Effect of purinergic agonists and antagonists on insulin secretion from INS-1 cells (insulinoma cell line) and rat pancreatic islets

The effects of purinergic agonists on insulin release are controversial in the literature. In our studies (mainly using INS-1 cells, but also using rat pancreatic islets), ATP had a dual effect on insulin release depending on the ATP concentration: increasing insulin release (EC50 approximately/= 0.0032 microM) and inhibiting insulin release (EC50 approximately/= 0.32 microM) at both 5.6 and 8.3 mM glucose. This is compatible with the view that either two different receptors are involved, or the cells desensitize and (or) the effect of an inhibitory degradation product such as adenosine (ectonucleotidase effect) emerges. The same dual effects of ATP on insulin release were obtained using rat pancreatic islets instead of INS-1 cells. ADPbetaS, which is less degradable than ATP and rather specific for P2Y1 receptors, had a dual effect on insulin release at 8.3 mM glucose: stimulatory (EC50 approximately/= 0.02 microM) and inhibitory (EC50 approximately/= 0.32 microM). The effectiveness of this compound indicates the possible involvement of a P2Y1 receptor. 2-Methylthio-ATP exhibited an insulinotropic effect at very high concentrations (EC50 approximately/= 15 microM at 8.3 mM glucose). This indicated that distinct P2X or the P2Y1 receptor may be involved in these insulin-secreting cells. UTP increased insulin release (EC50 approximately/= 2 microM) very weakly, indicating that a P2U receptor (P2X3 or possibly a P2Y2 or P2Y4) are not likely to be involved. Suramin (50 microM) antagonized the insulinotropic effect of ATP (0.01 microM) and UTP (0.32 microM). Since suramin is not selective, the data indicated that various P2X and P2Y receptors may be involved. PPADS (100 microM), a P2X and P2Y1,4,6 receptor antagonist, was ineffective using either low or high concentrations of ATP and ADPbetaS, which combined with the suramin data hints at a P2Y receptor effect of the compounds. Adenosine inhibited insulin release in a concentration-dependent manner. DPCPX (100 microM), an adenosine (A1) receptor antagonist, inhibited the inhibitory effects of both adenosine and of high concentrations of ATP. Adenosine deaminase (1 U/mL) abolished the inhibitory effect of high ATP concentrations, indicating the involvement of the degradation product adenosine. Repetitive addition of ATP did not desensitize the stimulatory effect of ATP. U-73122 (2 microM), a PLC inhibitor, abolished the ATP effect at low concentrations. The data indicate that ATP at low concentrations is effective via P2Y receptors and the PLC-system and not via P2X receptors; it inhibits insulin release at high concentrations by being metabolized to adenosine.     

β-Mannanase (Man26A) and α-galactosidase (Aga27A) synergism – A key factor for the hydrolysis of galactomannan substrates

This study investigated the behavior of mannan-degrading enzymes, specifically focusing on differences with respect to their substrate specificities and their synergistic associations with enzymes from different glycoside hydrolase (GH) families. Galactosidases from Cyamopsis tetragonolobus seeds (Aga27A, GH27) and Aspergillus niger (AglC, GH36) were evaluated for their abilities to synergistically interact with mannanases from Clostridium cellulovorans (ManA, GH5) and A. niger (Man26A, GH26) in hydrolysis of guar gum and locust bean gum. Among the mannanases, Man26A was more efficient at hydrolyzing both galactomannan substrates, while among the galactosidases; Aga27A was the most effective at removing galactose substituents on both galactomannan substrates and galactose-containing oligosaccharides. An optimal protein mass ratio of glycoside hydrolases required to maximize the release of both reducing sugar and galactose residues was determined. Clear synergistic enhancement of locust bean gum hydrolysis with respect to reducing sugar release was observed when both mannanases at 75% enzyme dosage were supplemented with 25% enzyme protein dosage of Aga27A. At a protein ratio of 75% Man26A to 25% Aga27A, the presence of Man26A significantly enhanced galactose release by 25% Aga27A (2.36 fold) with locust bean gum, compared to when Aga27A was used alone at 100% enzyme protein dosage. A dosage of Aga27A at 75% and ManA at 25% protein content liberated the highest reducing sugar release on guar gum hydrolysis. A dosage of Man26A and Aga27A at 75–25% protein content, respectively, liberated reducing sugar release equivalent to that when Man26A was used alone at 100% protein content. From the findings obtained in this study, it was observed that the GH family classification of an enzyme affects its substrate specificity and synergistic interactions with other glycoside hydrolases from different families (more so than its EC classification). The GH26 Man26A and GH27 Aga27A enzymes appeared to be more promising for applications that involve the hydrolysis of galactomannan containing biomass. This method of screening for maximal compatibility between various GH families can ultimately lead to a more rational development of tailored enzyme cocktails for lignocellulose hydrolysis.     

