1,2-Dioctanoylglycerol accelerates or retards mitotic progression in Tradescantia stamen hair cells as a function of the time of its addition

We have treated living, intact stamen hair cells from the spiderwort plant, Tradescantia virginiana, with 0.5 microgram/ml or 60 micrograms/ml 1,2-dioctanoylglycerol, a potent and permeant activator of protein kinase C, and have observed the rates of progression of mitosis from prophase through anaphase. We have found that in addition to the concentration used, the time of initial treatment with 1,2-dioctanoylglycerol defines the response by the cells. The cells rapidly undergo nuclear envelope breakdown when this diglyceride is added in very late prophase, 0 to approximately 8 min prior to the time of normal nuclear envelope breakdown. Anaphase onset occurs 28 min after nuclear envelope breakdown, rather than after the 33 min interval observed in untreated cells. Rapid progression through metaphase is also observed if cells are treated with 0.5 microgram/ml 1,2-dioctanoylglycerol during prometaphase, up to 15 min after nuclear envelope breakdown. The addition of 0.5 microgram/ml 1,2-dioctanoylglycerol in late metaphase, approximately 26 min after nuclear envelope breakdown, results in sister chromatid separation slightly ahead of its normal time, 33 min after nuclear envelope breakdown, and in precocious cell plate vesicle aggregation, 3-5 min earlier than that observed in untreated cells. Treatment of cells with 60 micrograms/ml of 1,2-dioctanoylglycerol at any point during the interval from 0 to approximately 5 min prior to nuclear envelope breakdown results in precocious entry into anaphase. If cells are treated with either 0.5 microgram/ml or 60 micrograms/ml 1,2-dioctanoylglycerol earlier than 20 min before nuclear envelope breakdown, they do not enter mitosis, but instead revert to interphase without dividing. When 1,2-dioctanoylglycerol is added at other times during mitosis, the rate of subsequent mitotic progression is dramatically slowed; the cells require greater than 55 min to progress from nuclear envelope breakdown to anaphase onset, though once in anaphase, the cells progress onward to cytokinesis at normal rates. Treatments o of cells with 1,3-dioctanoylglycerol at any point during prophase, prometaphase, or metaphase are without effect on the rate of subsequent mitotic progression. The shifts in response by cells treated at specific times with 1,2-dioctanoylglycerol during mid- and late metaphase may be indicative of the existence of one or more regulatory switch points (i.e., checkpoints) just prior to anaphase onset.     

Insulin-like growth factor-I stimulates diacylglycerol production via multiple pathways in Balb/c 3T3 cells

We previously reported that insulin-like growth factor-I (IGF-I) induced sustained calcium cycling across the plasma membrane in primed competent Balb/c 3T3 cells (Kojima, I., Matsunaga, H., Kurokawa, K., Ogata, E., and Nishimoto, I. (1989) J. Biol. Chem. 263, 16561-16567). The present study was conducted to examine whether IGF-I affected cellular metabolism of 1,2-diacylglycerol (1,2-DAG). In primed competent cells prelabeled with [3H]myristate, 1 nM IGF-I caused a 50% increase in [3H]DAG within 10 min. This increase in [3H]DAG was accompanied by 1) a decrease in radioactivity in the glycosylphosphatidylinositol fraction in [3H]glucosamine-labeled cells and a concomitant increase in [3H]inositol-glycan, and 2) a decrease in [3H]phosphatidylcholine and a concomitant elevation of [3H]phosphorylcholine in [3H]choline-labeled cells. When [3H]choline-labeled cells were treated with 10 nM 12-O-tetradecanoylphorbol-4-acetate (TPA), [3H]phosphatidylcholine was reduced by 50%. The TPA-induced reduction of [3H]phosphatidylcholine was completely blocked by 50 microM sphingosine and 50 microM H-7, inhibitors of protein kinase C. Both sphingosine and H-7 attenuated IGF-I-mediated reduction of [3H]phosphatidylcholine. In addition, treatment with IGF-I for 3 h or more resulted in sustained increase in 1,2-DAG mass, which was attenuated by cycloheximide. The increase in DAG mass was accompanied by enhanced incorporation of [14C]glucose into 1,2-DAG. These results indicate that, in primed competent Balb/c 3T3 cells, IGF-I stimulates 1,2-DAG production via multiple pathways and that IGF-I may induce breakdown of phosphatidylcholine by a mechanism involving protein kinase C.     

