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In the perfused rat liver the anaphylatoxin C5a enhanced glucose output, reduced flow, and elevated prostanoid overflow. Because hepatocytes (HCs) do not express C5a receptors, the metabolic C5a actions must be indirect, mediated by e.g. prostanoids from Kupffer cells (KCs) and hepatic stellate cells (HSCs), which possess C5a receptors. Surprisingly, the metabolic C5a effects were not only impaired by the prostanoid synthesis inhibitor, indomethacin, but also by the thromboxane A(2) (TXA(2)) receptor antagonist, daltroban, even though HCs do not express TXA(2) receptors. TXA(2) did not induce prostaglandin (PG) or an unknown factor release from KCs or sinusoidal endothelial cells (SECs), which express TXA(2) receptors, because (1) daltroban did neither influence the C5a-induced release of prostanoids from cultured KCs nor the C5a-dependent activation of glycogen phosphorylase in KC/HC cocultures and because (2) the TXA(2) analog, U46619, failed to stimulate prostanoid release from cultured KCs or SECs or to activate glycogen phosphorylase in KC/HC or SEC/HC cocultures. In the perfused liver, Ca(2+)-deprivation inhibited not only flow reduction but also glucose output elicited by C5a to similar extents as daltroban. Similarly, in the absence of extracellular Ca(2+), flow reduction and glucose output induced by U46619 were almost completely prevented, whereas glucose output induced by the directly acting PGF(2alpha) was only slightly lowered. Thus, in the perfused rat liver PGs released after C5a- stimulation from KCs and HSCs directly activated glycogen phosphorylase in HCs, and TXA(2) enhanced glucose output indirectly mainly by causing hypoxia as a result of flow reduction.
Botulinum neurotoxins (BoNTs) are potent neurotoxins produced by bacteria, which inhibit neurotransmitter release, specifically in their physiological target known as motor neurons (MNs). For the potency assessment of BoNTs produced for treatment in traditional and aesthetic medicine, the mouse lethality assay is still used by the majority of manufacturers, which is ethically questionable in terms of the 3Rs principle. In this study, MNs were differentiated from human induced pluripotent stem cells based on three published protocols. The resulting cell populations were analyzed for their MN yield and their suitability for the potency assessment of BoNTs. MNs produce specific gangliosides and synaptic proteins, which are bound by BoNTs in order to be taken up by receptor-mediated endocytosis, which is followed by cleavage of specific soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) proteins required for neurotransmitter release. The presence of receptors and substrates for all BoNT serotypes was demonstrated in MNs generated in vitro. In particular, the MN differentiation protocol based on Du et al. yielded high numbers of MNs in a short amount of time with high expression of BoNT receptors and targets. The resulting cells are more sensitive to BoNT/A1 than the commonly used neuroblastoma cell line SiMa. MNs are, therefore, an ideal tool for being combined with already established detection methods.
Botulinum neurotoxins (BoNTs) are potent neurotoxins produced by bacteria, which inhibit neurotransmitter release, specifically in their physiological target known as motor neurons (MNs). For the potency assessment of BoNTs produced for treatment in traditional and aesthetic medicine, the mouse lethality assay is still used by the majority of manufacturers, which is ethically questionable in terms of the 3Rs principle. In this study, MNs were differentiated from human induced pluripotent stem cells based on three published protocols. The resulting cell populations were analyzed for their MN yield and their suitability for the potency assessment of BoNTs. MNs produce specific gangliosides and synaptic proteins, which are bound by BoNTs in order to be taken up by receptor-mediated endocytosis, which is followed by cleavage of specific soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) proteins required for neurotransmitter release. The presence of receptors and substrates for all BoNT serotypes was demonstrated in MNs generated in vitro. In particular, the MN differentiation protocol based on Du et al. yielded high numbers of MNs in a short amount of time with high expression of BoNT receptors and targets. The resulting cells are more sensitive to BoNT/A1 than the commonly used neuroblastoma cell line SiMa. MNs are, therefore, an ideal tool for being combined with already established detection methods.