The effects of psychoactive agents on calcium uptake by preparations of rat brain mitochondria

The effects of chlorpromazine and other psychoactive agents on the uptake of calcium by partially purified preparations of mitochondria from rat brain were studied in vitro. Chlorpromazine at concentrations of about 0-1 ITIM caused a marked inhibition of mitochondrial calcium transport. Perphenazine also exhibited this action and was slightly more potent than chlorpromazine. Imipramine inhibited mitochondrial calcium uptake but higher concentrations were necessary than in experiments with chlorpromazine. The sulph-oxide of chlorpromazine did not inhibit calcium transport when tested at concentrations similar to those used with chlorpromazine. Up to concentrations of 20 mM, lithium ions did not influence mitochondrial calcium uptake.     

Inhibition of non-cytotoxic histamine liberation from isolated rat mast cells by antihistaminic preparations]

Phenothiazines (chlorpromazine and promethazine) and antihistaminic quinuclidine derivatives [phencarol, quinuclidyl-3-di-(o-tolyl) carbinol, hydrochloride quinuclidyl-3-di-(o-methoxyphenyl) carbinol--HQMC] at concentrations preceding the histamine-releasing ones inhibited the compound 48/80-induced histamine release from the isolated rat mast cells. HQMC inhibited histamine release induced by selective liberators (compound 48/80, MCD-peptide, specific antigen), but potentiated histamine release induced by nonselective liberators (chlorpromazine, tryton X-100). The inhibition by HQMC of histamine release induced by compound 48/80 increased during 1 min and was reversible. The inhibitory effect of all the compounds tested was partially counteracted by glucose.     

Influence of dietary lipids on the effect of chlorpromazine on membrane properties of rabbit red cells

The effect of diets containing different types of common natural oils on physical properties of red cells was investigated by using rabbits. The rabbits were fed for 18 months on a standard diet in which 8% of its energy content was provided by safflower oil and 32% energy by either more safflower oil or fish oil, linseed oil, olive oil or palm oil. Erythrocyte deformability was significantly decreased by the fish oil diet compared with each of the other diets. Osmotic fragility was significantly less (66 mM) for red cells from rabbits fed on the linseed oil diet, and significantly greater (71 mM) for red cells from rabbits on the fish oil diet, than for red cells from rabbits on the other three diets which did not differ significantly from each other (68 mM). With rabbits on the standard diet, the resistance of their erythrocytes to osmotic haemolysis was increased by chlorpromazine at concentrations below and decreased by concentrations above 30 microM. The dietary oils caused significant changes in the effects of chlorpromazine on osmotic fragility. The concentration at which the effect of chlorpromazine reversed from antihaemolytic to prohaemolytic was decreased by the safflower and linseed oil diets and increased by the fish oil diet, compared with the olive and palm oil diets. Analysis of the fatty acid compositions of the dietary oils on the one hand and of the red cell phospholipids on the other established, specifically, that in the presence of 30 microM chlorpromazine the percentage haemolysis was directly proportional to the linoleate content of the red cell phospholipids.     

