The endogenous cannabinoid anandamide inhibits cromakalim-activated K+ currents in follicle-enclosed Xenopus oocytes.

The effect of the endogenous cannabinoid anandamide on K(+) currents activated by the ATP-sensitive potassium (K(ATP)) channel opener cromakalim was investigated in follicle-enclosed Xenopus oocytes using the two-electrode voltage-clamp technique. Anandamide (1-90 microM) reversibly inhibited cromakalim-induced K(+) currents, with an IC(50) value of 8.1 +/- 2 microM. Inhibition was noncompetitive and independent of membrane potential. Coapplication of anandamide with the cannabinoid type 1 (CB(1)) receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR 141716A) (1 microM), the CB(2) receptor antagonist N-[(1S)endo-1,3,3-trimethyl bicyclo heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528) (1 microM), or pertussis toxin (5 microg/ml) did not alter the inhibitory effect of anandamide, suggesting that known cannabinoid receptors are not involved in anandamide inhibition of K(+) currents. Similarly, neither the amidohydrolase inhibitor phenylmethylsulfonyl fluoride (0.2 mM) nor the cyclooxygenase inhibitor indomethacin (5 microM) affected anandamide inhibition of K(+) currents, suggesting that the effects of anandamide are not mediated by its metabolic products. In radioligand binding studies, anandamide inhibited the specific binding of the K(ATP) ligand [(3)H]glibenclamide in the oocyte microsomal fractions, with an IC(50) value of 6.3 +/- 0.4 microM. Gonadotropin-induced oocyte maturation and the cromakalim-acceleration of progesterone-induced oocyte maturation were significantly inhibited in the presence of 10 microM anandamide. Collectively, these results indicate that cromakalim-activated K(+) currents in follicular cells of Xenopus oocytes are modulated by anandamide via a cannabinoid receptor-independent mechanism and that the inhibition of these channels by anandamide alters the responsiveness of oocytes to gonadotropin and progesterone.

Local granulocyte-macrophage colony-stimulating factor improves incisional wound healing in adriamycin-treated rats.

PURPOSE: Neoadjuvant treatment is often given for locally advanced malignancies; however, clinical and experimental studies have shown that some chemotherapeutic agents impair wound healing. It has been reported that granulocyte-macrophage colony-stimulating factor (GM-CSF) applied locally improves dermal wound healing. Thus, we investigated the effects of locally injected GM-CSF on abdominal wounds impaired by adriamycin, a widely used chemotherapeutic agent. METHODS: We divided 120 female Sprague-Dawley rats into five treatment groups of 24 rats. Group 1 received saline 8 mg/kg intravenously (i.v.) + laparotomy 14 days later (control); group 2 received 8 mg/kg i.v. adriamycin + laparotomy 14 days later; group 3 received adriamycin 8 mg/kg i.v. + laparotomy + local GM-CSF 50 microg 14 days later; group 4 received saline 8 mg/kg i.v. + laparotomy + local GM-CSF 50 microg 14 days later; and group 5 received adriamycin 8 mg/kg i.v. + laparotomy + systemic GM-CSF 50 microg 14 days later. Sutures were removed on postoperative day (POD) 7 in all five groups, and the abdominal bursting pressures were measured and recorded. Tissue samples were taken from the incision line for histopathological evaluation and hydroxyproline content measurement. RESULTS: The bursting pressure was significantly lower in groups 2 and 5 than in groups 1, 3, and 4. The hydroxyproline content and histopathological findings supported this result. CONCLUSION: The local injection of GM-CSF improved impaired wound healing in adriamycin-treated rats.

Effects of saturated long-chain N-acylethanolamines on voltage-dependent Ca2+ fluxes in rabbit T-tubule membranes.

The effects of saturated long-chain (C: 16-22) N-acylethanolamines and a series of saturated fatty acids with the same length of carbon chains were investigated on depolarization-induced (45)Ca(2+) fluxes mediated by voltage-dependent Ca(2+) channels in transverse tubule membrane vesicles from rabbit skeletal muscle. Vesicles were loaded with (45)Ca(2+) and membrane potentials were generated by establishing potassium gradients across the vesicle using the ionophore valinomycin. Arachidonoylethanolamide and docosaenoylethanolamide but not palmitoylethanolamide and stearoylethanolamide (all 10 microM) caused a significant inhibition of depolarization-induced (45)Ca(2+) fluxes and specific binding of [(3)H]Isradipine to transverse tubule membranes. On the other hand, saturated fatty acids including palmitic, stearic, arachidic, and docosanoic acids (all 10 microM) were ineffective in functional and radioligand binding experiments. Additional experiments using endocannabinoid metabolites suggested that whereas ethanolamine and arachidic acids were ineffective, arachidonoylethanolamide inhibited Ca(2+) effluxes and specific binding of [(3)H]Isradipine. Further studies indicated that only those fatty acids containing ethanolamine as a head group and having a chain length of more than 18 carbons were effective in inhibiting depolarization-induced Ca(2+) effluxes and specific binding of [(3)H]Isradipine. In conclusion, results indicate that depending on the chain length and the head group of fatty acid, N-acylethanolamines have differential effects on the function of voltage-dependent Ca(2+) channels and on the specific binding of [(3)H]Isradipine in skeletal muscle membranes.

