Increased binding activity at an antioxidant-responsive element in the metallothionein-1 promoter and rapid induction of metallothionein-1 and -2 in response to cerebral ischemia and reperfusion.

Metallothioneins (MTs) are cysteine-rich metal-binding proteins that are potentially involved in zinc homeostasis and free radical scavenging. The expression pattern of MT-1 and the binding activity of various MT-1 promoter elements were investigated after mild focal cerebral ischemia in the rat. Transient focal ischemia was induced by occluding both common carotid arteries and the right middle cerebral artery for 30 min. By the use of real-time quantitative PCR, a 10-fold increase in MT-1 and -2 mRNA levels was found in the cortex 24 hr after reperfusion. In situ hybridization and immunocytochemistry showed a rapid increase in MT-1 and -2 mRNA and MT protein in endothelial cells of microvessels at 6 hr after reperfusion, followed by an increased expression in astrocytes of the infarcted cortex at 24 hr after reperfusion. The early increase in MT expression preceded an increase in cerebral edema measured with T2-weighted magnetic resonance imaging. Gel shift assays were performed on nuclear extracts prepared from cortices before and at 6 and 24 hr after reperfusion. Increased binding activity was found at an antioxidant/electrophilic response element (ARE) sequence in the MT-1 promoter at 6 hr with a lower and variable binding activity at 24 hr after reperfusion. Constitutive binding activity was found for Sp1 and a metal response element in the MT-1 promoter that did not increase after ischemia and reperfusion. This study suggests a role of ARE-binding proteins in inducing cerebral MT-1 expression and implicates MT-1 as one of the early detoxifying genes in an endogenous defense response to cerebral ischemia and reperfusion.

VEGF antagonism reduces edema formation and tissue damage after ischemia/reperfusion injury in the mouse brain.

VEGF is mitogenic, angiogenic, and a potent mediator of vascular permeability. VEGF causes extravasation of plasma protein in skin bioassays and increases hydraulic conductivity in isolated perfused microvessels. Reduced tissue oxygen tension triggers VEGF expression, and increased protein and mRNA levels for VEGF and its receptors (Flt-1, Flk-1/KDR) occur in the ischemic rat brain. Brain edema, provoked in part by enhanced cerebrovascular permeability, is a major complication in central nervous system pathologies, including head trauma and stroke. The role of VEGF in this pathology has remained elusive because of the lack of a suitable experimental antagonist. We used a novel fusion protein, mFlt(1-3)-IgG, which sequesters murine VEGF, to treat mice exposed to transient cortical ischemia followed by reperfusion. Using high-resolution magnetic resonance imaging, we found a significant reduction in volume of the edematous tissue 1 day after onset of ischemia in mice that received mFlt(1-3)-IgG. 8-12 weeks after treatment, measurements of the resultant infarct size revealed a significant sparing of cortical tissue. Regional cerebral blood flow was unaffected by the administration of mFlt(1-3)-IgG. These results demonstrate that antagonism of VEGF reduces ischemia/reperfusion-related brain edema and injury, implicating VEGF in the pathogenesis of stroke and related disorders.

Developmental expression and co-localization of cyclin G1 and the B' subunits of protein phosphatase 2a in neurons.

