Role of membrane associated serine esterase in the activation of phospholipase A2 by calcium ionophore (A23187) in pulmonary arterial smooth muscle cells

Exposure of rabbit pulmonary arterial smooth muscle cells to 10 M of the calcium ionophore A23187 dramatically stimulates cell membrane-associated phospholipase A₂ activity and arachidonic acid release. In addition, A23187 also enhances cell membrane-associated serine esterase activity. Serine esterase inhibitors phenylmethylsulfonylfuoride and diisopropyl fluorophosphate prevent the increase in serine esterase and phospholipase A₂ activities and arachidonic acid release caused by A23187. A23187 still stimulated serine esterase and phospholipase A₂ activities and arachidonic acid release in cells pretreated with nominal Ca²⁺ free buffer. Treatment of the cell membrane with A23187 does not cause any appreciable change in serine esterase and phospholipase A₂ activities. Pretreatment of the cells with actinomycin D or cycloheximide did not prevent the increase in the cell membrane associated serine esterase and phospholipase A₂ activities, and arachidonic acid release caused by A23187. These results suggest that (i) a membrane-associated serine esterase plays an important role in stimulating the smooth muscle cell membrane associated phospholipase A₂ activity (ii) in addition to the presence of extracellular Ca²⁺, release of Ca²⁺ from intracellular storage site(s) by A23187 also appears to play a role in stimulating the cell membrane-associated serine esterase and phospholipase A₂ activities, and (iii) the increase in the cell membrane-associated serine esterase and phospholipase A₂ activities does not appear to require new RNA or protein synthesis.Abbreviations A23187 calcium ionophore - AA arachidonic acid - PMSF phenylmethyl sulfonylfuoride - DFP diisopropyl-fluorophosphate - DMEM Dulbecco's modified Eagles medium - FCS fetal calf serum - PBS phosphate buffered saline - HBPS Hank's buffered physiological saline - PLA₂ phospholipase A₂     

Hydrolysis of exogenous [3H]phosphatidylcholine by brain membranes and cytosol

Phosphatidylcholine, in addition to the widely studied inositol phospholipids, is cleaved to produce second messengers in neuronal signal transduction processes. Because of the difficulty in labelling and measuring the metabolism of endogenous phosphatidylcholine in brain tissue, we investigated the utility of measuring the hydrolysis of exogenous labelled substrate incubated with rat cerebral cortical cytosol and membrane fractions as has been successful in studies of phosphoinositide hydrolysis. In the cytosol [³H]phosphatidylcholine was hydrolyzed at a linear rate for 60 min of incubation and GTPS stimulated hydrolysis by 63%. The products of phospholipase C and phospholipase D, phosphorylcholine and choline, contributed only 44% of the [³H]phosphatidylcholine hydrolytic products in the cytosol, with phospholipase D activity slightly predominating. GTPS stimulated cytosolic phospholipase C and reduced phospholipase D activity. [³H]Phosphatidylcholine was hydrolyzed much more slowly by membranes than by cytosol. In membranes the production of [³H]phosphorylcholine and [³H]choline were approximately equal, contributing 27% of the total [³H]phosphatidylcholine hydrolysis, and GTPS only caused a slight stimulation of phospholipase C activity. Chronic lithium treatment (4 weeks) appeared to slightly reduce [³H]phosphatidylcholine metabolism in the cytosol and in membranes, but no statistically significant reductions were achieved. Cytosol and membrane fractions from postmortem human brain metabolized [³H]phosphatidylcholine slowly, and GTPS had no effects. In summary, exogenous [³H]phosphatidylcholine was hydrolyzed by brain cytosol and membranes, and this was stimulated by GTPS, but the complex contributions of multiple metabolic pathways complicates the application of this method for studying individual pathways, such as phospholipase D which contributes only a fraction of the total processes hydrolyzing exogenous [³H]phosphatidylcholine.     

