The melanophore aggregating response of isolated fish scales: a very rapid and sensitive diagnosis of whooping cough

Pertussis toxin (PT) has been found to block noradrenaline-induced pigment aggregation in fish melanophores, and, based on this, a rapid and highly sensitive assay for PT was developed. Some preliminary results have also indicated that it may be possible to detect PT-like activity in saliva samples from patients with clinically suspected pertussis. In the present study the diagnostic value of the fish melanophore method was evaluated in 70 patients suspected of having pertussis; culture, serology and physician diagnosis were used as reference methods. In 60 of the patients, pertussis was verified by at least one of the reference methods. The melanophore test showed PT-like activity in saliva samples from 58 of the patients. Three patients with reference-verified pertussis showed no PT-like activity in the test; among these, one patient had been immunized and had also been treated with erythromycin during 3 days immediately prior to visiting the hospital. The melanophore test has three major advantages: it allows detection of pertussis in the early and curable stage of the disease; it takes only 2 h to perform; and it requires no sophisticated equipment.     

A pertussis toxin resistant alpha 2-adrenoceptor pathway in fish melanophores

The effect of pertussis toxin (PT) on the aggregation of pigment granules in melanophores from cuckoo wrasse (Labrus ossifagus L.) was studied. The results indicate the presence of a PT resistant alpha 2-adrenoceptor signal transduction mechanism.     

The effect of pertussis toxin on alpha-2-adrenoceptor-mediated pigment migration in fish melanophores

The aggregation of melanin-granules within fish pigment cells (melanophores) can be elicited either by electrical stimulation of intrinsic nerves or by the addition of adrenergic agonists. The pigment aggregation seems to be mediated by alpha-2-adrenoceptors. In this investigation we have used various agonists and antagonists (noradrenaline, (+)- and (-)-adrenaline, isoprenaline, yohimbine and prazosin) to further characterize the pigment-aggregating receptor of Labrus ossifagus. All the results obtained support the notion of alpha-2-adrenoceptor-mediated pigment aggregation. The pertussis toxin, islet-activating protein (IAP), is known to inhibit the alpha-2-adrenoceptor-mediated signal transduction in mammals. We have used IAP to investigated whether fish melanophore alpha-2-adrenoceptors are also inhibited by this toxin. We found that IAP inactivated the alpha-2-adrenoceptor-mediated pigment aggregation in a dose-dependent manner. The inhibitory IAP-effect had a remarkably short onset-time in the melanophores (maximal effect was obtained within 10 min of incubation). Interestingly, binding of an agonist (noradrenaline) to the receptors prevented IAP from exerting its inhibitory action, whereas binding of an antagonist (yohimbine) gave no protection against the IAP-inactivation. In conclusion, the pigment-aggregating receptors of melanophores of L. ossifagus are very similar to the mammalian alpha-2-adrenoceptors. It is possible to inactivate the melanophore receptor system with IAP and this inactivation has a remarkably short onset-time. Stimulation of the alpha-2-adrenoceptors prevents IAP from inactivating the receptor system.     

Is Ca2+ the second messenger in the response to melatonin in cuckoo wrasse melanophores?

