Novel strategy for biofilm inhibition by using small molecules targeting molecular chaperone DnaK.

Biofilms are complex communities of microorganisms that attach to surfaces and are embedded in a self-produced extracellular matrix. Since these cells acquire increased tolerance against antimicrobial agents and host immune systems, biofilm-associated infectious diseases tend to become chronic. We show here that the molecular chaperone DnaK is important for biofilm formation and that chemical inhibition of DnaK cellular functions is effective in preventing biofilm development. Genetic, microbial, and microscopic analyses revealed that deletion of the dnaK gene markedly reduced the production of the extracellular functional amyloid curli, which contributes to the robustness of Escherichia coli biofilms. We tested the ability of DnaK inhibitors myricetin (Myr), telmisartan, pancuronium bromide, and zafirlukast to prevent biofilm formation of E. coli. Only Myr, a flavonol widely distributed in plants, inhibited biofilm formation in a concentration-dependent manner (50% inhibitory concentration [IC50] = 46.2 µM); however, it did not affect growth. Transmission electron microscopy demonstrated that Myr inhibited the production of curli. Phenotypic analyses of thermosensitivity, cell division, intracellular level of RNA polymerase sigma factor RpoH, and vulnerability to vancomycin revealed that Myr altered the phenotype of E. coli wild-type cells to make them resemble those of the isogenic dnaK deletion mutant, indicating that Myr inhibits cellular functions of DnaK. These findings provide insights into the significance of DnaK in curli-dependent biofilm formation and indicate that DnaK is an ideal target for antibiofilm drugs.

Haemodynamic differences between pancuronium and vecuronium in an experimental pig model.

OBJECTIVE: To compare baseline cardiovascular function in anesthetised pigs using either pancuronium or vecuronium as a neuromuscular blocker. STUDY DESIGN: Retrospective, non-randomized comparison. ANIMALS: Norwegian Land Race pigs (Sus scrofa domesticus) weighing mean 42 ± SD 3 kg. METHODS: One hundred and sixteen animals from four different research protocols premedicated with identical doses of ketamine, diazepam, atropine and isoflurane, and anaesthetised with pentobarbital, fentanyl, midazolam and N(2)O were arranged into three uniform groups with respect to neuromuscular blocking agent: pancuronium bolus of 0.063 mg kg(-1) followed by 0.14 mg kg(-1) hour(-1) (n = 54), low-dose vecuronium 0.4 mg kg(-1) /0.2 mg kg(-1) hour(-1) (n = 29) and high-dose vecuronium 0.6 mg kg(-1) /0.3 mg kg(-1) hour(-1) (n = 33). RESULTS: The majority of cardiovascular parameters demonstrated no significant differences between groups. For heart rate, there was an overall group difference, p = 0.036. Dromotropy was low in the pancuronium group, with an increased normalised PR-interval compared to the high-dose vecuronium group, median 0.200 interquartile range (0.190, 0.215) versus 0.182 (0.166, 0.199), p < 0.05. Left ventricular compliance was increased in pancuronium-treated animals, demonstrated as a reduction in the nonlinear end-diastolic pressure volume relationship ß compared to both vecuronium groups, 0.021 (0.016, 0.025) versus 0.031 (0.025, 0.046) and 0.031 (0.022, 0.048), p < 0.05. The linear end-diastolic pressure volume relationship EDPVR(lin) was reduced as well in the pancuronium group, compared to the low-dose vecuronium group, 0.131 (0.116, 0.169) versus 0.181 (0.148, 0.247), p < 0.05. CONCLUSIONS: There are only minor haemodynamic differences when using pancuronium compared to vecuronium in the fentanyl-pentobarbital-midazolam-N(2)O anesthetised domestic pigs. Furthermore, increasing doses of vecuronium have minimal haemodynamic effects. CLINICAL RELEVANCE: Experimental studies in pigs using either pancuronium or vecuronium as a neuromuscular blocking agent are comparable with regard to cardiac and haemodynamic performance.

The comparison of spontaneous breathing and muscle paralysis in two different severities of experimental lung injury.