Cell signalling through phospholipid breakdown

There is much evidence that G-proteins transduce the signal from receptors for Ca²⁺-mobilizing agonists to the phospholipase C that catalyzes the hydrolysis of phosphoinositides. However, the specific G-proteins involved have not been identified. We have recently purified a 42 kDa protein from liver that activates phosphoinositide phospholipase C and cross-reacts with antisera to a peptide common to G-protein -subunits. It is proposed that this protein is the a-subunit of the G-protein that regulates the phospholipase in this tissue.Ca²⁺-mobilizing agonists and certain growth factors also promote the hydrolysis of phosphatidylcholine through the activation of phospholipases C and D in many cell types. This yields a larger amount of diacylglycerol for a longer time than does the hydrolysis of inositol phospholipids. Consequently phosphatidylcholine breakdown is probably a major factor in long-term regulation of protein kinase C. The functions of phosphatidic acid produced by phospholipase D are speculative, but there is evidence that it is a major source of diacylglycerol in many cell types. The regulation of phosphatidylcholine phospholipases is multiple and involves direct activation by G-proteins, and regulation by Ca²⁺ protein kinase C and perhaps growth factor receptor tyrosine kinases.     

Parameters not influenced by vesamicol: Membrane potential,calcium uptake,and internal calcium concentration of synaptosomes

In our previous study vesamicol, an inhibitor of the acetylcholine transporter of the cholinergic vesicles, inhibited veratridine-evoked external Ca²⁺-dependent acetylcholine release from striatal slices but did not influence acetylcholine release observed in Ca²⁺-free medium (4). Here we examined if the effect of veratridine on membrane potential, Ca²⁺ uptake, and intracellular Ca²⁺ concentration of synaptosomes was altered by vesamicol in parallel with the inhibition of acetylcholine release. The depolarizing effect of 10 M veratridine (from 67±2.3 mV resting membrane potential to 50.7±2.5 mV) was not significantly influenced by vesamicol (1–20 M). Vesamicol (1–20 M) had no effect on either the overall curve of the veratridine-evoked⁴⁵Ca²⁺ uptake or the amount of Ca²⁺ taken up by synaptosomes. Veratridine caused a rise in intrasynaptosomal Ca²⁺ concentration as measured by Fura2 fluorescence, and the same increase both in characteristics and in magnitude was observed in the presence of vesamicol (20 M). The K⁺-evoked (40 mM) increase of Ca²⁺ uptake and of intracellular calcium concentration were also unaltered by vesamicol. In high concentration (50 M) vesamicol inhibited both the fall in membrane potential and the elevated Ca²⁺ uptake by veratridine, indicating a possible nonspecific effect on potential-dependent Na⁺ channels at this concentration. Vesamicol, in lower concentration (20 M) when neither of the above parameters was changed, completely prevented veratridine-evoked increase of [¹⁴C]acetylcholine release. This was observed only when vesamicol was present in the media throughout the experiment after loading the preparation with [¹⁴C]choline. The results suggest that vesamicol does not interfere with veratridine-induced changes in isolated nerve terminals other than with the release of acetylcholine, thus further supporting the involvement of a vesamicol-sensitive vesicular transmitter pool in Ca²⁺-dependent veratridine-elicited acetylcholine release.     

A role for G-proteins in the epidermal growth factor stimulation of phospholipase A2 in rat kidney mesangial cells

We have previously demonstrated phospholipase C (PLC) independent activation of phospholipase A₂(PLA₂) by epidermal growth factor (EGF) in glomerular mesangial cells in culture. In the current study using glass beads to permeabilize [³H]- or [¹⁴C]-arachidonate labelled mesangial cells we demonstrate that guanine nucleotides modulate the EGF-mediated stimulation of arachidonic acid release (75% inhibition with 100 M GDPS and 108% augmentation with 100 M GTPS). GTPS alone stimulated both the release of free arachidonic acid and production of diacylglycerol (DAG), while EGF itself neither stimulated DAG nor augmented the DAG response to GTPS. These findings suggest the intermediacy of a G-protein in PLC-independent stimulation of PLA₂ by a growth factor, and provide a model system for determining the relationship between G-protein intermediacy and the intrinsic tyrosine kinase activity of the growth factor receptor.Abbreviations EGF Epidermal Growth Factor - PLC phospholipase C - PLA₂ phospholipase A₂ - DAG Diacylglycerol - NEFA non-esterified fatty acid - GTPS guanosine-5-0-[3-thio]triphosphate - GDP\S guanosine-5-0-[2-thio]diphosphate     

