Biomedical subjects

R A Akhtar

Publications and source records attributed to R A Akhtar.

At least 19 recordsLinked to original sources

PubMed2005

Species differences in the effects of prostanoids on MAP kinase phosphorylation, myosin light chain phosphorylation and contraction in bovine and cat iris sphincter smooth muscle.

There is evidence from our own laboratory and that of others that EP-receptor ligands are strong contractile agonists in bovine iris sphincter and that FP-receptor agonists are strong contractile agonists in cat iris sphincter. Here, we have investigated the effects of prostaglandin (PG) receptor agonists of the FP-, EP-, TP- and DP-class on myosin light chain (MLC) phosphorylation, p42/p44 MAP kinase phosphorylation and contraction in the iris sphincter of bovine and cat. Using three signal transduction mechanism assays, namely MLC phosphorylation, MAP kinase phosphorylation and contraction, we demonstrated that in bovine iris sphincter the rank order of potency of the PG agonists in the contractile and MLC phosphorylation assays is as follows: E2>U46619>F2alpha>D2, and in cat F2alpha>D2>E2>U46619. In the MAP kinase assay, in bovine iris sphincter the rank order of potency is E2>F2alpha and in cat F2alpha>E2. These conclusions are supported by the following findings: (1) In the contractile assay, in the bovine sphincter the EC50s for PGF2alpha, PGE2, U46619 and PGD2 were found to be 1.4x10(-7), 5.0x10(-9), 9.0x10(-9) and 1.3x10(-6)M, respectively, and the corresponding values in the cat were 1.9x10(-8), 2.3x10(-7), 1.5x10(-6) and 6.9x10(-8)M, respectively. (2) In the MLC phophorylation assay, in the bovine sphincter PGF2alpha, PGE2, U46619 and PGD2 increased MLC phophorylation by 118%, 165%, 153% and 72%, respectively, and the corresponding values in cat were 175%, 99%, 90% and 95%, respectively. (3) In the MAP kinase assay, in the bovine iris sphincter PGF2alpha and PGE2, increased MAP kinase phosphorylation by 276% and 328%, respectively, and the corresponding values in cat were 308% and 245%, respectively. The data presented demonstrate pronounced species differences in the effects of the prostanoids on the MLC kinase signaling pathway in bovine and cat irides and furthermore confirm the existence of FP-receptors in that of the bovine.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed2001

Upregulation of phospholipase Cgamma1 activity during EGF-induced proliferation of corneal epithelial cells: effect of phosphoinositide-3 kinase.

PURPOSE: Previously, the authors showed that epidermal growth factor (EGF) stimulates phospholipase Cgamma1 (PLCgamma1) and phosphoinositide-3 kinase (PI3K) activities in confluent rabbit corneal epithelial cells (RCECs). The purpose of this study was to investigate whether PLCgamma1 activity is upregulated during EGF-induced proliferation of RCECs and to determine whether there is any cross-talk between PLCgamma1 and PI3K in these cells. METHODS: Simian virus (SV)-40-immortalized RCECs were cultured in the presence and absence of EGF and other agents. At prescribed time intervals, the cultures were terminated and the cells counted. PLCgamma1 activity in intact cells was assessed by measuring the production of [(3)H]IP(3) in [(3)H]myoinositol-labeled cells. The in vitro enzyme activity was assayed using immunoprecipitated PLCgamma1 and [(3)H]PI(4,5)P(2) as substrate. [(3)H]IP(3), the product of PLCgamma1, was analyzed by anion-exchange chromatography. The changes in protein content and level of phosphorylation of PLCgamma1 were determined by Western immunoblot analysis, with the appropriate antibodies. RESULTS: Addition of EGF (50 ng/ml) caused a time-dependent increase in proliferation of RCECS. The effect of EGF peaked at approximately 36 hours. Under the same experimental conditions, EGF stimulated PLCgamma1 activity with a time course similar to that of cell proliferation. Data from Western immunoblot analysis revealed that the EGF-stimulated PLCgamma1 activity was due to increased synthesis of the enzyme. Furthermore, during cell proliferation, tyrosine phosphorylation of PLCgamma1 increased in a time-dependent manner that corresponded closely with the expression of PLCgamma1. EGF exerted its effects both on cell proliferation and PLCgamma1 activation in a dose-dependent manner. Treatment of the cells with U-73122, a PLC inhibitor, or myr-GLYRKAMRLRY, a myristoylated PLCgamma1 inhibitor peptide, caused attenuation of both the EGF-stimulated cell proliferation and PLCgamma1 activity. Treatment of the cells with the PI3K inhibitors, wortmannin or LY294002, caused inhibition of both EGF-stimulated cell proliferation and PLCgamma1 activation. Addition of PI(3,4,5)P(3) to the in vitro PLCgamma1 assay mixture stimulated the enzyme activity in a dose-dependent manner. CONCLUSIONS: The data suggest a positive correlation between EGF-stimulated PLCgamma1 activation and cell proliferation in RCECS. The EGF-stimulated PLCgamma1 activity was mirrored by increased synthesis and tyrosine phosphorylation of the enzyme. The data also show that PLCgamma1 activation and cell proliferation were inhibited by PI3K inhibitors, suggesting a role for PI3K in EGF-stimulated proliferation of corneal epithelial cells.

