Cortical Homogenates Research Articles

Subchronic cadmium treatment affects the abundance and arrangement of cytoskeletal proteins in rat renal proximal tubule cells

Disfunction of proximal tubules (PT) in cadmium (Cd) nephrotoxicity in mammals results from the diminished functional capacity of brush-border membrane (BBM) caused by (a) direct inhibition of BBM transporters by Cd, (b) shortening and loss of microvilli, and (c) loss of specific BBM transporters. The loss of transporters may partially result from impaired intracellular vesicle recycling due to loss or/and inhibition of vacuolar H +-ATPase in the PT cell organelles. Cytoskeleton plays an important role in vesicle-mediated recycling and processing of BBM transporters in PT cells. Experiments in vitro have indicated that Cd may affect the state of polymerization of some cytoskeletal proteins. In this work we studied the in vivo effect of CdCl 2-treatment in rats (2 mg Cd/kg b. m., s.c., daily for 14 days) upon abundance and arrangement of actin filaments, actin-bundling protein villin, and microtubules (MT) in PT cells. Cd-treatment elicited a dramatic accumulation of Cd in the kidney cortex (200 μg/g tissue wet mass after 14 days) and a strongly increased abundance of metallothionein in PT cells. As revealed by immunocytochemistry in tissue cryosections, the staining intensity of actin and villin in PT cells of Cd-treated rats was generally decreased, without a marked change in their intracellular distribution, whereas MT became largely irregular, diminished in most cells, and lost in many cells. However, the immunoblots revealed an increased content of villin and α-tubulin in cortical tissue homogenates from Cd-treated rats, thus indicating an impaired bundling of actin and greatly depolymerized MT in cells intoxicated with Cd. The partial loss of apical actin and villin in PT cells of Cd-treated rats may reflect (or cause) shortening and loss of microvilli, whereas derangement and depolymerization of MT may contribute to the impairment of intracellular recycling of BBM proteins, and lead to the loss of BBM transporters.

Functional and toxicological characteristics of isolated renal mitochondria: impact of compensatory renal growth

Mitochondria were isolated from renal cortical homogenates from control rats and rats that had undergone uninephrectomy and compensatory renal growth (NPX rats). Activities of selected mitochondrial processes, including key enzymes of intermediary metabolism, glutathione-dependent enzymes, and glutathione transport, were measured, and the effects of three mitochondrial toxicants were assessed to test the hypothesis that compensatory renal growth is accompanied by increases in mitochondrial metabolism and that this is associated with increased susceptibility to injury from oxidants or other mitochondrial toxicants. Activities of malic and succinic dehydrogenases were significantly higher in mitochondria from NPX rats than in mitochondria from control rats. Although the rates of state 3 respiration were significantly higher in mitochondria from NPX rats, the rates of state 4 respiration and respiratory control ratios were not different between mitochondria from control and NPX rats. Activities of glutathione redox cycle enzymes did not differ significantly between mitochondria from control and NPX rats. However, the rates of uptake of glutathione into mitochondria were approximately 2.5-fold higher in tissue from NPX rats than in tissue from control rats. Incubation of mitochondria from NPX rats with three mitochondrial toxicants [ tert-butyl hydroperoxide, methyl vinyl ketone, and S-(1,2-dichlorovinyl)- l-cysteine] caused greater inhibition of state 3 respiration and larger increases in malondialdehyde formation than similar incubations of mitochondria from control rats. These results indicate that mitochondria from hypertrophied renal cells are more sensitive to oxidants or mitochondrial toxicants. Baseline levels of malondialdehyde were also significantly higher in mitochondria from NPX rats, suggesting that a basal oxidant stress exists in mitochondria from hypertrophied cells.

Lack of protective effect by intermittent hypoxia on MPTP-induced neurotoxicity in mice.

