Synthesis and evaluation of Strychnos alkaloids as MDR reversal agents for cancer cell eradication.

Natural products represent the fourth generation of multidrug resistance (MDR) reversal agents that resensitize MDR cancer cells overexpressing P-glycoprotein (Pgp) to cytotoxic agents. We have developed an effective synthetic route to prepare various Strychnos alkaloids and their derivatives. Molecular modeling of these alkaloids docked to a homology model of Pgp was employed to optimize ligand-protein interactions and design analogues with increased affinity to Pgp. Moreover, the compounds were evaluated for their (1) binding affinity to Pgp by fluorescence quenching, and (2) MDR reversal activity using a panel of in vitro and cell-based assays and compared to verapamil, a known inhibitor of Pgp activity. Compound 7 revealed the highest affinity to Pgp of all Strychnos congeners (Kd=4.4µM), the strongest inhibition of Pgp ATPase activity, and the strongest MDR reversal effect in two Pgp-expressing cell lines. Altogether, our findings suggest the clinical potential of these synthesized compounds as viable Pgp modulators justifies further investigation.

Syntheses of strychnine, norfluorocurarine, dehydrodesacetylretuline, and valparicine enabled by intramolecular cycloadditions of Zincke aldehydes.

A full account of the development of the base-mediated intramolecular Diels-Alder cycloadditions of tryptamine-derived Zincke aldehydes is described. This important complexity-generating transformation provides the tetracyclic core of many indole monoterpene alkaloids in only three steps from commercially available starting materials and played a key role in short syntheses of norfluorocurarine (five steps), dehydrodesacetylretuline (six steps), valparicine (seven steps), and strychnine (six steps). Reasonable mechanistic possibilities for this reaction, a surprisingly facile dimerization of the products, and an unexpected cycloreversion to regenerate Zincke aldehydes under specific conditions are also discussed.

Anesthesia in adult cardiac surgery without maintenance of muscle relaxants: a randomized clinical trial.

There may be no need for muscle paralysis during cardiac surgery when adequate anesthesia is provided. We studied intra- and post-operative conditions during cardiac surgery without maintenance muscle relaxant therapy. Eighty adult patients who were candidates for elective coronary artery bypass graft surgery were randomly allocated into two groups. In the noMR or study group (noMR group; n = 40) only an intubation dose of cisatracurium (0.15 mg kg(-1)) was administrated, as opposed to the control group (MR group; n = 40), who had a continuous infusion added to the intubation dose. The anesthesia level was maintained at a Bispectral score of 40-50 using a propofol infusion. A remifentanil infusion was titrated to control patient hemodynamic response. During surgery, any minor (fine body or respiratory muscle movements) or major (coarse body movements or bucking/caught) movements were recorded. Postoperatively, analgesia was provided by remifentanil. The surgical condition was classified into three states: good (no movement), acceptable (minor movements), or poor (major movements). Anesthesia, surgery and postoperative characteristics were compared between the two groups. Statistical analysis was performed in only 78 patients (noMR = 38, MR = 40). The demographic and preoperative characteristics of the two groups were comparable. Intra-operative propofol consumption was the same, but significantly more remifentanil was used in the noMR group (p = 0.001). Post-operative characteristics and complication rates did not differ between the two groups. There were no movements in the MR group patients, while in the noMR group one patient had major movement and three had minor movements. We concluded that omitting maintenance muscle relaxants in adult cardiac surgery or eliminating residual muscle paralysis at the end of the surgery without improving early outcome can increase patient intra-operative movement risk.

Efficient access to the core of the Strychnos, Aspidosperma and Iboga alkaloids. A short synthesis of norfluorocurarine.

An efficient anionic bicyclization of tryptamine-derived Zincke aldehydes forms the basis for a three-step route to the tetracyclic ABCE core of many Strychnos, Aspidosperma, and Iboga alkaloids. This powerful reaction is showcased in a five-step synthesis of the Strychnos alkaloid norfluorocurarine from tryptamine and pyridine.

Site selectivity of competitive antagonists for the mouse adult muscle nicotinic acetylcholine receptor.

