Suppressive effect of delta-9-tetrahydrocannabinol on herpes simplex virus infectivity in vitro.

Delta-9-Tetrahydrocannabinol (THC) was found to reduce the infectivity of herpes simplex virus and was without effect against adenovirus type 2 or poliovirus. The effective THC concentration resulting in an 80% decrement in virus viability was dependent upon the presence or absence of serum in the incubation mixture, as a 5% serum concentration decreased the drug activity by approximately 50-fold. THC-mediated inactivation of herpes simplex virus was both time and dose dependent and did not result in virion disassembly or clumping. The THC-related effect was not influenced by the pH of the suspending medium, suggesting that the mechanism of inactivation differed from that associated with the thermal inactivation of the virus. Thus, the data suggest that THC preferentially reduces the infectivity of the enveloped herpes simplex virus, and that this activity is modulated by the presence of serum proteins.     

In vivo inhibition of enterocyte metabolism by delta 9-tetrahydrocannabinol

Mice were given 10 to 100 mg/kg by stomach tube of delta 9-tetrahydrocannabinol (THC) in a single dose or for 4 consecutive days. [3H]Thymidine or [3H]glucosamine was given 3 or 24 hr before sacrifice. Enterocytes were isolated, and the incorporation of radioactivity into the acid insoluble fraction was measured. THC significantly inhibits in a dose-related fashion (from 10 to 90%) in vivo enterocyte metabolism. This inhibition is found in all enterocytes whatever their position in the intestinal tract; it is also independent of the state of differentiation of enterocytes. After a single ingestion of THC, crypt cells which synthesize DNA incorporate 37 to 45% less thymidine, and villus cells, which synthesize important amounts of glycoproteins, incorporate 15 to 39% less glucosamine. After 4 days of THC administration, the inhibition of thymidine incorporation is even more significant (up to 88%).     

Hypothermic action of delta 9-tetrahydrocannabinol, 11-hydroxy-delta 9-tetrahydrocannabinol and 11-hydroxy-delta 8-tetrahydrocannabinol in mice

The hypothermic activity of Δ⁹-tetrahydrocannabinol (Δ⁹-THC), a metabolite, 11-hydroxy-Δ⁹-tetrahydrocannabinol (11-OH-Δ⁹-THC) and 11-hydroxy-Δ⁸-tetrahydrocannabinol (11-OH-Δ⁸-THC) has been determined in male mice maintained at an ambient temperature of 20 ± 1°C. The mean body temperature of mice that received 2, 4, 16 or 32 mg/kg, i. v., of a tetrahydrocannabinol was significantly lower than that of vehicle treated mice (p <0.05) within 2 minutes after drug administration. Dose-response relationships show the intrinsic activity of Δ⁹-THC to be significantly greater than that of 11-OH-Δ⁹-THC or 11-OH-Δ⁸-THC in this system (p <0.05). The data indicate that the hypothermic activity of Δ⁹-THC cannot be explained entirely by metabolism to 11-OH-Δ⁹-THC.     

Deficient in vitro and in vivo phagocytosis of apoptotic T cells by resident murine alveolar macrophages

Apoptotic lymphocytes are readily identified in murine lungs, both during the response to particulate Ag and in normal mice. Because apoptotic lymphocytes are seldom detected in other organs, we hypothesized that alveolar macrophages (AMphi) clear apoptotic lymphocytes poorly. To test this hypothesis, we compared in vitro phagocytosis of apoptotic thymocytes by resident AMphi and peritoneal macrophages (PMphi) from normal C57BL/6 mice. AMphi were deficient relative to PMphi both in percentage containing apoptotic thymocytes (19.1 +/- 1% vs 96 +/- 2.6% positive) and in phagocytic index (0.23 +/- 0.02 vs 4.2 +/- 0.67). This deficiency was not due to kinetic differences, was seen with six other inbred mouse strains, and was not observed using carboxylate-modified polystyrene microbeads. Annexin V blockade indicated that both Mphi types cleared apoptotic T cells by a mechanism involving phosphatidylserine expression. By contrast, neither mAb blockade of a variety of receptors (CD11b, CD29, CD51, and CD61) known to be involved in clearance of apoptotic cells, nor the tetrapeptide RGDS (arginine-glycine-aspartic acid-serine) blocked ingestion by either type of macrophage. To confirm these studies, apoptotic thymocytes were given intratracheally or i.p. to normal mice, and then AMphi or PMphi were recovered 30-240 min later. Ingestion of apoptotic thymocytes by AMphi in vivo was significantly decreased at all times. Defective ingestion of apoptotic lymphocytes may preserve AMphi capacity to produce proinflammatory cytokines in host defense, but could contribute to development of autoimmunity by failing to eliminate nucleosomes.     

