The selective alpha7 nicotinic acetylcholine receptor agonist PNU-282987 [N-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride] enhances GABAergic synaptic activity in brain slices and restores auditory gating deficits in anesthetized rats.

Schizophrenic patients are thought to have an impaired ability to process sensory information. This deficit leads to disrupted auditory gating measured electrophysiologically as a reduced suppression of the second of paired auditoryevoked responses (P50) and is proposed to be associated with decreased function and/or expression of the homomeric alpha7 nicotinic acetylcholine receptor (nAChR). Here, we provide evidence that N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-282987), a novel selective agonist of the alpha7 nAChR, evoked whole-cell currents from cultured rat hippocampal neurons that were sensitive to the selective alpha7 nAChR antagonist methyllycaconitine (MLA) and enhanced GABAergic synaptic activity when applied to hippocampal slices. Amphetamine-induced sensory gating deficit, determined by auditory-evoked potentials in hippocampal CA3 region, was restored by systemic administration of PNU-282987 in chloral hydrate-anesthetized rats. Auditory gating of rat reticular thalamic neurons was also disrupted by amphetamine; however, PNU-282987 normalized gating deficit only in a subset of tested neurons (6 of 11). Furthermore, PNU-282987 improved the inherent hippocampal gating deficit occurring in a subpopulation of anesthetized rats, and enhanced amphetamine-induced hippocampal oscillation. We propose that the alpha7 nAChR agonist PNU-282987, via modulating/enhancing hippocampal GABAergic neurotransmission, improves auditory gating and enhances hippocampal oscillatory activity. These results provide further support for the concept that drugs that selectively activate alpha7 nAChRs may offer a novel, potential pharmacotherapy in treatment of schizophrenia.

Preclinical studies characterizing the anti-migraine and cardiovascular effects of the selective 5-HT1D receptor agonist PNU-142633.

The present study describes the preclinical pharmacology of a highly selective 5-HT1D receptor agonist PNU-142633. PNU-142633 binds with a Ki of 6 nm at the human 5-HT1D receptor and a Ki of> 18 000 nm at the human 5-HT1B receptor. The intrinsic activity of PNU-142633 at the human 5-HT1D receptor was determined to be 70% that of 5-HT in a cytosensor cell-based assay compared with 84% for that of sumatriptan. PNU-142633 was equally effective as sumatriptan and a half-log more potent than sumatriptan in preventing plasma protein extravasation induced by electrical stimulation of the trigeminal ganglion. Like sumatriptan, PNU-142633 reduced the increase in cat nucleus trigeminal caudalis blood flow elicited by electrical stimulation of the trigeminal ganglion compared with the vehicle control. The direct vasoconstrictor potential of PNU-142633 was evaluated in vascular beds. Sumatriptan increased vascular resistance in carotid, meningeal and coronary arteries while PNU-142633 failed to alter resistance in these vascular beds. These data are discussed in relation to the clinical findings of PNU-142633 in a phase II acute migraine study.

Coronary vasorelaxation by nitroglycerin: involvement of plasmalemmal calcium-activated K+ channels and intracellular Ca++ stores.

This study investigated nitroglygerin (NTG) relaxations in isolated dog coronary artery in comparison with other vascular preparations. Under maximal PNU-46619 precontraction, the coronary artery was significantly more sensitive to NTG than mesenteric artery, mesenteric vein and saphenous vein. In the coronary artery, NTG (1-100 nM) produced relaxations with EC50 = 9.4 nM. In KCl-contracted arteries (20-80 mM KCl), relaxation by NTG was progressively reduced. Relaxation responses to NTG also were inhibited significantly by potent calcium-activated K+ (BK) channel blockers, charybdotoxin (100 nM) and iberiotoxin (200 nM), but not by KATP blockers such as PNU-37883A (10 microM) or PNU-99963 (100 nM). Nitric oxide (0.1-30 nM) and acetylcholine (3-300 nM) also produced relaxations which were significantly attenuated by the BK blockers. In further experiments, NTG (1-100 nM) produced inhibition of PNU-46619-induced SR [Ca++]i release, with an IC50 of 8.5 nM, which was not affected by charybdotoxin. Furthermore, P1075 (50 nM), a KATP opener, did not inhibit agonist-stimulated SR [Ca++]i release. Ryanodine (10 microM), which acts on SR Ca++ release channels, did not alter NTG relaxations, whereas thapsigargin (0.1 microM), a selective inhibitor of SR Ca(++)-ATPase pump, produced pronounced inhibition of NTG relaxations. These results suggest that NTG, in the therapeutic concentration range, produces coronary relaxation primarily via two cellular mechanisms: plasmalemmal BK channel activation and stimulation of SR Ca(++)-ATPase to produce increased SR Ca++ accumulation. These two mechanisms apparently are equally important and act together to produce a unique vasorelaxation profile demonstrated by NTG-type coronary vasodilators.