Animals↗

PubMed2000

Genetic variation of basal iron status, ferritin and iron regulatory protein in mice: potential for modulation of oxidative stress.

Toxic and carcinogenic free radical processes induced by drugs and other chemicals are probably modulated by the participation of available iron. To see whether endogenous iron was genetically variable in normal mice, the common strains C57BL/10ScSn, C57BL/6J, BALB/c, DBA/2, and SWR were examined for major differences in their hepatic non-heme iron contents. Levels in SWR mice were 3- to 5-fold higher than in the two C57BL strains, with intermediate levels in DBA/2 and BALB/c mice. Concentrations in kidney, lung, and especially spleen of SWR mice were also greater than those in C57BL mice. Non-denaturing PAGE of hepatic ferritin from all strains showed a major holoferritin band at approximately 600 kDa, with SWR mice having > 3-fold higher levels than C57BL strains. SDS PAGE showed a band of 22 kDa, mainly representing L-ferritin subunits. A trace of a subunit at 18 kDa was also detected in ferritin from SWR mice. The 18 kDa subunit and a 500 kDa holoferritin from which it originates were observed in all strains after parenteral iron overload, and there was no major variation in ferritin patterns. Although iron uptake studies showed no evidence for differential duodenal absorption between strains to explain the variation in basal iron levels, acquisition of absorbed iron by the liver was significantly higher in SWR mice than C57BL/6J. As with iron and ferritin contents, total iron regulatory protein (IRP-1) binding capacity for mRNA iron responsive element (IRE) and actual IRE/IRP binding in the liver were significantly greater in SWR than C57BL/6J mice. Cytosolic aconitase activity, representing unbound IRP-1, tended to be lower in the former strain. SWR mice were more susceptible than C57BL/10ScSn mice to the toxic action of diquat, which is thought to involve iron catalysis. If extrapolated to humans, the findings could suggest that some people might have the propensity for greater basal hepatic iron stores than others, which might make them more susceptible to iron-catalysed toxicity caused by oxidants.

Absorption↗

PubMed2000

Epidermal growth factor stimulates phospholipase cgamma1 in cultured rabbit corneal epithelial cells.

Phospholipase Cgamma1 (PLCgamma1) catalyses hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol 1,4,5-trisphosphate (IP(3)), two second messengers which play important roles in cell proliferation and differentiation. The purpose of the current study was to identify PLCgamma1 in corneal epithelial cells and investigate whether epidermal growth factor (EGF) stimulates the activity of this enzyme. Addition of EGF to [(3)H]myo-inositol-labeled, cultured corneal epithelial cells stimulated production of IP(3), indicating activation of PLC. Western immunoblot analysis and an in vitro assay of PLC activity revealed that EGF activates gamma1 isoform of PLC, which is localized predominantly in the cytosolic fraction of the epithelial cells. EGF receptors were detected in the epithelial cells by EGF receptor antibody. Addition of EGF to the cells caused tyrosine phosphorylation of the receptors, translocation of PLCgamma1 from cytosol to plasma membrane, and phosphorylation of the enzyme at tyrosine residues. Addition of tyrphostin A-25, an inhibitor of receptor tyrosine kinase, attenuated the tyrosine phosphorylation of PLCgamma1 as well as its enzyme activity. These findings suggest that EGF stimulates PLCgamma1 in rabbit corneal epithelial cells, and that this effect is probably mediated by tyrosine phosphorylation of the enzyme.

Animals↗

PubMed2000

Effects of atrial natriuretic peptide and sodium nitroprusside on epidermal growth factor-stimulated wound repair in rabbit corneal epithelial cells.

