Evaluation of HIV-1 Tat induced neurotoxicity in rat cortical cell culture.

In a substantial number of cases, Human Immunodeficiency Virus type 1 (HIV-1) infection causes neuronal cell loss and leads to the development of AIDS associated dementia. Several studies have suggested that both host and viral factors contribute to neuronal loss. Here we studied the effect of HIV-1 Tat in primary rat neuronal cells as a model to understand mechanism of neuronal cell death. At nano molar concentration, recombinant Tat induced cell death in primary rat mixed cortical neurons. Tat could also induce uptake of calcium in primary rat cultures. When cells were incubated with NMDA receptor antagonists, MK-801 and D-CPP, cell death and 45Ca uptake were inhibited. Under similar conditions non-NMDA antagonists, NBQX, DNQX and CNQX, and sodium channel antagonist, TTX, did not inhibit Tat induced neuronal cell death. In a similar way HIV associated products from in vitro HIV-1 infected cells induced neuronal cell death which was inhibited by NMDA receptor antagonist. Results presented in this paper suggest that activation of NMDA receptors by HIV-1 Tat is responsible for neuronal cell death in primary rat cortical neurons.

Processing of cdk5 activator p35 to its truncated form (p25) by calpain in acutely injured neuronal cells.

Recently, it was shown that conversion of cdk5 activator protein p35 to a C-terminal fragment p25 promotes a deregulation of cdk5 activity, which may contribute to neurodegeneration in Alzheimer's disease. In this study, we present evidence that calpain is a protease involved in the conversion of p35 to p25. To activate calpain, rat cerebellar granule neurons were treated with maitotoxin (MTX). A C-terminus-directed anti-p35 antibody detected that p35 conversion to p25 paralleled the formation of calpain-generated alpha-spectrin (alpha-fodrin) breakdown products (SBDP's) in a maitotoxin-dose-dependent manner. Two calpain inhibitors (MDl28170 and SJA6017) reduced p35 processing but were unchanged when exposed to the caspase inhibitor carbobenzoxy-Asp-CH(2)OC(=O)-2, 6-dichlorobenzene or the proteasome inhibitors (lactacystin and Z-Ile-Glu(OtBu)Ala-Leu-CHO). p35 protein was also degraded to p25 when rat brain lysate was subjected to in vitro digestion with purified mu- and m-calpains. Additionally, in a rat temporary middle cerebral artery occlusion model, p35 processing to p25 again paralleled SBDP formation in the ischemic core. Lastly, in malonate-injured rat brains, the ipsilateral side showed a striking correlation of SBDP formation with p35 to p25 conversion and tau phosphorylation (at Ser202 and Thr205) increase. These data suggest that calpain is a major neuronal protease capable of converting p35 to p25 and might play a pathological role of activating cdk5 and its phosphorylation of tau in Alzheimer's disease.

Assessment of endothelin receptor subtype-mediated increases of [Ca2+]i in distinct rat cell types using fluorimetric imaging.

Activation of endothelin (ET) receptor subtypes by various agonists causes an increase in [Ca2+]i in different cell types. This effect can be readily monitored in a 96-well plate format by detecting 1-s fluorescence changes of cell-permeant, Ca(2+)-sensitive dyes (e.g., Calcium Green-1 AM) using a fluorimetric imaging plate reader. This device was used to assess the ET receptor subtypes in primary cultures of rat mixed neocortical neuronal/glial cells and aortic smooth-muscle cells. Pharmacologic experiments with several ET receptor agonists and antagonists indicated that the ETA receptor subtype was functionally responsive in the smooth-muscle cells and that the ETB receptor subtype had a similar role in the mixed neuronal/glial cells.

Sodium channel modulators prevent oxygen and glucose deprivation injury and glutamate release in rat neocortical cultures.

Neocortical cultures were deprived of oxygen and glucose to model ischemic neuronal injury. We used a graded series of periods of oxygen and glucose deprivation, providing graded insults. Cell death was measured by release of lactate dehydrogenase (LDH). One hundred and twenty to 240 min of deprivation caused graded increases in glutamate overflow, LDH release and 45Ca influx. Curves of LDH release with respect to deprivation time were shifted to longer intervals by treatment with tetrodotoxin (TTX; 3, 30 or 300 nM), phenytoin (10, 30 or 100 microM), lidocaine (10, 30 or 100 microM) or the N-methyl-D-aspartate antagonist CPP [3(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid, 3, 10, 30 or 100 microM]. Combined treatment with TTX and CPP caused pronounced rightward shifts of LDH deprivation curves. Our results indicate that Na+ channel blockade is neuroprotective in neocortex cultures. Our results also suggest that neuroprotection with Na+ channel blockers may be due to inhibition of glutamate release.

