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Biomedical subjects

M K Raizada

Publications and source records attributed to M K Raizada.

At least 127 records · Page 7Linked to original sources
PubMed1989

Regulation of rat brain/HepG2 glucose transporter gene expression by insulin and insulin-like growth factor-I in primary cultures of neuronal and glial cells.

We have demonstrated the expression of the rat brain/HepG2 glucose transporter gene in primary cultures of rat neuronal and glial cells by Northern blot analysis with a rat brain glucose transporter cDNA probe. Incubation of both neuronal and glial cells with insulin and insulin-like growth factor-I induced a time- and dose-dependent increase in the steady state levels of glucose transporter mRNA. The maximal response was achieved between 2-4 h and subsequently decreased. Both insulin and insulin-like growth factor-I at a dose of 1 ng/ml elicited an approximately 57% increase in glucose transporter mRNA levels in neuronal cultures after 90 min, suggesting that each peptide was acting through its own receptor. On the other hand, insulin stimulated [3H]2-deoxyglucose uptake in glial, but not neuronal, cells. These results suggest that insulin-like peptides regulate the expression of the rat brain/Hep G2 glucose transporter gene at both transcriptional and posttranscriptional levels, and that these regulatory mechanisms may be dissociated from each other. Insulin-like peptides may, therefore, participate in the control of brain energy metabolism.

Animals↗
PubMed1988

Effect of nitrogen dioxide on surface membrane fluidity and insulin receptor binding of pulmonary endothelial cells.

Nitrogen dioxide (NO2), an environmental oxidant pollutant, is known to peroxidize membrane lipids of lung cells. We evaluated the ability of NO2 to alter the surface membrane fluidity, lipid composition, and insulin receptor binding of porcine pulmonary artery endothelial cells in culture. After 3- to 24-hr exposure to 5 ppm NO2, cells were labeled with either 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), a cationic fluorescent aromatic hydrocarbon that anchors at the lipid-water interface, or fluorescamine, a fluorescent molecular probe that covalently binds with amino groups of surface phospholipids and proteins. Membrane fluidity was measured by monitoring changes in the steady-state fluorescence anisotropies (rs) for TMA-DPH and fluorescamine. Insulin specific receptor binding was monitored by measuring time-dependent binding of 125I-insulin. Following NO2 exposure, rs values for TMA-DPH and fluorescamine were increased significantly in a time-dependent fashion, with maximum increases at 24 hr (P less than 0.001). Similar increases in rs values were observed in isolated plasma membranes as well as in lipid vesicles prepared from total lipid extracts of endothelial cells or their plasma membranes. Phosphatidylethanolamine plus phosphatidylserine content in lipid extracts from 24-hr but not 3- to 12-hr NO2-exposed cells was increased significantly (P less than 0.01) compared to control cells. Specific binding of 125I-insulin to cells exposed to NO2 for 12 and 24 hr (but not 3 and 6 hr) was reduced significantly (P less than 0.05) compared to binding in control cells. Scatchard analysis of the binding data indicated that NO2 exposure caused a 5-fold reduction in insulin receptor binding sites in endothelial cells. Recovery was achieved 24 hr after NO2 exposure with, but not without, changing culture medium. These results indicate that NO2 exposure causes reversible changes in the physical state of lipids in the superficial lipid domains of the pulmonary endothelial cell plasma membrane, and these alterations may interfere with plasma membrane-dependent functions such as receptor-ligand interaction.

Amino Acids↗
PubMed1988

Increased expression of alpha 1-adrenergic receptors in the hypothalamus of spontaneously hypertensive rats.

The specificity and molecular weights of alpha 1-adrenergic receptors in various tissues of spontaneously hypertensive (SH) rat were compared with normotensive controls (Wistar-Kyoto; WKY) with the use of [125I]HEAT and [125I]azidoprazosin, specific alpha 1-adrenergic receptor antagonists. Binding of [125I]HEAT to membranes prepared from SH rat brain hypothalamus was significantly higher, due to a 75% increase in the Bmax, than the WKY control. In contrast, the Bmax and Kd of [125I]HEAT binding to brainstem and liver membranes from SH rats were not significantly different from those of WKY controls. Competition-inhibition data suggested similar pharmacological specificity with potencies in the order of prazosin greater than yohimbine greater than propranolol for both WKY and SH rat membranes prepared from liver, hypothalamus, brainstem and neuronal cultures. Photoaffinity labeling of alpha 1-adrenergic receptors from hypothalamus, brainstem and neuronal cultures using [125I]azidoprazosin followed by SDS-PAGE and autoradiography showed the presence of one major band with a molecular weight (MW) of 105,000 Da for both WKY and SH rats. In contrast, labeling of liver alpha 1-adrenergic receptors revealed one major band with a MW of 60,000 Da. Quantitation of the 105,000-Da band from SH rat hypothalamic membranes demonstrated a 52% higher intensity compared with WKY controls. Neuronal cultures prepared from 1-day-old SH rats showed a similarly greater intensity of the 105,000-Da band compared with WKY controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Angiotensinogen gene expression in neuronal and glial cells in primary cultures of rat brain.

