Background and Purpose: The primary cannabinoids, Δ9-tetrahydrocannabinol (Δ9-THC) and Δ8-tetrahydrocannabinol (Δ8-THC) are known to disturb the mitochondrial function and possess antitumor activities. These observations prompted us to investigate their effects on the mitochondrial O2 consumption in human oral cancer cells (Tu183). This epithelial cell line overexpresses bcl-2 and is highly resistant to anticancer drugs. Experimental Approach: A phosphorescence analyzer that measures the time-dependence of O2 concentration in cellular or mitochondrial suspensions was used for this purpose. Key Results: A rapid decline in the rate of respiration was observed when Δ9-THC or Δ8-THC was added to the cells. The inhibition was concentration-dependent, and Δ9-THC was the more potent of the two compounds. Anandamide (an endocannabinoid) was ineffective; suggesting the effects of Δ9-THC and Δ8-THC were not mediated by the cannabinoidreceptors. Inhibition of O2 consumption by cyanide confirmed the oxidations occurred in the mitochondrial respiratory chain. Δ9-THC inhibited the respiration of isolated mitochondria from beef heart. Conclusions and Implications: These results show the cannabinoids are potent inhibitors of Tu183 cellular respiration and are toxic to this highly malignant tumor.
A method previously described for measuring ACh in biological effluents has been simplified and extended for use with tissues. The tissue is homogenized in acetonitrile containing propionylcholine as the internal standard and after centrifugation the acetonitrile is removed by shaking with toluene. To the aqueous solution is added a solution of KIMZ to precipitate the quaternary compounds. The precipitate is dissolved in aqueous acetonitrile and then drawn through a small column of ion-exchange resin to convert the periodides of the quaternary compounds to chlorides which are then simultaneously pyrolysed and gas chromatographed. On the column the pyrolytic product of choline has a slower retention time than that of acetylcholine; under these circumstances the choline present in tissues does not obscure the measurement of acetylcholine. Specificity was demonstrated by several procedures including mass spectroscopy. The method can measure 25 ng (171 pmoles) of acetylcholine in extracts of brain, simply, and with high reproducibility. With the usual gas chromatograph, 16 samples can be run in a working day. The content of acetylcholine in rat brain was 26.4 nmol/g or almost precisely the values found with other gas chromatographic methods.The pyrolytic method was shown to be applicable to the detection of biologically interesting substances other than choline esters, including betaine, carnitine and the nonquaternary compound, y-aminobutyric acid, which is readily converted to a volatile compound (probably its methyl ester) when pyrolysed in the presence of tetramethylammonium hydroxide. Of additional general interest is the demonstration of the advantages of acetonitrile as a solvent for extracting water-soluble compounds from tissues. THE PYROLYSIS of acetylcholine to yield its demethylated product, a reaction first described for quaternary compounds by HOFMANN ( M a ; b), was combined with gas chromatography (SZILAGYI, SCHMIDT and GREEN, 1968) to yield a specific and sensitive method for measuring ACh in effluents from the guinea pig ileum (SCHMIDT, SZILAGYI, ALKON and GREEN, 1969; GREEN, SZILAGYI, SCHMIDT and ALKON, 1970a), rat phrenic nerve-diaphragm preparation (SCHMIDT, SZILAGYI, ALKON and GREEN, 1970; ALKON, SCHMIDT, GREEN and SZILAGYI, 1970) and rabbit vagus nerve-atrium preparation (GREEN, ALKON, SCHMIDT and SZILAGYI, 19706). The method has now been extended to the measurement of ACh in extracts of mammalian tissues and, in doing so, we have modified and simplified it. The method, previously presented in abstract form (SZILAGYI, 1970; GREEN, SZILAGYI and MARGOLIS, 1971), is fully described here.
A prejunctional mechanism involving an alpha 1-adrenergic receptor may exert control on the release of acetylcholine from parasympathetic nerve endings in the heart. To test this hypothesis in vivo, rats were prepared for electrical stimulation of the vagus nerves. Blood pressures and heart rates were monitored, and the animals were treated with alpha-agonists and alpha-antagonists. The alpha 1-selective agonist phenylephrine significantly attenuated vagally induced bradycardia in a dose-dependent fashion (ED50 = 19 micrograms/kg). This is consistent with the hypothesis that there is alpha-adrenergic inhibition of ACh release. In contrast, the alpha 2-selective agonist, BHT-920, caused no change in heart rate during vagal stimulation. The effects of phenylephrine to raise heart rate and blood pressure during vagal stimulation were blocked by the alpha 1-selective antagonist prazosin (ID50 approximately 1 microgram/kg) but not by the alpha 2-selective antagonists yohimbine and rauwolscine. This further supports an alpha 1 assignment to the prejunctional adrenergic receptor mechanism, which can regulate the release of acetylcholine from cardiac parasympathetic neurons.
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