In the study we report here, several lines of evidence support the preventive action of intermittent hypoxia against oxidative injuries in CNS. Our in vitro data showed that autooxidation and iron-induced lipid peroxidation were attenuated in cortical homogenates of intermittent hypoxia-treated animals. Furthermore, our preliminary study found that iron induced oxidative injuries were abolished in rat brain after intermittent hypoxic treatment (paper submitted). Several antioxidative defensive systems improve in response to intermittent hypoxia. Since attenuation of autooxidation and iron-induced lipid peroxidation were observed in cortical homogenates of intermittent hypoxia-treated mice, the lack of prevention by intermittent hypoxia of MPTP-induced neurotoxicity may be due to the MPTP action that is not oxidative related. Together with our previous studies, in which several antioxidants were shown to successfully prevent oxidative injuries, our data here suggest that intermittent hypoxia may offer a potential treatment for preventing CNS degenerative diseases.

NHE3 activity and trafficking depend on the state of actin organization in proximal tubule.

The present study was addressed to define the contribution of cytoskeleton elements in the kidney proximal tubule Na+/H+ exchanger 3 (NHE3) activity under basal conditions. We used luminal membrane vesicles (LMV) isolated from suspensions of rat cortical tubules pretreated with either colchicine (Colch) or cytochalasin D (Cyto D). Colch pretreatment of suspensions (200 microM for 60 min) moderately decreased LMV NHE3 activity. Cyto D pretreatment (1 microM for 60 min) elicited an increase in LMV NHE3 transport activity but did not increase Na-glucose cotransport activity. Cyto D pretreatment of suspensions did not change the apparent affinity of NHE3 for internal H+. In contrast, after Cyto D pretreatment of the suspensions, NHE3 protein abundance was increased in LMV and remained unchanged in cortical cell homogenates. The effect of Cyto D on NHE3 was further assessed with cultures of murine cortical cells. The amount of surface biotinylated NHE3 increased on Cyto D treatment, whereas NHE3 protein abundance was unchanged in cell homogenates. In conclusion, under basal conditions NHE3 activity depends on the state of actin organization possibly involved in trafficking processes between luminal membrane and intracellular compartment.

Gentamicin inhibits renal protein and phospholipid metabolism in rats: implications involving intracellular trafficking.

Studies were undertaken to characterize the mechanism of aminoglycoside-induced nephrotoxicity. Early time points in gentamicin treatment (1 to 3 d) were used to investigate the development of toxic events without the complication of gross morphologic cellular alterations. Enzyme activities of cortical homogenates and brush border membrane (BBM) preparations documented little effect on specific activities or the ability to isolate representative membrane fractions. In vivo protein synthesis experiments demonstrated that gentamicin reduced cellular protein synthesis after 2 d of treatment. This inhibition increased to 50% on the third day. Total cellular proteins synthesis was inhibited to the same extent as BBM protein synthesis. However, gentamicin had different effects on homogenate versus BBM phospholipids. The total phospholipid contents in cortical homogenates and BBM from treated animals were increased, compared with control animals. A significant decrease in phospholipid synthesis was observed only in homogenates from treated animals. When effects on individual phospholipids were investigated, only an increase in phosphatidylinositol levels was observed in cortical homogenates from treated rats. However, gentamicin treatment was demonstrated to increase the levels of phosphatidylinositol and phosphatidylcholine, while decreasing the level of sphingomyelin (SPH), in BBM. Incorporation of (32)P into SPH, phosphatidylserine, and phosphatidylethanolamine was inhibited in cortical homogenates from gentamicin-treated animals; among BBM phospholipids, however, a significant decrease was observed only for SPH synthesis. It was concluded that inhibition of phospholipid degradation was quantitatively the major contributor to the effects of gentamicin on phospholipid metabolism. Confocal microscopic studies, using tracer amounts of fluorescently labeled gentamicin, revealed gentamicin in large, mostly basal structures. Correlative electron microscopic studies, using photo-oxidation techniques, demonstrated that these structures consisted of lysosomal, Golgi complex, and mitochondrial structures. These observations suggest retrograde trafficking of gentamicin and indicate a general mechanism of gentamicin-induced nephrotoxicity.

