Application of bradykinin to neonatal rat dorsal root ganglion neurons caused a depolarization associated with an inward current and an increase in membrane conductance that was probably due to the opening of sodium channels. No hyperpolarization or outward current was detected. In addition, bradykinin increased the rate of 45Ca uptake into the neurons by a mechanism that was blocked by the dihydropyridine calcium channel antagonist nifedipine. Direct activation of protein kinase C (PKC) with phorbol esters mimicked the ability of bradykinin to depolarize the neurons and to increase the rate of 45Ca uptake. Down-regulation of PKC by prolonged treatment with phorbol esters and treatment of the cells with staurosporine, which inhibits PKC, blocked both bradykinin- and phorbol ester-induced 45Ca influx, and substantially reduced the proportion of cells that gave electrophysiological responses to either agent. Bradykinin also activated polyphosphoinositidase C in the dorsal root ganglion neurons, elevating levels of inositol(1,4,5)-trisphosphate and 1,2-diacylglycerol, an endogenous activator of PKC. It is suggested, therefore, that PKC may mediate some of the effects of bradykinin in sensory neurons.
Globally, there has been an increase in the use of herbal remedies including traditional Chinese medicine (TCM). There is a perception that products are natural, safe and effectively regulated, however, regulatory agencies are hampered by a lack of a toolkit to audit ingredient lists, adulterants and constituent active compounds. Here, for the first time, a multidisciplinary approach to assessing the molecular content of 26 TCMs is described. Next generation DNA sequencing is combined with toxicological and heavy metal screening by separation techniques and mass spectrometry (MS) to provide a comprehensive audit. Genetic analysis revealed that 50% of samples contained DNA of undeclared plant or animal taxa, including an endangered species of Panthera (snow leopard). In 50% of the TCMs, an undeclared pharmaceutical agent was detected including warfarin, dexamethasone, diclofenac, cyproheptadine and paracetamol. Mass spectrometry revealed heavy metals including arsenic, lead and cadmium, one with a level of arsenic >10 times the acceptable limit. The study showed 92% of the TCMs examined were found to have some form of contamination and/or substitution. This study demonstrates that a combination of molecular methodologies can provide an effective means by which to audit complementary and alternative medicines.
Neuroblastoma x glioma hybrid NG108-15 cells express a high-affinity IP prostanoid receptor. Saturation binding analysis of this receptor, using [3H]prostaglandin E1 ([3H]PGE1) as ligand, indicated that it was present at some 1.5 pmol/mg of membrane protein and displayed a dissociation constant for this ligand of 30-40 nM. Prolonged exposure of these cells either to PGE1 or to iloprost, which is a stable analogue of prostacyclin, caused a 40-70% decrease in levels of the receptor. The remaining receptors were capable of interacting with the stimulatory G-protein (Gs) of the adenylate cyclase cascade, as saturation analysis of the binding of [3H]PGE1 indicated that they had a similar affinity for the 3H-labelled ligand, and because the specific binding of [3H]PGE1 to these receptors was still sensitive to the presence of poorly hydrolysed analogues of GTP. We have recently demonstrated that prolonged exposure of NG108-15 ells to PGE1 causes a cyclic AMP-independent loss of Gs alpha-subunit (Gs alpha) from these cells [McKenzie & Milligan (1990) J. Biol. Chem. 265, 17084-17093]. Steady-state concentration of the larger 45 kDa form of Gs alpha (which is the predominant form expressed in these cells) was assessed to be 9.6 pmol/mg of membrane protein, and treatment with iloprost decreased levels of this polypeptide to some 3.0 pmol/mg of protein. Time courses of iloprost-mediated down-regulation of the IP prostanoid receptor, loss of Gs alpha protein as assessed by immunoblotting and loss of Gs alpha activity as assessed by the reconstitution of NaF stimulation of adenylate cyclase activity to membranes of S49 cyc- cells by sodium cholate extracts of NG108-15 cells were identical, suggesting that the loss of the IP prostanoid receptor and G-protein occurred in parallel. Each of these effects was half-maximal between 2 and 3 h of exposure to the agonist. Stoichiometry of loss of Gs alpha and IP prostanoid receptor was unchanged by the percentage receptor occupancy, and quantification indicated the loss of some 7-10 mol of Gs alpha/mol of receptor. This is the first report to demonstrate the temporal concurrence of loss of Gs alpha and of a receptor which interacts with this G-protein. Chronic activation of the IP prostanoid receptor on these cells results in the development of a heterologous form of desensitization to agents which function to activate adenylate cyclase [Kelly, Keen, Nobbs & MacDermot (1990) Br. J. Pharmacol. 99, 306-316]. Agonist regulation of Gs alpha levels in these cells may contribute to this process.