Pigment aggregation in melanophores of Labrus ossifagus is controlled by an alpha2-adrenoceptor and is somehow modulated by melatonin. The signal transduction mechanisms seem to involve both an attenuation of cAMP and an increase in intracellular Ca2+, inhibiting protein kinase A or activating a phosphatase, respectively. These effects result in dephosphorylation, which in turn induces aggregation. Various alpha2-adrenoceptor agonists attenuate cAMP levels or increase the concentration of intracellular Ca2+. Noradrenaline, for example, lowers cAMP but does not affect the calcium signal whereas B-HT 920, an alpha2-adrenoceptor specific agonist, does not induce a cAMP decrease but does appear to induce an increase in intracellular Ca2+. This later inference is drawn from experiments with BAPTA/AM, an intracellular calcium chelator, which counteracts the aggregation induced by B-HT 920. Interestingly, the very potent alpha2-adrenoceptor agonist medetomidine apparently activates both signal transduction pathways, which could explain its high efficacy in producing aggregation. Melatonin itself does not cause pigment aggregation, but it potentiates noradrenaline-induced aggregation. It has been suggested that melatonin receptors and alpha2-adrenoceptors follow the same signal transduction pathway, i.e. an attenuation of cAMP. In our experiments, melatonin did not reduce cAMP levels; instead it appears to increase Ca2+ concentration, since melatonin-potentiated aggregation was inhibited by BAPTA/AM. Thus, aggregation amplified by melatonin is probably not mediated by a further decrease in cAMP, but by the same signal transduction mechanism as B-HT 920, i.e. an increase in Ca2+. This further strengthens the suggestion that melatonin and B-HT 920 bind to the same site, but it is unclear if that particular site is on the melatonin receptor or the alpha2-adrenoceptor.     

MCH-induced pigment aggregation in teleost melanophores is associated with a cAMP reduction.

It has previously been shown that alpha 2-adrenoceptors are involved in noradrenaline-induced pigment aggregation within fish melanophores. In the present investigation, melanin concentrating hormone (MCH) elicited pigment aggregation (EC50 approximately 1 x 10(-7) M) that was associated with a significant reduction in the cAMP content; 1 x 10(-7) M MCH reduced the cAMP content from a basal level of 50.4 +/- 2.8 pmol/mg protein to 36.9 +/- 3.8 pmol/mg protein. Like the alpha 2-adrenoceptor-induced pigment aggregation, the MCH response was effectively blocked by the adenylate cyclase stimulator forskolin. These findings suggest that attenuation of cAMP may serve as an intracellular signal transduction mechanism for both MCH and noradrenaline.     

Multiple α2-Adrenoceptor Signalling Pathways Mediate Pigment Aggregation Within Melanophores

It has previously been shown that α₂-adrenoceptors (α₂-ARs) mediate pigment granule (melanosome) aggregation in melanophores of the teleost fish Labrus ossifagus. The present investigation scrutinized the signalling mechanisms of melanosome aggregation induced by sympathetic nerve stimulation or by exogenous addition of α-AR agonists and cAMP analogues. The following was observed: i) nerve-induced melanosome aggregation was associated with a rapid decrease in the cAMP level; ii) noradrenaline or medetomidine (an α₂-AR agonist) caused melanosome aggregation and reduced the cAMP content; iii) RP-S-CI-cAMP, a membrane-permeating inhibitor of protein kinase A induced melanosome aggregation; and iv) B-HT 920 (an α₂-AR agonist) and methoxamine (an α₁-AR agonist) induced melanosome aggregation, although they did not reduce cAMP. It has been suggested that in some teleost species α₁-ARs mediate melanosome aggregation by increasing the level of intracellular calcium. However, we found that the effect of methoxamine in melanophores from Labrus ossifagus could be blocked by yohimbine (an α₂-AR antagonist) but not by equimolar concentration of prazosin (an α₁-AR antagonist). Furthermore, 1 μM ionomycin (a calcium ionophore) did not induce melanosome aggregation. Our findings therefore do not indicate that α₁-ARs and/or an increase in intracellular calcium mediate melanosome aggregation in Labrus ossifagus. Our results suggest that α₂-AR-mediated melanosome aggregation is induced by multiple signalling pathways. One of these involves a reduction in cAMP, but none involves an increase in intracellular calcium.     

