Targeting acute ischemic stroke with a calcium-sensitive opener of maxi-K potassium channels.

During ischemic stroke, neurons at risk are exposed to pathologically high levels of intracellular calcium (Ca++), initiating a fatal biochemical cascade. To protect these neurons, we have developed openers of large-conductance, Ca++-activated (maxi-K or BK) potassium channels, thereby augmenting an endogenous mechanism for regulating Ca++ entry and membrane potential. The novel fluoro-oxindoles BMS-204352 and racemic compound 1 are potent, effective and uniquely Ca++-sensitive openers of maxi-K channels. In rat models of permanent large-vessel stroke, BMS-204352 provided significant levels of cortical neuroprotection when administered two hours after the onset of occlusion, but had no effects on blood pressure or cerebral blood flow. This novel approach may restrict Ca++ entry in neurons at risk while having minimal side effects.

Formation of intermediate-conductance calcium-activated potassium channels by interaction of Slack and Slo subunits.

Large-conductance calcium-activated potassium channels (maxi-K channels) have an essential role in the control of excitability and secretion. Only one gene Slo is known to encode maxi-K channels, which are sensitive to both membrane potential and intracellular calcium. We have isolated a potassium channel gene called Slack that is abundantly expressed in the nervous system. Slack channels rectify outwardly with a unitary conductance of about 25-65 pS and are inhibited by intracellular calcium. However, when Slack is co-expressed with Slo, channels with pharmacological properties and single-channel conductances that do not match either Slack or Slo are formed. The Slack/Slo channels have intermediate conductances of about 60-180 pS and are activated by cytoplasmic calcium. Our findings indicate that some intermediate-conductance channels in the nervous system may result from an interaction between Slack and Slo channel subunits.

Synthesis and biological evaluation of an iodinated iberiotoxin analogue, [mono-iodo-Tyr5, Phe36]-iberiotoxin.

The synthesis and iodination of a structural analogue of the specific large-conductance calcium-activated potassium (BK) channel blocker, iberiotoxin (IbTX), a 37-amino acid scorpion neurotoxin, is reported. The synthesis of this analogue, [Tyr5, Phe36]-IbTX, was accomplished using standard solid-phase Fmoc (9-fluorenylmethoxycarbonyl) chemistry protocols. The linear peptide was cyclized via the formation of three intramolecular disulfide bridges and subsequently iodinated at the Tyr5 position. Upon purification, the iodinated analogue, [mono-iodo-Tyr5, Phe36]-IbTX, exhibited comparable biological activity to native IbTX in blocking BK-mediated currents. These findings suggest the synthesis and use of an 125I labelled IbTX analogue for BK channel localization in autoradiography experiments.

Phenotypic alteration of a human BK (hSlo) channel by hSlobeta subunit coexpression: changes in blocker sensitivity, activation/relaxation and inactivation kinetics, and protein kinase A modulation.

A human homolog of the large-conductance calcium-activated potassium (BK) channel beta subunit (hSlobeta) was cloned, and its effects on a human BK channel (hSlo) phenotype are reported. Coexpression of hSlo and hSlobeta, in both oocytes and human embryonic kidney 293 cells, resulted in increased Ca2+ sensitivity, marked slowing of BK channel activation and relaxation, and significant reduction in slow inactivation. In addition, coexpression changed the pharmacology of the BK channel phenotype: hSlo-mediated currents in oocytes were more sensitive to the peptide toxin iberiotoxin than were hSlo + hSlobeta currents, and the potency of blockade by the alkaloid BK blocker tetrandrine was much greater on hSlo + hSlobeta- mediated currents compared with hSlo currents alone. No significant differences in the response to charybdotoxin or the BK channel opener NS1619 were observed. Modulation of BK channel activity by phosphorylation was also affected by the presence of the hSlobeta subunit. Application of cAMP-dependent protein kinase increased P(OPEN) of hSlo channels, but decreased P(OPEN)of most hSlo + hSlobeta channels. Taken together, these altered characteristics may explain some of the wide diversity of BK channel phenotypes observed in native tissues.

