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Biomedical subjects

M K Raizada

Publications and source records attributed to M K Raizada.

At least 109 records · Page 6Linked to original sources
PubMed1991

Developmental regulation of insulin-like growth factor-I-stimulated glucose transporter in rat brain astrocytes.

Astrocytic glial cells from 1- and 21-day-old rat brains were established in primary culture to study the expression of insulin-like growth factor-I (IGF-I) receptors and IGF-I-stimulated glucose transporter (Glut-1). Astrocytes from both age groups expressed specific high affinity IGF-I receptors, whose relative affinities for IGF-I, IGF-II, and insulin were comparable. However, the total number of binding sites and IGF-I receptor mRNA levels were 148% and 240% higher in astrocytes from 21-day-old compared with 1-day-old brains. IGF-I caused a dose-dependent stimulation of [3H]2-deoxy-D-glucose [( 3H]dGlc) uptake in astrocytes from 1-day-old brains. This was associated with increases in Glut-1 protein and mRNA levels. In contrast, astrocytes from 21-day-old brains exhibited a 58% decrease in the binding capacity and a 77% decrease in the steady state levels of Glut-1 protein and its mRNA. In addition, IGF-I failed to stimulate the Glut-1 system in these cells. This lack of IGF-I effect is not due to an alteration inherent to the Glut-1 system, since 12-O-tetradecanoyl-phorbol-13-acetate stimulated [3H]dGlc uptake and Glut-1 protein and its mRNA levels. These observations suggest that changes in basal and IGF-I-stimulated Glut-1 system in brain astrocytes may be developmentally regulated.

Animals↗
PubMed1991

Developmental expression of rat insulin-like growth factor binding protein-2 by astrocytic glial cells in culture.

Brain astrocytes were established in primary culture from postnatal and adult rats to characterize the developmental expression of secreted proteins. Astrocytes cultured from 21-day rat brain, but not 1-day rat brain, secreted a distinct group of proteins with Mr of 35,000 as determined by analysis of [35S]methionine-labeled proteins using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal amino acid sequence analysis of this protein group showed 100% identity to rat insulin-like growth factor binding protein-2 (rIGFBP-2), the BRL-3A IGFBP purified from a fetal rat liver cell line. An antiserum was generated against this astrocyte 35,000 Mr protein, and immunoblot analysis revealed a dramatic increase in rIGFBP-2 secretion in astrocytes cultured from 14-day, 21-day, and adult rat brain compared to astrocytes from 1-day and 7-day rat brain. Similar analysis of neonatal rat brain neurons in culture failed to show immunoreactive rIGFBP-2 in cell lysates or secreted protein. Ligand Western blot analysis demonstrated [125I]IGF-II binding to a single protein band which comigrated with a prominant rIGFBP-2 immunoreactive species in nonreduced conditioned medium from 21-day astrocytes. In comparison [125I]IGF-II binding proteins were detected only at low levels in medium from astrocytes cultured from 1-day rat brain and were undetectable in neuron-conditioned media. Northern blot analysis using a rIGFBP-2 complementary DNA revealed 5-fold greater messenger RNA levels in astrocytes from 21-day rat brain compared with astrocytes from 1-day brain, whereas neonate neurons showed no transcripts. Thus, rIGFBP-2 exhibits a pattern of developmental and cell-specific expression in cultured rat brain cells.

Aging↗
PubMed1990

Alpha 1-adrenergic receptors in the brain: characterization in astrocytic glial cultures and comparison with neuronal cultures.