Neuronal and glial cells in primary culture prepared from the hypothalamic-brain stem areas of one-day-old rat brains were analyzed for the presence of angiotensinogen messenger RNA (mRNA) to further confirm our previous conclusion that the brain contains an exclusive angiotensin system. Angiotensinogen mRNA was quantitated by Northern analysis using nick-translated angiotensinogen cDNA as the hybridization probe (Kunapuli and Kumar, 1987). Angiotensinogen mRNA sequences were present in the RNA isolated from both neuronal and glial cultures. Quantitative measurements of the mRNA using dot-blot analysis revealed that the level of angiotensinogen mRNA was three times higher in neuronal cultures compared with glial cultures. These observations provide the first evidence for the synthesis of angiotensinogen in neuronal as well as in glial cells form the brain.

Angiotensinogen↗
PubMed1988

Receptors for phorbol esters are primarily localized in neurons: comparison of neuronal and glial cultures.

Binding of [3H]PDB has been measured in the present study to determine the levels of protein kinase C in the neuronal and astrocytic glial cells in culture from rat brain. Binding of [3H]PDB to homogenates of cultured neuronal cells from the brains of normotensive and hypertensive rats was time-dependent and specific. The relative potency for competition by various phorbol esters to [3H]PDB binding was TPA greater than beta-PDD greater than POE greater than alpha-PDD greater than or equal to 4 alpha phorbol. Scatchard analysis showed that neuronal cultures from normotensive rat brains contained 2-3 fold more phorbol ester receptors compared with the glial cultures from the same brains. No differences in the Kd and Bmax were observed between neuronal cultures from normotensive and spontaneously hypertensive rat brains. These studies suggest that the phorbol ester receptors are primarily localized in neuronal cells.

Animals↗
PubMed1988

Insulin receptors in the brain: structural and physiological characterization.

The present study was conducted to characterize insulin receptors and to determine the effects of insulin in synaptosomes prepared from adult rat brains. Binding of 125I-insulin to synaptosome insulin receptors was highly specific and time dependent: equilibrium binding was obtained within 60 minutes, and a t1/2 of dissociation of 26 minutes. Cross-linking of 125I-insulin to its receptor followed by SDS-PAGE demonstrated that the apparent molecular weight of the alpha subunit of the receptor was 122,000 compared with 134,000 for the liver insulin receptor. In addition, insulin stimulated the dose-dependent phosphorylation of exogenous tyrosine containing substrate and a 95,000 MW plasma membrane associated protein, in a lectin-purified insulin receptor preparation. The membrane associated protein was determined to be the beta subunit of the insulin receptor. Incubation of synaptosomes with insulin caused a dose-dependent inhibition of specific sodium-sensitive [3H]norepinephrine uptake. Insulin inhibition of [3H]norepinephrine uptake was mediated by a decrease in active uptake sites without any effects in the Km, and was specific for insulin since related and unrelated peptides influenced the uptake in proportion to their structural similarity with insulin. These observations indicate that synaptosomes prepared from the adult rat brain possess specific insulin receptors and insulin has inhibitory effects on norepinephrine uptake in the preparation.

Animals↗
PubMed1988

Pharmacological characterization of high affinity choline transporters in primary neuronal cultures in rat brain.

High affinity transport of choline was investigated kinetically and pharmacologically in primary neuronal cultures and synaptosomes of the brain of the rat. Both preparations took up and acetylated [3H]choline in a similar high affinity, sodium-dependent manner. However, monoethylcholine mustard aziridinium ion (AF64A) (an irreversible inhibitor and potential neurotoxin) and hemicholinium-3 (a reversible inhibitor) were much less potent in the neuronal cultures than in synaptosomes. Antibodies, highly selective for ubiquitin, and able to block synaptosomal synthesis of acetylcholine, coupled to high affinity transport of choline had no effect of the synthesis of acetylcholine in intact cultured neurons, suggesting differential post-translational modification of the transporters in these two preparations.

