Step In Signal Transduction Research Articles

Activation of mast cells by incorporation of cholesterol into rafts.

IgE plus antigen-stimulated mast cells degranulate, synthesize leukotrienes and secrete cytokines. According to the coalescence model this process is initiated in specific membrane compartments termed rafts. There, enhanced levels of glycosphingolipids and cholesterol stabilize the interaction of FcepsilonRI and Lyn, and thus facilitate the first steps of signal transduction. Enforced changes in raft architecture by cholesterol deprivation and exogenous application of glycosphingolipids influence these early events by loss of tyrosine kinase activity or receptor-independent signal initiation respectively. Here we show that exogenously added cholesterol accumulates in rafts and activates mast cells. An investigation of the signaling events reveals that in contrast to IgE plus antigen-mediated stimulation, cholesterol triggers p38 mitogen-activated protein kinase and preferentially induces expression of FosB. Consequently, a comparative large-scale microarray analysis demonstrates that a number of IgE plus antigen-induced immediate early genes (peak expression at 30 min after induction) are repressed by cholesterol. These changes further translate into altered expression levels and time kinetics of a number of early genes (peak expression at 2 h after stimulation). As the most prominent example for cholesterol-dependent genes, we identified PAI1 (plasminogen activator inhibitor 1), a protein regarded as a risk factor for atherosclerosis.

Aluminum rapidly depolymerizes cortical microtubules and depolarizes the plasma membrane: evidence that these responses are mediated by a glutamate receptor.

Efforts to understand how plants respond to aluminum have focused on describing the symptoms of toxicity and elucidating mechanisms of tolerance; however, little is known about the signal transduction steps that initiate the plant's response. Here, we image cortical microtubules and quantify plasma-membrane potential in living, root cells of intact Arabidopsis seedlings. We show that aluminum depolymerizes microtubules and depolarizes the membrane, and that these responses are prevented by calcium channel blockade. Calcium influx might involve glutamate receptors, which in animals are ligand-gated cation channels and are present in the Arabidopsis genome. We show that glutamate depolymerizes microtubules and depolarizes the plasma membrane. These responses, and also the inhibition of root elongation, occur within the first few min of treatment, but are evoked more rapidly by glutamate than by aluminum. Microtubule depolymerization and membrane depolarization, induced by either glutamate or aluminum, are blocked by a specific antagonist of ionotropic glutamate receptors, 2-amino-5-phosphonopentanoate; whereas an antagonist of an aluminum-gated anion channel blocks the two responses to aluminum but not to glutamate. For growth, microtubule integrity, and membrane potential, responses to combined glutamate and aluminum were not greater than to glutamate alone. We propose that signaling in response to aluminum is initiated by efflux of a glutamate-like ligand through an anion channel and the binding of this ligand to a glutamate receptor.

The role of membrane-bound LBP, endotoxin aggregates, and the MaxiK channel in LPS-induced cell activation.

We have previously shown in patch-clamp experiments on excised outside-out cytoplasmic membrane patches from human macrophages that the activation of a high-conductance Ca(2+)- and voltage-dependent potassium channel, the MaxiK channel, is an early step in LPS-induced transmembrane signal transduction in macrophages. MaxiK can be activated by agonistically active LPS, and activation can be completely inhibited by LPS antagonists (e.g. synthetic compound 406) and by anti-CD14 antibodies. Furthermore, by inhibiting MaxiK with the specific MaxiK blocker paxilline, we could show that activation of MaxiK is essential for LPS-induced cytokine production. As shown by RT-PCR, blockade of MaxiK by paxilline also inhibits induction of the mRNA of TNF-alpha and IL-6. This observation together with the fact that all patch-clamp experiments were done on excised outside-out patches reveal that MaxiK activation is an early step in cell activation by endotoxins. Thus, since cells lacking TLR4 on their surface can also not be activated to produce cytokines, these data allow the conclusion that TLR4 and MaxiK are both essential for activation by LPS and may form a co-operative signaling complex. We have also shown that LBP not only exists as a soluble acute-phase serum protein, but is also incorporated as a transmembrane protein (mLBP) in the cytoplasmic membrane of MNC; in this configuration, it is obviously involved in the binding of endotoxin and its transfer to the transmembrane signaling proteins finally triggering cell activation. Complexation of soluble LBP and LPS in the serum prior to binding of LPS to mLBP, in contrast, leads to neutralization of LPS. Here, we provide evidence from fluorescence resonance energy transfer spectroscopy that endotoxin aggregates are intercalated into reconstituted membranes by mLBP. In addition, cell culture assays and patch-clamp experiments demonstrate that endotoxin activates macrophages and the MaxiK channel in the aggregated, but not in the monomeric, state at similar concentrations.

