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Pathogenic Yersinia enterocolitica produces two virulence plasmid-encoded cytotoxins, YopE and YopT, that are translocated into target cells where they disrupt the actin cytoskeleton. Here we show that infection of cells with wild type Y. enterocolitica and a yopE mutant, but not with a yopT mutant, induces an increase in the electrophoretic mobility of the small GTPase RhoA. As tested by isoelectric focusing, YopT-dependent modification resulted in an acidic shift of RhoA. Furthermore, RhoA modification induced by YopT was accompanied by redistribution of membrane-bound RhoA toward the cytosol. Finally, a yopE mutant of Y. enterocolitica expressing the cytotoxic activity of YopT specifically disrupted RhoA-controlled actin stress fibers. These findings provide evidence for inactivation of RhoA by the translocated Y. enterocolitica cytotoxin YopT and suggest a novel inhibitory modification of RhoA by a bacterial virulence factor.

The pharmacological and binding properties of four β-adrenomimetic drugs with N-alkyl substitutions (isoprenaline, terbutaline, salbutamol and soterenol) were compared with those of four corresponding drugs with N-aralkyl substitutions (protokylol, ME 506, salmefamol and zinterol). BD-40 A, a very powerful β2-agonist with a related chemical structure, was also included in this study. The β1- and β2-activities of these drugs were determined on guinea-pig atria and trachea, their α-adrenolytic activity was measured on rat aorta and their affinities (Ki) for α1- and α2-adrenoceptors on rat cortical membranes were assessed using [3H]prazosin and [3H]yohimbine. In this group of β-agonists, substitution of the N-alkyl by an N-aralkyl group had a variable effect on the β2-selectivity whereas α-adrenolytic properties were always enhanced. An increase of the affinities (Ki) for both α1- and α2-adrenoceptors was found but the effect was much more pronounced for α1-adrenoceptors. These results indicated that the α-adrenolytic activity observed with the N-aralkyl β-agonists was selective for α1-adrenoceptors.

1α‐Adrenoceptor stimulant activity was determined for noradrenaline (NA), clonidine and a series of para‐substituted derivatives of clonidine on rat aortic strips, a rat brain synaptosome preparation, and anaesthetized pithed rats. The effects on the blood pressure of intraventricular (i.c.v.) injections of para‐aminoclonidine were also determined in anaesthetized rats.2Para‐substituted derivatives of clonidine (amino‐, diethylamino‐, ethylamino‐, acetamido‐, bromoacetamido‐, N‐chloroethyl‐N‐methyl‐amino and N‐β‐chloroethyl‐N‐methylaminomethyl‐) retain α‐adrenoceptor stimulant activity.3pD2values determined on rat aortic strips were 11.2, 7.67 and 9.05 respectively for para‐aminoclonidine, clonidine and noradrenaline. The Kivalues of these agents, determined on a rat brain synaptosomal preparation with a radioreceptor assay using [3H]‐clonidine as ligand, were 1.3, 8.0 and 23 nmrespectively for para‐aminoclonidine, clonidine and NA. When given by i.c.v. injection in rats, para‐aminoclonidine lowered the blood pressure.4N‐β‐chloroethyl‐N‐methylaminomethylclonidine is an alkylating agent with an unusual agonist activity. It elicits contractions of the rat aorta that persist despite repeated washing.5α‐Adrenoceptor affinities are discussed in relation to their structural features.

The enteropathogenic bacterium Yersinia enterocolitica counteracts host defense mechanisms by interfering with eukaryotic signal transduction pathways. In this study, we investigated the mechanism by which Y. enterocolitica prevents macrophage tumor necrosis factor-alpha (TNFalpha) production. Murine J774A.1 macrophages responded to Y. enterocolitica infection by rapid activation of mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase (ERK), p38, and c-Jun NH2-terminal kinase (JNK). However, after initial activation, the virulent Y. enterocolitica strain harboring the Y. enterocolitica virulence plasmid caused a substantial decrease in ERK1/2 and p38 tyrosine phosphorylation. Simultaneously, the virulent Y. enterocolitica strain gradually suppressed phosphorylation of the transcription factors Elk-1, activating transcription factor 2 (ATF2), and c-Jun, indicating time-dependent inhibition of ERK1/2, p38, and JNK kinase activities, respectively. Analysis of different Y. enterocolitica mutants revealed that (i) MAPK inactivation parallels the inhibition of TNFalpha release, (ii) the suppressor effect on TNFalpha production, which originates from the lack of TNFalpha mRNA, is distinct from the ability of Y. enterocolitica to resist phagocytosis and to prevent the oxidative burst, (iii) the tyrosine phosphatase YopH, encoded by the Y. enterocolitica virulence plasmid, is not involved in the decrease of ERK1/2 and p38 tyrosine phosphorylation or in the cytokine suppressive effect. Altogether, these results indicate that Y. enterocolitica possesses one or more virulence proteins that suppress TNFalpha production by inhibiting ERK1/2, p38, and JNK kinase activities.

