Biomedical subjects

M K Raizada

Publications and source records attributed to M K Raizada.

At least 163 records · Page 9Linked to original sources

PubMed1985

Receptor-mediated inositide hydrolysis is a neuronal response: comparison of primary neuronal and glial cultures.

Cholinergic and adrenergic receptor-stimulated inositide hydrolysis was studied in neuronal and glial cells cultured from brains of 1-day-old Wistar-Kyoto rats. Incubation of the cells with [3H]inositol led to the incorporation of radioactivity specifically into inositol phospholipids. Labeling of the membrane lipids reached a maximum in 2-3 days. Receptor-stimulated breakdown of inositides was determined by following the accumulation of inositol phosphates after incubation of the labeled cells for 60 min with carbachol or norepinephrine in the presence of 10 mM lithium. Carbachol (1 mM) stimulated inositol phosphate production in neurons 30 times higher than that seen in glia. The response stimulated by norepinephrine (75 microM) was 6 times higher in neurons than glia. The response to carbachol was blocked by atropine, and the norepinephrine-induced response was inhibited by prazosin suggesting that the receptors mediating the responses were muscarinic and alpha 1-adrenergic, respectively. These results suggest that muscarinic cholinergic and alpha 1-adrenergic stimulated inositide hydrolysis is primarily a neuronal response and that this biochemical event may be important for transmembrane signaling which occurs during neurotransmission.

Animals↗

PubMed1985

Increased turnover of surface insulin receptors in fibroblastic cultures from genetically diabetic (DB/DB) mice.

The turnover of surface insulin receptors in fibroblastic cultures from genetically diabetic (db/db) mice and nondiabetic (m/m) littermates has been determined by combining a heavy isotope density shift technique with cross-linking of insulin to surface receptors. Our results indicate that the surface insulin receptors turn over faster in diabetic cells than in nondiabetic cells. In addition, fewer receptors are incorporated into the plasma membrane per hour in diabetic cells than in nondiabetic cells. It is possible to propose a model to account for the altered expression of surface insulin receptors in diabetic cells on the basis of abnormalities of receptor incorporation and turnover.

Animals↗

PubMed1985

Altered norepinephrine uptake in neuronal cultures from spontaneously hypertensive rat brain.

Uptake of [3H]norepinephrine (NE) has been characterized and compared in neuronal cultures prepared from the brains of 1-day-old normotensive (Wistar-Kyoto, WKY) and spontaneously hypertensive (SH) rats. In cultures from both strains total [3H]NE uptake consisted of a sodium-dependent portion and a sodium-independent portion. The sodium-dependent [3H]NE uptake was inhibited by NE uptake blockers such as maprotiline or desmethylimipramine (both at 0.5-100 microM). This sodium-dependent, NE uptake blocker-sensitive portion of the uptake was also stereospecific, preferring the l-isomer of NE. In contrast, the sodium-independent uptake was not sensitive to maprotiline or desmethylimipramine. Autoradiograms of cultures incubated with [3H]NE showed label concentrated in certain, but not all, neurites and in a few neuronal cell bodies. Cultures incubated with label in sodium-free buffer did not show any such localization of grains but instead showed a diffuse pattern. Incubation of neuronal WKY or SH brain cultures with various concentrations of l-[3H]NE and unlabeled l-NE in the presence or absence of sodium enabled the construction of saturation curves for sodium-dependent uptake in each culture type. In WKY cultures, Km and maximal velocity of uptake (Vmax) values of 0.37-0.45 microM and 0.58-0.69 pmol X mg protein-1 X min-1, respectively, were obtained for sodium-dependent uptake. In contrast, the Km and Vmax values for [3H]NE uptake in SH neuronal cultures were 1.4 microM and 1.31 pmol X mg protein-1 X min-1, respectively. Kinetic analyses of the results show that in SH neuronal cultures the [3H]NE uptake sites are of lower affinity but higher capacity compared with those in WKY neuronal cultures.

Animals↗

PubMed1985

Insulin stimulates macromolecular synthesis in cultured glial cells from rat brain.