Cyclin G1 is a recently cloned transcriptional target of p53, it is located in neurons and ventricular ependymal cells and is elevated in neurons after axotomy and cerebral ischemia. The biological function for cyclin G1 in differentiated neurons has thus far not been elucidated. Recently, cyclin G1 has been shown to interact with the B' subunits of serine/threonine protein phosphatase 2A (PP2A) in a rat fibroblast cell line [K. Okamoto, C., Kamibayashi, M. Serrano, C. Prives, M.C. Mumby, D. Beach, p53-dependent association between cyclin G and the B' subunit of protein phosphatase 2A, Mol. Cell. Biol. 16 (1996) 6593-6602]. To further explore whether a similar interaction between cyclin G1 and PP2A B' subunits exists in the central nervous system, the present study compared the regional and developmental expression pattern, subcellular distribution and complex formation between cyclin G1 and the PP2A B' regulatory subunits in the rat brain. In situ hybridization of cyclin G1 and the B'alpha and B'beta subunits of PP2A showed an overlapping distribution in neurons of the cerebral cortex, hippocampus and thalamus at embryonic and early postnatal ages, but their developmental regulation differed. Whereas mRNA and protein levels of PP2A B' subunits were high in the cortical plate, subiculum, hippocampal areas and thalamus at E20 and decreased with age, those of cyclin G1 increased with age and were maximal in the adult cortex and hippocampus. In rat 14-day-old embryonic cortical cultures, cyclin G1 and PP2A B'alpha protein co-localized in nuclear and perinuclear areas of neurons, and both proteins were highly expressed in nuclei of cortical and hippocampal pyramidal cells and the mitral cell layer of the neonatal olfactory bulb. Both cyclin G1 and the PP2A regulatory B'alpha subunits were specifically expressed in neurons and not in glial cells. Antibodies raised against the B'alpha subunits of PP2A immunoprecipitated cyclin G1 in adult cortical lysates, indicating the presence of a complex involving cyclin G1 and the B'alpha subunits of PP2A. This study shows that the regional and subcellular localization of PP2A B' regulatory subunits and cyclin G1 are very similar at early postnatal stages. We discuss the possible functions of a cyclin G1-PP2A B'alpha complex in neurons.

Increased expression of cyclin G1 and p21WAF1/CIP1 in neurons following transient forebrain ischemia: comparison with early DNA damage.

Oxidative stress affecting DNA integrity may be an important mediator of cell death induced by cerebral ischemia followed by reperfusion. Genes involved in the DNA repair processes may play an important role in cell viability. We studied the spatial expression of the DNA damage inducible gene p53 and its transcriptional targets p21WAF1/CIP1, cyclin G1, and Bax and compared their expression with markers of early DNA damage following 10 min of transient forebrain ischemia in rats. Cyclin G1 and p21WAF1/CIP1 mRNA levels increased significantly between 2.5 and 4-fold in neurons of the hippocampus, cortex, and striatum during the first 24 hr after reperfusion and decreased at 48 hr of reperfusion. Significant increases in the protein levels of Cyclin G1 and p21 WAF1/CIP1 were only seen in the striatum at 48 hr of reperfusion. The mRNA levels of the p21 family members p27KIP1 or p57KIP2 demonstrated no significant changes. p53, baxalpha, and bcl-xl mRNA levels increased in all areas of the hippocampus by 12 to 24 hr and decreased over the next 2 days of reperfusion. baxalpha mRNA was specifically induced in neurons of the outer cortical layers at 12 and 24 hr after reperfusion, and protein levels increased in the striatum at 48 hr. No changes in protein levels of p53, Bcl-xl, or Bcl-2 were detected in the cerebral cortex, hippocampus, or striatum at any time point following reperfusion. Single-stranded DNA breaks detected with DNA polymerase I-mediated in situ nick translation partly overlapped with nuclear cyclin G1 protein in the striatum, cortex, and hippocampus at 24 hr, however at 48 hr cyclin G1 remained elevated only in neurons bordering areas exhibiting DNA damage. Nuclear p53 protein, p21 mRNA, and baxalpha mRNA were absent in cells stained with the in situ nick translation method but p21 mRNA and baxalpha mRNA were increased in neurons adjacent to those with detectable DNA nick ends at 24 and 48 hr following reperfusion. The enhanced expression of cyclin G1, p21WAF1/CIP1, and baxalpha in neurons surviving transient forebrain ischemia may indicate their participation in an adaptive response to cerebral ischemia and reperfusion.

Tumor-suppressor p53 is expressed in proliferating and newly formed neurons of the embryonic and postnatal rat brain: comparison with expression of the cell cycle regulators p21Waf1/Cip1, p27Kip1, p57Kip2, p16Ink4a, cyclin G1, and the proto-oncogene Bax.