Rat adipose tissue possesses two Bordetella pertussis toxin (PTX) substrates and, in the same 39-41 kDa molecular mass range, positive immunoreactivity has also been reported with antibodies against the alpha subunit of Go, the major brain GTP-binding protein (G-protein). In this study, the presence of the brain Go alpha subunit at 39 kDa in adipocytes was reassessed, since direct correspondence between PTX substrates and Go alpha immunoreactivity has not yet been clearly established. On resolutive SDS/polyacrylamide-gel electrophoresis, the PTX substrates of human adipocytes were compared with the three PTX substrates found in brain. No ADP-ribosylated substrate at the level of the 39 kDa brain Go alpha could be detected in adipocyte membranes. Immunoblotting of human adipocyte membranes stained with our anti-Go alpha antibodies confirmed the presence of a positive immunoreactivity in this tissue, but the apparent molecular mass of the immunoreactive polypeptide in adipocytes was higher than that found in nervous tissues. Taken together, these results indicate that the brain Go alpha subunit is not present in adipose tissue. They also suggest the existence of a G-protein in adipocytes which is immunologically related to Go alpha but having a slightly higher molecular mass.

In muscle, it has been established that guanosine 5′-[gamma-thio]triphosphate (GTP[S]), a non-hydrolysable GTP analogue, elicits a rise in tension in chemically skinned fibres, and that pretreatment with Bordetella pertussis toxin (PTX) decreases GTP[S]-induced tension development [Di Virgilio, Salviati, Pozzan & Volpe (1986) EMBO J. 5, 259-262]. In the present study, G-proteins were analysed by PTX-catalysed ADP-ribosylation and by immunoblotting experiments at cellular and subcellular levels. First, the nature of the G-proteins present in neural and aneural zones of rat diaphragm muscle was investigated. PTX, known to catalyse the ADP-ribosylation of the alpha subunit of several G-proteins, was used to detect G-proteins. Three sequential extractions (low-salt-soluble, detergent-soluble and high-salt-soluble) were performed, and PTX was found to label two substrates of 41 and 40 kDa only in the detergent-soluble fraction. The addition of pure beta gamma subunits of G-proteins to the low-salt-soluble extract did not provide a way to detect PTX-catalysed ADP-ribosylation of G-protein alpha subunits in this hydrophilic fraction. In neural as well as in aneural zones, the 39 kDa PTX substrate, very abundant in the nervous system (Go alpha), was not observed. We then studied the nature of the G alpha subunits present in membranes from transverse tubules (T-tubules) purified from rabbit skeletal muscle. Only one 40 kDa PTX substrate was found in T-tubules, known to be the key element of excitation-contraction coupling. The presence of a G-protein in T-tubule membranes was further confirmed by the immunoreactivity detected with an anti-beta-subunit antiserum. A 40 kDa protein was also detected in T-tubule membranes with an antiserum raised against a purified bovine brain Go alpha. The presence of two PTX substrates (41 and 40 kDa) in equal amounts in total muscle extracts, compared with only one (40 kDa) found in purified T-tubule membranes, suggests that this 40 kDa PTX substrate might be involved in excitation-contraction coupling.