1,2-Diacylglycerol, protein kinase C, and pancreatic enzyme secretion

To determine the role of 1,2-diacylglycerol (1,2-DAG) and protein kinase C in pancreatic enzyme secretion, we measured the effect of various pancreatic secretagogues on the cellular mass of 1,2-DAG and amylase release in dispersed pancreatic acini from the guinea pig. In addition, we measured the effect of a recently described protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) (Hidaka, H., Inagaki, M., Kawamoto, S., and Sasaki, Y. (1984) Biochemistry 23, 5036-5041), on secretagogue-stimulated amylase release from the acini. Cholecystokinin-octapeptide (CCK-OP), cholecystokinintetrapeptide, and carbachol each increased 1,2-DAG 2-3-fold but the increases occurred only with concentrations of these secretagogues that were supramaximal for amylase release and that had an inhibitory effect on stimulated amylase release. Supramaximal concentrations of bombesin stimulated only a small increase in 1,2-DAG and did not cause inhibition of stimulated amylase release. When the action of carbachol was terminated with atropine or CCK-OP with dibutyryl cyclic GMP, stimulated amylase release ceased immediately but cellular 1,2-DAG required at least 15 min to return to the basal level. Increasing cytosolic free Ca2+ with the Ca2+ ionophore, A23187, in Ca2+-containing incubation media augmented amylase release stimulated by 4 beta-phorbol 12-myristate 13-acetate but inhibited amylase release stimulated by CCK-OP, carbachol, and bombesin without decreasing the cellular content of 1,2-DAG. H-7 inhibited protein kinase C activity in a pancreatic homogenate but augmented amylase release from acini stimulated by either CCK-OP, carbachol, or 4 beta-phorbol 12-myristate 13-acetate. These findings indicate that 1,2-DAG and protein kinase C do not have a stimulatory role in pancreatic stimulus-secretion coupling but may have an inhibitory one.     

Involvement of protein kinase C in translocation of desmoplakins from cytosol to plasma membrane during desmosome formation in human squamous cell carcinoma cells grown in low to normal calcium concentration

The intracellular signal transduction mechanism leading to desmosome formation in low-calcium-grown keratinocytes after addition of calcium to the medium was studied by immunofluorescence using antibodies to desmoplakins I and II (cytoplasmic desmosomal proteins) and by electron microscopy before and after addition of calcium; protein kinase C (PKC) activators 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol-12,13-dibutyrate (PDBu), and 1,2-dioctanoylglycerol (DOG); calcium ionophore A23187; selective PKC inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and staurosporine; and a Ca2+/calmodulin-dependent kinase inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). In previous studies using a low-calcium-grown human epidermal squamous cell carcinoma, we have shown that an increase in extracellular Ca2+ caused a four-fold increase in PKC activity and addition of TPA (10 ng/ml) induced a transient increase in membrane-bound PKC activity in association with cell-cell contact formation. The present study showed that TPA (10 ng/ml). PDBu (10 ng/ml), and DOG (1 mg/ml) induced a rapid cell-cell contact and redistribution of desmoplakins from cytoplasm to the plasma membrane with desmosome formation within 60-120 min, which was similar, although less marked, to the effect of increased Ca2+. The TPA-induced desmosome formation was inhibited by selective PKC inhibitors, H-7 (20 microM) or staurosporine (100 nM). On the other hand, calcium ionophore A23187 induced only a temporary increase in the number of desmoplakin-containing fluorescent spots in the cytoplasm and a temporary cell-cell attachment without desmosome formation. The calcium-induced desmosome formation was partially inhibited by 20-100 microM H-7 or 100 nM staurosporine; however, it was not inhibited by W-7 at a concentration of 25 microM, at which this agent selectively inhibits calmodulin-dependent protein kinase. These results suggest that PKC activation plays an important role in desmoplakin translocation from the cytoplasm to the plasma membrane as one of the processes of calcium-induced desmosome formation.     

The timing of protein kinase activation events in the cascade that regulates mitotic progression in Tradescantia stamen hair cells.