溶液环境对百日咳疫苗分离纯化的影响

针对百日咳疫苗在低盐条件下不稳定, 容易聚集而导致层析过程收率低、分离度低的难题, 实验中选择脲作为稳定剂来改善百日咳疫苗所处的溶液环境, 并采用离子交换层析和凝胶过滤层析进行百日咳疫苗的分离纯化, 通过ELISA抗原活性测定和还原性SDS-PAGE等方法研究了脲对百日咳疫苗分离纯化的影响。结果表明, 在流动相中加入 2 mol/L脲作为稳定剂, 能显著提高离子交换层析和凝胶过滤层析中的PT和FHA活性回收率、凝胶过滤层析的分离度、PT和FHA的纯度。这些结果对百日咳疫苗的分离纯化和层析工艺优化提供了重要的依据和参考。     

Regulation of organelle transport in melanophores by calcineurin

Previous studies have shown that pigment granule dispersion and aggregation in melanophores of the African cichlid, Tilapia mossambica, are regulated by protein phosphorylation and dephosphorylation, respectively (Rozdzial, M. M., and L. T. Haimo. 1986. Cell. 47:1061- 1070). The present studies suggest that calcineurin, a Ca2+/calmodulin- stimulated phosphatase, is the endogenous phosphatase that mediates pigment aggregation in melanophores. Aggregation, but not dispersion, is inhibited by okadaic acid at concentrations consistent with an inhibition of calcineurin activity. Inhibition of aggregation in melanophores that have been BAPTA loaded or treated with calmodulin antagonists implicate Ca2+ and calmodulin, respectively, in this process. Moreover, addition of calcineurin rescues aggregation in lysed melanophores which are otherwise incapable of aggregating pigment. Immunoblotting with an anticalcineurin IgG reveals that calcineurin is a component of the dermis, which contains the melanophores, and indirect immunofluorescence localizes calcineurin specifically to the melanophores. Finally, this antibody, which inhibits calcineurin's phosphatase activity (Tash, J. S., M. Krinks, J. Patel, R. L. Means, C. B. Klee, and A. R. Means. 1988. J. Cell Biol. 106:1625-1633), inhibits aggregation but has no effect on pigment granule dispersion. Together these studies indicate that retrograde transport of pigment granules to the melanophore cell center depends upon the participation of calcineurin.     

Pertussis toxin triggers rapid second messenger production in human T lymphocytes

Pertussis toxin (PT) is a known mitogen for T lymphocytes. The mechanism by which the toxin stimulates proliferation has remained obscure and paradoxical because, in some types of cells, the toxin also inhibits growth factor-mediated signal transduction. It has previously been shown that the adenosine-diphosphate ribosyltransferase activity of the toxin is not required to produce the mitogenic effect. A biochemical explanation for the mitogenic activity has therefore remained obscure. We investigated the biochemical basis for the mitogenic activity of PT by using the transformed human T cell line, Jurkat. PT stimulated a rapid rise in cytosolic-free [Ca2+] from both intra- and extracellular sources. This was associated with an increase in the cellular diacylglycerol and inositol triphosphate levels with a concomitant decrease in the levels of phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate. The half-maximal effective dose of PT was 1.7 nM. PT also stimulated the production of interleukin 2. Only the holotoxin or B-oligomer (the presumptive membrane-binding subunit) was capable of stimulating an increase in [Ca2+] in these cells. This activity of PT mimicked that of some anti-T3-T cell antigen receptor complex monoclonal antibodies that also stimulate increases in the second messengers, diacylglycerol and Ca2+. The effects of PT and anti-T3 complex antibody were identical and not additive in Jurkat cells, suggesting that both agents were activating the same signal transduction pathway. These data provide a mechanistic explanation for the mitogenic effects of PT and suggest that the toxin may be interacting with a specific receptor in the T lymphocyte plasma membrane.     

