phorbol myristate acetate (PMA) to the granules caused an increase of 32P incorporation from [gamma 32P]ATP in the DPI fraction, which can be catalyzed by PI kinase. This effect of PMA in the DPI synthesis was dose dependent and maximal effects were observed at 10 ng/ml.     

Atenolol,exaprolol and mast cell membranes

Lipophilic exaprolol and hydrophilic atenolol differ in their interaction with mast cell membranes. Exaprolol, as compared with atenolol, significantly decreased³²P incorporation into, but increased arachidonic acid liberation from, membrane phospholipids. Moreover, exaprolol significantly decreased phosphate incorporation in compound 48/80 and ConA-PS treated cells and decreased thromboxane formation in stimulated cells. On the other hand, atenolol decreased significantly only arachidonate liberation from stimulated mast cells. These results corroborate to some extent the effect of exaprolol and atenolol on histamine liberation which correlates with their membrane perturbing properties.     

Vascular reactivity in NISAG hypertensive rats

In vitro experiments on isolated segments of abdominal aorta of NISAG hypertensive rats with assessment of³²P incorporation showed that hypertension in these rats is developed as due to enhanced metabolism of phosphatidylinositol phosphates under the effect of norepinephrine resulting in higher vascular reactivity to norepinephrine. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 8, pp. 136–138, August, 1998     

Stimulation of phospholipid turnover by angiotensin II and phenylephrine in proximal convoluted tubules microdissected from mouse nephron

Angiotensin II and phenylephrine significantly increased phospholipid turnover in cortical tubule suspensions. To further localize this effect we studied the³²P incorporation into phosphatidylcholine (PC) and phosphatidylinositol (PI) in single dissected proximal convoluted tubules of mouse nephron. Both hormones significantly stimulated³²P labeling of PC and PI indicating that the proximal tubule is a target site of angiotensin II and phenylephrine action.     

Effect of endurance training on the phospholipid content of skeletal muscles in the rat

Only few data are available on the effect of training on phospholipid metabolism in skeletal muscles. The aim of the present study was to examine the effect of 6 weeks of endurance training on the content of particular phospholipid fractions and on the incorporation of blood-borne [¹⁴C]-palmitic acid into the phospholipids in different skeletal muscles (white and red sections of the gastrocnemius, the soleus and the diaphragm) of the rat. Lipids were extracted from the muscles and separated using thin-layer chromatography into the following fractions: sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, cardiolipin and neutral lipids (this fraction being composed mostly of triacylglycerols). It was found that training did not affect the content of any phospholipid fraction in soleus muscle. It increased the content of sphingomyelin in white gastrocnemius muscle, cardiolipin and phosphatidylethanolamine in red gastrocnemius muscle and phosphatidylinositol in white gastrocnemius muscle and diaphragm. The total phospholipid content in red gastrocnemius muscle of the trained group was higher than in the control group. Training reduced the specific activity of sphingomyelin and cardiolipin in all muscles, phosphatidylcholine in soleus, red, and white gastrocnemius muscles, phosphatidylserine in all muscles, phosphatidylinositol in all except the soleus muscle, and phosphatidylethanolamine in hindleg muscles, but not in the diaphragm compared to the corresponding values in the sedentary group. It was concluded that endurance training affects skeletal muscle phospholipid content and the rate of incorporation of the blood-borne [¹⁴C]-palmitic acid into the phospholipid moieties.     

Polyamines stimulate the phosphorylation of phosphatidylinositol in rat mast cell granules

Rat mast cell granules were obtained by homogenization of highly purified rat mast cells and isolated in a Percoll gradient. Diphosphoinositide (DPI) synthesis in rat mast cell granules was assayed by measuring the incorporation of 32P from [gamma 32P] ATP into DPI in the absence of exogenous phosphatidylinositol (PI). Lipids were isolated with methanol/chloroform/HCl and were separated by thin-layer chromatography on oxalic acid impregnated silica gel plates. DPI areas were identified by staining with iodine, scraped and measured for 32P radioactivity. The addition of polyamines, spermine and spermidine, to the granules caused an increase of DPI synthesis, which can be catalyzed by PI kinase. This effect of polyamines in the DPI synthesis was in a dose-dependent manner and maximal effects were observed at 1 mM spermine and 10 mM spermidine, respectively.     

Enhanced phospholipid metabolism in rat mast cells stimulated to release histamine

Incorporation of 32PO4 into the major phospholipids was examined in purified rat peritoneal mast cells stimulated to secrete histamine. Dog anti-rat IgE, concanavalin A, compound 48/80 and the calcium ionophore A23187 all caused significant increases of incorporation of 32PO4 into phosphatidic acid (PA), phosphatidylinositol (PI) and phosphatidylcholine (PC) at concentrations inducing non-cytotoxic release of histamine. The degree of 32PO4 incorporation into these phospholipids paralleled the dose-response curves for histamine release. For anti-IgE and Con A stimulation, histamine release and 32PO4 incorporation into PA, PI and PC were potentiated by passively sensitizing the mast cells with rat IgE or by the addition of phosphatidylserine (PS). These results suggest that enhanced phospholipid metabolism may be an important step in the biochemical sequence of events leading to release of histamine.     