The role of membrane-bound LBP, endotoxin aggregates, and the MaxiK channel in LPS-induced cell activation

We have previously shown in patch-clamp experiments on excised outside-out cytoplasmic membrane patches from human macrophages that the activation of a high-conductance Ca2+ - and voltage-dependent potassium channel, the MaxiK channel, is an early step in LPS-induced transmembrane signal transduction in macrophages. MaxiK can be activated by agonistically active LPS, and activation can be completely inhibited by LPS antagonists ( e.g. synthetic compound 406) and by anti-CD14 antibodies. Furthermore, by inhibiting MaxiK with the specific MaxiK blocker paxilline, we could show that activation of MaxiK is essential for LPS-induced cytokine production. As shown by RT-PCR, blockade of MaxiK by paxilline also inhibits induction of the mRNA of TNF-α and IL-6. This observation together with the fact that all patch-clamp experiments were done on excised outside-out patches reveal that MaxiK activation is an early step in cell activation by endotoxins. Thus, since cells lacking TLR4 on their surface can also not be activated to produce cytokines, these data allow the conclusion that TLR4 and MaxiK are both essential for activation by LPS and may form a co-operative signaling complex. We have also shown that LBP not only exists as a soluble acute-phase serum protein, but is also incorporated as a transmembrane protein (mLBP) in the cytoplasmic membrane of MNC; in this configuration, it is obviously involved in the binding of endotoxin and its transfer to the transmembrane signaling proteins finally triggering cell activation. Complexation of soluble LBP and LPS in the serum prior to binding of LPS to mLBP, in contrast, leads to neutralization of LPS. Here, we provide evidence from fluorescence resonance energy transfer spectroscopy that endotoxin aggregates are intercalated into reconstituted membranes by mLBP. In addition, cell culture assays and patch-clamp experiments demonstrate that endotoxin activates macrophages and the MaxiK channel in the aggregated, but not in the monomeric, state at similar concentrations.

The NFP locus of Medicago truncatula controls an early step of Nod factor signal transduction upstream of a rapid calcium flux and root hair deformation.

Establishment of the Rhizobium-legume symbiosis depends on a molecular dialogue, in which rhizobial nodulation (Nod) factors act as symbiotic signals, playing a key role in the control of specificity of infection and nodule formation. Using nodulation-defective (Nod-) mutants of Medicago truncatula to study the mechanisms controlling Nod factor perception and signalling, we have previously identified five genes that control components of a Nod factor-activated signal transduction pathway. Characterisation of a new M. truncatula Nod- mutant led to the identification of the Nod Factor Perception (NFP) locus. The nfp mutant has a novel phenotype among Nod- mutants of M. truncatula, as it does not respond to Nod factors by any of the responses tested. The nfp mutant thus shows no rapid calcium flux, the earliest detectable Nod factor response of wild-type plants, and no root hair deformation. The nfp mutant is also deficient in Nod factor-induced calcium spiking and early nodulin gene expression. While certain genes controlling Nod factor signal transduction also control the establishment of an arbuscular mycorrhizal symbiosis, the nfp mutant shows a wild-type mycorrhizal phenotype. These data indicate that the NFP locus controls an early step of Nod factor signal transduction, upstream of previously identified genes and specific to nodulation.