Binding of [125I]HEAT to membranes prepared from primary cultures of astrocytic glial cells was time-dependent and 70-85% specific. Various adrenergic agonists and antagonists competed for [125I]HEAT binding according to the potencies of prazosin greater than, yohimbine greater than or equal to, clonidine, norepinephrine (NE), and propranolol. Scatchard analysis showed the Bmax of 209 fmol/mg protein and a Kd of 184 pM for [125I]HEAT binding by astrocytic glial membranes. Pretreatment of astrocytes with NE resulted in a dose-dependent downregulation of [125I]HEAT binding sites with a maximal response observed after 8 h at 100 microM NE. Removal of NE from cultures after pretreatment resulted in a time- and protein synthesis-dependent recovery of binding sites to control levels within 120 h. Incubation of astrocytic glial cultures with NE stimulated phosphoinositide (PI) hydrolysis in a time- and dose-dependent manner with a maximal stimulation of 2-fold observed in 60 min by 100 microM NE. Clonidine expressed differential effects on alpha 1-adrenergic receptors of the neuronal and astrocytic glial cultures. Pretreatment with 10 microM clonidine caused a 40% decrease in the Bmax of [125I]HEAT binding without influencing the Kd value in neuronal cultures. This downregulatory effect of clonidine was associated with a reduction in the ability of NE to stimulate PI hydrolysis in clonidine pretreated cells. In contrast to neuronal cultures, clonidine neither downregulated [125I]HEAT binding sites nor stimulated PI hydrolysis in glial cultures.

Adrenergic alpha-Agonists↗
PubMed1990

Phorbol esters increase insulin binding in astrocytic glial but not neuronal cells in primary culture from the brain.

In this study we used differential culturing techniques to study the effects of phorbol esters on insulin receptors on neuronal and astrocytic glial cells in primary culture from the brain. 12-O-Tetradecanoyl-phorbol-13-acetate (TPA), a potent activator of protein kinase C (PKC), increased [125I]insulin binding in a time- and concentration-dependent manner with a maximally effective dose of 50 nM TPA for 2 h in glial cells. Treatment with TPA did not affect [125I]insulin binding in neuronal cells. The TPA effect on glial [125I]insulin binding was specific as evidenced by the observation that potencies of phorbol ester analogs to increase [125I]insulin binding were similar to their abilities to stimulate PKC. Competitive-inhibition experiments indicated that this effect of TPA was due primarily to an increase in the number of high affinity insulin binding sites on glial cells. Removal of the TPA after pretreatment resulted in a recovery from its effects within 6 h. The increase in glial insulin binding was not accompanied by an increase in insulin-sensitive glucose uptake, suggesting that TPA inactivates the glial cell receptors as it increases their numbers.

Animals↗
PubMed1990

Alpha-1-adrenergic receptors in the nucleus tractus solitarii region of rats with experimental and genetic hypertension.

The binding of the alpha 1-adrenergic receptors antagonist, 125I-HEAT, to membranes of nucleus tractus solitarii (NTS) regions of the brains of neurogenic hypertensive, spontaneously hypertensive (SH), and deoxycorticosterone (DOCA)/salt hypertensive rats and their respective controls was studied to quantitate the expression of alpha 1-adrenergic receptors. Scatchard analysis of the binding studies revealed a 1.8-fold increase in the Bmax of alpha 1-adrenergic receptors in NTS region membranes of SH rats when compared to their Wistar-Kyoto (WKY) control without significant difference in the Kd for 125I-HEAT. A decrease in the Kd with no difference in Bmax of receptors for 125I-HEAT was observed in the NTS region membranes of neurogenic hypertensive rats when compared with their sham-operated controls. In contrast, comparison of the Bmax and Kd values for 125I-HEAT binding in NTS region membrane of the DOCA/salt hypertensive rats and its sham-operated control showed no significant differences. We suggest that alterations in baroreceptor afferent activity may be related to alterations in central alpha 1-adrenergic receptors binding in SH rats and rats with neurogenic hypertension.

Adrenergic alpha-Antagonists↗
PubMed1990

The influence of angiotensin II on catecholamine synthesis in neuronal cultures from rat brain.