Effects of channel modulators on cloned large-conductance calcium-activated potassium channels.

Through expression of the cloned mouse (mSlo) or human (hSlo) large-conductance (BK) Ca(2+)-activated K+ channel in Xenopus laevis oocytes and HEK 293 cells, we characterized the effects of reported blockers and openers of BK channels to initiate the study of the molecular determinants of BK channel modulation. In oocytes, iberiotoxin and charybdotoxin, peptidyl scorpion toxins, were both equally effective blockers of BK current, although iberiotoxin was significantly more potent than charybdotoxin. The structurally related peptide kaliotoxin was not a potent blocker of BK current. Paxilline, a fungal tremorgenic alkaloid, was an effective but complex blocker of BK current. Tetrandrine, a putative blocker of type II BK channels, and ketamine were relatively ineffective. The putative BK openers NS004 and NS1619, phloretin, niflumic acid, flufenamic acid, and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) increased BK current in oocytes at microM concentrations; many of these produced biphasic concentration-response relationships. Coapplication of representative blockers and openers revealed several patterns of interaction, including competitive and noncompetitive antagonism. NS1619, niflumic acid, and phloretin were tested by using excised inside-out membrane patches from HEK 293 cells and were found to increase the activity of hSlo BK channels and produce a leftward shift in the G/Gmax-versus-voltage relationship of these channels. These results represent the first comprehensive examination of the molecular pharmacology of BK channels.

The effects of the adenosine reuptake inhibitor soluflazine on synaptic potentials and population hypoxic depolarizations in area CA1 of rat hippocampus in vitro.

Adenosine has recently been shown to play a potentially important role in the regulation of synaptic excitability during experimental hypoxia in the hippocampus of the rat. Endogenous adenosine, rapidly released at the initiation of a hypoxic episode, produced synaptic depression, which could protect sensitive neurons. In the present experiments, an inhibitor of the reuptake of adenosine, soluflazine (R64719) was employed to increase the levels of endogenous adenosine under normoxic and hypoxic conditions in slices of the hippocampus of the rat. Soluflazine produced a slow-onset, concentration-dependent depression of population excitatory postsynaptic potentials, which was reversed by the specific A1 adenosine receptor antagonist, 8-cyclopentyltheophylline. During severe N2-induced hypoxia, soluflazine significantly delayed hypoxic depolarization. These results suggest that inhibition of the reuptake of adenosine may have therapeutic potential in the amelioration of hypoxic/ischemic neuronal damage, particularly in the hippocampus.

A role for sigma binding in the antipsychotic profile of BMY 14802?

BMY 14802 was identified as a potential antipsychotic drug in traditional model systems, and this identification was confirmed in modern behavioral and electrophysiological systems. The drug appears to be atypical as an antipsychotic in its lack of activity in models predictive of the potential to produce extrapyramidal side effects and tardive dyskinesia. Indeed, this suggestion is corroborated by clinical findings to date. The atypical profile of BMY 14802 extends to its neurochemical actions and appears to find its basis in regionally selective, indirect modulation of the dopamine system. Furthermore, BMY 14802 exhibits interactions with sigma binding sites in vitro and in vivo, a notion supported by data from neurophysiological, behavioral, and biochemical investigations. BMY 14802 also appears to be neuroprotective in some model systems and may have utility in the treatment of stroke (Boissard et al. 1991). BMY 14802 appears to interact with 5-HT1A receptors, but this interaction does not seem to contribute significantly to the potential antipsychotic actions of the drug. Moreover, the formation of active metabolites of BMY 14802 does not appear to occur in animals or humans to an extent of physiological or behavioral relevance. If clinically efficacious, BMY 14802 may treat the symptoms of schizophrenia by a mechanism novel for antipsychotic drugs: regionally selective, indirect modulation of dopaminergic systems by specific interaction at sigma sites.

Age-dependence of effects of A1 adenosine receptor antagonism in rat hippocampal slices.