Studies on the Cytosolic Phospholipase A2 in Immortalized Astrocytes (DITNC) Revealed New Properties of the Calcium Ionophore,A23187

Besides playing an important role in the maintenance of cell membrane phospholipids, phospholipases A₂ (PLA₂) are responsible for the release of arachidonic acid (AA) which is a precursor for prostaglandin biosynthesis. The cytosolic PLA₂ has been the focus of recent studies, probably due to its ability to respond to protein kinases and changes in intracellular calcium levels. In this study, we examined agents for stimulation of the cytosolic phospholipase A₂ in immortalized astrocytes (DITNC). Incubation of DITNC cells with [¹⁴C]arachidonic acid (AA) resulted in a time-dependent uptake of the label into phospholipids (PL) and neutral glycerides. In prelabeled cells, release of labeled AA could be stimulated by calcium mobilizing agents such as calcium ionophore A23187 (4–20 M) and thimerosal (100 M), and by phorbol myristate acetate (PMA, 100 nM), an agent for activation of protein kinase C. The release of AA could also be stimulated by ATP (200 M), probably through activation of the purinergic receptor but not by glutamate (1 mM). The stimulated release of AA was dependent on extracellular Ca²⁺ and was inhibited by mepacrine (50 M), a non-specific PLA₂ inhibitor. Western blot analysis further confirmed the presence of an 85 kDa cPLA₂ in both membrane and cytosol fractions of these cells and stimulation by A23187 resulted in translocation of this protein to the membrane fraction. Besides labeled fatty acids, A23187 also stimulated the concomitant release of labeled PL into the culture medium and this event was accompanied by the increased release in lactate dehydrogenase (LDH). Results thus revealed that besides activation of cPLA₂, the calcium ionophore A23187 is capable of perturbating cell membrane integrity.     

Exendin-4 Protects Mitochondria from Reactive Oxygen Species Induced Apoptosis in Pancreatic Beta Cells

Objective

Mitochondrial oxidative stress is the basis for pancreatic β-cell apoptosis and a common pathway for numerous types of damage, including glucotoxicity and lipotoxicity. We cultivated mice pancreatic β-cell tumor Min6 cell lines in vitro and observed pancreatic β-cell apoptosis and changes in mitochondrial function before and after the addition of Exendin-4. Based on these observations, we discuss the protective role of Exendin-4 against mitochondrial oxidative damage and its relationship with Ca²⁺-independent phospholipase A2.

Methods

We established a pancreatic β-cell oxidative stress damage model using Min6 cell lines cultured in vitro with tert-buty1 hydroperoxide and hydrogen peroxide. We then added Exendin-4 to observe changes in the rate of cell apoptosis (Annexin-V-FITC-PI staining flow cytometry and DNA ladder). We detected the activity of the caspase 3 and 8 apoptotic factors, measured the mitochondrial membrane potential losses and reactive oxygen species production levels, and detected the expression of cytochrome c and Smac/DLAMO in the cytosol and mitochondria, mitochondrial Ca₂-independent phospholipase A2 and Ca²⁺-independent phospholipase A2 mRNA.

Results

The time-concentration curve showed that different percentages of apoptosis occurred at different time-concentrations in tert-buty1 hydroperoxide- and hydrogen peroxide-induced Min6 cells. Incubation with 100 µmol/l of Exendin-4 for 48 hours reduced the Min6 cell apoptosis rate (p<0.05). The mitochondrial membrane potential loss and total reactive oxygen species levels decreased (p<0.05), and the release of cytochrome c and Smac/DLAMO from the mitochondria was reduced. The study also showed that Ca²⁺-independent phospholipase A2 activity was positively related to Exendin-4 activity.