Vascular Endothelial Growth Factor-dependent Down-regulation of Flk-1/KDR Involves Cbl-mediated Ubiquitination: CONSEQUENCES ON NITRIC OXIDE PRODUCTION FROM ENDOTHELIAL CELLS

Ligand-stimulated degradation of receptor tyrosine kinase (RTK) is an important regulatory step of signal transduction. The vascular endothelial growth factor (VEGF) receptor Flk-1/KDR is responsible for the VEGF-stimulated nitric oxide (NO) production from endothelial cells. Cellular mechanisms mediating the negative regulation of Flk-1 signaling in endothelial cells have not been investigated. Here we show that Flk-1 is rapidly down-regulated following VEGF stimulation of bovine aortic endothelial cells (BAECs). Consequently, VEGF pretreatment of endothelial cells prevents any further stimulation of Flk-1, resulting in decreased NO production from subsequent VEGF challenges. Ubiquitination of RTKs targets them for degradation; we demonstrate that activation of Flk-1 by VEGF leads to its polyubiquitination in BAECs. Furthermore, VEGF stimulation of BAECs or COS-7 cells transiently transfected with Flk-1 results in the phosphorylation of the ubiquitin ligase Cbl, the enhanced association of Cbl with Flk-1, and the relocalization of Cbl to vesicular structures in BAECs. Overexpression of Cbl in COS-7 cells enhances VEGF-induced ubiquitination of Flk-1, whereas a Cbl mutant lacking the ubiquitin ligase RING finger domain, 70Z/3-Cbl, does not. Moreover, expression of Cbl in contrast to 70Z/3-Cbl inhibits the Flk-1-dependent activation of eNOS and, thus, NO release. In BAEC overexpressing Cbl, the degradation of Flk-1 upon VEGF stimulation is accelerated compared with cells transfected with a control vector (green fluorescent protein). Our findings demonstrate that Flk-1 is rapidly down-regulated following sustained VEGF stimulation and identify Cbl as a negative regulator of Flk-1 signaling to eNOS. Cbl thus plays a role in the regulation of VEGF signaling by mediating the stimulated ubiquitination and, consequently, degradation of Flk-1 in endothelial cells.

Bacterial chemotaxis: a new player in response regulator dephosphorylation.

Escherichia coli chemotaxis is arguably the best understood of all biological behaviors, but even this relatively “simple” system continues to offer up new molecular insights from Evolution’s bag of signaling tricks. The latest surprise comes from Silversmith et al. (15), who report in this issue how the X-ray structure of a mutant CheY protein led them to the discovery of a CheY residue that plays a special role in this response regulator’s phosphorylation activities. Their story not only clarifies a mechanistically murky step in chemotactic signal transduction but also provides us with a nice example of molecular detective work.

Interactions of Insulin-like Growth Factors and Estradiol in Rat Pituitary Gonadotrophs

Insulin-like growth factors are involved in the regulation of gonadotropin secretion. Insulin-like growth factor I (IGF-I) has an augmenting effect on gonodotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) release from female rat gonadotrophs that is facilitated by estradiol. To identify the underlying mechanisms, we investigated whether IGF-I influences total LH pool and the production of intracellular inositol phosphate. In another series of experiments we tested whether IGF-II and estradiol affect LH release of gonadotrophs. Pituitary cells were incubated with 100 pM IGF-I and/or 100 pM estradiol for 24 h. They were stimulated, partially in the presence of Wortmannin, an inhibitor of phosphoinositide 3-kinase, with 330 pM GnRH for 3 h. Subsequently, total LH pool (released and remaining hormone content in lysed cells) in cultures was measured. Intracellular inositol trisphosphate of ! T3-1 cells, a gonadotrope cell line, treated with estradiol and IGF-I as described before and stimulated with 100 nM GnRH for 15 min was analyzed by ion exchange chromatography. To determine the interaction of IGF-II and estradiol on GnRH-stimulated LH secretion, cells were treated with increasing concentrations of IGF-II (0.05 pM-10 nM) and 100 pM estradiol. IGF-I significantly increased the accumulation of inositol trisphosphate in basal and GnRH-stimulated cells. IGF-I, estradiol, or their combinations did not change total LH pool, although they enhanced LH secretion. Wortmannin abolished the positive effects of IGF-I and estradiol on LH secretion. IGF-II alone increased basal, but not GnRH-induced LH secretion at low concentrations (0.05 pM). Additional estradiol treatment further increased basal, but not GnRH-induced LH secretion. In conclusion, our results suggest that increased LH secretion from female anterior pituitary cells after IGF treatment is due to the amplification of early signal transduction steps rather than changes in LH pool. The inositol trisphosphate signaling pathway is involved in the regulation of LH secretion from gonadotrophs treated with IGF-I. It is not likely that IGF-II plays an important role in the regulation of gonadotropin secretion.

