In vivo measurement of blood-brain barrier permeability.

This unit describes various protocols for the in vivo quantitation of drug permeability across the rodent blood - brain barrier. Methods for the measurement of drug influx or efflux are described, and support protocols are provided for determining intravascular capillary volume and cerebral perfusion flow. An in situ perfusion technique is also provided for assessing whether transport of a test compound occurs by carrier-mediated or saturable transport.

A critical evaluation of the brain efflux index method as applied to the nitric oxide synthase inhibitor, aminoguanidine.

The Brain Efflux Index (BEI) method is an in vivo procedure designed to quantitate saturable efflux mechanisms resident at the blood--brain barrier (BBB). The present work utilized the BEI method to assess the BBB efflux mechanisms of [(14)C]aminoguanidine, a nitric oxide synthase inhibitor. The BEI for [(14)C]aminoguanidine was >100% (relative to [(3)H]inulin diffusion) over a range of 41-184 pmol after 40 min. The unusually high retention (>100%) of [(14)C]aminoguanidine suggested brain parenchymal sequestration, either by neuronal uptake or tissue protein binding. The uptake of [(14)C]aminoguanidine in dendritic neuronal endings (synaptosomes) showed a saturable concentration dependency, consistent with a carrier-mediated process. Nonlinear least-squares regression yielded the following Michaelis--Menten and diffusional (k(ns)) parameters for synaptosomal [(14)C]aminoguanidine uptake: V(max)=118.50 +/- 28.77 pmol x mg protein(-1)/3 min; K(m)=58.34 +/- 8.33 muM; k(ns)=0.15 +/- 0.029 pmol x mg protein(-1)/3 min/muM; mean +/- SEM; n=3 concentration profiles). Protein binding studies using brain tissue showed negligible binding. In summary, this work identified three principle findings: (1) An apparent lack of quantifiable aminoguanidine BBB efflux; (2) a previously undescribed synaptosomal accumulation process for aminoguanidine; and (3) an interesting limitation of the BEI technique where unusual brain parenchymal sequestration yields values >100%.

Blood-brain barrier transport studies of organic guanidino cations using an in situ brain perfusion technique.

Blood-brain barrier (BBB) transport of essential polar substrates is mediated by specific, carrier-mediated transport proteins. The BBB transport mechanisms for polar compounds with terminal guanidino functional groups (R-NHC(NH)NH(2)) are not well defined. The goal of the present work was to investigate the BBB transport mechanism(s) for terminal guanidino substrates using an in situ brain perfusion technique. Brain region radiotracer influx clearance (Cl(in)) was calculated for representative guanidino substrates, [14C]L-arginine, [14C]aminoguanidine and [14C]guanidine, in the presence or absence of excess terminal guanidino analogues. The Cl(in) for [14C]L-arginine (0.21+/-0.0094 cm(3)/min/g wet brain weight, mean+/-S.E.M., n=four rats) was significantly decreased by 1000x concentrations of unlabeled L-arginine, N(G)-methyl-L-arginine, N(G)-,N(G)-dimethyl-L-arginine and N(G)-amino-L-arginine by approximately 83% (P<0.01; n=4-5), whereas 1000x concentrations of nitro-L-arginine, aminoguanidine and guanidine were without effect. In contrast, the respective Cl(in) of [14C]aminoguanidine and [14C]guanidine (0.0085+/-0.00039 and 0.015+/-0.0015 cm(3)/min/g, n=4, respectively) were not significantly decreased by 1000x concentrations of unlabeled aminoguanidine or guanidine. The Cl(in) values for all [14C]guanidino probes were significantly greater (P<0. 05) from that of [3H]inulin, a marker of cerebrovascular blood volume. These data suggest that the hydrophilic guanidino cations aminoguanidine and guanidine penetrate the BBB by a minor diffusional process with no appreciable transport via saturable processes. In contrast, BBB penetration of L-arginine occurs via the saturable basic amino acid transporter that has specificity for amino acid analogues possessing cationic terminal guanidino groups.

Nitric oxide redox species exert differential permeability effects on the blood-brain barrier.