OBJECTIVES: The benefits of spontaneous breathing over muscle paralysis have been proven mainly in mild lung injury; no one has yet evaluated the effects of spontaneous breathing in severe lung injury. We investigated the effects of spontaneous breathing in two different severities of lung injury compared with muscle paralysis. DESIGN: Prospective, randomized, animal study. SETTING: University animal research laboratory. SUBJECTS: Twenty-eight New Zealand white rabbits. INTERVENTIONS: Rabbits were randomly divided into the mild lung injury (surfactant depletion) group or severe lung injury (surfactant depletion followed by injurious mechanical ventilation) group and ventilated with 4-hr low tidal volume ventilation with spontaneous breathing or without spontaneous breathing (prevented by a neuromuscular blocking agent). Inspiratory pressure was adjusted to control tidal volume to 5-7 mL/kg, maintaining a plateau pressure less than 30 cm H2O. Dynamic CT was used to evaluate changes in lung aeration and the regional distribution of tidal volume. MEASUREMENTS AND RESULTS: In mild lung injury, spontaneous breathing improved oxygenation and lung aeration by redistribution of tidal volume to dependent lung regions. However, in severe lung injury, spontaneous breathing caused a significant increase in atelectasis with cyclic collapse. Because of the severity of lung injury, this group had higher plateau pressure and more excessive spontaneous breathing effort, resulting in the highest transpulmonary pressure and the highest driving pressure. Although no improvements in lung aeration were observed, muscle paralysis with severe lung injury resulted in better oxygenation, more even tidal ventilation, and less histological lung injury. CONCLUSIONS: In animals with mild lung injury, spontaneous breathing was beneficial to lung recruitment; however, in animals with severe lung injury, spontaneous breathing could worsen lung injury, and muscle paralysis might be more protective for injured lungs by preventing injuriously high transpulmonary pressure and high driving pressure.

Allosteric inhibition of muscle-type nicotinic acetylcholine receptors by a neuromuscular blocking agent pancuronium.

Muscle relaxants are indispensable for surgical anesthesia. Early studies suggested that a classical non-depolarizing muscle relaxant pancuronium competitively binds to the ligand binding site to block nicotinic acetylcholine receptors (nAChR). Our group recently showed that nAChR which has two distinct subunit combinations are expressed in zebrafish muscles, αßδε and αßδ, for which potencies of pancuronium are different. Taking advantage of the distinct potencies, we generated chimeras between two types of nAChRs and found that the extracellular ACh binding site is not associated with the pancuronium sensitivity. Furthermore, application of either 2 µM or 100 µM ACh in native αßδε or αßδ subunits yielded similar IC50 of pancuronium. These data suggest that pancuronium allosterically inhibits the activity of zebrafish nAChRs.

Effect of preoperative ivabradine on hemodynamics during elective off-pump CABG.