Incubation of primary neuronal cultures prepared from the hypothalamus and brainstem of neonatal rats with angiotensin II (Ang-II) resulted in a concentration-dependent effect on the incorporation of [3H]-tyrosine ([3H]-Tyr) into [3H]-catecholamines ([3H]-CA). At concentrations of 1 nM-1 microM, Ang-II (60 min. incubation) caused significant decreases (31-52%) in neuronal [3H]-CA content compared with controls. Conversely, higher concentrations of Ang-II (10-100 microM; 60 min.) caused significant increases (20-60%) in neuronal [3H]-CA content compared with controls. Both of these effects were blocked by co-incubation with the Ang-II receptor antagonist Sar1Ile8-Ang-II. These observations demonstrate that neuronal cells in primary culture have the ability to synthesize [3H]-CA from [3H]-Tyr, and that Ang-II has a receptor-mediated biphasic influence on newly synthesized [3H]-CA (norepinephrine and dopamine).

1-Sarcosine-8-Isoleucine Angiotensin II↗
PubMed1990

Effects of phorbol ester on immunoreactive protein kinase C, insulin binding, and glucose uptake in astrocytic glial and neuronal cells from the brain.

Phorbol esters, potent stimulators of protein kinase C (PKC), stimulate [3H]2-deoxy-D-glucose (dGlc) uptake and [125I] insulin binding in cultured glial cells but not neuronal cells from neonatal rat brains. Using an antibody to the alpha and beta forms of PKC we have demonstrated that both neuronal and glial cells contain an immunoactive PKC of Mr approximately 80 kD, although the PKC level in neurons is greater than 4-fold that in glia. The majority of immunoactive PKC (63%) is cytosolic in glial cells although the reverse is true in neuronal cells, in which 88% of the PKC is membrane-bound in the basal state. The most potent phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulates a redistribution of this enzyme in neuronal and glial cells. The TPA-stimulated translocation of PKC from cytosol to membrane precedes TPA's effects on [3H]dGlc uptake and insulin binding in glial cells.

Animals↗
PubMed1990

Immunoreactive atrial natriuretic peptide in neuronal and glial cells of spontaneously hypertensive rat brain.

Neuronal and glial cultures from the hypothalamic-brain stem areas of 1-day-old normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SH) rat brains stained positively with atrial natriuretic peptide (ANP)-specific antibodies. The endogenous levels of the ANP immunoreactivity in WKY neuronal and glial cultures were 17.0 +/- 2.2 and 14.3 +/- 2.7 pg/mg, respectively. Comparable neuronal and glial cultures from SH rat brains contained a 48 to 70% decrease in the endogenous ANP immunoreactivity levels. Culture media from both brain cell types also contained ANP immunoreactivity, the levels of which are significantly higher than those found in the cells. However, similar to endogenous levels, the media levels of immunoreactive ANP in SH neuronal and glial cultures were significantly reduced compared with WKY brain cultures. These observations demonstrate that endogenous ANP-like immunoreactivity is found in neuronal and glial cells and is released into the media. The levels of peptide are reduced in cultures of SH compared with WKY cultures, suggesting a genetically controlled difference between the hypertensive and normotensive rat strains long before hypertension develops.

Animals↗
PubMed1990

Regulation of rat brain/HepG2 glucose transporter gene expression by phorbol esters in primary cultures of neuronal and astrocytic glial cells.

We have demonstrated regulation of the rat brain/Hep G2 glucose transporter gene (GT1) by Northern blot analysis with a rat brain glucose transporter cDNA probe. Incubation of both neuronal and glial cells derived from neonatal rats with 12-O-tetradecanoyl-phorbol-13-acetate induced a time- and dose-dependent increase in the steady state levels of GT1 mRNA. In glial cells, this corresponded to an increase in both the level of GT1 protein and glucose transporter activity, as demonstrated by Western blot analysis and [3H]2-deoxyglucose (dGlc) uptake studies. In contrast, in neuronal cells 12-O-tetradecanoyl-phorbol-13-acetate had no effect on either the concentration/level of the GT or [3H]dGlc uptake. These results suggest that phorbol esters regulate dGlc uptake at the transcriptional level in both neuronal and glial cells, but that the increase in expression of the GT1 gene is dissociated from posttranscriptional events involved in dGlc uptake in neuronal cells.