Effects of Odorants on Pigment Aggregation and cAMP in Fish Melanophores

Odor perception within olfactory neuroepithelium and pigment translocation within melanophores both seem to rely on a cAMP-based second messenger system. From studies on cultured frog melanophores, Lerner et al. (Proc. Natl. Acad. Sci. USA 85:261-264, 1988) suggested that some aspect of odor perception may be mediated by a nonspecific mechanism whose signal is transduced by a cAMP-based second messenger system. In the present study, odorants (β-ionone, benzylaldehyde, cineole, cinnamaldehyde, and octanol), which previously have been shown to stimulate formation of cAMP in the olfactory neuroepithelium, were investigated for possible pigment dispersing and cAMP-increasing effects. Pretreatment of fish melanophores with the adenylate cyclase activator forskolin (1 μM) resulted in an approximately 300% increase in cAMP and an almost complete blockage of noradrenaline-induced pigment aggregation. However, none of the tested odorants were able to increase the cAMP level and only cinnaldehyde and β-ionone were found to have any pigment dispersing activity.     

Comparative studies on nerve- and noradrenaline-induced melanosome aggregation within different species of fish

1. The aggregation of melanosomes within melanophores of the cuckoo wrasse (Labrus ossifagus; belonging to the family Labridae) has, on pharmacological grounds, been shown to be mediated by postsynaptic alpha 2-adrenoceptors which in turn act via an inhibitory control of adenylate cyclase. 2. In the present paper we have investigated some American species belonging to the Labridae, Haemulidae, Embiotocidae, Clinidae and Pleuronectidae. 3. In all instances, except in the case of sargo (Haemulidae), we could demonstrate that melanosome aggregation probably was mediated by postsynaptic alpha 2-adrenoceptors which mediate their effect by inhibiting the adenylate cyclase of the melanophores. 4. Although these receptors apparently, on pharmacological grounds, may be classified as alpha 2-adrenoceptors it was also concluded that there is a phylogenetic divergence among these receptors.     

Suppression of the cytotoxic T-lymphocyte response in mice by pertussis toxin

Pertussis toxin (PT), the major toxin produced by Bordetella pertussis, has been reported both to enhance and to suppress immune responsiveness. These findings suggested that PT contributes to the virulence of B. pertussis through mechanisms involving immune regulation. We report that PT suppressed both the primary and the secondary cytotoxic T-lymphocyte (CTL) responses of mouse spleen cells cultured against two different allogeneic stimulator spleen cells in vitro. This suppression was dependent on the dose of PT used. PT must be present during the initial stages (within the first 24 hr) of CTL generation. Soluble factor(s) obtained from spleen cells preexposed to PT did not suppress the CTL response. Suppression of the CTL response observed was not due to depletion of the antigen by PT. The cytotoxic activity of CTL clones could not be suppressed by PT. The analysis of responder spleen cells, fractionated by anti-immunoglobulin panning techniques, provided evidence that L3T4-, Lyt 2+ cells mediate the PT-induced immunosuppression. We propose that suppression of the CTL response by PT is generated through the activation of L3T4-, Lyt 2+ suppressor T lymphocytes.     

Signal transduction pathway for IL-1. Involvement of a pertussis toxin-sensitive GTP-binding protein in the activation of adenylate cyclase

Human Il-1 alpha induces the synthesis of kappa Ig L chains by the pre-B cell line 7OZ/3, IL-2R alpha by the human NK cell line YT, and PGE2 by human rheumatoid synovial cells. Pertussis toxin (PT) markedly inhibited all three IL-1-induced activation events. The inhibition by PT was associated with a decrease in IL-1-mediated cAMP production. PT also inhibited IL-1-stimulated cAMP production in crude membrane fractions from 7OZ/3, YT, and 3T3 fibroblasts. In addition, IL-1 stimulated GTPase activity present in the membranes IL-1-responsive cells. Furthermore, the IL-1-induced GTPase activity was sensitive to PT. PT induced the ADP-ribosylation of a 46-kDa substrate in membrane preparations from IL-1-responsive cells. Cholera toxin also induced the ADP-ribosylation of a 46-kDa substrate in the same membrane preparations. The present findings indicate that the IL-1R is linked to a PT-sensitive G protein that stimulates the activity of adenylate cyclase.     