Conclusion

Exendin-4 reduces Min6 cell oxidative damage and the cell apoptosis rate, which may be related to Ca²-independent phospholipase A2.     

Effect of chemical modifiers of amino acid residues on proton conduction by the H+-ATPase of mitochondria

The effect of chemical modifiers of amino acid residues on the proton conductivity of H⁺-ATPase in inside out submitochondrial particles has been studied. Treatment of submitochondrial particles prepared in the presence of EDTA (ESMP) with the arginine modifiers, phenylglyoxal or butanedione, or the tyrosine modifier, tetranitromethane, caused inhibition of the ATPase activity. Phenylglyoxal and tetranitromethane also caused inhibition of the anaerobic release of respiratory H⁺ in ESMP as well as in particles deprived of F₁ (USMP). Butanedione treatment caused, on the contrary, acceleration of anaerobic proton release in both particles. The inhibition of proton release caused by phenylglyoxal and tetranitromethane exhibited in USMP a sigmoidal titration curve. The same inhibitory pattern was observed with oligomycin and withN,N-dicyclohexylcarbodiimide. In ESMP, relaxation of H⁺ exhibited two first-order phases, both an expression of the H⁺ conductivity of the ATPase complex. The rapid phase results from transient enhancement of H⁺ conduction caused by respiratory H⁺ itself. Oligomycin,N,N-dicyclohexylcarbodiimide, and tetranitromethane inhibited both phases of H⁺ release, and butanedione accelerated both. Phenylglyoxal inhibited principally the slow phase of H⁺ conduction. In USMP, H⁺ release followed simple first-order kinetics. Oligomycin depressed H⁺ release, enhanced respiratory H⁺, and restored the biphasicity of H⁺ release. Phenylglyoxal and tetranitromethane inhibited H⁺ release in USMP without modifying its first-order kinetics. Butanedione treatment caused biphasicity of H⁺ release from USMP, introducing a very rapid phase of H⁺ release. Addition of soluble F₁ to USMP also restored biphasicity of H⁺ release. A mechanism of proton conduction by F _o is discussed based on involvement of tyrosine or other hydroxyl residues, in series with the DCCD-reactive acid residue. There are apparently two functionally different species of arginine or other basic residues: those modified by phenylglyoxal, which facilitate H⁺ conduction, and those modified by butanedione, which retard H⁺ diffusion.     

Action of phosphatidylinositol specific phospholipase C on platelets: nonlytic release of acetylcholinesterase, effect on thrombin and PAF induced aggregation

Phosphatidylinositol (PI) specific phospholipase C treatment of rabbit platelets caused 95% release of acetylcholinesterase in the supernatant and 4 to 6% hydrolysis of membrane PI in 2 min. Under these conditions there was no cell lysis as monitored by lack of lactate dehydrogenase activity in the medium. The phospholipase C had no activity towards phosphatidylinositol-4- phosphate and phosphatidylinositol-4,5-bis phosphate. Platelets pretreated with the phospholipase C responded normally to thrombin and platelet activating factor. It is concluded that acetylcholinesterase exists in specific interaction with PI in platelet membranes. Further, the membrane protein release phenomenon caused by the PI-specific phospholipase C did not effect the physiological responsiveness of platelets. Possible implications of these findings to the linkage between PI and membrane enzyme are also discussed.     