Trifluoperazine and chlorpromazine inhibit phosphatidylcholine biosynthesis and CTP:phosphocholine cytidylyltransferase in HeLa cells

The influence of chlorpromazine and trifluoperazine on phosphatidylcholine biosynthesis in HeLa cells was investigated. HeLa cells were prelabeled with [Me-3H]choline for 1 h. The cells were subsequently incubated with various concentrations of drugs. Both compounds were potent inhibitors of phosphatidylcholine biosynthesis, with 50% inhibition by 5 micron of either drug. Analysis of the radioactivity in the soluble precursors indicated a block in the conversion of phosphocholine to CDPcholine catalyzed by CTP:phosphocholine cytidylyltransferase (CTP:cholinephosphate cytidylyltransferase, EC 2.7.7.15). Inhibition by these drugs was slowly reversed after incubation for more than 2 h, or was immediately abolished when 0.4 mM oleate was included in the cell medium or when the drug-containing medium was removed. The subcellular location of the cytidylyltransferase was unaffected by either drug, nor did the drugs alter the rate of release of cytidylyltransferase from HeLa cells by digitonin treatment. The drugs had a direct inhibitory effect on cytidylyltransferase activity in HeLa cell postmitochondrial supernatants. Half-maximal inhibition was achieved with 30 microM trifluoperazine and 50 microM chlorpromazine. These drugs did not change the apparent Km of the cytidylyltransferase for CTP or phosphocholine. Inhibition of cytidylyltransferase by these compounds was reversible with exogenous phospholipid or oleate in the enzyme assay. The data indicate that both drugs inhibit phosphatidylcholine synthesis by an effect on the cytidylyltransferase. The mechanism of action remains unknown at this time.     

Investigations of the inhibitory effect of propranolol,chlorpromazine, quinine,and dicyclohexylcarbodiimide on the swelling of yeast mitochondria in potassium acetate. Evidences for indirect effects mediated by the lipid phase

The mode of action of propranolol, chlorpromazine, and quinine, three cationic drugs inhibiting swelling of yeast mitochondria in potassium acetate, was investigated by looking at their effect on fluorescent probes of the polar heads and of the nonpolar moiety of the membranes, under inhibitory conditions of swelling. As expected, propranolol and chlorpromazine exhibited specificity for anionic phospholipids since they increased the binding of the anionic probe 1-anilino 8-naphthalenesulfonate (ANS). Although propranolol did not release 1,6-diphenyl-1,3,5-hexatriene (DPH) from the hydrophobic moiety of the membrane, it increased the excimer/ monomer fluorescence ratio of 10-(1-pyrene)decanoate, suggesting that it induced a limitation in the movements of the aliphatic chains of phospholipids. Opposite to propranolol, chlorpromazine removed DPH from the membrane, suggesting that it bound essentially to the hydrophobic moiety. However, chloramphenicol, which was also able to remove DPH but did not increase the binding of ANS, did not inhibit swelling. Inhibition by chlorpromazine therefore appeared to be related to its binding to the hydrophobic moiety of anionic phospholipids. Quinine had no effect on membrane properties: at inhibitory concentrations of swelling in potassium acetate, it did not inhibit swelling in ammonium phosphate (mediated by the phosphate/H⁺ cotransporter), whereas propranolol and chlorpromazine did, suggesting a more specific effect of quinine on (a) protein(s) involved in the K⁺/H⁺ exchange. Dicyclohexylcarbodiimide (DCCD), which irreversibly inhibits the swelling in potassium acetate, bound to ethanolamine heads; despite this effect, DCCD had no major consequences on the binding of the probes. Consequently, propranolol and chlorpromazine are of no help for characterizing protein(s) catalyzing the K⁺/H⁺ exchange, although their effect on lipids seems to involve limited zones of the inner mitochondrial membrane. Quinine and DCCD, although they also bind to lipids, may inhibit the activity by acting on a limited number of proteins.     

Differential effects of chlorpromazine on secretion, protein phosphorylation and phosphoinositide metabolism in stimulated platelets.

