John D. Wagstaff

Isolation and characterization of a novel Conus peptide with apparent antinociceptive activity

Cone snails are tropical marine mollusks that envenomate prey with a complex mixture of neuropharmacologically active compounds. We report the discovery and biochemical characterization of a structurally unique peptide isolated from the venom of Conus marmoreus. The new peptide, mr10a, potently increased withdrawal latency in a hot plate assay (a test of analgesia) at intrathecal doses that do not produce motor impairment as measured by rotarod test. The sequence of mr10a is NGVCCGYKLCHOC, where O is 4-trans-hydroxyproline. This sequence is highly divergent from all other known conotoxins. Analysis of a cDNA clone encoding the toxin, however, indicates that it is a member of the recently described T-superfamily. Total chemical synthesis of the three possible disulfide arrangements of mr10a was achieved, and elution studies indicate that the native form has a disulfide connectivity of Cys1-Cys4 and Cys2-Cys3. This disulfide linkage is unprecedented among conotoxins and defines a new family of Conus peptides.

Dopamine D2-receptors regulate neurotensin release from nucleus accumbens and striatum as measured by in vivo microdialysis

The present study was undertaken to examine the role of dopamine D2-receptors in the regulation of neurotensin release. Through a modification of the methods described by Maidment et al. (Neuroscience, 45 (1991) 81-93), we have developed a highly reproducible method of measuring changes in extracellular NT in the striatum and nucleus accumbens by in vivo microdialysis in awake animals. It was observed that calcium-dependent release of NT was evoked in both structures by infusing a high concentration of potassium. In addition, systemic administration of the D2 agonist quinpirole (5 mg/kg) induced a rapid increase of approximately 200% in extracellular NT levels in the lateral caudate and 30-40% in the nucleus accumbens. Conversely, treatment with the D2 antagonist eticlopride (0.5 mg/kg) reduced extracellular NT in the medial anterior caudate and nucleus accumbens 20-30%, but had no effect in the lateral anterior caudate. These data demonstrate for the first time that D2-receptors are important in the dopaminergic regulation of extrapyramidal and limbic NT release in conscious animals.

Endogenous neurotensin antagonizes methamphetamine-enhanced dopaminergic activity

Neurotensin (NT) has been proposed to be an endogenous neuroleptic based on observations that i.c.v. administration of this peptide antagonizes dopamine-mediated behavior. Because NT influences dopamine activity, this peptide may contribute to the pathogenesis of psychotic disorders such as schizophrenia; however, the precise physiological effects of NT remain speculative. In order to elucidate the function of endogenous NT, a selective NT antiserum (NTAS) was administered i.c.v. through a push-pull cannula in unanesthetized, freely moving rats in combination with dopamine activation caused by methamphetamine (METH). Locomotor and rearing activities induced by a low dose of METH (0.5 mg/kg) were substantially enhanced (4-5-fold) in rats receiving NTAS compared to control animals receiving METH alone. Similarly raised antiserum to vasoactive intestinal polypeptide (VIP) did not alter METH-induced effects. To determine a possible mechanism for these observations, perfusate delivered into the cerebral ventricular space was collected by push-pull cannulae and assayed for dopamine release. METH-induced dopamine release was enhanced 4-5-fold by co-administration of NTAS but not VIP antiserum. To verify these observations, and to identify the site of dopamine release, this experiment was repeated utilizing microdialysis and the recently described NT antagonist, SR-48692. Results from this experiment were similar to those found using NTAS. Like NTAS, co-administration of the NT antagonist enhanced the behavioral responses to a low dose of METH. These studies with SR-48692 also revealed that blockade of NT receptors increased METH-induced release of dopamine from the nucleus accumbens. These findings are the first to demonstrate directly that endogenous NT antagonizes stimulated dopamine pathways and its inactivation substantially enhances METH-induced DA release and related behaviors.

An assessment of the antinociceptive efficacy of intrathecal and epidural contulakin-G in rats and dogs.

Contulakin-G is a novel conopeptide with an incompletely defined mechanism of action. To assess nociceptive activity we delivered Contulakin-G as a bolus intrathecally (0.03, 0.1, 0.3, 3 nmol) or epidurally (10, 30, 89 nmol) in rats. Intrathecal Contulakin G significantly decreased Phase II and, to a lesser degree, Phase I paw flinching produced by intradermal formalin. Intrathecal and epidural doses of ED50s were 0.07 nmol and 45 nmol, respectively, giving an epidural/intrathecal ED50 ratio = 647). In dogs, intrathecal Contulakin-G (50-500 nmoL) produced a dose-dependent increase in the thermally evoked skin twitch latency by 30 min after administration, as did morphine (150 and 450 nmol). Epidural morphine (750 and 7500 nmol), but not epidural 1000 nmol Contulakin-G, also significantly decreased skin twitch in dogs. No changes in motor function were seen in any rats or dogs receiving these doses of Contulakin-G. In dogs, no physiologically significant dose-dependent changes in motor function, heart rate, arterial blood pressure, or body temperature were found. Contulakin-G is a potent antinociceptive drug when delivered intrathecally with no observable negative side effects in rats or dogs and may provide an alternative to opioid spinal analgesics.