Effect of bacterial toxins on the GTPase activity of transducin from bovine rod outer segments

The effects of choleragen- and pertussis toxin (PT)-induced ADP-ribosylation on the GTP-binding protein transducin (TD) from retinal rod outer segments (ROS) have been studied. It has been shown that both toxins cause inhibition of the TD GTPase activity. PT inhibited the GTPase by 30-40% in "native" ROS and by 70-80% in homogeneous TD. Choleragen, in contrast with PT, had no effect on the GTPase activity of homogeneous TD, but was as effective as PT in membrane preparations. The effects of both toxins on the GTPase activity of TD were found to be dependent on the chemical structure of the guanyl nucleotide present in the vehicle. The data obtained suggest that PT and choleragen differ in their specificity for the TD-guanyl nucleotide complex. The former can interact with free TD as well as with the TD-GDP complex, while the latter affects only the TD-GTP complex.     

Pertussis Toxin, an Inhibitor of G(αi) PCR, Inhibits Bile Acid- and Cytokine-Induced Apoptosis in Primary Rat Hepatocytes

Excessive hepatocyte apoptosis is a common event in acute and chronic liver diseases leading to loss of functional liver tissue. Approaches to prevent apoptosis have therefore high potential for the treatment of liver disease. G-protein coupled receptors (GPCR) play crucial roles in cell fate (proliferation, cell death) and act through heterotrimeric G-proteins. G(αi)PCRs have been shown to regulate lipoapoptosis in hepatocytes, but their role in inflammation- or bile acid-induced apoptosis is unknown. Here, we analyzed the effect of inhibiting G(αi)PCR function, using pertussis toxin (PT), on bile acid- and cytokine-induced apoptosis in hepatocytes. Primary rat hepatocytes, HepG2-rNtcp cells (human hepatocellular carcinoma cells) or H-4-II-E cells (rat hepatoma cells) were exposed to glycochenodeoxycholic acid (GCDCA) or tumor necrosis factor-α (TNFα)/actinomycin D (ActD). PT (50-200 nmol/L) was added 30 minutes prior to the apoptotic stimulus. Apoptosis (caspase-3 activity, acridine orange staining) and necrosis (sytox green staining) were assessed. PT significantly reduced GCDCA- and TNFα/ActD-induced apoptosis in rat hepatocytes (-60%, p<0.05) in a dose-dependent manner (with no shift to necrosis), but not in HepG2-rNtcp cells or rat H-4-II-E cells. The protective effect of pertussis toxin was independent of the activation of selected cell survival signal transduction pathways, including ERK, p38 MAPK, PI3K and PKC pathways, as specific protein kinase inhibitors did not reverse the protective effects of pertussis toxin in GCDCA-exposed hepatocytes. Conclusion: Pertussis toxin, an inhibitor of G(αi)PCRs, protects hepatocytes, but not hepatocellular carcinoma cells, against bile acid- and cytokine-induced apoptosis and has therapeutic potential as primary hepatoprotective drug, as well as adjuvant in anti-cancer therapy.     

Natural contamination with mycotoxins in forage maize and green coffee in Nayarit State (Mexico)

The presence of mycotoxins in forage maize (zearalenone, fumonisin B1, T-2 toxin and diacetoxyscirpenol) and green coffee (ochratoxin A) from Nayarit State (Mexico) has been studied. All maize samples analyzed showed fumonisin B1 contamination, with an average concentration of 2,541 microg/kg. Fifteen percent of the samples contained zearalenone, with an average concentration of 1,610 microg/kg. Only one sample showed T-2 toxin contamination (7 microg/kg), and no diacetoxyscirpenol was detected. Sixty-seven per cent of green coffee samples were contaminated with ochratoxin A, with an average concentration of 30.1 microg/kg. This is the first study about mycotoxins developed in Nayarit and it has shown that mycotoxin contamination is a real problem in both foodstuffs studied.     

A quantitative analysis for the ADP-ribosylation activity of pertussis toxin: an enzymatic-HPLC coupled assay applicable to formulated whole cell and acellular pertussis vaccine products.