The tumor-suppressor protein p53 has been implicated in cell cycle arrest and apoptotic cell death in dividing cells (Yonish-Rouach et al. [1991] Nature 352:342-347. To elucidate possible functions of p53 in the regulation of cell division and cell death during development of the rat central nervous system, we compared the spatial and temporal expressions of p53 mRNA and protein with those of its transcriptional targets Bax, p21Waf1, and cyclin G1 and with the cyclin-dependent kinase inhibitors p27Kip1, p57Kip2, and p16Ink4a. From embryonic day 14 until the second postnatal week, p53 mRNA and protein were found predominantly in proliferating zones, including the cells of the emerging external granular layer of the cerebellum, and the ventricular and the subventricular zones of the forebrain. At all postnatal ages, there was a high expression of p53 mRNA and protein in cells of the rostral migratory stream, a well-defined pathway along which precursor cells of olfactory interneurons migrate from the ventricular and subventricular zones toward the olfactory bulb. In addition to its expression in proliferating cell populations, p53 was expressed in postmitotic cells of the cerebral cortex and hippocampus at embryonic and early postnatal stages. p53, p27Kip1, p16Ink4a, and bax alpha mRNA colocalized in the ventricular and subventricular zones at embryonic and early postnatal stages, but the distribution of p53 differed from that of its transcriptional targets cyclin G1 and p21Waf1 and from that of the cyclin-dependent kinase inhibitor p57Kip2, which were predominantly expressed in fully differentiated neurons. Double-labeling studies showed that p53 mRNA and protein were absent in cells undergoing developmental cell death, as identified by the presence of single- or double-stranded nuclear DNA breaks. Protein levels of p53 decreased during development in all brain areas studied. These results indicate a role for p53 in the control of cell division and early differentiation rather than in the control of cell death during rat brain development. The nonoverlapping temporal and spatial expression patterns of p53 and its transcriptional targets Bax, cyclin G1 and p21Waf1 suggest that each of these gene products fulfill independent roles in brain morphogenesis.

Cell cycle-related gene expression in the adult rat brain: selective induction of cyclin G1 and p21WAF1/CIP1 in neurons following focal cerebral ischemia.

The present studies were initiated to investigate whether p53 transactivated target genes are induced in a rat model of focal cerebral ischemia. Therefore, we applied in situ hybridization, immunocytochemistry and western blotting to study the temporal and spatial expression of p53 and its transcriptional targets Bax, p21 and cyclin G1 following permanent middle cerebral artery occlusion in the rat. Cyclin G1 immunoreactivity was constitutively expressed in the nuclei of cells in the choroid plexus and ependymal cell layer and in the cytoplasm of cell bodies and dendrites of pyramidal neurons of the cerebral cortex. Cyclin G1 messenger RNA and protein levels transiently increased to 150% of contralateral levels in neurons of the ipsilateral frontal and parietal cortex and striatum 3 h following middle cerebral artery occlusion. A low level of constitutively expressed p21 messenger RNA and protein was found in nuclei of cells in the choroid plexus, oligodendrocytes and neurons. p21 messenger RNA and protein levels gradually increased to 250% and 140% of contralateral levels in areas bordering the infarct core up to 6 h following middle cerebral artery occlusion. In contrast, p53 and Bax messenger RNA and protein levels, and protein levels of p27, cyclin-dependent kinase 5, p35 and cyclin E decreased in the infarct core and border areas with time after middle cerebral artery occlusion. The selective up-regulation of cyclin G1 and p21 in neurons in the border zone of a focal ischemic infarct indicates their involvement in an adaptive response to ischemic injury. The possible participation of cyclin G1 and p21 in a signal transduction pathway associated with ischemia-induced cellular stress is discussed.

Analysis of microspore-specific promoters in transgenic tobacco.