We have previously identified two isoforms of Go alpha in membranes of N1E-115 neuroblastoma cells, using an antibody raised against the purified Go alpha subunit; one isoform of the Go alpha subunit (pI 5.80) is present in undifferentiated cells, whereas a more acidic isoform (pI 5.55) appears during differentiation [J. Neurochem. 54:1310-1320 (1990)]. Recently, the Go alpha gene has been shown to encode, by alternative splicing, two polypeptides, Go1 alpha and Go2 alpha, which differ only in their carboxyl-terminal part. To determine unambiguously whether the two Go alpha subunits detected in neuroblastoma cells were actually the products of different mRNAs, rabbit polyclonal antibodies were generated against synthetic peptides (amino acids 291-302) of both sequences. Specificity of the two affinity-purified antipeptide antibodies was assessed on Western blots by comparing their immunoreactivities with those of other G alpha antibodies. On a blotted mixture of purified brain guanine nucleotide-binding proteins, the anti-alpha o1 and anti-alpha o2 peptide antibodies only recognized the 39-kDa Go alpha subunit. Furthermore, the immunological recognition of brain membranes from 15-day-old mouse fetuses by antipeptide antibodies could be specifically blocked by addition of the corresponding antigen. When membrane proteins from differentiated neuroblastoma cells and mouse fetus brain were blotted after two-dimensional gel electrophoresis, the anti-alpha o1 and anti-alpha o2 peptide antibodies labeled a 39-kDa subunit focused at a pI value of 5.55 or 5.80, respectively. Study of the ontogenesis of both Go alpha subunits revealed the predominance of Go2 alpha in the frontal cortex at day 15 of gestation. Thereafter, there was a progressive decline of the Go2 alpha polypeptide to a very low level, concomitant with an increase in the Go1 alpha protein, which plateaued about 15 days after birth to a level 8 times higher than at gestational day 15. Similarly, on neuroblastoma cells, the Go2 alpha subunit was almost exclusively present in undifferentiated cells, and differentiation induced the appearance of the Go1 alpha subunit, with a reduction in the amount of Go2 alpha polypeptide. Thus, the evolution of the two Go alpha subunits during cell differentiation, unambiguously identified with specific antibodies, suggests that neuronal differentiation is responsible for the on/off switch of the expression of the Go alpha isoforms and indicates that Go1 alpha, rather than Go2 alpha, is involved in neurotransmission.

The peptide hormones bradykinin and kallidin (Lys-bradykinin), as well as their analogues [des-Arg9]-bradykinin, a selective B1 agonist, [des-Arg9,Leu8]-bradykinin, a selective B1 antagonist, and [Thi5,8,D-Phe7]-bradykinin and D-Arg0-[Hyp3,D-Phe7]-bradykinin, two selective B2 antagonists, induced rapid histamine release from purified rat peritoneal mast cells. In contrast, the N-terminal fragment bradykinin-(1-5) was inactive. These peptides also activate the GTPase activity of GTP-binding proteins (G proteins) (Go/Gi) purified from calf brain, with an order of potency identical to that observed on mast cells, [Thi5,8,D-Phe7]-bradykinin much greater than kallidin greater than bradykinin greater than D-Arg0-[Hyp3,D-Phe7]-bradykinin greater than [des-Arg9]-bradykinin greater than [des-Arg9,Leu8]-bradykinin greater than bradykinin-(1-5). This correlation suggested that G proteins are the targets of kinins in mast cells. Accordingly, the concomitant increase in inositol trisphosphates and release of histamine elicited by kinins were inhibited by pertussis toxin pretreatment of mast cells. The inhibitory effect of benzalkonium chloride showed that the G proteins involved belong to the Gi type. GTPase activity was measured in the supernatant of homogenized mast cells but not in the membranous fraction. This activity was stimulated by kinins and by the venom peptide mastoparan. The potency of peptides was similar to that observed with purified bovine G proteins. Sodium dodecyl sulfate-gel electrophoresis of mast cell supernatant revealed pertussis toxin-induced ADP-ribosylation of two proteins, in the Mr 41,000 and 40,000 range, i.e., similar to purified alpha-subunits of Gi1 and Gi2 or Gi3 subtypes. The data support the proposal that bradykinin and analogues act like mastoparan, substance P, and compound 48/80, interacting first with sialic acid residues of the cell surface and then with Gi-like proteins, inducing phospholipase C activation and intracellular calcium mobilization.

Bovine brain contains two GTP-binding proteins, Gi and Go, which are substrates for ADP ribosylation by pertussis toxin. The Gi protein mediates hormone and GTP inhibition of adenylate cyclase, but the function and the precise tissue distribution of Go are unknown. To immunologically probe the localization of Go, we have purified the Go alpha and G beta, gamma subunits of Go and have raised antibodies against them. The polyclonal anti-Go alpha antibodies obtained were very selective for Go alpha compared to Gi alpha or Gs alpha. The positive Go alpha and G beta, gamma immunoreactivities were investigated in different tissues of vertebrates and invertebrates on immunoblots after gel electrophoresis of the crude membranes. The anti-G beta, gamma antibodies recognized a 35-36-kDa protein in brain of vertebrates such as mammals (rat), avians (pigeon), amphibians (frog), fish (trout), and reptiles (turtle) but not in the invertebrates such as molluscs (snail) and insects (locust). With the anti-Go alpha antibodies a high level of immunoreactivity was detected at molecular weights of 39,000-40,000 in the brain of invertebrates as well as in the central nervous system of vertebrates. Moreover, ADP ribosylation with pertussis toxin occurred in the nervous system of invertebrates. These results suggest that the GTP-binding proteins of invertebrates either are devoid of G beta, gamma subunit or, more probably, possess immunologically different G beta, gamma subunits when compared to those of vertebrates. In the vertebrates, Go alpha immunoreactivity was also present in the peripheral nervous system in areas such as the superior cervical ganglia and sciatic nerve. When examined with the anti-Go alpha antibodies, the neuro-and adenohypophysis exhibited a similar immunoreactivity which was about 6 times lower than in brain. Our antibodies also recognized a 40-kDa protein in human adipocytes but at a concentration 17 times lower than that recognized in brain. Taken together, these data show that the Go alpha subunit is well conserved through evolution and, furthermore, confirm that Go alpha is not strictly limited to the nervous system. This suggests that the protein Go ensures a function required for neuronal activity but also present in some other non-nervous tissues.

