Pharmacologic rescue of motor and sensory function by the neuroprotective compound P7C3 following neonatal nerve injury.

Nerve injuries cause pain, paralysis and numbness that can lead to major disability, and newborns often sustain nerve injuries during delivery that result in lifelong impairment. Without a pharmacologic agent to enhance functional recovery from these injuries, clinicians rely solely on surgery and rehabilitation to treat patients. Unfortunately, patient outcomes remain poor despite application of the most advanced microsurgical and rehabilitative techniques. We hypothesized that the detrimental effects of traumatic neonatal nerve injury could be mitigated with pharmacologic neuroprotection, and tested whether the novel neuroprotective agent P7C3 would block peripheral neuron cell death and enhance functional recovery in a rat neonatal nerve injury model. Administration of P7C3 after sciatic nerve crush injury doubled motor and sensory neuron survival, and also promoted axon regeneration in a dose-dependent manner. Treatment with P7C3 also enhanced behavioral and muscle functional recovery, and reversed pathological mobilization of spinal microglia after injury. Our findings suggest that the P7C3 family of neuroprotective compounds may provide a basis for the development of a new neuroprotective drug to enhance recovery following peripheral nerve injury.

Comparative actions of synthetic omega-grammotoxin SIA and synthetic omega-Aga-IVA on neuronal calcium entry and evoked release of neurotransmitters in vitro and in vivo.

The effects of synthetic omega-grammotoxin SIA (omega-GsTxSIA) and synthetic omega-Aga-IVA were tested in in vitro and in vivo neurochemical assays that are reflective of voltage-sensitive calcium channel function. Synthetic omega-GsTx SIA inhibited K(+)-evoked rat and chick synaptosomal 45Ca2+ flux, K(+)-evoked release of [3H]D-aspartate and [3H]norepinephrine from rat hippocampal brain slices and K(+)-evoked release of [3H]norepinephrine from chick cortical brain slices with potency values that were comparable to those found previously with omega-GsTx SIA purified from the venom of the tarantula spider Grammostola spatulata. These results indicate that trace contaminants do not account for the pharmacology of purified omega-GsTx SIA. omega-GsTx SIA caused a complete inhibition of rat synaptosomal 45Ca2+ flux and hippocampal slice [3H]D-aspartate release, whereas omega-Aga-IVA caused a maximal inhibition of approx 75%. omega-GsTx SIA and omega-Aga-IVA caused an identical partial inhibition of K(+)-evoked increases of intracellular calcium in cortical neurons in primary culture. The addition of nitrendipine to either omega-GsTx SIA or omega-Aga-IVA resulted in an additive and virtually complete inhibition of the cortical neuron intracellular calcium response. In in vivo microdialysis studies, the K(+)-evoked release of glutamate from hippocampus of awake freely moving rats was inhibited with the following rank order of potency: omega-conotoxin GVIA > omega-GsTx SIA > omega-Aga-IVA. Complete inhibition of K(+)-evoked hippocampal glutamate release was observed with 300 nM omega-conotoxin GVIA and 3 microM omega-GsTx SIA. In urethane anesthetized rats, omega-CgTx GVIA caused a partial inhibition, whereas omega-GsTx SIA caused a concentration-dependent and complete inhibition, of basal serotonin release in the hippocampus. Therefore, omega-GsTx SIA was shown to inhibit responses that are sensitive to omega-conotoxin GVIA, omega-Aga-IVA and omega-conotoxin MVIIC, consistent with the notion that omega-GsTx SIA inhibits N-, P- and Q-type high threshold voltage-sensitive calcium channels.

Differential inhibition of neuronal calcium entry and [3H]-D-aspartate release by the quaternary derivatives of verapamil and emopamil.