Brain microsomal oxidation of delta 8- and delta 9-tetrahydrocannabinol

Brain microsomes of mice, rats, guinea pigs and rabbits catalyzed the oxidation of delta 8- and delta 9-tetrahydrocannabinol to their monohydroxylated metabolites. The most prominent metabolite was the 4'-hydroxylated metabolite on the pentyl side chain of the cannabinoids in all species tested, except that the 5'-hydroxylation of delta 9-tetrahydrocannabinol was most abundant in the guinea pig. These results are quite different from the metabolic profile of the cannabinoids with hepatic microsomes.     

Anticonvulsant properties of delta 9-tetrahydrocannabinol and other cannabinoids

Anticonvulsant doses of Δ⁹-tetrahydrocannabinol (Δ⁹-THC) markedly lower body temperature in mice at an ambient temperature of 22°C, but there is little such effect at 30°C. The anticonvulsant properties of Δ⁹-THC are as follows: The drug abolishes hind-limb extension in a maximal electroshock (MES) test, elevates both the MES (extensor) and 6-Hz-electroshock thresholds, exerts no effect on the 60-Hz-electroshock threshold, and enhances minimal seizures caused by pentylenetetrazol. All anticonvulsant properties studied, with the exception of the 60-Hz-electroshock threshold, were unaffected by the hypothermia resulting at 22°C. Additional experiments with Δ⁹-THC indicated that chronic treatment results in the development of tolerance, as determined by the MES test with rats. The four principal naturally occurring cannabinoids, Δ⁹-THC, Δ⁸-THC, cannabinol and cannabidiol, display anticonvulsant activity, as does the major, primary metabolite of Δ⁹-THC, 11-hydroxy-Δ⁹-THC. Of all agents investigated in mice, the synthetic cannabinoids, dimethylheptylpyran and its isomers, are the most potent anticonvulsants. The results of a study of the relative motor toxicity and anticonvulsant activity of the cannabinoids demonstrate that these properties are at least partially separable among the various agents.     

Teratologic evaluation of synthetic delta 9-tetrahydrocannabinol in rabbits.

Synthetic delta 9-tetrahydrocannabinol (THC) was dissolved in undiluted propylene glycol and administered in daily subcutaneous doses of 15.0, 30.0 or 60.0 mg/kg to pregnant New Zealand white rabbits on days 7--19 of gestation. Maternal food consumption and weight gain were markedly reduced at all dose levels. Embryotoxicity and embryocidal effects were observed in the form of reduced litter weight and number of viable fetuses, respectively, in offspring from pregnant mothers treated with THC. However, on the basis of extensive external, visceral and skeletal examination of all fetuses it may be concluded that THC is not teratogenic in the New Zealand white strain rabbit following subcutaneous administration of doses as high as 60.0 mg/kg/day during the critical period of organogenesis (days 7--19 of gestation). On the other hand, an oral dose of thalidomide (200.0 mg/kg/day), the positive control used in this study, was both embryocidal and teratogenic.     

Nonspecificity of the phenomenon of influenza virus phagocytosis by murine macrophages

Peritoneal macrophage cultures from intact mice and those immune to influenza virus A/PR/8/34 (HON1) were infected with homologous virus or influenza virus A/England/42/72 (H3N2) whereupon virus was isolated from chick embryos. It was established that in intact macrophages, both viruses duplicated similarly. Macrophages immune to virus HON1 equally disintegrated both in homologous virus and heterologous influenza virus H3N2.     

Effect of delta 9-tetrahydrocannabinol on cyclic nucleotides in synchronously dividing Tetrahymena

Cyclic nucleotide levels were determined in division-synchronized Tetrahymena and the effect of delta 9-tetrahydrocannabinol (THC) on the cyclic nucleotide levels was studied. In non-drug-treated division-synchronized cells, there was no statistically significant variation in the level of cAMP and cGMP during the G2 period, preceding the first division. During the free running cell cycle (the interval of time between the first and second synchronous division) the twofold increase in the level of cAMP was statistically significant; however the variation in the level of cGMP was not statistically significant. THC caused a lowering of cAMP and cGMP levels throughout the 4-experimental treatment. The suppression of cAMP and cGMP levels altered the cyclic nucleotide pattern of the cell cycle. The cAMP pattern was changed particularly in the G2 period preceding the first synchronous division, and immediately after division during the free running cell cycle. THC treatment caused division delays of approximately 8-15 min in the onset of the first and second synchronous division. However, the duration of the free running cell cycle (110-120 min) was unchanged. The suppression of cyclic nucleotide levels resulting from THC treatment is discussed in relation to delays in the division schedule.     

