Cannabinoid induced degranulation of rabbit neutrophils

We have examined the effects of various cannabinoids on the degranulation of rabbit peritoneal neutrophils. Several cannabinoids were found to cause a dose-dependent and noncytotoxic release of lysosomal enzymes from the neutrophils. The degranulation induced by cannabidiol is rapid ($\text{t}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 2.3 min}$), and enhanced by extracellular calcium and cytochalasin B. In addition to their intrinsic activity, cannabinoids also modulate the neutrophils' responses to the chemotactic peptide formyl-methionyl-leucyl-phenylalanine. This investigation represents the initial step toward the characterization of the effect of cannabinoids on the excitation-activation coupling sequence of hormonally responsive cells.     

Anti-inflammatory properties of cannabichromene

It was not known if Cannabichromene (CBC), which is a major constituent of drug types of $\text{Cannabis}$, has anti-inflammatory properties as do other cannabinoids. CBC was tested $\text{in vivo}$ using the rat paw edema test and $\text{in vitro}$ using the erythrocyte membrane stabilization assay. CBC was as effective as phenylbutazone (PBZ) at equivalent doses. Since CBC is less toxic than PBZ, larger doses may be given to produce a greater therapeutic effect.     

Cannabinoids stimulate and inhibit testosterone production in vitro and in vivo

The major psychoactive Δ⁹-tetrahydrocannabinol (THC) and the non-psychoactive cannabinol (CBN) of cannabidiol (CBD) can both stimulate and inhibit testicular testosterone (T) production $\text{in vitro}$ and $\text{in vivo}$. At nanomolar concentrations, these cannabinoids stimulate T production by decapsulated mouse testes while, in micromolar amounts, the effects are markedly inhibitory.     

The physiologic disposition of marihuana in man

Marihuana and hashish are the most widely used illicit drugs. They are derived from the hemp plant, $\text{Cannabis sativa}$. Among the diverse chemicals in the plant, more than 20 so-called cannabinoids have been isolated and their chemical structures elucidated (1). In 1965, Mechoulam and coworkers (2,3) isolated △⁹- tetrahydrocannabinol (△⁹-THC) from cannabis extract and demonstrated that it was responsible for the psychopharmacologic effects of cannabis in animals. Later, Isbell (4) and Hollister $\text{et al.}$ (5) confirmed these findings in man. This paper reviews the physiologic disposition of △⁹-THC in man; the disposition of △⁹-THC in animals has been reviewed elsewhere (6, 7, 8).     

Presence of peptide hormones that control gonadotropin secretion in extrahypothalamic areas of the brain.

The hypocholesterolemic drug clofibrate (ethyl-α-$\text{p}$-chlorophenoxyisobutyrate) was found to strongly suppress 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) activity in cultured mouse L cells at concentrations of 20 – 50 μg/ml. The half-life ($\text{t1}\text{2}$) of the reductase (approximately 120 min) was strongly reduced when L cells were incubated with cycloheximide plus a maximal inhibitory concentration of clofibrate (50 μg/ml), resulting in a $\text{t1}\text{2}$ value of 10 min. Preliminary kinetic analysis of the inhibition suggested that clofibrate increased the rate of inactivation (or degradation) of the reductase without affecting the rate of enzyme synthesis.     

Allylbenzene analogs of Δ9-tetrahydrocannabinol as tumor growth inhibitors

A series of substituted allylbenzene derivatives was tested $\text{in vitro}$ for ability to inhibit cell growth in the KB cell line. None of the compounds was as active as Δ⁹-tetrahydrocannabinol. It is suggested that inhibition of cell growth by cannabinoids requires a polycyclic system which may act by an intercalaction mechanism.     

Coliphage T7 receptors are present in Pseudomonasaeruginosa rough lipopolysaccharides

Lipopolysaccharides (LPS) isolated from various rough derivatives of $\text{Pseudomonas}\text{aeruginosa}$ strains were found to neutralize coliphage T7. Concentrations of 0.4 – 17 μg LPS/ml were sufficient for 50% inactivation of T7 within 1 hour. From the LPS analyses of the mutants, it is believed that T7 may be binding to the heptose region of $\text{P.}\text{aeruginosa}$ LPS, suggesting a similarity in structure between the heptose regions of $\text{P.}\text{aeruginosa}$ and $\text{Escherichia}\text{coli}$ LPS.     