Genetic and cytogenetic mapping of DMI1, DMI2, and DMI3 genes of Medicago truncatula involved in Nod factor transduction, nodulation, and mycorrhization.

The DMI1, DMI2, and DMI3 genes of Medicago truncatula, which are required for both nodulation and mycorrhization, control early steps of Nod factor signal transduction. Here, we have used diverse approaches to pave the way for the map-based cloning of these genes. Molecular amplification fragment length polymorphism markers linked to the three genes were identified by bulked segregant analysis. Integration of these markers into the general genetic map of M. truncatula revealed that DMI1, DMI2, and DMI3 are located on linkage groups 2, 5, and 8, respectively. Cytogenetic studies using fluorescent in situ hybridization (FISH) on mitotic and pachytene chromosomes confirmed the location of DMI1, DMI2, and DMI3 on chromosomes 2, 5, and 8. FISH-pachytene studies revealed that the three genes are in euchromatic regions of the genome, with a ratio of genetic to cytogenetic distances between 0.8 and 1.6 cM per microm in the DMI1, DMI2, and DMI3 regions. Through grafting experiments, we showed that the genetic control of the dmi1, dmi2, and dmi3 nodulation phenotypes is determined at the root level. This means that mutants can be transformed by Agrobacterium rhizogenes to accelerate the complementation step of map-based cloning projects for DMI1, DMI2, and DMI3.

Molecular basis of transmembrane signalling by sensory rhodopsin II-transducer complex.

Microbial rhodopsins, which constitute a family of seven-helix membrane proteins with retinal as a prosthetic group, are distributed throughout the Bacteria, Archaea and Eukaryota. This family of photoactive proteins uses a common structural design for two distinct functions: light-driven ion transport and phototaxis. The sensors activate a signal transduction chain similar to that of the two-component system of eubacterial chemotaxis. The link between the photoreceptor and the following cytoplasmic signal cascade is formed by a transducer molecule that binds tightly and specifically to its cognate receptor by means of two transmembrane helices (TM1 and TM2). It is thought that light excitation of sensory rhodopsin II from Natronobacterium pharaonis (SRII) in complex with its transducer (HtrII) induces an outward movement of its helix F (ref. 6), which in turn triggers a rotation of TM2 (ref. 7). It is unclear how this TM2 transition is converted into a cellular signal. Here we present the X-ray structure of the complex between N. pharaonis SRII and the receptor-binding domain of HtrII at 1.94 A resolution, which provides an atomic picture of the first signal transduction step. Our results provide evidence for a common mechanism for this process in phototaxis and chemotaxis.

Signalling in Rhizobacteria‐Induced Systemic Resistance in Arabidopsis thaliana

Abstract: To protect themselves from disease, plants have evolved sophisticated defence mechanisms in which the signal molecules salicylic acid, jasmonic acid and ethylene often play crucial roles. Elucidation of signalling pathways controlling disease resistance is a major objective in research on plant‐pathogen interactions. The capacity of a plant to develop a broad spectrum, systemic acquired resistance (SAR) after primary infection with a necrotizing pathogen is well‐known and its signal transduction pathway extensively studied. Plants of which the roots have been colonized by specific strains of non‐pathogenic fluorescent Pseudomonas spp. develop a phenotypically similar form of protection that is called rhizobacteria‐mediated induced systemic resistance (ISR). In contrast to pathogen‐induced SAR, which is regulated by salicylic acid, rhizobacteria‐mediated ISR is controlled by a signalling pathway in which jasmonic acid and ethylene play key roles. In the past eight years, the model plant species Arabidopsis thaliana was explored to study the molecular basis of rhizobacteria‐mediated ISR. Here we review current knowledge of the signal transduction steps involved in the ISR pathway that leads from recognition of the rhizobacteria in the roots to systemic expression of broad‐spectrum disease resistance in aboveground foliar tissues.

Molecular cloning of actin filament-associated protein: a putative adaptor in stretch-induced Src activation.

