Phencyclidine [PCP; (1-phencyclohexyl)piperidine] is a general anesthetic and hallucinogen that has prolonged duration of action and is also psychotomimetic (1, 2). It potentiates the direct and indirect evoked muscle twitch, an effect that seems to be related to a marked blockade of potassium conductance (3). In addition, after an initial twitch potentiation the neuromuscular transmission is blocked as a result of a direct effect of PCP on the ionic channel of the acetylcholine (AcCho) receptor. PCP decreases the peak amplitude of the end-plate current (epc) in a voltage-and time-dependent manner, causes significant nonlinearity of the current-voltage relationship and marked acceleration of the decay time of both epc and miniature end-plate current (mepc), and concomitantly shortens the mean channel lifetime without alteration of the single channel conductance (4,5 MATERIALS AND METHODS Electrophysiological Techniques. Experiments were performed at room temperature (20-22°C) on sciatic nerve sartorius muscle preparations of the frog Rana pipiens, except that extrajunctional AcCho sensitivity measurements were made on 10-to 15-day denervated rat soleus muscles. The Ringer solutions used, nerve stimulation, intracellular recordings of the resting membrane potential, action potential, end-plate potential (epp), and miniature end-plate potential (mepp), delayed rectification, and microiontophoresis of AcCho were as described (6, 10, 11). The voltage-clamp circuitry and recording and analysis of the epc fluctuations in response to iontophoretic application of AcCho were similar to those described previously and were analyzed by an on-line PDP-11 computer. The drugs were superfused from micropipets by pressure with close application to the end-plate region.Biochemical Techniques. Membrane preparation from the electric organ of T. ocellata was as described (8). [piperidyl-3,4-3H(N)

SummaryNatural ( -)-perhydrohistrionicotoxin (6a), its unnatural (t )-antipode 6b, (-)-2-depentylperhydrohistrionicotoxin (7a) and its (+ )-antipode 7b have been prepared and characterized. Kishi's lactam 8 reacted with optically active isocyanates, and the mixture of diastereomeric carbamates so obtained was separated and hydrolyzed yielding the optical antipodes of Kishi's lactam in optically pure form. Reduction with LiAlH4 yielded the optically active 2-depentyl analogs, while another sequence already developed in the racemic series afforded the natural toxin and its (+ )-antipode. Some electrophysiological properties of these compounds are presented.Natural histrionicotoxin (1, (-)-HTX) was isolated from extracts of skins of the Columbian poison frog Dendrobates histrionicus by Witkop et al. [l]. HTX represents a spiropiperidine, substituted at C ( 2 ) and C (7) with side-chains containing cis-ene-yne unsaturation (Scheme 1).The absolute configuration of 1 shown in Scheme 1, was deduced from data obtained by a single crystal X-ray analysis of isodihydrohistrionicotoxin hydro- ')

5-Hydroxytryptamine (5-HT) is sequestered and released by endothelial cells, acts as an endothelial cell mitogen, promotes the release of nitric oxide (NO), and has been associated with the p44/p42 mitogen-activated protein kinase (MAPK) cascade. NO also acts as a cell mitogen and promotes signals that culminate in the phosphorylation of MAPK. The aim of this study was to test whether endothelial 5-HT receptors stimulate dual (tyrosyl- and threonyl-) phosphorylation of MAPK through a mitogen-activated protein kinase kinase-1 (MEK-1) and eNOS-dependent pathway in bovine aortic endothelial cells (BAECs). As shown by Western blot analysis, 5-HT and the 5-HT1B-selective agonist 5-nonyloxytryptamine (5-NOT) stimulate time- and concentration-dependent (0.001-10 microM) phosphorylation of MAPK in these cells. The agonist-stimulated phosphorylation of MAPK was blocked by the 5-HT1b-receptor antagonist isamoltane (0.01-10 p3M) and the MEK-1 inhibitor PD 098059 ([2-(2'-amino-3'-methoxy-phenyl)-oxanaphthalen-4-one]; 0.01-10 microM¿. The eNOS inhibitor L-N(omega)-iminoethyl-L-ornithine (L-NIO; 0.01-10 microM) failed to block the 1 microM 5-NOT-stimulated responses. Our findings suggest that the 5-HT receptors (specifically 5-HT1B) mediate signals to MEK-1 and subsequently to MAPK through an eNOS-independent pathway in BAECs.