The effect of insulin on macromolecular synthesis in glial cells cultured from brains of 1-day-old rats was studied to investigate the role of insulin in brain growth. Insulin caused a dose-dependent stimulation of protein synthesis (measured by [3H]valine incorporation into protein) that became significant by 7 nM insulin. Maximal stimulation of protein synthesis of 145% of control occurred with 18 nM insulin. Long-term protein synthesis was also stimulated to 136% of control by insulin in a dose-dependent manner after 6 days of insulin incubation. Insulin also stimulated net RNA and DNA synthesis (measured by [3H]uridine and [3H]thymidine incorporation into RNA or DNA, respectively) with significant stimulation by 2 nM insulin. Net RNA synthesis stimulation was maximal at 120% of control by 18 nM insulin. Plateau stimulation of DNA synthesis of 175% of control was reached by 200 nM insulin. The effects of insulin on glial protein and RNA synthesis appear to be mediated completely by the insulin receptor. Insulin, in physiological concentrations, stimulated glial DNA synthesis via its interaction with the insulin receptor (46% of total response). At supraphysiological concentrations insulin may have stimulated DNA synthesis via its cross-reactivity with the insulinlike growth factor I receptor (54% of total response). Thus insulin, at concentrations known to exist in the brain, stimulates the processes necessary for growth in the glial cell and is an important growth factor in the developing rat brain.

Animals↗

PubMed1984

Insulin binds to specific receptors and stimulates 2-deoxy-D-glucose uptake in cultured glial cells from rat brain.

The kinetics of 125I-insulin binding and physiological activity of insulin on glial cells cultured from brains of 1-day-old rats have been studied. Binding of 125I-insulin to cultured glial cells was specific, reversible, and time-dependent. Porcine and chicken insulin competed equally for 125I-insulin binding while other hormones or insulin analogs competed in proportion to their insulin-like biological activity. Incubation of glial cultures with insulin resulted in a time- and dose-dependent stimulation of 2-deoxy-D-glucose uptake. Maximal stimulation (190% of control) was observed with 18 nM insulin, and 0.1 nM insulin caused half-maximum effect. The stimulatory effect of insulin on 2-deoxy-D-glucose uptake was due to its effect on Vmax without affecting the Km. These observations suggest that insulin stimulates glucose uptake in glial cells cultured from rat brain, the effect mediated by insulin specific receptors.

Animals↗

PubMed1984

Insulin inhibits pyramidal neurons in hippocampal slices.

Recent studies have confirmed the presence of insulin receptors in the rat brain although their function has still not been well defined. The present study explores the possibility that insulin receptors in the brain can alter or contribute to central neurotransmission. Insulin caused a dose-dependent inhibition of hippocampal pyramidal neurons. The pattern of inhibition mirrored the binding kinetics of insulin in the hippocampus. Two related peptides, proinsulin and desoctapeptide insulin, had neuronal effects consistent with their binding to insulin receptors in the brain. Proinsulin was effective in doses 30-fold greater than insulin, whereas desoctapeptide insulin had little or no effect. These observations indicate that the inhibitory effect of insulin in this tissue may be insulin receptor-mediated and support a previously suggested functional role of insulin in the central nervous system.

Animals↗

PubMed1984

The effects of insulin and tunicamycin on insulin receptors of cultured fibroblasts.

The effect of down-regulation on the intracellular pool of insulin receptors and the role of glycosylation in recovery from down-regulation have been studied in fibroblastic cultures from the skin of non-diabetic mice. In control cultures, 55% of the total specific [125I]insulin-binding activity was in the intracellular compartment. Insulin caused a time- and concentration-dependent decrease in the number of cell surface insulin receptors, with no significant change in total insulin receptors. This decrease in surface receptors was accompanied by an increase in the specific binding of [125I]insulin in the intracellular compartment. Removal of insulin from down-regulated cells resulted in a time-dependent increase in the binding of [125I]insulin to surface receptors, reaching 90% of that in controls by 12 h. The recovery of surface insulin receptors after removal of insulin was blocked by incubation of cultures with tunicamycin, but not by cycloheximide. These results indicate that down-regulation of surface insulin receptors by insulin is associated with translocation of receptors into the intracellular pool and suggest that protein glycoslylation is important in insulin receptor recycling and externalization.

Animals↗

PubMed1984

Catecholamine-angiotensin II receptor interaction in primary cultures of rat brain.

Neuron-enriched primary cultures from 1-day-old rat brains have been used to study the influence of catecholamines on angiotension II receptors. Treatment of cultures with alpha-methyl-p-tyrosine caused a time- and concentration-dependent increase in the specific binding of 125I-angiotensin II and a decrease in neuronal norepinephrine and dopamine contents. A maximum increase of 75% in the binding and a decrease of 67% in catecholamine content was observed with 400 microM alpha-methyl-p-tyrosine. Removal of alpha-methyl-p-tyrosine from cultures resulted in the recovery of catecholamine levels and decrease in 125I-angiotensin II binding to control levels. In contrast, treatment of cultures with pargyline caused decreases in the binding of 125I-angiotensin II and increases in catecholamine levels in neuronal cultures. Scatchard analysis demonstrated that an increase in 125I-angiotensin II binding by alpha-methyl-p-tyrosine was due to an increase in the affinity of receptors for angiotensin II without changes in the number of angiotensin II binding sites. The results obtained here indicate that angiotensin II binding to neuron-enriched cultures is reciprocally related to the levels of neuronal catecholamines.