1. Verapamil and emopamil are structurally related phenylalkylamine calcium channel/5-HT2 receptor antagonists that differ in their anti-ischaemic properties in experimental studies. The quaternary ammonium derivatives of these compounds were prepared and tested in assays of neuronal voltage-sensitive calcium channel (VSCC) function to determine whether the compounds act at intra- or extracellular sites. 2. The compounds were tested in K(+)-evoked: (1) rat brain synaptosomal 45Ca2+ influx, (2) release of [3H]-D-aspartate from rat hippocampal brain slices and (3) increase of intracellular calcium in rat cortical neurones in primary culture. 3. Verapamil, emopamil and the emopamil quaternary derivative caused concentration-dependent and comparable (IC50 values approximately 30 microM) inhibition of synaptosomal 45Ca2+ influx and [3H]-D-aspartate release. The verapamil quaternary derivative was considerably less active in these assays (IC50 > 300 microM). 4. The evoked increase of intracellular calcium in cortical neurones was inhibited with the following rank order of potency (IC50 value, microM): emopamil (3.6) > verapamil (17) > emopamil quaternary derivative (38) > verapamil quaternary derivative (200). 5. The results suggest that verapamil and emopamil inhibit nerve terminal VSCC function (synaptosomal 45Ca2+ influx and [3H]-D-aspartate release) by acting at distinct intracellular and extracellular sites, respectively. Verapamil and emopamil may inhibit cell body VSCC function (evoked increase of intracellular calcium in neocortical neurones) by acting at both intracellular and extracellular sites. 6. The different 'sidedness' of action of emopamil and verapamil on nerve terminal VSCC function and/or the preferential inhibition of cell body VSCC function by emopamil may at least partially explain the relatively greater neuroprotective efficacy of emopamil in experimental models of ischaemia.

Isolation and pharmacological characterization of omega-grammotoxin SIA, a novel peptide inhibitor of neuronal voltage-sensitive calcium channel responses.

omega-Grammotoxin SIA, a peptidergic blocker of voltage-sensitive calcium channel (VSCC) responses, was purified from Grammostola spatulata (tarantula spider) venom by reverse phase high performance liquid chromatography. Protease-sensitive biological activity was monitored by determining the inhibition of K(+)-stimulated influx of 45Ca2+ into rat brain synaptosomes. Electrospray mass spectrometry indicated an average molecular mass of 4109.2 Da for the native peptide. Chemical reduction of omega-grammotoxin SIA indicated the presence of three disulfide bridges. Primary sequence data confirmed the existence of six cysteine residues and 36 residues in total, with an average theoretical molecular mass of 4109.7 Da for the amidated carboxyl-terminal species. The biological profile of omega-grammotoxin SIA indicated virtually complete blockade of presynaptic vertebrate N-type as well as P-type VSCC responses. Specifically, omega-grammotoxin SIA caused a concentration-dependent and virtually complete inhibition of K(+)-evoked influx of 45Ca2+ into either rat or chick brain synaptosomes. Similar inhibition profiles were generated for the inhibition of release of either D-[3H]aspartate or [3H]norepinephrine from rat hippocampal or [3H]norepinephrine from chick cortical brain slice preparations evoked by K+ depolarization. As reported earlier, omega-grammotoxin SIA did not inhibit 125I-omega-conotoxin GVIA, [3H]PN 200-110, or [3H]desmethoxyverapamil binding to neuronal membrane fragments. To our knowledge, omega-grammotoxin SIA is the first ligand identified to block putative N-channel function without displacement of 125I-omega-conotoxin GVIA. omega-Grammotoxin SIA thus represents a novel vertebrate VSCC antagonist that inhibits neuronal N- and P-type VSCC responses.

Effects of stimulus intensity on the inhibition by omega-conotoxin GVIA and neomycin of K(+_-evoked [3H]norepinephrine release from hippocampal brain slices and synaptosomal calcium influx.

The effects of various K+ concentrations on the inhibition of [3H]norepinephrine release from rat hippocampal brain slices and evoked synaptosomal 45Ca2+ influx by omega-conotoxin GVIA (omega-CgTx) and neomycin were examined. K+ (15-75 mM) caused a concentration-dependent release of [3H]norepinephrine that was greater than 90% dependent on extracellular calcium. The ability of omega-CgTx to inhibit [3H]norepinephrine release was optimal at 25 mM K+ and was reduced substantially at higher concentrations of K+. omega-CgTx maximally inhibited [3H]norepinephrine release by 49% (15 mM K+), 58% (25 mM K+), 22% (50 mM K+), and 12% (75 mM K+). In contrast, neomycin caused a concentration-dependent and virtually complete inhibition of [3H]norepinephrine release at all concentrations of K+, with IC50 values of 210 microM (15 mM K+), 150 microM (25 mM K+), 450 microM (50 mM K+), and 1500 microM (75 mM K+). omega-CgTx (1 microM) had little effect (10% or less inhibition) on hippocampal synaptosomal 45Ca2+ influx at any concentration of K+, whereas 3 mM neomycin caused at least 75% inhibition of 45Ca2+ influx, with the largest inhibition (96%) occurring at 25 mM K+. The results suggest that increasing stimulus intensity decreases the contribution of N-type voltage-sensitive calcium channels (VSCC) in mediating K(+)-evoked release of [3H]norepinephrine. The comparative absence of omega-CgTx-sensitive synaptosomal 45Ca(2+)-influx sites suggests that N-type calcium channels are a small subset of channels in rat hippocampal synaptosomes. The demonstration that neomycin can inhibit omega-CgTx-sensitive and -insensitive neurotransmitter release and calcium influx suggests that neomycin may block N-type VSCC as well as non-N-type VSCC.