Acetylcholine↗
PubMed1988

Biosynthesis of angiotensinogen and angiotensins by brain cells in primary culture.

This study focuses on the ability of primary rat brain cells in culture to synthesize angiotensinogen, angiotensin I, and angiotensin II. HPLC in combination with radioimmunoassay was used to characterize these compounds. Following incubation with 3H-labeled isoleucine, radioactively labeled angiotensinogen with an approximate molecular weight of 25,000 was identified in both glial and neuronal cells. Other molecular weight forms of angiotensinogen with molecular weights of about 300 and 160,000 were present in both cell types. In addition to angiotensinogen, radioactively labeled angiotensin I and angiotensin II were also synthesized by neuronal and glial cells. These results suggest that glial and neuronal cells can synthesize angiotensinogen, angiotensin I, and angiotensin II in a similar manner shown for the peripheral renin angiotensin system.

Angiotensin I↗
PubMed1988

Phorbol ester-induced upregulation of angiotensin II receptors in neuronal cultures is potentiated by a calcium ionophore.

Previous studies have suggested that protein kinase C is important in the regulation of angiotensin II receptors in neuronal cultures, because the C-kinase agonists, phorbol esters, are able to increase the number of these receptors. In the present study, we have further investigated the role of protein kinase C in angiotensin II receptor regulation. This enzyme is calcium dependent, and so we investigated the effects of A23187, a calcium ionophore, on phorbol ester-stimulated and basal angiotensin II receptor regulation. A23187, at concentrations that increased 45Ca2+ influx, caused a dose-dependent potentiation of phorbol-12-myristate-13-acetate (TPA)-stimulated upregulation of angiotensin II receptors. This potentiation by A23187 was a further increase in angiotensin II receptor number and was abolished in calcium-free medium. In the absence of TPA, A23187 caused a decrease in angiotensin II receptor number, an effect not observed in calcium-free medium. The results suggest at least two pathways for angiotensin II receptor regulation in neuronal cells: (a) by calcium-dependent protein kinase C and (b) via an influx of calcium into the cell.

Angiotensin II↗
PubMed1988

Immunocytochemical and biochemical characterization of angiotensin I and II in cultured neuronal and glial cells from rat brain.

Neuronal and glial cells cultured from neonatal rat brains showed staining for both angiotensin I and II using the peroxidase-antiperoxidase method. In glial cell extracts of normotensive Wistar-Kyoto rats, the concentrations of angiotensin I and II were 12.47 +/- 2.71 (n = 4) and 66.73 +/- 13.28 fmol/mg protein (n = 4). Angiotensin I and II found in neuronal cell extracts of normotensive Wistar-Kyoto rats were 11.29 +/- 2.99 (n = 4) and 60.25 +/- 12.77 fmol/mg protein (n = 4). No significant difference was found in the concentration of angiotensin I and II in both cell types from the same rat strain. Angiotensin I concentrations of 16.83 +/- 3.43 fmol/mg protein (n = 5) determined in neuronal cell extracts derived from spontaneously hypertensive rats did not differ significantly from those found in neuronal cell extracts of Wistar-Kyoto rats. However, neuronal cell extracts from spontaneously hypertensive rats revealed values of 25.19 +/- 4.31 fmol angiotensin II/mg protein (n = 4). This was significantly different (p less than 0.05) and represented a 58% reduction in the angiotensin II levels in neuronal cells from spontaneously hypertensive rats compared to Wistar-Kyoto rat cultures. Angiotensin I and II measured in the growth medium containing 10% plasma-derived horse serum was below the detection limit of both radioimmunoassays. No difference in the angiotensin I and II levels was found in cells kept in serum-free medium. The angiotensin I and II immunoreactive material determined in the cell extracts could be characterized on reversed-phase high pressure liquid chromatography as (Ile5)-angiotensin I and II. (Ile5)-angiotensin III was not detectable.

Angiotensin I↗
PubMed1988

Effects of beta-naphthoflavone on insulin receptor binding and protein kinase activity in rat liver and placenta.