Angiotensin II↗

PubMed1984

Angiotensin II in neuronal cultures from brains of normotensive and hypertensive rats.

Primary neuronal cultures from 1-day-old rat brains, which contain angiotensin II (ANG II) immunoreactivity within the neurons and are capable of de novo synthesis of this immunoreactivity, have been used in this study to determine the nature of this immunoreactivity by high-performance liquid chromatography (HPLC). Neuronal cultures from the brains of normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SH) rats were found to contain ANG II immunoreactivity, which co-migrated with authentic ANG II on HPLC. The angiotensin detected in brain cultures was not derived from the growth medium, and its level was significantly decreased by incubating the cultures with captopril. A 71% decrease in the levels of ANG II was observed in neuronal cultures of SH rat brains compared with those from WKY controls. These observations show that differences in brain angiotensin between SH and WKY are present before differences in blood pressure are manifested.

Angiotensin II↗

PubMed1984

Increased angiotensin II receptors in neuronal cultures from hypertensive rat brain.

Binding of 125I-angiotensin II (ANG II) to neuronal cultures made from the brains of 1-day-old normotensive (Wistar-Kyoto, WKY) and spontaneously hypertensive (SH) rats was time dependent, saturable, reversible, and 90-95% specific. Neuronal cultures from SH rats bound 50-70% more 125I-ANG II compared with their WKY controls. Scatchard analysis revealed that the increase in the specific binding of ANG II to SH rat neuronal cultures was due to an increase in the number of binding sites per cell rather than change in the affinity of receptors for ANG II. Light-microscopic autoradiographic analysis showed that ANG II specific binding sites were located on neuronal cell bodies and neurites. Treatment of neuronal cultures from both strains of rats with alpha-methyl-p-tyrosine caused a 50-60% decrease in the endogenous levels of norepinephrine (NE) and dopamine (DA). This decrease was associated with increases in the specific binding of 125I-ANG II in neuronal cultures from WKY rat brain. In contrast, ANG II binding in neuronal cultures from SH rat brain failed to respond to changes in NE and DA levels. These observations suggest that ANG II specific receptors are increased and that they are not under a negative-feedback control by catecholamines in SH rat brain neuronal cultures.

Angiotensin II↗

PubMed1983

Cycloheximide causes accumulation of insulin receptors at the cell surface of cultured fibroblasts.

Incubation of confluent cultures of mouse fibroblasts with cycloheximide caused a time-dependent increase in the binding of 125I-insulin. The increase was concentration-dependent between 0.05 and 3.5 microM cycloheximide and showed a high correlation (r = 0.97) between the increase in 125I-insulin binding and the inhibition of protein synthesis. In the presence of 3.5 microM cycloheximide, insulin binding increased to 236% of control and incorporation of [3H]valine into proteins fell to 10-20% of control. Scatchard analysis of the binding data indicated that cycloheximide-treated cultures had a total of 6.6 X 10(4) binding sites/cell compared to 3.9 X 10(4) sites/cell in untreated cultures. No significant changes in affinity were observed. Other protein synthesis inhibitors also caused an increase in 125I-insulin binding. With 25 mM ethionine and 2 mM sodium fluoride, binding was 155 and 245% of control and incorporation of [3H]leucine into proteins was decreased to 41 and 47% of control, respectively. These results suggest that the accumulation of insulin receptors at the cell surface following treatment with cycloheximide results from inhibition of synthesis of proteins involved in insulin receptor turnover.

Animals↗

PubMed1983

Angiotensin II stimulates changes in the norepinephrine content of primary cultures of rat brain.

Interactions between norepinephrine and angiotensin II were investigated in neuron-enriched primary brain cell cultures, which have been demonstrated to contain catecholamines, angiotensin II-like immunoreactivity and specific receptors for angiotensin II. Angiotensin II (7.5 and 15.0 micrograms/ml) caused significant increases in both neuronal and growth media norepinephrine levels, which were inhibited by saralasin. These observations suggest that angiotensin acts at its specific receptors to alter neuronal norepinephrine levels.

Angiotensin II↗

PubMed1983

Rat brain cells in primary culture: visualization and measurement of catecholamines.