Cortical Na+,K+-ATPase Activity, Abundance and Distribution after in vivo Renal Ischemia without Reperfusion in Rats

The aim of our work was to study the changes in activity, abundance and distribution of sodium, potassium-adenosine triphosphatase (Na⁺,K⁺-ATPase) in membranes of cortical tubular cells in an in vivo model of ischemic injury without reperfusion. Na⁺,K⁺-ATPase, alkaline phosphatase (AP) activities and their distribution in membranes isolated from renal cortex using a Percoll gradient were studied after different ischemic periods. Na⁺,K⁺-ATPase α-subunit protein abundance was analysed by Western-blot. Plasma urea and cortical adenosine 5’triphosphate (ATP) were also measured. In cortical homogenates 5 min of ischemia promoted a diminution in ATP content. Na⁺,K⁺-ATPase activity diminished after 40 min and AP after 100 min of ischemia. Na⁺,K⁺-ATPase activity in the Percoll gradient fractions after 5 min peaked at a higher density and was significantly decreased after 40 min. AP activity was decreased in typically enriched apical membranes after both times of ischemia. At each time studied Na⁺,K⁺-ATPase abundance was increased in cortical homogenates and membranes. Our results showed opposite effects of ischemia on Na⁺,K⁺-ATPase activity and abundance. Increased levels of Na⁺,K⁺-ATPase protein were observed. The enzyme would be rapidly delivered to membrane domains and become inactivated as ischemia persists.

Effects of reboxetine and sertraline treatments alone and in combination on the binding properties of cortical NMDA and beta1-adrenergic receptors in an animal model of depression.

Changes to the binding properties of cortical N-methyl-D-aspartic acid (NMDA) and beta-adrenergic receptors have both been reported as potential indicators of antidepressant activity. In the present investigation we examined the effects of the noradrenaline reuptake inhibitor, reboxetine, the serotonin reuptake inhibitor, sertraline, alone and in combination on the binding properties of cortical NMDA receptors and cortical beta1-adrenoceptors following 14 days of treatment in the olfactory bulbectomized rat model of depression. A decrease in the potency of glycine to displace the strychnine insensitive glycine antagonist [3H] 5,7 dichlorokynurenic acid (5,7 DCKA) was observed in cortical homogenates of OB rats when compared to sham-operated controls. Similarly, treatment with the combination of reboxetine and sertraline for 14 days produced a decrease in the potency of glycine when compared to vehicle treated controls. By contrast neither olfactory bulbectomy or drug treatment significantly altered basal or glycine enhanced binding of the non-competitive NMDA antagonist [3H] MK-801 in cortical homogenates. Reboxetine alone, and in combination with sertraline, down-regulated [3H]-CGP 12177 (a selective beta-adrenoceptor antagonist) binding in both OB and sham-operated animals. The lack of a bulbectomy effect in the [3H] CGP-12177 binding assay, and the fact that olfactory bulbectomy and antidepressant treatments produce a similar change to the potency of glycine at the NMDA receptor, suggests that these tests do not provide a neurochemical marker for either the behavioral hyperactivity deficit or antidepressant response in the model.

Na +–Ca 2+ exchanger isoforms in rat neuronal preparations: different changes in their expression during postnatal development

We examined the relative amounts of Na +–Ca 2+ exchanger (NCX) isoform mRNAs in cultured neurons, astrocytes and developmental rat brain. NCX1 transcript was predominant in neurons and astrocytes, but NCX2 transcript was about four-fold higher than NCX1 or NCX3 transcript in adult rat cortex. NCX2 transcript in the cortex increased markedly during postnatal development, whereas NCX1 and NCX3 transcripts decreased. Na +-dependent 45Ca 2+ uptake in the cortical homogenate increased significantly during postnatal development.

Effect of picrotoxin-induced seizures on lipid composition of cortical tissue homogenate and its nuclear fraction in rats

Effect of picrotoxin-induced seizures on lipid composition of cortical tissue homogenate and its nuclear fraction in rats

2,7-Dioxo-2,3,4,5,6,7-hexahydro-1H-benzo[h][1,4]diazonine as a new template for the design of CCK(2) receptor antagonists.