Regulation of the production of the agglutination substances responsible for sexual agglutination in Saccharomyces cerevisiae: Changes associated with conjugation and temperature shift

Summary Isolated zygotes showed self-agglutination caused by the sex-specific glycoproteins, the agglutination substances responsible for sexual agglutination. The agglutination substances of both a and mating types were detected in the extracts obtained by the autoclave method from zygotes. Although the first diploid daughter cells from zygotes showed self-agglutinability, the self-agglutinability decreased gradually in the successive diploid daughter cells. The self-agglutination in diploid cells was also brought about by the complementary binding of the sex-specific agglutination substances of opposite mating types.The constitutive sexual agglutinability in a and cells was lost with concomitant loss of the agglutination substances in both cell wall and cytoplasmic fractions when cultured at a temperature higher than 35°C.The repression of the production of the agglutination substances was reversed by the opposite mating type pheromones even at the repressive temperature, 36°C, associated with the appearance of sexual agglutinability. The sex pheromones, a substance-I and substance-I, and the binding substance for substance-I were produced even at 36°C, repressive for the production of the agglutination substances.     

Alterations in Ca2+/Mg2+ ATPase activity upon treatment of heart sarcolemma with phospholipases

In order to examine the role of phospholipids in the activation of membrane bound Ca²⁺/Mg²⁺ ATPase, the activities of Ca²⁺ ATPase and Mg²⁺ ATPase were studied in heart sarcolemma after treatments with phospholipases A, C and D. The Mg²⁺ ATPase activity was decreased upon treating the sarcolemmal membranes with phospholipases, A, C and D; phospholipase A produced the most dramatic effect. The reduction in Mg², ATPase activity by each phospholipase treatment was associated with a decrease in the V_max value without any changes in the K_a value. The depression of Mg²⁺ ATPase in the phospholipase treated preparations was not found to be due to release of fatty acids in the medium and was not restored upon reconstitution of these membranes by the addition of synthetic phospholipids such as lecithin, lysolecithin or phosphatidic acid. In contrast to the Mg²⁺ ATPase, the sarcolemmal Ca²⁺ ATPase was affected only slightly by phospholipase treatments. The greater sensitivity of Mg^- ATPase to phospholipase treatments was also apparent when deoxycholate-treated preparations were employed. These results indicate that glycerophospholipids are required for the sarcolemmal Mg²⁺ ATPase activity to a greater extent in comparison to that for the Ca²⁺ ATPase activity and the phospholipids associated with Mg²⁺ ATPase are predominantly exposed at the outer surface of the membrane.     

Optimized Binding of [35S]GTPγS to Gq-Like Proteins Stimulated with Dopamine D1-Like Receptor Agonists

Subtypes of dopamine D₁-like receptors are coupled through the G proteins Gs or Gq to stimulate either adenylate cyclase or phospholipase C signaling cascades. In the present study, we have uncovered the marked enhancement by sodium deoxycholate of D1-like agonist-stimulated [³⁵S]GTPS binding to Gq-like G proteins in brain membranes, and determined the optimal experimental conditions for assessing agonist effects on [³⁵S]GTPS binding in the presence of the detergent. Factors and their optimal levels that were found to significantly enhance the sensitivity and robustness of the agonist-stimulated [³⁵S]GTPS binding reaction include protein concentration at 40 g/ml, cationic concentrations of 120 mM Na⁺, 1.8 mM K⁺, and 20 mM Mg²⁺, a molar guanine nucleotide ratio of 100,000 GDP to [³⁵S]GTPS, the presence of 1 mM deoxycholate, and an overall incubation duration of 30–120 min. Under the optimized conditions, the D₁-like agonist SKF38393 induced potent and highly efficacious (up to 1000%) stimulation of [³⁵S]GTPS binding in membrane preparations from the striatum and other rat brain regions. In striatal membranes incubated with drug for 2 h, immunoprecipitation of the [³⁵S]GTPS-bound proteins with specific G antibodies showed that at least 70% of SKF38393-stimulated [³⁵S]GTPS binding was to Gq. The present reaction parameters are consistent with conditions previously found to support dopaminergic stimulation of phospholipase C-mediated signaling in brain slice preparations. These results imply that different but equally physiologically relevant conditions can be obtained under which subtypes of dopaminergic receptors may couple preferentially to Gs and the adenylate cyclase pathway or to Gq and the phospholipase C pathway.     

