Inositol 1,4,5 trisphosphate is inactivated by a 5-phosphatase in stamen hair cells of Tradescantia.

Inositol 1,4,5 trisphosphate [Ins(1,4,5)P3] is produced from the hydrolysis of phosphatidylinositol 4,5 bisphosphate, and as part of a second-messenger signal transduction mechanism, induces release of Ca2+ from internal stores in both plant and animal systems. It is less well established how the active Ins(1,4,5)P3 is inactivated. Studies in animal cells have demonstrated two separate metabolic pathways. Ins(1,4,5)P3 can be hydrolyzed by a 5-phosphatase or phosphorylated by a 3-kinase, resulting in the formation of Ins(1,4)P2 and Ins(1,3,4,5)P4, respectively, neither of which is able to mobilize intracellular Ca2+. Plant cell extracts have been reported to have hydrolytic and kinase activities that produce Ins(1,4)P2, and Ins(4,5)P2 and Ins(1,4,5,6)P4 from Ins(1,4,5)P3. These results offer little insight into the enzyme activities in the intact plant cell since the observed activities might be confined to intracellular compartments that have little if any impact on the signaling events within the cytosol that require Ins(1,4,5)P3. To resolve the mechanism of Ins(1,4,5)P3 inactivation, we microinjected stamen hair cells of Tradescantia virginiana L. with nonhydrolysable analogs of Ins(1,4,5)P3 that have been previously shown to cause Ca2+ release from intracellular stores. Our results indicate a sustained cytosolic [Ca2+] increase when cells were injected with the 5-phosphatase-insensitive 5-monophosphorothioate derivative of Ins(1,4,5)P3, in contrast to a brief transient when injected with the 3-kinase-insensitive 3-fluoro-3-deoxy Ins(1,4,5)P3 analog. We conclude that the 5-phosphatase pathway is the preferred pathway for Ins(1,4,5)P3 inactivation in the stamen hair cells of Tradescantia.

The role of phosphoinositide-derived second messengers in oxytocin-stimulated prostaglandin F2 alpha release from endometrium of pigs.

The mechanism for prostaglandin (PG) F2 alpha release from pig endometrium after oxytocin (OT) treatment is unknown. OT may rapidly stimulate inositol (1,4,5)-trisphosphate (IP3) and diacylglycerol (DAG) formation, consistent with the concept of rapid activation of a second-messenger system. In support of this hypothesis, endometrial IP3 levels were increased (P < 0.05) within 0.5 min after treatment with 0.1 microM OT. In contrast, increased DAG formation was not detected after treatment with OT. However, similar to the stimulation of endometrial PGF2 alpha secretion observed after OT treatment (P < 0.001), PGF2 alpha release was increased (P < 0.01) after treatment with phorbol-12-myristate-13-acetate (PMA), which mimics DAG activation of protein kinase C. Further, stimulation of endometrial PGF2 alpha secretion did not result from cell death induced by PMA or OT because lactate dehydrogenase, a cytosolic marker of cellular integrity, did not leak into the medium after PMA or OT treatment. In contrast, 0.5% saponin (positive control for cell death and concomitant release of lactate dehydrogenase) increased PGF2 alpha secretion (P < 0.05) and lactate dehydrogenase release (P < 0.001). These results indicate that OT induces endometrial IP3 production in a rapid manner indicative of a second-messenger system. The finding that increased DAG was not also detected after OT treatment may reflect rapid metabolism or compartmentalized production of DAG involved in the second-messenger stimulation of phospholipase C. The high background of DAG used in the biosynthesis of cellular lipids would obscure the rather small spatially localized changes in DAG levels resulting from the activation of phospholipase C. The finding that DAG was present at approximately 10 to 20-fold higher levels than IP3 in resting cells was consistent with this conclusion.

The phosphotyrosine phosphatase inhibitor-phenylarsine oxide restores defective phosphoinositide hydrolysis response in anergic C3H-gld/gld lymphocytes.

