The small GTP-binding protein RhoA regulates serotonin-induced Na+-current response in the neurons of Aplysia

Application of serotonin (5-HT) induces a slow inward current response in identified neurons of Aplysia ganglia under voltage clamp. The 5-HT-induced current response was depressed in Na+-free media, but augmented in Ca2+-free media, and unaffected by a change in external K+. The 5-HT-induced response was markedly blocked by intracellular injection of guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS). After the injection of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), the responses to 5-HT gradually and significantly increased at the initial period, reached its plateau, and finally decreased. Intracellular injection of Clostridium difficile toxin B, a blocker of small G-protein Rho family members such as Rho (RhoA, RhoB and RhoC), Rac and Cdc42, markedly depressed the 5-HT-induced response. Intracellular injection of Clostridium botulinum C3 exoenzyme, a specific blocker of RhoA, RhoB, RhoC, exhibited a similar depressing effect observed with toxin B. In contrast, intracellular injection of recombinant L63RhoA, a constitutively active form of RhoA, significantly augmented the 5-HT-induced response without affecting the resting membrane. These results suggested that the 5-HT-induced Na+-current response might be facilitated by the activation of Aplysia Rho which is closely homologous to RhoA, RhoB or RhoC in mammalian neuron.     

Effects of Clostridium difficile toxin A and toxin B on phospholipase D activation in human promyelocytic leukemic HL60 cells.

The possible involvement of Rho family GTP-binding proteins in the regulation of phospholipase D (PLD) activity has recently been demonstrated. In the present study, to further examine the role of Rho family proteins in PLD activation of human promyelocytic leukemic HL60 cells, we used toxin A and toxin B from the anaerobic bacterium Clostridium difficile, which was shown to glucosylate Rho family proteins and inhibit their interaction with effectors. Pretreatment of [3H]oleic acid-labeled HL60 cell lysates with either one of the toxins resulted in a remarkable inhibition of membrane PLD activity stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). The magnitude of inhibition of PLD activity was correlated well with the extent of toxin A- or B-induced glucosylation of 22-kDa RhoA in HL60 cells, toxin B being more effective than toxin A. GTPgammaS-stimulated PLD activation measured with the exogenous substrate containing phosphatidylinositol 4,5-bisphosphate was also inhibited by toxin B. Toxin B had no effect on GTP-gammaS-induced translocation of RhoA from cytosol to membranes. Furthermore, the toxin B pretreatment also suppressed PLD activation induced by 4beta-phorbol 12-myristate 13-acetate in HL60 cell lysates. Thus, it was indicated that Rho family proteins play a key role in GTPgammaS- and 40-phorbol 12-myristate 13-acetate-induced PLD activity in HL60 cells. In addition, the results obtained here indicate that C. difficile toxins are a useful tool for researching the regulation of the Rho family protein-mediated PLD activation and also provide a clue toward understanding the pathogenic background of pseudomembranous colitis from the viewpoint of signal transduction.     

Multiple effects of dopamine on layer V pyramidal cell excitability in rat prefrontal cortex

The mechanisms underlying the inhibitory effects of dopamine (DA) on layer V pyramidal neuron excitability in the prelimbic region of the rat medial prefrontal cortex were investigated. Under control conditions, DA depressed both action potential generation (driven by somatic current injection) and input resistance (R(N)). The presence of GABA(A) receptor antagonists blocked DA-induced depression of action potential generation and revealed a delayed increase in excitability that persisted for the duration of experimental recording, up to 20 min following the washout of DA. In contrast to spike generation, disinhibition did not affect the transient depression of R(N) produced by DA, suggesting independent actions of DA on spike generation and R(N). Consistent with the hypothesis that DA acts to decrease pyramidal cell output via a GABAergic mechanism, DA increased the frequency of spontaneous inhibitory postsynaptic currents in both the absence and presence of TTX. Furthermore focal application of GABA to a perisomatic region mimicked the inhibitory effect of DA on spike production without affecting R(N). Focal application of bicuculline to the same location reversed the inhibitory effect of bath-applied DA on spike generation, while again having no effect on R(N). The depression of R(N) by DA was both occluded and mimicked by the Na(+) channel blocker TTX, suggesting the involvement of a Na(+) conductance in reducing pyramidal cell R(N) during the acute presence of DA. Together these data demonstrate that the acute presence of DA decreases pyramidal neuron excitability by two independent mechanisms. At the same time DA triggers a delayed and longer-lasting increase in excitability that is partially masked by synaptic inhibition.     