Stamen hair cells of the spiderwort plant Tradescantia virginiana exhibit unusually predictable rates of progression through mitosis, particularly from the time of nuclear envelope breakdown (NEBD) through the initiation of cytokinesis. The predictable rate of progression through prometaphase and metaphase has made these cells a useful model system for the determination of the timing of regulatory events that trigger entry into anaphase. A number of studies suggest that the elevation of one or more protein kinase activities is a necessary prerequisite for entry into anaphase. The current experiments employ two strategies to test when these elevations in protein kinase activity actually occur during metaphase. In perfusions, we added the protein kinase inhibitors K-252a, staurosporine, or calphostin C to living stamen hair cells for 10-min intervals at known times during prometaphase or metaphase and monitored the subsequent rate of progression into anaphase. Metaphase transit times were altered as a function of the time of addition of K-252a or staurosporine to the cells; metaphase transit times were extended significantly by treatments initiated in prometaphase through early metaphase and again late in metaphase. Transit times were normal after treatments initiated in mid-metaphase, approximately 15 to 21 min after NEBD. Calphostin C had no significant effect on the metaphase transit times. In parallel, cells were microinjected with known quantities of a general-purpose protein kinase substrate peptide, VRKRTLRRL, at predefined time points during prometaphase and metaphase. At a cytosolic concentration of 100 nM to 1 microM, the peptide doubled or tripled the metaphase transit times when injected into the cytosol of mitotic cells within the first 4 min after NEBD, at any point from 7.5 to 9 min after NEBD, at any point from 14 to 16 min after NEBD, at 21 min after NEBD, or at 24 min after NEBD. At the concentration used and during these brief intervals, the peptide appeared to act as a competitive inhibitor to reveal inflection points when protein kinase activation was occurring or when endogenous substrate levels approached levels of the peptide. The timing of these inflection points coincides with the changes in protein kinase activities during prometaphase and metaphase, as indicated by our perfusions of cells with the broad spectrum kinase inhibitors. Collectively, our results suggest that the cascade that culminates in anaphase is complex and involves several successive protein kinase activation steps punctuated by the activation of one or more protein phosphatases in mid-metaphase.     

Mitotic progression in stamen hair cells of Tradescantia is accelerated by treatment with ruthenium red and Bay K-8644

The normally predictable duration of metaphase in stamen hair cells from the spiderwort, Tradescantia virginiana, is shortened significantly by treatment during prometaphase with either ruthenium red or Bay K-8644. Ruthenium red is an inhibitor of Ca2+ translocation and Bay K-8644 is a Ca2+-channel agonist. Their action on mitotic progression appears to involve a rise in the cytosolic Ca2+ level that in turn has a pronounced effect on the duration of metaphase. The timing of addition of ruthenium red for accelerated progression through metaphase is less critical than that for Bay K-8644 which will promote metaphase progression only if added 0 to 12 min after nuclear envelope breakdown. In contrast, ruthenium red can be added at any time from approximately 10 min prior to nuclear envelope breakdown up to 25 min afterward. A reduction of extracellular Ca2+ is sufficient by itself to prolong the duration of metaphase in stamen hair cells, but the duration of metaphase by ruthenium red or Bay K-8644 is significantly shortened in identical solutions with Ca2+ buffered at levels greater than 1 microM. Metaphase progression rates with either agent are independent of changes in extracellular Mg2+ levels. Correlated with the precocious entry into anaphase was rapid formation of the spindle and a marked reduction in spindle rotation during metaphase. Interestingly, we observed a modest increase in the rate of anaphase chromosome separation, but the appearance of cell plate vesicles at the site of incipient cell plate formation occurred normally approximately 19 min after anaphase onset. Similarly, the initial appearance of cell plate vesicles in Bay K-8644 was normal, approximately 19 min after the onset of anaphase. These results further implicate shifts in cytosolic Ca2+ in the regulation of mitotic events.     

Diacylglycerol induces deacylation of phosphatidylinositol and mobilization of arachidonic acid in mouse macrophages. Comparison with induction by phorbol diester.

1,2-Dioctanoyl-sn-glycerol (2-50 microM) was found, like phorbol myristate acetate (greater than or equal to 3 nM) to stimulate phospholipase A-type cleavage of phosphatidylinositol and the release of arachidonic acid from macrophage phospholipids. The 1,3 isomer of dioctanoylglycerol was inactive, whereas racemic 1,2-dioctanoylglycerol was half as potent as the 1,2-sn enantiomer. Dioctanoylglycerol-induced deacylation of phosphatidylinositol was only partly dependent on extracellular calcium but was more severely inhibited by depletion of intracellular calcium. Chlorpromazine inhibited the deacylation of phosphatidylinositol, whereas inhibitors of cyclo-oxygenase and lipoxygenase were ineffective. Since both phorbol myristate acetate and 1,2-dioctanoyl-sn-glycerol are known to activate protein kinase C, the results suggest that this kinase is involved in the sequence of events leading to release of arachidonic acid in macrophages.     