Mice homozygous for the gld (generalized lymphoproliferative disease) mutation develop both lymphadenopathy and autoimmune disease. CD4-CD8- (double negative, DN) T cells comprise the major population of T cells in mature C3H-gld/gld peripheral lymphoid tissues. These DN T cells are unresponsive to many forms of stimuli and have previously been shown to exhibit abnormally elevated levels of membrane phosphotyrosine phosphatase (PTPase) activity. In the present study, we demonstrate that IP3 production in response to mitogenic stimulation with Con A or anti-CD3 mAb (145-2CII) is significantly diminished in C3H-gld/gld lymphocytes when compared to that in congenic C3H(-)+/+ cells. The capacity to produce this second-messenger can be restored by pretreating C3H-gld/gld cells with the PTPase inhibitor, phenylarsine oxide (PAO). Although the inhibition of PTPase activity by treatment with PAO did restore C3H-gld/gld cell ability to produce IP3, the signal did not lead to lymphocyte proliferation, but instead to cell death. Our results suggest that the altered phosphoinositide hydrolysis observed in the mutant cells is related to their elevated membrane PTPase activity and that the anergy in these cells is at least in part related to the abnormally high activity of endogenous PTPases.

Abscisic Acid-Induced Phosphoinositide Turnover in Guard Cell Protoplasts of Vicia faba.

Guard cell protoplasts of Vicia faba treated with 10 [mu]M (+)abscisic acid (ABA) in the light exhibited a 20% decrease in diameter within 1.5 h, from 24.1 to 19.6 [mu]m. Within 10 s of administration of ABA, a 90% increase in levels of inositol 1,4,5-trisphosphate was observed, provided that cells were treated with Li+, an inhibitor of inositol phosphatase activity, prior to incubation. Concomitantly, levels of 32P-labeled phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate decreased 20% compared to levels in control cells; levels of label in the membrane lipids phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol did not change significantly in response to ABA treatment. These results show that phosphoinositide turnover is activated in response to ABA in guard cells. We conclude that phosphoinositide signaling is likely to be a step in the biochemical cascade that couples ABA to guard cell shrinking and stomatal closure.

Inositol 1,4,5-trisphosphate may mediate closure of K+ channels by light and darkness in Samanea saman motor cells.

Leaflet movements of Samanea saman (Jacq.) Merr. depend in part upon circadian-rhythmic, light-regulated K+ fluxes across the plasma membranes of extensor and flexor cells in opposing regions of the leaf-moving organ, the pulvinus. We previously showed that blue light appears to close open K+ channels in flexor protoplasts during the dark period (subjective night) (Kim et al., 1992, Plant Physiol 99; 1532-1539). In contrast, transfer to darkness apparently closes open K+ channels in extensor protoplasts during the light period (subjective day) (Kim et al., 1993, Science 260; 960-962). We now report that both these channel-closing stimuli increase inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] levels in the appropriate protoplasts. If extensor cells are given a pulse of red light followed by transfer to darkness, channels still apparently close (Kim et al., 1993) but changes in Ins(1,4,5)P3 levels are complex with an initial decrease under red light followed by accumulation. Neomycin, an inhibitor of polyphosphoinositide hydrolysis, inhibits both blue-light-induced Ins(1,4,5)P3 production and K(+)-channel closure in flexor protoplasts and both dark-induced Ins(1,4,5)P3 production and K+ channel closure in extensor protoplasts. The G-protein activator, mastoparan, mimics blue light and darkness in that it both increases Ins(1,4,5)P3 levels and closes K+ channels in the appropriate cell type at the appropriate time. These results indicate that phospholipase C-catalyzed hydrolysis of phosphoinositides, possibly activated by a G protein, is an early step in the signal-transduction pathway by which blue light and darkness close K+ channels in S. saman pulvinar cells.

A proposed role for oxytocin in regulation of endometrial prostaglandin F2 alpha secretion during luteolysis in swine.