An enhanced incorporation of fatty acid into phosphatidyl choline that parallels histamine discharge in mast cells

Summary Purified rat peritoneal and pleural mast cells preincubated briefly with radioactively labeled fatty acid were treated with A23187, which bypasses primary receptors in stimulating exocytosis. An enhanced incorporation of fatty acid into phosphatidyl choline (PC) that occurred in parallel with histamine release at 24–25°C was observed and was initially proportional to the total amount of histamine discharged. Enhanced PC labeling and histamine secretion were also proportional at temperatures ranging from 17–37°C. Both radioactive linoleic and palmitic acids were incorporated selectively at the -position of the glycerol backbone of PC. PC labeling by [³H]choline was not detectably different in control and stimulated cells, and phosphatidic acid did not exhibit selectively enhanced -acylation. Thus, the stimulated labeling in A23187-treated cells may occur secondary to the action of a phospholipase A₂ that favors PC as a substrate.Other peritoneal cell types exhibit a very similar A23187-stimulated selective labeling of PC. Therefore, autoradiography has been used to provide a direct demonstration that in purified preparations, mast cells are the principal cell type engaged in A23187-elicited incorporation of fatty acid into PC. The efficacy of this approach has relied on special procedures devised to obtain significantly different autoradiographic grain densities between control and stimulated preparations that can be attributed to differences in the level of [³H]palmitate-labeled PC.Preliminary tests using compound 48/80 as a secretory stimulus for mast cells have identified a similar selectively enhanced PC labeling. In either case, however, consideration of possible relationships between PC metabolism and the secretory process are premature since they have not been tested directly.     

Production of mouse monoclonal antibodies using a continuous cell culture fermenter and protein G affinity chromatography

The production of anti--fetoprotein monoclonal antibodies for diagnostic use was carried out in a stirred tank fermenter equipped with a double membrane stirrer for bubble free aeration and continuous medium perfusion. A serum-free medium supplemented with 4 mM L-glutamine and 2.0 g/l glucose with a protein content of only 780 g/ml was used for the production process. The harvested antibodies were concentrated 50-fold using a tangential ultrafiltration system and were then purified in a one step purification process by protein G affinity chromatography. The purity of the final product (90%) was controlled by SDS-polyacrylamide gel electrophoresis, gel exclusion chromatography and isoelectric focussing. For further quality controls of the product the immunoglobulin subclass and the isoelectric point were determined and the specificity of the purified mAb was tested by RIA using¹²⁵I labelled -fetoprotein.1.87 g of purified monoclonal antibodies were produced (90% purity) within 2 weeks. It was found that the use of this type of stirred tank fermenter combined with a one step purification process using protein G affinity chromatography represents a suitable method for the fast production of medium scale quantities (500 mg–5 g) of monoclonal antibodies for diagnostic use.Abbreviations AFP -Fetoprotein - BSA bovine serum albumine - FCS Fetal calf serum - HRP horseradish peroxidase - OPD o-phenylenediamine dihydrochloride - I.P. isoelectric point - IEF isoelectric focussing - PBS Phosphate buffered saline     

Stable ploidy levels in long-term callus cultures of loblolly pine

Ploidy levels were calculated for callus cultures of loblolly pine (Pinus taeda L.), based on nuclear DNA content measured by Feulgen cytophotometry. The nuclear DNA content of initial stem explants showed a predominant 2C condition with less 3C and 4C, in ratios approximating those expected from diploid cells as they replicate DNA in the mitotic cell cycle. Cells with higher ploidy were produced during callus initiation, as indicated by a sharp reduction in the 2C population and a concomitant increase in higher DNA levels up to 8C. A gradual decrease in the higher ploidy levels occurred in subsequent subculture intervals, so that by 18 weeks the diploid nuclear DNA distribution was again observed, with complete elimination of DNA levels greater than 4C. Established callus cultures derived from stem or embryo explants and cultured on three different nutrient media for 48–76 weeks also showed the diploid nuclear DNA distribution with no indication of polyploid cells.Abbreviations BAP benzylaminopurine - NAA -naphthaleneacetic acid - BL Brown and Lawrence's medium - BLG modified BL medium - LM Litvay's medium Paper No. 11952 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh NC 27695-7643, USA     