Mechanical stretch-induced activation of c-Src is an important step for signal transduction of stretch-induced fetal rat lung cell proliferation. This process appears to be mediated through actin filament-associated protein (AFAP), encoded by a gene originally cloned from the chicken. In the present study, we cloned the rat AFAP gene from fetal rat lungs. Its mRNA and protein are differentially expressed among various tissues. The protein is colocalized with actin filaments in fetal rat lung epithelial cells and fibroblasts. Mechanical stretch increased tyrosine phosphorylation of rat AFAP and its binding to c-Src within the initial several minutes. Src SH2 and SH3 binding motifs are highly conserved in the AFAP proteins (from chicken, rat to human). On the basis of the molecular structure of AFAP protein, we speculate that it is an adaptor in mechanical stretch-induced activation of c-Src. A novel model of mechanoreception is proposed.

Alteration of thrombine-signaling mechanism by heptachlor in human platelets.

Heptachlor is a persistent organochlorine insecticide that has been detected in human tissues and fluids. The ability of heptachlor to interfere with platelet phosphoinositides metabolism and related signaling events stimulated by thrombin was evaluated. In vitro incubations with a concentration range of 1-100 microM heptachlor, prior to platelets activation, were performed. Experiments showed that 10 microM increased protein Kinase C (PKC) activity and phosphatidylinositolbiphosphate and phosphatidic acid phosphorylation. Simultaneously phosphatidylcholine and phosphatidylethanolamine breakdown were prevented. Similar effects were observed with HC 1 microM. However, heptachlor 100 microM increased phosphatidylinositolbiphosphate phosphorylation but reduced serine/threonine kinases activity. We propose that signal transduction steps downstream phospholipase C (PLC) are unphysiologically activated by heptachlor and facilitated by the increase in phosphatidylinositolbiphosphate, the substrate for PLC activity, thus producing an accumulation of phosphatidic acid. The elevated level of this compound itself or the transient increase in diacylglycerol produced may cause calcium mobilization and the activation of PKC. In contrast with the alterations observed in phospholipids and protein phosphorylation, no changes in aggregation properties were observed.

Early steps of the intramolecular signal transduction in rhodopsin explored by molecular dynamics simulations.

We present molecular dynamics simulations of bovine rhodopsin in a membrane mimetic environment based on the recently refined X-ray structure of the pigment. The interactions between the protonated Schiff base and the protein moiety are explored both with the chromophore in the dark-adapted 11-cis and in the photoisomerized all-trans form. Comparison of simulations with Glu181 in different protonation states strongly suggests that this loop residue located close to the 11-cis bond bears a negative charge. Restrained molecular dynamics simulations also provide evidence that the protein tightly confines the absolute conformation of the retinal around the C12-C13 bond to a positive helicity. 11-cis to all-trans isomerization leads to an internally strained chromophore, which relaxes after a few nanoseconds by a switching of the ionone ring to an essentially planar all-trans conformation. This structural transition of the retinal induces in turn significant conformational changes of the protein backbone, especially in helix VI. Our results suggest a possible molecular mechanism for the early steps of intramolecular signal transduction in a prototypical G-protein-coupled receptor.

Mechanisms of phosphate-induced disease resistance in cucumber

Certain phosphate salts are known inducers of systemic acquired resistance (SAR). In the present study, a local spray application of dipotassium hydrogenphosphate (K2HPO4) was effective in inducing a high level of systemic protection in cucumber plants against anthracnose caused by Colletotrichum lagenarium. Resistance induction by K2HPO4 was associated with localized cell death in cucumber leaves treated with the phosphate salt. The cell death observed, subsequently resulted in the appearance of macroscopically visible, necrotic spots. Appearing lesions resembled those provoked by tobacco necrosis virus (TNV) during a hypersensitive response (HR) that leads to pathogen-induced activation of SAR. Phosphate-mediated cell death was preceeded by a rapid generation of superoxide and hydrogen peroxide. As a further consequence of phosphate application, a local and systemic increase in free and conjugated salicylic acid (SA) levels was detected. The phosphate-induced responses were also identified with a similar time range in cucumber leaves that had been pre-inoculated with TNV. In contrast, none of these responses was triggered by application of the commercial plant activator benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester (BTH), which nevertheless was highly effective in inducing SAR in cucumber against anthracnose. In conclusion, the chemical SAR inducer K2HPO4 and the biological inducer TNV share some common early steps in signal transduction leading to SAR in cucumber, which differ from those involved in BTH-mediated SAR.