PURPOSE: Treatment of rabbit corneal epithelial cells (RCEC) with epidermal growth factor (EGF) stimulates cell proliferation and wound repair in a cell culture model system. Studies have also shown that atrial natriuretic peptide (ANP) and sodium nitroprusside (SNP), a nitric oxide-generating agent, inhibit proliferation of a variety of cell types. The aim of the present work was to examine whether ANP or SNP has any effect on EGF-stimulated proliferation of RCEC involved in wound repair. METHODS: The SV-40 immortalized RCEC were cultured in 24-well plates until they became confluent. Wounds of uniform size (8 mm diameter) were created and the cells allowed to grow in the presence and absence of EGF and/or other agents. At prescribed time intervals, the cells were stained by Giemsa and the wound areas digitized and quantified by Sigma Image Scan System. The cGMP contents in RCEC, treated with or without ANP or SNP, were measured by radioimmunoassay. RESULTS: Addition of EGF (1-100 ng/ml) to RCEC stimulated cell proliferation which significantly reduced the time required for wound closure. Addition of ANP (1 nM to 10 microM) or SNP (10 microM to 1 mM), in the presence of EGF, dose-dependently inhibited the growth factor-stimulated wound closure in RCEC. When added alone to the cells, ANP or SNP increased cGMP accumulation in a dose-dependent manner. Addition of ANP (1 microM) or SNP (1 mM) to primary corneal epithelial cells, in the presence and absence of EGF, also inhibited the wound closure with a corresponding increase in cGMP contents. Treatment of the cells with ODQ (10 nM to 10 microM), a soluble guanylate cyclase inhibitor, dose-dependently decreased the SNP-induced accumulation of cGMP, and reversed the inhibitory effect of SNP on EGF-stimulated wound closure. Addition of membrane-permeable cGMP analog, 8-bromo-cGMP, to RCEC inhibited the EGF-stimulated wound closure in a dose-dependent manner. Treatment of RCEC with mitomycin C (5 microM) exerted a marked inhibitory effect on wound closure in the presence and absence of EGF, and also abrogated the inhibitory effect of 8-bromo-cGMP on wound closure in the EGF-treated and untreated cells. CONCLUSIONS: The results demonstrate that ANP and SNP inhibit the EGF-stimulated wound repair in RCEC. The effect of these agents is mediated via activation of guanylate cyclases that generate cGMP. Cyclic GMP appears to exert its inhibitory effect at the level of cell proliferation and not cell migration. The data suggest an important role for cGMP-dependent protein kinase in proliferation of RCEC stimulated by EGF.

Animals↗

PubMed1999

Expression of phosphatidylinositol 3-kinase during EGF-stimulated wound repair in rabbit corneal epithelium.

PURPOSE: To investigate the effect of epidermal growth factor (EGF) on the induction of phosphatidylinositol 3-kinase (PI 3- kinase) gene expression during rabbit corneal epithelial wound repair. METHODS: Epithelial wounds (6 mm in size) were created in rabbit corneas and EGF (2 microg) applied every 8 hours to one eye, and the other eye served as a control. The wound repair was monitored by staining the tissue with fluorescein followed by photography. The wound area was quantified with a computer program. At different time intervals, the rabbits were killed and the corneal epithelium used for estimation of PI 3-kinase activity, western blot analysis, or reverse transcription-polymerase chain reaction (RT-PCR). For in situ hybridization, the whole corneas were sectioned and the sections processed with PI 3-kinase mRNA probes. RESULTS: In the untreated eye, the epithelial wound progressively healed in a time-dependent manner, with 75% of the wound closed at 48 hours post wounding. Application of EGF to the corneal epithelium further stimulated wound repair at all time intervals, and the wound was completely closed at 48 hours. Analysis of PI 3-kinase showed a time-dependent increase in its enzyme activity that was maximally increased at 36 hours, the time when the wound was nearly closed. Western blot analysis revealed increased amounts of PI 3- kinase protein during the course of wound repair. Analysis of RT-PCR products from epithelial tissues, taken at different times during wound repair, showed increased PI 3-kinase expression that was maximum at 48 hours post wounding. A visible increase in PI 3-kinase gene expression was also detected by in situ hybridization during the course of the wound repair. This expression was increased maximally by EGF at 48 hours post wounding. CONCLUSIONS: The results indicate a temporal correlation between increased activation and expression of PI 3- kinase and the epithelial wound repair. Topical application of EGF further stimulates the activity and expression of PI 3- kinase. It is suggested that PI 3- kinase and its products may play a role in EGF-induced cell proliferation during corneal epithelial wound repair.

Animals↗

PubMed1998

Epidermal growth factor stimulates phospholipase D independent of phospholipase C, protein kinase C or phosphatidylinositol-3 kinase activation in immortalized rabbit corneal epithelial cells.

