Visualization of the same PtK2 cytoskeletons by both immunofluorescence and low power electron microscopy.

A procedure is described which allows the examination of the cytoskeleton of a single PtK2 cell first by immunofluorescence and then by electron microscopy after staining with uranyl acetate. The immunofluorescent patterns of these detergent resistant cytoskeletons elicited with various monospecific antibodies closely resemble the patterns found in whole cells. Comparison of the immunofluorescence and electron micrographs directly supports the previous assignments of actin, myosin, filamin, α-actinin and tropomyosin as proteins associated with microfilament bundles in non-muscle cells. Actin is also found associated with a fine lattice-like structure present both in the ruffles and lying above the microfilament bundles in the cell body. The tonofilament bundles present in PtK2 cytoskeletons are not decorated by antibodies directed against the proteins associated with microfilament bundles. Antibodies directed against tonofilaments decorate specifically this system and not the microfilament bundles.     

Control of microtubule nucleation and stability in Madin-Darby canine kidney cells: the occurrence of noncentrosomal, stable detyrosinated microtubules

The microtubule-nucleating activity of centrosomes was analyzed in fibroblastic (Vero) and in epithelial cells (PtK2, Madin-Darby canine kidney [MDCK]) by double-immunofluorescence labeling with anti-centrosome and antitubulin antibodies. Most of the microtubules emanated from the centrosomes in Vero cells, whereas the microtubule network of MDCK cells appeared to be noncentrosome nucleated and randomly organized. The pattern of microtubule organization in PtK2 cells was intermediate to the patterns observed in the typical fibroblastic and epithelial cells. The two centriole cylinders were tightly associated and located close to the nucleus in Vero and PtK2 cells. In MDCK cells, however, they were clearly separated and electron microscopy revealed that they nucleated only a few microtubules. The stability of centrosomal and noncentrosomal microtubules was examined by treatment of these different cell lines with various concentrations of nocodazole. 1.6 microM nocodazole induced an almost complete depolymerization of microtubules in Vero cells; some centrosome nucleated microtubules remained in PtK2 cells, while many noncentrosomal microtubules resisted that treatment in MDCK cells. Centrosomal and noncentrosomal microtubules regrew in MDCK cells with similar kinetics after release from complete disassembly by high concentrations of nocodazole (33 microM). During regrowth, centrosomal microtubules became resistant to 1.6 microM nocodazole before the noncentrosomal ones, although the latter eventually predominate. We suggest that in MDCK cells, microtubules grow and shrink as proposed by the dynamic instability model but the presence of factors prevents them from complete depolymerization. This creates seeds for reelongation that compete with nucleation off the centrosome. By using specific antibodies, we have shown that the abundant subset of nocodazole-resistant microtubules in MDCK cells contained detyrosinated alpha-tubulin (glu tubulin). On the other hand, the first microtubules to regrow after nocodazole removal contained only tyrosinated tubulin. Glu-tubulin became detectable only after 30 min of microtubule regrowth. This strongly supports the hypothesis that alpha-tubulin detyrosination occurs primarily on "long lived" microtubules and is not the cause of the stabilization process. This is also supported by the increased amount of glu-tubulin that we found in taxol-treated cells.     

Tetrahydrocannabinol treatment suppresses growth restriction of Legionella pneumophila in murine macrophage cultures.

Legionella pneumophila is a facultative intracellular pathogen which readily grows in human and guinea pig macrophages and in peritoneal exudate macrophages from A/J mice. Macrophage cultures capable of supporting the growth of Legionella can be used to test the potency of biologically active substances suspected of modulating host mechanisms of resistance to infection. Accordingly, this model was used to evaluate the influence of delta-9-tetrahydro-cannabinol (THC) on macrophage resistance to infection with an intracellular pathogen. Pretreatment of the macrophages with THC in the concentration range of 2.5 micrograms/ml (8 microM) to 5.0 micrograms/ml (16 microM) had little if any effect on the ability of the macrophages to either ingest or support the replication of Legionella. However, THC treatment of cells following Legionella infection resulted in increased numbers of bacteria recoverable from the macrophage cultures. Stimulation of the macrophage cultures with the activating agent lipopolysaccharide (LPS) was effective in reducing the ability of Legionella to grow in the cells. However, treatment of the LPS activated macrophages with THC resulted in greater growth of the Legionella in the cultures, indicating that the drug abolished the LPS induced enhanced resistance. These results demonstrate that THC treatment of macrophages following infection rather than before infection with Legionella promotes the replication of the bacteria within the macrophages. In addition, drug treatment suppresses the growth restricting potential of macrophages activated by LPS.     