The muscle-type nicotinic acetylcholine receptor has two nonidentical binding sites for ligands. The selectivity of acetylcholine and the competitive antagonists (+)-tubocurarine and metocurine for adult mouse receptors is known. Here, we examine the site selectivity for four other competitive antagonists: cisatracurium, pancuronium, vecuronium, and rocuronium. We rapidly applied acetylcholine to outside-out patches from transfected BOSC23 cells and measured macroscopic currents. We have reported the IC(50) of the antagonists individually in prior publications. Here, we determined inhibition by pairs of competitive antagonists. At least one antagonist was present at a concentration producing > or =67% receptor inhibition. Metocurine shifted the apparent IC(50) of (+)-tubocurarine in quantitative agreement with complete competitive antagonism. The same was observed for pancuronium competing with vecuronium. However, pancuronium and vecuronium each shifted the apparent IC(50) of (+)-tubocurarine less than expected for complete competition but more than expected for independent binding. The situation was similar for cisatracurium and (+)-tubocurarine or metocurine. Cisatracurium did not shift the apparent IC(50) of pancuronium or vecuronium, indicating independent binding of these two pairs. The data were fit to a two-site, two-antagonist model to determine the antagonist binding constants for each site, L(alphaepsilon) and L(alphadelta). We found L(alphaepsilon)/L(alphadelta) = 0.22 (range, 0.14-0.34), 20 (9-29), 21 (4-36), and 1.5 (0.3-2.9) for cisatracurium, pancuronium, vecuronium, and rocuronium, respectively. The wide range of L(alphaepsilon)/L(alphadelta) for some antagonists may reflect experimental uncertainties in the low affinity site, relatively poor selectivity (rocuronium), or possibly that the binding of an antagonist at one site affects the affinity of the second site.

Curariform antagonists bind in different orientations to acetylcholine-binding protein.

Acetylcholine-binding protein (AChBP) recently emerged as a prototype for relating structure to function of the ligand binding domain of nicotinic acetylcholine receptors (AChRs). To understand interactions of competitive antagonists at the atomic structural level, we studied binding of the curare derivatives d-tubocurarine (d-TC) and metocurine to AChBP using computational methods, mutagenesis, and ligand binding measurements. To account for protein flexibility, we used a 2-ns molecular dynamics simulation of AChBP to generate multiple snapshots of the equilibrated dynamic structure to which optimal docking orientations were determined. Our results predict a predominant docking orientation for both d-TC and metocurine, but unexpectedly, the bound orientations differ fundamentally for each ligand. At one subunit interface of AChBP, the side chain of Tyr-89 closely approaches a positively charged nitrogen in d-TC but is farther away from the equivalent nitrogen in metocurine, whereas, at the opposing interface, side chains of Trp-53 and Gln-55 closely approach the metocurine scaffold but not that of d-TC. The different orientations correspond to approximately 170 degrees rotation and approximately 30 degrees degree tilt of the curare scaffold within the binding pocket. Mutagenesis of binding site residues in AChBP, combined with measurements of ligand binding, confirms the different docking orientations. Thus structurally similar ligands can adopt distinct orientations at receptor binding sites, posing challenges for interpreting structure-activity relationships for many drugs.

Interaction of d-tubocurarine analogs with mutant 5-HT(3) receptors.

d-Tubocurarine is a potent competitive antagonist of both the muscle-type nicotinic acetylcholine receptor (AChR) and the serotonin type-3 receptor (5HT(3)R). We have previously used a series of structural analogs of d-tubocurarine to demonstrate that the ligand-binding domains of both receptors share common structural features. We have now extended these studies to examine the interaction of a series of d-tubocurarine analogs with 5HT(3)Rs containing mutations at either of two residues within the ligand-binding domain of the receptor (W90F and R92A). The W90F mutation results in an approximately 2-4-fold decrease in the affinity of the analogs relative to wild-type receptors, while the R92A results in an approximately 8-10-fold increase in affinity. However, since the effect of a given mutation is more or less equivalent for all analogs, neither residue W90 nor R92 is likely to make a specific interaction with d-tubocurarine itself. Rather, these two residues are likely to play a role in determining both the geometry of the binding site, as well as the overall environment that a ligand encounters in the binding site.