Altered Postreceptor Signal Transduction of Formyl-Met-Leu-Phe Receptors in Polymorphonuclear Leukocytes of Patients with Non-Insulin-Dependent Diabetes Mellitus

The signal transduction of the formyl-Met-Leu-Phe (FMLP) receptor in polymorphonuclear leukocytes (PMNLs) from patients with non-insulin-dependent diabetes mellitus (NIDDM) was compared to that of PMNLs obtained from healthy volunteers. According to our previous studies in this group of patients neither the decrease in insulin binding capacity nor the enhanced insulin-degrading enzyme activity was involved. In control PMNLs, 10 nMFMLP induced a pertussis toxin-sensitive increase in phosphatidyl inositol (PI) cleavage and a subsequent Ca²⁺signaling from the intracellular pools. On the other hand, the FMLP-induced protein kinase C (PKC) activation and translocation into the membrane could not be detected in these cells via the measurement of³²P incorporation into histone. In contrast, in PMNLs of this special group of patients suffering from NIDDM the FMLP stimulus produced a significantly low increase in PI cleavage and Ca²⁺signaling from the intracellular pools. Moreover, in resting PMNLs of these patients with NIDDM, not only the [Ca²⁺]_ibut also the membrane-bound PKC activity was found to be significantly increased. In addition, PKC translocation into the cell membrane of diabetic PMNLs could be further triggered with FMLP as judged by the measurement of³²P incorporation into histone. Based on these results, it appears that the signaling of FMLP receptors in PMNLs of some NIDDM patients may have an alternative pathway through Ca²⁺influx from extracellular medium, arachidonic acid cascade, and PKC activation.     

Effect of phorbol myristate acetate (PMA) on diphosphoinositide (DPI) synthesis in rat mast cell granules

Rat mast cell granules were obtained by homogenization of highly purified rat mast cells and isolated in a Percoll gradient. DPI synthesis in rat mast cell granules was assayed by measuring the incorporation of 32P from [gamma 32P] ATP into DPI in the absence of exogenous phosphatidylinositol (PI). Lipids were isolated with methanol/chloroform/HC1 and were separated by thin-layer chromatography on oxalic acid impregnated silica gel plates. DPI areas were identified by staining with iodine, scraped and measured for 32P radioactivity. The addition of PMA to the granules caused an increase of DPI synthesis, which can be catalysed by PI kinase. Neither an inactive phorbol ester, 4-alpha-phorbol-12, 13-didecanoate, nor dimethyl sulfoxide (DMSO) used as a solvent for PMA had any effect. The effect of PMA in the DPI synthesis was dose-dependent and maximal effects were observed at 10-100 ng/ml. Dose-response curves of the effects of PMA in DPI synthesis in the granules corresponded to those of other biochemical effects of PMA in rat mast cells, such as mediator release mediated through the activation of protein kinase C. These results suggest that PMA may directly affect PI kinase or indirectly regulate its activity in rat mast cell granules.     

Protein phosphorylation in isolated plasma membranes and postsynaptic junctional structures from brain synapses.

The intrinsic protein kinase and phosphoprotein phosphatase activities of rat brain synaptic plasma membranes were investigated by following the incorporation of³²P from [γ-³²P]adenosine 5′-triphosphate into endogenous membrane proteins. The relationship of both these enzymes and of the phosphorylated proteins to brain synaptic junctions was determined by comparing the distribution of both the enzyme activities and the³²P-labelled polypeptides in subcellular fractions containing either synaptic membranes, isolated postsynaptic densities or the postsynaptic junctional lattice, a structure composed of interlacing protein fibres which underlies the postsynaptic density. After labelling synaptic membranes and then isolating the two junctional subfractions the specific activity of proteinbound³²P was two-fold higher in the postsynaptic density than in synaptic membranes and more than three-fold higher in the postsynaptic lattice. The enzyme activities were elevated two-fold in the postsynaptic density compared to synaptic membranes but in the postsynaptic lattice they were less than half the synaptic membrane level. The low levels of both enzymes in the postsynaptic lattice could be the result of inactivation by exposure to the sodium deoxycholate used to prepare the lattice fraction rather than indicating that the enzyme molecules are absent.Each subcellular fraction was phosphorylated and individual labelled polypeptides were identified by electrophoresis and autoradiography. All the³²P-labelled bands in synaptic membranes were also present in the postsynaptic density and postsynaptic lattice with the exception of material of molecular weight 47,000 which, although shown to be present by protein staining, was not phosphorylated in either junctional fraction. A³²P-labelled band of molecular weight 26,000 which was found in both postsynaptic junctional fractions was below the limit of detection in synaptic membranes. The two lowest molecular weight polypeptides, of 21,000 and 19,000, were labelled with specific activities respectively nine-fold and six-fold higher in isolated postsynaptic densities than in the synaptic membranes from which they were derived. Most of the postsynaptic density polypeptides exhibited a higher incorporation of³²P when labelling was performed with the addition of 50μm cyclic adenosine monophosphate. However, the three smallest (molecular weights: 19,000, 21,000 and 26,000) showed an inverse response, the incorporation of³²p into them being markedly depressed when cyclic adenosine monophosphate was present.Thus most of the [³²P]phosphate incorporated into synaptic membranes via the intrinsic kinase is associated with the postsynaptic junctional lattice, which is probably a structural framework for the postsynaptic organelle (MATUS &;WALTERS, 1975). This suggests that rather than being exclusively involved in synaptic transmission the phosphorylation of postsynaptic junctional proteins may be part of a more general metabolic response to afferent stimulation.