Tissue and species variation in the vascular receptor binding of 3H-P1075, a potent KATP opener vasodilator.

A high-affinity receptor site for 3H-P1075 previously observed in rat aorta has been proposed to mediate the vasorelaxation effects of P1075 and other ATP-sensitive K+ channel (KATP) openers. We tested this hypothesis by correlating the receptor binding of 3H-P1075 with its vasorelaxation effects in several isolated vascular preparations from three species: rat, rabbit and dog. In rat aorta and mesenteric artery, 3H-P1075 (1-5 nM) showed high amounts of specific binding (5-10 fmol/mg tissue), which was 48 to 79% of total binding. In contrast, little (< or = 17%) to no specific binding of 3H-P1075 (1-5 nM) was observed in dog coronary artery, dog mesenteric artery or rabbit mesenteric artery. However, all vascular preparations studied relaxed with P1075 (1-100 nM), showing maximal relaxations at 30 to 100 nM. The P1075 relaxation EC50 values in rat aorta, rabbit mesenteric artery and dog coronary artery ranged from 7.5 to 24.1 nM depending on the level of contractile activation. Thus, the pharmacological effect of P1075 could be correlated with the presence of specific receptor binding sites only in rat vascular preparations. These data show that there are significant differences in the characteristics of the proposed specific receptor site for 3H-P1075 in different vascular preparations from different species, and they raise questions regarding the pharmacological significance of this KATP opener binding site. Until such questions are resolved, it appears that the study of functional significance of this receptor site as well as further biochemical characterization of this receptor site may necessitate the use of only the rat vascular preparations.

Pharmacological characterization of novel cyanoguanidines as vascular KATP channel blockers.

KATP blockers derived from cyanoguanidine KATP opener (P1075) chemistry were characterized in isolated rabbit mesenteric artery and evaluated functionally by their ability to antagonize maximal relaxation induced by pinacidil (1 microM) of norepinephrine (5 microM) contraction. PNU-89692, PNU-97025E and PNU-99963 were identified as KATP blockers with IC50 values of 860, 83 and 18 nM, respectively. Studies with selected chiral compounds demonstrated that the (R)-enantiomers were more potent as KATP blockers than the (S)-enantiomers. Further studies demonstrated that PNU-99963 (1) inhibited relaxations by other KATP openers, such as cromakalim (0.5 microM) and minoxidil sulfate (5 microM); (2) was more potent than the other known vascular KATP blockers (glyburide and PNU-37883A); and (3) acted as a KATP blocker in isolated rat aorta as well as dog coronary artery. PNU-99963 actions were selective because PNU-99963 (100 nM) was without any inhibitory effect on relaxations induced by forskolin (0.5 microM), nitroglycerin (1 microM), D600 (25 or 500 nM) or 15 mM K+-induced relaxations of NE contractions in K+-free PSS. The discovery of KATP blockers and openers from the same chemical series is a first for the K+ channel field. The close structural similarity between P1075 (KATP opener) and PNU-99963 (KATP blocker), stereospecificity of action and potency and selectivity all suggest that these molecules may prove to be valuable tools in understanding the structure and function of the KATP channel complex in vascular smooth muscle.