Ceramide and glucosamine antagonism of alternate signaling pathways regulating insulin- and osmotic shock-induced glucose transporter 4 translocation.

In addition to insulin, hyperosmolarity induces glucose transporter 4 (GLUT4) translocation in 3T3-L1 adipocytes. However, in contrast to insulin this stimulation is independent of PI3K/Akt. In this study we assessed whether ceramide and/or glucosamine, two known insulin-signaling antagonists, also affected the PI3K/Akt-independent signal. Insulin, but not hyperosmolarity, clearly increased the activities of PI3K and Akt. C2-ceramide did not alter insulin-stimulated PI3K activity, but did decrease the ability of insulin to activate Akt and GLUT4 translocation. Consistent with osmotic shock-mediated GLUT4 translocation being independent of PI3K/Akt, GLUT4 translocation induced by hyperosmolarity was not altered by C2-ceramide. In contrast to the specific C2-ceramide-induced attenuation of insulin-stimulated GLUT4 translocation, overexpression of glutamine:fructose-6-phosphate amidotransferase, the rate-limiting enzyme in the synthesis of UDP-N-acetylglucosamine, and/or pretreatment of cells with glucosamine, a precursor of UDP-N-acetylglucosamine, inhibited both insulin- and hyperosmolarity-stimulated GLUT4 translocation. Glucosamine did not alter any of the known proximal insulin signal transduction events. These data suggest that although the hyperosmolarity-induced signal bypasses the initial insulin signal transduction steps, it is likely to induce GLUT4 translocation through activation of a common convergent signal transduction step, targeted by UDP-N-acetylglucosamine, downstream of and/or in parallel to PI3K/Akt.

Ceramide and Glucosamine Antagonism of Alternate Signaling Pathways Regulating Insulin- and Osmotic Shock-Induced Glucose Transporter 4 Translocation

In addition to insulin, hyperosmolarity induces glucose transporter 4 (GLUT4) translocation in 3T3-L1 adipocytes. However, in contrast to insulin this stimulation is independent of PI3K/Akt. In this study we assessed whether ceramide and/or glucosamine, two known insulin-signaling antagonists, also affected the PI3K/Akt-independent signal. Insulin, but not hyperosmolarity, clearly increased the activities of PI3K and Akt. C2-ceramide did not alter insulin-stimulated PI3K activity, but did decrease the ability of insulin to activate Akt and GLUT4 translocation. Consistent with osmotic shock- mediated GLUT4 translocation being independent of PI3K/Akt, GLUT4 translocation induced by hyperosmolarity was not altered by C2-ceramide. In contrast to the specific C2-ceramide-induced attenuation of insulin-stimulated GLUT4 translocation, overexpression of glutamine:fructose-6-phosphate amidotransferase, the rate-limiting enzyme in the synthesis of UDP-N-acetylglucosamine, and/or pretreatment of cells with glucosamine, a precursor of UDP-N-acetylglucosamine, inhibited both insulin- and hyperosmolarity-stimulated GLUT4 translocation. Glucosamine did not alter any of the known proximal insulin signal transduction events. These data suggest that although the hyperosmolarity-induced signal bypasses the initial insulin signal transduction steps, it is likely to induce GLUT4 translocation through activation of a common convergent signal transduction step, targeted by UDP-N-acetylglucosamine, downstream of and/or in parallel to PI3K/Akt.

A novel voltage-sensitive Na + and Ca 2+ channel blocker, NS-7, prevents suppression of cyclic AMP-dependent protein kinase and reduces infarct area in the acute phase of cerebral ischemia in rat