Nifedipine reversibly arrests mitosis in stamen hair cells of tradescantia

Mitotic stamen hair cells of Tradescantia virginiana (cv. Zwanenburg Blue) become arrested in metaphase following a 30-min treatment with 10 to 100 microM nifedipine, a Ca2+-channel entry blocker. The time interval between nuclear envelope breakdown and anaphase onset in untreated cells is approximately 33 min +/- 4 min; nifedipine extends this "metaphase transit time" beyond 70 min. Nifedipine can be photoreversed in situ by exposure to 365 nm light. UV illumination inactivates the drug, its inhibitory effect on Ca2+ is abolished, and cells arrested in metaphase enter anaphase within 3 to 18 min of UV exposure if CaCl2 is present in the medium. The interval between UV illumination and anaphase onset is inversely related to the extracellular concentration of CaCl2. If CaCl2 is not added to the medium, the interval between UV exposure and anaphase onset is usually longer than 18 min. The sole addition of 100 microM CaCl2 to the medium is insufficient to reverse nifedipine inhibition; unless the cells are exposed to UV light, anaphase will not commence. The threshold concentration of free Ca2+ for rapid anaphase onset (less than 10 min after UV photoreversal) is between 1 and 10 microM. These results suggest that an influx of Ca2+ from the extracellular medium to the cytosolic compartment is necessary for normal progression from metaphase to anaphase and that this influx may serve as a trigger for chromosome separation.     

Platelet-derived growth factor stimulates synthesis of 1,2-diacylglycerol from monoacylglycerol in Balb/c 3T3 cells.

1,2-Diacylglycerol (1,2,-DAG) plays an important role in the protein kinase C-mediated signal-transduction system. Several reports have shown that 1,2-DAG is generated through various pathways other than classical phospholipid hydrolysis. We observed a rapid incorporation of [3H]myristate into 1,2-DAG in platelet-derived-growth-factor (PDGF)-treated Balb/c 3T3 cells. [14C]Palmitate was similarly incorporated into 1,2-DAG. The effect of PDGF, which was inhibited by cycloheximide, became maximal after 60 min treatment with PDGF, and disappeared 300 min after removal of PDGF. Treatment with triacylglycerol lipase revealed that labelled saturated fatty acid was incorporated into the sn-1 position. PDGF barely stimulated incorporation of [3H]glycerol or [14C]glucose into 1,2-DAG. Incorporation of [3H]myristate into 1,2-DAG preceded that into triacyglycerol and phospholipids. These results suggest that synthesis of 1,2-DAG from monoacylglycerol is enhanced in PDGF-treated cells. However, there is no significant difference in the activity of monoacylglycerol acyltransferase measured in vitro in quiescent and PDGF-treated cells. The reason for this discrepancy is discussed.     

Calcium restriction prolongs metaphase in dividing Tradescantia stamen hair cells

Agents that lower extracellular calcium concentration (EGTA) or modulate calcium transport (lanthanum or D600) have been applied to dividing stamen hair cells of Tradescantia and analyzed for their ability to change the following: (a) the time required to progress from nuclear envelope breakdown to the onset of anaphase (metaphase transit time), (b) the time required to progress from anaphase to the initiation of the cell plate, and (c) the rate of chromosome motion in anaphase. Control cells complete metaphase in 32 min, initiate a cell plate in 19 min, and display a chromosome motion rate of 1.45 micron/min. If cells are treated with a calcium-EGTA buffer (pCa 8) for 4 h, the metaphase transit time is increased to 53 min without any change in the time of cell plate formation or the rate of chromosome motion. Lanthanum and D600, under conditions in which their access to the plasmalemma has been facilitated by pretreating the cells with cutinase, also markedly extend metaphase and in several instances permanently arrest cells. Lanthanum, however, produce little or no change in cell plate initiation or the rate of chromosome motion. Microscopic observations of the mitotic apparatus in calcium-stressed cells reveal normal chromatin condensation and metaphase progression. Chromosomes partly untwine but remain attached at their kinetochores. It is suggested that a flux of calcium, derived from the extracellular compartment, may cause the final splitting of sister chromosomes and trigger the onset of anaphase. However, once anaphase has begun, chromosome motion and cell plate initiation proceed normally even under conditions of extracellular calcium restriction.     