Bacterial protein toxins inhibiting low-molecular-mass GTP-binding proteins

The Rho GTPases, which belong to the Ras superfamily of low-molecular-mass GTP-binding proteins, are the preferred intracellular targets of bacterial protein toxins. The Rho GTPases RhoA/B/C, Rac1/2 and Cdc42 are the master regulators of the actin cytoskeleton. Clostridium difficile toxins A and B, the causative agents of the antibiotic-associated pseudomembranous colitis, are intracellularly acting cytotoxins which mono-glucosylate the Rho GTPases. Clostridium botulinum C3 toxin, which is not related to the clostridial neurotoxins, catalyses ADP-ribosylation of RhoA/B/C but not of other Rho GTPases. Glucosylation as well as ADP-ribosylation result in functional inactivation of Rho causing disassembly of the actin cytoskeleton.     

Direct inhibition of substantia gelatinosa neurones in the rat spinal cord by activation of dopamine D2-like receptors

Dopaminergic innervation of the spinal cord is largely derived from the brain. To understand the cellular mechanisms of antinociception mediated by descending dopaminergic pathways, we examined the actions of dopamine (DA) on nociceptive transmission by using behavioural studies and whole-cell patch-clamp recordings from substantia gelatinosa (SG) neurones in the spinal cord. Intrathecal administration of DA increased the mechanical nociceptive threshold and this effect was mimicked by a D2-like receptor agonist, quinpirole, but not by a D1-like receptor agonist, SKF 38393. In current-clamp mode of patch-clamp recordings, bath application of DA hyperpolarized the membrane potential of SG neurones and suppressed action potentials evoked by electrical stimulation of a dorsal root. In voltage-clamp mode, DA induced an outward current that was resistant to TTX, was blocked by the addition of Cs⁺ or GDP-β-S in the pipette solution, and was inhibited in the presence of Ba⁺. The DA-induced current reversed its polarity at a potential close to the equilibrium potential of the K⁺ channel calculated from the Nernst equation. The DA-induced outward current was mimicked by quinpirole, but not by SKF 38393. The DA-induced outward current was suppressed by a D2-like receptor antagonist, sulpiride, but not by a D1-like receptor antagonist, SCH 23390. In contrast, DA did not cause any significant change in amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs). These results indicate that DA mainly acts on postsynaptic SG neurones to induce an outward current via G-protein-mediated activation of K⁺ channels through D2-like receptors. This may be a possible mechanism for antinociception by the descending dopaminergic pathway.     

Electrophysiological evaluation of the time-course of dopamine uptake inhibition induced by intravenous cocaine at a reinforcing dose

Cocaine effectively inhibits dopamine (DA) uptake and this action appears to be the primary cause for increased DA transmission following systemic cocaine administration. Although this action had been reliably demonstrated in vivo with cocaine at high doses, data on the extent and the time-course of DA uptake inhibition induced by i.v. cocaine at low, reinforcing doses remain controversial. To clarify this issue, we examined how cocaine affects striatal neuronal responses to repeated iontophoretic DA applications in urethane-anesthetized rats. Because most striatal neurons during anesthesia have low, sporadic activity, DA tests were performed on cells tonically activated by continuous glutamate application. DA phasically decreased the activity of most dorsal and ventral striatal neurons; these responses in control conditions (i.v. saline) were current (dose) -dependent and remained highly stable following repeated DA applications at the same currents. DA also consistently decreased the activity of striatal neurons after i.v. cocaine (1 mg/kg); the magnitude of DA-induced inhibition slowly increased from approximately 5 min, became significantly larger from approximately 9 min, and peaked at 13-15 min after a single i.v. injection. Then, the difference in the DA response slowly decreased toward the pre-cocaine baseline. A similar enhancement of DA induced-inhibition was also seen after i.p. cocaine administration at a high dose (15 mg/kg). In this case, the DA response became significantly stronger at 7-9 min and remained enhanced vs. a pre-drug control up to 24-26 min after the injection. Both regimens of cocaine treatment did not result in evident changes in either onset or offset of the DA-induced inhibitions. Our data confirm that cocaine at low, reinforcing doses inhibits DA uptake, resulting in potentiation of DA-induced neuronal inhibitions, but they suggest that this effect is relatively weak and delayed from the time of i.v. injection. These slow and prolonged effects of i.v. cocaine on DA-induced neuronal responses are consistent with previous binding and our electrochemical evaluations of DA uptake, presumably reflecting the total time necessary for i.v.-delivered cocaine to reach brain microvessels, cross the blood-brain barrier, passively diffuse within brain tissue, interact with the DA transporters, and finally inhibit DA uptake.     

