Structure-activity relationship of N-methyl-N-aralkyl-peptidylamines as novel N-type calcium channel blockers.

Selective N-type voltage sensitive calcium channel (VSCC) blockers have shown efficacy in several animal models of stroke and pain. In the process of searching for small molecule N-type calcium channel blockers, we have identified a series of N-methyl-N-aralkyl-peptidylamines with potent functional activity at N-type VSCCs. The most active compound discovered in this series is PD 173212 (11, IC50 = 36 nM in the IMR-32 assays). SAR and pharmacological evaluation of this series are described.

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.

K(+)-stimulated 45Ca2+ flux into rat neocortical mini-slices is blocked by omega-Aga-IVA and the dual Na+/Ca2+ channel blockers lidoflazine and flunarizine.

High-threshold neuronal voltage-sensitive Ca2+ channels (VSCCs) have been classified into at least three subtypes, including L, N, and P, based on biophysical and pharmacological criteria. We examined K(+)-induced 45Ca2+ flux into rat neocortical mini-slices to determine which of these subtype(s) might be involved in this phenomenon. Neither the L-type Ca2+ channel antagonist isradipine at 10 microM nor the N-type antagonist omega-conotoxin GVIA at 1 microM were effective antagonists of 45Ca2+ flux in this model. However, the P-type Ca2+ channel antagonist, omega-Aga-IVA, blocked 70% of flux at 200 nM, with an IC50 of 17 nM, strongly implicating P-type Ca2+ channel involvement in K(+)-stimulated Ca2+ entry into mammalian nerve terminals. About 30% of the flux response was resistant to the action of omega-Aga-IVA, suggesting that a still uncharacterized subtype of VSCC is involved in Ca2+ entry into mammalian nerve terminals. Both the omega-Aga-IVA sensitive and insensitive components of 45Ca2+ flux were blocked by the diphenylalkylpiperazines, lidoflazine and flunarizine (IC50 = 6.4 microM and 11 microM, respectively), which have dual Na+/Ca2+ channel blocking actions.

Endogenous extracellular glutamate accumulation in rat neocortical cultures by reversal of the transmembrane sodium gradient.

Glutamate excites receptors located on neurons that cause calcium and sodium influx involved in excitatory synaptic transmission. During ischemia, excess glutamate is present in the extracellular space of brain tissue, leading to abnormal levels of calcium influx and eventually to cell death. In mixed neuronal/glial cell cultures we have found that reduction of extracellular sodium concentration below approximately 10 mM causes marked increases in glutamate and aspartate in medium collected 10 min after changing to low sodium. Various data indicate that the accumulated glutamate comes from reversal of normal cellular glutamate uptake, a process also thought to occur during ischemia.

Changes in alpha2 adrenoreceptors in various areas of the rat brain after long-term administration of "mu" and "kappa" opiate agonists.

Clonidine, an alpha 2 adrenoreceptor agonist, is used to treat opiate dependent individuals who are experiencing the signs and symptoms of withdrawal. Changes in the apparent number of alpha 2 adrenoreceptors in specific areas of the rat brain have been observed after chronic morphine administration. In the present study, the effects of chronically administered morphine sulfate upon alpha 2 adrenoreceptors were compared to those of UM-1072, (+/-)-5,9-alpha-dimethyl-2-hydroxy-2-tetrahydro-furfuryl-6, 7-benzomorphan HCl, a "kappa" agonist which does not produce typical morphine-like dependence. The maximum number of specific binding sites (Bmax) and dissociation constants (KD's) for 3H-clonidine were measured with neural membranes isolated from saline or drug-treated rats. Rats were injected with saline, morphine or UM-1072, i.p., every 8 hr for 14 days. Doses of morphine ranged from 10 mg/kg, t.i.d., on the first three days to 100 mg/kg, t.i.d., on the last two days. Doses of UM-1072 covered a similar range. In control experiments, the Bmax's for specific binding of 3H-clonidine were (in fmoles/mg protein): hypothalamus, 142 +/- 7; amygdala, 141 +/- 3; brainstem, 70 +/- 2; parietal cortex, 130 +/- 4; hippocampus, 94 +/- 2; and caudate nucleus, 62 +/- 3. After chronic morphine treatment, the Bmax's were decreased significantly in all areas except the hippocampus. After chronic UM-1072 treatment, the Bmax's were decreased significantly in all areas studied. Neither treatment altered appreciably the KD's for 3H- clonidine. This study suggests that "mu" and "kappa" agonists might have similar actions upon noradrenergic systems in the brain.