For the five principal prostanoids PGD2, PGE2, PGF2alpha, prostacyclin and thromboxane A2 eight receptors have been identified that belong to the family of G-protein-coupled receptors. They display an overall homology of merely 30%. However, single amino acids in the transmembrane domains such as an Arg in the seventh transmembrane domain are highly conserved. This Arg has been identified as part of the ligand binding pocket. It interacts with the carboxyl group of the prostanoid. The aim of the current study was to analyze the potential role in ligand binding of His-81 in the second transmembrane domain of the rat PGF2alpha receptor, which is conserved among all PGF2alpha receptors from different species. Molecular modeling suggested that this residue is located in close proximity to the ligand binding pocket Arg 291 in the 7th transmembrane domain. The His81 (H) was exchanged by site-directed mutagenesis to Gln (Q), Asp (D), Arg (R), Ala (A) and Gly (G). The receptor molecules were N-terminally extended by a Flag epitope for immunological detection. All mutant proteins were expressed at levels between 50% and 80% of the wild type construct. The H81Q and H81D receptor bound PGF2alpha with 2-fold and 25-fold lower affinity, respectively, than the wild type receptor. Membranes of cells expressing the H81R, H81A or H81G mutants did not bind significant amounts of PGF2alpha. Wild type receptor and H81Q showed a shallow pH optimum for PGF2alpha binding around pH 5.5 with almost no reduction of binding at higher pH. In contrast the H81D mutant bound PGF2alpha with a sharp optimum at pH 4.5, a pH at which the Asp side chain is partially undissociated and may serve as a hydrogen bond donor as do His and Gln at higher pH values. The data indicate that the His-81 in the second transmembrane domain of the PGF2alpha receptor in concert with Arg-291 in the seventh transmembrane domain may be involved in ligand binding, most likely not by ionic interaction with the prostaglandin's carboxyl group but rather as a hydrogen bond donor.
1) During orthograde perfusion of rat liver human anaphylatoxin C3a caused an increase in glucose and lactate output and reduction of flow. These effects could be enhanced nearly twofold by co-infusion of the carboxypeptidase inhibitor MERGETPA, which reduced inactivation of C3a to C3adesArg. 2) During retrograde perfusion C3a caused a two- to threefold larger increase in glucose and lactate output and reduction of flow than in orthograde perfusions. These actions tended to be slightly enhanced by MERGETPA. 3) The elimination of C3a plus C3adesArg immunoreactivity during a single liver passage was around 67%, irrespective of the perfusion direction and the presence of the carboxypeptidase inhibitor MERGETPA; however, less C3adesArg and more intact C3a appeared in the perfusate in the presence of MERGETPA in orthograde and retrogade perfusions It is concluded that rat liver inactivated human anaphylatoxin C3a by conversion to C3adesArg and moreover eliminated it by an additional process. The inactivation to C3adesArg seemed to be located predominantly in the proximal periportal region of the liver sinusoid, since C3a was less effective in orthograde perfusions, when C3a first passed the proximal periportal region before reaching the predominant mass of parenchyma as its site of action, than in retrograde perfusions, when it first passed the perivenous area. These data may be evidence for a periportal scavenger mechanism, by which the liver protects itself from systemically released mediators of inflammation that interfere with the local regulation of liver metabolism and hemodynamics.
The complement fragments C3a and C5a were purified from zymosan-activated human serum by column chromatographic procedures after the bulk of the proteins had been removed by acidic polyethylene glycol precipitation. In the isolated in situ perfused rat liver C3a increased glucose and lactate output and reduced flow. Its effects were enhanced in the presence of the carboxypeptidase inhibitor DL-mercaptomethyl-3-guanidinoethylthio-propanoic acid (MERGETPA) and abolished by preincubation of the anaphylatoxin with carboxypeptidase B or with Fab fragments of an anti-C3a monoclonal antibody. The C3a effects were partially inhibited by the thromboxane antagonist BM13505. C5a had no effect. It is concluded that locally but not systemically produced C3a may play an important role in the regulation of local metabolism and hemodynamics during inflammatory processes in the liver.