Differential effects of endogenous and synthetic cannabinoids on voltage-dependent calcium fluxes in rabbit T-tubule membranes: comparison with fatty acids.

The effects of cannabinoid receptor ligands including 2-arachidonoylglycerol, R-methanandamide, Delta9-THC (Delta9-tetrahydrocannabinol), WIN 55,212-2 [4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenylcarbonyl)-6H-pyrrolo[3,2,1ij]quinolin-6-one], CP 55,940 ([1alpha,2beta-(R)-5alpha]-(-)-5-(1,1-dimethyl)-2-[5-hydroxy-2-(3-hydroxypropyl) cyclohexyl-phenol]) and a series of fatty acids on depolarization-induced Ca2+ effluxes mediated by voltage-dependent Ca2+ channels were investigated comparatively in transverse tubule membrane vesicles from rabbit skeletal muscle. Vesicles were loaded with 45Ca2+ and membrane potentials were generated by establishing potassium gradients across the vesicle using the ionophore valinomycin. Endocannabinoids, 2-arachidonoylglycerol and R-methanandamide (all 10 microM), inhibited depolarization-induced Ca2+ effluxes and specific binding of [3H]PN 200-110 (isradipine) to transverse tubule membranes. On the other hand, synthetic cannabinoids, including CP 55,940, WIN 55,212-2, and Delta9-THC (all 10 microM), were ineffective. Additional experiments using endocannabinoid metabolites suggested that whereas ethanolamine and glycerol were ineffective, arachidonic acid inhibited Ca2+ effluxes and specific binding of [3H]PN 200-110. Further studies indicated that only those fatty acids containing two or more double bonds were effective in inhibiting depolarization-induced Ca2+ effluxes and specific binding of [3H]PN 200-110. These results indicate that endocannabinoids, but not synthetic cannabinoids, directly inhibit the function of voltage-dependent calcium channels (VDCCs) and modulate the specific binding of calcium channel ligands of the dihydropyridine (DHP) class.

Risk factors associated with postoperative pulmonary complications following oncological surgery.

The purpose of our study was to determine the incidence of different postoperative pulmonary complications (PPCs) and their associated risk factors in patients who have undergone various elective surgical procedures in an oncological surgery center. Ninety five adult patients were studied prospectively for one year period. For the study group, predictors of pulmonary complications of interest were determined as age, gender, body mass index, co morbid conditions (preexisting history of chronic obstructive pulmonary disease, asthma, bronchiectasis, restrictive lung disease), site and type of the operation, smoking history, The American Society of Anesthesiologists (ASA) physical status, physical examination and chest X- Ray findings, pulmonary function tests, type and duration of anesthesia, surgical incision site and length and presence of nasogastric tube suction. The PPC rate of our study group was 40% (38/95). Atelectasis and bronchospasm were the most frequently observed PPCs (13.7%) Among all the risk factors taken into consideration, only three were found to be significant independent predictors of pulmonary complications according to multivariate analysis as follows: incision location concerning abdomen (p= 0.008), duration of anesthesia per hour (p= 0.0001), values of FEV1 < 50% (p= 0.007). Our data revealed that the incidence of PPCs was high in our study group when compared to results of general population. Application of major resection surgeries for cancer patients can be an explanation for this result. Shortening the duration of surgery, avoiding general anaesthesia in selected group of patients may reduce the risk of PPCs.

Cocaine inhibits cromakalim-activated K+ currents in follicle-enclosed Xenopus oocytes.

The effect of cocaine on K+ currents activated by the KATP channel opener cromakalim was investigated in follicular cells of Xenopus oocytes. The results indicate that cocaine in the concentration range of 3-500 microM reversibly inhibits cromakalim-induced K+ currents. The IC50 value for cocaine was 96 microM. Inhibition of the cromakalim-activated K+ current by cocaine was noncompetitive and voltage independent. Pretreatment with the Ca2+ chelator BAPTA did not modify the cocaine-induced inhibition of cromakalim-induced K+ currents, suggesting that Ca2+-activated second messenger pathways are not involved in the actions of cocaine. Outward K+ currents activated by the application of 8-Br-cAMP or forskolin were also inhibited by cocaine. The EC50 and slope values for the activation of K+ currents by cromakalim were 184+/-19 microM and 1.14 in the absence of cocaine as compared to 191+/-23 microM and 1.03 in the presence of cocaine (300 microM). Cocaine also blocked K+ currents mediated through C-terminally deleted form of Kir6.2 (KirDeltaC26) in the absence of sulfonylurea receptor with an IC50 value of 87 microM, suggesting that cocaine interacts directly with the channel forming Kir6.2 subunit. Radioligand binding studies indicated that cocaine (100 microM) did not affect the binding characteristics of the KATP ligand, [3H]glibenclamide. These results demonstrate that cromakalim-activated K+ currents in follicular cells of Xenopus oocytes are modulated by cocaine.