Lithium alters mitotic progression in stamen hair cells of Tradescantia in a time-dependent and reversible fashion

Stamen hair cells of Tradescantia exhibit remarkable precision in the timing of their mitotic events. This precision is altered dramatically with treatment in 50 microM to 1 mM LiCl, an inhibitor of the polyphosphoinositide cycle. Mitotic progression is altered as a function of the time of treatment with LiCl. If cells are treated during late prophase, greater than 80% fail to enter metaphase. Most of the cells that undergo nuclear envelope breakdown become arrested in metaphase. Treatment with LiCl earlier in prophase also results in metaphase arrest. Metaphase arrest can be reversed by the addition of 10 microM myo-inositol or 100 microM CaCl2 to the extracellular medium. The timing of reversal by myo-inositol takes 10 to 14 min while CaCl2 promotes anaphase onset in 2 to 5 min. The difference in kinetics for reversal between these two treatments suggests that myo-inositol addition overrides a biochemical pathway while Ca2+ addition supplants a phosphoinositide-mediated rise in the cation that may be necessary for anaphase onset. Buffer without myo-inositol or CaCl2 is insufficient for reversal. If the cells are treated with LiCl in mid-late-metaphase, at least 5 min prior to the expected time of anaphase onset, sister chromatids split at the normal time, 33 +/- 4 min after nuclear envelope breakdown, but further chromosome separation is arrested. Anaphase chromosome movement can be restored by treatment with either 10 microM myo-inositol or 100 microM CaCl2 in the medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein tyrosine phosphorylation in rabbit peritoneal neutrophils.

Protein tyrosine phosphorylation in rabbit peritoneal neutrophils was examined by immunoblotting with antibodies specific for phosphotyrosine. Stimulation of the neutrophils with chemotactic factor fMet-Leu-Phe (10 nM) caused rapid increases of tyrosine phosphorylation of several proteins with apparent molecular masses of (Group A) 54-58 kDa and 100-125 kDa and (Group B) 36-41 kDa. Stimulation of Group A proteins was observed by fMet-Leu-Phe (10 nM, maximum at 20 s) and A23187 (1 microM, 1 min). Stimulation of Group B proteins was observed by fMet-Leu-Phe (ED50 0.15 nM, 1 min), leukotriene B4 (ED50 0.15 nM, 1 min), phorbol 12-myristate 13-acetate (PMA) (ED50 25 ng/ml, 10 min) and partially by ionophore A23187 (1 microM, 1 min). Pretreatment of the cell with the protein kinase inhibitor H-7 (25 microM, 5 min) and PMA (0.1 microgram/ml, 3 min) partially inhibited the fMet-Leu-Phe effect. However, pretreatment of the cells with quin 2/AM (20 microM, 10 min) completely inhibited the fMet-Leu-Phe effect. The results indicate that rapid regulation of tyrosine phosphorylation is an early event occurring in stimulated neutrophils. Furthermore the effect of fMet-Leu-Phe on tyrosine phosphorylation may require Ca2+ mobilization and may partially require the activity of H-7-sensitive protein kinases.     

Diacylglycerol and calcium induce rapid enhancement of A-system amino acid transport by independent mechanisms in human T-lymphocytes

Sn-1,2-diacylglycerols (DAG) and ionized-free calcium can act as intracellular second messengers for cell activation. Traditionally, T-lymphocyte activation is assessed by measurements of DNA synthesis or lymphokine production, but these responses require several days to occur and involve multiple intermediary regulatory steps. In contrast, we have found that T-lymphocytes demonstrate rapid enhancement of A-(alanine-favoring) system amino acid uptake when treated with DAG or ionomycin. A 30-40% increase in the initial velocity of uptake (vi) of the synthetic A-system specific amino acid, methylamino-isobutyric acid (MeAIB), was measured following 5 min of exposure to DAG or ionomycin. The vi was enhanced 60% from 12 to 19 mumol/liter cell water per min after 30 min exposure of T-cells to optimal concentrations of dioctanoylglycerol (30 microM), oleoylacetylglycerol (30 microM), or ionomycin (5 microM) (P less than .01 for each agent). A 50-fold excess of non-radioactive MeAIB inhibited 80% of [14C]MeAIB uptake in both unstimulated and stimulated cells, indicating that uptake remained largely carrier-mediated on treatment with these agents. Cycloheximide, 100 micrograms/ml, inhibited protein synthesis but did not block the A-system amino acid transport enhancement induced by DAG or ionomycin. The DAG-induced increase in the vi was blocked 40% with 100 microM H-7, an inhibitor of protein kinase C. H-7 treatment did not inhibit the ionomycin-induced A-system enhancement. A marked increase in cytoplasmic free calcium was measured when T-lymphocytes were exposed to ionomycin but not on DAG exposure, and the A-system effect of ionomycin but not DAG was blocked by extracellular EGTA. These data are compatible with two pathways for rapid enhancement of A-system amino acid uptake in T-lymphocytes. DAG stimulation is mediated via protein kinase C whereas ionomycin produces an A-system effect of similar magnitude independent of protein kinase C by an increase in cytoplasmic calcium.     