A novel series of nonpeptide CCK(2) receptor antagonists has been prepared, in which 2,7-dioxo-2,3,4,5,6,7-hexahydro-1H-benzo[h][1, 4]diazonine (5) was used as a chemical template. This uncommon ring system was obtained in a highly substituted form and in high yield by ozonolysis of the enamine bond of 1,2,3,4-tetrahydro-9H-pyrido[3, 4-b]indole derivatives (4), in which the configuration of the substituents was established stereoselectively via the Pictet-Spengler reaction. Further structural manipulation was guided by molecular modeling through comparison of fieldpoint-based structures of candidate compounds with a selected low-energy conformation of the representative CCK(2) receptor antagonist 5-[[[(1S)-[[(3, 5-dicarboxyphenyl)amino]carbonyl]-2-phenylethyl]amino]carbonyl]-6- [[( 1-adamantylmethyl)amino]carbonyl]indole (JB93182 (3)). By this approach compounds such as (3R, 5S)-4-acetyl-3-(1-adamantyl)methyl-1-(2-chlorobenzyl)-5-carboxymet hyl aminocarbonyl-2,7-dioxo-2,3,4,5,6,7-hexahydro-1H-benzo[h][1, 4]diazonine (32) were prepared. Compound 32 behaved as a competitive CCK(2) receptor antagonist in vitro as judged by its inhibition of pentagastrin-stimulated acid secretion in an isolated, lumen-perfused, immature rat stomach assay (pK(B) = 6.74 +/- 0.27) and by its displacement of [(125)I]CCK-8S from CCK(2) sites in mouse cortical homogenates (pK(i) = 6.99 +/- 0.05). Compound 32 was 100-fold selective for CCK(2) over CCK(1) receptors based on the affinity estimate obtained in a guinea pig pancreas radioligand binding assay (pK(i) = 5.0).

Metabolism alters the selectivity of angiotensin-(1-7) receptor ligands for angiotensin receptors.

The present study examined whether metabolism of the putative angiotensin-(1-7) receptor agonist and antagonist [angiotensin-(1-7) and D-alanine(7) angiotensin-(1-7), respectively] altered their ability to interact with angiotensin AT(1), AT(2), and AT(4) receptor subtypes. Both angiotensin-(1-7) and D-alanine(7) angiotensin-(1-7) competed with low affinity for (125)I-sarcosine(1), isoleucine(8) angiotensin II binding to AT(1) and AT(2) receptors in rat liver and adrenal medulla membranes, respectively, and competed with low affinity for (125)I-angiotensin IV binding to AT(4) receptors in bovine kidney epithelial cell membranes. In vitro renal metabolism of the angiotensin-(1-7) receptor ligands (incubating peptides with rat cortical tissue homogenates) had minimal influence on low-affinity binding to AT(1) and AT(2) receptors, yet caused a significant and dramatic shift toward high-affinity binding for AT(4) receptors. Low-affinity angiotensin II binding to the AT(4) receptor was also shifted toward high-affinity binding following renal metabolism of the peptide. Conversely, angiotensins with high affinity for the AT(4) receptor (e.g., angiotensin IV) were shifted toward low-affinity binding states following peptide metabolism. Incubation of (125)I-angiotensin-(1-7) with rat cortical tissue generated the high-affinity AT(4) receptor ligand (125)I-angiotensin-(3-7), whereas the renal metabolism of (125)I-angiotensin II generated both (125)I-angiotensin-(3-7) and (125)I-angiotensin IV. These results reveal that renal metabolism of angiotensin-(1-7) receptor ligands and angiotensin II yields products that have high affinity for the AT(4) receptor and could potentially contribute to the biologic actions of the parent peptide in the kidney.