HP 749 enhances calcium-independent release of [3H]norepinephrine from rat cortical slices and synaptosomes

Previous studies have shown that, at concentrations of 1 M and 10 M, HP 749 increased electrically-stimulated release of [³H]norepinephrine (NE) from rat cortical slices. These effects were Ca²⁺-dependent, indicating an effect on release from vesicular stores. At 100 M, HP 749 had two effects. In addition to enhancing the Ca²⁺-dependent electrically-evoked release, it also induced a rise in the basal efflux (spontaneous release) of [³H]NE, which was observed in both cortical slices and synaptosomes. The spontaneous release effect was (1) not blocked by the reuptake inhibitor nomifensine, (2) not affected by removal of external calcium, (3) not blocked by vesicular depletion with reserpine, and (4) not inhibited by the sodium channel blocker tetrodotoxin (TTX). As would be expected, the spontaneous [³H]NE release induced by the cytoplasmic releaser tyramine and the sodium channel activator veratridine were blocked by nomifensine and TTX, respectively. Notably, however, the Ca²⁺-independent veratridine-induced release was completely blocked by 100 M HP 749. The mechanism of spontaneous release of [³H]NE caused by 100 M HP 749 is unresolved at present; however, the data are consistent with this release originating from a cytoplasmic source.     

Regulation of phospholipase D from human hepatocarcinoma cell line by purine nucleotides and protein kinase A

The regulation of phosphatidylcholine-specific phospholipase D by purine nucleotides and protein kinase A were studied in vitro using an enzyme preparation partially purified from the membranous fraction of 7721 hepatocarcinoma cells. It was found that the enzyme activity was elevated by low concentrations of some purine nucleotides, but the activating effects were decreased when the concentrations of the nucleotides were higher. The optimal concentrations of GTP, GTP[S] , GDP and ATP for maximal activation were 0.1mM, 5M,1 mM and 1 mM respectively. The activation caused by 1mM ADP was lower. The enzyme was not activated by 1mM AMP, but significant activation was observed by the addition of 1mM cAMP. The latter was mediated by protein kinase A, as a specific inhibitor of protein kinase A ablished the activation. There were synergic effects between ATP and GTP, ATP and PIP₂, but not between ATP and GTP[S] , or PIP₂ and GTP[S]. The activating effects of GTP and ATP were abolished by neomycin, a PIP₂ scavenger. These results suggest that phospholipase D is regulated by GTP-binding protein and the presence of PIP₂ is required for the activation induced by GTP. Protein kinase A may be another protein kinase in addition to protein kinase C and protein tyrosine kinase which regulate the activity of phospholipase D, when the intracellular concentration of cAMP is increased.     

Unisite Hydrolysis of [γ32P]ATP by Soluble Mitochondrial F1ATPase and Its Release by Excess ADP and ATP. Effect of Trifluoperazine

Some of the characteristics of unisite hydrolysis of [³²P]ATP as well as the changes that occur on the transition to multisite catalysis were further studied. It was found that a fraction of [³²P]ATP bound at the catalytic sites of F₁ under unisite conditions undergoes both hydrolysis and release induced by medium nucleotides upon addition of millimolar concentrations of ADP or ATP. The fraction of [³²P]ATP that undergoes release is similar to the fraction that undergoes hydrolytic cleavage, indicating that the rates of the release and hydrolytic reactions of bound [³²P]ATP are in the same range. As part of studies on the mechanisms through which trifluoperazine inhibits ATP hydrolysis, its effect on unisite hydrolysis of [³²P]ATP was also studied. Trifluoperazine diminishes the rate of unisite hydrolysis by 30–40%. The inhibition is accompanied by a nearly tenfold increase in the ratio of [³²P]ATP/³²Pi bound at the catalytic site and a 50% diminution in the rate of ³²Pi release from the enzyme into the media. Trifluoperazine also induces heterogeneity of the three catalytic sites of F₁ in the sense that in a fraction of F₁ molecules, the high-affinity catalytic site has a turnover rate lower than the other two. Trifluoperazine does not modify the release of previously bound [³²P]ATP induced by medium nucleotides. The latter indicates that hindrances in the release of Pi do not necesarily accompany alterations in the release of ATP even though both species lie in the same site.     