Background: Ivabradine is a specific heart rate (HR)-lowering agent which blocks the cardiac pacemaker If channels. It reduces the HR without causing a negative inotropic or lusitropic effect, thus preserving ventricular contractility. The authors hypothesized that its usefulness in lowering HR can be utilized in patients undergoing off-pump coronary artery bypass (OPCAB) surgery. Objective: To study the effects of preoperative ivabradine on hemodynamics (during surgery) in patients undergoing elective OPCAB surgery. Methods: Fifty patients, New York Heart Association (NYHA) class I and II, were randomized into group I (control, n = 25) and group II (ivabradine group, n = 25). In group I, patients received the usual anti-anginal medications in the preoperative period, as per the institutional protocol. In group II, patients received ivabradine 5 mg twice daily for 3 days before surgery, in addition to the usual anti-anginal medications. Anesthesia was induced with fentanyl, thiopentone sodium, and pancuronium bromide as a muscle relaxant and maintained with fentanyl, midazolam, pancuronium bromide, and isoflurane. The hemodynamic parameters [HR and mean arterial pressure (MAP)] and pulmonary artery (PA) catheter-derived data were recorded at the baseline (before induction), 3 min after the induction of anesthesia at 1 min and 3 min after intubation and at 5 min and 30 min after protamine administration. Intraoperatively, hemodynamic data (HR and MAP) were recorded every 10 min, except during distal anastomosis of the coronary arteries when it was recorded every 5 min. Post-operatively, at 24 hours, the levels of troponin T and brain natriuretic peptide (BNP) were measured. This trial's CTRI registration number is CTRI/005858. Results: The HR in group II was lower when compared to group I (range 59.6-72.4 beats/min and 65.8-80.2 beats/min, respectively) throughout the study period. MAP was comparable [range (78.5-87.8 mm Hg) vs. (78.9-88.5 mm Hg) in group II vs. group I, respectively] throughout the study period. Intraoperatively, 5 patients received metoprolol in group I to control the HR, whereas none of the patients in group II required metoprolol. The incidence of preoperative bradycardia (HR <60 beats/min) was higher in group II (20%) vs. group I (8%). There was no difference in both the groups in terms of troponin T and BNP level after 24 hours, time to extubation, requirement of inotropes, incidence of arrhythmias, in-hospital morbidity, and 30-day mortality. Conclusion: Ivabradine can be safely used along with other anti-anginal agents during the preoperative period in patients undergoing OPCAB surgery. It helps to maintain a lower HR during surgery and reduces the need for beta-blockers in the intraoperative period, a desirable and beneficial effect in situations where the use of beta-blockers may be potentially harmful. Further studies are needed to evaluate the beneficial effects of perioperative Ivabradine in patients with moderate-to-severe left ventricular dysfunction.

Adenosine A(2A) receptor antagonists are broad facilitators of antinicotinic neuromuscular blockade monitored either with 2 Hz train-of-four or 50 Hz tetanic stimuli.

1. The 2 Hz train-of-four ratio (TOF(ratio)) is used to monitor the degree of patient curarization. Using a rat phrenic nerve-hemidiaphragm preparation, we showed that antinicotinic agents, such as hexamethonium, d-tubocurarine and pancuronium, but not cisatracurium, decreased contractions produced by physiological nerve activity patterns (50 Hz) more efficiently than those caused by 2 Hz trains. Uncertainty about the usefulness of the TOF(ratio) to control safe recovery from curarization prompted us to investigate the muscarinic and adenosine neuromodulation of tetanic (50 Hz) fade induced by antinicotinic agents at concentrations that cause a 25% reduction in the TOF(ratio) (TOF(fade)). 2. Tetanic fade caused by d-tubocurarine (1.1 µmol/L), pancuronium (3 µmol/L) and hexamethonium (5.47 mmol/L) was attenuated by blocking presynaptic inhibitory muscarinic M(2) and adenosine A(1) receptors with methoctramine (1 µmol/L) and 1,3-dipropyl-8-cyclopentylxanthine (2.5 nmol/L), respectively. These compounds enhanced rather than decreased tetanic fade induced by cisatracurium (2.2 µmol/L), but they consistently attenuated cisatracurium-induced TOF(fade). The effect of the M(1) receptor antagonist pirenzepine (10 nmol/L) on fade produced by antinicotinic agents at 50 Hz was opposite to that observed with TOF stimulation. Blockade of adenosine A(2A) receptors with ZM 241385 (10 nmol/L) attenuated TOF(fade) caused by all antinicotinic drugs tested, with the exception of the 'pure' presynaptic nicotinic antagonist hexamethonium. ZM 241385 was the only compound tested in this series that facilitated recovery from tetanic fade produced by cisatracurium. 3. The data suggest that distinct antinicotinic relaxants interfere with fine-tuning neuromuscular adaptations to motor nerve stimulation patterns via activation of presynaptic muscarinic and adenosine receptors. These results support the use of A(2A) receptor antagonists together with atropine to facilitate recovery from antinicotinic neuromuscular blockade.

Pancuronium dose refinement in experimental pigs used in cardiovascular research.