Selective priming of rate and duration of the respiratory burst of neutrophils by 1,2-diacyl and 1-O-alkyl-2-acyl diglycerides. Possible relation to effects on protein kinase C

Both 1,2-diacyl- and 1-O-alkyl-2-acyl-sn-glycerols are released during stimulation of human polymorphonuclear leukocytes (PMNL). 1,2-Diacylglycerols have received intense interest as intracellular "second messengers" due to their ability to activate protein kinase C (Ca2+ phospholipid-dependent enzyme). However, little is known about bioactivities of the alkylacylglycerols. This study compared the ability of 1,2-diacyl- and 1-O-alkyl-2-acylglycerols to modulate the respiratory burst of stimulated PMNL, a response which depends on the activation of an NADPH oxidase to generate bactericidal species of reduced oxygen. Direct stimulation by N-formyl-Met-Leu-Phe caused an abrupt release of H2O2 which ceased within 2.5 min. Preincubation with diacylglycerols (1-oleoyl-2-acetylglycerol,5-30 microM, and 1,2-dioctanoylglycerol,2-5 microM) caused a decrease in lag time, 3-fold increase in initial rate of H2O2 release, and marked prolongation of the response to N-formyl-Met-Leu-Phe (features characteristic of a priming effect). Preincubation with alkylacylglycerols (1-O-delta 9-octadecenyl-2-acetylglycerol, 5-30 microM, and 1-O-octyl-2-octanoylglycerol, 20-50 microM) primed initiation (shortened lag time and increased velocity) but, in contrast to diacylglycerols, did not alter duration of H2O2 release. While low concentrations of diacylglycerols (5-30 microM) primed PMNL, higher concentrations (greater than or equal to 70 microM) stimulated the cells directly. In contrast, higher (70-100 microM) concentrations of alkylacylglycerols did not prime the responses but, in fact, inhibited priming (especially of duration) induced by diacylglycerol. The high concentrations of alkylacylglycerol also inhibited direct stimulation induced by high concentrations of diacylglycerol. Direct stimulation by high concentrations of diacylglycerol probably involves activation of protein kinase C, whereas alkylacylglycerol was found to inhibit activation of protein kinase C by diacylglycerol in vitro. Thus, diacylglycerols are complete priming agonists, altering both rate and duration of the response. In contrast, alkylacylglycerols may have biphasic, concentration-related effects in modulation of functions of PMNL. At low concentrations, they may facilitate initiation of functional events; however, as their concentration increases, they may serve to terminate responses. The distinct priming effects of these diglycerides also reveal that priming can involve at least two distinct events: 1) initiation and 2) prolongation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activation of protein kinase C modulates the adenylate cyclase effector system of B-lymphocytes

