Biomedical subjects

E K Matthews

Publications and source records attributed to E K Matthews.

At least 19 recordsLinked to original sources

PubMed2000

A key role for the mitochondrial benzodiazepine receptor in cellular photosensitisation with delta-aminolaevulinic acid.

The aim of this study was to determine the part played by the mitochondrial benzodiazepine receptor in cellular photosensitisation with the protoporphyrin IX precursor, delta-aminolaevulinic acid. Evaluation of the delta-aminolaevulinic acid-concentration dependence and kinetics of fluorescent protoporphyrin IX accumulation in monolayers of rat AR4 2J pancreatoma cells established a basis for assessing pharmacological modulation of the biosynthetic pathways for protoporphyrin IX production and photocytotoxicity. Iron chelation enhanced the accumulation of photo-active protoporphyrin IX whereas 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxam ide (PK11195), dipyridamole, or 7-(dimethylcarbamoloxy)-6-phenylpyrrolo-[2,1-d]benzothiazepi ne (DPB), competitive ligands of the mitochondrial benzodiazepine receptor, all diminished protoporphyrin IX accumulation, as did acifluorfen, a mitochondrial protoporphyrinogen oxidase inhibitor. In addition to protoporphyrin IX (Em(max): 630 nm), delta-aminolaevulinic acid-treated cells also generated a fluorophore of Em(max) 580 nm; this compound was identified as Zn-protoporphyrin IX. Mitochondrial benzodiazepine receptor ligands increased the formation of the zinc porphyrin whilst decreasing that of protoporphyrin IX. The involvement of the mitochondrial benzodiazepine receptor in the translocation of porphyrins and the formation of Zn-protoporphyrin IX have wide implications for the use of delta-aminolaevulinic acid in photodynamic therapy.

Aminolevulinic Acid↗

PubMed2000

Photosensitization of pancreatic tumour cells by delta-aminolaevulinic acid esters.

A series of straight chain, branched and cyclo-delta-aminolaevulinic acid (ALA) esters have been synthesized and their photosensitizing properties analysed using an in vitro system of rat pancreatoma cells. Structurally favourable ALA esters not only induced the formation of more of the endogenous photosensitizer, protoporphyrin IX (PpIX), but they did so at a faster rate than ALA itself. This action was reflected in a substantial increase in photocytotoxicity of some 270 times, using the more potent ALA esters. An important structural feature was identified in two of the ALA esters which greatly limited PpIX production, i.e. a branch point located next to the site of ester cleavage. Experiments on the transport of ALA and of ALA esters across the cell membrane showed that ALA, but not ALA esters, gain access to the cell via the di- and tripeptide transporter, PEPTI. Finally, these results show that the esterification of ALA can greatly increase its cellular uptake, so generating more intracellular PpIX, improved tumour cell photosensitization and enhanced photocytotoxicity.

Aminolevulinic Acid↗

PubMed1998

Photodynamic modulation of cellular function.

Photodynamic action with a large number of photosensitisers has important practical implications such as photodynamic cancer therapy. But the cellular and molecular mechanisms involved have been rather poorly understood. In this paper, photodynamic modulation of cell signal transduction and the resultant changes in cellular function are reviewed, with a particular emphasis on smooth muscle and the pancreas.

Animals↗

PubMed1996

Cytotoxic, nuclear, and growth inhibitory effects of photodynamic drugs on pancreatic carcinoma cells.

The light-activated drugs AlPcS4 and T4MPyP were studied in a pancreatic carcinoma cell line for their effects on DNA integrity, cell division, proliferation, and survival. The micronucleus assay measured nuclear changes and also the number of actively dividing cells while, under similar conditions, the MTT assay measured cell survival. When tumour cells were exposed to light, pre-treatment with AlPcS4 induced more micronuclei than did T4MPyP at the same levels of cell division and survival. Both drugs showed a correlation between phototoxicity and changes to DNA integrity so establishing micronuclei formation as an important indicator of photodynamic drug action on tumour cells.