Inhibition of cell-associated herpes simplex virus type 2 glycoproteins by delta 9-tetrahydrocannabinol

This study was conducted to define the effect of micromolar concentrations of delta 9-tetrahydrocannabinol (delta 9-THC) on the biosynthesis and expression of herpes simplex virus type 2 (HSV2)-specified glycoproteins. Dose-related reductions in all species of virus glycoproteins were recorded by one-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography of [14C]glucosamine-labeled infected Vero cells treated with 10(-7) to 10(-5) M delta 9-THC. A drug dose-related depletion of the mature HSV2 major envelope glycoprotein complex (119-kDa average molecular weight), accompanied by accumulation of immature unglycosylated species, was demonstrated by two-dimensional SDS-PAGE in concert with Western immunoblotting or autoradiography. Light and electron microscopy immunoperoxidase staining revealed that delta 9-THC effected depletion of 119-kDa determinants from the infected cell surface. This depletion occurred concomitantly with accumulation of 119-kDa components at the perinucleus. However, the expression of 119-kDa glycoproteins on the virion envelope was not affected. These results indicate that delta 9-THC inhibits the synthesis, maturation, and cellular transport of HSV2-specified glycoproteins. Decreased expression of virus glycoproteins on the infected cell surface may affect host immune responsiveness to HSV2.     

Protein synthesis by attached pulmonary macrophages effect of phagocytosis

We studied the effect of phagocytosis of polystyrene latex beads on protein synthesis by pulmonary macrophages. To do this we determine the specific radioactivity of extracellular and intracellular free phenylalanine and of phenylalanine released from tRNA and used this information in calculating the rates of protein synthesis. Phagocytosis resulted in an increased rate of protein synthesis irrespective of which precursor specific radioactivity was used in the calculation. The rate of protein synthesis was increased per μg polyribosomal RNA; but there was no increase in the amount of polyribosomal RNA in phagocytizing macrophages. The increase in the rate of protein synthesis (1.4-fold) was almost identical to the increase (1.3-fold) in the rate of ribosome transit in phagocytizing compared to nonphagocytizing macrophages. The decreased ribosome transit time during phagocytosis occurred without a fall in the average molecular weight of macrophage proteins. We conclude that phagocytosis increases the rate of protein synthesis in attached pulmonary macrophages and that this increased rate of synthesis can be accounted for almost completely by an increased rate of polypeptide chain elongation and/or termination.     

Effects of delta 9-tetrahydrocannabinol on testosterone production in vitro: influence of Ca++, Mg++ or glucose

The major psychoactive component of marihuana, delta 9-tetrahydrocannabinol (THC), influences testicular function. In the present experiments, the addition of THC to incubations of whole decapsulated mouse testes altered testosterone (T) production differentially, depending on the specific gonadotropin used, the dose of THC and/or the amount of divalent cation present in the media. In the presence of luteinizing hormone (LH; 10 ng/ml), and a dose of 25 micrograms THC/ml, T production was significantly decreased, compared to that by testes incubated with LH and vehicle at all Ca++ levels, except at 0.127 or 1.0 mM Ca++. The production of T by these paired testes exposed to either THC or vehicle (ethanol; ETOH), increased as Ca++ concentration approached physiological levels. In contrast, in the presence of follicle-stimulating hormone (FSH; 1 microgram/ml), THC-induced suppression of T production was significant in the absence of Ca++ from the media, and at 12.7 mM Ca++. However, it appeared that the levels of Ca++ did not differentially affect T production in the presence of FSH, whether or not THC was also added. In the presence of human chorionic gonadotropin (hCG; 12.5 mIU/ml), a lower dose of THC (25 ng/ml), stimulated T production at 0.25 to 1 mM Ca++, but had no effect as Ca++ reached 2.5 mM. Without additional Ca++ in the media, this dose of THC significantly reduced T secretion. In contrast, in the presence of hCG, a higher THC dose (25 micrograms/ml), suppressed T accumulation at 0.127, and from 1.0 to 12.7, but had no effect at 0.25 mM, or in the absence of Ca++. In the presence of hCG, the high 25 micrograms/ml dose of THC stimulated T production, in the absence of additional Mg++, and at 0.01 mM Mg++, but THC had no effect at 0.1 mM Mg++, but inhibited T production at 1.1 mM Mg++. In the presence of hCG, 25 micrograms THC/ml produced a consistent suppression of T production across glucose concentrations examined. These findings suggest that the mechanisms by which THC effects testicular steroidogenesis may involve Ca++- and/or Mg++-dependent processes. Differential requirements for these divalent cations by the gonadotropins may explain the interactive effects of THC with LH, hCG or FSH.     