The majority of the biological effects of pertussis toxin (PT) are the result of a toxin-catalyzed transfer of an adenosine diphosphate-ribose (ADP-ribose) moiety from NAD(+)to the alpha-subunits of a subset of signal-transducing guanine-nucleotide-binding proteins (G-proteins). This generally leads to an uncoupling of the modified G-protein from the corresponding receptor and the loss of effector regulation. This assay is based on the PT S1 subunit enzymatic transfer of ADP-ribose from NAD to the cysteine moiety of a fluorescent tagged synthetic peptide homologous to the 20 amino acid residue carboxyl-terminal sequence of the alpha-subunit of the G(i3)protein. The tagged peptide and the ADP-ribosylated product were characterized by HPLC/MS and MS/MS for structure confirmation. Quantitation of this characterized ADP-ribosylated fluorescently tagged peptide was by HPLC fluorescence using Standard Addition methodology. The assay was linear over a five hr incubation period at 20 degrees C at PT concentrations between 0.0625 and 4.0 microg/ml and the sensitivity of the assay could be increased several fold by increasing the incubation time to 24 h. Purified S1 subunit of PT exhibited 68.1+/-10.1% of the activity of the intact toxin on a molar basis, whereas the pertussis toxin B oligomer, the genetically engineered toxoid, (PT-9K/129G), and several of the other components of the Bordetella pertussis organism possessed little (<0.6%) or no detectable ribosylation activity. Commonly used pertussis vaccine reference materials, US PV Lot #11, BRP PV 66/303, and BRP PV 88/522, were assayed by this method against Bordetella pertussis Toxin Standard 90/518 and demonstrated to contain, respectively, 0.323+/-0.007, 0.682+/-0.045, and 0.757+/-0.006 microg PT/ml (Mean+/-SEM) or in terms of microg/vial: 3.63, 4.09 and 4.54, respectively. A survey of several multivalent pertussis vaccine products formulated with both whole cell as well as acellular components indicated that products possessed a wide range of ribosylation activities. The pertussis toxin S1 subunit catalyzed ADP- ribosylation of the FAC-Galpha(i3)C20 peptide substrate and its subsequent quantitation by HPLC was demonstrated to be a sensitive and quantitative method for measuring intrinsic pertussis toxin activity. This methodology not only has the potential to be an alternative physicochemical method to replace existing bioassay methodology, but has the added advantage of being a universal method applicable to the assay of pertussis toxin in both whole cell and acellular vaccines as well as bulk and final formulated vaccine products. Acceptance of this method by regulatory agencies and industry as a credible alternative to existing methods would, however, require validation in an international collaborative study against the widely accepted bioassay methods.     

Pertussis toxin effects on T lymphocytes are mediated through CD3 and not by pertussis toxin catalyzed modification of a G protein

Pertussis toxin (PT) has been shown to have a variety of effects on T lymphocyte function, and its activity has been used to suggest the involvement of a G protein in the early events of T lymphocyte activation. In this report, the effects of PT on T lymphocytes have been investigated in detail. PT at a concentration of 10 micrograms/ml rapidly stimulated early events that are normally induced by occupancy of the TCR complex in Jurkat cells and cloned, murine CTL including increased intracellular Ca2+ concentration, serine esterase release, and induction of Ag non-specific target cell lysis. However, 1-h treatment with this concentration of PT induced a state that was refractory to further receptor stimulation in Jurkat cells but not cloned CTL although substrate membrane proteins were modified to a similar extent in both cell lines. The functional effects of PT were mimicked by the B oligomer of PT which did not, however, catalyze ADP-ribosylation of membrane proteins. In addition, overnight exposure of Jurkat cells to a lower concentration of PT also modified substrate membrane proteins but did not inhibit receptor stimulation. These findings indicate that PT catalyzed ADP-ribosylation of a G protein does not account for the actions of the toxin on T lymphocytes. Finally, direct stimulation of increased intracellular Ca2+ concentration by PT and the B oligomer only occurred in T lymphocytes expressing CD3. This suggests that the mitogenic effect of PT holotoxin is mediated by the interaction of the B oligomer with CD3 and that this may account for many of the effects of PT holotoxin both in vivo and in vitro.     