Animals↗

PubMed1995

Molecular characterisation of recombinant green fluorescent protein by fluorescence correlation microscopy.

The cDNA for the green fluorescent protein (GFP) of Aequorea victoria has been expressed in transformed cells of Saccharomyces cerevisiae and the recombinant GFP isolated. Protonation and deprotonation of the cloned and purified GFP produced major effects on its spectral absorption characteristics with an increase in pH enhancing the fluorescence emission of the GFP more than twofold. Finally, molecular characterisation of GFP by fluorescence correlation microscopy in a minimal target volume of 1 fL yielded a translational diffusion coefficient (DT) of 8.7 x 10(-7) cm2.sec-1, equivalent to a Stokes radius of 2.82nm for a monodisperse globular protein of 27kDa.

Amino Acid Sequence↗

PubMed1995

Modification of the photodynamic action of delta-aminolaevulinic acid (ALA) on rat pancreatoma cells by mitochondrial benzodiazepine receptor ligands.

We have shown that addition of exogenous delta-aminolaevulinic acid (ALA) to rat pancreatoma AR4-2J cells in culture leads to the increased production of porphobilinogen (PBG) and the accumulation of photoactive protoporphyrin IX (PPix) in these cells. Exposure to light (lambda > 400 nm) at an intensity of 0.2 mW cm-2 for 8 min resulted in an ALA dose-dependent cytolysis of the cells, with an EC50 of 6.6 +/- 0.7 microM. This cytolytic effect was light intensity dependent, with greater cell destruction after exposure to light at an intensity of 0.47 mW cm-2 than at 0.2 mW cm-2; it was also dependent on the duration of illumination, cell survival decreasing with increasing illumination times. The photodestruction of the AR4-2J cells following exposure to ALA can be attributed to the production of endogenous PPix, a photoactive porphyrin that we have shown to generate singlet oxygen upon illumination, whereas ALA itself does not. Further investigation of the molecular mechanisms underlying the photodynamic action of ALA demonstrated the involvement of the mitochondrial (peripheral) benzodiazepine receptor (MBR), a high-affinity recognition site for dicarboxylic porphyrins, and especially PPix. The centrally acting benzodiazepine compounds clonazepam and flumazenil, which have negligible affinities for the MBR, had no effect on ALA-mediated phototoxicity. In contrast, both the isoquinoline carboxamide PK11195 and the benzodiazepine Ro 5-4864 ligands, displaying a high affinity for the MBR, did affect ALA-mediated phototoxicity, each markedly increasing the EC50 for cell photodestruction and thus exerting a photoprotective effect. It is concluded that the MBR may play an important role in the expression of ALA-mediated PPix phototoxicity and that MBR ligands, by diminishing the actions of endogenous PPix, have the potential to rescue cells from porphyrin-induced photolysis.

Aminolevulinic Acid↗

PubMed1994

The effects of a photosensitive nitric oxide donor on basal and electrically-stimulated dopamine efflux from the rat striatum in vitro.

The reported effects of nitric oxide (NO) on dopamine release from the striatum are variable and its precise effect on striatal nerve terminals is unclear. In the present study a novel method of applying NO to brain tissue in situ was employed. Photo-activation of Roussin's Black Salt (RBS), retained in isolated perfused brain tissue, was used to release NO at will upon illumination. Basal and electrically-stimulated dopamine efflux from the rat striatum in vitro was measured in real time using fast cyclic voltammetry. Illumination of an RBS pre-treated brain slice elicited a light intensity-related increase in basal dopamine efflux. Concomitantly there was a decrease in the level of electrically-stimulated dopamine efflux. Illumination in the absence of RBS pre-treatment had no effect on basal or stimulated dopamine efflux. The increase in basal dopamine efflux upon photo-activation of RBS was reduced by the presence of 10 microM oxyhaemoglobin, but was insensitive to the removal of extracellular calcium or the addition of 1 microM sulpiride. The decrease in electrically-stimulated dopamine efflux following illumination was not affected by the presence of either oxyhaemoglobin or sulpiride. It is concluded that NO, produced by photo-activation of RBS, releases dopamine from the rat striatum in vitro by a mechanism independent of extracellular calcium entry.