Selective reduction of anaphase B in quinacrine-treated PtK1 cells

Quinacrine, an acridine derivative which competitively binds to ATP binding sites, has previously been shown to cause the reorganization of metaphase spindle microtubules (MTs) due to changes in interactions of non-kinetochore microtubules (nkMTs) of opposite polarity (Armstrong and Snyder: Cell Motil. Cytoskeleton 7:10-19, 1987). In the study presented here, mitotic PtK1 cells were treated in early anaphase with concentrations of quinacrine ranging from 2 to 12 microM to determine energy requirements for chromosome motion. The rate and extent of chromosome-to-pole movements (anaphase A) were not affected by these quinacrine treatments. The extent of anaphase B (kinetochore-kinetochore separation) was reduced with increasing concentrations of quinacrine. Five micromolar quinacrine reduced the extent of kinetochore-kinetochore separation by 20%, and addition of 12 microM quinacrine reduced the kinetochore-kinetochore separation by 40%. To determine the role of nkMTs in anaphase spindle elongation, quinacrine-treated metaphase cells were treated with hyperosmotic sucrose concentrations, and spindle elongation was measured (Snyder et al.: Eur J. Cell Biol. 39:373-379, 1985). Metaphase cells treated with 2-10 microM concentrations of quinacrine for 2-5 min reduced spindle lengths by 10-50% prior to 0.5 M sucrose treatment for 5 min. This treatment showed a significant reduction in the ability of sucrose to induce spindle elongation in cells pretreated with quinacrine. As spindle length and birefringence was reduced by quinacrine treatment, sucrose-induced elongation was concomitantly diminished. These data suggest that quinacrine-sensitive linkages are necessary for anaphase B motions. Reduction in these linkages and/or MT length in the nkMT continuum may reduce the ability of the nkMTs to hold compression at metaphase. This form of energy is thought to drive a significant proportion of normal anaphase B in PtK1 cells and sucrose-induced metaphase spindle elongation.     

Cannabinoids reduce fertility of sea urchin sperm

Cannabinoids are potent pharmacological substances derived from marihuana. The effects of delta 9-tetrahydrocannabinol (THC), cannabinol (CBN), and cannabidiol (CBD) on fertilization in the sea urchin Strongylocentrotus purpuratus were investigated. Insemination of THC-treated eggs (5-400 microM) with excess sperm did not result in polyspermic fertilization. At minimal sperm densities, THC (0.1-10 microM) inhibited fertilization in a dose-dependent manner. Pretreatment of eggs with THC did not reduce their receptivity to sperm. Pretreatment of sperm with THC reduced their fertilizing capacity. The concentration of THC required to reduce sperm fertility by 50% was 1.1 +/- 1.1 microM. The fertilizing capacity of THC-treated sperm depended on concentration of sperm and duration of pretreatment. The fertility of sperm at minimal densities was reduced by 50% at 129.3 +/- 43 s treatment with 10 microM THC. The adverse effect of THC on sperm fertility was reversible. CBN and CBD at comparable concentrations (0.1-10 microM) inhibited fertilization in a manner similar to THC. First division was not delayed in zygotes that were fertilized with sperm pretreated with 10 microM THC. These studies show that cannabinoids directly affect the process of fertilization in sea urchins by reducing the fertilizing capacity of sperm.     

Cytoskeletal organization following cannabinoid treatment in undifferentiated and differentiated PC12 cells.

