S. Scott Bowersox

Sympatholysis after neuron-specific, N-type, voltage-sensitive calcium channel blockade : First demonstration of N-channel function in humans

SNX-111 is the first neuronal N-type, voltage-sensitive calcium channel (VSCC) blocker to enter clinical drug development. Areas of potential therapeutic utility include treatment of nociceptive and neuropathic pain and neuroprotection after ischemic brain injury. The data presented demonstrate that SNX-111 is biologically active in humans and indicate for the first time a neurophysiologic function of N-type VSCCs in humans.

Brain dopamine receptor levels elevated in canine narcolepsy

Concentrations of dopamine D2 receptors in discrete brain areas differed significantly between dogs with the genetically transmitted form of narcolepsy, and age- and breed-matched controls. D2 receptors were assayed and quantified with Scatchard analysis using [3H]spiperone. Receptor densities in the nucleus accumbens, rostral caudate, and amygdala were consistently higher in narcoleptic animals. In amygdala, dopamine receptor abnormalities were associated with elevated dopamine and 3,4-dihydroxyphenylacetic acid concentrations, but no change in 3-methoxytyramine or homovanillic acid concentrations. These data indicate mesolimbic system involvement in canine narcolepsy and point to impaired dopamine release as a possible etiologic factor.

Role of central alpha-1 adrenoceptors in canine narcolepsy.

The role of central alpha-1 adrenergic receptors in cataplexy was investigated in genetically narcoleptic Doberman pinschers. Treatment of narcoleptic dogs with 25-600 micrograms/kg prazosin, a selective alpha-1 adrenergic receptor blocker, exacerbated cataplexy, whereas treatment with the alpha-1 agonist, methoxamine, ameliorated it. Subsequent studies showed that the beneficial effects of classical treatments of human narcolepsy (amphetamines and tricyclic antidepressants) are antagonized by prazosin, suggesting that these drugs are active through an indirect alpha-1 stimulation (via an increase of norepinephrine in the synaptic cleft). Other studies confirmed that the observed effects were not due to peripheral alpha-1 cardiovascular involvement. Atropine, a central anticholinergic agent, but not methylatropine, a peripheral one, completely suppressed the prazosin effect, which suggests that adrenergic and cholinergic systems act sequentially and not independently to generate cataplexy. Little is known about the physiological role of central alpha-1 adrenoceptors. This series of experiments implicates these receptors in narcolepsy-cataplexy.

Effect of α1-adrenoceptors blockade with prazosin in canine narcolepsy ☆

The role of central α1-adrenergic receptors in cataplexy was investigated in 4 narcoleptic poodles and 6 genetically narcoleptic Doberman pinschers. Treatment of narcoleptic dogs with prazosin, a selective α1-adrenergic receptor blocker, exacerbated cataplexy in both narcoleptic dog breeds. Control and heterozygous Dobermans were not affected by the drug. Binding studies using [3H]prazosin revealed an increase in α1-receptor binding apparently limited to the amygdala. The present study suggests that central α1-adrenoceptors, whose role is still mostly unknown, play a fundamental role in controlling mechanisms involved in cataplexy and REM sleep.

Selective blockade of N-type voltage-sensitive calcium channels protects against brain injury after transient focal cerebral ischemia in rats

The neuroprotective efficacy of the selective N-type voltage-sensitive calcium channel blocker, SNX-111, was evaluated in spontaneously hypertensive rats subjected to 60 min of focal cerebral ischemia by permanent ligation of the right common carotid artery and temporary occlusion of the right middle cerebral artery. Intravenous infusion of 167 microg/kg per min SNX-111 for 30 min (5 mg/kg), initiated immediately after reperfusion, significantly reduced cortical infarct volumes measured 24 h after the ischemic insult.

CNS monoamines and their metabolites in canine narcolepsy: a replication study.

In two separate studies a significantly greater concentration of DA (dopamine) and its metabolite, DOPAC (3,4-dihydroxyphenylacetic acid), was observed in the amygdala of narcoleptic canines. DOPAC was also significantly elevated in the reticularis parvicellularis, whereas NE (norepinephrine) was significantly elevated in the reticularis oralis, but depressed in the preoptic hypothalamus. No changes were observed in concentrations of serotonin or its metabolite, 5-HIAA (5-hydroxyindoleauric acid) in any region in the narcoleptic canine brain. Results of the two studies were similar, except that previously observed differences between narcoleptic and control canines in DOPAC levels in the caudate and reticularis oralis failed to replicate. Thus, steady state measures of neurotransmitter/metabolite tissue concentrations suggest region-specific alterations in DA and NE metabolism, rather than a global deficit in catecholamine neurotransmission in canine narcolepsy.

Canine narcolepsy is associated with an elevated number of α2-receptors in the locus coeruleus

alpha 2-Receptors in the canine brain were pharmacologically characterized using [3H]yohimbine binding. Competition studies revealed a single class of binding sites in frontal cortex but two distinct subtypes in nucleus caudatus. The role of central alpha 2-receptors in narcolepsy was investigated in 5 normal and 5 narcoleptic Doberman pinschers. Scatchard analysis of [3H]yohimbine binding in different brain areas revealed an increase in the number of alpha 2-binding sites limited to the locus coeruleus. This suggests that altered autoinhibition of norepinephrine release may be associated with the narcoleptic symptomatology.