In order to modify the early stages of pollen development in a transgenic context microspore-specific promoters are required. We tested two putatively microspore-specific promoters, the Bp4 promoter from rapeseed and the NTM19 promoter from tobacco. Expression of the gus and barnase reporter genes under the control of these two promoters was studied in transgenic tobacco. Contrary to expectations, the Bp4 promoter became active only after the first pollen mitosis, and not in the microspores. The NTM19 promoter turned out to be highly microspore-specific and directed very high levels of gus expression to the unicellular microspores. The NTM19-barnase transgene caused cell-autonomous death at the mid-unicellular microspore stage, whereas Bp4-barnase induced cell ablation of early to mid-bicellular pollen. Both promoter-barnase transgenes did not affect the sporophyte and were inherited through the female germline. These results show that both the NTM19 and Bp4 promoters are expressed only in the male germline, and that the NTM19 promoter is an excellent tool to direct high levels of transgene expression exclusively to the microspores. This may have important biotechnological applications.

Restoration of the cAMP second messenger pathway enhances cardiac preservation for transplantation in a heterotopic rat model.

Current organ preservation strategies subject graft vasculature to severe hypoxia (PO2 approximately 20 Torr), potentially compromising vascular function and limiting successful transplantation. Previous work has shown that cAMP modulates endothelial cell (EC) antithrombogenicity, barrier function, and leukocyte/EC interactions, and that hypoxia suppresses EC cAMP levels. To explore the possible benefits of cAMP analogs/agonists in organ preservation, we used a rat heterotopic cardiac transplant model; dibutyryl cAMP added to preservation solutions was associated with a time- and dose-dependent increase in the duration of cold storage associated with successful graft function. Preservation was also enhanced by 8-bromo-cAMP, the Sp isomer of adenosine 3',5'monophosphorothioate, and types III (indolidan) and IV (rolipram) phosphodiesterase inhibitors. Neither butyrate alone nor 8-bromoadenosine were effective, and the cAMP-dependent protein kinase antagonist Rp isomer of adenosine 3',5'monophosphorothioate prevented preservation enhancement induced by 8-bromo-cAMP. Grafts stored with dibutyryl cAMP demonstrated a 5.5-fold increase in blood flow and a 3.2-fold decreased neutrophil infiltration after transplantation. To explore the role of cAMP in another cell type critical for vascular homeostasis, vascular smooth muscle cells were subjected to hypoxia, causing a time-dependent decline in cAMP levels. Although adenylate cyclase activity was unchanged, diminished oxygen tensions were associated with enhanced phosphodiesterase activity (59 and 30% increase in soluble types III and IV activity, respectively). These data suggest that hypoxia or graft ischemia disrupt vascular homeostasis, at least in part, by perturbing the cAMP second messenger pathway. Supplementation of this pathway provides a new approach for enhancing cardiac preservation, promoting myocardial function, and maintaining vascular homeostatic properties.

8-Chloroadenosine 3',5'-monophosphate inhibits the growth of Chinese hamster ovary and Molt-4 cells through its adenosine metabolite.

8-Chloroadenosine 3',5'-monophosphate has been reported to inhibit growth of various mammalian cell lines at micromolar concentrations. We have used Chinese hamster ovary cell lines with mutated cyclic AMP-dependent protein kinase or altered cyclic nucleotide metabolism to show that a metabolite, 8-chloroadenosine, is formed in the medium and is the active inhibitor of cell growth in Chinese hamster ovary cells. Adding adenosine deaminase to the Chinese hamster ovary cell growth media removes the inhibition of cell growth attributed to 8-chloroadenosine 3',5'-monophosphate. Adenosine deaminase or dipyridamole also protects Molt-4 lymphoblasts from the growth-inhibitory effects of 8-chloroadenosine 3',5'-monophosphate.

Control of cAMP-induced gene expression by divergent signal transduction pathways.