Deep sedation and mechanical ventilation without paralysis for 3 weeks in normal beagles: exaggerated resistance to metocurine in gastrocnemius muscle.

BACKGROUND: Patients in the intensive care unit may have muscle weakness in the recovery phase, and disuse atrophy may play a role in this weakness. To assess this problem, the authors measured changes in the potency of the nondepolarizing neuromuscular blocking agent metocurine in a canine model that involved 3 weeks of intensive care, nonparalyzing anesthesia with pentobarbital, and positive-pressure ventilation. METHODS: Six dogs were anesthetized with pentobarbital to a sufficient depth that spontaneous and reflex muscle movements were absent. Their tracheas were intubated, their lungs were mechanically ventilated, and they received round-the-clock intensive medical and nursing care for 3 weeks. Transduced gastrocnemius muscle responses to metocurine were determined weekly. A 4- to 15-min infusion of 148-4,300 microg/min (longer durations and greater concentrations on progressive weeks) yielded more than 80% paralysis. Serial metocurine plasma concentrations during the onset of the block and recovery provided data to determine pharmacokinetics using NONMEM. Metocurine plasma concentrations and the degree of paralysis were used to model the effect compartment equilibration constant, and the Hill equation was used to yield the slope factor and potency within the effect compartment. RESULTS: The metocurine effect compartment concentration associated with a 50% diminution of twitch height after 3 weeks was 1,716+/-1,208 ng/ml (mean +/- SD), which was significantly different from 257+/-34 ng/ml, the value on day 0. There were no pharmacokinetic differences. CONCLUSION: The absence of muscle tone and reflex responsiveness for 3 weeks was associated with exaggerated resistance to the neuromuscular blocker metocurine.

Interaction of D-tubocurarine analogs with the 5HT3 receptor.

D-Tubocurarine is a potent competitive antagonist of two members of the ligand-gated ion channel family, the muscle-type nicotinic acetylcholine receptor (AChR) and serotonin type-3 receptor (5HT3R). We have used a series of analogs of D-tubocurarine to determine the effects of methylation, stereoisomerization and halogenation on the interaction of D-tubocurarine with the 5HT3R. The affinities of the analogs for the 5HT3R span a 200-fold concentration range and fall into three broad groups. The first group, with affinity constants (Ki) < 150 nM, consists of D-tubocurarine and analogs modified at the nitrogens or 7' hydroxyl. The fact that these compounds all have high affinity for the 5HT3R suggests that these portions of the ligand do not make interactions with the receptor that are critical for high-affinity binding. The second group, with Ki's in the 1-5 microM range, consists of analogs modified at the 12'-hydroxyl or the adjacent 13'-carbon, which suggests that this portion of the ligand makes interactions that are important for high-affinity binding. The third, very low affinity, group is a compound with altered stereoconfiguration at the 1 carbon, demonstrating the importance of proper configuration of the antagonist in ligand-receptor interactions. For the most part, this pattern of selectivity is similar to that for the AChR, suggesting that the structures of the ligand-binding sites of these two receptors share common structural features.

Interaction of d-tubocurarine analogs with the mouse nicotinic acetylcholine receptor. Ligand orientation at the binding site.