Dopamine presynaptically depresses fast inhibitory synaptic transmission via D4 receptor-protein kinase A pathway in the rat dorsolateral septal nucleus

The lateral septal nucleus receives a diffuse dopaminergic input originating from the ventral tegmental area of the brain stem. We examined whether dopamine (DA) modulates synaptic transmission in the slice preparation of the rat dorsolateral septal nucleus (DLSN). Bath application (10-15 min) of DA (30 muM) markedly depressed the amplitude of fast and slow inhibitory postsynaptic potentials (IPSPs) in DLSN neurons, while it produced only a minor depression of the amplitude of excitatory postsynaptic potentials (EPSPs) obtained in the presence of bicuculline. DA (30 muM) depressed the monosynaptic fast IPSP to approximately 50% of control, but did not depress the inward current (I(GABA)) induced by exogenous gamma-aminobutyric acid (GABA). DA decreased the frequency of miniature fast IPSPs (m-fIPSPs) without significantly changing their amplitude. PD 168077, a selective D4 receptor agonist, depressed the fast and slow IPSPs but not the EPSP and decreased the frequency of m-fIPSPs. Both DA and PD 168077 increased the paired-pulse ratio of the monosynaptic fast IPSP. The inhibitory effect of DA on the fast IPSP was significantly attenuated by L-741,742, an antagonist at D4 receptors, but not by SCH 23390 and sulpiride, a D1-like and a D2-like receptor antagonist, respectively. N-ethylmaleimide, a blocker of pertussis toxin (PTX)-sensitive G protein (G(i/o)), attenuated the DA-induced depression of the fast IPSP. N-[2-((p-bromocinnamyl) amino)ethyl]-5-isoquinoline sulfonamide, a protein kinase A (PKA) inhibitor, attenuated the DA-induced depression of the fast IPSP. These results suggest that DA inhibits spontaneous and evoked release of GABA via the D4 receptor-G(i)-protein-PKA system in DLSN neurons.     

Transient expression of RhoA, -B, and -C GTPases in HeLa cells potentiates resistance to Clostridium difficile toxins A and B but not to Clostridium sordellii lethal toxin.

The bacterial pathogen Clostridium difficle synthesizes two high-molecular-weight toxins (A and B), which exhibit toxic effects in vivo and in vitro. Here, we present evidence that the major intracellular targets of these two toxins are the Rho GTPases. Overexpression of RhoA, RhoB, or RhoC GTPases in transfected HeLa cells conferred an increased resistance to toxins A and B, indicating that these toxins cause their cytopathic effects primarily by affecting Rho proteins. In addition, toxin A and B treatment appeared to result in modification of Rho, since Rho isolated from toxin-treated cells had a decreased ability to be ADP-ribosylated by Clostridium botulinum C3 exoenzyme. In contrast, the lethal toxin (LT) of Clostridium sordellii, although structurally and immunologically related to C. difficile toxin B, appeared to induce cytopathic effects independently of the Rho GTPases. Overexpression of RhoA in transfected HeLa cells did not protect them from the effect of LT, and Rho isolated from lysates of LT-treated cells was not resistant to modification by C3. Immunofluorescence studies showed that LT treatment caused a cytopathic effect that was very different from those described for C. difficile toxins A and B, resulting in an increase in cortical F-actin, with a concomitant decrease in the number of stress fibers, and in the formation of numerous microvilli containing the actin-bundling protein fimbrin/plastin.     