Pulsatile secretion of endometrial prostaglandin (PG)F2 alpha is stimulated by oxytocin (OT) during late diestrus in domestic ruminants (i.e., cattle, sheep and goats) and results in corpus luteum (CL) regression leading to the onset of a new estrous cycle. Pulsatile PGF2 alpha release is also responsible for CL regression in swine, but the stimulus for its secretion from the uterine endometrium is not known. We propose that OT binds to specific OT receptors (OTR) on the endometrium to stimulate phosphoinositide (PI) hydrolysis, thereby activating the inositol trisphosphate (IP3)-diacylglycerol (DAG) second-messenger system to promote pulsatile PGF2 alpha secretion. Exogenous OT administered to cyclic gilts during late diestrus (days 10-16) decreased interestrous interval in three of four experiments. However, OT did not promote CL regression in hysterectomized gilts indicating that the effect of OT was uterine-dependent. Circulating concentrations of 13,14-dihydro-15-keto PGF2 alpha (the major stable metabolite of PGF2 alpha) were increased (p < 0.01) 10 min after i.v. injection of OT on days 14 and 16 in cyclic gilts and on days 10-16 in pregnant gilts, but the magnitude of the response to OT on all days in pregnant gilts was markedly reduced compared to the response in cyclic gilts on days 14 and 16. Mean density and Kd of OTR detected on endometrium of cyclic pigs 15 days post-estrus were 29.2 +/- 5.5 fmol/mg protein and 1.59 +/- 0.23 nM, respectively. Density of OTR was correlated with OT-stimulated PI hydrolysis (r = 0.83, p < 0.05) and PGF2 alpha secretion (r = 0.87, p < 0.10). Endometrial IP3 was increased within 30 seconds after OT treatment and preceded the increase in PGF2 alpha release stimulated by OT. Endometrial PI hydrolysis and PGF2 alpha secretion were similarly increased by AIF4-(phospholipase C activator), but not by cholera toxin (adenylyl cyclase activator). Although OT binding to OTR could be displaced by lysine-vasopressin and lysine-vasopressin stimulated PI hydrolysis, lysine-vasopressin did not stimulate PGF2 alpha release. Distinct receptors for OT and lysine-vasopressin on pig endometrium were confirmed by treatment with 100 nM OT + 100 nM lysine-vasopressin which stimulated PI hydrolysis more than 100-200 nM OT or lysine-vasopressin alone. These results support the hypothesis that OT stimulates phospholipase C to hydrolyze PI, yielding IP3 and DAG second-messengers which promote endometrial PGF2 alpha release during CL regression in pigs.

Exposure of phosphatidylcholine and phosphatidylinositol in plasma membranes from rat brain synaptosomes treated with phospholipase A2 toxins (beta-bungarotoxin, notexin) and enzymes (Naja nigricollis, Naja naja atra).

Phospholipase A2 (PLA2) toxins act presynaptically to block acetylcholine release and are much more potent and specific in their actions than PLA2 enzymes even though they have lower enzymatic activity. Since their mechanism of action is not completely understood, it was of interest to examine the toxins' effects on phospholipid asymmetry as changes in asymmetry are associated with changes in membrane functioning. Rat brain synaptosomes were treated with the PLA2 toxins beta-bungarotoxin (beta-BuTx) and notexin and with the PLA2 enzymes Naja nigricollis and Naja naja atra under relatively non-disruptive conditions as judged by leakage of lactate dehydrogenase (LDH) and levels of phospholipid hydrolysis. The exposure of phosphatidylcholine (PC) and phosphatidylinositol (PI) on the synaptosomal surface was investigated by means of a specific PC-exchange protein (PCEP) and a PI-specific phospholipase C (PI-PLC), respectively. Treatment of the synaptosomes with N. nigricollis PLA2, beta-BuTx and notexin did not affect the availability of PC to exchange by PCEP, but significantly increased the exposure of PI to hydrolysis by PI-PLC. In contrast, N. n. atra PLA2 slightly decreased the exposure of PC and did not affect that of PI. The differences between N. n. atra PLA2, on the one hand, and N. nigricollis PLA2, beta-BuTx and notexin, on the other hand, parallel differences in their pharmacological activities. Our earlier studies showed that PLA2 enzymes, and possibly PLA2 toxins, have a pharmacological site separate from the enzymatic site. Since in the present study the effect on PI was abolished by EDTA, the presence of an enzymatic site in addition to the pharmacological site may be required or alternatively divalent cations may be required for the effects on PI asymmetry independent of the inhibition of PLA2 by EDTA.