N ‐methyl‐3‐(1‐hydroxy‐5‐[ 123 I]iodopent‐4‐enyl)‐4‐acetoxypiperidine, a novel candidate of acetylcholinesterase activity imaging agent

A novel acetylcholine radioanalog, N-methyl-3-(1-hydroxy-5-[ 123 I]iodopent-4-enyl)-4-acetoxypiperidine, was prepared by radioiodination of the corresponding tributylstannyl precursor that was synthesized in eight steps from 4-piperidone. The tracer has three asymmetric carbons giving eight optical isomers. Two optical isomers were isolated in the precursor synthesis by diastereomeric and enantiomeric separation. In the incubation experiments using rat cerebral cortical homogenate, one optical isomer was hydrolyzed by acetylcholinesterase with high reactivity and selectivity. The tracer is a candidate for mapping cerebral regional acetylcholinesterase activity by single photon emission computed tomography.

Melatonin suppresses iron-induced neurodegeneration in rat brain

The antioxidative action of melatonin on iron-induced neurodegeneration in the nigrostriatal dopaminergic system was evaluated in vivo. Intranigral infusion of iron chronically degenerated the dopaminergic transmission of the nigrostriatal system. An increase in lipid peroxidation in the infused substantia nigra and reductions in dopamine levels and dopaminergic terminals in the ipsilateral striatum were observed 7 d after iron infusion. Whereas local infusion of melatonin (60 μg/μl, 1 μl) alone did not alter dopaminergic transmission, coinfusion of melatonin with iron suppressed iron-induced oxidative damages. Systemic infusion of melatonin via osmotic pumps had no effect on iron-induced neurodegeneration. However, repetitive intraperitoneal injections of melatonin (10 mg/kg) prevented iron-induced oxidative injuries. The ratio of glutathione (GSH)/oxidized glutathione (GSSG) was moderately increased in the lesioned substantia nigra of the melatonin-treated rats compared to that of the lesioned group in control rats. The antioxidative effect of melatonin was verified in cortical homogenates. Melatonin dose-dependently suppressed autoxidation and iron-induced lipid peroxidation. Melatonin was as effective as GSH and was less effective than Trolox (a water-soluble analogue of vitamin E) in inhibiting iron-elevated lipid peroxidation of brain homogenates. Our data suggest that melatonin is capable of at least partially preventing the iron-induced neurodegeneration in the nigrostriatal dopaminergic system.

Hepatic α2μ-globulin localizes to the cytosol of rat proximal tubule cells

Alpha 2 mu-Globulin (A2), an 18.6 kD protein of hepatic origin, accumulates in the proximal tubule as an abundant, 15.5 kD cleavage product termed "A2-fragment" (A2-f). A2-f facilitates proximal tubule fatty acid oxidation, presumably by binding hydrophobic ligands. This requires some A2-f to enter the cytosol of the renal epithelial cell (REC). The localization of A2/A2-f in the proximal tubule cell was evaluated in this study. Immunoblot analysis of renal cortical homogenates separated by differential centrifugation and quantitative immunoelectron microscopy (IEM) was performed to localize A2/A2-f using an affinity-purified antibody that detects both proteins. To evaluate A2 as a physiologically relevant ligand, the accumulation of A2-f in the female rat kidney (normally devoid of A2-f) was examined after the induction of hepatic A2 synthesis. Ligand binding, uptake, and degradation assays were used to assess A2 processing by RECs in vitro. Although A2 and A2-f were detected in the "lysosomal" fraction, only A2-f was found in the soluble protein fraction. IEM confirmed the presence of significant signal in the vesicular and lysosomal as well as the cytosolic compartments. In contrast, both beta 2 mu globulin (B2) and cathepsin B were restricted to endosomes. In the female rat, induction of hepatic A2 production resulted in A2-f accumulation in the renal cortex. In RECs in culture, uptake of A2 and B2 demonstrated nonsaturable, nondisplacable surface binding and similar uptake rates. Compared with B2, A2 was markedly resistant to degradation. A fraction of A2 escapes lysosomal degradation, permitting A2-f to accumulate in the cytosol of the proximal tubule epithelial cell. A2 may represent an unusual example of a physiologic protein capable of accumulating in a distant cell type.