Animals↗

PubMed1994

Calcium-dependent photodynamic action of di- and tetrasulphonated aluminium phthalocyanine on normal and tumour-derived rat pancreatic exocrine cells.

Important differences exist in the responses to photodynamic agents of normal and tumour-derived pancreatic acinar cells. In the present study amylase release has been used to assess the mechanisms by which the photodynamic drugs tetra- and disulphonated aluminium phthalocyanine (A1PcS4, A1PcS2) act on pancreatic cells via energy and calcium-dependent activation and transduction pathways. The photodynamic release of amylase was found to be energy dependent and inhibited by the chelation of free cytoplasmic calcium but not by the removal of extracellular calcium. In contrast to their effects on normal acinar cells, the photodynamic action of A1PcS4 and A1PcS2 was to inhibit amylase secretion from pancreatoma AR4-2J cells. Removal of extracellular calcium reversed this inhibitory effect on AR4-2J cells and produced a significant increase in amylase release, but chelation of free cytoplasmic calcium did not affect the inhibitory photodynamic action of the phthalocyanines on amylase release from the tumour cells. Overall, these results demonstrate further important distinctions between the photodynamic action of sulphonated aluminium phthalocyanines on normal versus tumour exocrine cells of the pancreas and indicate that calcium plays an important role in photodynamic drug action, since these agents affected intracellular calcium mobilisation at some distal point in the membrane signal transduction pathway for regulated secretion. Furthermore, the photodynamic inhibition of constitutive secretion in tumour cells may involve a calcium-dependent membrane target site or modulation of membrane calcium channels by activation of protein kinase C.

Amylases↗

PubMed1994

Photon pharmacology of an iron-sulphur cluster nitrosyl compound acting on smooth muscle.

1. The mechanisms of action on smooth muscle of the iron-sulphur cluster nitrosyl compound, heptanitrosyl-tri-mu 3-thioxotetraferrate (1-), (RBS), a photosensitive nitric oxide donor, have been investigated in the guinea-pig taenia caeci (coli) in vitro. 2. After exposure to RBS (50 microM) for 30 min, and subsequent washout, a sustained contraction was recorded in the absence of light to either the agonist carbachol (50 microM) or a depolarizing concentration of KCl (23.5 mM). Photon irradiation (> 400 nm) caused a prompt relaxation of precontracted RBS-treated muscle, the magnitude of which depended upon the intensity (1.1 x 10(3) to 1.1 x 10(5) lux), duration (30 s to 20 min) and wavelength (400 to 800 nm), of the incident illumination. 3. Repeated periods of illumination at 1.1 x 10(4) lux produced a reversible relaxation of both carbachol and KCl-evoked tone in muscle pretreated with RBS (50 microM). These photorelaxations were reproducible at 10 min intervals for several hours with a maximal relaxation amounting to 80 to 90% that of the tone produced by carbachol (50 microM). 4. The nitric oxide synthase inhibitor, NG-nitro-L-arginine (60 microM), caused no inhibition of the photon-induced relaxation of RBS-treated muscle. In contrast, N-methylhydroxylamine (2 mM), L-cysteine (10 mM), DL-dithiothreitol (2 mM), methylene blue (30 microM), and haemoglobin (20 microM), all reversibly but significantly inhibited (P < 0.001) the photorelaxation response. However, neither the aminothiol N-acetyl-L-cysteine (10 mM) nor the tripeptide glutathione (10 mM) blocked the RBS-induced photorelaxation. 5 The photolytic cleavage of RBS depended on the intensity and duration of illumination; it was accompanied by a corresponding decrease in absorbance and by the liberation of NO as measured by the Griess diazo reaction with sulphanilic acid. L-Cysteine (10 mM) prevented the decrease in absorbance and the photolytic liberation of NO.6 It is concluded that (i) sequestered or bound RBS, when photon-activated, liberates NO by a process which can be controlled by the wavelength, intensity and duration of the incident light, (ii) the photon-released NO rapidly relaxes the smooth muscle cells of the taenia coli primarily via cyclic GMP-dependent pathways which can be blocked by use of appropriate inhibitors, and (iii) the RBS-induced photorelaxation effect does not involve the activation of NO synthase. RBS is therefore a valuable photosensitive NO donor for establishing the functional and pharmacological significance of NO.