A compilation of literature data and recent experiments led to the following conclusions regarding cyclic adenosine 3':5' monophosphate (cAMP) regulation of gene expression. Several classes of cAMP-induced gene expression can be discriminated by sensitivity to stimulation kinetics. The aggregation-related genes respond only to nanomolar cAMP pulses. The prestalk-related genes respond both to nanomolar pulses and persistent micromolar stimulation. The prespore specific genes respond only to persistent micromolar stimulation. The induction of the aggregation- and prestalk-related genes by nanomolar cAMP pulses may share a common transduction pathway, which does not involve cAMP, while involvement of the inositol 1,4,5-trisphosphate (IP3)/Ca2+ pathway is unlikely. Induction of the expression of prespore and prestalk-related genes by micromolar cAMP stimuli utilizes divergent signal processing mechanisms. cAMP-induced prespore gene expression does not involve cAMP and probably also not cyclic guanosine 3'.5' monophosphate (cGMP) as intracellular intermediate. Involvement of cAMP-induced phospholipase C (PLC) activation in this pathway is suggested by the observation that IP3 and 1,2-diacylglycerol (DAG) can induce prespore gene expression, albeit in a somewhat indirect manner and by the observation that Li+ and Ca2+ antagonists inhibit prespore gene expression. Cyclic AMP induction of prestalk-related gene expression is inhibited by IP3 and DAG and promoted by Li+, and is relatively insensitive to Ca2+ antagonists, which indicates that PLC activation does not mediate prestalk-related gene expression. Neither prespore nor prestalk-related gene expression utilizes the sustained cAMP-induced pHi increase as intracellular intermediate.

Enzymatic synthesis of the cAMP antagonist (Rp)-adenosine 3',5'-monophosphorothioate on a preparative scale.

(Rp)-Adenosine 3',5'-monophosphorothioate ((Rp)-cAMPS) is a highly specific antagonist of the cAMP-dependent protein kinase from eukaryotic cells and is a very poor substrate for phosphodiesterases. It is therefore a useful tool for investigating the role of cAMP as a second messenger in a variety of biological systems. Taking advantage of stereospecific inversion of configuration around the alpha-phosphate during the adenylate cyclase reaction, we have developed a method for the preparative enzymatic synthesis of the Rp diastereomer of adenosine 3',5'-monophosphorothioate ((Rp)-cAMPS) from the Sp diastereomer of adenosine 5'-O-(1-thiotriphosphate) ((Sp)-ATP alpha S). The adenylate cyclase from Bordetella pertussis, partially purified by calmodulin affinity chromatography, cyclizes (Sp)-ATP alpha S approximately 40-fold more slowly than ATP, but binds (Sp)-ATP alpha S with about 10-fold higher affinity than ATP. The triethylammonium salt of the reaction product can be purified by elution from a gravity flow reversed-phase C18 column with a linear gradient of increasing concentrations of methanol. Yields of the pure (Rp)-cAMPS product of a synthesis with 2 mg of substrate are about 75%.

Cyclic AMP responses are suppressed in mammalian cells expressing the yeast low Km cAMP-phosphodiesterase gene.

A genomic DNA fragment from Saccharomyces cerevisiae which contains the SRA5 (=PDE2) gene, coding for a low Km cAMP-phosphodiesterase, was transfected into Chinese hamster ovary cells. Clones carring the cAMP-phosphodiesterase gene were capable of growth in the presence of cholera toxin, which slows the growth of untransfected cells by elevating their cAMP levels. The cholera toxin-resistant transfected cell lines expressed high levels of cAMP-phosphodiesterase mRNA and cAMP-phosphodiesterase activity. Basal intracellular cAMP levels were not significantly affected by the presence of the yeast cAMP-phosphodiesterase gene, but elevation of cAMP levels in response to cholera toxin or prostaglandin E1 was suppressed. Induction of the cAMP-responsive tyrosine aminotransferase promoter by cholera toxin was also blocked in cell lines carrying the yeast cAMP-phosphodiesterase gene. Cholera toxin-resistant transfected cell lines were sensitive to the growth inhibitory effects of N6,02'-dibutyryladenosine 3',5'-monophosphate, which can be used to bypass the effects of the yeast cAMP-phosphodiesterase.