CHO cells stably transfected with cDNA encoding the human M1 muscarinic acetylcholine (HM1) receptor were treated with the cholinergic agonist carbachol at various concentrations for differing times. Levels of the HM1 receptor and of a range of G-proteins were subsequently measured. Carbachol treatment of the transfected cells caused a substantial down-regulation of cellular levels of the alpha subunit of Gq (Gq alpha), but did not significantly alter cellular levels of the alpha subunits of Gs or Gi2. A small decrease in levels of G-protein beta-subunit was also produced. Parallel assessment of agonist-induced down-regulation of the HM1 receptor demonstrated that it was lost in concert with the G-protein. Similar concentrations of carbachol (5 microM) were required to produce half-maximal stimulation of inositol phosphate generation and loss of each of the HM1 receptor and Gq alpha, and half-maximal losses of both receptor and Gq alpha were produced by 3 h of treatment with 1 mM-carbachol. By contrast, treatment of the non-transfected parental CHO cells, which do not express detectable levels of the receptor, with carbachol had no effect on cellular Gq alpha levels. Concurrent treatment of the HM1-expressing CHO cells with carbachol and cycloheximide indicated that suppression of protein synthesis de novo did not mimic the effect of carbachol, and hence even complete inhibition of transcription of the Gq alpha gene and/or translation of pre-existing Gq alpha mRNA could not account for the agonist-induced effect. We have previously noted that cellular levels of both Gs alpha [McKenzie and Milligan (1990) J. Biol. Chem. 265, 17084-17093] and the alpha subunits of the pertussis-toxin-sensitive G-proteins Gi1, Gi2 and Gi3 [Green, Johnson and Milligan (1990) J. Biol. Chem. 265, 5206-5210] can be regulated in certain cell systems by agonist activation of receptors expected to interact with these G-proteins. These results demonstrate that the same is true of Gq alpha and suggest that agonist-induced co-ordinate loss of receptors and associated G-proteins may be a more common feature than has been appreciated to date.
Rat-1 fibroblasts were transfected with a cDNA encoding the mouse delta opioid receptor. Two separate clones, D2 (which expressed some 6 pmol of the receptor/mg of membrane protein) and DOE (which expressed some 0.2 pmol/mg of membrane protein), were examined in detail. With membranes from both clones, the opioid agonist [D-Ala2]leucine enkephalin (DADLE) caused stimulation of high-affinity GTPase activity and of the binding of guanosine 5'-[gamma-[35S]thio]triphosphate, and inhibition of forskolin-amplified adenylate cyclase activity. DADLE also induced phosphorylation and activation of both the p42MAPK (42 kDa isoform) and p44MAPK (44 kDa isoform) members of the mitogen-activated protein kinase (MAP kinase) family. All of these effects of DADLE were prevented in both clones by pretreatment of the cells with pertussis toxin. The maximal response that could be produced by DADLE in direct assays of G-protein activation were substantially greater in clone D2 than in clone DOE, but in both clones essentially full phosphorylation of both p42MAPK and p44MAPK could be achieved. EC50 values for DADLE stimulation of GTPase activity and for activation of p44MAPK were substantially lower in clone D2 than in clone DOE. Moreover, in both clones the EC50 value for DADLE stimulation of p44MAPK was substantially lower than that for stimulation of GTPase activity, and the Hill coefficients for agonist activation of p44MAPK (h > 1) displayed marked co-operativity whereas those for G-protein activation did not (h 0.8-1.0). DADLE activation of p44MAPK showed more sustained kinetics in clone D2 than in clone DOE. By contrast, lysophosphatidic acid, acting at an endogenously expressed G-protein-coupled receptor, also activated p44MAPK in both clones in a pertussis toxinsensitive manner, but both the kinetics and the concentration-response curve for activation of p44MAPK by this ligand were similar. As with other systems, maintained cellular levels of a cAMP analogue prevented the effects of both G-protein-coupled receptors on activation of p44MAPK. These results demonstrate for the first time that an opioid receptor, at least when expressed in Rat-1 fibroblasts, is able to initiate activation of the MAP kinase cascade in a G1-dependent manner, and show that only a very small proportion of the cellular G1 population is required to be activated to result in full phosphorylation of the p42MAPK and p44MAPK MAP kinases.