Antibodies to surface immunoglobulins activate inositol phospholipid hydrolysis in B-lymphocytes, but very little is known concerning their effects on cAMP levels. In other cells, products from the hydrolysis of phosphatidylinositol 4,5-bisphosphate can increase and/or potentiate cAMP accumulation. In this study we have examined whether goat anti-mouse IgM (mu-chain-specific) stimulates and/or potentiates increases in the cAMP levels of splenocytes from athymic nude mice. Goat anti-mouse IgM, by itself, stimulated a 60% increase in cAMP within 2 min. Pretreating the cell suspensions at 37 degrees C with anti-IgM produced opposite effects on the forskolin- and prostaglandin E1 (PGE1)-induced increase in cAMP. Anti-IgM (25 micrograms/ml) potentiated the rise in cAMP induced by 100 microM forskolin 76%, but it decreased the response to 50 nM PGE1 by 30%. Direct activation of protein kinase C (Ca2+/phospholipid-dependent enzyme) by 12-O-tetradecanoylphorbol 13-acetate and/or sn-1,2-dioctanoylglycerol resulted in a similar pattern of responses. A 3-min preincubation with 97 nM 12-O-tetradecanoylphorbol 13-acetate potentiated the forskolin-induced response from 1.7 +/- 0.1 to 4.3 +/- 0.6 pmol of cAMP/10(6) cells but reduced the PGE1 response from 0.98 +/- 0.06 to 0.51 +/- 0.03 pmol of cAMP/10(6) cells. Similarly, preincubating the cells for 3 min with 5 microM sn-1,2-dioctanoylglycerol increased the forskolin response from 1.7 +/- 0.1 to 5.1 +/- 0.2 pmol of cAMP/10(6) cells but reduced the response to PGE1 from 1.15 +/- 0.03 to 0.75 +/- 0.04 pmol of cAMP/10(6) cells. Thus, activation of protein kinase C by hydrolysis products of inositol phospholipids, 12-O-tetradecanoylphorbol 13-acetate, or exogenous diacylglycerols modified adenylate cyclase itself and sites upstream of adenylate cyclase such as the receptor or G proteins coupling the receptor to the cyclase. Furthermore, modification of the PGE1 response by anti-IgM provides a mechanism by which antigen can differentially regulate T- and B-cells responding to macrophage-produced prostaglandins during an immune response.     

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.     

Phorbol ester and 1-oleoyl-2-acetylglycerol inhibit angiotensin activation of phospholipase C in cultured vascular smooth muscle cells

Angiotensin II acts on cultured rat aortic vascular smooth muscle cells (VSMC) to induce the rapid, phospholipase C-mediated generation of inositol trisphosphate from phosphatidylinositol 4,5-bisphosphate and mobilization of intracellular Ca2+. sn-1,2-Diacylglycerol, the other major product of inositol phospholipid breakdown, is known to activate protein kinase C, but its role in angiotensin II action on VSMC has not been defined. We report herein that, in cultured VSMC prelabeled with [3H]myoinositol, brief incubations (2-5 min) with 4 beta-phorbol 12-myristate 13-acetate (PMA) (1-100 nM) or 1-oleoyl-2-acetylglycerol (10-100 microM), two potent activators of protein kinase C, inhibit subsequent angiotensin II (100 nM)-induced increases in phosphatidylinositol 4,5-bisphosphate breakdown and inositol trisphosphate formation. In addition, pretreatment of VSMC with either PMA (IC50 approximately 1 nM) or 1-oleoyl-2-acetylglycerol (IC50 approximately 7.5 microM) also markedly inhibits angiotensin II (1 nM)-stimulated increases in cytosolic free Ca2+, as measured with the calcium-sensitive fluorescent indicator quin 2, or 45Ca2+ efflux. Neither PMA nor 1-oleoyl-2-acetylglycerol initiated phosphatidylinositol 4,5-bisphosphate breakdown or Ca2+ flux by itself. PMA treatment (10 nM, 5 min) did not influence the number or affinity of 125I-angiotensin II-binding sites in intact cells. These data suggest that one function of angiotensin II-generated sn-1,2-diacylglycerol in vascular smooth muscle may be to modulate, by protein kinase C-mediated mechanisms, angiotensin II receptor coupling to phospholipase C.     

Differential regulation of phosphatidylcholine biosynthesis by 12-O-tetradecanoylphorbol-13-acetate and diacylglycerol in NG108-15 neuroblastoma x glioma hybrid cells

12-O-Tetradecanoylphorbol-13-acetate (TPA), a tumor promoter and potent activator of protein kinase C, stimulates [3H]choline incorporation into phosphatidylcholine (PtdCho) in NG108-15 cells (Liscovitch, M., Freese, A., Blusztajn, J. K. and Wurtman, R. J. (1986) J. Neurochem. 47, 1936-1941). In the present study we demonstrate that two cell-permeant diacylglycerols, sn-1-oleoyl-2-acetylglycerol and sn-1,2-dioctanoylglycerol, also stimulate [3H]choline incorporation into PtdCho. However, the effect of diacylglycerol is additional to that produced by a maximally effective concentration of TPA (0.5 microM), suggesting that the two agents may not act via the same mechanism. In addition, the protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (at 200 microM) inhibits the action of TPA by 59% while not affecting that of diacylglycerol. Finally, preincubation of the cells with TPA (0.1 microM) for 24 h reduces protein kinase C activity in the cells and completely abolishes the effect of additional TPA on choline incorporation. In contrast, diacylglycerol-induced stimulation of PtdCho biosynthesis was not inhibited in the cells that were desensitized to TPA. These results suggest that the effect of the two cell-permeant diacylglycerols on PtdCho biosynthesis either is not mediated by protein kinase C activation, or, is mediated by a TPA-insensitive isoenzyme of protein kinase C.     