Animals↗

PubMed1993

Photodynamic drug action on isolated rat pancreatic acini. Mobilization of arachidonic acid and prostaglandin production.

Chloro-aluminium phthalocyanine sulphonate (SALPC) when photon-activated generates singlet oxygen, elicits amylase release and causes plasma membrane permeabilization of pancreatic acinar cells (Matthews and Cui, Biochem Pharmacol 39: 1444-1457, 1990). Amylase release precedes membrane permeabilization suggesting that the initial release of amylase may be due to direct stimulation by singlet oxygen of secretagogue receptors or their coupled guanine nucleotide binding proteins (G-proteins) and effector systems including phospholipase A2 (PLA2). The aim of the experiments reported here was to establish the extent to which PLA2 activation, arachidonic acid mobilization, and prostaglandin production are involved in the photon-induced action of SALPC on dispersed, perifused acini isolated from the rat pancreas. The mobilization of arachidonic acid by a major secretory stimulant of pancreatic exocrine cells, cholecystokinin octapeptide, was also assessed: it produced a time- and concentration-dependent (10(-10)-10(-6) M) stimulation of arachidonic acid output from acini prelabelled with [1-14C]arachidonic acid. In contrast, the kinetics of arachidonic acid mobilization with photon-activated SALPC 1 microM, 4500 or 18,400 lux light intensity (lambda > 570 mm), was biphasic, an intensity-dependent stimulation being preceded by a more immediate initial inhibition of output. Light activation of SALPC and singlet oxygen generation may evoke the stimulatory phase of arachidonic acid release by an action on G-proteins, or by PLA2 activated directly, or via calcium influx, because NaF 20 mM, mellitin 2 mg/mL and the calcium ionophore A23187 1 microM caused a 2.9-, 33- and 5-fold increase, respectively, in arachidonic acid output. However, not only was the arachidonate stimulation delayed in response to SALPC but in other experiments designed to gain more insight into the turnover of arachidonic acid and its metabolites, the photodynamic release of amylase preceded maximum prostaglandin E2 (PGE2) output and amylase release was completely unaffected when PGE2 production was blocked by the cyclo-oxygenase inhibitor, indomethacin 10 microM. It is therefore likely that the rapid initial photodynamic release of amylase from pancreatic acini induced by SALPC is mediated by activation of the signal transduction pathway involving the release of intracellular calcium; arachidonic acid mobilization and prostanoid production may then be linked to the longer-term, cytolytic action of SALPC, especially in tumour cells.

Amylases↗

PubMed1993

Photodynamic action of aluminium phthalocyanine tetrasulphonate (A1PcS4) on smooth muscle: effects of thiols and a cyclic GMP analogue.