1. Population excitatory postsynaptic potentials (EPSPs) and population spikes evoked in area CA1 of hippocampal slices from aged Fischer 344 rats were significantly smaller in amplitude than responses obtained in slices from young Fischer 344 rats. 2. The A1 adenosine receptor antagonist 8-cyclopentyltheophylline (8-CPT) produced a concentration-dependent increase in synaptic potentials in slices from both young and aged rats. Low concentrations (1 nM) of 8-CPT were effective in producing increases in both population spike amplitudes and population EPSP slopes in young and aged rat slices. Response increases were maximized by 100 nM 8-CPT in slices from rats of both age groups. 3. Adenosine antagonism produced greater average increases in synaptic responses in hippocampal slices from aged rats at all concentrations tested (1.0 nM-1.0 microM). A qualitative age-related difference in the response to 8-CPT was also observed; 8-CPT produced a late component, consisting of multiple population spikes, in evoked responses in slices obtained from aged but not young rats. 4. Adenosine antagonism significantly increased the maximum evocable response (both spike amplitude and EPSP slope) in slices from aged rats, relative to increases observed in slices from young rats. This suggested that smaller synaptic potentials seen in slices from aged rats were in part due to greater levels of "tonic" adenosinergic inhibition. 5. Slices from young and aged rats were incubated in the adenosine reuptake inhibitor soluflazine (R64719; 1.0, 10, and 100 microM) and the inhibition of population EPSPs was observed for 60 min. No difference was observed in the rate of inhibition or the maximal level of inhibition produced by soluflazine, in slices from rats of either age group. 6. Application of (+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cyclo-hepten- 5,10-imine hydrogen maleate (MK-801) and 2-amino-5-phosphonopentanoic acid (2-AP5), antagonists of N-methyl-D-aspartate (NMDA) excitatory amino acid (EAA) receptors, reduced the late multiple population spike component in slices from aged rats incubated in 8-CPT. A smaller direct effect of the NMDA antagonists was observed in slices from aged rats in the absence of 8-CPT treatment at maximal response levels. No effect of NMDA receptor antagonism was observed in slices from young rats under either condition. 7. Hippocampal tissue, from young and old rats utilized in the electrophysiological experiments, was assayed for A1 adenosine binding site density with a saturating concentration of radiolabeled agonist and antagonist. Guanine nucleotide modulation of agonist binding was also measured.(ABSTRACT TRUNCATED AT 400 WORDS)

Hypoxia produces cell death in the rat hippocampus in the presence of an A1 adenosine receptor antagonist: an anatomical and behavioral study.

Endogenous adenosine depresses synaptic transmission in rat hippocampal slices during periods of hypoxia, a potentially neuroprotective cellular response that is attenuated by the adenosine antagonist 8-cyclopentyltheophylline. In this study, rats were exposed to moderate hypoxic conditions (5% O2- 95% N2, 40 min x three days) in the presence or absence of 8-cyclopentyltheophylline, and the effects of reducing adenosinergic inhibition during hypoxia were assessed histologically and behaviorally. Half the rats exposed to low levels of oxygen in the presence of 8-cyclopentyltheophylline (10 mg/kg) suffered unilateral or bilateral hippocampal damage largely limited to the CA1 subfield. Animals which had suffered hippocampal damage were also impaired in their performance of a working memory version of the Morris Water Maze, but not a passive avoidance task (step-through). Hypoxia alone did not result in neuronal damage or behavioral impairment. These results provide further evidence that endogenous adenosine provides an important level of neuronal protection during even prolonged periods of hypoxia.

Suppressant effect of REM sleep deprivation on neophobia in normal rats and in rats with selective DSP-4 induced damage of locus coeruleus neurons.