Polyunsaturated fatty acids modulate stomatal aperture and two distinct K+ channel currents in guard cells.

Regulation of stomatal aperture is critical for both CO2 uptake and water retention by plants. Stomatal opening is produced by osmotic water flow into guard cells, which follows K+ transport across the plasma membrane. We report here that linolenic acid and arachidonic acid, but not several other fatty acids, enhance stomatal opening and inhibit stomatal closing. In patch clamped guard cell protoplasts, linolenic and arachidonic acid rapidly potentiated inward K+ currents and inhibited outward K+ currents, which are carried via distinct K+ channels. These results suggest that certain fatty acids regulate stomatal aperture by modulation of two different K+ channels and may act as second messengers for stimuli that regulate CO2 uptake and water retention by plants.

Deflagellation of Chlamydomonas reinhardtii follows a rapid transitory accumulation of inositol 1,4,5-trisphosphate and requires Ca2+ entry.

C. reinhardtii sheds its flagella in response to acidification. Previously, we showed correlations between pH shock, deflagellation, and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] production, but 100% of cells deflagellated by 5 s, which was the earliest that Ins(1,4,5)P3 accumulation could be accurately measured by techniques available to us at that time (Quarmby, L. M., Y. G. Yueh, J. L. Cheshire, L. R. Keller, W. J. Snell, and R. C. Crain. J. Cell Biol. 1992. 116:737-744). To learn about the causal relationship between Ins(1,4,5)P3 accumulation and deflagellation, we extended these studies to early times using a continuous-flow rapid-quench device. Within 1 s of acidification to pH 4.3-4.5, 100% of cells deflagellated. A transient peak of Ins(1,4,5)P3 was observed 250-350 ms after pH shock, preceding deflagellation. Preincubation with 10 microM neomycin, which prevents hydrolysis of phosphatidylinositol 4,5-bisphosphate, inhibited both the transient production of Ins(1,4,5)P3 and the subsequent deflagellation. The nonspecific Ca2+ channel blockers La3+ and Cd2+ prevented flagellar excision induced by mastoparan without inhibiting rapid Ins(1,4,5)P3 production. Likewise, the Ins(1,4,5)P3-gated channel inhibitors ruthenium red and heparin blocked deflagellation in response to mastoparan. These studies were extended to mutants defective in flagellar excision. Fa-1, a mutant defective in flagellar structure, produced Ins(1,4,5)P3 but failed to deflagellate. These results support a model in which acid pH activates a putative cellular receptor leading to G-protein dependent activation of phospholipase C and accumulation of Ins(1,4,5)P3. These events are upstream of Ins(1,4,5)P3-dependent Ca2+ entry from the medium, and of deflagellation.

Potassium Channels in Samanea saman Protoplasts Controlled by Phytochrome and the Biological Clock.

Leaflet movement in legumes depends on rhythmic, light-regulated ion fluxes in opposing regions of the leaf-moving organ. In flexor and extensor protoplasts from Samanea saman Merrill, opening and closing of K(+) channels were rhythmic in constant darkness. When channels were open in flexor protoplasts they were closed in extensor protoplasts, and vice versa. The rhythms were shifted by a delay in the onset of constant darkness, a response typical of endogenous circadian rhythms. During the light period, the channels in flexor protoplasts were sensitive to red light that was followed by premature darkness; phytochrome was implicated as the photoreceptor.