Animals↗
PubMed1989

Effects of phorbol esters and a calcium ionophore on angiotensin II binding in rat brain synaptosomes.

In previous studies we determined that protein kinase C (PKC) and calcium are important intracellular regulators of neuronal angiotensin II (Ang II) binding sites. In the present study we investigated the effects of the protein kinase C (PKC) agonist phorbol esters (PE) and also a calcium ionophore (A23187) on the specific binding of [125I]Ang II to brain synaptosomes prepared from rats of different ages. The rationale was to determine whether the large changes in the level of brain Ang II specific binding observed in different age rats are due to changes in the regulation of these sites by PKC or by calcium. The present data indicate no qualitative differences in the effects of PE or A23187 on [125I]Ang II specific binding to hypothalamic or brain stem synaptosomes, from either 2-5 or 70-day-old rats, i.e. the active PE TPA increased while A23187 decreased Ang II binding in all situations. Thus, the dramatic differences in brain Ang II specific binding seen with age appear not to be due to changes in regulation by PKC or calcium.

Aging↗
PubMed1989

Insulin and IGF-I stimulate phosphorylation of their respective receptors in intact neuronal and glial cells in primary culture.

Previous studies have shown that insulin and IGF-I bind to their respective receptors and stimulate autophosphorylation of the receptor beta subunits in detergent extracts of neuronal and glial cells. In the present study, intact neuronal and glial cells in primary culture have been utilized to characterize insulin- and IGF-I-stimulated phosphorylation of their receptors. Following [32P]orthophosphate labelling and stimulation by insulin or IGF-I, the cells were solubilized and the phosphorylated receptors were partially purified on wheat germ agglutinin--agarose columns, and immunoprecipitated using anti-phosphotyrosine or anti-insulin receptor antibodies. Insulin stimulated the phosphorylation of its receptor beta subunit (95 kD phosphoprotein) in a dose-dependent manner, within at least 20 seconds in both neuronal and glial cells. Additionally, a 102-kD phosphoprotein was observed in insulin-stimulated neuronal cells. Maximal stimulation of receptor phosphorylation occurred at 1 minute for the glial cells, and 10 minutes for the neuronal cells. IGF-I stimulated the phosphorylation of two phosphoproteins in intact neuronal and glial cells; a 95-kD protein and a 102-kD protein, in a dose-dependent manner. These observations demonstrate that both insulin and IGF-I stimulate the phosphorylation of the beta subunits of their respective receptors in brain cells in a similar fashion to their effects on receptors from nonneural tissues.

Animals↗
PubMed1989

Chromatographic methods for characterization of angiotensin in brain tissue.

Ang II antiserum with high sensitivity and specificity was produced. The native Ang II antiserum was purified by affinity chromatography on Affi-Gel 102 with covalently coupled [Ile5]Ang II, and purified Ang II antiserum was covalently coupled to Affi-Gel 10. The column with the covalently coupled Ang II antiserum was used for the specific enrichment of Ang II from brain extracts. The efficiency and usefulness of affinity chromatography for the purification of Ang II from biological sources were tested with 125I-labeled, 3H-labeled, and synthetic [Ile5]Ang II added to rat brains prior to extraction. In addition, the methodology was used for the purification of endogenous Ang II from pig brain. The described three-step procedure for the isolation and purification of Ang II including extraction, affinity chromatography, and HPLC is rapid and highly specific with high loading capacity. We have applied the method to the peptide Ang II in brain, but the methodology may also be used in general for the rapid purification of other neuropeptides. A combination of HPLC with specific radioimmunoassays for Ang I and Ang II was utilized to demonstrate that rat brain cells in culture devoid of the influence of the peripheral RAS were able to synthesize radioactively labeled Ang I and Ang II after incubation with [3H]isoleucine. And, finally, an HPLC system capable of separating Ang I, Ang II, and its metabolites was used to obtain insight into the degradation pattern of angiotensin peptides in the brain. Aminopeptidases appear to be the major angiotensin-degrading enzymes, and endopeptidases do not appear to be involved.