Confocal microscopy in association with three-dimensional reconstruction was used to examine the changes in the microtubules and microfilaments following cannabinoid treatment of PC12 cells. Microtubules and microfilaments were disrupted in a dose-dependent manner following treatment with 10-30 microM delta 9-tetrahydrocannabinol (THC). A disruption of microtubules and microfilaments was observed following treatment with 30 microM cannabidiol and cannabinol. The amount of microtubules and microfilaments was reduced in a dose-dependent manner following treatment with 10 and 20 microM THC. Cannabidiol and cannabinol reduced the amount of microtubules and microfilaments; however, the reduction was less than that observed with THC treatment. Following the addition of nerve growth factor, differentiated PC12 cells were generally more sensitive to cannabinoid treatments than undifferentiated cells. The possible mechanisms that may account for the changes in microtubules and microfilaments following cannabinoid treatment are discussed.     

Role of actin-filament disassembly in lamellipodium protrusion in motile cells revealed using the drug jasplakinolide.

BACKGROUND: In motile cells, protrusion of the lamellipodium (a type of cell margin) requires assembly of actin monomers into actin filaments at the tip of the lamellipodium. The importance of actin-filament disassembly in this process is less well understood, and is assessed here using the actin drug jasplakinolide, which has two known activities - inhibition of filament disassembly and induction of an increase in actin polymer. RESULTS: In cells the two activities of jasplakinolide were found to be separable; 1 microM jasplakinolide could permeate cells, bind cellular filamentous actin (F-actin) and inhibit filament disassembly within 3.5 minutes, but significant increase in actin polymer was not detected until 60 minutes of treatment. In live, permeabilised cells, jasplakinolide did not inhibit filament assembly from supplied, purified actin monomers. In migrating chick fibroblasts, lamellipodium protrusion was blocked within 1-5 minutes of treatment with 1 microM jasplakinolide, without any perturbation of actin organisation. In non-migrating chick fibroblasts, there was a delay in the onset of jasplakinolide-induced inhibition of lamellipodium protrusion, during which lamellipodium length increased linearly with no increase in protrusion rate. Motility of the bacterium Listeria in infected PtK2 cells was reduced 2.3-fold within 3 minutes of treatment with 1 microM jasplakinolide. CONCLUSIONS: Actin-filament disassembly is tightly coupled to lamellipodium protrusion in migrating chick fibroblasts and motility of Listeria in PtK2 cells. One simple interpretation of these data is a situation whereby ongoing actin-filament assembly uses free actin monomer derived from filament disassembly, in preference to stored monomer.     

Novel time-dependent vascular actions of Delta9-tetrahydrocannabinol mediated by peroxisome proliferator-activated receptor gamma

Cannabinoids have widespread effects on the cardiovascular system, only some of which are mediated via G-protein-coupled cell surface receptors. The active ingredient of cannabis, Delta9-tetrahydrocannabinol (THC), causes acute vasorelaxation in various arteries. Here we show for the first time that THC also causes slowly developing vasorelaxation through activation of peroxisome proliferator-activated receptors gamma (PPARgamma). In vitro, THC (10 microM) caused time-dependent vasorelaxation of rat isolated arteries. Time-dependent vasorelaxation to THC was similar to that produced by the PPARgamma agonist rosiglitazone and was inhibited by the PPARgamma antagonist GW9662 (1 microM), but not the cannabinoid CB1 receptor antagonist AM251 (1 microM). Time-dependent vasorelaxation to THC requires an intact endothelium, nitric oxide, production of hydrogen peroxide, and de novo protein synthesis. In transactivation assays in cultured HEK293 cells, THC-activated PPARgamma, transiently expressed in combination with retinoid X receptor alpha and a luciferase reporter gene, in a concentration-dependent manner (100 nM-10 microM). In vitro incubation with THC (1 or 10 microM, 8 days) stimulated adipocyte differentiation in cultured 3T3L1 cells, a well-accepted property of PPARgamma ligands. The present results provide strong evidence that THC is a PPARgamma ligand, stimulation of which causes time-dependent vasorelaxation, implying some of the pleiotropic effects of cannabis may be mediated by nuclear receptors.     