An alpha-mercaptoacrylic acid derivative is a selective nonpeptide cell-permeable calpain inhibitor and is neuroprotective.

Overactivation of calcium-activated neutral protease (calpain) has been implicated in the pathophysiology of several degenerative conditions, including stroke, myocardial ischemia, neuromuscular degeneration, and cataract formation. Alpha-mercaptoacrylate derivatives (exemplified by PD150606), with potent and selective inhibitory actions against calpain, have been identified. PD150606 exhibits the following characteristics: (i) Ki values for mu- and m-calpains of 0.21 microM and 0.37 microM, respectively, (ii) high specificity for calpains relative to other proteases, (iii) uncompetitive inhibition with respect to substrate, and (iv) it does not shield calpain against inactivation by the active-site inhibitor trans-(epoxysuccinyl)-L-leucyl-amido-3-methylbutane, suggesting a nonactive site action for PD150606. The recombinant calcium-binding domain from each of the large or small subunits of mu-calpain was found to interact with PD150606. In low micromolar range, PD15O6O6 inhibited calpain activity in two intact cell systems. The neuroprotective effects of this class of compound were also demonstrated by the ability of PD150606 to attenuate hypoxic/hypoglycemic injury to cerebrocortical neurons in culture and excitotoxic injury to Purkinje cells in cerebellar slices.

A specific inhibitor of calcium/calmodulin-dependent protein kinase-II provides neuroprotection against NMDA- and hypoxia/hypoglycemia-induced cell death.

Calcium/calmodulin-dependent protein kinase-II (CamK-II) is a major neuronal protein which plays a significant role in the cellular process of long-term potentiation (LTP), and vesicular release of neurotransmitters. Here, we show that KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4- phenylpiperazine, a specific cell-permeable inhibitor of CamK-II substantially protected neurons from (1) acute NMDA toxicity and (2) hypoxia/hypoglycemia-induced neuronal injury in fetal rat cortical cultures. KN-62 did not directly inhibit glutamate, kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), glycine, or [piperidyl-3,4-(N)]-(N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine) (TCP) binding to rat brain membranes. Finally, KN-62 significantly reduced cellular calcium accumulation following either NMDA challenge or hypoxia/hypoglycemia insult. Our results show that CamK-II plays a key role in mediating some of the biochemical events leading to cell death following an acute excitotoxic insult.

Synthesis and pharmacological evaluation of phenylacetamides as sodium-channel blockers.

The synthesis and structure-activity relationships of a series of phenylacetamides related to N-[3-(2,6-dimethyl-1-piperidinyl)propyl]-alpha-phenylbenzeneacetamide (1) (PD85639) acting at the voltage-dependent Na+ channel are described. All structural variations for this study were made in the phenylacetic acid portion of these molecules, and the compounds were synthesized by coupling the appropriately substituted phenylacetic acid derivative with 3-[1-(2,6-dimethyl)piperidinyl]-propanamine using standard methods of amide formation. Compounds were tested as inhibitors of [3H]batrachtoxinin binding in rat neocortical membranes and also as inhibitors of veratridine-induced Na+ influx in Chinese hamster ovary cells expressing type IIA Na+ channels. Diphenylacetic acid derivatives with halogenated aromatic rings (12-15) were very potent in both assays, while alkoxy and alkyl substitution did not affect activity (16 and 17). Selected compounds were tested as potential neuroprotective agents in two cell culture assays involving inhibition of veratridine-induced and hypoxia-induced lactate dehydrogenase release. Compound 15 was equipotent with flunarizine, a reference compound in both neuroprotection assays.

Synthesis and pharmacological evaluation of 4a-phenanthrenamine derivatives acting at the phencyclidine binding site of the N-methyl-D-aspartate receptor complex.