Increasing concentrations of chlorpromazine (30-500 microM) caused a progressive lysis of gel-filtered platelets, as monitored by the extracellular appearance of cytoplasmic ([14C]adenine-labelled) adenine nucleotides. The chlorpromazine-induced lysis was markedly enhanced by thrombin and phorbol ester, and complete cytolysis was found at chlorpromazine concentrations of 100 microM and above in the presence of thrombin. At non-lytic concentrations, chlorpromazine caused a dramatic increase in the thrombin- or phorbol ester-mediated incorporation of 32P into phosphatidylinositol 4-phosphate and, to a lesser extent, into phosphatidylinositol 4,5-bisphosphate in platelets pulse-labelled with [32P]Pi. Chlorpromazine alone also caused an incorporation of 32P into the phosphoinositides. Non-lytic concentrations of chlorpromazine had no effect on the phosphorylation of the 47 kDa protein (regarded as the substrate for protein kinase C), but markedly inhibited the accompanying secretion of ATP + ADP and beta-hexosaminidase when platelets were incubated with 0.17 microM-phorbol ester or 0.1-0.2 unit of thrombin/ml. At lower concentrations of thrombin, chlorpromazine did not inhibit, but slightly enhanced, secretion. A protein of 82 kDa was phosphorylated during the interaction of platelets with thrombin and phorbol ester, and this phosphorylation was enhanced by chlorpromazine (non-lytic). These results suggest that the previously reported inhibition of protein kinase C by chlorpromazine is probably non-specific and due to cytolysis. However, since non-lytic concentrations of chlorpromazine inhibit secretion, but not protein kinase C, in platelets, activation of protein kinase C is not involved in the stimulation-secretion coupling, or chlorpromazine acts at a step after kinase activation. Possible mechanisms of this inhibition by chlorpromazine are discussed in the light of its effect on phosphoinositide metabolism and protein phosphorylation.     

Cocaine induced secretion of ACTH, beta-endorphin, and corticosterone

The effect of intraperitoneal administration of cocaine on the concentrations of hypothalamic corticotropin releasing factor like-immunoreactivity (CRF-LI), plasma ACTH, beta-endorphin, and corticosterone was investigated. Groups of rats were injected with 20 mg/kg cocaine HCI or 0.9% NaCl and then killed 0, 10, 20, 30 or 60 minutes later. Hypothalamic CRF-LI, plasma ACTH, beta-endorphin, and corticosterone concentrations were determined by radioimmunoassay. A significant increase in plasma ACTH, beta-endorphin, and corticosterone concentrations was observed after cocaine administration. In contrast, cocaine had no significant effect on hypothalamic CRF-LI concentrations. Intravenous administration of 0.5 and 2.0 mg/kg cocaine to rats in which the endogenous release of CRF was blocked by chlorpromazine, morphine, and pentobarbital elicited a significant increase in plasma corticosterone concentrations. These results demonstrate that cocaine induces the release of ACTH, beta-endorphin, and corticosterone and suggest that this response is mediated at the pituitary level.     

Modulation of an acetylcholine receptor responsiveness by filipin and chlorpromazine studied in neurons ofAplysia californica

The responsiveness of Aplysia acetylcholine receptors (AChR) was studied using a polyene antibiotic, filipin, which specifically complexes cholesterol, and another compound, chlorpromazine (CPZ), which inserts at the proteolipidic interface. Both substances enhanced the evoked postsynaptic responses or responses to iontophoretic application of carbachol only on the H-type receptor (opening a Cl-permeability), whereas at the same concentrations filipin was without effect on the D-type receptor (opening a cationic permeability) while CPZ depressed the D-type response. The facilitation observed specifically for the H-type receptor was similar to that previously described after acetylcholinesterase (AChE) inhibition or when low concentrations of detergents were applied to this preparation. No additive effect was obtained after the addition of chlorpromazine following a maximal potentiation obtained with an anticholinesterase agent. Since at Aplysia central neurons, AChE is a membranal protein, we propose that the facilitation of H-type responses is attributable to the removal of a modulatory action of AChE on AChR. Filipin or chlorpromazine might disrupt the interaction between AChR and AChE.     

Prostaglandin F2α (PGF2α) and prolactin secretion in rats

Plasma prolactin and F-prostaglandins (PGF) were measured in anesthetized male Sprague-Dawley rats before and at 15, 30, 45 and 60 minutes following i.v. injection of either PGF_2α (4 mg/kg), chlorpromazine, 1 mg/kg or chlorpromazine (1 mg/kg) after pretreatment with i.p. indomethacin (2 mg/kg). Following PGF_2α administration, plasma prolactin levels increased significantly only at 15 and 30 minutes in spite of extremely high PGF levels throughout 60 minutes. Besides the expected rise in plasma prolactin, chlorpromazine caused a transient but statistically significant increase in PGF. Indomethacin blocked the chlorpromazine-induced PGF rise but not prolactin increase. Animals stressed with ether anesthesia showed elevation of plasma prolactin, which was not blocked by indomethacin although PGF concentration fell. These results indicate that PGF_2α can stimulate prolactin release. This effect does not appear to be physiologic since very high PGF levels are required. Furthermore, blockade of prostaglandin synthesis by indomethacin does not prevent the release of prolactin in response to chlorpromazine or stress. Our findings do not support a possible role of PGFs as intermediaries in prolactin release. However, it is possible that PGFs may work through other mechanisms not investigated in our study.     