1. The smooth muscle system of the guinea-pig taenia caeci has been used in vitro to characterize the photodynamic action of aluminium phthalocyanine tetrasulphonate (A1PcS4) in the presence or absence of the thiol reductants L-cysteine (Cys), N-acetyl-L-cysteine (NAC), DL-dithiothreitol (DTT) or reduced glutathione (GSH). 2. In all photodynamic experiments the muscle was exposed to A1PcS4 (10(-5) M) for 30 min, followed by a 30 min washout period before photon irradiation at 32,000 lux (lambda > 570 nm) for 30 min. Photodynamic contractions were measured relative to the contractile response to carbachol (5 x 10(-5) M) and relaxation responses were determined in muscle precontracted with either carbachol 5 x 10(-5) M or KCl 23.5 mM. 3. Photon-activation of A1PcS4-sensitized smooth muscle evoked a triphasic response: an initial transient contraction and subsequent relaxation followed by a secondary sustained contraction. Cys 10 mM, NAC 10 mM and DTT 5 mM had no effect on the initial photodynamic contraction but significantly decreased the magnitude of the sustained contraction from mean values of 98% to 18%, 95% to 72% and 93% to 6% of the standard carbachol contraction (5 x 10(-5) M), respectively; GSH 10 mM was without significant effect on either the initial or sustained contraction. 4. In the absence of extracellular calcium the A1PcS4-sensitized smooth muscle did not respond to photon activation but re-introduction of calcium after cessation of illumination produced a sustained contraction which was markedly inhibited by Cys 10 mM. 5. In precontracted AlPcS4-treated muscle preparations photon activation produced a triphasic relaxation response, i.e. a rapid relaxation followed by a transient contraction and a secondary more sustained relaxation. The sustained phase of photodynamic relaxation was potentiated significantly by Cys 10 mM,NAC 10 mM, DTT 5 mM and GSH 10 mM, the relaxation being approximately doubled in magnitude from mean values of 34% to 68%, 30% to 73%, 34% to 68%, and 48% to 77%, respectively, relative to the standard carbachol (5 x l0-5 M) response.6. The cyclic GMP analogue, 8-(4-chlorophenylthio)-guanosine-3':5'-cyclic monophosphate (8-PCPTcGMP)(2 x 10-4 M) alone caused a triphasic relaxation response similar to that produced by photon activation of an AIPcS4-sensitized precontracted preparation in the presence of thiol reductants. The pattern of 8-PCPT-cGMP-induced relaxation was similar in muscle precontracted with carbachol 5 x 10-5M or KCI 23.5 mM.7. It is concluded that the rapid generation of reactive intermediates by photon-activation of boundAlPcS4 leads to membrane permeabilization, calcium entry and muscle contraction. These effects may be opposed by a direct stimulatory action of singlet oxygen on guanylate cyclase which is enhanced by the action of thiol reagents and mimicked by the cyclic GMP analogue, 8-PCPT-cGMP.

Animals↗

PubMed1990

Photodynamic action of sulphonated aluminium phthalocyanine (SALPC) on isolated rat pancreatic acini.

The photodynamic action of SALPC has been investigated on dispersed, perifused, acini isolated from the rat pancreas. Stimulation of secretion was assessed by measuring amylase release and membrane permeabilization determined by the leakage of cytoplasmic lactate dehydrogenase (LDH) and by the efflux of 86Rb from preloaded acini. Light alone (greater than 570 nm, less than or equal to 18,400 lux), or SALPC (less than or equal to 1 microM) in the absence of light, had no effect on pancreatic acini but cellularly bound SALPC when illuminated caused a dose-dependent, light intensity-dependent and temperature-dependent release of amylase. Singlet oxygen generated by photon-activation of SALPC was measured by the formation of an imidazole adduct and bleaching of the secondary substrate, RNO. Whereas illumination caused a rapid increase in photodynamically-evoked pancreatic amylase release, the efflux of 86Rb and loss of cytosolic LDH were markedly delayed in onset: similar results were obtained with monochromatic laser light (633 nm). In contrast, the muscarinic agonist bethanechol evoked a rapid increase in amylase release but with an almost immediate efflux of 86Rb. Finally, electron microscopy confirmed that the structural integrity of the pancreatic acinar cells was maintained after the photodynamic action of SALPC. It is concluded that the stimulation of amylase secretion and membrane permeabilization by SALPC is due to the generation of singlet oxygen. However, the consistent difference between the time course of amylase secretion and membrane permeabilization makes it likely that an initial stage in photodynamic drug action involves oxidation of plasma membrane protein and activation of secretagogue receptors or the G-proteins and their effector systems.

Amylases↗

PubMed1990

Photodynamic action of sulphonated aluminium phthalocyanine (SALPC) on AR4-2J cells, a carcinoma cell line of rat exocrine pancreas.