Direct and indirect actions of dopamine on the membrane potential in medium spiny neurons of the mouse neostriatum

Many studies have shown dopamine (DA) to have a modulatory effect on neuronal excitability, which cannot be simply classified as excitatory or inhibitory in the neostriatum. To clarify whether the responses to DA (10-30 microM) are excitatory or inhibitory in the mouse medium spiny neurons, we examined the effects of DA agonists on the synchronous potential trajectory from the resting potential to the subthreshold potential. The DA-induced potential changes, which were estimated at the subthreshold potential (approximately -60 mV), were summarized as the combination of three kinds of responses: an initial hyperpolarization lasting approximately 1 min and a slow depolarization and/or hyperpolarization lasting more than 20 min. A D(1)-like receptor agonist, R(+)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF81297, 1 microM) mainly induced the initial hyperpolarization and slow depolarization. A D(2)-like receptor agonist, trans-(-)-4aR-4,4a,5,6,7,8,8a,9-octahydro-5-propyl-1H-pyrazolo[3,4-g]quinoline hydrochloride (quinpirole, 1 microM), mainly induced the initial hyperpolarization and slow hyperpolarization. D(1)-like receptor antagonist R(+)-7-chloro-8-hydroxy-3-methyl1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH23390, 1 microM) depressed both the initial hyperpolarization and slow depolarization. D(2)-like receptor antagonist sulpiride (1 microM) depressed all the DA-induced responses except for the slow depolarization. TTX (0.5 microM) abolished all the DA-induced responses. Bicuculline (20 microM) and atropine (1 microM) abolished the DA-induced initial hyperpolarization and slow depolarization, respectively. Either DL-2-amino-5-phosphonopentanoic acid (AP5; 100 microM) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 microM) blocked both the initial hyperpolarization and slow depolarization. The application of exogenous glutamate (Glu) mimicked the initial hyperpolarization and slow depolarization. These results suggest that the initial hyperpolarization is mainly due to GABA release via the cooperative action of D(1)- and D(2)-like receptors and Glu receptors in GABAergic interneurons, whereas the slow depolarization is mediated by acetylcholine (ACh) release via the cooperative action of mainly D(1)-like receptors and Glu receptors in cholinergic interneurons. The potential oscillation was generated at the subthreshold level in a Ba(2+)-, AP5-, CNQX-, bicuculline-, and atropine-containing medium. The oscillation depressed after the addition of TTX, Co(2+), or DA. In DA agonists, quinpirole rather than SKF81297 had a more depressive effect on the potential oscillation. These results indicate that the slow hyperpolarization is due to the suppression of noninactivating Na(+)-Ca(2+) conductances via mainly D(2)-like receptors in the medium spiny neurons. In conclusion, the DA actions on the medium spiny neurons show a transient inhibition by the activation of D(1)- and D(2)-like receptors in mainly GABAergic interneurons and a tonic excitation and/or inhibition by the activation of mainly D(1)-like receptors in cholinergic interneurons and by the activation of mainly D(2)-like receptors in the medium spiny neurons, respectively.     

Involvement of Rho and Rac small G proteins and Rho GDI in Ca2+-dependent exocytosis from PC12 cells

Background : The Rho small G protein family, which includes the Rho, Rac and Cdc42 subfamilies, is implicated in various cell functions such as cell shape change, cell motility and cytokinesis, through the reorganization of actin filaments. Rho GDI is an inhibitory regulator of the Rho small G protein family and inhibits the Rho family dependent cell functions. Reorganization of actin filaments is also known to regulate Ca²⁺-dependent exocytosis.
Results: We have examined here whether the Rho family members are also involved in Ca²⁺-dependent exocytosis. We have found, by the use of the human growth hormone (GH) co-expression assay system on PC12 cells, that overexpression of Rho GDI inhibits high K⁺-induced, Ca²⁺-dependent GH release. This inhibitory action of Rho GDI is restored by co-expression of a dominant active mutant of RhoA or Rac1, but not of a dominant active mutant of Cdc42. C3 transferase, known to ADP-ribosylate Rho and to inhibit its function, also inhibits this GH release. Overexpression of a dominant active mutant of RhoA or Rac1 alone shows only a small effect on GH release. Moreover, immunocytochemical studies show that the overexpression of Rho GDI prevents a partial disruption of the cortical actin network which accompanies exocytosis.
Conclusions: These results suggest that RhoA, Rac1 and Rho GDI are involved in Ca²⁺-dependent exocytosis at least partly through the reorganization of actin filaments, and that the activation of RhoA or Rac1 alone is not sufficient for this reaction.     