Characterization of the yeast low Km cAMP-phosphodiesterase with cAMP analogues. Applications in mammalian cells that express the yeast PDE2 gene.

The essential interactions between cAMP and the yeast low Km cAMP-phosphodiesterase have been analyzed using cAMP analogues and phosphodiesterase inhibitors. cAMP specificity is conferred by hydrogen bonding at the N-6 and N-7 positions. In contrast to the other yeast phosphodiesterase, (Rp)-adenosine 3',5'-monophosphorothioate is not hydrolyzed. Eleven standard phosphodiesterase inhibitors were not highly effective. In Chinese hamster ovary (CHO) cells that express the yeast cAMP-phosphodiesterase (PDE2) gene, cAMP levels cannot be raised by cholera toxin. cAMP analogues that are efficiently hydrolyzed by the yeast cAMP-phosphodiesterase had no effect on the growth of CHO cells that express the PDE2 gene, even though they block the growth and alter the morphology of control cells. cAMP analogues that are not hydrolyzed by the yeast enzyme inhibited the growth and changed the morphology of both control and PDE2 expressing CHO cells. We have developed a method for creating cell lines in which cAMP levels can be reduced by expression of an exogenous cAMP-phosphodiesterase gene. By employing cAMP analogues that are not hydrolyzed by this phosphodiesterase, the inhibitory effects of the enzyme can be bypassed.

Lithium ions induce prestalk-associated gene expression and inhibit prespore gene expression in Dictyostelium discoideum.

We investigated the effect of Li+ on two types of cyclic AMP-regulated gene expression and on basal and cyclic AMP-stimulated inositol 1,4,5-triphosphate (Ins(1,4,5)P3) levels. Li+ effectively inhibits cyclic AMP-induced prespore gene expression, half-maximal inhibition occurring at about 2 mM-LiCl. In contrast, Li+ (1-3 mM) promotes the cyclic AMP-induced increase of cysteine proteinase-2 mRNA levels, and induces the expression of this prestalk-associated gene in the absence of cyclic AMP stimuli. At concentrations exceeding 4-5 mM, LiCl inhibits cysteine proteinase-2 gene expression. LiCl reduces basal Ins(1,4,5)P3 levels and decreases the cyclic AMP-induced accumulation of Ins(1,4,5)P3; both effects occur half-maximally at 2-3 mM-LiCl. These results indicate that the induction of the cysteine proteinase-2 gene by Li+ is not due to elevated levels of Ins(1,4,5)P3. It is, however, possible that inhibition of prespore gene expression by Li+ is caused by Li+-induced reduction of basal and/or stimulated Ins(1,4,5)P3 levels.

Chemoattractant and guanosine 5'-[gamma-thio]triphosphate induce the accumulation of inositol 1,4,5-trisphosphate in Dictyostelium cells that are labelled with [3H]inositol by electroporation.

The analysis of the inositol cycle in Dictyostelium discoideum cells is complicated by the limited uptake of [3H]inositol (0.2% of the applied radioactivity in 6 h), and by the conversion of [3H]inositol into water-soluble inositol metabolites that are eluted near the position of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] on anion-exchange h.p.l.c. columns. The uptake was improved to 2.5% by electroporation of cells in the presence of [3H]inositol; electroporation was optimal at two 210 microseconds pulses of 7 kV. Cells remained viable and responsive to chemotactic signals after electroporation. The intracellular [3H]inositol was rapidly metabolized to phosphatidylinositol and more slowly to phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. More than 85% of the radioactivity in the water-soluble extract that was eluted on Dowex columns as Ins(1,4,5)P3 did not co-elute with authentic [32P]Ins(1,4,5)P3 on h.p.l.c. columns. Chromatography of the extract by ion-pair reversed-phase h.p.l.c. provided a good separation of the polar inositol polyphosphates. Cellular [3H]Ins(1,4,5)P3 was identified by (a) co-elution with authentic [32P]Ins(1,4,5)P3 and (b) degradation by a partially purified Ins(1,4,5)P3 5-phosphatase from rat brain. The chemoattractant cyclic AMP and the non-hydrolysable analogue guanosine 5'-[gamma-thio]triphosphate induced a transient accumulation of radioactivity in Ins(1,4,5)P3; we did not detect radioactivity in inositol 1,3,4-trisphosphate or inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. In vitro, Ins(1,4,5)P3 was metabolized to inositol 1,4- and 4,5-bisphosphate, but not to Ins(1,3,4,5)P4 or another tetrakisphosphate isomer. We conclude that Dictyostelium has a receptor- and G-protein-stimulated inositol cycle which is basically identical with that in mammalian cells, but the metabolism of Ins(1,4,5)P3 is probably different.