Role of LPS-induced microfilament depolymerization in MIP-2 production from rat pneumocytes

We have previously demonstrated that lipopolysaccharide (LPS) induces production of macrophage inflammatory protein-2 (MIP-2), a C-X-C chemokine for neutrophil recruitment and activation, in primary cultured rat lung alveolar epithelial cells. We have also demonstrated that LPS depolymerizes microfilaments in rat alveolar epithelial cells. To determine whether the polymerization status of microfilaments affects LPS-induced MIP-2 production, we treated rat alveolar epithelial cells with cytochalasin D (CytoD), a microfilament-disrupting agent, before and during LPS stimulation. A lower concentration (0.1 microM) of CytoD inhibited LPS-induced MIP-2 production without affecting microfilament polymerization. In contrast, LPS-induced MIP-2 production was enhanced by a higher concentration (10 microM) of CytoD, which disrupted the filamentous structure of actin. Jasplakinolide (1 nM to 1 microM), a polymerizing agent for microfilaments, decreased LPS-induced MIP-2 secretion. Jasplakinolide (1 microM) also blocked LPS-induced depolymerization of microfilaments. These results suggest that, in alveolar epithelial cells, LPS-induced MIP-2 production is at least partially regulated by microfilament depolymerization.     

Primary cilia cycle in PtK1 cells: effects of colcemid and taxol on cilia formation and resorption

The effects of colcemid (0.16-1.0 microM) and taxol (10 microM) on the primary cilia cycle in PtK1 cells were studied by antitubulin immunofluorescence microscopy and by high-voltage electron microscopy of serial 0.25-micron sections. Although these drugs induce a fully characteristic rearrangement (taxol) or disassembly (colcemid) of cytoplasmic microtubules, neither affects the structure of primary cilia formed prior to the treatment or the resorption of primary cilia during the initial stages of mitosis. Cells arrested in mitosis by taxol or colcemid remain in mitosis for 5-7 h at 37 degrees C and then form 4N "micronucleated" restitution nuclei. Formation of primary cilia in these micronucleated cells is blocked by colcemid in a concentration-dependent fashion: normal cilia with expanded (ie, bulbed) distal ends form at the lower (0.16-0.25 microM) concentrations, while both cilia formation and centriole replication are inhibited at the higher (greater than or equal to 1.0 microM) concentrations. However, even in the presence of 1.0 microM colcemid, existing centrioles acquire the appendages characteristically associated with ciliating centrioles and attach to the dorsal cell surface. Continuous treatment with colcemid thus produces a population of cells enriched for the early stages of primary cilia formation. Micronucleated cells formed from a continuous taxol treatment contain two normal centriole pairs, and one or both parenting centrioles possess a primary cilium. Taxol, which has been reported to stabilize microtubules in vitro, does not inhibit the cell-cycle-dependent assembly and disassembly of axonemal microtubules in vivo.     

The role of calcium ions during mitosis

Calcium-containing solutions were microinjected into dividing PtK1 cells to assess the effect of calcium ion concentration on the morphology and physiology of the mitotic spindle. Solutions containing 50 microM or more CaCl2 are immediately and irreversibly toxic to PtK1 cells. Those containing 5-10 microM CaCl2 cause reversible reduction in spindle birefringence followed by normal anaphase and cytokinesis. Microinjection of 5 microM or less CaCl2 into anaphase PtK1 cells has no detectable effect on the rate or extent of chromosome movement. Metaphase cells tend to enter anaphase 4-5 min after injection with 1-10 microM CaCl2, compared with an average of 16 min after injection with calcium-free buffer. Reducing the intracellular calcium concentration by injection of EGTA-CaCl2 buffers increases the lag between injection and anaphase to 20 min or more. Microinjection of calcium solutions does not promote precocious chromatid separation in nocodazole-arrested metaphase cells, indicating that the increase in calcium concentration does not induce centromere separation directly. An increase in the concentration of free calcium ions during metaphase appears to stimulate the onset of anaphase. Such an increase, regulated by the cell itself, may contribute to the initiation of chromosome separation in mammalian cells.     