Studies investigating the effects of beta-naphthoflavone (beta NF) on insulin receptor binding and its intrinsic protein kinase activity in rat liver and placenta were performed. Membranes were prepared from maternal liver and placenta on gestation day 11 and used for [125I]insulin radioreceptor assay. Scatchard analysis showed that association constants (Ka) for high affinity binding sites were similar for placental and liver membrane. The administration of beta NF, 15 mg/kg, 1 day before study did not alter the specific binding of insulin to liver membranes, whereas ligand binding to placental preparations was decreased 40% from control. Scatchard analysis of binding to placental membranes suggests that beta NF treatment was associated with a change in the number of high affinity binding sites. In further studies membrane receptors were solubilized and partially purified by wheat germ agglutinin affinity chromatography for protein kinase assay. Insulin stimulated the phosphorylation of the Mr 95,000 subunit of the receptor in lectin-purified membrane proteins from liver and placenta. In liver receptor preparations, beta NF treatment was associated with a nearly 3-fold increase in the insulin-stimulated phosphorylation of the 95-kD protein. In contrast, placental receptor preparations showed a 40% decrease in the extent of autophosphorylation following beta NF treatment. Insulin-stimulated phosphorylation of an exogenous substrate poly(Glu4, Tyr) also showed a divergent pattern of changes in liver and placental receptors following beta NF treatment. In studies during late gestation (day 18), beta NF treatment was also associated with an increase in liver receptor kinase activity, whereas placental receptors showed a decrease in autophosphorylation. Thus, acute treatment with beta NF during mid and late gestation was associated with significant alterations in insulin receptor protein kinase activity, and data suggest that fetal insulin receptors may respond in a different manner than maternal receptors to polyaromatic compounds like beta NF. The observed effects of beta NF on liver and placental receptor kinase activity may be related to alterations in insulin function in the regulation of pregnancy and fetoplacental growth.

Animals↗
PubMed1987

Presence of renin in primary neuronal and glial cells from rat brain.

Immunocytochemical and biochemical techniques have been utilized in the present study to characterize renin in brain cell cultures. With the use of renin-specific antibody, positive renin staining was seen in neuronal and in astrocytic glial cells using the peroxidase-antiperoxidase method. Renin concentration was pH-dependent with highest concentrations at 5.5, decreasing from pH 6.0 to 6.5. At pH 7.4 no renin was detectable in either glial or neuronal cells. The contribution of cathepsin D to the measured renin was about 10% at pH 5.5; 7% at pH 6.0 and 3% at pH 6.5. Comparison of glial with neuronal cells from WKY rats revealed significantly elevated renin at pH 5.5 in glial cells. No difference was seen between glial and neuronal renin levels in WKY rats at pH 6.0 and 6.5. At pH 5.5 and 6.0 renin was significantly increased in neuronal cells of SHR compared to WKY, whereas at pH 6.5 no difference was observed. The renin concentration in cells kept for 2 days in serum-free medium did not differ from those measured in cells kept in serum-containing medium. The generated peptide was identified as [Ile5]Angiotensin I on reversed-phase HPLC.

Angiotensin I↗
PubMed1987

Phorbol esters inhibit agonist-stimulated phosphoinositide hydrolysis in neuronal primary cultures.

The effects of phorbol esters on neurotransmitter-stimulated phosphoinositide (PI) hydrolysis in neurons in primary culture were investigated. Ten-day-old neuronal cultures were incubated with [3H]inositol for 2-3 days, exposed to phorbol esters, and the release of [3H]inositol phosphates was measured in the presence of 10 mM lithium. Pretreatment of the neuronal cultures with 1 microM phorbol myristate acetate (PMA) inhibited alpha 1, muscarinic, and glutamate receptor-mediated PI hydrolysis in a time-dependent manner with maximal inhibition observed after a 20-30 min preincubation. The active beta-phorbol didecanoate inhibited stimulated PI hydrolysis, but its stereo-isomer alpha-phorbol didecanoate was without effect at 1 microM. PMA was about 10 times more potent at inhibiting PI hydrolysis stimulated by norepinephrine and glutamate compared to carbachol. The order of potency of the various phorbol esters for inhibition of stimulated PI hydrolysis and the differences between active and inactive stereoisomers suggests that the activation of protein kinase C may mediate the inhibitory effects. Thus, stimulation of neuronal protein kinase C may represent a mechanism for the regulation of agonist-stimulated PI hydrolysis.

Animals↗
PubMed1987

Phorbol esters stimulate 2-deoxyglucose uptake in glia, but not neurons.