Activation of endothelial nitric oxide synthase (eNOS) results in the production of nitric oxide (NO) that mediates the vasorelaxing properties of endothelial cells. The goal of this project was to address the possibility that 5-hydroxytryptamine (5-HT) stimulates eNOS activity in bovine aortic endothelial cell (BAEC) cultures. Here, we tested the hypothesis that 5-HT receptors mediate eNOS activation by measuring agonist-stimulated [3H]L-citrulline ([3H]L-Cit) formation in BAEC cultures. We found that 5-HT stimulated the conversion of [3H]L-arginine ([3H]L-Arg) to [3H]L-Cit, indicating eNOS activation. The high affinity 5-HTl receptor agonist, 5-nonyloxytryptamine (5-NOT)-stimulated [3H]L-Cit turnover responses were concentration-(0.01 nM to 100 pM) and time-dependent. Maximal responses were observed within 10 min following agonist exposures. These responses were effectively blocked by the 5-HT,, receptor antagonist, isamoltane, the 5-HT,$5-HT2 receptor antagonist, methiothepin, and the eNOS selective antagonists (0.01-10 pM): L-N"-monornethyl-L-arginine (L-NMMA) and L-Nu-iminoethyl-L-ornithine (L-NIO). Pretreatment of BAEC cultures with pertussis toxin (PTX; 1-100 ng/ml) for 16 hr resulted in significant inhibition of the agoniststimulated eNOS activity, indicating the involvement of Gi proteins. These findings lend evidence of a 5-HTlB receptor/eNOS pathway, accounting in part for the activation of eNOS by 5-HT. Further investigation is needed to determine the role of other vascular 5-HT receptors in the stimulation of eNOS activity. [P.S.E.B.M. 1999, Vol 2211everal vasoactive substances elicit the release of nitric oxide (NO) from endothelium following activation of S the calciumhalmodulin-dependent enzyme endothelial nitric oxide synthase (eNOS) (l), which has been cloned in both cultured and native bovine aortic endothelial cell (BAEC) cultures (2). The diffusion of NO into vascular smooth muscle cells (VSMCs) results in activation of soluble guanylyl cyclase (solGC) and subsequent vasorelaxation (1). 5-Hydroxytryptamine (5-HT) stimulates endothe-This project was supported by grants. NSF/RIMI 9550699 and NIH T32 HL077809 I To whom requests for reprints should be addressed

Activation of endothelial nitric oxide synthase (eNOS) results in the production of nitric oxide (NO) that mediates the vasorelaxing properties of endothelial cells. The goal of this project was to address the possibility that 5-hydroxytryptamine (5-HT) stimulates eNOS activity in bovine aortic endothelial cell (BAEC) cultures. Here, we tested the hypothesis that 5-HT receptors mediate eNOS activation by measuring agonist-stimulated [3H]L-citrulline ([3H]L-Cit) formation in BAEC cultures. We found that 5-HT stimulated the conversion of [3H]L-arginine ([3H]L-Arg) to [3H]L-Cit, indicating eNOS activation. The high affinity 5-HT1B receptor agonist, 5-nonyloxytryptamine (5-NOT)-stimulated [3H]L-Cit turnover responses were concentration-(0.01 nM to 100 microM) and time-dependent. Maximal responses were observed within 10 min following agonist exposures. These responses were effectively blocked by the 5-HT1B receptor antagonist, isamoltane, the 5-HT1B/5-HT2 receptor antagonist, methiothepin, and the eNOS selective antagonists (0.01-10 microM): L-Nomega -monomethyl-L-arginine (L-NMMA) and L-N omega-iminoethyl-L-ornithine (L-NIO). Pretreatment of BAEC cultures with pertussis toxin (PTX; 1-100 ng/ml) for 16 hr resulted in significant inhibition of the agonist-stimulated eNOS activity, indicating the involvement of Gi proteins. These findings lend evidence of a 5-HT1B receptor/eNOS pathway, accounting in part for the activation of eNOS by 5-HT. Further investigation is needed to determine the role of other vascular 5-HT receptors in the stimulation of eNOS activity.