Suppression by cannabinoids of a cloned cell line with natural killer cell activity

Preincubation of a cloned cell line with natural killer (NK) cell activity, as well as splenic mononuclear cells with either delta 9-tetrahydrocannabinol (THC) or 11-hydroxy-delta 9-tetrahydrocannabinol (11-OH-THC) suppressed NK cytolytic activity against YAC-1 target cells in a dose-dependent manner. THC was more inhibitory for cloned cells than 11-OH-THC and suppressed the lytic activity of these cells without reducing cell viability in the concentration range of 5 micrograms/ml (16 microM) to 10 micrograms/ml (32 microM). THC also inhibited proliferation of cloned NK cells, but this inhibitory effect was reversible in that extensive washing of cells following cannabinoid pretreatment eliminated the suppressive effect. Single-cell analysis revealed that THC did not inhibit the binding of cloned NK cells to target cells and further showed that NK cells freshly isolated from mouse spleen were restricted in killing capacity following binding to target cells. Therefore, THC and 11-OH-THC appear to directly inhibit NK cell cytolytic activity in a postbinding stage.     

Stress fiber dynamics as probed by antibodies against myosin

The dynamics of microfilament bundles (stress fibers) in tissue culture cells were studied by microinjecting an affinity-purified polyclonal antibody against chicken gizzard myosin. This antibody cross-reacted exclusively with the light chains of nonmuscle myosin and should therefore bind to the head portion of myosin molecules. When injected in high concentrations (13-26 mg/ml), it disrupted stress fibers in a high proportion (60-80%) of rat and chicken embryo fibroblasts, as well as in PtK2 cells. Myosin was found collected in large aggregates probably comprising protein: antibody precipitates, while actin and alpha-actinin were not localized in any defined structures in stress fiber depleted cells. Fibroblasts rounded up, probably because of lack of tension-generating microfilament bundles. After several hours, stress fibers were seen to regrow again in the afflicted cells, even when myosin precipitates and excess antibody were still present. The extent of stress fiber disruption and the time point of their reappearance were dependent on the concentration of the injected antibody.     

Blockade of first ovulation in pubertal rats by delta-9-tetrahydrocannabinol: requirement for advanced treatment due to early initiation of the critical period

Successful blockade of ovulation in pubertal rats by delta-9-tetrahydrocannabinol (THC) required earlier treatment during proestrus than was required in adults under the same conditions. Only 1 of 8 adult rats ovulated after treatment with THC (10 mg/kg body weight, i.p.) at 1400 h proestrus, whereas 77% of pubertal rats released full sets of ova following similar treatment during proestrus of the first or second vaginal cycle. When treatment of pubertal rats was advanced to 1300 h, only 2 of 10 THC-treated rats exhibited full ovulation, an incidence significantly lower than the 80% ovulation rate observed in vehicle-treated animals (p less than 0.05). To determine whether the requirement for earlier THC treatment in pubertal rats was related specifically to THC or reflected possible age-associated differences in timing of the critical period, the ovulation-blocking efficacy of atropine sulfate (ATR) was tested in pubertal rats for comparison with that of THC. The serum concentrations of luteinizing hormone (LH) during the first proestrus (1200-1900 h) were determined in pubertal rats that remained untreated. The incidence of ovulation in rats treated with ATR (350 mg/kg, s.c.) at 1400 h proestrus was not significantly reduced from that in vehicle-treated rats; however, after ATR treatment at 1300 h, only 2 of 11 animals released full sets of ova whereas all vehicle-treated rats ovulated (p less than 0.025). The mean serum LH concentration in untreated pubertal rats was not significantly increased over baseline at 1300 h proestrus, but was markedly elevated by 1400 h (1009 +/- 375 ng/ml; p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of cytokinesis and altered contractile ring morphology induced by cytochalasins in synchronized PtK2 cells