Excessive production of nitric oxide (NO) in the central nervous system is suspected to contribute to neurodegenerative diseases. Previous studies showed that excessive central nervous system NO increased the permeability of the blood-brain barrier (BBB) during experimental meningitis. The present work hypothesizes that the various NO redox forms (NO(*), NO(+), NO(-)) differentially mediate disruption of the BBB. Pharmacological agents that release NO redox forms (i.e., NO prodrugs) were selected based on the rate of NO release and the liberated NO redox form. An in situ rodent brain perfusion preparation was used to administer NO prodrugs into the cerebrovascular circulation, followed by brain perfusion with [(14)C]sucrose, a marker of BBB integrity. Cerebrovasculature infusion of certain NO prodrugs caused a significant, 2- to 5-fold BBB permeability increase in all forebrain regions (P <.01). The NO prodrug rank order of BBB disruption was S-nitroso-N-acetylpenicillamine-beta-cyclodextrin (releases NO(*), NO(+), and NO(-)) > Angeli's salt (NO(*), NO(-)) > MAHMA NONOate approximately diethylamine NONOate (NO(*)) > spermine NONOate (NO(*)) > DETA NONOate approximately Piloty's acid (negligible NO redox release) approximately saline. When normalized to BBB disruption caused by hyperosmotic mannitol (100%), S-nitroso-N-acetylpenicillamine-beta-cyclodextrin (NO(*), NO(+), and NO(-)) elicited approximately 45% disruption, Angeli's salt (NO(*), NO(-)) elicited approximately 18% disruption, and the NONOates (NO(*)) ranged from approximately 0 to 8% disruption. Cerebral blood flows and intracranial pressures were within normal limits for each tested NO prodrug, thereby suggesting that BBB disruption was not secondary to altered cerebral perfusion. Collectively, the results of this work identify that NO(*) alone exerts modest BBB disruption compared with the specie combination of NO(*) and NO(-), and the greatest disruption is exerted by the combination of NO(*), NO(-), and NO(+).

Cellular transport processes of aminoguanidine, a nitric oxide synthase inhibitor, in the opossum kidney cell culture line.

Aminoguanidine has potential pharmacologic utility for diabetes and nitric oxide - mediated inflammation. Because aminoguanidine is positively charged at physiologic pH (pK(a) approximately 10), it is unlikely that simple diffusion is a predominant mechanism for cellular penetration. This study sought to determine the transport processes by which aminoguanidine, a cationic compound, traverses across cellular membranes. In cultured opossum kidney (OK) cell monolayers, aminoguanidine transport involved both saturable and non-saturable diffusion processes. At passage numbers below 67, the observed V(max) and K(m) for saturable influx were significantly lower than that observed at passages greater than 79 (V(max): low passage, 21.2+/-7.8 pmol/(min*mg protein), n=3; versus high passage, 129.7+/-24.3 pmol/(min*mg protein), n=3, P<0.05; K(m): low passage, 23.7+/-10.8 microM, n=3; versus high passage, 101.7+/-5.6 microM, n=3, P<0.05; mean+/-S.E.M.). Nonsaturable processes were not statistically different (k(ns): low passage, 1.6+/-0.1 pmol/(min*mg protein*microM), n=3; high passage, 1.1+/-0.2 pmol/(min*mg protein*microM) n=3). Saturable influx was temperature dependent, and independent of ATP energy, sodium gradients or changes in membrane potential. Other organic cations competitively inhibited and trans-stimulated saturable influx. Aminoguanidine influx was increased in the presence of an outwardly-directed proton gradient and was inhibited in the presence of an inwardly-directed proton gradient. Correspondingly, aminoguanidine efflux was trans79) express a saturable, bi-directional carrier-mediated process to transport aminoguanidine across cellular membranes.

Theoretical pharmacokinetic and pharmacodynamic simulations of drug delivery mediated by blood--brain barrier transporters.