Actions of neomycin on neuronal L-, N-, and non-L/non-N-type voltage-sensitive calcium channel responses.

The effects of neomycin on neuronal voltage-sensitive calcium channel (VSCC) responses were investigated by evaluating its effects on calcium-dependent neuronal responses that are sensitive and insensitive to the N-type voltage-sensitive calcium channel antagonist omega-conotoxin GVIA and the L-type VSCC antagonist nitrendipine. Chick synaptosomal 45Ca2+ influx and K(+)-evoked release of [3H]norepinephrine from chick cortical brain slices were omega-conotoxin GVIA sensitive and nitrendipine insensitive, suggesting that these responses are mediated predominantly by N-type VSCC. The K(+)-evoked increase of intracellular calcium in cortical neurons and the K(+)-evoked release of [3H]norepinephrine from rat brain cortical slices was partially sensitive to omega-conotoxin GVIA and nitrendipine, suggesting that these responses are mediated by N-, L- and non-L/non-N-type VSCC. Rat synaptosomal 45Ca2+ influx and the K(+)-evoked release of [3H]D-aspartate from rat hippocampal slices were completely insensitive to omega-conotoxin GVIA and nitrendipine, suggesting that these responses were mediated predominantly by non-L/non-N-type VSCC. Neomycin caused a concentration-dependent and virtually complete inhibition of all response parameters, with IC50 values ranging from 90 to 400 microM. The results suggest that neomycin is a nonselective inhibitor of neuronal responses mediated by L-, N-, and non-L/non-N-type VSCC.

Inhibition of K(+)-evoked [3H]D-aspartate release and neuronal calcium influx by verapamil, diltiazem and dextromethorphan: evidence for non-L/non-N voltage-sensitive calcium channels.

The effects of inhibitors of voltage-sensitive calcium channels (VSCC) on K(+)-evoked [3H]D-aspartate release from rat hippocampal slices and the K(+)-evoked increase in intracellular calcium in neocortical neurons in primary culture were examined. K+ caused a concentration-dependent release of [3H]D-aspartate that was approximately 85% dependent on the presence of extracellular calcium. Neither the marine snail toxin, omega-conotoxin GVIA, nor the dihydropyridine VSCC antagonist, nitrendipine, had any effect on K(+)-evoked release of [3H]D-aspartate. omega-Conotoxin GVIA and nitrendipine caused a relatively small (20-30%) inhibition of K(+)-evoked increase in intracellular calcium in neocortical neurons in primary culture. This suggests that K(+)-evoked [3H]D-aspartate release is not dependent on L- or N-type VSCC, whereas K(+)-evoked neuronal calcium influx was only partially dependent on L- and N-type VSCC. Verapamil, dextromethorphan and diltiazem caused a concentration-dependent inhibition of K(+)-evoked release of [3H]D-aspartate with IC50 values of 30, 100 and 120 microM, respectively. The K(+)-evoked increase in intracellular calcium was inhibited with essentially the same rank order of potency, but with slightly greater potencies (IC50 values for verapamil, diltiazem and dextromethorphan were 20, 50 and 50 microM, respectively). At 300 microM, neither verapamil, diltiazem nor dextromethorphan inhibited [3H]D-aspartate release evoked by the calcium ionophore ionomycin, suggesting that these compounds are not acting intracellularly to inhibit the ability of free cytosolic calcium to evoke release.(ABSTRACT TRUNCATED AT 250 WORDS)

Stereoselectivity for the (R)-enantiomer of HA-966 (1-hydroxy-3-aminopyrrolidone-2) at the glycine site of the N-methyl-D-aspartate receptor complex.