The small GTP-binding proteins Rho and Rac induce T cell adhesion to the mucosal addressin MAdCAM-1 in a hierarchical fashion.

Here we report that an activator (AIF4-) of heterotrimeric GTP-binding proteins (G-proteins) and inhibitors (lovastatin and C3 exoenzyme) of small GTP-binding proteins regulate the induction of alpha4beta7-mediated adhesion of TK-1 T lymphoma cells (alpha4+beta7+beta1-) to the mucosal addressin cell adhesion molecule MAdCAM-1. Activation of cell adhesion by AIF4- was abrogated by lovastatin, thereby establishing a link between heterotrimeric G-proteins and small GTP-binding proteins in the regulation of alpha4beta7-mediated cell adhesion. Increased numbers of cells bound MAdCAM-1-coated microspheres following activation with AIF4-, discounting an obligatory role for cell spreading in alpha4beta7-mediated cell adhesion. MAdCAM-1-Fc dimers triggered ligand-induced clustering of alpha4beta7 in response to AIF4- and Mn2+-induced activation of integrins. Hence alpha4beta7 cluster formation may be responsible, at least in part, for inducing cell adhesion in response to both extracellular and intracellular signals that impact on integrin function. Electroporation of constitutively active V14RhoA and V12Rac1 recombinant proteins into TK-1 cells revealed that both RhoA and Rac1 induce alpha4beta7 adhesion to MAdCAM-1. Activation is hierarchical since Rac1 is unable to directly activate alpha4beta7, but induces cell adhesion via RhoA, whereas the transient induction of cell adhesion mediated by RhoA is dependent on the activities of protein tyrosine kinases and protein kinase(s) C.     

Tyrosine kinase-regulated small GTPase translocation and the activation of phospholipase D in HL60 granulocytes

We focus on the mechanisms of regulation of phospholipase D (PLD) activity. Three agonists known to stimulate PLD activity, fMet-Leu-Phe (fMLP), phorbol 12-myristate 13-acetate (PMA) and V4+-OOH, induced a differential translocation of ADP-ribosylation factor (ARF), RhoA, and protein kinase Calpha (PKCalpha), all cofactors for PLD activation. Whereas fMLP recruited all three proteins to membranes, V4+-OOH only elicited RhoA translocation and PMA induced ARF and PKCalpha translocation. Three tyrosine kinases inhibitors, ST-638, methyl 2,5-dihydroxycinnamate, and genistein reduced fMLP-stimulated PLD activity by up to 80%. Furthermore, tyrosine kinase inhibitors reduced the fMLP-induced increase of GTPgammaS-stimulated PLD activity in membranes and recruitment of ARF, RhoA, and PKCalpha to the membrane fraction. The data suggest that a tyrosine phosphorylation event is located upstream of the translocation of ARF, RhoA, and PKCalpha in the signaling pathway leading to PLD activation by fMLP. RO 31-8220, a specific inhibitor of PKC, reduced PMA-induced PLD activity by 80% in intact HL60 granulocytes but enhanced fMLP-stimulated PLD activity by 60%. Although PMA alone had no effect on RhoA recruitment to the membrane fraction, in the presence of RO 31-8220 the levels of membrane-bound RhoA were increased. The levels of membrane-bound ARF and PKCalpha were unaffected by RO 31-8220 during PMA stimulation. In contrast, fMLP-induced recruitment of ARF and RhoA was insensitive to RO 31-8220 but PKCalpha translocation was increased. We propose that RhoA translocation may be regulated by PKC in an ATP-independent manner. Furthermore, increased fMLP-induced PKCalpha translocation in the presence of RO 31-8220 may partially account for the synergistic activation of PLD observed when both fMLP and RO 31-8220 are used together in intact HL60 cells.     