OBJECTIVE: To quantify the dose of pancuronium required to obtain moderate neuromuscular blockade as monitored by acceleromyography (NMB(mod) : train-of-four count of ≤2) as a part of a balanced anaesthetic protocol in pigs used in cardiovascular research. STUDY DESIGN: Prospective cross-sectional study. ANIMALS: Five pigs (median body weight: 60 (range 60-63) kg). METHODS: Anaesthesia was induced with xylazine, ketamine, atropine and midazolam and maintained with isoflurane in O(2) :air and fentanyl. Pigs received 0.1 mg kg(-1) pancuronium initial bolus to reach NMB(mod) followed by 0.1 mg kg(-1) hour(-1) constant rate infusion (CRI). During anaesthesia a twitch count of 3 or measureable T4/T1 ratio indicated unsatisfactory NMB. In this case additional 0.4 mg boluses of pancuronium were administered IV to effect in addition to the CRI. Descriptive statistical analysis was performed to express the median and range of the bolus and CRI dose of pancuronium in pigs. Cardiovascular parameters were analyzed at selected time points with Friedman Repeated Measures Analysis on Ranks. Spearman Rank test was used to evaluate correlation between parameters. RESULTS: Acceleromyographic monitoring of NMB is feasible in anaesthetized pigs. The median initial dose and rate of pancuronium required to achieve NMB(mod) were 0.10 (range 0.10-0.13) mg kg(-1) and 0.11 (range 0.10-0.21) mg kg(-1) hour(-1) , respectively. The administration rate showed considerable individual variation. CONCLUSIONS AND CLINICAL RELEVANCE: These pancuronium doses can be used as a guideline to achieve NMB(mod) in pigs as part of a balanced anaesthetic protocol. Instrumental NMB monitoring is essential because of individual kinetic variations and compliance to monitoring guidelines.

Variability in the muscle composition of rat esophagus and neural pathway of lower esophageal sphincter relaxation.

Several studies from our laboratory show that axial stretch of the lower esophageal sphincter (LES) in an oral direction causes neurally mediated LES relaxation. Under physiological conditions, axial stretch of the LES is caused by longitudinal muscle contraction (LMC) of the esophagus. Because longitudinal muscle is composed of skeletal muscle in mice, vagal-induced LMC and LES relaxation are both blocked by pancuronium. We conducted studies in rats (thought to have skeletal muscle esophagus) to determine if vagus nerve-mediated LES relaxation is also blocked by pancuronium. LMC-mediated axial stretch on the LES was monitored using piezoelectric crystals. LES and esophageal pressures were monitored with a 2.5-Fr solid-state pressure transducer catheter. Following bilateral cervical vagotomy, the vagus nerve was stimulated electrically. LES, along with the esophagus, was harvested after in vivo experiments and immunostained for smooth muscle (smooth muscle α-actin) and skeletal muscle (fast myosin heavy chain). Vagus nerve-stimulated LES relaxation and esophageal LMC were reduced in a dose-dependent fashion and completely abolished by pancuronium (96 µg/kg) in six rats (group 1). On the other hand, in seven rats, LES relaxation and LMC were only blocked completely by a combination of pancuronium (group 2) and hexamethonium. Immunostaining revealed that the longitudinal muscle layer was composed of predominantly skeletal muscle in the group 1 rats. On the other hand, the longitudinal muscle layer of group 2 rats contained a significant amount of smooth muscle (P < 0.05). Our study shows tight coupling between axial stretch on the LES and relaxation of the LES, which suggests a cause and effect relationship between the two. We propose that the vagus nerve fibers that cause LMC induce LES relaxation through the stretch-sensitive activation of inhibitory motor neurons.

Effects of common anesthetics on eye movement and electroretinogram.