Stomatal Opening Is Induced in Epidermal Peels of Commelina communis L. by GTP Analogs or Pertussis Toxin.

Pretreatment with pertussis toxin or microinjection of guanosine- 5[prime]-(3-thiotriphosphate) (GTP-[gamma]-S) into guard cells in peeled epidermis of Commelina communis L. promoted stomatal opening under subsaturating white light. Guanosine-5[prime]-(2-thiodiphosphate) (GDP-[beta]-S) and adenosine-5[prime]-(3-thiotriphosphate) (ATP-[gamma]-S) did not change stomatal aperture under identical conditions. These results indicate that G proteins may be involved in the regulation of stomatal opening.

The role of calcium in the Chlamydomonas reinhardtii mating reaction.

The mating reaction of Chlamydomonas reinhardtii entails a rapid series of cell-cell interactions leading to cell fusion. We have demonstrated (Pasquale, S. M., and U. Goodenough. 1987. J. Cell Biol. 105:2279-2293) that cAMP plays a key role in this process: gametic flagellar adhesion elicits a sharp increase in intracellular cAMP, and presentation of dibutyryl-cAMP to unmated gametes elicits all known mating responses. The present study evaluates the role of Ca2+ in this system. We document that the mating-induced increase in cAMP, and hence the mating responses themselves, are blocked by a variety of drugs known to interfere with Ca(2+)-sensitive processes. These data suggest that Ca(2+)-mediated events may couple adhesion to the generation of cAMP. Such events, however, appear to be localized to the flagellar membrane; we find no evidence for the mating-related increase in cytosolic free Ca2+ that has been postulated by others. Indeed, by monitoring the length of the Ca(2+)-sensitive centrin-containing nucleus-basal body connector, we show that cytosolic free Ca2+ levels, if anything, decrease in response to cAMP signaling. We confirm a previous report that Ca2+ levels increase in the mating medium, but document that this represents a response to augmented cAMP levels and not a prelude. Finally, we show that IP3 levels remain constant throughout the mating reaction. These results are discussed in terms of the various signal transduction systems that have now been identified in Chlamydomonas.

The asymmetric distribution of phosphatidylcholine in rat brain synaptic plasma membranes.

The distribution of phosphatidylcholine between inner and outer monolayers of rat brain synaptic plasma membrane was investigated by means of a phosphatidylcholine specific exchange protein. About 70% of the total membranal phosphatidylcholine was in the outer leaflet, 33% of which was exposed and readily exchanged in intact synaptosomes while the remainder was exchangeable following osmotic shock. Permeabilization of the synaptic plasma membranes by overnight incubation in buffer or by saponin (< 0.08%) exposed an additional 30% of phosphatidylcholine to exchange, presumably from the inner cytoplasmic leaflet. Phosphatidylcholine is therefore asymmetrically distributed in the synaptosomal plasma membrane, as it is in other plasma membranes.

Artifactual Elevation of the Apparent Levels of Phosphatidic Acid and Phosphatidylinositol 4,5-Bisphosphate during Short-Term Labeling of Plant Tissue with Radioactive Precursor.

Some pulvini of Samanea saman Mehr. labeled with radioactive phosphate show apparent remarkable elevations of the levels of phosphatidic acid and phosphatidylinositol 4,5-bisphosphate. The elevated levels, however, appear to be illusory and to result from rapid initial incorporation of label into these phospholipids relative to others. These results demonstrate the need for caution in interpreting apparent changes in the levels of phosphatidic acid or inositol phospholipids in cultures or plants labeled with radioactive precursors.

Effects of Light on the Membrane Potential of Protoplasts from Samanea saman Pulvini : Involvement of K Channels and the H -ATPase.