The photodynamic effects of sulphonated aluminium phthalocyanine (SALPC) have been compared on cultured AR4-2J cells of a pancreatic carcinoma cell line and on exocrine cells of the normal phenotype freshly isolated from the rat pancreas; a multi-channel perifusion system was used for this kinetic study in vitro. Whereas light alone or SALPC alone was without effect on either cell type, photon activation of cellularly-bound SALPC with light greater than 570 nm permeabilised the cells and caused an increase in amylase secretion from normal acinar cells but a dose-dependent inhibition (10(-7) to 10(-5) M) of amylase release from AR4-2J cells. In contrast, direct permeabilisation of the plasma membrane with digitonin, 10 micrograms ml-1, evoked a marked release of amylase from both types of cell. Elevation of [Ca2+]i by the ionophore A23187, 10(-6) M, elicited secretion of amylase from normal cells but had little effect on AR4-2J cells. Finally, it was established that the differential photodynamic effects of SALPC on amylase release were not attributable to any topographical differences in the microanatomical organisation of normal or tumour-derived cells; furthermore, the structural integrity of normal and AR4-2J cells was maintained after the photodynamic action of SALPC. It is concluded that the generation of singlet oxygen is responsible for permeabilisation of both types of cell and that photon-activated SALPC has functionally distinct effects on the constitutive secretion of amylase of tumour cells and the regulated secretory pathway of normal cells. These observations may be important in the development of drugs with a selective photodynamic action on pancreatic tumour cells.

Amylases↗

PubMed1989

Photodynamic action of rose bengal on isolated rat pancreatic acini: stimulation of amylase release.

The halogenated fluorescein derivative, rose bengal, upon photon activation, elicits amylase secretion from isolated, perifused pancreatic acini. This effect is due to production of highly reactive singlet delta oxygen which can permeabilize the cell membrane and may also react chemically with secretagogue receptors, or other functional components of the membrane such as the G-proteins. The profile of photodynamically induced amylase secretion is anion-dependent: it becomes biphasic when the chloride ion is substituted by the glutamate ion, an effect attributed to the action of glutamate on the ionic transport systems of the zymogen granule membrane.

Amylases↗

PubMed1988

Cyclic GMP inhibits protein kinase C-mediated secretion in rat pancreatic acini.

Dibutyryl cyclic GMP (Bu2cGMP) inhibited agonist-induced secretion of amylase from isolated rat pancreatic acini. In contrast to previous studies, this inhibitory action was not confined to butyryl derivatives of cyclic GMP, since the membrane-permeant cyclic GMP analogues Bu2cGMP and cyclic 8-bromo-GMP (8-Br-cGMP) were equipotent (IC50 2 nM) in their inhibition of amylase secretion stimulated by cholecystokinin-(26-33)-octapeptide (CCK8): at extracellular concentrations up to 1 mM, cyclic GMP itself was devoid of inhibitory activity. Both Bu2cGMP and 8-Br-cGMP also potently inhibited secretion stimulated by 4 beta-phorbol 12-myristate 13-acetate (PMA) (IC50 6 nM), but only partially inhibited responses elicited by bethanechol or bombesin and were without effect on A23187-evoked secretion. Furthermore, agents that are known to raise intracellular cyclic GMP levels (MB22948 (2-o-propoxyphenyl-8-azapurin-6-one) or nitroprusside) or antagonize the actions of protein kinase C (4 alpha-PMA or staurosporine), also inhibited CCK8- or PMA-stimulated secretion but not secretion elicited by bombesin, bethanechol, or A23187. It is concluded from these and other observations reported here that protein kinase C is the major intracellular mediator of amylase secretion stimulated by CCK8 and that this pathway may be regulated by cyclic GMP at a step that follows protein kinase C activation.

Amylases↗

PubMed1988

Effect of leukotriene C4 on electromechanical activity and Ca2+ uptake in taenia coli.