High-resolution magnetic resonance imaging (MRI) provides non-invasive images of retinal anatomy, physiology, and function with depth-resolved laminar resolution. Eye movement and drift, however, could limit high spatial resolution imaging, and anesthetics that minimize eye movement could significantly attenuate retinal function. The aim of this study was to determine the optimal anesthetic preparations to minimize eye movement and maximize visual-evoked retinal response in rats. Eye movements were examined by imaging of the cornea with a charge-coupled device (CCD) camera under isoflurane, urethane, ketamine/xylazine, and propofol anesthesia at typical dosages in rats. Combination of the paralytic pancuronium bromide with isoflurane or ketamine/xylazine anesthesia was also examined for the eye movement studies. Visual-evoked retinal responses were evaluated using full-field electroretinography (ERG) under isoflurane, ketamine/xylazine, urethane, and ketamine/xylazine + pancuronium anesthesia in rats. The degree of eye movement, measured as displacement per unit time, was the smallest under 1% isoflurane + pancuronium anesthesia. The ketamine/xylazine groups showed larger dark-adapted ERG a- and b-waves than other anesthetics tested. The isoflurane group showed the shortest b-wave implicit times. Photopic ERGs in the ketamine/xylazine groups showed the largest b-waves with the isoflurane group showing slightly shorter implicit times at the higher flash intensities. Oscillatory potentials revealed an early peak in the isoflurane group compared with ketamine/xylazine and urethane groups. Pancuronium did not affect the a- and b-wave, but did increase oscillatory potential amplitudes. Compared with the other anesthetics tested here, ketamine/xylazine + pancuronium was the best combination to minimize eye movement and maximize retinal function. These findings should set the stage for further development and application of high-resolution functional imaging techniques, such as MRI, to study retinal anatomy, physiology, and function in anesthetized rats.

Presynaptic facilitatory adenosine A2A receptors mediate fade induced by neuromuscular relaxants that exhibit anticholinesterase activity.

1. Pancuronium, cisatracurium and vecuronium are antinicotinic agents that, in contrast with d-tubocurarine and hexamethonium, exhibit anticholinesterase activity. Pancuronium-, cisatracurium- and vecuronium-induced fade results from blockade of facilitatory nicotinic receptors on motor nerves, but fade produced by such agents also depends on the presynaptic activation of inhibitory muscarinic M2 receptors by acetylcholine released from motor nerve terminals and activation of inhibitory adenosine A1 receptors by adenosine released from motor nerves and muscles. The participation of presynaptic facilitatory A2A receptors in fade caused by pancuronium, cisatracurium and vecuronium has not yet been investigated. In the present study, we determined the effects of ZM241385, an antagonist of presynaptic facilitatory A2A receptors, on fade produced by these neuromuscular relaxants in the rat phrenic nerve-diaphragm (PND) preparation. 2. The muscles were stimulated indirectly at 75±3Hz to induce a sustained tetanizing muscular contraction. The lowest concentration at which each antinicotinic agent produced fade without modifying initial tetanic tension (presynaptic action) was determined. 3. d-Tubocurarine-induced fade occurred only at 55 nmol/L, a concentration that also reduced maximal tetanic tension (post-synaptic action). At 10 nmol/L, ZM 241385 alone did not produce fade, but it did attenuate pancuronium (0.32 µmol/L)-, cisatracurium (0.32 µmol/L)- and vecuronium (0.36 µmol/L)-induced fade. 4. The fade induced by the 'pure' antinicotinic agents d-tubocurarine (55 nmol/L) and hexamethonium (413 µmol/L) was not altered by 10 nmol/L ZM 241385, indicating that presynaptic adenosine A2A receptors play a significant role in the fade produced by antinicotinic agents when such agents have anticholinesterase activity.

Role of 5-HT1A receptors in control of lower urinary tract function in anesthetized rats.