Effects of α,β-methylene-adenosine-5′-diphosphate on blood platelet aggregation

The effects on platelet aggregation of α,β-methylene-adenosine-5′-diphosphate (Ado-PCP) have been investigated. Using human citrated platelet-rich plasma it has been shown that: (i) at concentrations of 10^?3 M or higher Ado-PCP is able to induce platelet aggregation; (ii) the rate of Ado-PCP-induced aggregation increases on raising the pH of platelet-rich plasma above the pK_a for the secondary phosphonyl dissociation of Ado-PCP; (iii) at concentrations from 1 · 10^?4 to 5 · 10^?4 M Ado-PCP does not cause platelet aggregation itself, but it inhibits ADP-induced aggregation. This inhibition is also observed in washed platelet suspensions. The data suggest that Ado-PCP acts at the same site on the platelet membrane as does ADP and that ADP to AMP transformation is not a prerequisite for the process of aggregation. The observed effect of pH on the rate of Ado-PCP induced aggregation suggests that the ionization state of a nucleotide terminal acid group is important in the process of aggregation.     

Membrane stabilizers inhibit potassium efflux from Staphylococcus aureus strain No. U2275

The effect of different categories of membrane stabilizers on K⁺ loss and growth has been characterized in a culture of Staphylococcus aureus. Chlorpromazine, thiopental and tetracaine at low concentrations produced a marked inhibition of K⁺ loss and an equivalent increase in the K⁺ contents of S. aureus. Whereas the inhibitory effect of chlorpromazine on K⁺ loss was observed at lower than bacteriostatic concentrations of the drug, thiopental had no effect on growth in the concentration range where K⁺ loss was maximally inhibited. It is concluded that the bacteriostatic action of chlorpromazine is probably not related to its membrane stabilizing effect only.     

Effects of hyperoxia on microvascular cells in vitro

Summary Microvascular cells are most vulnerable to direct oxygen damage. Using an in vitro model system we have investigated the effect of elevated oxygen on the proliferation, morphology, and integrity of microvascular endothelial cells (EC) and pericytes. Cultivation of these cells at oxygen concentrations of 40% for 1 wk resulted in the inhibition of EC proliferation but had no effect on the growth of the pericytes. Similarly, hyperoxia induced a dramatic change in the shape of the EC, increasing their spread area by close to six-fold. Under the same conditions, the spread area of the pericytes was unaffected. To understand the effect of the hyperoxic treatment on the cells, the integrity of various membrane systems was assessed.⁵¹Chromium release was used to monitor plasma membrane integrity. There was no difference in chromium release by EC and pericytes over the 7 d of growth under normoxic and hyperoxic conditions. Mitochondrial integrity was examined by staining the cells with Rhodamine 123, which is selectively accumulated by the mitochondria. The staining pattern of the mitochondria of both EC and pericytes was altered by growth in the elevated oxygen. Finally, the lysosomes were visualized using acridine orange. The acridine orange staining pattern revealed enlarged and perinuclear lysosomes in the EC but no change in the pericyte lysosomal staining pattern. Thus, the cells of the microvasculature seem to be differentially affected by hyperoxia, a fact that may be significant in the etiology of reperfusion injury, ischemic disease, and pathologies associated with prematurity. This research was supported by grant E4-05318 from the National Institutes of Health, Bethesda, MD.     