The binding of d-tubocurarine and several of its analogs to the mouse nicotinic acetylcholine receptor (AChR) was measured by competition against the initial rate 125I-alpha-bungarotoxin binding to BC3H-1 cells. The changes in affinity due to methylation or halogenation at various functional groups on d-tubocurarine was measured to both the high affinity (alphagamma-site) and the low affinity site (alphadelta-site). We show that quaternization by methylation of the 2'-N ammonium group enhances the affinity for both the acetylcholine binding sites of mouse AChR, whereas this change does not affect affinity for the Torpedo AChR sites. The effect of N-methylation suggests the presence of interactions with the ammonium moiety that cannot be readily attributed to the known conserved residues thought to stabilize this functional group. Methylation of both the 7'- and 12'-phenols produced net affinity changes at both sites. The changes resulted from contributions at both the 7'- and the 12'-positions; however, these effects were dependent on whether the ammoniums were also methylated. Substitution of bromine or iodine at the 13'-position decreased the affinity considerably to the high affinity alphagamma-site of mouse AChR, whereas the affinity for the Torpedo alphagamma-site was slightly increased. Furthermore, binding to the mouse AChR was unaffected by the conformational state, whereas these ligands strongly preferred the desensitized conformation of the Torpedo AChR. Comparison of binding changes upon 13'-halogenation to the changes in amino acid residues at the ACh binding sites of the mouse and Torpedo AChR shows mouse residue Ile-gamma116 as likely to be involved in interacting with the 13'-position of d-tubocurarine. It is predicted that this residue is involved in the conformational equilibrium between the resting and desensitized conformations.

Determinants involved in the affinity of alpha-conotoxins GI and SI for the muscle subtype of nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors from muscle contain two functionally active and pharmacologically distinct acetylcholine-binding sites located at the alpha/gamma and alpha/delta subunit interfaces. The alpha-conotoxins are competitive antagonists of nicotinic receptors and can be highly site-selective, displaying greater than 10,000-fold differences in affinities for the two acetylcholine-binding sites on a single nicotinic receptor. The higher affinity site for alpha-conotoxins GI, MI, and SI is the alpha/delta site on mouse muscle-derived BC3H-1 receptors. However, alpha-conotoxins GI and MI exhibit higher affinity for the other site (alpha/gamma site) on nicotinic receptors from Torpedo californica electric organ. alpha-Conotoxin SI does not distinguish between the two acetylcholine-binding sites on Torpedo receptors. In this study, alpha-conotoxins [K10H]SI and [K10N]SI displayed wild-type affinity for the two acetylcholine-binding sites on BC3H-1 receptors but a 10-20-fold decrease in apparent affinity at one of the two acetylcholine-binding sites on Torpedo receptors. alpha-Conotoxin [P9K]SI displayed a selective and dramatic increase in the apparent affinity for the alpha/delta site of BC3H-1 receptors and for the alpha/gamma site of Torpedo receptors. alpha-Conotoxin [R9A]GI displayed a reduction in affinity for both acetylcholine-binding sites on BC3H-1 receptors, although the extent of its selectivity for the alpha/delta site was retained. alpha-Conotoxin [R9A]GI also displayed a loss of affinity for the two acetylcholine-binding sites on Torpedo receptors, but its site-selectivity was apparently abolished. These results indicate that positions 9 and 10 in alpha-conotoxins GI and SI are involved in complex species- and subunit-dependent interactions with nicotinic receptors.

The effect of non-depolarizing neuromuscular blocking agents on the release of acetylcholine from the right atrium of the guinea pig.

The effect of various non-depolarizing neuromuscular blocking agents (gallamine, pancuronium, vecuronium, d-tubocurarine, metocurine, atracurium and pipecuronium) on [3H]acetylcholine release in the response to field electrical stimulation was investigated in vitro in preparations of the guinea pig right atrium. In this preparation, atropine enhanced and oxotremorine, a muscarinic agonist, reduced the release of [3H] acetylcholine. Atropine reversed the inhibitory effect of oxotremorine in a concentration dependent manner, indicating that there is negative feedback modulation of acetylcholine release from the vagal nerve. While pancuronium, gallamine and atracurium enhanced the release of [3H]acetylcholine, d-tubocurarine, metocurine, vecuronium and pipecuronium did not affect it. Pancuronium and gallamine also reduced the inhibitory effect of oxotremorine and the Kd value of pancuronium for muscarinic receptors located on cholinergic nerve terminals was 2.31 microM. These findings indicate that pancuronium and gallamine enhanced the release of acetylcholine from the atrial parasympathetic nerve, probably by inhibiting presynaptic muscarinic receptors.