Pharmacokinetic/pharmacodynamic simulations were performed to assess the feasibility of central nervous system (CNS) drug delivery via endogenous transporters resident at the blood-brain barrier (BBB). Pharmacokinetic models were derived for intravenous bolus dosing of a hypothetical drug in the absence or presence of an endogenous, competing transport inhibitor. These models were linked to CNS pharmacodynamic models where the effect sites were either cell surface receptors or intracellularly localized enzymes. The response of the dependent parameter, the duration of effect (t(dur)), was examined in relationship to changes in the independent parameters, i.e. dose, elimination rate constant (k(e1)), BBB transport parameters (K(m1) and V(max1)) and EC(50) (effective concentration that elicits a 50% response). As expected, t(dur) increased with (a) increases in drug doses, (b) decreases in k(e1) or (c) decreases in EC(50), irrespective of the effect site. Surprisingly, endogenous transport inhibition produced decreases in drug terminal half-life and corresponding decreases in t(dur). Interestingly, t(dur) was independent of assigned transporter K(m) and V(max) when the dose/EC(50) ratio (dose/EC(50)) was >1 (irrespective of endogenous transport inhibition), but highly dependent on K(m1) and V(max1) when dose/EC(50) was (a) <1 (no endogenous transport inhibition) or (b) equal to 1 (with endogenous transport inhibition). Oral input of the endogenous transport inhibitor produced a decrease in t(dur) when the dose/EC(50) range was 0.1-10. These simulations highlight that (a) systemic pharmacokinetic and BBB transport parameters influence t(dur), (b) drug terminal half-life is inversely related to circulating levels of endogenous inhibitors, and (c) oral ingestion of endogenous transport inhibitor(s) reduces t(dur). Overall, these simulations provide insight for the feasibility of rational CNS drug design/delivery via endogenous transporters.

Quantification of iNOS mRNA with reverse transcription polymerase chain reaction directly from cell lysates.

Inducible nitric oxide synthase (iNOS) is a member of a family of primary inflammatory response genes. Quantitative measurement of iNOS mRNA levels is important for the study of gene expression of this enzyme during the process of inflammation. We report here a method for quantitative measurement of iNOS mRNA levels with rtPCR directly from cells lysed with a single step phenol/chloroform/ether extraction. Using a mouse macrophage cell line, J774.2, which expresses iNOS mRNA upon LPS + IFN-gamma treatment as the model, the effects of the extraction on iNOS mRNA recovery and cytosolic RNase removal have been studied. The cells are lysed and RNases denatured and removed by phenol/chloroform extraction. Trace amounts of the phenol partitioned in the samples are then removed by ether extraction. After the extraction, the samples can be used directly for reverse transcription and PCR without further purification of RNA. The recovery of specific mRNA is not affected by the extraction procedure and externally added iNOS cRNA shows no degradation by the extracted cell lysates. Measurement of iNOS mRNA with this procedure is linear using serially double-diluted cells in the range from 94 to 6000 cells. The efficiencies of rtPCR of iNOS wild-type and deletion cRNAs are also compared in our study. By controlling the molecular size of the deletion construct to within 10% of that of the wild type and maintaining PCR cycling below 25 cycles, the rtPCR efficiencies of iNOS wild type and deletion are identical. The detection of rtPCR products is enhanced by hybridization with specific probes. Under these conditions, iNOS mRNA concentration can directly be calculated from the internal standard in each tube without a standard curve. We conclude that our procedure provides an accurate method for quantitative measurement of iNOS mRNA from limited amount of cells without complete RNA isolation.

Improved high-performance liquid chromatographic assay for nimesulide.

An improved, validated HPLC assay was developed for the non-steroidal anti-inflammatory agent, nimesulide. In contrast to previous methods, the present assay requires smaller plasma volumes (0.2 ml) and utilizes a commercially available, structurally similar analogue of nimesulide, NS-398. The method involves a liquid-liquid extraction procedure that can be completed within 4 h, followed by reversed-phase HPLC analysis. Briefly, the extraction protocol required toluene extraction of acidified plasma samples, followed by back-extraction of the retained toluene phase with aqueous base. The retained aqueous alkaline phase was concentrated by toluene re-extraction. The retained toluene phase was evaporated to dryness and reconstituted with 100 microl of mobile phase. Extracted samples were injected (50 microl) onto a Shandon Hypersil BDS C18 column (5 microm particle size; 250x4.6 mm) equilibrated with 1.0 ml/min of 68:32 (v/v) methanol-citrate (0.08 M)-phosphate (0.04 M) buffer (pH 3.0) at room temperature, with detection at 240 nm. The chromatographic run time was 12 min with retention times of 5.9 min and 9.1 min for nimesulide and NS-398, respectively. The analytical method was successfully utilized for a pilot pharmacokinetic study.