Somatic embryogenesis and plantlet regeneration from cell suspension cultures of palmarosa grass (Cymbopogon martinii)

A cell suspension culture was established from nodal callus ofCymbopogon martinii (Roxb.) Wats in a liquid medium containing Murashige and Skoog (1962) basal salts, vitamins, 100 mg 1^–1 myo-inositol and 20 g l^–1 of sucrose (MS) that was supplemented with 13.6 M 2,4-dichlorophenoxyacetic acid and 1.15 M kinetin. An initial inoculum density of 2 x 10⁴ cells ml^–1exhibited optimum cell growth. Calli were obtained 12–15 days after the suspension was plated onto semisolid medium of a similar composition. When calli were transferred to semisolid regeneration medium containing MS + 6.7 M N ⁶-benzyl-adenine + 1.15 M kinetin, somatic embryogenesis and plantlet regeneration occurred after 10–25 days. There was no significant decrease in the regeneration potential of the calli even when the cultures were initiated from 47-week-old cell suspensions. Chromosome counts of cells in suspensions, calli and somatic embryos derived from cultures of different ages revealed the presence of diploids, tetraploids and octaploids. However, the 33 regenerated plants tested were all diploid, indicating that only diploid cells were capable of regeneration in vitro.Abbreviations MS Murashige and Skoog (1962) basal salts with vitamins (100 mg1^–1 myo-inositol, 20 g1^–1 sucrose) - 2,4-D 2,4-dichlorophenoxyacetic acid - BA N ⁶-benzyl-adenine - Kn kinetin - MSC MS + 13.6 M 2,4-D + 1.15 M Kn - MSR MS +6.7 M BA + 1.15 M Kn     

Na+ transport by rabbit urinary bladder,a tight epithelium

Summary By in vitro experiments on rabbit bladder, we reassessed the traditional view that mammalian urinary bladder lacks ion transport mechanisms. Since the ratio of actual-to-nominal membrane area in folded epithelia is variable and hard to estimate, we normalized membrane properties to apical membrane capacitance rather than to nominal area (probably 1 F 1 cm² actual area). A new mounting technique that virtually eliminates edge damage yielded resistances up to 78,000 F for rabbit bladder, and resistances for amphibian skin and bladder much higher than those usually reported. This technique made it possible to observe a transport-related conductance pathway, and a close correlation between transepithelial conductance (G) and short-circuit current (I _sc) in these tight epithelia.G andI _sc were increased by mucosal (Na⁺) [I _sc0 when (Na⁺)0], aldosterone, serosal (HCO ₃ ^– ) and high mucosal (H⁺); were decreased by amiloride, mucosal (Ca⁺⁺), ouabain, metabolic inhibitors and serosal (H⁺); and were unaffected by (Cl^–) and little affected by antidiuretic hormone (ADH). Physiological variation in the rabbits' dietary Na⁺ intake caused variations in bladderG andI _sc similar to those caused by the expectedin vivo changes in aldosterone levels. The relation betweenG andI _sc was the same whether defined by diet changes, natural variation among individual rabbits, or most of the above agents. A method was developed for separately resolving conductances of junctions, basolateral cell membrane, and apical cell membrane from thisG–I _sc relation. Net Na⁺ flux equalledI _sc. Net Cl^– flux was zero on short circuit and equalled only 25% of net Na⁺ flux in open circuit. Bladder membrane fragments contained a Na⁺–K⁺-activated, ouabain-inhibited ATPase. The physiological significance of Na⁺ absorption against steep gradients in rabbit bladder may be to maintain kidney-generated ion gradients during bladder storage of urine, especially when the animal is Na⁺-depleted.