Characteristics of penicillinase release by washed cells of Bacillus licheniformis

Saline-washed cells of Bacillus licheniformis strain 749/C (constitutive for penicillinase) were able to release exopenicillinase in the presence of concentrations of chloramphenicol that prevented protein synthesis completely. The release reaction was strongly pH-dependent, occurring at a faster rate at alkaline pH in anionic or cationic buffers than at neutral pH. A strongly pH-dependent release reaction was noted in growing cells also. The reaction in washed cells can be stopped completely by changing the pH to 6.0. Within 30 min at pH 9.0, about 55% of the cell-bound penicillinase was released; thereafter, release continued at a greatly reduced rate. Suspensions of washed cells retained their capacity to release penicillinase at pH 9.0 for 90 min. Penicillinase released at pH 9.0 from either cells or protoplasts was not readsorbed over a 60-min period after changing the pH to 6.0. The release reaction was strongly temperature-dependent. We examined the effect of a large number of metabolic inhibitors and other compounds on the pH-dependent release phenomenon. Quinacrine hydrochloride, chloroquine diphosphate, and chlorpromazine hydrochloride reduced secretion substantially at 10(-4)m. Deoxycholate and Triton X-100 were active at 10(-3)m, but tungstate, arsenate, and molybdate had small effects at 10(-1)m. The rate of exopenicillinase release at pH 9.0 from fully stabilized protoplasts was one-half that of intact cells. Protoplasts lysed in hypotonic media or detergents showed even greater reduction in releasing activity. Penicillinase released from washed cells at pH 7.5 or 9.0 appeared to be derived from the periplasmic tubule and vesicle fraction that was released by protoplast formation.     

The varied responses of different F1-ATPases to chlorpromazine

The effects of chlorpromazine on various properties of the F1-ATPases from bovine heart mitochondria (MF1), the plasma membranes of Escherichia coli (EF1), and plasma membranes of the thermophilic bacterium PS3 (TF1) have been examined. While chlorpromazine inhibited MF1 with an I0.5 of about 50 microM and EF1 with an I0.5 of about 150 microM at 23 degrees C, the ATPase activity of TF1 was stimulated by chlorpromazine concentrations up to 0.6 mM at this temperature. Maximal activation of about 20% was observed at 0.2 mM chlorpromazine at 23 degrees C. Chlorpromazine concentrations greater than 0.6 mM inhibited TF1 at 23 degrees C. At 37 degrees C the ATPase activity of TF1 was doubled in the presence of 0.5 mM chlorpromazine, the concentration at which maximal stimulation was observed at this temperature. Chlorpromazine inhibited the rate of inactivation of EF1 by dicyclohexylcarbodiimide (DCCD) at 23 degrees C and pH 6.5. Concentrations of chlorpromazine which inhibited the ATPase activity of TF1 at pH 7.0 accelerated the rate of inactivation of the enzyme by DCCD at pH 6.5, while lower concentrations of the phenothiazine, which stimulated the ATPase, had no effect on DCCD inactivation. Chlorpromazine concentrations up to 1.0 mM had no effect on the rate of inactivation of TF1 by DCCD at 37 degrees C and pH 6.5. Chlorpromazine at 0.5 mM accelerated the rate of inactivation of MF1 by 5'-p-fluorosulfonylbenzoyladenosine (FSBA), while it slowed the rate of inactivation of EF1 by FSBA. The inactivation of TF1 by FSBA in the absence of chlorpromazine was complex and was not included in this comparison. Chlorpromazine protected MF1 and EF1 against cold inactivation. Whereas 100 microM chlorpromazine afforded about 90% stabilization of MF1 at 4 degrees C, only about 30% stabilization of EF1 was observed under the same conditions in the presence of 400 microM chlorpromazine. Each of the ATPases was inactivated by the structural analog of chlorpromazine, quinacrine mustard. Whereas 5 mM ATP and 5 mM ADP protected MF1 and TF1 against inactivation by 0.5 mM quinacrine mustard, the rate of inactivation of EF1 by quinacrine mustard was accelerated fourfold by 5 mM ATP and slightly accelerated by 5 mM ADP.     

Controlled Release Matrix Tablets of Zidovudine: Effect of Formulation Variables on the <Emphasis Type="BoldItalic">In Vitro</Emphasis> Drug Release Kinetics