The interaction of nucleotides with pertussis toxin. Direct evidence for a nucleotide binding site on the toxin regulating the rate of ADP-ribosylation of Ni, the inhibitory regulatory component of adenylyl cyclase

The interaction of nucleotides with pertussis toxin (PT), and their effects on the ability of the toxin to ADP-ribosylate pure Ni, were evaluated. [32P]ATP (10 nM) bound directly to dithiothreitol-activated PT. This binding was competitively inhibited by nucleotides and anions with the following IC50 concentrations in order of decreasing potency: ATP = ATP gamma S (adenosine-5'-O-(3-thiotriphosphate)) = 0.2-0.3 microM, GDP beta S (guanosine-5'-O-(2-thiodiphosphate)) = 2-3 microM, GTP gamma S (guanosine-5'-O-(3-thiotriphosphate)) = 10-15 microM, ADP = 20-25 microM, GTP = 30-40 microM, GMP-P(NH)P (guanyl-5'-yl imidodiphosphate) = 100-150 microM, GDP = 150-200 microM, Pi = SO4(2-) = 20 mM and Cl- = acetate = 30-35 mM. Treatment of PT with ATP, AMP-P(NH)P, GTP, GDP, or GDP beta S, resulted in a stimulated state of NAD+-Ni ADP-ribosyltransferase activity. Addition of ATP, AMP-P(NH)P (adenyl-5'-yl imidodiphosphate), GTP, GDP, and GDP beta S to the ADP-ribosylation reactions resulted in increased rates of ADP-ribosyl-Ni formation. It is concluded that these effects on the nucleotides are due to their action to stimulate the activity of PT. At concentrations of PT between 0.04 and 0.4 microgram/ml, the stimulation of ADP-ribosylation of Ni effected by nucleotides was hysteretic in nature, exhibiting an approximately 25-min long lag when GDP was used as the activating nucleotide. These lags decreased with increasing concentrations of PT, and were abolished by pretreatment of the toxin with GDP or ATP. Preliminary incubation of Ni with GDP had no effect on the lag in its ADP-ribosylation by non-nucleotide treated PT. Addition of divalent cations (Mg2+, Mn2+, and Ca2+) inhibited formation of ADP-ribosyl-Ni, possibly by causing aggregation and denaturation of Ni. This is the first demonstration that both adenine and guanine nucleotides interact directly with PT and act to stimulate its activity to ADP-ribosylate Ni, and that guanine nucleotides do so regardless of whether they are nucleoside di- or triphosphates.     

Pertussis toxin inhibits stimulated motility independently of the adenylate cyclase pathway in human melanoma cells

The human melanoma cell line, A2058, has previously been shown to respond to an autocrine motility factor (AMF). We have studied biochemical pathways that may be involved in the generation of such a motile response. Pertussis toxin (PT) caused a profound, rapid decrease in stimulated motility that was both dose and time-dependent. Preincubation of cells for 2 hr with as little as 1 ng/ml of PT significantly inhibited motility. A concentration of PT (0.5 microgram/ml) that completely eliminated migration after a 30 min. preincubation had a markedly reduced effect when added 1 hr after the start of the assay. In contrast, agents which selectively modulate or have a role in the adenylate cyclase pathway, e.g., cholera toxin, forskolin, the cAMP analogue 8-bromoadenosine 3':5'-cyclic monophosphate and the cyclase inhibitor 2',5'-dideoxyadenosine, all had negligible effect upon motility. These data are consistent with the presence of a receptor coupled to a PT sensitive G protein initiating motility independently of the adenylate cyclase system.