PURPOSE: Activation of phospholipase D (PLD) is believed to be an important signaling pathway involved in cell growth and differentiation in several tissues, in response to a variety of mitogens. The aim of the present study was to investigate the effect of epidermal growth factor (EGF) on PLD activity in rabbit corneal epithelial cells (RCEC). We have also examined whether the EGF effect is dependent on concurrent activation of phospholipase C (PLC), protein kinase C (PKC) or phosphatidylinositol 3-kinase (PI-3-kinase) in these cells. METHODS: RCEC, immortalized with adenovirus SV-40, were cultured until they became confluent. The cells were labeled with [3H]myristic acid and incubated with or without EGF or other agents for specified time intervals. PLD activity was measured by quantifying [3H]phosphatidylethanol in cells incubated in the presence of ethanol. PLC activity was determined by measuring the radioactivity in inositol trisphosphate in myo[3H]inositol-labeled RCEC. PI 3-kinase activity was assessed by measuring the production of PIP3 in 32P-labeled cells. RESULTS: Addition of EGF to RCEC stimulated PLD activity in a time- and dose-dependent manner. The maximal effect was observed with 150 ng/ml EGF and at 10 min of incubation. The PLD activity was also stimulated when phorbol myristate acetate (PMA) was added to the cells. Treatment of the cells with EGF stimulated PLC activity which was inhibited by U73122, a PLC inhibitor. Under the same experimental conditions, the inhibitor had no effect on EGF-stimulated PLD activity. Down-regulation of PKC or treatment of the cells with RO31-8220, a PKC inhibitor, inhibited the PMA- but not EGF-stimulated PLD activity. Incubation of the cells with wortmannin, a PI 3-kinase inhibitor, abolished the EGF-stimulated PI 3-kinase activity, but potentiated the EGF-stimulated PLD activity. The EGF effect was inhibited by treatment of the cells with tyrphostin B42, a receptor tyrosine kinase inhibitor. CONCLUSIONS: These results indicate that EGF stimulates PLD activity in RCEC by a mechanism that involves tyrosine phosphorylation of a protein(s) in the cascade of biochemical reactions initiated by EGF-receptor interaction, and it is not dependent on concurrent activation of PKC, PLC, or PI 3-kinase in these cells.

Animals↗

PubMed1998

Chromosomal linkage analysis of porphyria in mice induced by hexachlorobenzene-iron synergism: a model of sporadic porphyria cutanea tarda.

Genetic susceptibility to toxic chemicals is of major importance but most studies concentrate on candidate genes and searches for unknown susceptibility genes are uncommon. Human sporadic porphyria cutanea tarda is usually precipitated by alcohol, oestrogens, hepatitis viruses, HIV or haemodialysis. The mechanism is not known but there is a role for iron metabolism and an underlying genetic predisposition is suspected. A similar porphyria in humans has also been caused by hexachlorobenzene. These human porphyrias can be modelled in iron-loaded mice exposed to hexachlorobenzene, in which C57BL/10ScSn is a prototype susceptible strain whereas DBA/2 mice are extremely resistant. A search for susceptibility genes was undertaken using complex trait analysis with DNA microsatellite markers of 'high' and 'low' responders from an F2 intercross. Correlation of markers with susceptibility, defined as accumulation of uroporphyrin in the liver, was assessed by chi-squared test for the proportion of C57BL/10ScSn and DBA/2 alleles present. Susceptibility loci on chromosomes 12, 14 and 17 were identified. Further analysis of markers on chromosomes 14 and 17 by MAPMAKER/EXP and MAPMAKER/QTL gave LOD scores of 7.3 and 3.6, respectively. Typing of chromosome 12 for the Ahr gene, using a restriction fragment length polymorphism distinguishing between the b-1 and d alleles, gave significant but not perfect linkage. However, no strong association between alleles or expression of Cyp1a1/2 genes, regulated by Ahr, and susceptibility for porphyria was detected. The results demonstrate that the porphyria induced by hexachlorobenzene in C57BL/10ScSn mice is a complex trait determined by at least three genes, which may be of relevance to susceptibility in the development of sporadic porphyria cutanea tarda and unknown aspects of liver damage.

Animals↗

PubMed1997

Inhibition of muscarinic-stimulated polyphosphoinositide hydrolysis and Ca2+ mobilization in cat iris sphincter smooth muscle cells by cAMP-elevating agents.