Synthesis of α,ω-bis(diphenylphosphino)alkane and α,ω-bis(diphenylphosphino)(poly)ether ligands and complexes of rhodium(I)

α,ω-Bis(diphenylphosphino)alkane and α,ω-bis(diphenylphosphino)(poly)ether ligands can be prepared in very high yields via reaction of the appropriate dihalide with two equivalents of LiPPh₂. For the [Rh(COD)($\text{P P}$)][ClO₄] complexes of these ligands, the $\text{P P}$ ligands with five or less atoms in the alkane or ether bridge form monomeric complexes via η²-coordination. In general the ligands with eight or more atoms in the bridge give di- or polynuclear species. In addition the long chain diphosphino-polyethers form – to a small extent – monomeric species by η²-coordination.     

Binding of scorpion and sea anemone neurotoxins to a common site related to the action potential Na+ ionophore in neuroblastoma cells.

The protein neurotoxin II from the venom of the scorpion $\text{Androctonus}\text{australis}$ Hector was labeled with ¹²⁵I by the lactoperoxidase method to a specific radioactivity of about 100 μCi/μg without loss of biological activity. The labeled neurotoxin binds specifically to a single class of non intereacting binding sites of high affinity (K_D = 0.3 – 0.6 nM) and low capacity (4000 – 8000 sites/cell) to electrically excitable neuroblastoma cells. Relation of these sites to the action potential Na⁺ channel is derived from identical concentration dependence of scorpion toxin binding and increase in duration and amplitude of action potential. The protein neurotoxin II from the sea anemone $\text{Anemona sulcata}$ also affects the closing of the action potential Na⁺ ionophore in nerve axons. The unlabelled sea anemone toxin modifies ¹²⁵I-labeled scorpion toxin II binding to neuroblastoma cells by increasing the apparent K_D for labeled scorpion toxin without modification of the number of binding sites. It is concluded that both $\text{Androctonus}$ scorpion toxin II and $\text{Anemona}$ sea anemone toxin II interact competitively with a regulatory component of the action potential Na⁺ channel.     

Circular dichroism study of 2'-5' dinucleoside monophosphates modified with N-2-acetylaminofluorene.

The conformational properties of four 2′ – 5′ dinucleoside monophosphates modified with $\text{N}$-2-acetylaminofluorene have been studied by circular dichroism spectroscopy. Covalent binding of this chemical carcinogen at the C₈ position of guanosine in the 2′ – 5′ dinucleoside monophosphates induces striking changes in their circular dichroic spectra depending on their base sequence and composition. The changes in CD spectra, redshift of the extrema and change of their polarity, not observed in the spectra of corresponding 3′ – 5′ derivatives modified with $\text{N}$-2-acetylaminofluorene are correlated with the difference in the configuration of 2′ – 5′ and 3′ – 5′ dinucleoside monophosphates and discussed in respect to the intramolecular stacking interactions.     

Inability of detect cyclic AMP in vegetative or sporulating cells or dormant spores of Bacillus megaterium

Cyclic AMP was not found in vegetative cells or sporulating cells or dormant spores of $\text{Bacillus}\text{megaterium}$ using an assay which would have detected an $\text{in}\text{vivo}$ concentration of 1 – 2 × 10^?9 M. Adenyl cyclase and cyclic AMP phosphodiesterase were also not detected in sonicates of vegetative or sporulating $\text{B.}\text{megaterium}$ cells.     

pH dependence of magnesium ion binding to prothrombin fragment 1 and gamma-carboxyglutamic acid-containing peptides via 25Mg NMR.

The binding of magnesium ions to two tripeptides, $\text{L}$-Arg-$\text{D}$-Gla-$\text{D}$-Gla-OMe and Z-$\text{L}$-Arg(NO₂)-$\text{D}$-Gla-$\text{D}$-Gla-OMe, and to bovine prothrombin fragment 1 as a function of pH has been monitored by ²⁵Mg NMR spectroscopy. Binding to the tripeptide was dependent on peptide ionizations occurring at pH 4.6 – 4.8. The pH dependence of magnesium ion binding to fragment 1 reveals two inflection points 4.2 may be attributed to the deprotonation of the third side chain carboxylic acid group of the double γ-carboxyglutamic acid sequence. The origin of the increased binding of magnesium ions to fragment 1 at pH values above 7 is unknown.     