Catecholamines have been visualized and quantified in primary cultures of whole rat brain. Twenty-one-day old cultures treated with glyoxylic acid and viewed under a fluorescence microscope revealed neurons stained specifically with blue-green catecholamine fluorescence. Brightly stained multipolar cell bodies were seen, along with stained neurites and varicosities, and there was no staining associated with the non-neuronal portion of the culture. Twenty-one-day-old non-neuron-enriched cultures contained 10-20 times less norepinephrine and dopamine than cytosine arabinoside-treated neuron-enriched cultures. The latter cultures contained 10-12 times more norepinephrine than 1-day-old rat brains, demonstrating maturation and differentiation of the cultured neurons. Norepinephrine levels of neuron-enriched cultures were, however, 3 times less than those in 21-day-old rat brains. The cultured neurons had the ability to synthesize catecholamines since levels were decreased with alpha-methyl-p-tryosine. On the other hand, the growth medium contained significant amounts of norepinephrine, but did not have the ability to synthesize catecholamines. It may be concluded that the cellular catecholamines are not derived from the medium in any great amounts. This study provides the basis of a system in which to examine catecholaminergic neurotransmission and peptide catecholamine interactions at the cellular level under semi-defined conditions.

Animals↗

PubMed1983

Localization of insulin-like immunoreactivity in the neurons from primary cultures of rat brain.

Brains from 1-day-old rats were dissociated with trypsin and the cells were maintained in culture for 3-4 weeks. These primary cultures contained insulin-like immunoreactivity in the limited populations of neurons. Typically, the fluorescent staining pattern observed in the soma was homogeneous and varicosity-like structures were observed on the neurites of the majority of insulin-like immunoreactive neurons. Serum deprivation of brain cell cultures did not reduce immunoreactivity, whereas cycloheximide caused approx. 80% decrease in the number of insulin-like immunoreactive neurons. Incubation of these cultures with [3H]valine resulted in the incorporation of radioactivity into immunoprecipitable insulin. These results suggest that insulin-like immunoreactivity present in the Central Nervous System (CNS) neurons may be synthesized by brain cell cultures.

Animals↗

PubMed1983

Effects of insulin and tunicamycin on neuronal insulin receptors in culture.

We have examined the effect of insulin and tunicamycin, which cause decreases in cell surface insulin receptor numbers in peripheral tissues, on insulin receptors in neuron-enriched brain cell cultures. Incubation with 0.016-0.83 microM insulin for 24 h failed to decrease the specific binding of 125I-insulin in neuron-enriched cultures prepared from whole brains of 1-day-old rats. In contrast, these concentrations of insulin produced a dose-dependent decrease in the binding of 125I-insulin in fibroblastic cultures. Tunicamycin, an antibiotic which inhibits glycosylation of proteins and greatly reduced insulin binding via a reduction in apparent receptor numbers in fibroblastic cultures, caused no decrease in the binding of 125I-insulin in neuron-enriched cultures. These results indicate that brain insulin receptors are not down regulated by insulin and that glycosylation of insulin receptors may not be an important step in the regulation of their surface expression in the brain.

Animals↗

PubMed1983

Primary cultures from fetal rat brain incorporate [3H]-isoleucine and [3H]-valine into immunoprecipitable angiotensin II.

Primary cultures from fetal rat brain contain angiotensin II immunoreactivity in neurons. To study the origin of this immunoreactivity, primary cultures were incubated with [3H]-valine or [3H]-isoleucine. A time-dependent incorporation of radioactivity into immunoprecipitable angiotensin II was observed. Incubation of cultures with Captopril, an inhibitor of the converting enzyme, resulted in a significant inhibition of [3H]-valine incorporation into immunoreactive [3H]-angiotensin II. These results demonstrate that primary cultures from fetal rat brain can synthesize an angiotensin II-like immunoreactivity and support a concept that angiotensin II may be synthesized in fetal rat brain.

Angiotensin II↗

PubMed1982

Adult-level insulin binding is present in term fetal rat CNS membranes.

Insulin binding was measured using partially-purified membranes prepared from the brains of term fetal and adult rats as the source of receptors. The membranes from the fetal rat brains bound at least as much insulin as did those from the adult rats at all concentrations of insulin used. This finding supports our hypothesis that insulin contributes to the postnatal growth of the rat brain.

Animals↗

PubMed1982

Uptake of prolactin from cerebrospinal fluid in rat brain.

The fate of prolactin in the cerebrospinal fluid was studied by the use of fluorescein labelled prolactin. The distribution was compared to the immunocytochemical distribution of endogenous prolactin. High intensity fluorescence was found in the ependyma of the area postrema, rostral dorsolateral cerebral aqueduct close to the subcommissural organ, and in some cells of the floor of the cerebral aqueduct. This distribution was not seen when excess unlabelled prolactin was injected. The results suggest prolactin uptake from CSF at specific sites which correspond to sites of localization of immunoreactive prolactin.

Animals↗