The effects of carbachol (CCh) on inositol 1,4,5-trisphosphate (IP3) production and intracellular calcium ([Ca2+]i) mobilization, and their regulation by cAMP-elevating agents were investigated in SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. CCh produced time- and dose-dependent increases in IP3 production; the t1/2 and EC50 values were 68 s and 0.5 microM, respectively. The muscarinic agonist provoked a transient increase in [Ca2+]i which reached maximum within 77 s, and increased [Ca2+]i mobilization in a concentration-dependent manner with an EC50 of 1.4 microM. Thapsigargin, a Ca(2+)-pump inhibitor, caused a rapid rise in [Ca2+]i and subsequent addition of CCh was without effect. Both CCh-induced IP3 production and CCh-induced [Ca2+]i mobilization were more potently antagonized by 4-DAMP, an M3 muscarinic receptor antagonist, than by pirenzepine, an M1 receptor antagonist, suggesting that both responses are mediated through the M3 receptor subtype. Treatment of the cells with U73122, a phospholipase C (PLC) inhibitor, resulted in a concentration-dependent decrease in both CCh-stimulated IP3 production and [Ca2+]i mobilization. These data indicate close correlation between enhanced IP3 production and [Ca2+]i mobilization in these smooth muscle cells and suggest that the CCh-stimulated increase in [Ca2+]i could be mediated through increased IP3 production. Isoproterenol (ISO) inhibited CCh-induced IP3 production (IC50 = 80 nM) and [Ca2+]i mobilization (IC50 = 0.17 microM) in a concentration-dependent manner. Microsomal fractions isolated from SV-CISM-2 cells contained phospholipase C (PLC) which was stimulated by CCh (10 microM) and GTP gamma S (0.1 microM). Pretreatment of the cells with ISO or forskolin, 5 microM each, produced membrane fractions in which CCh-stimulated PLC activity was significantly attenuated. Furthermore, when microsomal fractions isolated from SV-CISM-2 cells were phosphorylated with Protein kinase A (PKA), the CCh- and GTP gamma S-stimulated IP3 production were significantly inhibited. It can be concluded from these studies that in SV-CISM-2 cells, activation of M3 muscarinic receptors results in stimulation of PLC-mediated PIP2 hydrolysis, generating IP3 which mobilizes [Ca2+]i. Furthermore, elevation of cAMP may inhibit IP3 production and [Ca2+]i mobilization through mechanisms involving PKA-dependent phosphorylation of PLC, G-proteins, IP3 receptor and/or IP3 metabolizing enzymes.

Adrenergic beta-Agonists↗

PubMed1997

Epidermal growth factor stimulation of phosphatidylinositol 3-kinase during wound closure in rabbit corneal epithelial cells.

PURPOSE: To determine whether there is an association between epidermal growth factor (EGF)-induced activation of phosphatidylinositol 3-kinase (PI 3-kinase) and stimulation of wound closure in rabbit corneal epithelial cells. METHODS: Immortalized rabbit corneal epithelial cells were cultured in 24-well plates until they became confluent. Circular wounds were created in confluent cultures by cell denudation and then incubated in the absence and presence of EGF for varying intervals. Wound closure was monitored by staining the cells with Giemsa and quantifying the wound area with SigmaS can computer program. Cell proliferation during wound repair was estimated by measuring the incorporation of [3H]thymidine into nuclear DNA. Changes in PI 3-kinase activity were assessed by measuring the production of phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P3] in 32P-labeled cells as well as by immunoprecipitating and assaying PI 3-kinase activity with phosphatidylinositol 4,5-bisphosphate and [gamma-32P]ATP as substrates. The enzyme product, PIP3, was analyzed by a combination of thin-layer and high-pressure liquid chromatography. RESULTS: Addition of 10 ng/ml EGF to the wounded corneal epithelial cells stimulated wound closure in a time-dependent manner, and the wound closed completely within 48 hours. The effect of EGF was dose dependent, and maximal wound closure occurred at 10 ng/ml EGF. As the epithelial cells were undergoing EGF-stimulated wound closure, there was a time-dependent increase in PI 3-kinase activity. The enzyme activity increased maximally at 24 hours and then decreased gradually as the incubation was continued to 48 hours. When the cells were treated with wortmannin, a PI 3-kinase inhibitor, the EGF-stimulated PIP3 formation as well as the wound closure were inhibited significantly. Treatment of the cells with genistein or tyrphostin B42 also decreased both EGF-stimulated PIP3 formation and wound closure in a dose-dependent manner. Concomitant with stimulation of wound repair, the growth factor increased [3H]thymidine incorporation into nuclear DNA, and this effect was inhibited by pretreatment of the cell with wortmannin. CONCLUSIONS: The data suggest a close correlation between EGF-stimulated wound closure and activation of PI 3-kinase in corneal epithelial cells. It can be concluded that PI 3-kinase might be an important component in signal transduction cascade initiated by EGF-receptor interaction, which leads to mitosis and cell proliferation during wound closure in corneal epithelial cells.

Androstadienes↗

PubMed1996

Uroporphyria induced by 5-aminolaevulinic acid alone in Ahrd SWR mice.