Inactivation of 5-HT 1A receptors in hippocampal and cortical homogenates

5-HT 1A receptor function can be assessed in rat hippocampal and cortical membrane preparations as agonist-stimulated [ 35S]GTPγS binding. Membranes were preincubated in vitro with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). R(+)-8-hydroxy-2-(di- n-propylamino)tetralin [ R(+)-8-OH-DPAT]-stimulated [ 35S]GTPγS binding and [ 3H]8-OH-DPAT binding assays were used to assess 5-HT 1A receptor function and density, respectively. EEDQ decreased both R(+)-8-OH-DPAT-stimulated [ 35S]GTPγS and [ 3H]8-OH-DPAT binding in hippocampal and cortical membranes. The E max but not the EC 50 of R(+)-8-OH-DPAT to stimulate [ 35S]GTPγS binding was decreased by EEDQ in both preparations. Additionally, the IC 50 for EEDQ to reduce R(+)-8-OH-DPAT-stimulated [ 35S]GTPγS and [ 3H]8-OH-DPAT binding was the same for both brain regions in both assays. In contrast to EEDQ alone, agonist-stimulated [ 35S]GTPγS binding was not reduced in hippocampal membranes preincubated with EEDQ and the 5-HT 1A receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]- N-2-pyridinyl-cyclohexanecarboxamide maleate (WAY 100,635), suggesting that EEDQ acts directly on the receptor. Due to parallel reductions in receptor density and maximal functional response, it is concluded that there is little or no reserve for 5-HT 1A receptor coupling to G α in these preparations. In addition, the sensitivity of hippocampal and cortical 5-HT 1A receptors to inactivation by EEDQ in vitro is the same.

Matrix metalloproteinases contribute to brain damage in experimental pneumococcal meningitis.

The present study was performed to evaluate the role of matrix metalloproteinases (MMP) in the pathogenesis of the inflammatory reaction and the development of neuronal injury in a rat model of bacterial meningitis. mRNA encoding specific MMPs (MMP-3, MMP-7, MMP-8, and MMP-9) and the inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) were significantly (P < 0.04) upregulated, compared to the beta-actin housekeeping gene, in cortical homogenates at 20 h after infection. In parallel, concentrations of MMP-9 and TNF-alpha in cerebrospinal fluid (CSF) were significantly increased in rats with bacterial meningitis compared to uninfected animals (P = 0.002) and showed a close correlation (r = 0.76; P < 0. 001). Treatment with a hydroxamic acid-type MMP inhibitor (GM6001; 65 mg/kg intraperitoneally every 12 h) beginning at the time of infection significantly lowered the MMP-9 (P < 0.02) and TNF-alpha (P < 0.02) levels in CSF. Histopathology at 25.5 +/- 5.7 h after infection showed neuronal injury (median [range], 3.5% [0 to 17.5%] of the cortex), which was significantly (P < 0.01) reduced to 0% (0 to 10.8%) by GM6001. This is the first report to demonstrate that MMPs contribute to the development of neuronal injury in bacterial meningitis and that inhibition of MMPs may be an effective approach to prevent brain damage as a consequence of the disease.

A novel serotonin transporter ligand: (5-Iodo-2-(2-dimethylaminomethylphenoxy)-benzyl alcohol

The serotonin transporters (SERT) are the primary binding sites for selective serotonin reuptake inhibitors, commonly used antidepressants such as fluoxetine, sertraline, and paroxetine. Imaging of SERT with positron emission tomography and single photon emission computed tomography in humans would provide a useful tool for understanding how alterations of this system are related to depressive illnesses and other psychiatric disorders. In this article the synthesis and characterization of [ 125I]ODAM [(5-iodo-2-(2-dimethylaminomethylphenoxy)-benzyl alcohol, 9)] as an imaging agent in the evaluation of central nervous system SERT are reported. A new reaction scheme was developed for the preparation of compound 9, ODAM, and the corresponding tri-n-butyltin derivative 10. Upon reacting 10 with hydrogen peroxide and sodium[ 125I]iodide, the radiolabeled [ 125I]9 was obtained in good yield (94% yield, radiochemical purity >95%). In an initial binding study using cortical membrane homogenates of rat brain, ODAM displayed a good binding affinity with a value of K i = 2.8 ± 0.88 nM. Using LLC-PK 1 cells specifically expressing the individual transporter (i.e. dopamine [DAT], norepinephrine [NET], and SERT, respectively), ODAM showed a strong inhibition on SERT (K i = 0.12 ± 0.02 nM). Inhibition constants for the other two transporters were lower (K i = 3.9 ± 0.7 μM and 20.0 ± 1.9 nM for DAT and NET, respectively). Initial biodistribution study in rats after an intravenous (IV) injection of [ 125I]ODAM showed a rapid brain uptake and washout (2.03, 1.49, 0.79, 0.27, and 0.07% dose/organ at 2, 30, 60, 120, and 240 min, respectively). The hypothalamus region where the serotonin neurons are located exhibited a high specific uptake. Ratios of hypothalamus-cerebellum/cerebellum based on percent dose per gram of these two regions showed values of 0.35, 0.86, 0.86, 0.63, and 0.34 at 2, 30, 60, 120, and 240 min, post-IV injection, respectively. The specific uptake in hypothalamus can be effectively blocked by pretreatment of known SERT ligands. The results suggest that this novel ligand displays desirable in vitro and in vivo properties as a potential SERT imaging agent.