Trifluoperazine stimulates the coordinate degradation of sphingomyelin and phosphatidylcholine in GH3 pituitary cells

Prior studies demonstrated that 1,2-diacylglycerols stimulated degradation of the choline-containing phospholipids, phosphatidylcholine and sphingomyelin, in GH3 pituitary cells by a phospholipase A2 and a sphingomyelinase, respectively (Kolesnick, R. N. (1987) J. Biol. Chem. 262, 16759-16762). The present studies demonstrate that the phenothiazine trifluoperazine also stimulates degradation of these phospholipids. Trifluoperazine (25 microM) reduced phosphatidylcholine and sphingomyelin levels to 81 and 58% of control, respectively, after 30 min in cells labeled for 48 h with [3H] choline. Choline-containing metabolites were released specifically into the cytosolic fraction. The level of cytosolic phosphocholine, but not choline or CDP-choline, increased to 150% of control. These events were not mediated by inhibition of phosphatidylcholine synthesis. The level of 1,2-diacylglycerols, but not lysophosphatidylcholine or glycerol-3-phosphocholine, also increased. These data are most consistent with phosphatidylcholine degradation via a phospholipase C. Trifluoperazine-stimulated sphingomyelin degradation was accompanied by quantitative generation of ceramides consistent with activation of a sphingomyelinase. In contrast to trifluoperazine, choline-containing metabolites were released into the medium during stimulation by the 1,2-diacylglycerol 1,2-dioctanoyl-glycerol. Although both trifluoperazine and 1,2-dioctanoylglycerol increased ceramide levels, only 1,2-dioctanoylglycerol increased the sphingoid base level from 24 to 43 pmol/10(6) cells. Hence, trifluoperazine appears to deplete an intracellular pool of phosphatidylcholine and sphingomyelin by a different mechanism than 1,2-diacylglycerols. This is the first report of phenothiazine-induced degradation of choline-containing phospholipids.     

Protein kinase C activators suppress stimulation of capillary endothelial cell growth by angiogenic endothelial mitogens

The intracellular events regulating endothelial cell proliferation and organization into formalized capillaries are not known. We report that the protein kinase C activator beta-phorbol 12,13-dibutyrate (PDBu) suppresses bovine capillary endothelial (BCE) cell proliferation (K50 = 6 +/- 4 nM) and DNA synthesis in response to human hepatoma-derived growth factor, an angiogenic endothelial mitogen. In contrast, PDBu has no effect on the proliferation of bovine aortic endothelial cells and is mitogenic for bovine aortic smooth muscle and BALB/c 3T3 cells. Several observations indicate that the inhibition of human hepatoma-derived growth factor-stimulated BCE cell growth by PDBu is mediated through protein kinase C. Different phorbol compounds inhibit BCE cell growth according to their potencies as protein kinase C activators (12-O-tetradecanoylphorbol 13-acetate greater than PDBu much greater than beta-phorbol 12,13-diacetate much much greater than beta-phorbol; alpha-phorbol 12,13-dibutyrate; alpha-phorbol 12,13-didecanoate). PDBu binds to a single class of specific, saturable sites on the BCE cell with an apparent Kd of 8 nM, in agreement with reported affinities of PDBu for protein kinase C in other systems. Specific binding of PDBu to BCE cells is displaced by sn-1,2-dioctanoylglycerol, a protein kinase C activator and an analog of the putative second messenger activating this kinase in vivo. The weak protein kinase C activator, sn-1,2-dibutyrylglycerol, does not affect PDBu binding. A cytosolic extract from BCE cells contains a calcium/phosphatidylserine-dependent protein kinase that is activated by sn-1,2-dioctanoylglycerol and PDBu, but not by beta-phorbol. These findings indicate that protein kinase C activation can cause capillary endothelial cells to become desensitized to angiogenic endothelial mitogens. This intracellular regulatory mechanism might be invoked during certain phases of angiogenesis, for example when proliferating endothelial cells become differentiated to organize into nongrowing tubes.