The role of 5-hydroxytryptamine (5-HT) 1A (5-HT1A) receptors in lower urinary tract function was examined in urethane-anesthetized female Sprague-Dawley rats. Bladder pressure and the external urethral sphincter electromyogram (EUS EMG) activity were recorded during continuous-infusion transvesical cystometrograms (TV-CMGs) to allow voiding and during transurethral-CMGs (TU-CMGs) which prevented voiding and allowed recording of isovolumetric bladder contractions. 8-Hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT), a 5-HT1A receptor agonist, decreased volume threshold (VT) for initiating voiding and increased contraction amplitude (CA) during TU-CMGs but decreased CA during TV-CMGs. 8-OH-DPAT prolonged EUS bursting as well as the intrabursting silent periods (SP) during voiding. N-[2-[4-(2-methoxyphenyl)-1- piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamine trihydrochloride (WAY-100635), a 5-HT1A antagonist, increased VT, increased residual volume, markedly decreased voiding efficiency, decreased the amplitude of micturition contractions recorded under isovolumetric conditions, and decreased the SP of EUS bursting. These results indicate that activation of 5-HT1A receptors by endogenous 5-HT lowers the threshold for initiating reflex voiding and promotes voiding function by enhancing the duration of EUS relaxation, which should reduce urethral outlet resistance.

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.

Gallamine triethiodide (flaxedil): tetraethylammonium- and pancuronium-like effects in myelinated nerve fibers.

Gallamine triethiodide (Flaxedil) is commonly used as a neuromuscular blocking agent. Voltage-clamp studies show that gallamine also directly affects amphibian and mammalian myelinated nerve fibers. Externally, gallamine is about five times more potent than tetraethylammonium in blocking potassium conductance, where this is present, but has no effect on the sodium channel. Internal application slows sodium inactivation, which in addition is often incomplete. At positive potentials, gallamine can occlude sodium channels, thereby almost eliminating outward sodium currents.

Curare and pancuronium compared: effects on previously undepressed mammalian myoneural junctions.

Curare and pancuronium have multiple effects on previously undepressed rat diaphragm; these include depression of transmitter output and prolongation of the refractory period of prejunctional structures. The effect of curare on motor nerve terminals is greater than that of pancuronium. Both drugs depress postjunctional receptors; but curare, in addition, raises the threshold for the generation of muscle action potentials. In addition, these results raise questions about the validity of statistical methods used to calculate transmitter output.

Investigation of the relaxant effects of pancuronium, rocuronium, vecuronium and mivacurium on rat thoracic aorta.

BACKGROUND AND OBJECTIVE: Pancuronium, vecuronium, mivacurium and rocuronium are nondepolarizing neuromuscular blocking agents, which are competitive antagonists against acetylcholine at nicotinic receptors, and considered to have no direct actions on vascular smooth muscle. We aimed to investigate the relaxant effects and possible underlying mechanisms of these agents on isolated rat thoracic aorta. METHODS: The preparations were precontracted with prostaglandin F2alpha (10(-7) mol l(-1)) and pancuronium (10(-7)-10(-4) mol l(-1)), rocuronium (10(-7)-10(-4) mol l(-1)), vecuronium (10(-7)-10(-4) mol l(-1)) and mivacurium (10(-7)-10(-4) mol l(-1)) added at cumulative concentrations in the presence or absence of a prostaglandin synthesis inhibitor, indomethacin (10(-6) M), and a nitric oxide synthesis inhibitor, N(omega)-nitro-L-arginine methylester (3 x 10(-5)). The same protocol was applied to both endothelia (+) and endothelia (-) aortic rings. The preparations precontracted with prostaglandin F2alpha (10(-7) mol l(-1)) were stimulated with electrical field stimulation at a frequency of 10 Hz as square-wave pulses of 50 V (0.2 ms) in the presence of a noradrenaline reuptake inhibitor desipramine (10(-7) mol l(-1)) and a nonselective beta-blocker propranolol (10(-6) mol l(-1)). Drugs were added at ineffective concentration of 10(-7) mol l(-1). Tetrodotoxin (10(-7) mol l(-1)) was added to test whether the changes were dependent on the neuronal response. RESULTS: Pancuronium and rocuronium relaxed aortic rings precontracted by prostaglandin F2alpha in a dose-dependent manner, but vecuronium and mivacurium did not. The relaxation effect of pancuronium and rocuronium was endothelium independent because there was not a significant response difference from the endothelium-denuded group. CONCLUSION: In conclusion, their relaxation effect may be due to an increase in prostaglandin synthesis. The increased relaxation effect of these agents at electrical field stimulation may be by the decreasing effect of noradrenaline reuptake from nerve endings because a noradrenaline reuptake inhibitor desipramine did not change this effect. Also, these neuromuscular agents may affect beta-receptors, because a nonselective beta-blocker agent, propranolol, decreased their electrical field stimulation-induced relaxations.