In mice, depression of hepatic uroporphyrinogen decarboxylase (UROD) leading to porphyrin accumulation (uroporphyria) occurs with chlorinated ligands of the aryl hydrocarbon (AH) receptor especially after iron overload. However, in the absence of chlorinated ligands, iron itself will eventually cause uroporphyria, but this response is not associated with the Ahr genotype. These effects are potentiated by administration of the haem precursor 5-aminolaevulinate (ALA). The aim of this study was to investigate the effects of ALA alone. Prolonged administration of 2 mg ALA/mL in the drinking water to SWR mice also led to decarboxylase insufficiency (11% of control) and uroporphyria by 8 weeks, whereas DBA/2 mice did not show reduced enzyme activity. Both strains are considered AH nonresponsive and analysis of the Ahr gene using restriction fragment length polymorphism was consistent with SWR, like DBA/2, possessing the Ahrd allele. Exposure of isolated hepatocytes to ALA (150-500 microM) for up to 48 hr showed a significant accumulation of both uroporphyrin and coproporphyrin in the medium, which for uroporphyrin particularly was significantly greater with SWR than with DBA/2 cells. Basal in vivo CYP1A2 activity, measured as microsomal methoxyresorufin dealkylation, was significantly greater in SWR than in DBA/2 mice (1.3-fold), but it was unclear whether this was sufficient to explain the marked difference in sensitivities of the two strains. Despite SWR mice being AH nonresponsive, uroporphyria and decarboxylase depression after an initial iron overload and ALA for 3 weeks were greatly potentiated by a single dose (100 mg/kg) of hexachlorobenzene (a weak AH ligand). The results demonstrate that there is a genetic difference in mice independent of the Ahr genotype and response to iron, which influences the susceptibility to ALA-induced uroporphyria. Thus chemicals, iron and ALA can act independently, but also together, to cause porphyria in susceptible individuals.

Aminolevulinic Acid↗

PubMed1996

Effect of epidermal growth factor on phosphatidylinositol 3-kinase activity in rabbit corneal epithelial cells.

We investigated the effect of epidermal growth factor (EGF) on phosphatidylinositol 3-kinase (PI 3-kinase) activity in SV-40 transformed rabbit corneal epithelial cells (RCEC) and in normal corneal epithelial cells grown in primary culture. The enzyme products, 3-phosphorylated phosphoinositides, were identified by TLC and by deacylation followed by separation on HPLC. Addition of 30 ng ml-1 EGF to 32P-labeled SV-40 transformed or primary epithelial cell culture, increased the radioactivity in phosphatidylinositol 3,4,5-trisphosphate (PIP3) by about 96% over that of control incubations. The growth factor also significantly increased the radioactivity in phosphatidylinositol 4-phosphate [PI(4)P], phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and phosphatidic acid which was quite similar in the two cell types. The EGF-mediated increase in PIP3 formation in RCEC was dose- and time-dependent. Treatment of RCEC lysate with anti-PI 3-kinase antibody immunoprecipitated proteins that phosphorylated PI, PI(4)P and PI(4,5)P2 to corresponding 3-phosphorylated phosphoinositides. Incubation of RCEC with EGF resulted in a dose- and time-dependent increase in PI 3-kinase activity associated with the immunoprecipitate. Nonionic detergent, Nonidet P-40, strongly inhibited the PI 3-kinase activity in the immunoprecipitate, but it stimulated the PI 4-kinase activity in the cell lysate. Wortmannin, a fungal metabolite, also exerted a dose-dependent inhibitory effect on the activity of PI 3-kinase. Analysis of the PI 3-kinase immunoprecipitate by Western blot did not show any increase in concentration of the 85-kDa subunit of PI 3-kinase in EGF-treated cells. However, probing the immunoblot with anti-phosphotyrosine antibody showed a large increase (145%) in phosphorylation of the 85-kDa subunit of the enzyme. It can be concluded from these data that PIP3 and other 3-phosphoinositides exist in RCEC, and that EGF stimulates the production of these lipids by activation of PI 3-kinase, probably by increased tyrosine phosphorylation of the 85-kDa subunit of the enzyme.

Androstadienes↗

PubMed1996

Effects of endothelin on phospholipases and generation of second messengers in cat iris sphincter and SV-CISM-2 cells.

In both immortalized cat iris sphincter smooth muscle cells (SV-CISM-2 cells) and cat iris sphincter, endothelin-1 (ET-1) markedly increased the activities of phospholipase A2 (PLA2), as measured by the release of arachidonic acid (AA), phospholipase C (PLC), as measured by the production of inositol trisphosphate (IP3), and phospholipase D (PLD), as measured by the formation of phosphatidylethanol (PEt). In SV-CISM-2 cells, ET-1 induced AA release, IP3 production and PEt formation in a dose- and time-dependent manner. The dose-response studies showed that the peptide is more potent in activating PLD (EC50 = 1.2 nM) than in activating PLC (EC50 = 1.5 nM) or PLA2 (EC50 = 1.7 nM). The time course studies revealed that ET-1 activated the phospholipases in a temporal sequence in which PLA2 was stimulated first (t1/2 = 12 s), followed by PLC (t1/2 = 48 s) and lastly PLD (t1/2 = 106 s). In SV-CISM-2 cells, in contrast to the intact iris sphincter, sarafotoxin-c, an ETB receptor agonist, had no effect on the phospholipases, and indomethacin, a cyclooxygenase inhibitor, had no effect on the stimulatory effect of ET-1 on the phospholipases. These results suggest that in this smooth muscle cell line, ET-1 interacts with the ETA receptor subtype to activate, via G proteins, phospholipases A2, C and D in a temporal sequence.