HA-966 (1-hydroxy-3-aminopyrrolidone-2) is an antagonist at the glycine allosteric site of the N-methyl-D-aspartate receptor ionophore complex. Unlike presently known glycine antagonists, HA-966 is chiral. We report stereoselectivity for the (R)-enantiomer at the glycine antagonist site. In [3H]glycine binding, the (R)-enantiomer has an IC50 of 4.1 +/- 0.6 microM. The racemic mixture has an IC50 of 11.2 +/- 0.5 microM, whereas (S)-HA-966 has an IC50 greater than 900 microM. In glycine-stimulated [3H]1-[1-(2- thienyl)cyclohexyl]piperidine binding, the (R)-enantiomer inhibits with an IC50 of 121 +/- 61 microM, whereas the racemic mixture has an IC50 of 216 +/- 113 microM and (S)-HA-966 is inactive. The inhibition by (R)-HA-966 can be prevented by the addition of glycine. (R)-HA-966 and racemic HA-966, but not (S)-HA-966, also prevent N-methyl-D-aspartate cytotoxicity in cortical cultures. The (R)-enantiomer and, less potently, the (S)-enantiomer inhibit N-methyl-D-aspartate-evoked [3H]norepinephrine release from rat hippocampal slices (IC50 values of about 0.3 mM and 1.6 mM, respectively), but only the inhibition by (R)-HA-966 is reversed by added glycine. In glutamate-evoked contractions of the guinea pig ileum, (R)-HA-966 causes a glycine-reversible inhibition (IC50 of about 150 microM), whereas (S)-HA-966 is much less potent (IC50 of greater than 1 mM). These results demonstrate stereoselectivity of the glycine antagonist site of the N-methyl-D-aspartate receptor complex in a variety of tissues and assays. The stereoselectivity also confirms the specificity of N-methyl-D-aspartate receptors in glutamate-evoked contractions of the guinea pig ileum, and supports their similarity to central N-methyl-D-aspartate receptors.

6,7-Dinitroquinoxaline-2,3-dione blocks the cytotoxicity of N-methyl-D-aspartate and kainate, but not quisqualate, in cortical cultures.

Based on radioligand binding and electrophysiological studies, quinoxalinediones such as 6,7-dinitroquinoxaline-2,3-dione (DNQX) have been shown to be potent competitive antagonists at the quisqualate and kainate subtypes of the glutamate receptor. In this report we have examined the effects of DNQX on excitatory amino acid neurotoxicity and evoked neurotransmitter release. DNQX was found to be a potent neuroprotective agent against glutamate and N-methyl-D-aspartate (NMDA) neurotoxicity. The data suggest that this neuroprotective activity of DNQX is due to its antagonism of the coagonist activity of glycine at the NMDA receptor-channel complex. The specificity of DNQX for the glycine site associated with the NMDA receptor-channel complex was confirmed in radioligand binding and neurotransmitter release studies. DNQX also prevented kainate neurotoxicity and kainate-evoked neurotransmitter release, presumably by direct competition for the kainate receptor. DNQX, however, did not prevent quisqualate neurotoxicity, suggesting that a novel quisqualate-preferring receptor insensitive to DNQX may mediate quisqualate toxicity.

Glycine-evoked neurotransmitter release from rat hippocampal brain slices: evidence for the involvement of glutaminergic transmission.