Angiotensin II↗
PubMed1989

Metabolism of angiotensin peptides by neuronal and glial cultures from rat brain.

The degradation pattern and rate of [Ile5]-Angiotensin (Ang) I, II, and III were studied in neuron-enriched and glia-enriched cells in primary cultures from rat brain. Metabolites were separated by HPLC, and their identities were evaluated by comparison of their retention times with those of synthetic Ang peptide fragments and by analysis of their amino acid composition. Major metabolites were identified as des-Asp1-[Ile5]-Ang I, des-Asp1-[Ile5]-Ang II, [Ile5]-Ang II (3-8) hexapeptide, [Ile5]-Ang II (4-8) pentapeptide, and [Ile5]-Ang II (5-8) tetrapeptide. Glia-enriched cells degraded [Ile5]-Ang I and [Ile5]-Ang III significantly faster than neuron-enriched cells, whereas no difference between the two types of cells was found in the degradation rate of [Ile5]-Ang II. Although the half-lives of [Ile5]-Ang I and [Ile5]-Ang III in neuron-enriched cells from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were not significantly different, neuron-enriched cultures from WKY rats metabolized [Ile5]-Ang II about 2.6 times faster than neuron-enriched cells derived from SHR.

Angiotensin I↗
PubMed1989

Alpha 2-adrenergic receptors in neuronal and glial cultures: characterization and comparison.

Membranes prepared from either neuronal or glial cultures contain alpha 2-adrenergic receptors as determined by the characteristics of [3H]yohimbine [( 3H]YOH) binding. The binding was rapid, reversible, saturable, dependent on the protein concentration used, and reached equilibrium by 5 min in membranes from both neuronal and glial cultures. Scatchard analyses of saturation isotherms revealed similar KD values of 13.7 +/- 1.35 nM (n = 10) for neuronal cultures and 18.42 +/- 2.34 nM (n = 10) for glial cultures. Glial cultures contained many more binding sites for [3H]YOH than neuronal cultures, having a Bmax of 1.6 +/- 0.33 pmol/mg protein (n = 10) compared with 0.143 +/- 0.018 pmol/mg protein (n = 10) in neurons. Drugs selective for alpha 2-adrenergic receptors were the most effective displacers of [3H]YOH binding in both neuronal and glial cultures, i.e., the alpha 2-adrenergic antagonists rauwolscine and yohimbine were better displacers than the other catecholamine antagonists prazosin, corynanthine, or propranolol. The agonists showed the same pattern with the alpha 2-selective drugs clonidine and naphazoline being the most effective competitors for the [3H]YOH site. GTP and its nonhydrolyzable analog. 5'-guanylyl-imidodiphosphate, were able to lower the affinity of the alpha 2-receptors for agonists but not antagonists in membranes from both neuronal and glial cultures, suggesting that the receptors are linked to a G protein in both cell types. The presence of alpha 2-adrenergic receptors in neuronal cultures was also substantiated by light microscopic autoradiography of [3H]YOH binding. In summary, we have demonstrated that both neuronal and glial cultures contain alpha 2-adrenoceptors.

Animals↗
PubMed1989

Angiotensin receptors in pulmonary arterial and aortic endothelial cells.