Rho prevents apoptosis through Bcl-2 expression: Implications for interleukin-2 receptor signal transduction

Here we describe a Rho-mediated apoptosis suppression pathway driven by Bcl-2 expression in the interleukin (IL)-4- or IL-2-dependent murine T cell line TS1αβ. IL-2, but not IL-4, induces Bcl-2 expression through RhoA activation which is inhibited by the specific Rho family inhibitor, Clostridium difficile Toxin B, as well as by a dominant negative RhoA mutant. Using transient transfections of RhoA mutants tagged with the vesicular stomatitis virus glycoprotein, we show that a constitutively active RhoA mutant induces Bcl-2 expression and prevents apoptosis upon IL-4 withdrawal. Finally, we have identified the signaling pathway involved together with RhoA in Bcl-2 induction and show compelling evidence for the implication of phosphatidylinositol 3 kinase and protein kinase C.     

Mechanisms of dopamine activation of fast-spiking interneurons that exert inhibition in rat prefrontal cortex

Prefrontal cortical dopamine (DA) modulates pyramidal cell excitability directly and indirectly by way of its actions on local circuit GABAergic interneurons. DA modulation of interneuronal functions is implicated in the computational properties of prefrontal networks during cognitive processes and in schizophrenia. Morphologically and electrophysiologically distinct classes of putative GABAergic interneurons are found in layers II-V of rat prefrontal cortex. Our whole cell patch-clamp study shows that DA induced a direct, TTX-insensitive, reversible membrane depolarization, and increased the excitability of fast-spiking (FS) interneurons. The DA-induced membrane depolarization was reduced significantly by D1/D5 receptor antagonist SCH 23390, but not by the D2 receptor antagonist (-)sulpiride, D4 receptor antagonists U101958 or L-745870, alpha1-adrenoreceptor antagonist prazosin, or serotoninergic receptor antagonist mianserin. The D1/5 agonists SKF81297 or dihydrexidine, but not D2 agonist quinpirole, also induced a prolonged membrane depolarization. Voltage-clamp analyses of the voltage-dependence of DA-sensitive currents, and the effects of changing [K(+)](O) on reversal potentials of DA responses, revealed that DA suppressed a Cs(+)-sensitive inward rectifier K(+) current and a resting leak K(+) current. D1/D5, but not D2 agonists mimicked the suppressive effects of DA on the leak current, but the DA effects on the inward rectifier K(+) current were not mimicked by either agonist. In a subgroup of FS interneurons, the slowly inactivating membrane outward rectification evoked by depolarizing voltage steps was also attenuated by DA. Collectively, these data showed that DA depolarizes FS interneurons by suppressing a voltage-independent 'leak' K(+) current (via D1/D5 receptor mechanism) and an inwardly rectifying K(+) current (via unknown DA mechanisms). Additional suppression of a slowly inactivating K(+) current led to increase in repetitive firing in response to depolarizing inputs. This D1-induced increase in interneuron excitability enhances GABAergic transmission to PFC pyramidal neurons and could represent a mechanism via which DA suppresses persistent firing of pyramidal neurons in vivo.     

Up-regulation of small GTPases,RhoA and RhoC,is associated with tumor progression in ovarian carcinoma