Inhibition of cromakalim-activated K+ current by ethanol in follicle-enclosed Xenopus oocytes.

Ethanol has been reported to modulate arterial dilation and insulin secretion. ATP-inhibited K+ channels (K(ATP)) are reported to have regulatory roles during these events. In the present study, the effect of ethanol on K+ currents activated by the K(ATP) channel opener cromakalim was investigated in follicular cells of Xenopus oocytes. The results indicate that ethanol in the concentration range of 10-300 mM (approximately 0.06%-2% v/v) reversibly inhibits the cromakalim-induced K+ currents. The 50% of maximal ethanol effect was obtained at 53 mM. Inhibition of cromakalim-activated K+ current by ethanol was non-competitive. In oocytes treated with the Ca2+-chelator BAPTA, inhibition of cromakalim-induced K+ currents by ethanol was not altered, suggesting that Ca2+-activated second messenger pathways were not involved in the actions of ethanol. Similarly, currents activated by 8-Br-cAMP were also inhibited by ethanol, but the ethanol inhibition of 8-Br-cAMP-activated currents was significantly less than inhibition of cromakalim-activated currents by ethanol. These results indicate that cromakalim-activated K+ currents in follicular cells of Xenopus oocytes were modulated by ethanol.

A closed chamber method for performing biochemical experiments at accurate concentrations of volatile agents.

Experiments with volatile agents such as general anesthetics present difficulties in maintaining defined concentrations of these agents during in-vitro experimental conditions. In conventional filtration apparatuses, due to their partition between liquid and vapor phases (to open air or headspaces of the incubation vehicles), some degree of inaccuracy in calculated concentrations of these agents may occur in experiments using these types of chambers. In the present study, a method is described which permits the performance of biochemical experiments in a closed system in which the concentrations of a volatile agent, desflurane, in the liquid phase of the assay environment can be maintained constant for a relatively long time period.

Comparison of the effects of xenon and halothane on voltage-dependent Ca(2+) fluxes in rabbit T-tubule membranes.

The effects of xenon and halothane on depolarization-induced (45)Ca(2+) fluxes mediated by voltage-dependent Ca(2+) channels were investigated in transverse tubule membrane vesicles from rabbit skeletal muscle. Halothane, in the concentration range of 0.5-2 mM, caused a significant inhibition of (45)Ca(2+) fluxes. Xenon tested in the range of 60%-100% did not affect the (45)Ca(2+) fluxes. Radioligand binding studies indicated that xenon and halothane have different effects on the specific binding of [(3)H]Isradipine to transverse tubule membranes. Halothane caused a significant inhibition on the specific binding of [(3)H]Isradipine. In controls and in presence of 0.5 mM halothane, B(max) values were 26.9 pmole/mg and 15.1 pmole/mg, and K(D) values were 238 pM and 247 pM, respectively. On the other hand, there was no effect of xenon (60%-100%) on the characteristics of [(3)H]Isradipine binding. In conclusion, results indicate that xenon and halothane differ in their effects on the function of voltage-dependent Ca(2+) channels and on the specific binding of [(3)H]Isradipine in skeletal muscle membranes.

Effects of isoflurane on voltage-dependent calcium fluxes in rabbit T-tubule membranes: comparison with alcohols.

The effects of racemic (+/-) and (+)- and (-)-stereoisomers of isoflurane on depolarization-induced (45)Ca(2+) fluxes mediated by voltage-dependent Ca(2+) channels were investigated in transverse tubule membrane vesicles from rabbit skeletal muscle. In the concentration range 0.5 to 2 mM, (+/-)-isoflurane inhibited (45)Ca(2+) fluxes and functionally modulated the effects of the Ca(2+) channel antagonist nifedipine (1-10 microM). Isoflurane-induced inhibition of (45)Ca(2+) fluxes was not significantly affected by pretreatment with either pertussis toxin (5 microg/ml) or phorbol 12-myristate 13-acetate (50 nM). Further experiments indicated that there were no significant differences between (+)- and (-)-stereoisomers of isoflurane with respect to the extent of inhibition of (45)Ca(2+) fluxes. Radioligand binding studies indicated that racemic and (+)- and (-)-isoflurane were equally effective in displacing the specific binding of [(3)H]PN 200-110 to transverse tubule membranes. There were no apparent differences between the effects of (+)- and (-)-isoflurane on the characteristics of [(3)H]PN 200-110 binding. Although the concentrations of isoflurane for the inhibitions of (45)Ca(2+) fluxes and radioligand bindings were similar, the concentrations of n-alcohols required for the inhibition of (45)Ca(2+) fluxes were lower than those for the displacement of radioligand. Comparison of the data for the displacement of [(3)H]PN 200-110 binding and the inhibition of (45)Ca(2+) fluxes by isoflurane and by n-alcohols suggested that both isoflurane and n-alcohols may have more than a single binding site. In conclusion, results indicate that isoflurane, independent of intracellular Ca(2+) levels, nonstereospecifically inhibits the function of voltage-dependent Ca(2+) channels and this effect is mediated through multiple binding sites.