Nitric oxide and prostaglandin E2 formation parallels blood-brain barrier disruption in an experimental rat model of bacterial meningitis.

During meningitis, the host produces a plethora of signaling agents as part of a coordinated defense mechanism against invading pathogens. Nitric oxide (NO) and prostaglandin E2 (PGE2) are two such inflammatory mediators produced in response to bacterial endotoxins. Disruption of the blood-brain barrier (BBB) is one of many pathophysiological consequences of meningitis. The present objective was to examine the time-course of NO and PGE2 production in relationship to BBB permeability alterations during experimentally-induced meningitis. Meningeal inflammation was elicited by intracisternal administration of the bacterial endotoxin, lipopolysaccharides (LPS; 200 microg), and NO, PGE2, and BBB integrity were monitored over the next 24 h. Meningeal NO production was assessed by headspace chemiluminescence; cerebrospinal fluid PGE2 was determined by enzyme-linked immunosorbent assay (ELISA) immunoassay; and BBB integrity was determined by the brain accumulation of 14C-sucrose. Similar time-course profiles for NO and PGE2 were observed, with a peak effect for both inflammatory mediators observed within 6-8 h after intracisternal LPS dosing. Statistically significant (p < 0.05) disruption of the BBB was observed in various brain regions. Strikingly similar temporal relationships were observed for NO and PGE2 production and BBB disruption. These results suggest the hypothesis that NO and PGE2 may act in conjunction to disrupt the BBB during experimental meningitis.

Chronic dosing with 1-aminocyclopropanecarboxylic acid, a glycine partial agonist, modulates NMDA inhibition of muscarinic-coupled PI hydrolysis in rat cortical slices.

Chronic dosing with the glycine partial NMDA agonist, 1-aminocyclopropanecarboxylic acid (ACPC) elicited an altered allosteric regulation of cortical NMDA receptor binding. The present study hypothesized that these allosteric receptor binding changes would be manifest as pharmacologically functional reductions in NMDA receptor activity following chronic ACPC dosing. NMDA inhibition of carbachol-induced phosphoinositide (PI) hydrolysis was used as a functional assay to assess NMDA receptor function in rat cerebral cortex. NMDA inhibition of stimulated PI turnover was similar in naive (46% +/- 4.5%; mean +/- SE; n = 34) and ACPC dosed rats (39% +/- 2.3%; n = 34). While ACPC reversed NMDA's inhibitory effects in naive rats (80% +/- 13%; n = 9), it was ineffective (P < 0.05) in ACPC pretreated rats (48% +/- 9.8%; n = 9). In contrast, the NMDA antagonists, MK-801 (ion channel), 7-chlorokynurenic acid (glycine site) and AP-7 (glutamate site), effectively reversed NMDA's inhibitory effects in naive and ACPC treated rats. The potency of these antagonists were unaltered by prior ACPC dosing. Thus, chronic ACPC therapy does not alter the functioning of the NMDA ion channel or glutamate receptor sites, but elicits functional tolerance to ACPC's actions in the cortical NMDA complex.

Chronic administration of NMDA glycine partial agonists induces tolerance in the Porsolt swim test.

The Porsolt swim test (PST) was used to assess behavioral effects following acute or chronic treatment with two N-methyl-D-aspartate (NMDA) glycine partial agonists, 1-aminocyclopropanecarboxylic acid (ACPC), and D-cycloserine (DCS). Consistent with previous findings in mice, single intravenous doses of ACPC in rats produced a significant, dose-dependent reduction in immobility in the PST compared to saline. Single dose DCS also elicited significant dose-dependent reductions in PST immobility times. Single-dose ACPC or DCS (200 mg/kg) reduced immobility (p < 0.05) by 26 or 30%, respectively, compared to saline. However, multiple dosing with either ACPC or DCS (6 daily doses, 200 mg/kg) produced an apparent behavioral adaptation, as the immobility data were indistinguishable from chronic saline administration. Moreover, pretreatment with a 5-day course of ACPC or DCS promoted the development of a behavioral cross-tolerance following a sixth dose of DCS or ACPC, respectively. The development of a behavioral tolerance in the PST following chronic therapy of these drugs appears to be a general feature of glycine partial agonists. In toto, these findings support the hypothesis that chronic administration of NMDA glycine partial agonists produces a behavioral tolerance putatively through an adaptation of the NMDA receptor complex.