The influence of REM sleep deprivation (REMD) on open field behavior of normal and locus coeruleus (LC)-damaged animals was investigated under the assumption that REMD suppresses neophobia in rats. REMD (for 24 or 72 hr, water tank technique) produced marked changes in behavior of rats encountering a novel object (white cube) in the center of the open field. REMD induced an increase in activity of treated rats; latency to the object approach was shorter, the number of center entries, time spent in object exploration, frequency of ambulation and rearing were significantly higher than in controls, also defecation was nearly abolished. LC-damage (using DSP-4, a selective central noradrenergic neurotoxin) induced neophobic-like reactions manifested by significantly prolonged latency, tendency to decreased object exploration, center entries and reduction of ambulation and rearing. This "neophobic" behavior of DSP-4 rats was counteracted by REMD as well as by subchronic, but not acute treatment, with antidepressant oxaprotiline (2 X daily for 8 days, 10 mg/kg, IP). The results provide strong support for antineophobic activity of REMD. In addition, they indicate possible similarity of REMD and subchronic oxaprotiline action on neophobia-like behavior in rats with damaged LC-neurons.

Effects of apomorphine, TL-99 and 3-PPP on yawning in rats.

Dopaminergic agonists, apomorphine (APO) (0.025-0.25 mg/kg, s.c.), TL-99 (0.5-3 mg/kg, s.c.) and 3-PPP (0.15-10 mg/kg, s.c.) elicited yawning in rats and the dose-response curves of all 3 compounds showed a bell-shaped form. Haloperidol (0.02 mg/kg, s.c.) reduced the yawning induced by DA-agonists to about 50%. The potencies of the DA-agonists in inducing yawning were APO greater than TL-99 greater than 3-PPP (comparable to potencies obtained in other in vivo tests, determining DA-ergic activity). The findings support the validity of the yawning phenomenon as a screening test for DA-agonists. Additionally, it was found that apomorphine induced yawning was significantly and dose-dependently enhanced by the beta-agonist, formoterol. This effect was counteracted by scopolamine, not changed by metergoline and further increased by l-propranolol. These data support the hypothesis of cholinergic involvement in yawning and indicate a role, though unclear at present, of beta-receptors in this behaviour.

The effects of single and repeated doses of maprotiline, oxaprotiline and its enantiomers on foot-shock induced fighting in rats.

Foot-shock induced fighting behavior (SIF) in rats was tested after single and repeated dose-treatment (10 mg/kg IP twice daily for 10 days) of maprotiline, oxaprotiline and of (+)- and (-)-enantiomers of oxaprotiline. Marked facilitation of SIF was observed after repeated but not single administration of all drugs including the NA-uptake inactive (-)-enantiomer of oxaprotiline. No enhancement of SIF was seen after multiple dose-treatment with promethazine, an antihistaminic, or atropine. The mechanism of the facilitation of SIF induced by antidepressants maprotiline and oxaprotiline as well as by its enantiomers is unclear. The clear-cut dissociation of the effect of (+)- and (-)-oxaprotiline on the rate of NA-disappearance, but their similar enhancing effect on SIF challenges the assumption of a primary importance of central NA-system in this behavior. By contrast, the increase in jumping behavior recorded additionally to SIF, seems to be a great extent dependent on NA-uptake inhibiting properties of tested drugs.

Facilitation of shock-induced fighting in the rat after DSP-4, a selective noradrenergic neurotoxin.

Rats treated with DSP-4 showed a marked enhancement of shock-induced fighting (SIF). Administration of 1-propranolol attenuated or completely counteracted SIF in control animals, but only the highest dose (10 mg/kg) of this beta-adrenergic antagonist was effective in reducing SIF in DSP-4 animals. Other behavioral experiments indicated that the responsiveness of DSP-4 rats to dopaminergic, serotoninergic, and cholinergic agonists was unchanged compared to that of control rats. The results confirm the participation of the noradrenergic system in SIF, and substantiate an involvement of beta-adrenergic receptors in this kind of aggression.

Altered hindlimb extension in the rat after DSP-4: a useful marker of central noradrenergic depletion.

Rats treated with DSP-4, a selective noradrenergic neurotoxin, usually exhibited an attenuation of hindlimb extension when suspended by the tail. Those animals showing the reduced extension had no post-decapitation reflex (PDR), whereas those animals having a normal extension had the PDR. On this basis, rats injected with DSP-4 can be readily screened for an effective or ineffective noradrenergic lesion. Preliminary evidence suggests that the alpha 2-adrenoceptor is involved in the regulation of hindlimb extension.