Identification of residues in the adult nicotinic acetylcholine receptor that confer selectivity for curariform antagonists.

We identify residues in the epsilon and delta subunits of the adult nicotinic acetylcholine receptor that give the alphaepsilon and alphadelta binding sites different affinities for the curariform antagonist dimethyl d-tubocurarine (DMT). By constructing epsilon-delta subunit chimeras, coexpressing them with complementary subunits, and measuring DMT binding, we identify two pairs of residues, Ileepsilon58/Hisdelta60 and Aspepsilon59/Aladelta61, responsible for DMT site selectivity in the adult receptor. The two determinants contribute approximately equally to the binding site and interact in contributing to the site. Exchange of these residues from one subunit to the other exchanges the affinities of the resulting binding sites. These determinants in the adult receptor are far from those that confer site selectivity in the fetal receptor; determinants in the fetal receptor are Ilegamma116/Valdelta118, Tyrgamma117/Thrdelta119, and Sergamma161/Lysdelta163. Thus, alternative residues confer DMT selectivity in fetal and adult acetylcholine receptors.

Interaction of d-tubocurarine analogs with the Torpedo nicotinic acetylcholine receptor. Methylation and stereoisomerization affect site-selective competitive binding and binding to the noncompetitive site.

Analogs of d-tubocurarine were used to determine the individual effects of methylation, stereoisomerization, and halogenation of d-tubocurarine on the affinity for each of the two acetylcholine (ACh) binding sites of the Torpedo nicotinic acetylcholine receptor (AChR) and for the noncompetitive antagonist site. Eight analogs were synthesized, including three new compounds: 7'-O-methyl-chondocurarine, 12'-O-methyl-chondocurarine, and 13'-bromo-d-tubocurarine. The two ACh sites differ in their affinities for d-tubocurarine by 400-fold, as shown by inhibition of [3H]ACh binding, whereas the affinity ratio for metocurine, the trimethylated derivative of d-tubocurarine, is reduced to 30 due to a decreased affinity for the high affinity site. Binding analysis of five d-tubocurarine analogs demonstrates that methylation of the phenols alone is responsible for the observed changes in affinity. Substitution with bromine or iodine at the 13'-position affected affinity at both sites with a net increase in site selectivity. Stereoisomers of d-tubocurare had decreased affinity for only the high affinity ACh site. Thus, the ring systems, including the 12'- and 13'-positions and the 1-position stereocenter, appear to be important in discriminating between the two ACh binding sites. Desensitization of the AChR was measured by increased affinity for [3H]phencyclidine. Binding to only the single, high affinity acetylcholine binding site, comprised by the alpha gamma-subunits, was required for partial desensitization of the AChR by d-tubocurarine and its analogs. Stronger desensitization, to the same extent observed in the presence of the agonist carbamylcholine, occurred upon binding by iodonated or brominated d-tubocurarine. Interaction of the analogs at the noncompetitive antagonist site of the AChR was also measured by [3H]phencyclidine binding. The bis-tertiary ammonium analogs of either the d- or l-stereoisomers bound to the noncompetitive antagonist binding site of the AChR with 100-fold higher affinity than the corresponding quaternary ammonium analogs.

Metocurine pharmacokinetics and pharmacodynamics in goats.

Non-depolarizing muscle relaxants can facilitate surgery and anaesthesia in numerous species. and volatile inhalational anaesthetics such as isoflurane potentiate their action. We studied the effect of isoflurane on the pharmacodynamics and pharmacokinetics of metocurine in six goats. Each was studied twice: once during barbiturate-opiate anaesthesia and once during isoflurane anaesthesia. The evoked response to sciatic nerve stimulation was measured using a force transducer attached to the hoof. Metocurine was infused until approximately 80-90% blockade. Plasma metocurine concentration was determined by high-performance liquid chromatography. Isoflurane increased the potency of metocurine significantly; IC50 (the concentration in the effect compartment at 50% paralysis) was 70 +/- 15 ng/mL during isoflurane anaesthesia and 129 +/- 42 ng/mL during barbiturate-opiate anaesthesia (P < 0.03). Volume of distribution (63 +/- 18 mL/kg), clearance (1.6 +/- 0.4 mL/min.kg) and elimination half-life (99 +/- 9 min) during barbiturate-opiate anaesthesia were not significantly different during isoflurane anaesthesia: 64 +/- 25 mL/kg, 1.5 +/- 0.7 mL/kg.min, 116 +/- 16 min respectively. We conclude that, relative to barbiturate-opiate anaesthesia, isoflurane potentiates metocurine in goats.