Inhibition of nitric oxide synthase attenuates blood-brain barrier disruption during experimental meningitis.

Increased permeability of the blood-brain (B-B) barrier is observed during meningitis. Preventing B-B barrier alterations is important because adverse neurological outcomes are correlated with breeches in barrier integrity. It was hypothesized that pathological production of nitric oxide (NO) contributes to B-B barrier disruption during meningitis in the rat. Experimental meningitis was induced by intracisternal (i.c.) administration of lipopolysaccharides (LPS) or vehicle. Groups of rats were concomitantly infused intravenously (i.v.) with saline or the NO synthase inhibitor, aminoguanidine (AG). Eight h after i.c. dosing, B-B barrier alterations were quantitated pharmacokinetically using [14C]sucrose. Serum and regional brain tissues were obtained 0-30 min after tracer dosing and sucrose influx transfer coefficients (Kin(app)) were calculated from the brain tissue data. Compared to the control groups (i.c. vehicle/i.v. saline), the Kin(app) of the i.c. LPS/i.v. saline group increased 1.6-2.1-fold in various brain regions, thus confirming previous observations of increased [14C]sucrose barrier penetration during meningeal inflammation. Remarkably, i.v. administration of AG to i.c. LPS-treated rats significantly inhibited meningeal NO synthesis and decreased Kin (app) permeability alterations in the B-B barrier, compared to i.c. LPS/i.v. saline-treated rats. Regional brain Kin (app) estimates in the i.c. LPS/i.v. AG group were similar to control groups (i.c. vehicle/i.v. AG and i.c. vehicle/i.v. saline). In conclusion, these data suggest the general concept that excessive NO production during neuroinflammatory diseases contributes to disruption of the blood-brain barrier.

Pharmacological characterization of nitric oxide production in a rat model of meningitis.

Depending on its concentration and target site, nitric oxide (NO) is an intracellular messenger or inflammatory mediator. Recent research supports an expanded role for NO in the pathophysiology of neuroinflammatory diseases. Using analytical and pharmacological techniques, the present study identifies NO as a potential inflammatory mediator in experimental meningitis in the rat. Intracisternal administration of lipopolysaccharide (LPS) induced NO synthesis from the lateral and third ventricle choroid plexi and surface meninges but not from systemic white blood cells, suggesting that meningeal inflammation was restricted to the central nervous system. The time course of NO production revealed at 3 hr lag after intracisternal LPS, followed by a peak of 8 hr and subsequent decrease to baseline 24 hr after LPS dosing. The pharmacological rank order of NO synthase inhibitors in meningeal preparations (NG-aminoarginine > NG-methylarginine approximately aminoguanidine) was slightly different than the rank order for the LPS-stimulated monocyte-macrophage cell line, J774A.1 (NG-aminoarginine approximately NG-methylarginine > aminoguanidine). A prolonged inhibition of NO production was observed in cultured meningeal preparations or J774A.1 cells briefly exposed to and washed free from NO synthase inhibitors. These findings implicate NO as an inflammatory mediator during experimental meningitis, and suggest that NO synthase inhibitors might be potentially useful agents for meningeal inflammation.

Cerebrovascular permeability changes during experimental meningitis in the rat.

Alterations in the blood-brain and blood-cerebrospinal fluid barriers occur during bacterial meningitis. Preventing barrier alterations is important as the increases in barrier permeability are thought to contribute to adverse neurological outcomes. The objective of this study was to characterize pharmacokinetically cerebrovascular permeability alterations during meningeal inflammation. 14C-Sucrose was used as a quantitative marker of cerebrovascular integrity 8 hr after induction of experimental meningitis by intracisternal injection of 0, 25 or 200 micrograms lipopolysaccharides. Serum and brain tissues were obtained after tracer dosing. 14C-Sucrose influx transfer coefficients (Kin(app)) and cerebrovascular volumes (Vbr) were calculated for each brain region. Regional Vbr values were unaffected by lipopolysaccharide pretreatment. However, statistically significant increases in 14C-sucrose K(in)(app) values were observed in various brain regions (1.6- to 3.3-fold from control; P < .05). These permeability alterations cannot be attributed to changes in the systemic pharmacokinetics of 14C-sucrose as total clearance and the volume of distribution were unaffected by lipopolysaccharide treatment. This approach can be used in future studies to examine the contribution of various inflammatory mediators to altered cerebrovascular permeabilities during experimental meningitis.