Activation of protein kinase C by phorbol esters caused a time- and dose-dependent stimulation (270% of control) of glucose uptake in cultured glia, but not in neurons from rat brain. The phorbol ester stimulation of Vmax of glial glucose uptake occurred only in glia despite nearly 2.5-fold greater phorbol ester binding in neurons. These differences in cellular responses to protein kinase C activation may be the key to understanding brain glucose regulation.

Animals↗
PubMed1987

Insulin receptors and insulin action in dissociated brain cells.

The present study was conducted to characterize insulin receptors and insulin action in rat brain cells. Binding of [125I]insulin to cells obtained by mechanically dissociating rat brains was 86% specific, time-dependent and reached equilibrium within 90 min. The t1/2 of association was 14 min and t1/2 of dissociation was 8 min. Scatchard analysis demonstrated the typical curvilinear plot providing high affinity (0.03 nM) and low affinity (6.6 nM) binding sites. The total number of binding sites were 0.15 pmol/mg protein. Crosslinking of [125I]insulin to its receptors on dissociated brain cells followed by SDS-PAGE and autoradiography showed that the alpha-subunit of the receptor had a molecular weight of 122,000. This was in contrast with a molecular weight of 134,000 for the liver alpha-subunit. Incubation of dissociated brain cells with insulin resulted in a concentration-dependent inhibition of total [3H]norepinephrine (NE) uptake. This inhibitory effect of insulin on [3H]NE uptake was sodium ion-dependent suggesting that 80-90% of the sodium ion-dependent uptake was insulin-sensitive. Incubation of lectin-purified insulin receptors with insulin resulted in a time- and concentration-dependent stimulation of phosphorylation of the tyrosine residue of an exogenous substrate poly (Glu, Tyr) (4:1). In addition, insulin also stimulated the autophosphorylation of the beta-subunit of the insulin receptors. These observations corroborate our contention that insulin exerts neuromodulatory effects mediated by the specific insulin receptors in the brain.

Animals↗
PubMed1987

Insulin-like growth factor I receptors in neuronal and glial cells. Characterization and biological effects in primary culture.

Primary cultures of neuronal and glial cells from 1-day-old neonatal rats contain high affinity receptors for insulin-like growth factor I (IGF-I). The IC50 for displacement of 125I-IGF-I binding by unlabeled IGF-I was 3 nM for neuronal cells and 4 nM for glial cells. Unlabeled insulin was 20-50 times less potent. Apparent molecular mass of the alpha subunits of the IGF-I receptor was 125 kDa in neuronal and 135 kDa in glial cells. IGF-I induced autophosphorylation of the IGF-I receptor beta subunit in lectin-purified membrane preparations in a dose-dependent manner. The major phosphoamino acid of the beta subunit in both cell types was tyrosine in the IGF-I-stimulated state and serine in the basal state. Apparent molecular mass of the beta subunits of the IGF-I receptors was 91 kDa for neuronal and 95 kDa for glial cells. Tyrosine kinase activity of the IGF-I receptors was demonstrated by IGF-I-induced phosphorylation of the exogenous substrate poly(Glu, Tyr) 4:1 in both cell types. IGF-I had no effect on 2-deoxyglucose uptake in neuronal cells. In contrast, in glial cells, IGF-I stimulated 2-deoxyglucose uptake at very high doses, presumably acting via the insulin receptor. The effect of IGF-I as a neurotrophic growth factor in both neuronal and glial cells was demonstrated by its stimulation of [3H]thymidine incorporation. These findings suggest the IGF-I is an important growth factor in nervous tissue-derived cells.

Animals↗
PubMed1987

Angiotensin II inhibits the K+-evoked release of [3H]norepinephrine from hypothalamic synaptosomes of the spontaneously hypertensive rat.

The effect of angiotensin II on the basal and K+-evoked release of [3H]norepinephrine was examined in hypothalamic and brainstem synaptosomes from adult male normotensive (Wistar-Kyoto, WKY, and Sprague-Dawley, SD), and spontaneously hypertensive (SH) rats. Angiotensin II attenuated the [3H]norepinephrine release caused by maximal depolarizing concentrations of K+ (75 mM) in hypothalamic synaptosomes of the SH rat, but had no effect on basal [3H]norepinephrine release. Angiotensin II had no effect on either the basal or K+-evoked release of [3H]norepinephrine in brain synaptosomes prepared from either WKY or SD adult male rats. The results suggest a distinct role of angiotensin II in the modulation of catecholamine release in the SH rat.

Angiotensin II↗