To clarify the role of small GTPases Rho in the biologic behavior of ovarian carcinoma, we first examined the mRNA expression of RhoA, RhoB, and RhoC in benign, borderline, and malignant ovarian tumors using RT-PCR and real-time RT-PCR. The expression and localization of RhoA protein were also analyzed by Western blotting and immunohistochemistry. Finally, we examined whether up-regulation of Rho enhances the invasiveness of ovarian cancer cells in vitro. Analysis of mRNA levels of the Rho family genes revealed that levels of both RhoA and RhoC were significantly higher in carcinomas than in benign tumors (RhoA, p = 0.0035; RhoC, p = 0.0006). According to histologic subtype, both RhoA and RhoC mRNA levels in serous carcinomas were significantly higher than those in other histologic types. With regard to the International Federation of Gynecological and Obstetrics stage classification, both of RhoA and RhoC mRNA levels were significantly higher in tumors of Stages III+IV than in those of Stages I+II (RhoA, p = 0.0200; RhoC, p = 0.0057). In addition, analysis of matched pairs of primary and disseminated lesions demonstrated that expression of both RhoA and RhoC mRNA was significantly higher in metastatic than in primary tumors. Examination of the protein level showed that expression of RhoA was also increased in advanced ovarian carcinomas, especially those of serous histology. Accordingly, we hypothesized that up-regulation of Rho GTPases plays an important role in the progression of ovarian carcinoma. Matrigel invasion assay using the ovarian cancer cell line, SKOV3, showed that up-regulation and activation after treatment with lysophosphatidic acid was associated with enhanced invasion of the cancer cells. This increase in invasiveness was suppressed by the addition of C3, a specific inhibitor of Rho. These findings suggest that up-regulation of Rho GTPases is important in the tumor progression of ovarian carcinoma and that Rho family proteins could be a molecular target in cancer therapy.     

Depolarization of rat locus coeruleus neurons by adenosine 5'-triphosphate.

Intracellular recordings were performed in a pontine slice preparation of the rat brain containing the locus coeruleus. The enzymatically stable P2-purinoceptor agonist alpha,beta-methylene ATP increased the firing rate without altering the amplitude or shape of action potentials; the afterhyperpolarization following a spike was not changed either. When locus coeruleus neurons were hyperpolarized by current injection in order to prevent spontaneous firing, alpha,beta-methylene ATP produced depolarization and a slight increase in the apparent input resistance. A combined application of kynurenic acid and bicuculline methiodide failed to alter the alpha,beta-methylene ATP-induced depolarization, and tetrodotoxin only slightly depressed it. A gradual shift of the membrane potential by hyperpolarizing current injection led to a corresponding decrease, but no abolition or reversal of the alpha,beta-methylene ATP effect. In the hyperpolarized region, the current-voltage curve of alpha,beta-methylene ATP came into close approximation with, but did not cross, the control curve. Elevation of the external K+ concentration, or the intracellular application of Cs+ by diffusion from the microelectrode, depressed the response to alpha,beta-methylene ATP; external tetraethylammonium was also inhibitory. External Ba2+ and Cs+ had no effect or only slightly decreased the alpha,beta-methylene ATP-induced depolarization. A low Na+, or a low Ca2+ high Mg2+ medium, as well as the presence of Co2+ in the medium, markedly reduced or even abolished the depolarization by alpha,beta-methylene ATP. ATP itself did not produce consistent changes in the membrane potential or input resistance. However, in the presence of the P1-purinoceptor antagonist 8-cyclopentyl-1,3-dipropylxanthine, ATP consistently increased the firing rate and evoked an inward current. In conclusion, P2-purinoceptor activation appears to depolarize locus coeruleus neurons by inhibiting a persistent potassium current, and at the same time opening calcium-sensitive sodium channels or calcium-sensitive non-selective cationic channels.     

Sema4D-plexin-B1 implicated in regulation of dendritic spine density through RhoA/ROCK pathway

Plexin-B1, Sema4D receptor, mediates retraction and extension signals in axon guidance by associating with PDZ-containing Rho guanine nucleotide exchange factors (PDZ-RhoGEFs) which can activate a small Rho GTPase RhoA. RhoA is implicated in spine formation by rearranging actin cytoskeleton. Exogenous application of Sema4D to cultured neurons caused activation of RhoA, increase of spine density and changes in spine shape. Sema4D-induced changes in spine density were blocked by either Rho-kinase (a downstream of RhoA, ROCK) inhibitor Y-27632 or by overexpression of plexin-B1 mutant lacking the C-terminus which no longer associates with PDZ-RhoGEFs. This study suggests that Sema4D-plexin-B1 play a crucial role in spine formation by regulating RhoA/ROCK pathway.     