Binding of cyclic AMP to the regulatory subunit of cyclic AMP-dependent protein kinase (PKA) is an essential step in cyclic AMP-mediated intracellular signal transduction. This binding is, however, rapidly inhibited in the acute phase of cerebral ischemia, indicating that the signal transduction via PKA is very vulnerable to ischemia, although this signal pathway is very important for neuronal survival in the brain. Several lines of evidence suggest that the activation of voltage-sensitive Na + and Ca 2+ channels is an important mediator of acute ischemic brain damage. In the present study, therefore, we examined the effect of a novel Na + and Ca 2+ channel blocker, NS-7 (4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride), on changes in the binding activity of PKA to cyclic AMP in permanent focal cerebral ischemia, which was induced by occlusion of the middle cerebral artery by the intraluminal suture method for 5 h in the rat. NS-7 (1 mg/kg) or saline was intravenously infused 5 min after occlusion. The binding activity of PKA to cyclic AMP and local cerebral blood flow were assessed by the in vitro [ 3H]cyclic AMP binding and the [ 14C]iodoantipyrine methods, respectively. NS-7 significantly suppressed inhibition of the binding activity of PKA to cyclic AMP in the ischemic regions such as the frontal and parietal cortices and the medial region of the caudate-putamen without affecting cerebral blood flow or arterial blood pressure. Infarct area measured in the brain slices stained with cresyl violet was significantly smaller in animals treated with NS-7 than in those treated with saline. Blockade of voltage-sensitive Na + and Ca 2+ channels by NS-7 was expected to reduce ischemia-induced depolarization and thus prevent a massive formation of free radicals, which is known to inhibit the binding activity of PKA to cyclic AMP. These data clearly indicate that NS-7 provides very efficient neuroprotection in the acute phase of cerebral ischemia, and sustains the normal function of PKA.

Effects of bethanechol on canine urinary bladder smooth muscle function

We studied whether the effects of bethanechol are mediated via a muscarinic receptor, the role of extracellular calcium on bladder contraction, and down-regulation of bladder contraction by bethanechol after activation with potassium chloride (KCl) and acetylcholine (Ach). Smooth muscle strips of normal urinary bladder were studied with standard methods to measure isometric force. Bethanechol caused a dose-dependent increase in bladder contraction. The potency of bethanechol is higher than Ach, as shown by higher peak active isometric stress (Pmax) and lower half-maximal contraction (ED50) (P<0.01). The contractile responses to bethanechol were diminished in the presence of atropine, nifedipine and in calcium-free medium as shown by Pmaxdecreased by 58%, 87% and 65% andED50 increased by 314-, 24- and 16-fold, respectively. When bladder strips were stimulated with KCl and Ach, pre-treatment with bethanechol reduced the responses to KCl by 116–242% (P<0.05), while the contractile responses to Ach were unaltered. Thus, bethanechol induces bladder contraction via muscarinic receptor activation while both intracellular and extracellular calcium play a crucial role on bladder smooth muscle contraction. The mechanisms of down-regulation by bethanechol may be related to interference with calcium influx into the smooth muscle cells, rather than the desensitisation of muscarinic receptors or post-receptor steps of signal transduction following bethanechol binding to the receptor.

CGMP abolishes agonist-induced [Ca(2+)](i) oscillations in human bladder epithelial cells.

Cytosolic calcium oscillations may permit cells to respond to information provided by increases in intracellular Ca(2+) concentration ([Ca(2+)](i) ) while avoiding prolonged exposure to constantly elevated [Ca(2+)](i). In this study, we demonstrated that agonists could induce Ca(2+) oscillations in human bladder epithelial cells. Application of 10 microM acetylcholine or 200 nM bradykinin triggered an initial Ca(2+) transient that was followed by periodic [Ca(2+)](i) oscillations. The oscillations did not depend on extracellular Ca(2+). 8-Bromoguanosine 3',5'-cyclic monophosphate abolished acetylcholine- or bradykinin-induced oscillations. Elevation of cellular cGMP by dipyridamole, an inhibitor of cGMP-specific phosphodiesterase, also terminated the [Ca(2+)](i) oscillations. The inhibitory effect of cGMP could be reversed by KT-5823, a highly specific inhibitor of protein kinase G (PKG), suggesting that the action of cGMP was mediated by PKG. Comparison of the effect of cGMP with that of xestospongin C, an inhibitor of the inositol 1,4,5-trisphosphate (IP(3)) receptor, revealed similarities between the action of cGMP and xestospongin C. Therefore, it is likely that cGMP and PKG may target a signal transduction step(s) linked to IP(3) receptor-mediated Ca(2+) release.