Central α1 adrenoceptor subtypes in narcolepsy-cataplexy: a disorder of REM sleep

The present study suggests the specific involvement within the central nervous system of an α1 adrenoceptor subtype in a behavior, the control of cataplexy, a pathological analogue of rapid eye movement (REM) sleep atonia. Experiments have shown that prazosin, an α1 antagonist, dramatically aggravates canine narcolepsy-cataplexy through a central mechanism, and that [ 3H]prazosin binding sites are increased in the amygdala of narcoleptic dogs16. However, the corresponding Scatchard plots were curvilinear and best fit was obtained with a two-site model, suggesting the existence of two [ 3H]prazosin binding sites18. These two sites (high and low affinity [ 3H]prazosin binding sites) met the criteria for authentic receptors and were respectively very similar to the α1a an α1b (high and low affinity for WB4101, respectively) subtypes recently described in the rat and rabbit. Our results of in vivo pharmacology and in vitro [ 3H]prazosin binding in canine narcolepsy now clearly implicate the low affinity [ 3H]prazosin binding site (αb) in canine narcolepsy: (1) Prazosin, an α1 antagonist with similar affinity for both subtypes, was much more potent in increasing cataplexy than WB4101, a compound with more affinity for the α1a receptor. (2) Chlorethylclonidine and phenoxybenzamine, two irreversible blockers of the α1 receptors with more affinity for the α1b receptors, aggravate cataplexy for up to two weeks. (3) The α1 receptor upregulation previously reported by our group in the amygdala of narcoleptic dogs was due to a selective increase in the low affinity [ 3H]prazosin binding sites. A role for noradrenaline in REM sleep regulation has been suspected for many years, but has never been clearly elucidated. Our data provide evidence for its specific involvement through a specific central α1-adrenergic receptor subtype.

Sleep-wakefulness patterns in the aged cat

This investigation compared the 24 h sleep-wake characteristics of young adult (2-4 years) and old (10-11 years) cats in order to determine whether there were significant age- and/or gender-related differences. Aged animals had more brief (6-14 sec) awakenings, less REM sleep, and more NREM sleep than young adults. There were no significant age- or gender-related differences with respect to either the daily numbers or the mean durations of sleep and waking episodes. Compared with the young adults, aged males had substantially less DSWS and fewer numbers of short-duration (less than or equal to 2.5 min) LSWS bouts. Both males and females showed age-related differences with respect to the sequencing of state-patterns. The expression of these patterns in relation to time of day was comparable for young and old animals. Overall, these findings compare favorably with those commonly reported in the elderly human.

Evidence for multiple [3H]prazosin binding sites in canine brain membranes.

Two classes of alpha 1 adrenoceptors were identified in canine brain and liver using conventional radioligand binding methods. Scatchard plots of specific [3H]prazosin binding to brain and liver membranes prepared from 100-150-day-old Doberman pinscher dogs were consistently curvilinear and best fit a two-site binding model (frontal cortex, Kd1 = 57.7 +/- 10.0 pM, Bmax1 = 64.6 +/- 17.1 fmol/mg protein, Kd2 = 1.5 +/- 0.5 nM, Bmax2 = 159 +/- 37.6 fmol/mg protein; liver, Kd1 = 82.6 +/- 36 pM, Bmax1 = 7.0 +/- 5.1 fmol/mg protein, Kd2 = 0.8 +/- 0.2 nM, Bmax2 = 62.1 +/- 8.7 fmol/mg protein). Kinetically derived affinity constants from association and dissociation experiments agreed with those obtained by Scatchard analyses of equilibrium binding data. Binding sites were saturable, heat labile, bound ligand reversibly, and appeared to be appropriately distributed in relation to endogenous catecholamine. [3H]Prazosin also bound with high affinity to two classes of binding site in porcine and bovine brain membrane but [3H]prazosin binding in monkey and rat brain was best described by a single-site binding model. Affinities obtained were in between values obtained for high and low affinity Kds in the other species. Competitions for [3H]prazosin binding sites in canine frontal cortex were conducted with the following antagonists: WB-4101, corynanthine, phentolamine, benoxathian, phenoxybenzamine, chlorethylclonidine, thymoxamine, prazosin, yohimbine and agonists: methoxamine, (-)-norepinephrine, and clonidine. All ligands but prazosin, norepinephrine and clonidine competed for specific [3H]prazosin binding in a statistically significant biphasic manner. Benoxathian and WB-4101 displayed the highest affinities (benoxathian: Ki1 = 0.26 nM, WB-4101: Ki1 = 0.20 nM) and selectivity (high affinity/low affinity: benoxathian = 1640, WB-4101 = 13204) for the high affinity [3H]prazosin binding site; chlorethylclonidine had highest affinity (Ki2 = 91 nM) and selectivity (low affinity/high affinity = 405) for the lower affinity [3H]prazosin binding site. As defined, the two sites were similar to the alpha 1a and alpha 1b recently described in the rat and rabbit. A noticeable difference was that the subtypes described in dog brain had a 30-fold difference in affinity for prazosin.