Regulation of the small GTPase RhoA by syndecan-4 and integrins in focal adhesion formation

Introduction The transmembrane heparan sulfate proteoglycan, syndecan‐4, and integrins are important receptors for focal adhesion (FA) formation on fibronectin (FN) substrates. The small GTPase RhoA is also known to regulate FA and stress fiber formation. It has been suggested that syndecan‐4 and integrins co‐operatively regulate the assembly of FA in a Rho‐dependent manner, but the mechanism is unclear. Here, we examined the function of RhoA and the Rho effector kinases ROCKs in syndecan‐4 signalling on the process of FA formation and the possible mechanism by which syndecan‐4 may regulate RhoA activity. Methods Primary rat embryonic fibroblasts (REFs) were seeded on FN or ‘RGD’‐containing integrin‐binding domain of FN and lysed at various time points. The amount of active form of RhoA in each lysate was analysed by pull‐down experiments. Results and discussion The relative activities of RhoA showed one peak in the process of FA formation on FN, whereas no peak was obtained on the integrin‐binding domain. The one peak of RhoA activity on integrin‐binding domain was restored by addition of heparin‐binding domain into medium. These results suggested that a signal through syndecan‐4 link to the Rho pathway. Both ROCK‐I and ‐II isozymes were present in REF cell lysates and each could be specifically immunoprecipitated. The ROCK kinase activities in immunoprecipitates were analysed using GST‐myosin light chain as a substrate. The amount of ROCK‐I and ‐II activities changed through the adhesion process on FN and appeared to be independently regulated. Therefore, one or both ROCKs may be downstream of a syndecan‐4‐mediated signalling response through RhoA. The core protein of syndecan‐4 can directly bind to and activate PKC‐α. We found that PKC‐α could phosphorylate Rho‐Guanine Nucleotide Dissociation Inhibitor (GDI) in vitro. It has been suggested that PKC‐α‐mediated phosphorylation of Rho GDI stimulates GDI dissociation, thereby resulting in Rho activation. It is possible that syndecan‐4 regulates Rho/ROCK pathway through PKC‐α activation on the process of FA formation.     

Rho mediates calcium-dependent activation of p38alpha and subsequent excitotoxic cell death

Excitotoxic neuronal death contributes to many neurological disorders, and involves calcium influx and stress-activated protein kinases (SAPKs) such as p38alpha. There is indirect evidence that the small Rho-family GTPases Rac and cdc42 are involved in neuronal death subsequent to the withdrawal of nerve growth factor (NGF), whereas Rho is involved in the inhibition of neurite regeneration and the release of the amyloidogenic Abeta(42) peptide. Here we show that Rho is activated in rat neurons by conditions that elevate intracellular calcium and in the mouse cerebral cortex during ischemia. Rho is required for the rapid glutamate-induced activation of p38alpha and ensuing neuronal death. The ability of RhoA to activate p38alpha was not expected, and it was specific to primary neuronal cultures. The expression of active RhoA alone not only activated p38alpha but also induced neuronal death that was sensitive to the anti-apoptotic protein Bcl-2, showing that RhoA was sufficient to induce the excitotoxic pathway. Therefore, Rho is an essential component of the excitotoxic cell death pathway.     

Gi2 proteins couple somatostatin receptors to low-conductance K+ channels in rat pancreatic α-cells

Somatostatin hyperpolarized rat pancreatic alpha-cells and inhibited spontaneous electrical activity by activating a low-conductance K+ channel (0.9 pS with physiological ionic gradients). This channel was insensitive to tolbutamide (a blocker of ATP-sensitive K+ channels) and apamin (an inhibitor of small-conductance Ca(2+)-activated K+ channels). Channel activation was prevented by pre-treating the cells with pertussis toxin, indicating the involvement of G-proteins. A direct interaction between an inhibitory G-protein and the somatostatin-activated K+ channel is suggested by the finding that intracellular application of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma-S) and the G beta gamma subunit of G-proteins resulted in a transient stimulation of the current. Activation of the K+ current by somatostatin was inhibited by intracellular dialysis with specific antibodies to Gi1/2 and was not seen in cells treated with antisense oligonucleotides against G-proteins of the subtype Gi2. We conclude that somatostatin suppresses alpha-cell electrical activity by a Gi2-protein-dependent mechanism, which culminates in the activation of a sulphonylurea- and apamin-insensitive low-conductance K+ channel.