Animals↗

PubMed1995

Purification and properties of D-myo-inositol 1,4,5-trisphosphate 3-kinase from bovine iris sphincter smooth muscle: effects of protein phosphorylation in vitro and in intact muscle.

Stimulation of bovine iris sphincter muscle with carbachol (10 microM) increased accumulation of Ins(1,4,5)P3 (InsP3) and Ins(1,3,4,5)P4 (InsP4) by 86 and 32% respectively. Addition of isoproterenol (5 microM) to muscle pretreated with carbachol reduced the 3H-radioactivity in InsP3 by 30% and increased that of InsP4 by 41%. InsP3 3-kinase was predominantly localized in the soluble fraction (110,000 g supernatant) of the iris sphincter. The enzyme was purified from this fraction by sequential chromatography on DEAE-cellulose, calmodulin (CAM)-agarose affinity, and Mono-Q anion-exchange columns. The specific activity of the purified enzyme was 1.94 mumol/min per mg protein with a purification of 114-fold, compared with the cytosolic fraction of the muscle. SDS/PAGE showed the enzyme to be associated with a protein band corresponding to 50 kDa. In the presence of 10 microM Ca2+, CaM dose-dependently stimulated the enzyme. InsP3 3-kinase specifically phosphorylated InsP3 with an apparent K(m) of 0.56 microM and a Vmax. of 2.5 mumol/min per mg protein. The stimulatory effect of CaM was due to a change in Vmax. and not in its K(m). The enzyme was maximally active at pH 7.0-7.5. Phosphorylation of the purified InsP3 3-kinase with protein kinase A increased its activity; in contrast, phosphorylation with protein kinase C inhibited the enzyme activity. Treatment of the intact iris sphincter with isoproterenol or phorbol 12,13-dibutyrate resulted in stimulation of InsP3 3-kinase activity in the soluble fraction and this activation was preserved on SDS/PAGE and renaturation. These results indicate that the bovine iris sphincter contains a Ca-CaM-dependent InsP3 3-kinase which can be differentially regulated, both in vitro and in intact muscle, by protein kinases A and C.

Animals↗

PubMed1995

Effect of carbachol on phospholipase C-mediated phosphatidylinositol 4,5-bisphosphate hydrolysis, and its modulation by isoproterenol in rabbit corneal epithelial cells.

The effects of carbachol (CCh) on phospholipase C(PLC)-mediated phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and its modulation by isoproterenol were investigated in SV40-adenovirus transformed rabbit corneal epithelial cells (RCEC). When examined under light microscope, these cells exhibited a cobblestone-like appearance typical of the corneal epithelial cells grown in primary culture. Addition of CCh (0.1 mM) for 30 min to RCEC, prelabeled with 32Pi, decreased the radioactivity in phosphatidylinositol 4-phosphate and PIP2 by 15 and 27%, respectively, and concomitantly increased the radioactivity in phosphatidylinositol and phosphatidic acid by 14 and 38%, respectively. When the concentration of CCh was increased to 1 mM, the changes in radioactivity were even more pronounced. Addition of CCh (0.1 mM) to the cells, prelabeled with myo[3H]inositol, increased the accumulation of [3H]inositol 1,4,5-trisphosphate ([3H]InsP3) by 115%, indicating stimulation of PLC-mediated PIP2 hydrolysis. Similar increases were also observed in [3H]InsP1 and [3H]InsP2. The effects of CCh on inositol phosphate accumulation were time- and dose-dependent, and were inhibited by atropine (10 microM), suggesting that the observed effects of CCh were mediated by activation of muscarinic cholinergic receptors. The effects of CCh were antagonized more potently by 4-diphenylacetoxy N-methyl-piperidine than by pirenzepine, indicating that the muscarinic receptors involved in PLC activation are probably of M3 type. By Western immunoblotting analysis with various anti-PLC antibodies, the RCEC were shown to contain PLC gamma 1 and PLC delta 1 in the soluble fraction and PLC beta 1 in the microsomal fraction. Addition of isoproterenol to RCEC, increased cAMP both in a time- and dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Agonists↗

PubMed1995

Effects of surgical sympathetic denervation on G-protein levels, alpha and beta-adrenergic receptors, cAMP production and adenylate cyclase activity in the smooth muscles of rabbit iris.