Effects of delta9-tetrahydrocannabinol on ATPases in mouse brain subcellular fractions.

The effect of (?)-Δ⁹-THC on the activities of Mg²⁺?, Na⁺?K⁺? and Mg²⁺Ca²⁺-ATPases were studied in mouse brain subcellular fractions. $\text{In vitro}$treatment with Δ⁹-THC produced a dose dependent stimulation of Mg²⁺ ATPase in the crude mitochondrial fraction and its subfractions and a dose-related inhibition of this activity in the microsomal fraction. Na⁺-K⁺- and Mg²⁺-Ca²⁺-ATPase activities were inhibited in a dose-related manner in all subcellular fractions studied.     

Action of delta 9-tetrahydrocannabinol on the pool of acid soluble nucleotides

The effects of delta 9-tetrahydrocannabinol (THC) treatment on acid soluble pools of uridine nucleoside and nucleotides were investigated in Tetrahymena pyriformis and in isolated mouse lymphocytes and spermatogenic cells. In THC treated Tetrahymena and mouse lymphocytes the uptake of labelled precursor into acid soluble pools of uridine nucleoside and nucleotides fluctuated, whereas in pachytene spermatocytes and round spermatid cells the labelled pool was reduced. The reduction in the labelled pool measured in mouse spermatogenic cells was attributed primarily to a reduction in radioactively labelled uridine nucleoside. Treatment of Tetrahymena in high concentrations of THC (960 and 3,200 microM) resulted in an increase of labelled uridine nucleoside and a reduction in the amount of labelled uridine nucleotides. Expansion of the acid soluble pool with radioactive uridine resulted in small differences in labelled nucleoside and nucleotides in control and THC treated Tetrahymena and mouse lymphocytes. The results are discussed in terms of the effects of THC on macromolecular synthesis in various cellular systems.     

Potentials evoked in the intermediolateral column by hypothalamic stimulation - suppression by delta 9-tetrahydrocannabinol

Δ⁹-Tetrahydrocannabinol (THC), the primary psychoactive component of marihuana produces pronounced effects on the cardiovascular system including bradycardia and hypotension. A decrease in sympathetic activity may contribute to these actions. In chloralose urethane anesthetized cats, THC (2 mg/kg, i.v.) produced significant bradycardia, hypotension and attenuation of threshold pressor responses induced by hypothalamic stimulation. Evoked potentials recorded in the intermediolateral cell column (ILC) by stimulation of these hypothalamic pressor sites were significantly altered after THC. Hypotension induced by histamine administration (5 μg/kg, i.v.) altered ILC potentials before and after THC. These results support the hypothesis that THC reduces sympathetic outflow and reversibly resets the level of central cardiovascular homeostasis.     

Calcium transients during Fc receptor-mediated and nonspecific phagocytosis by murine peritoneal macrophages

Studies with populations of macrophages have produced conflicting results concerning the possibility that the concentration of intracellular ionized calcium [( Ca2+]i) may act as an important mediator for phagocytosis. Since asynchronous changes in [Ca2+]i in individual cells undergoing phagocytosis may be averaged to undetectability in population studies, we studied single adhering murine macrophages using fura-2 and our previously described digital imaging system. The proportion of macrophages phagocytosing IgG-coated latex beads was greater than for uncoated beads (percent phagocytosing cells: 71 +/- 7 vs. 27 +/- 7, P less than 0.01). Phagocytosis of IgG-coated and uncoated beads was always associated with a calcium transient that preceded the initiation of phagocytosis. No calcium transients were detected in cells that bound but did not phagocytose beads. Four major differences between Fc receptor-mediated and nonspecific phagocytosis were detected: (a) the duration of calcium transients was longer for nonspecific phagocytosis compared with Fc receptor-mediated phagocytosis (69.9 +/- 10.2 vs. 48.7 +/- 4.7 s, P less than 0.05) and the magnitude of calcium transients was less for nonspecific phagocytosis (178 +/- 43 vs. 349 +/- 53 nM, P less than 0.05); (b) removal of extracellular calcium abolished the calcium transients associated with nonspecific phagocytosis but had no effect on those associated with receptor-mediated phagocytosis; (c) in the absence of extracellular calcium, buffering intracellular calcium with a chelator reduced Fc receptor-mediated phagocytosis but had no additive inhibitory effect on nonspecific phagocytosis; and (d) inhibition of protein kinase C (PKC) with staurosporine inhibited nonspecific phagocytosis but had no effect on receptor-mediated phagocytosis. Our observations suggest that despite both types of phagocytosis being associated with intracellular calcium transients, the role played by intracellular calcium in the signaling pathways may differ for Fc receptor-mediated and nonspecific phagocytosis by elicited murine macrophages.