Characterization of L-arginine and aminoguanidine uptake into isolated rat choroid plexus: differences in uptake mechanisms and inhibition by nitric oxide synthase inhibitors.

Recent reports suggest that nitric oxide (NO) may contribute to several neurodegenerative diseases, e.g., focal cerebral ischemia, N-methyl-D-aspartate-mediated neurotoxicity, and experimental autoimmune encephalomyelitis. Accordingly, an understanding of the CNS transport processes of NO synthase (NOS) inhibitors has important therapeutic implications. The objective of the present study was to characterize the in vitro transport processes governing the uptake of L-[14C]arginine and the NOS inhibitor [14C]aminoguanidine in rat choroid plexus tissue. Consistent with previous reports, the uptake of L-[14C]arginine was mediated by both saturable and nonsaturable processes and was inhibited by the NOS inhibitors NG-methyl-L-arginine, NG-amino-L-arginine, and N5-imidoethyl-L-ornithine. L-[14C]Arginine uptake was not inhibited by aminoguanidine or NG-nitro-L-arginine. Because aminoguanidine is an organic cation that bears some structural similarity to L-arginine, aminoguanidine might be transported by either an organic cation transporter or by the basic amino acid transporter governing arginine uptake. However, there was no evidence of a saturable uptake process for [14C]aminoguanidine in isolated rat choroid plexus, in contrast to that observed for L-[14C]arginine.

Inhibition of nitric oxide synthase partially attenuates alterations in the blood-cerebrospinal fluid barrier during experimental meningitis in the rat.

The permeability of the blood-cerebrospinal fluid (blood-CSF) barrier is increased upon exposure to lipopolysaccharides during bacterial meningitis. Lipopolysaccharides induce nitric oxide (NO) synthase in a variety of cells. Increased meningeal NO production and blood-CSF barrier permeability were observed in a rat model of meningitis. Administration of aminoguanidine, an inhibitor of NO synthase, blocked meningeal NO production and significantly attenuated permeability changes in the blood-CSF barrier. It is hypothesized that pathological production of NO may contribute to the disruption of the blood-CSF barrier during meningitis.

Desensitization of the NMDA receptor complex by glycinergic ligands in cerebellar granule cell cultures.

Glutamate neurotoxicity was examined in cultured cerebellar granule neurons following both prolonged (20-24 h) and brief (45 min) exposure to compounds acting at strychnine-insensitive glycine receptors. Glutamate neurotoxicity was reduced in a concentration-dependent fashion by brief exposure to the glycine partial agonists 1-aminocyclopropanecarboxylic acid (ACPC) and (+-)-3-amino-1-hydroxy-2-pyrrolidone (HA-966) and the competitive antagonist, 7-chlorokynurenic acid (7-CK) with a rank order efficacy: 7-CK > HA-966 > ACPC. Neither D-cycloserine (D-CS) nor glycine affected neurotoxicity produced by maximum glutamate concentrations, while glycine but not D-CS augmented the effects of submaximum glutamate concentrations. Prolonged exposure of cultures to either full (glycine) or partial agonists (ACPC, D-CS, HA-966) abolished the neuroprotective effects of ACPC and significantly diminished the neuroprotective effects of HA-966. In contrast, the neuroprotective effects of 7-CK were only marginally reduced by prolonged exposure to glycinergic ligands, while the neuroprotection afforded by compounds acting at other loci on the NMDA receptor complex (e.g. 2-amino-5-phosphonopentanoate (APV) and dizocilpine (MK-801)) were unaltered. These effects may represent homologous desensitization of the NMDA receptor complex at its strychnine-insensitive glycine receptor induced by prolonged exposure to glycinergic agonists and partial agonists. Nonetheless, levels of the NMDA receptor subunit zeta 1 mRNA were unaffected by prolonged exposure to ACPC, indicating the apparent desensitization could involve a post-translational modification of the NMDA receptor complex.

Microglial-produced nitric oxide and reactive nitrogen oxides mediate neuronal cell death.