The changing pharmacodynamics of metocurine identify the onset and offset of canine gastrocnemius disuse atrophy.

BACKGROUND: Immobilization of skeletal muscle results in disuse atrophy and resistance to nondepolarizing muscle relaxants. We studied the pharmacodynamics of metocurine (MTC) to identify the development and recovery of disuse-related resistance to MTC. METHODS: Nineteen dogs underwent cast immobilization of a hind limb for as long as 3 weeks. Before, during, and after casting, dogs were intermittently anesthetized with thiamylal-N2O-fentanyl. The blood concentration of MTC and the corresponding degree of paralysis after a brief infusion were recorded and were used to characterize the pharmacokinetics and pharmacodynamics of MTC. RESULTS: Pharmacodynamic study of the response to MTC demonstrated resistance by the 4th day of casting. The effect-site concentration associated with 50% paralysis of twitch increased after 3 weeks from approximately 250 to 750 ng/ml. After cast removal, resistance persisted for 2 more weeks. Six weeks after cast removal, the effect-site concentration associated with 50% paralysis of twitch was normal in every dog. CONCLUSIONS: Within the context of this study of immobilization disuse atrophy, pharmacokinetic and pharmacodynamic characterization of antagonist responses can be used to infer muscle disuse-related changes in acetylcholine receptors.

Allometry of pharmacokinetics and pharmacodynamics of the muscle relaxant metocurine in mammals.

We investigated the effects of body size on the pharmacokinetics and pharmacodynamics of the renally cleared muscle relaxant metocurine. We hypothesized that pharmacokinetics of the drug would change allometrically in proportion to physiological time [infinity Mb0.25, where Mb is body mass] and that pharmacodynamics would be independent of size because of the highly conserved structure of the acetylcholine receptor. Metocurine effects during general anesthesia were examined in 17 rats, 8 cats, 6 dogs, 5 pigs, 7 sheep, and 12 horses. Allometric analysis demonstrated size dependence for pharmacokinetics, which were affected by physiological time (Mb0.25). Pharmacodynamics were size independent, except for the value for effect compartment concentration associated with 50% twitch paralysis (IC50). Data from individual species had a bimodal distribution that was significant: pigs and sheep were more sensitive than other large species, and their IC50 appeared size independent. IC50 was size dependent in more active species (horse, dog, cat, rat). Although the mechanism is unknown, we speculate that this trend might relate to receptor density within the end plate. Thus pharmacokinetics changed in proportion to physiological time, and pharmacodynamics were in part size independent.

Pretreatment with sedative-hypnotics, but not with nondepolarizing muscle relaxants, attenuates alfentanil-induced muscle rigidity.

STUDY OBJECTIVE: To evaluate and compare the efficacy of various pretreatment agents to attenuate or prevent opioid-induced muscle rigidity using a well-established, previously described clinical protocol. DESIGN: Prospective, controlled, single-blind, partially randomized study. SETTING: Large medical center. PATIENTS: ASA physical status I-III patients undergoing elective surgical procedures of at least 3 hours' duration. INTERVENTIONS: The effect of pretreatment with nondepolarizing muscle relaxants (atracurium 40 micrograms/kg or metocurine 50 micrograms/kg), benzodiazepine agonists (diazepam 5 mg or midazolam 2.5 mg), or thiopental sodium 1 mg/kg on the increased muscle tone produced by alfentanil 175 micrograms/kg was compared with a control group (given no pretreatment). MEASUREMENTS AND MAIN RESULTS: Rigidity was assessed quantitatively by measuring the electromyographic activity of five muscle groups (biceps, intercostals, abdominals, quadriceps, and gastrocnemius). Rigidity also was rated qualitatively by attempts to initiate and maintain mask ventilation, attempts to flex an extremity, and the occurrence of myoclonic movements. Pretreatment with the two nondepolarizing muscle relaxants had no effect on the severe muscle rigidity produced by high-dose alfentanil. Whereas thiopental was only mildly effective, the benzodiazepines midazolam and diazepam significantly attenuated alfentanil rigidity (p < 0.05). CONCLUSION: This study suggests that benzodiazepine pretreatment is frequently, but not always, effective in preventing opioid-induced muscle rigidity.