A novel series of octahydrophenanthrenamines and their heterocyclic analogues have been synthesized as potential noncompetitive antagonists of the N-methyl-D-aspartate (NMDA) receptor complex. The compounds were evaluated for their affinity at the phencyclidine (PCP) binding site by determining their ability to displace [3H]TCP from crude rat brain synaptic membranes. A wide range of affinities were observed, with the most potent analogs possessing IC50's equivalent to that of the reference agent MK-801 (3, dizocilpine). NMDA antagonist activity was demonstrated by prevention of glutamate-induced accumulation of [45Ca2+] in cultured rat cortical neurons. Selected compounds were also studied in vivo to determine their ability to prevent the lethal effects of systemically injected NMDA in the mouse. In general, the SAR of the phenanthrenamine series may be summarized as follows: (a) for the amino group at C4a, NHMe > NH2 > NHEt >> NC5H10; (b) for the B-ring substitution, X = CH2 > S > O; (c) unsaturation of the C ring decreases receptor affinity; (d) cis-ring fusion between the B and C rings is desirable; (e) 6-hydroxy or 6-methoxy substitution of the phenanthrenamine system identified an additional hydrogen bonding interaction that substantially increased receptor affinity; (f) spiro analogues (such as 55, IC50 = 3400 nM), which altered the point of attachment of the C ring, caused a substantial reduction in PCP-site affinity. Molecules from this series were useful for refining a pharmacophore model consistent with previous models of the PCP site. In this model, the (R)-(+)-phenanthrenamine 13 superimposes closely onto MK-801 (3), and the angular 4a-amino group is believed to hydrogen bond with a putative receptor site atom. In the phenanthrenamine and thiaphenanthrenamine series, the (R)-(+)-enantiomers (9, 13, and 44) are more potent by approximately 5-10-fold than their corresponding (S)-(-)-enantiomers with respect to their affinity for the PCP site, their ability to prevent accumulation of [45Ca2+] in cultured neuronal cells, and their protection against the lethal effects of NMDA in mice. In general, there was no separation between the dose that prevented NMDA lethality and the dose that produced ataxia in mice, except in the case of the thiaphenanthrenamines 41 and 43. We have not yet obtained evidence that this small separation in activity offers a therapeutic advantage in the treatment of cerebral ischemia or other neurodegenerative disorders.

Exploration of N-phosphonoalkyl-, N-phosphonoalkenyl-, and N-(phosphonoalkyl)phenyl-spaced alpha-amino acids as competitive N-methyl-D-aspartic acid antagonists.

A series of N-substituted alpha-amino acids containing terminal phosphonic acid groups has been synthesized as potential N-methyl-D-aspartate (NMDA) receptor antagonists. NMDA receptor affinity was determined by displacement of a known ligand ([3H]CPP) from crude rat brain synaptic membranes; an antagonist action was demonstrated by the inhibition of glutamate-induced accumulation of [45Ca2+] in cultured rat cortical neurons. Receptor affinity was significantly correlated with antagonist activity (Figure 1). Moderate affinity (IC50 = 1-2 microM) was retained for analogues (31 and 32, Table I; and 59 and 66, Table II) with reduced flexibility in their phosphonate side chains and is consistent with entropy playing a role in determining receptor affinity. Modeling studies suggest a folded conformation that brings the distal phosphonic acid group into close proximity with the alpha-carboxylate is required for binding. Each of the active analogues possess entropy-limiting features (double bonds, phenyl rings) in their side chains that allows the superposition of their key NH2, alpha-COOH, and distal PO3H2 groups with those of known competitive antagonists. Affinity decreased for analogues with alpha-carbon substitution, presumably because the alpha-substituent inhibits the folding of these structures into a bioactive conformation and occupies receptor-excluded volume. A complete description of the NMDA antagonist pharmacophore model is provided in a companion paper.

Treatment with conotoxin, an 'N-type' calcium channel blocker, in neuronal hypoxic-ischemic injury.