The role of inflammatory cytokines in the pathogenesis of neurological diseases is not well understood. The neurotoxic effects of cytokines could be mediated by immunostimulation of glial cells to produce toxic concentrations of nitric oxide (NO) and reactive nitrogen oxides. Cultured microglia and meningeal fibroblasts, but not Type 1 astrocytes, were induced by lipopolysaccharides and cytokines to synthesize NO and reactive nitrogen oxides from L-arginine. In co-cultures of immunostimulated microglia and cerebellar granule neurons, neurotoxicity was blocked by an inhibitor of NO synthase, NG-nitroarginine, and by oxyhemoglobin, which inactivates NO. Microglial-induced neurotoxicity was also partially attenuated by the N-methyl-D-aspartate (NMDA) receptor antagonists, MK-801 and 2-amino-5-phosphovalerate (APV). Superoxide dismutase, which stabilizes NO through inactivation of superoxide anion, augmented microglial-mediated neurotoxicity either alone or in combination with MK-801 or APV. Hence, immunostimulated microglia mediate neurotoxicity by NO, reactive nitrogen oxides, superoxide anion and NMDA-like substances. These findings suggest a novel role for microglial-produced NO and reactive nitrogen oxides as a neurotoxic agent in neurodegenerative disease states.

High-performance liquid chromatographic assay for 1-aminocyclopropanecarboxylic acid from plasma and brain.

A reversed-phase high-performance liquid chromatographic method for the analysis of 1-aminocyclopropanecarboxylic acid (ACPC) from plasma or brain tissue is described. Samples were deproteinized with perchloric acid, centrifuged, alkalinized with potassium hydroxide and recentrifuged. The supernatants were derivatized with o-phthaldialdehyde and injected onto a C18 3-microns column (100 mm x 4 mm I.D.) pumped with 1 ml/min methanol-acetonitrile-0.1 M sodium phosphate buffer pH 6.0 (28:5:67, v/v). The retention times for ACPC and the internal standard were 15 and 31 min, respectively. The minimum detectable amount of ACPC was 0.08 nmol. The extraction recovery of ACPC (2.7-270 nmol) from spiked plasma or brain tissue ranged from 88 to 109%. The intra- and inter-day coefficients of variation for 27 nmol ACPC were 3.9 and 4.9%, respectively. This method was utilized to obtain preliminary pharmacokinetic parameters following ACPC administration to mice.

Strychnine-insensitive glycine receptors in embryonic chick retina: characteristics and modulation of NMDA neurotoxicity.

In the mammalian brain, the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is coupled to a cation channel and a strychnine-insensitive glycine receptor. The present paper demonstrates the presence of NMDA receptor-coupled strychnine-insensitive glycine receptors in embryonic chick retina. Both glycine and 1-aminocyclopropanecarboxylic acid (ACPC) exhibited similar potencies (271 +/- 39 vs 247 +/- 39 nM, respectively) as inhibitors of strychnine-insensitive [3H]glycine binding to retinal membranes. Moreover, glycine and ACPC enhanced [3H]MK-801 binding to sites within the NMDA-coupled cation channel in retinal membranes with potencies comparable to those reported in rat brain. While the potency of ACPC was significantly higher than glycine (EC50 54 +/- 12 vs 256 +/- 57 nM, P < 0.02) in this measure, there were no significant differences in the maximum enhancement (efficacy) of [3H]MK-801 binding by these compounds. Since glycine appears to be required for the operation of NMDA-coupled cation channels, we examined the effects of glycine and ACPC on NMDA-induced acute excytotoxicity in the 14-day embryonic chick retina. Histological evaluation of retina revealed that either ACPC (10-100 microM) or glycine (200 microM) attenuated NMDA-induced (200 microM) retinal damage and a combination of these agents produced an enhanced protection against acute NMDA toxicity. ACPC (100 microM), but not MK-801 (1 microM) also afforded a modest protection against kainate-induced (25 microM) retinal damage. These findings demonstrate that while strychnine-insensitive glycine receptors are present in embryonic chick retina, occupation of these sites does not augment the cytotoxic actions of NMDA. Moreover, the ability of ACPC and glycine to attenuate NMDA-induced cytotoxicity does not appear to be mediated through occupation of these sites.