Incubation of the neuroblastoma x glioma hybrid cell line NG108-15 in tissue culture with dibutyryl cyclic AMP (1 mM) for up to 8 days produced a morphological differentiation of the cells, during which they extended neurite-like processes. Pertussis-toxin-catalysed ADP-ribosylation indicated that amounts of guanine-nucleotide-binding proteins (G-proteins), which are substrates for this toxin, were approximately doubled in membranes from the 'differentiated' cells in comparison with the control cells. Immunoblotting of membranes derived from either untreated or dibutyryl cyclic AMP-treated cells with anti-peptide antisera specific for the alpha subunits of the pertussis-toxin-sensitive G-proteins Gi and Go demonstrated that amounts of these G-proteins were reciprocally modulated during the differentiation process. In comparison with the untreated cells, the amount of Gi in the 'differentiated' cells was decreased, whereas the amount of Go was substantially increased. Stimulation of high-affinity GTPase activity in response to opioid peptides, which in this cell line interact with an opioid receptor of the delta subclass, was much decreased, and inhibition of adenylate cyclase activity was almost entirely attenuated in the 'differentiated'-cell membranes in comparison with membranes of untreated cells. Opioid receptor number was also decreased in membranes of the dibutyryl cyclic AMP-treated cells in comparison with the control cells. These data demonstrate that relatively small changes in the observed pattern of pertussis-toxin-catalysed ADP-ribosylation of membranes can mask more dramatic alterations in amounts of the individual pertussis-toxin-sensitive G-proteins, and further demonstrate the importance of methodologies able to discriminate between the different gene products.
Three distinct antipeptide antisera generated against synthetic peptides that represent parts of the primary sequence of the alpha-subunit of the (pertussis toxin-sensitive) guanine nucleotide binding protein G0 were used in two-dimensional immunoblots of membranes of neuroblastoma X glioma (NG108-15) cells. Each antiserum identified two distinct polypeptides of some 39 kDa. These had apparent isoelectric points of 5.5 and 5.8. Differentiation of NG108-15 cells in response separately to dibutyryl cyclic AMP (cAMP), 8-bromo cAMP, forskolin, and prostaglandin E1 produced elevated levels of G0 alpha, as has previously been noted in one-dimensional immunoblots. Two-dimensional analysis demonstrated that the cAMP-induced increases in levels of G0 alpha were only of the more acidic isoform. The two isoforms were both substrates for pertussis toxin-catalysed ADP-ribosylation and did not appear to represent differentially phosphorylated forms of the same polypeptide. Separation of the two forms of G0 alpha could be achieved in one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis when 4 M deionized urea was included in the resolving gel. The more slowly migrating band was the acidic form and corresponded exactly in mobility with the major form of G0 from both rat and mouse brain. There was no equivalent in brain of the more rapidly migrating form of G0 from the cells. In agreement with the data from two-dimensional gels, only the more slowly migrating form was expressed in considerably higher amounts following cAMP-induced differentiation of NG108-15 cells. Of these two forms of "G0," the acidic species is equivalent to G0 from brain, but the basic form is not identical with G0*, which has been purified from bovine brain.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.