Presynaptic M1, M2, and A1 receptors play roles in tetanic fade induced by pancuronium or cisatracurium.

PURPOSE: We investigated whether presynaptic facilitatory M1 and/or inhibitory M2 muscarinic receptors contributed to pancuronium- and cisatracurium-induced tetanic fade. METHODS: Phrenic nerve-diaphragm muscle preparations of rats were indirectly stimulated with tetanic frequency (75 +/- 3.3 Hz; mean +/- SD). Doses of pancuronium, cisatracurium, hexamethonium, and d-tubocurarine for producing approximately 25% fade were determined. The effects of pirenzepine and methoctramine, blockers of presynaptic M1 and M2 receptors, respectively, on the tetanic fade were investigated. RESULTS: The concentrations required for approximately 25% fade were 413 microM for hexamethonium (26.8 +/- 2.4% 4% fade), 55 nM for d-tubocurarine (28.7 +/- 2.55% fade), 0.32 microM for pancuronium (25.4 +/- 2.2% fade), and 0.32 microM for cisatracurium (24.7 +/- 0.8% fade). Pirenzepine or methoctramine alone did not produce the fade. Methoctramine, 1 microM, attenuated the fade induced by hexamethonium (to 16.0 +/- 2.5% fade), d-tubocurarine (to 6.0 +/- 1.6 fade), pancuronium (to 8.0 +/- 4.0% fade), and cisatracurium (to 11.0 +/- 3.3% fade). 10 nM pirenzepine attenuated only the fades produced by pancuronium (to 5.0 +/- 0.11% fade) and cisatracurium (to 13.3 +/- 5.3% fade). Cisatracurium (0.32 microM) showed antiacetylcholinesterase activity (in plasma, 14.2 +/- 1.6%; 6%; in erythrocyt 17.2 +/- 2.66%) similar to that of pancuronium (0.32 microM). The selective A1 receptor blocker, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 2.5 nM), also attenuated the fades induced by pancuronium and cisatracurium. CONCLUSION: The tetanic fades produced by pancuronium and cisatracurium depend on the activation of presynaptic inhibitory M2 receptors; these agents also have anticholinesterase activities. The fades induced by these agents also depend on the activation of presynaptic inhibitory A1 receptors through the activation of stimulatory M1 receptors by acetylcholine.

Deep Tissue Incision Enhances Spinal Dorsal Horn Neuron Activity During Static Isometric Muscle Contraction in Rats.

Translational correlates to pain with activities after deep tissue injury have been rarely studied. We hypothesized that deep tissue incision causes greater activation of nociception-transmitting neurons evoked by muscle contraction. In vivo neuronal activity was recorded in 203 dorsal horn neurons (DHNs) from 97 rats after sham, skin-only, or skin + deep muscle incision. We evaluated DHN responses to static, isometric muscle contractions induced by direct electrical stimulation of the muscle. The effect of pancuronium on DHN response to contractions was also examined. Approximately 50% of DHNs with receptive fields in the hindpaw were excited during muscle contraction. One-second .5- and 1.0-g muscle contractions produced greater DHN activity after skin + deep muscle incision (median [interquartile range], 32 [5-39] impulses, P = .021; and 36 [26-46] impulses, P = .006, respectively) than after sham (6 [0-21] and 15 [8-32] impulses, respectively). Neuromuscular blockade with pancuronium inhibited the muscle contractions and DHN activation during electrical stimulation, demonstrating contraction-induced activation. The greater response of spinal DHNs to static muscle contraction after skin + deep muscle incision may model and inform mechanisms of dynamic pain after surgery. PERSPECTIVE: Completion of various activities is an important milestone for recovery and hospital discharge after surgery. Skin + deep muscle incision caused greater activation of nociception-transmitting DHNs evoked by muscle contraction compared with skin-only incision. This result suggests an important contribution of deep muscle injury to activity-evoked hyperalgesia after surgery.