Angiotensin II (ANG II) is formed from angiotensin I by the action of angiotensin-converting enzyme located on the luminal surface of vascular endothelial cells. We determined whether binding sites specific for ANG II exist on pulmonary artery and aortic endothelial cells. The binding of 125I-ANG II to pulmonary artery and aortic endothelial cells was time dependent, saturable, and reversible. Scatchard analysis indicated a single class of high-affinity binding sites with equilibrium dissociation constants (Kd) of 0.85 and 0.81 nM and total binding capacities of 70 and 73 fmol/mg protein in pulmonary artery and aortic endothelial cells, respectively. Angiotensin analogues [Sar1,Ile8]ANG II and [Sar1,Ala8]ANG II, as well as angiotensin I and angiotensin III, competitively displaced 125I-ANG II in both pulmonary artery and aortic endothelial cells. The degree of inhibition of 125I-ANG II binding by these angiotensin analogues and antagonists was comparable except that [Sar1,Ala8]ANG II was 65% less potent than the other antagonists in both cell types. The binding of 125I-ANG II in pulmonary artery and aortic endothelial cells was not affected by vasopressin, substance P, or insulin, suggesting the presence of specific angiotensin receptors on these cells. These receptors appear to recognize the general configuration of angiotensin peptide rather than being specific to ANG II with no major differences between endothelial cells from pulmonary arterial or aortic vessels.

Angiotensin II↗
PubMed1989

Release of immunoreactive angiotensin II from neuronal cultures: adrenergic influences.

The effects of adrenergic drugs on the release of immunoreactive angiotensin II (ANG II-ir) from brain cells in culture were examined. In neuronal cultures, basal release of Ang II-ir was 43.65 +/- 7.44 pg/5-min incubation period (n = 14 experiments; 52 individual determinations), and in astrocytic glial cultures, it was 21.76 +/- 5.7 pg (n = 8 experiments; 24 individual determinations) when cells were exposed to buffer alone. Incubation of neuronal cultures with the alpha 2-adrenergic antagonist yohimbine (0.1-50 microM, 5 min) caused concentration-dependent increases in ANG II-ir release above basal levels. Analysis of the released material by high-pressure liquid chromatography revealed that authentic ANG II was present. No increase in the release of ANG II-ir was seen from glial cells. Experiments using neuronal cultures revealed that the yohimbine-induced release of ANG II-ir may be secondary to increased norepinephrine (NE) release. Incubation of neuronal cultures with NE (10 nM-50 microM) caused concentration-dependent increases in the release of ANG II-ir. This effect of NE was not inhibited by the alpha 1-adrenergic blocker prazosin. However, a weaker release of ANG II-ir from neuronal cultures was stimulated by the beta-adrenergic agonist isoproterenol at 100 microM. These data show that ANG II-ir can be released from neuronal but not glial cell cultures by adrenergic receptor-mediated mechanisms.

Adrenergic alpha-Agonists↗
PubMed1989

Characterization of epidermal growth factor receptors in astrocytic glial and neuronal cells in primary culture.

Studies characterized the structure and function of epidermal growth factor (EGF) receptors in astrocytic glial cells and neuronal cells in primary culture from neonatal rat brain. [125I]EGF binding to membranes prepared from glial and neuronal cultures was specific and dependent on protein concentration; however, glial preparations bound 5-fold more [125I]EGF per mg protein. Unlabeled EGF competed for binding to both glial and neuronal membranes with an IC50 of 5 nM, whereas insulin, insulin-like growth factor I, and nerve growth factor failed to compete. Scatchard plot analysis of binding data for glial cells yielded a curvilinear plot with dissociation constants of 7.12 nM for high affinity and 6.2 microM for low affinity sites. The higher level of binding in glial compared to neuronal membranes reflected a greater number of binding sites rather than differences in receptor affinity. In glial membranes, [125I]EGF covalently cross-linked to one major protein with a mol wt of 170,000, and EGF stimulated the phosphorylation of a 170,000 protein which was half-maximal at 20 nM. In contrast, neither covalent cross-linking nor receptor autophosphorylation could be detected in neuronal membranes. Culture of glial cells in the presence of EGF stimulated [35S]methionine incorporation into both cellular and secreted proteins, whereas no effect of EGF was observed in neuronal cultures. The addition of EGF to glial cultures produced a dose-dependent stimulation of [3H]thymidine incorporation as well as the multiplication of cells over a 6-day period. These observations show that functional EGF receptors in the neonatal brain are predominantly localized in glial cells.

Animals↗