Noradrenergic lesion antagonizes desipramine-induced adaptation of NMDA receptors

Repeated administration of the tricyclic antidepressant, desipramine, for 28 days to mice effected a decrease in the potency of glycine to displace [ 3H]5,7-dichlorokynurenic acid (5,7-DCKA) in mouse cortical homogenates. Pre-treatment with the noradrenergic neurotoxin DSP-4, while having no effect alone, attenuated the desipramine-induced effect. The present findings support a norepinephrine-dependent adaptation of the NMDA receptor complex in vivo following chronic desipramine treatment. The inter-relationship of norepinephrine and glutamate transmission may provide insight into the mechanism underlying the action of antidepressant drugs.

Characterization of a Selective Protein Kinase C Substrate Derived from the MARCKS Phosphorylation Site Domain for Use in Brain Tissue Homogenates

Protein kinase C (PKC) isozymes play crucial roles in neuronal signal transduction and can regulate transmitter release, ion channels, neural development, and plasticity. In vitro assays of PKC are frequently used to associate PKC activity with cellular function, and the availability of selective PKC substrates can facilitate such studies. We have characterized a commercially available 12 amino acid peptide derived from the myristoylated alanine-rich C kinase substrate (MARCKS-PSD, Calbiochem) for use in crude rat brain homogenates. Assays were performed at 25°C for 10 min (linear up to 12 min) using optimal concentrations of calcium and lipid cofactors. Kinetic analysis of MARCKS-PSD phosphorylation by PKC purified from rat brain gave a Km of 2.3 μM, which was similar to the Km of 2.8 μM obtained using rat brain cortical homogenates. The selective PKC inhibitor bisindolylmaleimide reduced phosphorylation of MARCKS-PSD in a concentration-dependent manner, with greater than 95% inhibition at 1.0 μM. MARCKS-PSD was more potent than another widely used selective PKC substrate (neurogranin(28–43) and was a good substrate for human recombinant PKC α, δ, and ϵ but not ζ. The ontogeny of PKC activity was examined in the cortex and cerebellum. PKC activity was low at birth and reached adult levels by 21 days of age in both regions. Calcium-independent PKC activity in brain homogenates could be measured with MARCKS-PSD and accounted for approximately 25 and 10% of total activity in 1-day-old and adult rat cortex, respectively. These results suggest that the MARCKS-PSD peptide can be used as a selective PKC substrate in rat brain homogenates.

Effect of picrotoxin-induced seizures on lipid composition of cortical tissue homogenate and its nuclear fraction in rats.

The content of lipids in rat cortical tissue homogenate and fraction of neuronal nuclei was determined under normal conditions and after picrotoxin-induced seizures. Changes in lipid composition of homogenate and nuclear fraction differed considerably. In homogenate, the content of sphingomyelin, lysophosphatidylcholine, and total phospholipids increased, while the content of free fatty acid remained unchanged; in the nuclear fraction the total phospholipid content decreased, while the concentration of free fatty acids increased.