The purpose of this research was to design oral controlled release (CR) matrix tablets of zidovudine (AZT) using hydroxypropyl methylcellulose (HPMC), ethyl cellulose (EC) and carbopol-971P (CP) and to study the effect of various formulation factors on in vitro drug release. Release studies were carried out using USP type 1 apparatus in 900 ml of dissolution media. Release kinetics were analyzed using zero-order, Higuchi’s square root and Ritger–Peppas’ empirical equations. Release rate decreased with increase in polymer proportion and compression force. The release rate was lesser in formulations prepared using CP (20%) as compared to HPMC (20%) as compared to EC (20%). No significant difference was observed in the effect of pH of dissolution media on drug release from formulations prepared using HPMC or EC, but significant difference was observed in CP based formulations. Decrease in agitation speed from 100 to 50 rpm decreased release rate from HPMC and CP formulations but no significant difference was observed in EC formulations. Mechanism of release was found to be dependent predominantly on diffusion of drug through the matrix than polymer relaxation incase of HPMC and EC formulations, while polymer relaxation had a dominating influence on drug release than diffusion incase of CP formulations. Designed CR tablets with pH independent drug release characteristics and an initial release of 17–25% in first hour and extending the release up to 16–20 h, can overcome the disadvantages associated with conventional tablets of AZT.     

Dual regulation of arachidonic acid release by P2U purinergic receptors in dibutyryl cyclic AMP-differentiated HL60 cells.

ATP promoted biphasic effects on both basal and fMLP-stimulated arachidonic acid (AA) release in neutrophil-like HL60 cells: stimulation in the micromolar range (EC50 = 3.2 +/- 0.9 microM) and inhibition at higher concentrations (EC50 = 90 +/- 11 microM). ATP also inhibited UTP- and platelet activating factor-stimulated AA release. Only stimulatory effects of ATP on basal or fMLP-stimulated phospholipase C were observed. The inhibitory effect of ATP on AA release was not due to reacylation of released AA, chelation of extracellular Ca2+, cell permeabilization, or changes in the rise of [Ca2+]i induced by agonist. The inhibition was rapid, being detected within 5-15 s. The inhibitory effect of ATP on fMLP-stimulated AA release could be desensitized by pretreatment of the cells with 2 mM ATP, but not 20 microM ATP, the concentration that resulted in maximal release of AA and inositol phosphates. The inhibition by ATP was neither dependent on generation of adenosine by ATP hydrolysis nor the result of direct interaction of ATP with P1 purinergic receptors. Among other nucleotides tested (CTP, GTP, ITP, TTP, XTP, adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP), adenyl-5'-yl imidodiphosphate (AMP-P(NH)P), ADP, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), and UTP), only UTP and ATP gamma S displayed biphasic effects with potencies and efficacies almost identical to those of ATP. The other nucleotides only exhibited stimulatory effects (EC50 = 60-300 microM). The results are consistent with a model of dual regulation of AA release by two distinct subtypes of P2U receptors in HL60 cells.     

Possible involvement of calmodulin in maturation and activation of Chaetopterus eggs

We report the isolation of calmodulin from oocytes of Chaetopterus pergamentaceus. The identification of this protein is based on (1) activation of beef heart cAMP phosphodiesterase, (2) heat stability, (3) sensitivity to chlorpromazine, and (4) electrophoretic mobility identical to that of porcine brain calmodulin after sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of either Ca2+ or EGTA. We treated oocytes with chlorpromazine and W-7 to investigate the involvement of calmodulin in meiosis initiation and egg activation. Very low concentrations of chlorpromazine inhibited germinal vesicle breakdown (GVBD). This effect was shown to be dependent upon bright indirect light, since the drug was much less effective at GVBD inhibition under conditions of very low illumination. Higher concentrations of chlorpromazine and W-7 (100 microM) inhibited GVBD and activated eggs with intact germinal vesicles as determined by fertilization envelope formation and the onset of ameboid activity. Neither egg activation nor inhibition of calmodulin stimulation of phosphodiesterase activity in vitro was affected by light. These results are consistent with a role for calmodulin in egg activation and GVBD, but suggest that chlorpromazine in bright light may prevent GVBD by some mechanism other than calmodulin inhibition.     

Dopamine release from nerve endings induced by polysialogangliosides

Polysialogangliosides but not monosialoganlioside or a neutral glycosphingolipid induce release of [³H] -dopamine from synaptosomes in presence of Ca⁺⁺, presumably by exocytosis. This effect is discussed in relation to the ability of polysialogangliosides to induce membrane fusion in chicken erythrocytes and to their behaviour in lipid monolayers. It is suggested that characteristic interactions with phosphatidylcholine involving decreases of surface potential are participating in the polysialoganglioside-induced neurotransmitter release.