Conantokins: Peptide Antagonists of NMDA Receptors

Conantokins are small peptides (17-27 amino acids) found in the venoms of cone snails (Conus sp.) that inhibit the activity of N-methyl-D-aspartate (NMDA) receptors. Unlike most of the peptides characterized from cone snail venom that contain multiple disulfide bridges, conantokins are linear peptides that possess a high degree of alpha-helicity in the presence of divalent cations, and contain gamma-carboxyglutamic acid residues. Four naturally occurring conantokins have been identified and characterized to date, conantokin-G, conantokin-T, conantokin-R, and conantokin-L. The most extensively characterized, conantokin-G, is selective for subtypes of NMDA receptors containing the NR2B subunit. The conantokins have been synthesized and characterized in a number of animal models of human pathologies including pain, convulsive disorders, stroke, and Parkinsons disease. The potential pharmacological selectivity of the conantokins, coupled with their efficacy in preclinical models of disease and favorable safety profiles indicate that these peptides represent both novel probes for NMDA receptor function as well as an important class of compounds for continued investigation as human therapeutics.

Dynamic dopaminergic regulation of neuropeptide Y systems in discrete striatal and accumbens regions

In this study we evaluated the effects of multiple administrations of selective dopamine D1 and D2 receptor agonists and antagonists on striatal, nigral, accumbens, pallidal and cortical neuropeptide Y systems. Treatment with the D1 receptor agonist, SKF 38393, decreased, while that with the D1 receptor antagonist, SCH 23390, increased neuropeptide Y-like immunoreactivity in the globus pallidus and several regions within the caudate-putamen. SCH 23390 did not change accumbens neuropeptide Y-like immunoreactivity levels but SKF 38393 increased neuropeptide Y-like immunoreactivity levels in anterior and decreased neuropeptide Y-like immunoreactivity levels in the posterior nucleus accumbens. Interestingly, reductions in neuropeptide Y-like immunoreactivity content occurred in response to administrations of both D2 receptor agonist, quinpirole, or antagonist, sulpiride, in all identified regions of each structure at some time point. These data suggest that the neuropeptide Y systems studied may be regulated by selective activity at postsynaptic or presynaptic dopamine receptors. They further suggest that within structures such as the caudate-putamen and nucleus accumbens are multiple distinct neuropeptide Y systems which are uniquely influenced by dopamine receptors.

The pharmacokinetics of the conopeptide Contulakin-G (CGX-1160) after intrathecal administration: An analysis of data from studies in beagles

BACKGROUND:The synthetic peptide agent Contulakin-G (CGX-1160), isolated from the toxin of the snail Conus Geographus, produces significant analgesia in animals. Its peptide structure requires intrathecal administration for effectiveness, therefore we determined the intrathecal pharmacokinetics of CGX-1160 after bolus dose and multiple day infusions to beagles. METHODS:For the bolus dose study, eight animals received a dose ranging from 16.7 to 1000 nmol under isoflurane anesthesia. Cerebral spinal fluid sampling for drug assay occurred up to 24 h. For the multiple day infusion study, three animals received infusions of 10, 40, and 160 &mgr;g/h respectively for 24 h at each rate. Cerebral spinal fluid sampling occurred during the infusion rate and the washout period after the 72 h of cumulative drug delivery. Data from the two study designs were modeled separately using NONMEM. RESULTS:The results showed a biexponential disposition profile for both experiments with a rapid rate constant that was an order of magnitude greater than the slow rate constant. The bolus results showed a nonlinear dependence of the slow rate constant on administered dose due to the large bolus range used in the study. CONCLUSION:These data, coupled with clinical pharmacology results, provide a basis for determining appropriate dosing strategies to achieve therapeutic intrathecal concentrations of Contulakin-G.

Psychostimulants and neuropeptide response.

Publisher Summary Neurotensin (NT) receptors play a significant role in the regulation of the associated nigral-striatal and mesolimbic dopaminergic pathways. The role of NT systems in methamphetamine (METH)-mediated responses is determined by blocking extracellular NT activity with either NT-directed antibodies or a selective NT antagonist and measuring the effect on METH-induced behavior. An interesting dose-dependent response suggesting distinctive high- and low-dose effects by METH on NT systems has been observed. High doses of METH temporarily increased NT tissue content predominantly in the medial caudate and nucleus accumbens. The elevation in NT levels is selectively blocked by dopaminergic D1-, but not D2-, receptor antagonists. In contrast, a low dose of METH decreases NT tissue content in lateral caudate and in the nucleus accumbens. These changes are consistent with a D2-receptor mechanism, because the selective D2 agonist, quinpirole, similarly reduces NT tissue content. These data suggest that extrapyramidal and limbic NT systems are differentially influenced by low and high doses of METH. The elevation in tissue levels is mediated by increased synthesis, because this METH treatment also increases the mRNA for the precursor in medial caudate and accumbens regions. However, release of NT is not altered in these structures after a high dose of METH and appears not to exert the regulatory impact observed with low doses of METH. This lack of NT release may lead to an exaggerated reaction of dopaminergic pathways to METH treatment.

Isolation and Characterization of a NovelConusPeptide with Apparent Antinociceptive Activity

Cone snails are tropical marine mollusks that envenomate prey with a complex mixture of neuropharmacologically active compounds. We report the discovery and biochemical characterization of a structurally unique peptide isolated from the venom of Conus marmoreus. The new peptide, mr10a, potently increased withdrawal latency in a hot plate assay (a test of analgesia) at intrathecal doses that do not produce motor impairment as measured by rotarod test. The sequence of mr10a is NGVCCGYKLCHOC, where O is 4-trans-hydroxyproline. This sequence is highly divergent from all other known conotoxins. Analysis of a cDNA clone encoding the toxin, however, indicates that it is a member of the recently described T-superfamily. Total chemical synthesis of the three possible disulfide arrangements of mr10a was achieved, and elution studies indicate that the native form has a disulfide connectivity of Cys1-Cys4 and Cys2-Cys3. This disulfide linkage is unprecedented among conotoxins and defines a new family of Conus peptides.