The relative densities of a number of G protein subunits were quantified in membranes prepared from iris sphincter and dilator muscles of rabbits that have undergone sympathetic denervation and from contralateral innervated controls by immunoblotting with specific polyclonal antibodies against Gs alpha, Gi alpha and Gq alpha protein subunits. In addition, alpha and beta-adrenergic receptor densities, basal and isoproterenol (ISO)-stimulated cAMP production, and basal and ISO+GTP gamma S-stimulated adenylate cyclase (AC) activities were measured in the same tissues. Densitometric analysis of the immunoblot data revealed a 32% reduction in the level of Gi alpha in the denervated sphincter, a 26% increase in the level of Gi alpha in the denervated dilator, and no changes due to denervation were found in the levels of Gs alpha and Gq alpha. Sympathetic denervation had no effect on the densities of alpha- and beta-adrenergic receptors in these tissues, however, it did induce a significant decrease in the KD values of alpha-adrenergic receptors in both dilator and sphincter, and in beta-adrenergic receptors in the sphincter. The basal- and ISO-stimulated cAMP production in the sphincter was 4-fold as high as that of the dilator, and at 0.05 microM ISO the denervation supersensitivity for cAMP production in the sphincter increased by 118% as compared to 36% in the dilator. Sympathetic denervation increased by 19-47% the basal- and ISO-stimulated activity of AC in these tissues, however, it had no effect on the phospholipase C activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗

PubMed1994

Studies on the properties of myo-inositol-1,4,5-trisphosphate 5-phosphatase and myo-inositol monophosphatase in bovine iris sphincter smooth muscle: effects of okadaic acid and protein phosphorylation.

In bovine iris sphincter, myo-inositol 1,4,5-trisphosphate (IP3) 5-phosphatase and myo-inositol 1-phosphate (IP1) monophosphatase are mainly localized in the microsomal and soluble fractions, respectively. Studies on the properties of these enzymes can be summarized as follows. (1) The microsomal IP3 5-phosphatase hydrolyzed IP3 to myo-inositol 1,4-bisphosphate with an apparent Km of 28 microM and Vmax of 32 nmol/min per mg protein. The IP1 monophosphatase in the soluble fraction hydrolyzed IP1 into free inositol with an apparent Km of 89 microM and Vmax of 7 nmol/min per mg protein. (2) IP3 5-phosphatase and IP1 monophosphatase had optimal pH values at 8.0 and 7.0, respectively. (3) Both enzymes required Mg2+ and their highest specific activities were at a cation concentration of 2 mM. (4) Ca2+ (> 0.5 microM) exerted an inhibitory effect on IP3 5-phosphatase activity, and marked inhibition (47%) was observed at a concentration of 10 microM. Higher concentrations of the cation (> 100 microM) were required to inhibit IP1 monophosphatase. (5) IP1 monophosphatase, but not IP3 5-phosphatase, was inhibited by Li+. Li+ had no effect on the contractile response in this smooth muscle. (6) Both enzymes were inhibited by ATP and by the thiol-blocking agent, disulfiram. In addition, thimerosal, a thiol reagent, also inhibited the IP3 5-phosphatase activity. (7) Protein phosphorylation of the microsomal and soluble fractions with PKA or PKC had no effect on the activities of these enzymes. (8) Okadaic acid, a protein phosphatase inhibitor, had no effect on the activity of IP3 5-phosphatase. However, in the intact iris sphincter the toxin significantly reduced the carbachol-induced IP3 production, 1,2-diacylglycerol formation, measured as phosphatidic acid, and caused muscle relaxation.

Animals↗

PubMed1994

Identification of phosphoinositide-specific phospholipase C-beta 1 and GTP-binding protein, Gq alpha, in bovine iris sphincter membranes: characteristics of the phospholipase and its coupling to cholinergic muscarinic receptors.

Previously, we have established that treatment of iris sphincter smooth muscle with carbachol (CCh) results in increased phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) and in muscle contraction. To throw more light on the mechanism of muscarinic stimulation of PLC in this tissue we have investigated the properties of this enzyme and its regulation by GTP analogs and protein phosphorylation in bovine iris sphincter membranes. The data obtained can be summarized as follows: (1) the presence of PLC-beta 1 and a GTP-binding protein, Gq alpha, was detected in the microsomal (membrane) fraction by anti-PLC beta 1 and anti-Gq alpha antibodies, respectively. The membrane PLC hydrolysed exogenously added PIP2 and this hydrolysis was increased dose-dependently by Ca2+ (1-10 microM) but the enzyme activity was inhibited by Mg2+. (2) Addition of guanosine 5'-O-thiotriphosphate (GTP gamma S, 0.1 microM) to the membrane fraction increased PIP2 hydrolysis by 30%, whereas addition of CCh (10 microM) was without effect. However, when added together, CCh and GTP gamma S increased PIP2 hydrolysis by 46%. This effect was significantly inhibited by atropine and by the anti-PLC-beta 1 and anti-Gq alpha antibodies. (3) Removal of PLC-beta 1 from the membranes with 2 M KCl resulted in a significant reduction of the CCh-induced PIP2 hydrolysis, and this effect of the muscarinic agonist was restored when the membrane fraction was supplemented with PLC-beta 1 purified from bovine brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