Glycine caused a concentration-dependent evoked release of [3H]norepinephrine from rat hippocampal brain slices. Other amino acids evoked [3H]norepinephrine release with a rank order of potency: L-serine greater than or equal to glycine greater than beta-alanine greater than D-serine. Strychnine inhibited [3H]norepinephrine release evoked by both glycine and L-serine, but was less effective in inhibiting the release evoked by N-methyl-D-aspartate (NMDA) and kainic acid. Inhibitors of the NMDA receptor/ionophore complex, MK-801, CPP and Mg++, as well as the strychnine-insensitive glycine receptor antagonist, HA-966, caused an incomplete inhibition (maximum approximately 60%) of glycine-evoked [3H]norepinephrine release. The potencies with which MK-801, CPP and Mg++ inhibited glycine- and NMDA-evoked [3H]norepinephrine release were very similar. The combination of MK-801 plus kynurenic acid, a nonselective glutamate receptor antagonist, caused no greater inhibition of glycine-evoked release than MK-801, alone. omega-Conotoxin GVIA, an inhibitor of neuronal L- and N-type voltage-sensitive calcium channels, inhibited glycine-evoked [3H]norepinephrine release by approximately 50%, whereas the L-channel inhibitor PN 200-110 had no significant effect. The combination of MK-801 plus omega-conotoxin GVIA caused only a slightly greater inhibition (P greater than .05) of glycine-evoked release than MK-801 alone. Tetrodotoxin inhibited glycine-evoked release of [3H]norepinephrine by approximately 75%. The inhibitory effects of tetrodotoxin and omega-conotoxin GVIA suggest that voltage-sensitive sodium channels and N-type voltage-sensitive calcium channels are important mediators of glycine-evoked release of [3H]norepinephrine.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of N-methyl-D-aspartate- and kainic acid-induced neurotransmitter release by omega-conotoxin GVIA.

1. The role of voltage-sensitive calcium channels (VSCC) in N-methyl-D-aspartate (NMDA)- and kainic acid (KA)-evoked neurotransmitter release from rat cortical and hippocampal brain slices was evaluated by determining the effects of omega-conotoxin GVIA, an inhibitor of neuronal L- and N-type VSCC, and PN 200-110, a selective inhibitor of L-type VSCC. 2. Selective antagonists of the NMDA receptor ionophore complex, Mg2+, CPP and MK-801, inhibited NMDA- but not KA-evoked release of [3H]-noradrenaline from hippocampal and cortical brain slices. This suggests that cortical and hippocampal receptors are similar and that NMDA and KA act at distinct excitatory amino acid receptor subtypes. 3. [3H]-noradrenaline release induced by both NMDA and KA was similarly inhibited (approximately 30%) by omega-conotoxin GVIA. In contrast, PN 200-110 had no significant effect, although there was a tendency towards inhibition. 4. The results suggest that although NMDA- and KA-receptors are pharmacologically distinct, the N-type, but not the L-type, VSCC plays a small but significant role in neurotransmitter release induced by both NMDA and KA. It remains to be determined whether the N-type VSCC are involved in the physiological and/or pathological manifestations of excitatory amino acid receptor stimulation.

HA-966 acts at a modulatory glycine site to inhibit N-methyl-D-aspartate-evoked neurotransmitter release.

The role of endogenous glycine in supporting N-methyl-D-aspartate (NMDA)-evoked neurotransmitter release was investigated. HA-966 (1-hydroxy-3-aminopyrrolidone-2) inhibited NMDA-evoked release of [3H]norepinephrine from rat hippocampal brain slices, but was much less effective in inhibiting [3H]norepinephrine release evoked by kainic acid (KA). Glycine (1 mM) reversed the HA-966 (1 mM) antagonism of NMDA-evoked release of [3H]norepinephrine. Strychnine (10 microM) had no effect on the ability of glycine to reverse HA-966 antagonism of NMDA-evoked neurotransmitter release. Other amino acids were also capable of reversing the HA-966 antagonism of NMDA-evoked [3H]norepinephrine release with a rank order of potency: D-serine greater than or equal to glycine much greater than L-serine approximately beta-alanine. These same compounds inhibited strychnine-insensitive [3H]glycine binding to rat cortical membrane fragments with a rank order of potency: glycine greater than D-serine much greater than L-serine greater than or equal to beta-alanine. In addition, HA-966 inhibited [3H]glycine binding (IC50 = 8.5 microM). The results suggest that HA-966 antagonism of NMDA-evoked neurotransmitter release is due to the inhibition of endogenous glycine acting at a strychnine-insensitive modulatory glycine site associated with the NMDA receptor/ionophore complex.

Characterization of the effects of omega-conotoxin GVIA on the responses of voltage-sensitive calcium channels.