Cyclic-AMP-induced elevation of intracellular pH precedes, but does not mediate, the induction of prespore differentiation in Dictyostelium discoideum.

Prespore gene expression in Dictyostelium is induced by the interaction of cAMP with cell surface cAMP receptors. We investigated whether intracellular pH (pHi) changes mediate the induction of prespore gene expression by cAMP. It was found that cAMP induces a 0.15 unit increase in pHi within 45 min after stimulation. The cAMP-induced pHi increase precedes the induction of prespore gene expression, measured by in vitro transcription, by about 15-30 min. Cyclic-AMP-induced pHi changes can be bypassed or clamped by addition of, respectively, the weak base methylamine, which increases pHi, or the weak acid 5,5-dimethyl-2,4-oxazolidinedione (DMO), which decreases pHi. Bypass of the cAMP-induced increase of pHi with methylamine does not induce the expression of prespore genes, while inhibition of the pHi increase with DMO does not inhibit the induction of prespore gene expression. Cyclic-AMP-induced prespore protein synthesis and the proportion of prespore cells in multicellular aggregates are also not affected by bypassing or inhibiting the cAMP-induced pHi increase. These results show that although a morphogen-induced pHi increase precedes the induction of prespore gene expression, this increase does not mediate the effects of the extracellular cAMP signal on the transcription or translation of prespore genes in Dictyostelium discoideum.

Regulation of diacylglycerol kinase in the transition from quiescence to proliferation in Dictyostelium discoideum.

Diacylglycerol kinase and phosphatidylinositol kinase were examined in stationary phase D. discoideum amoeba induced to synchronously proliferate by dilution into fresh medium. Membrane bound diacylglycerol kinase activity showed a rapid and transitory 3-5 fold increase in the preproliferative interphase while phosphatidylinositol kinase activity was kept quite constant during the same period. The changes in diacylglycerol kinase activity seem to be due to a translocation of the enzyme from the soluble to the particulate cell compartments.

Lithium respecifies cyclic AMP-induced cell-type specific gene expression in Dictyostelium.

We investigated the effect of LiCl on pattern formation and cAMP-regulated gene expression in Dictyostelium discoideum. In intact slugs, 5 mM LiCl induces an almost complete redifferentiation of prespore into prestalk cells. We found that LiCl acts by interfering with the transduction of extracellular cAMP to cell-type-specific gene expression; LiCl inhibits the induction of prespore-specific gene expression by cAMP, while it promotes the induction of prestalk-associated gene expression by cAMP. Our results indicate that two divergent pathways transduce the extracellular cAMP signal to, respectively, prestalk and prespore gene expression.

Cyclic AMP induces a transient alkalinization in Dictyostelium.

In a wide range of cell types, stimulus-response coupling is accompanied by a rise in cytoplasmic pH (pHi). It is shown that stimulation of developing Dictyostelium discoideum cells with the chemoattractant cAMP also results in a rise in pHi. About 1.5 min after stimulation, pHi starts increasing from pHi approximately 7.45 to pHi approximately 7.60, as is revealed independently by two different pH null-point methods. The rise in pHi is transient, also with a persistent stimulus, and effectively inhibited by diethylstilbestrol (DES), strongly suggesting that the rise in pHi is accomplished by the DES-sensitive plasma membrane proton pump which has been demonstrated in D. discoideum.