Ethanol directly inhibits Δ5-3β-hydroxysteroid dehydrogenase-isomerase activity in rat testis interstitial cells

Acute ethanol exposure has been demonstrated to inhibit testosterone synthesis both $\text{in vivo}$ and $\text{in vitro}$; however, the precise step(s) affected is controversial. Using intact collagenase-dispersed interstitial cells or 10,000xg supernatants of interstitial cell homogenates, studies were undertaken to determine whether ethanol specifically inhibited Δ⁵-3β-hydroxysteroid dehydrogenase-isomerase activity. In both cellular preparations, varing concentrations of ethanol (2.2 – 652 mM) inhibited this enzyme activity. Because alcohol dehydrogenase activity was identified specifically in Leydig cells and because the inhibition of Δ⁵-3β-hydroxysteroid dehydrogenase-isomerase activity by concentrations of ethanol normally observed in circulation of alcoholic men (2.2 – 65 mM) could be reversed by saturating concentrations of NAD⁺ (0.2 mM) or by 4-methylpyrazole (2 mM), these results suggest that the mechanism of this inhibition is by limitation of available cofactor.     

RHC 80267 inhibits thyrothopin-stimulated prostaglandin release from rat thyroid lobes

In the present report, we studied the effect of the diglyceride (DG) lipase inhibitor, RHC 80267 on basal and thyrotropin (TSH) - stimulated prostaglandin (PG) release from rat thyroid lobes Further, we tested the effect of RHC 80267 on phosphatidylinositol phospholipase C (PIPLC), DG lipase, and arachidonate cyclo-oxygenase acdtivities in rat thyroid cytosol, plasma membrane, and whole homogenate preparations, r espectively. Whereas RHC 80267 inhibited DG lipase activity in a dose - re;ated manner from 0.5 – 10 μM (17 – 80% inhibition), it failed either PIPLC or arachidonate cyclo-oxygenase activities by more than 9% when tested at 5 and 10 μM (n = 3). RHC 80267 reduced TSH-stimulated 6-keto-PGF_1α and PGE_2α relase by 100 ± 14% and 57 ± 12%, respectively 9$\text{x}\text{+}\text{S.E.}\text{;}\text{p}\text{< 0.01}$ for both; n = 10 – 12; the diglyceride lipase inhibitor did not reduce basal release of either PG. These data provide additional evidence which implicate a PIPLC - DG lipase pathway in TSH-stimulated PG synthesis in thyroid.     

The effect of embryo quality at freezing on subsequent development of thawed cow embryos

Day 7 to 9 embryos were frozen by a rapid two-step method to ?38°C before being plunged into liquid nitrogen. Glycerol was used as the cryoprotectant and, following thawing, the embryos were cultured for 12 – 24 hours in PBS + 15% heat-treated steer serum. In Experiment 1, embryos were frozen in 2.0 ml glass ampoules or 0.5 ml Cassou straws. Two levels of glycerol (1.0M and 1.4M) gave comparable $\text{in vitro}$ survival rates ($\text{12}\text{20}$ and $\text{13}\text{25}$, respectively). A greater proportion of embryos developed in culture after freezing in straws. In Experiment 2, embryos were classified morphologically before and after freezing into 5 grades (1 = excellent; 2 = good; 3 = fair; 4 = poor; 5 = degenerate). Only embryos of grade 1, 2 and 3 were frozen. The post-thaw survival rates for embryos graded 1, 2 and 3 before freezing were 100% ($\text{11}\text{11}$), 86% ($\text{24}\text{28}$) and 83% ($\text{20}\text{24}$), respectively. Furthermore, the porportion of surviving embryos estimated to be of poor quality (grade 4) was greater for embryos graded 3 before freezing ($\text{13}\text{20}$) than for embryos graded 2 ($\text{6}\text{24}$) or 1 ($\text{1}\text{11}$). The percentage of embryos which developed normally after $\text{in vitro}$ culture for each of the pre-freezing grades 1, 2 and 3 was 91% ($\text{10}\text{11}$), 50% ($\text{14}\text{28}$) and 29% ($\text{7}\text{24}$), respectively. Of the total number of frozen-thawed embryos which developed in culture, $\text{5}\text{31}$ (16%) were of poor quality. The proportion of poor quality developing embryos was greater inembryos graded 3 before freezing ($\text{3}\text{7}$) than those graded 2 ($\text{2}\text{14}$). All of the embryos graded 1 before freezing and which developed in culture were of good quality. Results indicate that, if high post-thaw survival rates are to be obtained, stringent embryo selection processes will be required.     

Further studies on the pharmacology of a false cholinergic transmitter, (2-hydroxyethyl) methyldiethylammonium (diethylcholine).