The actions of leukotriene C4 (LTC4) on electromechanical activity and 45Ca2+ uptake in guinea pig taenia coli were investigated. The contractile action of LTC4 was abolished by the removal of extracellular Ca2+. LTC4 concentrations eliciting a maximal contraction in normal medium produced no response in preparations depolarized with KCl. In single sucrose gap studies, LTC4 increased both the frequency of electrical spiking and tension. These effects were blocked by the dihydropyridine Ca2+-channel antagonist PY 108-068 and by the leukotriene receptor antagonist FPL 55712. In double sucrose gap experiments, LTC4 caused a small depolarization without measurable change in membrane conductivity; increased spontaneous electrical activity was again accompanied by an increase in tension. LTC4 caused a detectable increase in 45Ca2+ uptake only at extracellular Ca2+ concentrations less than 1 mM, and this was again inhibited by PY 108-068 or FPL 55712. It is concluded that the contractile effects of LTC4 in guinea pig taenia coli occur as a consequence of its ability to open voltage-sensitive Ca2+ channels, an effect that may occur independently of membrane depolarization.

Animals↗

PubMed1987

Permeabilization of thymocytes by photon activation of erythrosin.

Thymocytes previously loaded with quin 2 were rapidly permeabilized by the photon activation of erythrosin and the rate of permeabilization monitored by measuring fluorimetrically the increasing saturation of quin 2 with calcium. The extent of permeabilization was assessed also by the loss of [3H]quin 2 from the thymocytes and penetration of the cells by eosin and trypan blue. Lactate dehydrogenase leakage from the permeabilized cells was markedly delayed compared to the rapid increase in permeability to calcium and quin 2. The rate of permeabilization was dependent upon the concentration of erythrosin, the duration of illumination, the presence of oxygen, and the temperature. These results are consistent with the rapid photochemical generation of highly reactive singlet oxygen which alters thymocyte membrane structure and permeability.

Aminoquinolines↗

PubMed1987

Effects of a cytosolic protein on the interaction of rat pancreatic zymogen granules in vitro.

Photon correlation spectroscopy has been used to study the kinetics of aggregation of isolated rat pancreatic zymogen granules in vitro by monitoring time-dependent changes in mean particle size derived from the photon count autocorrelation function, g2(tau). Isolated granules were stable in isotonic sucrose (pH 5.4-7.0). At pH 6.0 they maintained a mean diameter of 1225 +/- 18 nm with a polydispersity index of 0.199 +/- 0.007. The mean granule diameter showed a limited decrease (approx. 20%) with increasing pH within the range 5.4-7.0, but the polydispersity index was unaltered. At pH greater than 7.0 granule instability was indicated by a rapid reduction in total photon counts. In solutions of monovalent cations ([M+] greater than 10 mM) and divalent cations ([M2+] greater than 0.5 mM) zymogen granules aggregated at a rate dependent upon both ion and granule concentration. These effects were consistent with the bimolecular nature of the interaction mechanism and were clearly distinguishable from the limited size changes associated with osmolarity. At concentrations of Na+ or K+ salts greater than 50 mM granule aggregation was accompanied by anion-dependent solubilisation. A soluble protein fraction separated from the pancreatic acinar cell cytosol by gel filtration reduced the mean diameter and polydispersity index of zymogen granules suspended in isotonic sucrose, inhibited cation-induced aggregation and stabilised granules to solubilisation induced by raising pH greater than 7.0 or exposure to high ionic strength media. The inhibitory effects of this protein were apparent at concentrations less than or equal to 10 micrograms X ml-1 (i.e. at inhibitor: granule protein ratios less than 1:20) and could not be mimicked by bovine serum albumin, the Ca2+-binding proteins calmodulin and troponin C (less than or equal to 100 micrograms X ml-1), nor the highly negatively charged polymer polyglutamate (less than or equal to 10 micrograms X ml-1). Inhibitory activity was also absent from fractions of rat liver cytosol prepared identically to pancreatic acinar cytosol. These observations are consistent with the presence in pancreatic acinar cells of a specific cytosolic granule stabilisation factor (or factors) that normally restricts zymogen granule interaction and may therefore play an important role in the regulation of granule mobility and exocytosis.

Animals↗