The labelling of rat cerebral cortex a2-adrenoceptors with [3H]-yohimbine ([3H]-YOH) was investigated. At 25° C, binding equilibrium was reached in about 10 min and dissociation occurred with a half time of about 1 min. Saturation experiments gave an equilibrium KD value of 10.13±1.95 nM and a maximum number of sites of 254±22 fmol/mg protein. The [3H]-YOH binding sites exhibited a2-adrenergic receptor specificity; the order of potency for the antagonists was rauwolscine > yohimbine » prazosin > corynanthine. For the agonists, the order was: oxymetazoline > clonidine > (-)-adrenaline > (-)-noradrenaline » (-)-phenylephrine. Agonists exhibited shallow curves in inhibiting [3H]-YOH binding, with pseudo-Hill coefficients (nH) of less than 1.0. These curves were shifted to lower overall affinity and steepened in the presence of 100 µM GTP. Antagonist competition curves were also shallow but GTP had no significant effect.

We examined the expression and activity of inducible nitric oxide synthase (iNOS) in both gamma interferon (IFN-gamma)-treated and untreated murine macrophages infected with the gram-negative bacterium Brucella suis. The bacteria were opsonized with a mouse serum containing specific antibrucella antibodies (ops-Brucella) or with a control nonimmune serum (c-Brucella). The involvement of the produced NO in the killing of intracellular B. suis was evaluated. B. suis survived and replicated within J774A.1 cells. Opsonization with specific antibodies increased the number of phagocytized bacteria but lowered their intramacrophage development. IFN-gamma enhanced the antibrucella activity of phagocytes, with this effect being greater in ops-Brucella infection. Expression of iNOS, interleukin-6, and tumor necrosis factor alpha (TNF-alpha) mRNAs was induced in both c-Brucella- and ops-Brucella-infected cells and was strongly potentiated by IFN-gamma. In contrast to that of cytokine mRNAs, iNOS mRNA expression was independent of opsonization. Similar levels of iNOS mRNAs were expressed in IFN-gamma-treated cells infected with c-Brucella or ops-Brucella; however, expression of iNOS protein and production of NO were detected only in IFN-gamma-treated cells infected with ops-Brucella. These discrepancies between iNOS mRNA and protein levels were not due to differences in TNF-alpha production. The iNOS inhibitor N omega-nitro-L-arginine methyl ester increased B. suis multiplication specifically in IFN-gamma-treated cells infected with ops-Brucella, demonstrating a microbicidal effect of the NO produced. This observation was in agreement with in vitro experiments showing that B. suis was sensitive to NO killing. Together our data indicate that in B. suis-infected murine macrophages, the posttranscriptional regulation of iNOS necessitates an additive signal triggered by macrophage Fcgamma receptors. They also support the possibility that in mice, NO favors the elimination of Brucella, providing that IFN-gamma and antibrucella antibodies are present, i.e., following expression of acquired immunity.

5-Hydroxytryptamine (5-HT) stimulates the accumulation of inositol-trisphosphate in WRK1 cells, a cell line originating from a rat mammary tumor. 5-HT acts via a single receptor type for which it has an affinity constant estimated to be 1.27 microM. A series of agonists known to act at 5-HT2 receptors are partial agonists in this system and have a rank order of relative intrinsic efficacies corresponding to that seen in other systems possessing 5-HT2 receptors. There is an essentially linear occupancy-response relationship for 5-HT and other agonists indicating the absence of a strong amplification mechanism between receptor activation and inositol phosphate formation. The selective blockade of the 5-HT response by nanomolar concentrations of 5-HT2 selective antagonists but not by drugs acting at other 5-HT receptor subtypes suggest that the receptor in WRK1 cells is of the 5-HT2 type. Additionally, we demonstrate that in WRK1 membranes 5-HT acts via the 5-HT2 receptor to elicit a GTP dependent increase in the production of inositol-bisphosphate and inositol-trisphosphate. These properties of the WRK1 cell line indicate that it is a useful model with which to study the nature of 5-HT receptor coupling to the putative second messenger(s), the inositol phosphates.