The pharmacology of a possible false cholinergic transmitter, (2-hydroxyethyl) methyldiethylammonium (diethylcholine, DEC) was studied with various preparations. It was found to inhibit the neuromuscular transmission of frog sciatic nerve-gastrocnemius muscle $\text{in}$$\text{vitro}$ with ED₅₀ of 1.93 (0.66 - 5.79) × 10⁻⁴ M. DEC was also found to inhibit dog chorda tympani-Wharton's duct (postganglionic parasympathetic neuro-effector junction) and cat superior cervical ganglionnictitating membrane (sympathetic ganglion) preparations $\text{in}$$\text{vivo}$ with ED₅₀'s of 6.2 (1.8 – 21.1) mg/kg and 12.0 (5.7 - 25.2) mg/kg, respectively. After blockade of these preparations with DEC, the former was still responsive to intravenous injection of pilocarpine (1 mg/kg) and choline (10 mg/kg) and the latter to close arterial injection of acetylcholine (100 μg/injection) and choline (3 mg/min infusion). These results support the idea that DEC paralyzes cholinergic neurons possibly through false cholinergic transmission without blocking the cholinergic receptor at the post-junctional membrane.     

Metabolites of estradiol-17β in guinea pig uterus late in pregnancy

The metabolism of estradiol-17β by the guinea pig uterus late in pregnancy was studied $\text{in vivo}$ and $\text{in vitro}$.Whole uteri were examined for estrogen metabolites one hour following an intravenous injection of [³H]-estradiol-17β or uterine sections were examined after incubation for one hour at 37°C in medium containing [³H]-estradiol-17β.In both instances uterine tissue metabolized estradiol-17g to five products: estrone, estrone-3-sulfate, 17β-estradiol-3-sulfate, estrone-3-glucuronide and 17β-estradiol-3-glucuronide. Of the total radioactive products 11 – 43% were glucuronides, 17 – 26% were sulfates and 4 – 17% was estrone. These results indicate that the guinea pig uterus actively transforms estradiol-17β into glucuronides and sulfates late in pregnancy.     

Esterase activity of liver alcohol dehydrogenase.

Liver alcohol dehydrogenase is found to possess, in addition to its dehydrogenase and dismutase activities, the ability to hydrolyze octanoate esters at a rate approximately $\text{1}\text{500}\text{–}\text{1}\text{1000}$ of that of the dehydrogenase reaction. The esterase and dehydrogenase activities exhibit an identical isozyme pattern indicating that the same protein catalyzes both reactions. Inhibition studies suggest that the esterase activity presumably shares the catalytic domain with the dehydrogenase activity.     

Coordination of adenylate energy charge and phosphorylation state during ischemia and under physiological conditions in rat liver and kidney

The relation of the adenylate energy charge $\text{(}\text{ATP}\text{+}\text{1}\text{2}\text{ADP/ATP}\text{+}\text{ADP}\text{+}\text{AMP}\text{)}$ to the phosphorylation state (ATP)/(ADP)(HPO₄^2?) in rat liver and kidney was analyzed. Under physiological conditions and in ischemia, the two regulatory parameters, calculated from reported values for adenine nucleotides and inorganic phosphate (P_i) and from new observations, were closely coordinated. Energy charge was an inverse linear function of P_i and -log (1 - energy charge) was a positive linear function of log phosphorylation state. To evaluate experimental data with known energy charge, but unknown P_i, and to determine the theoretical relation between energy charge and phosphorylation state, P_i was estimated from a) the regression equation: P_i, μmol/g wet wt tissue = 1.05 - energy charge/0.073 and b) the empirical relationship: (P_i/2P_a) + energy charge = k, where P_a = σAMP + 2ADP + 3ATP and k = 1. With both estimates, the relation between phosphorylation state and energy charge for the experimental data was, within error, the same as that observed with measured P_i and concordant with theoretical values. Over the physiological range of energy charge (～0.85 – 0.95, log phosphorylation state ～3.3 – 4.3), apparent ΔG_ATP (×2) was closer to the range of ΔG observed by Wilson $\text{et al}$ (Biochem. J. $\text{140}$:57, 1974) for transfer of two electrons from mitochondrial NAD to the cytochrome c couple than the ΔG_ATP (×2) they reported, supporting their conclusion that near-equilibrium exists between the mitochondrial respiratory chain and the cytoplasmic phosphorylation state under physiological conditions. From evidence presented, it is postulated that the phosphorylation state is regulated by the adenylate energy charge.     

Localization of Cu and heme a on cytochrome c oxidase polypeptides.

After mild dissociation of cytochrome $\text{c}$ oxidase protomers, and polyacrylamide gel electrophoresis, copper was found predominantly in polypeptides of Bands V (m.w. 12,100) and VII (m.w. 3,400), and heme a predominantly in polypeptides of Bands I (m.w. 35,300) and II (m.w. 21,000). Some copper was found in Band II – III, and heme a in Band V.