1. omega-conotoxin GVIA (omega-CT) caused a potent (IC50 approximately 2nM) but less than maximal (55%) inhibition of [3H]-noradrenaline release from cortical brain slices induced by K+. At 0.1 microM, omega-CT inhibited [3H] gamma-aminobutyric acid (GABA) and [3H]-acetylcholine release by approximately 40%. 2. K+-evoked [3H]-noradrenaline release from cortical brain slices was also characterized with respect to the effects of PN 200-110 (dihydropyridine L-channel antagonist), BAY K8644 (L-channel VSCC agonist), and Cd++ (an inorganic L- and N-channel antagonist). 10 microM Cd++ and 1 microM PN 200-110 inhibited K+-evoked [3H]-noradrenaline release by 52% and 17%, respectively. 10 microM Bay K 8644 enhanced K+-evoked [3H]-noradrenaline release by 22%, and this enhancement was blocked by 1 microM PN 200-110. 3. omega-CT caused a near-maximal inhibition of the electrically evoked twitch responses of the rat vas deferens (IC50 approximately 10 nM) and guinea-pig ileum (IC50 approximately 60 nM), but had no effect on the postjunctional contractile responses of noradrenaline (vas deferens) or carbachol (ileum). At concentrations as high as 1 microM, omega-CT had no effect on the K+-induced contraction of the rat aorta. 4. Neither the equilibrium binding of [3H]-(+)-PN 200-110 nor the allosteric regulation of [3H]-(+)-PN 200-110 binding by tiapamil or diltiazem were altered by omega-CT (0.1 microM). 5. These observations support the notion that the N-type voltage-sensitive calcium channel plays a major role in coupling neuronal excitation with neurotransmitter release.

Heart contains two substrates (Mr = 40,000 and 41,000) for pertussis toxin-catalyzed ADP-ribosylation that co-purify with Ns.

Two peptides (Mr = 40,000 and 41,000) in membranes of rabbit heart are radiolabeled when the membranes are incubated in the presence of activated pertussis toxin and [32P]NAD+. The 41,000-Mr peptide appears to be the alpha subunit of the inhibitory regulatory protein of adenylate cyclase, Ni. The 40,000-Mr substrate for pertussis toxin in the heart was investigated. Purification of the stimulatory regulatory protein of adenylate cyclase, Ns, results in the co-purification of the alpha subunits of both Ns and Ni, the putative beta- (Mr = 35,000) and gamma- (Mr approximately equal to 15,000) subunits of Ns and Ni, and the additional 40,000-Mr peptide that is ADP-ribosylated by pertussis toxin. This 40,000-Mr substrate for pertussis toxin action appears to be a major N-protein of mammalian heart.

Fat cell adenylate cyclase system. Enhanced inhibition by adenosine and GTP in the hypothyroid rat.

Hypothyroidism is associated with an enhanced sensitivity of rat fat cells to the inhibitory action of adenosine and adenosine agonists. The sensitivity of the forskolin-stimulated cyclic AMP response of rat fat cells to the adenosine agonist N6-phenylisopropyladenosine is amplified 3-fold by hypothyroidism. Forskolin-stimulated adenylate cyclase activity is more sensitive to inhibition by this adenosine agonist in membranes of fat cells isolated from hypothyroid as compared to euthyroid rats. Hypothyroidism does not significantly alter the number of affinity of binding sites for N6-cyclohexyl[3H]adenosine or N6-phenylisopropyladenosine in membranes of rat fat cells. GTP-induced inhibition of forskolin-stimulated adenylate cyclase was markedly enhanced in the hypothyroid state, suggesting an alteration in the inhibitory regulatory component (Ni)-mediated control of adenylate cyclase. Incubating membranes with [alpha-32P]NAD+ and preactivated pertussis toxin results in the radiolabeling of two peptides with Mr = 40,000 and 41,000 as visualized in autoradiograms of polyacrylamide gels run in sodium dodecyl sulfate. The amount of label incorporated by pertussis toxin into these two peptides (putative subunits of Ni) per mg of protein of membrane is increased 2-3-fold in the hypothyroid state. The amount of the stimulatory regulatory component, Ns, in fat cell membranes is not altered by hypothyroidism (Malbon, C. C., Graziano, M. P., and Johnson, G. L. (1984) J. Biol. Chem. 259, 3254-3260). The amplified response of hypothyroid rat fat cells to the inhibitory action of adenosine appears to reflect a specific increase in the activity and abundance of Ni.