PtK2 cells and antigen affinity-purified antibodies to actin and tubulin were used to study the effects on mitosis of cytochalasin B (CB) and dihydrocytochalasin B (H₂CB). PtK2 cells were synchronized in S phase by a double thymidine block and CB or H₂CB was added at various concentrations at the time of release from the block. CB- and H₂CB-treated populations, and control populations not treated with either drug, progressed synchronously through G2 and into mitosis with similar time courses. By both phase contrast and immunofluorescence microscopy, CB- and H₂CB-treated cells appeared normal in terms of chromosome condensation, spindle formation and spindle dynamics throughout prophase, metaphase and early anaphase. At late anaphase, contractile ring staining with actin antibody was not normal. High actin antigenicity remained localized in the region of the contractile ring; however, it appeared atypically as a punctate line of fluorescence across the midzone. Although some degree of furrowing was often seen to occur, at suitable concentrations of CB or H₂CB only binucleate G1 cells formed. Scanning electron microscopy (SEM) of normal and CB- and H₂CB-treated cells verified that cleavage furrowing did not proceed normally in treated cells. Large numbers of microvilli and surface blebs occurred in the normally smooth furrow region in these treated populations. These results suggest that intact microfilament function is not necessary for progression from S phase into mitosis, for spindle formation or for chromosome movement. They indicate that CB and H₂CB lead to formation of binucleated cells by causing aberrant cleavage furrowing and inhibition of contractile ring microfilaments.     

The cytoskeleton and rat granulosa cell steroidogenesis: possible involvement of microtubules and microfilaments

The participation of both microtubules and microfilaments in granulosa cell steroidogenesis was assessed by monitoring the effects of colchicine (0-250 microM) and/or cytochalasin B (0-10 micrograms/ml) or dihydrocytochalasin B (0-2.0 micrograms/ml) on cellular morphology and production of progestins during 24 h of culture. Both colchicine and the cytochalasins increased granulosa cell production of progesterone and of 20 alpha-hydroxy-pregn-4-en-3-one (20 alpha-OH-progesterone) in a dose-dependent manner. The largest increase in steroidogenesis (about 2- to 3-fold) was observed at 4-250 microM colchicine and at 2-10 micrograms/ml cytochalasin. Those concentrations of the inhibitors of microtubule or microfilament polymerization that stimulated basal progestin production also markedly influenced cell spreading. Whereas cells cultured for 24 h in medium alone became very flattened with numerous cytoplasmic extensions, those cultured with colchicine (0.2-250 microM) or cytochalasin (0.4-2 micrograms/ml) were much less spread and progressively became more rounded and regular in outline. These changes in cell morphology were reflected by decreases in the mean area occupied by the cells on the culture surface of up to 60-65% and reductions in mean contour index values from 5.7 +/- 0.1 (control) to 3.9 +/- 0.1 (250 microM colchicine), 4.2 +/- 0.1 (2 micrograms/ml cytochalasin B), or 4.1 +/- 0.1 (2 micrograms/ml dihydrocytochalasin B). Cultures containing both colchicine and cytochalasin B exhibited a greater steroidogenic response than that elicited by either inhibitor alone. For example, granulosa cell progesterone production was stimulated almost 2-fold by 4 microM colchicine or 2 microM/ml cytochalasin B, but 5.5-fold by 4 microM colchicine plus 2 micrograms/ml cytochalasin B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of reduced levels of the LDL receptor of Ehrlich ascites tumor cells on cholesterol uptake and cell proliferation: a coculture study with baby hamster kidney cells