Therapeutic efficacy of calcium channel blockers in stroke remains controversial, but previously used agents bind almost exclusively to L-type calcium channels. The newly-discovered N-type calcium channel is specific to neurons, and therapy involving blockade of this site has not been previously attempted. We assessed the neuroprotective effect of omega-conotoxin GVIA (CgTx), a blocker of N-type calcium channels, using both in vitro hypoxic injury to rat cortical neurons and an in vivo model of reversible spinal cord ischemia in the rabbit. In cell cultures, CgTx inhibited hypoxia-induced 45Ca accumulation and neuronal injury minimally, compared to the NMDA antagonist ketamine. In vivo, the duration of spinal cord ischemia which produced permanent paraplegia in 50% of control animals (ET50) was 24.0 +/- 2.6 min. Animals treated 2 h prior to ischemia with 0.5 nmol CgTx in the subarachnoid space had an ET50 of 26.9 +/- 1.8 min (P = 0.36). Animals treated 24 h prior to ischemia (all had persistent systemic tremor) had a ET50 of 28.9 +/- 1.8 min (P = 0.13). We conclude that pharmacologic modulation of the N-type calcium channel does not provide a significant protective effect against neuronal hypoxic-ischemic injury.

Hypoxic neuronal injury in tissue culture is associated with delayed calcium accumulation.

Calcium accumulation and neuronal injury were studied after hypoxia in cerebrocortical cell cultures in vitro. Neuronal injury was associated with a delayed calcium accumulation, which was greatest 5-7 hours after hypoxic exposure. Antiexcitotoxic treatments with tetrodotoxin and magnesium chloride or the selective N-methyl-D-aspartate antagonist (+/-)-4-(3-phosphonopropyl)-2-piperazinecarboxylic acid prevented hypoxic calcium accumulation and neuronal injury even when added 3 hours after hypoxia, during reoxygenation. Rescue of the neurons after hypoxia by blocking the delayed calcium accumulation in this cell culture preparation suggests a "therapeutic window" determined by calcium entry.

Exploration of phenyl-spaced 2-amino-(5-9)-phosphonoalkanoic acids as competitive N-methyl-D-aspartic acid antagonists.

To investigate the preferred spatial relationship of the distal phosphonic acid to the alpha-amino acid group of the established competitive N-methyl-D-aspartic acid (NMDA) antagonists APH (1) and APV (2), we have prepared a series of ortho-, meta-, and para-substituted (phosphonoalkyl)phenylglycine and -phenylalanine derivatives. With use of a [3H]CPP receptor binding assay, significant binding activity was observed to be critically dependent on both the position of substitution and length of alkyl spacing groups. Two compounds, 4-(phosphonomethyl)-phenylglycine (6, PD 129635) and 3-(phosphonomethyl)phenylalanine (15, PD 130527), displayed receptor-binding affinity comparable to that of APH. Like APH, these compounds were also effective in antagonizing both the proconvulsant and lethal action of NMDA-administered retrobulbar in the mouse. Data are also provided which compare directly the binding efficacy of these compounds against that disclosed recently for the related NMDA antagonist 18 (NPC 451). A preliminary comparison of the structures showing good receptor-binding affinity and in vivo antagonist activity suggests that the NMDA receptor prefers a "folded" rather than "extended" conformation.

Effects of antiarthritic compounds on type II collagen-induced arthritis in rats.

The therapeutic effects of a variety of agents on established lesions of type II collagen arthritis in rats were tested. Drug activity was determined using an Arthritic Index, which consisted of a rank score ranging from 0 to 8 (0 to 4 in each hind paw) based on hind paw swelling and redness, and by measuring titers of antibody to type II collagen. Four of five non-steroidal antiinflammatory drugs (NSAIDs), phenylbutazone, benoxaprofen, indomethacin and isoxicam, reduced swelling. Of the NSAIDs, only acetylsalicylic acid was inactive. None of these drugs reduced levels of antibody to type II collagen as determined by an ELISA procedure. One of two immunosuppressive drugs, cyclophosphamide, and a steroid, hydrocortisone acetate, significantly inhibited hind paw inflammation. The other immunosuppressive, azathioprine, did not significantly reduce swelling. Also, cyclophosphamide lowered levels of antibody to type II collagen. Five antirheumatic agents, auranofin, levamisole, D-penicillamine, chloroquine and aurothioglucose, were tested and none inhibited the disease process. Of these five, only auranofin inhibited antibody titer to type II collagen. In conclusion, although rat type II collagen arthritis did not detect standard antirheumatic drugs, this model detected the activity of several standard NSAIDs, an immunosuppressive, and a steroid. In addition, the level of antibody to type II collagen was neither a selective nor sensitive indicator of the efficacy of standard antiarthritic agents.