Rhythmic light-sensitive movements of the leaflets of Samanea saman depend upon ion fluxes across the plasma membrane of extensor and flexor cells in opposing regions of the leaf-movement organ (pulvinus). We have isolated protoplasts from the extensor and flexor regions of S. saman pulvini and have examined the effects of brief 30-second exposures to white, blue, or red light on the relative membrane potential using the fluorescent dye, 3,3'-dipropylthiadicarbocyanine iodide. White and blue light induced transient membrane hyperpolarization of both extensor and flexor protoplasts; red light had no effect. Following white or blue light-induced hyperpolarization, the addition of 200 millimolar K(+) resulted in a rapid depolarization of extensor, but not of flexor protoplasts. In contrast, addition of K(+) following red light or in darkness resulted in a rapid depolarization of flexor, but not of extensor protoplasts. In both flexor and extensor protoplasts, depolarization was completely inhibited by tetraethylammonium, implicating channel-mediated movement of K(+) ions. These results suggest that K(+) channels are closed in extensor plasma membranes and open in flexor plasma membranes in darkness and that white and blue light, but not red light, close the channels in flexor plasma membranes and open them in extensor plasma membranes. Vanadate treatment inhibited hyperpolarization in response to blue or white light, but did not affect K(+) -induced depolarization. This suggests that white or blue light-induced hyperpolarization results from activation of the H(+) -ATPase, but this hyperpolarization is not the sole factor controlling the opening of K(+) channels.

Inositol phospholipid metabolism may trigger flagellar excision in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii cells shed their flagella in response to environmental stress. Under favorable conditions, flagella are quickly regrown. To learn more about the signals that trigger flagellar excision and regrowth we have investigated inositol phospholipid metabolites, molecules implicated in signal transduction in several other systems. After deflagellation by low pH or mastoparan, a potent activator of G proteins, there was a rapid increase in levels of inositol 1,4,5-trisphosphate measured by use of receptor-binding assays and HPLC. This increase was concomitant with a decrease in levels of phosphatidylinositol 4,5-bisphosphate and was followed by an increase in phosphatidic acid, results consistent with activation of phospholipase C and diacylglycerol kinase. Additional experiments suggest that this activated phospholipase C is not important for flagellar regrowth but plays a role in informing the excision apparatus of the environmental stress. Addition of neomycin (an inhibitor of phospholipase C) before exposure of cells to low pH or mastoparan prevented the increase in inositol 1,4,5-trisphosphate and also prevented deflagellation. Addition of neomycin after deflagellation blocked increases in inositol 1,4,5-trisphosphate that normally followed deflagellation, but did not block flagellar assembly. Furthermore, a flagellar excision-defective mutant, fa-1, did not shed its flagella in response to low pH or mastoparan, yet both of these agents activated phospholipase C in these cells. The results suggest that activation of phospholipase C, possibly via a G protein, is a proximal step in the signal transduction pathway inducing deflagellation in Chlamydomonas.

Characterization of a Polyphosphoinositide Phospholipase C from the Plasma Membrane of Avena sativa.

A phosphoinositide-specific phospholipase C activity was identified in oat root (Avena sativa, cv Victory) plasma membranes purified by separation in an aqueous two-phase polymer system. The enzyme is highly active toward inositol phospholipids but only minimally active toward phosphatidylethanolamine and phosphatidylcholine. Activity approaches maximal levels at 200 micromolar phosphatidylinositol 4-phosphate (PIP) and is highly dependent on calcium; it is inhibited by 1 millimolar EGTA and is activated by calcium with an apparent activation constant of 2 micromolar. At 10 micromolar calcium and 200 micromolar inositol phospholipid, the enzyme is specific for phosphatidylinositol 4,5-bisphosphate (PIP(2)) and PIP, which are hydrolyzed at 10 and 4 times, respectively, the rate of phosphatidylinositol (PI) hydrolysis. The principle water soluble products of hydrolysis, as determined by high performance liquid chromatography, are inositol 1,4,5-trisphosphate from PIP(2), inositol 1,4-bisphosphate from PIP, and inositol phosphate from PI.