We have introduced a heterologous coculture model between Ehrlich ascites tumor (EAT) and baby hamster kidney cells (PtK2), and we have studied the influence of PtK2 cells and their newly synthesized cholesterol on uptake and tumor cell proliferation. PtK2 cells produce about five times more cholesterol as compared to EAT cells, and they support tumor cell growth in coculture experiments. This growth stimulation is reduced by 80% when EAT cells are cultured in PtK2 cell-derived medium in the presence of a monoclonal anti-low-density lipoprotein receptor (anti-LDL(r)) antibody. Freshly synthesized cholesterol by PtK2 cells is taken up by EAT cells in a time-dependent manner amounting to a threefold increase after 24 h. Alternatively, cholesterol transfer to EAT cells is decreased between 28% and 35%, when PtK2 cell cholesterol synthesis is inhibited in the presence of mevinolin, the specific inhibitor of the hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase. Furthermore, lower levels of EAT cell LDL receptor induced by antisense technology reduces cholesterol uptake and cell proliferation. These data demonstrate a metabolic interaction between normal and tumor cells mediated via the exchange of cholesterol, an important membrane constituent.     

Reduction of the fertilizing capacity of sea urchin sperm by cannabinoids derived from marihuana. I. Inhibition of the acrosome reaction induced by egg jelly.

delta 9-Tetrahydrocannabinol (THC) and two other major cannabinoids derived from marihuana--cannabidiol (CBD) and cannabinol (CBN)--inhibit fertilization in the sea urchin Strongylocentrotus purpuratus by reducing the fertilizing capacity of sperm (Schuel et al., 1987). Sperm fertility depends on their motility and on their ability to undergo the acrosome reaction upon encountering the egg's jelly coat. Pretreatment of S. purpuratus sperm with THC prevents triggering of the acrosome reaction by solubilized egg jelly in a dose (0.1-100 microM) and time (0-5 min)-dependent manner. Induction of the acrosome reaction is inhibited in 88.9 +/- 2.3% of sperm pretreated with 100 microM THC for 5 min, while motility of THC-treated sperm is not reduced compared to solvent (vehicle) and seawater-treated controls. The acrosome reaction is inhibited 50% by pretreatment with 6.6 microM THC for 5 min and with 100 microM THC after 20.8 sec. CBN and CBD at comparable concentrations inhibit the acrosome reaction by egg jelly in a manner similar to THC. THC does not inhibit the acrosome reaction artificially induced by ionomycin, which promotes Ca2+ influx, and nigericin, which promotes K+ efflux. THC partially inhibits (20-30%) the acrosome reaction induced by A23187, which promotes Ca2+ influx, and NH4OH, which raises the internal pH of the sperm. Addition of monensin, which promotes Na+ influx to egg jelly or to A23187, does not overcome the THC inhibition. Inhibition of the egg jelly-induced acrosome reaction by THC produces a corresponding reduction in the fertilizing capacity of the sperm. The adverse effects of THC on the acrosome reaction and sperm fertility are reversible.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

纯氧与空气环境复苏对低氧新生大鼠大脑皮质神经元凋亡的影响

目的应用无创性低氧动物模型,比较纯氧环境(pure oxygen environment,POE,100%氧)与空气环境(room air environment,RAE,21%氧)复苏对低氧新生大鼠大脑皮质神经元凋亡的影响。方法 30只SpragueDawley(SD)7日龄乳鼠在建立低氧2.5 h模型后,分别于纯氧环境与空气环境中进行复苏。实验分为正常对照组(无预缺氧和复氧实验,n=10)、纯氧环境组(POE,n=10)和空气环境组(RAE,n=10),依复苏后时间点每组又分为24 h组和72 h组,每亚组5只。按时间点取各组乳鼠大脑皮质行尼氏体染色、原位末端标记(TUNEL)技术、图像分析和透射电镜观察神经元凋亡。结果与正常对照组相比,低氧乳鼠复氧后各组大脑皮质内锥体层均可见散在的、单个的、成群的或2～4个排列成串的凋亡锥体神经元,细胞凋亡现象RAE 72 h组最明显,POE 72 h组凋亡细胞个数和面密度都较RAE 72 h组少(P0.01)。电镜下RAE 72 h组类似凋亡细胞核也较其他各组多见。结论在本动物模型POE复苏较RAE复苏早期可更能减少细胞凋亡,缓解低氧致神经元的损伤,表明纯氧早期对新生儿低氧性脑损伤有一定的保护作用。