The kinetics of competitive antagonism of nicotinic acetylcholine receptors at physiological temperature.

Detailed information about the ligand-binding site of nicotinic acetylcholine receptors has emerged from structural and mutagenesis experiments. However, these approaches provide only static images of ligand-receptor interactions. Kinetic measurements of changes in protein function are needed to develop a more dynamic picture. Previously, we measured association and dissociation rate constants for competitive inhibition of current through embryonic muscle acetylcholine receptor channels at 25 degrees C. Little is known about competitive antagonism at physiological temperatures. Here, we performed measurements at 37 degrees C and used thermodynamics to estimate the energetics of antagonism. We used rapid solution exchange protocols to determine equilibrium and kinetics of inhibition of acetylcholine-activated currents in outside-out patches by (+)-tubocurarine, pancuronium and cisatracurium. Kinetic rates as high as 600 s(-1) were resolved by this technique. Binding was primarily enthalpy driven. The 12 degrees C increase in temperature decreased equilibrium antagonist binding by 1.7- to 1.9-fold. In contrast, association and dissociation rate constants increased 1.9- to 6.0-fold. Activation energies for dissociation were 90 +/- 6, 106 +/- 8 and 116 +/- 10 kJ mol(-1) for cisatracurium, (+)-tubocurarine and pancuronium, respectively. The corresponding apparent activation energies for association were 38 +/- 6, 85 +/- 6 and 107 +/- 13 kJ mol(-1). The higher activation energy for association of (+)-tubocurarine and pancuronium compared with cisatracurium is notable. This may arise from either a more superficial binding site for the large antagonist cisatracurium compared to the other ligands, or from a change in receptor conformation upon binding of (+)-tubocurarine and pancuronium but not cisatracurium. Differences in ligand desolvation and ligand conformation are not likely to be important.

Dose-response effect of serum butyrylcholinesterase activity after clinical doses of pancuronium.

INTRODUCTION: Low-dose pancuronium is known to affect serum cholinesterase activity (BChE); however, the dose-response effect of clinical doses of pancuronium on BChE has not been investigated. METHODS: Thirteen ASA I-II patients scheduled for elective surgery requiring muscle relaxation were enrolled in this study. All patients had normal BChE before surgery. Incremental doses of pancuronium (10, 20, 50, and 100 microg/kg) were injected in accordance with surgical needs every 45 min. BChE was measured 3 min after injection by an automatic colorimetric method. RESULTS: BChE decreased significantly in all except one patient in comparison to the baseline (P < 0.05). However all values remained within normal clinical range. A dose of 100 microg/kg yielded significant decrease in comparison to 10 microg/kg but not to other dosages. Linear regression was not significant for the dose-response relationship (P = 0.05). CONCLUSION: After clinical incremental doses of pancuronium, BChE remained within clinical range.

Allosteric interaction of the neuromuscular blockers vecuronium and pancuronium with recombinant human muscarinic M2 receptors.

Neuromuscular blocking drugs produce muscle weakness by interaction with nicotinic-acetylcholine receptors. Cardiovascular side effects have been reported. In this study the neuromuscular blocking drug vecuronium and the controls gallamine and pancuronium slowed the rate of atropine induced [(3)H]N-methylscopolamine dissociation from Chinese hamster ovary cells expressing recombinant human muscarinic M2 receptors K(off) values min(-1); vecuronium (125 nM), atropine 0.45+/-0.07+blocker 0.04+/-0.02; gallamine (21 nM), atropine 0.42+/-0.05+blocker 0.15+/-0.04; pancuronium(21 nM), atropine 0.36+/-0.03+blocker 0.03+/-0.01). These data indicate that vecuronium, gallamine and pancuronium interact with an allosteric site on the muscarinic M2 receptor (located on the heart) and this may explain some of their cardiac side effects.