Competitive antagonists bridge the alpha-gamma subunit interface of the acetylcholine receptor through quaternary ammonium-aromatic interactions.

We recently demonstrated that conserved tyrosines Tyr198 of the alpha subunit and Tyr117 of the gamma subunit of the acetylcholine receptor stabilize binding of the curariform antagonist dimethyl-d-tubocurarine (DMT). To test the hypothesis that DMT interacts directly with these tyrosines, and therefore bridges the alpha-gamma subunit interface, we introduced point mutations into these key positions and expressed one or both mutant subunits in alpha 2 beta gamma 2 acetylcholine receptors in 293 HEK cells. Binding of DMT, measured by competition against the initial rate of 125I-alpha-bungarotoxin binding, shows high affinity for aromatic mutations, reduced affinity for polar mutations, and lowest affinity for arginine mutations. Similar side chain dependences were observed for both Tyr alpha 198 and Tyr gamma 117, indicating interaction of these residues with two symmetrical chemical groups in DMT. Two more bisquaternary antagonists, pancuronium and gallamine, show side chain dependences similar to that of DMT, indicating that the primary stabilizing interactions are aromatic-quaternary in both subunits. For the rigid ligands DMT and pancuronium, co-expressing mutant alpha and gamma subunits revealed independent contributions by each determinant, but strict independence was not observed for the flexible ligand gallamine. The free energy contributed by each aromatic-quaternary interaction was estimated to be 2-4 kcal/mol, as determined from the free energy difference between aromatic and alkyl hydroxyl mutations. Our results suggest that bis-quaternary competitive antagonists bridge the alpha-gamma subunit interface by fitting into a pocket bounded by tyrosines at positions 198 of the alpha subunit and 117 of the gamma subunit.

Anesthesia blocks nonshivering thermogenesis in the neonatal rabbit.

Nonshivering thermogenesis (NST) is a normal physiological response of the neonate to cold exposure, characterized by increased blood flow to metabolically active brown fat stores. It is standard practice during neonatal surgery to warm the ambient environment in order to avoid consuming vital energy stores. While NST has been well-studied in the neonate, the response during anesthesia and paralysis has not been fully characterized. Rabbit pups (aged 1 to 7 days) were randomized into several groups. The experimental groups consisted of animals mechanically ventilated and administered either metocurine, pancuronium, curare, fentanyl, nitrous oxide (N2O), or halothane. The controls were spontaneously breathing animals. Oxygen consumption (VO2), an index of metabolic activity, was measured at thermoneutrality (39 degrees C) and after cold exposure (25 degrees C). Control and metocurine animals had a significant increase in VO2 in response to cold exposure. The increase in VO2 was not noted in animals that received curare, pancuronium, fentanyl, N2O, or halothane. To test the effect of anesthetic withdrawal during cold exposure on VO2, additional series of animals were studied. One group received continuous halothane throughout the period of cold exposure; the other had cessation of the halothane during cold exposure. Both groups were rewarmed subsequently. The animals that had withdrawal of halothane during cold exposure had a marked and significant increase in VO2 compared with the control group (continuous halothane). VO2 returned to near-baseline levels upon rewarming. The authors conclude that many commonly used anesthetic and paralyzing agents inhibit the thermogenic response to cold exposure. However, cessation of anesthesia (halothane) in a cold environment results in a marked increase in metabolic activity.