In the isolated rat liver perfused in situ stimulation of the nerve bundles around the portal vein and the hepatic artery caused an increase of urate formation that was inhibited by the α1-blocker prazosine and the xanthine oxidase inhibitor allopurinol. Moreover, nerve stimulation increased glucose and lactate output and decreased perfusion flow. Infusion of noradrenaline had similar effects. Compared to nerve stimulation infusion of glucagon led to a less pronounced increase of urate formation and a twice as large increase in glucose output but a decrease in lactate release without affecting the flow rate. Insulin had no effect on any of the parameters studied.
In perfused rat livers, infusion of prostaglandin F₂α (PGF₂α) or noradrenaline increased glucose and lactate output and reduced flow. Glucagon increased glucose output and decreased lactate output without influence on flow. Infusion of phorbol 13-myristate 14-acetate (PMA) for 20 min prior to these stimuli strongly inhibited the metabolic and hemodynamic effects of noradrenaline, reduced the metabolic actions of PGF₂α but did not alter the effects of glucagon. In isolated rat hepatocytes PGF₂α, noradrenaline and glucagon activated glycogen phosphorylase but only PGF₂α and noradrenaline increased intracellular inositol 1,4,5-1risphosphalc (InsP₃). The noradrenaline- or PGF₂α-elicited activation of glycogen phosphorylase and increase in InsP₃ were largely reduced after preincubation of the cells for 10 min with PMA, whereas the glucagon-mediated enzyme activation was not affected. In contra\t to PMA, the phorbol ester 4a-phorbol 13,14-didecanoate. which does not activate protein kinase C, did not attenuate the PGF₂α- and noradrenaline-elicited stimulation of glucose output, glycogen phosphorylase and InsP, formation. Stimulation of InsP₃ formation by AlF₄⁻, which activates phospholipase C independently of the receptor, was not attenuated by prior incubation with PMA. Plasma membranes purified from isolated hepatocytes had both a high-capacity, low-affinity and a low-capacity, high-affinity binding site for PGF₂α. The Kd of the high-capacity, low-affinity binding site was close to the concentration of PGF₂α that increased glycogen phosphorylase activity halfmaximally. Binding to the high-capacity, low-affinity binding site was enhanced by guanosine 5'- 0-(3-thio)triphosphate (GTP[S]). This high-capacity, low-affinity site might thus represent the receptor. The Bmax and Kd of the high-capacity site, as well as the enhancement by GTP[S] of PGF₂α binding to this site, remained unaffected by PMA pretreatment. It is concluded that, in hepatocytes, activation of protein kinase C by PMA interrupted the InsP₃-mediated signal pathway from PGF₂α via a PGF₂α receptor and phospholipase C to glycogen phosphorylase at a point distal of the receptor prior to phospholipase C.
Human placenta is surprisingly rich in post-proline dipeptidyl peptidase activity. Among various cell fractions, microsomes have the highest specific activity. A homogeneous enzyme preparation is obtained in a six-step purification procedure. The final preparation appears homogeneous upon dodecyl sulfate electrophoresis, but analytical isoelectric focussing reveals various active bands with isoelectric points in the range of pH 3 - 4. The enzyme is a glycoprotein containing about 30% carbohydrate. Treatment with neuraminidase lowers the isoelectric points but does not reduce the heterogeneity of the band pattern. The subunit molecular weight is 120000 as estimated by dodecyl sulfate electrophoresis, whereas Mr of the native enzyme is > 200000, as can be concluded from gel filtration experiments. The purified dipeptidyl peptidase cleaves various synthetic and natural peptides, including substance P, kentsin, casomorphin and a synthetic renin inhibitor. In general, the specificity of the placenta peptidase is similar to that of post-proline dipeptidyl peptidase from other sources. Phenylalanylprolyl-P-naphthylamide (Km = 0.02 mM, I/ = 92 Ujmg) is the best substrate among various synthetic peptide derivatives. Only peptides with a free N-terminal amino group and proline, hydroxyproline, or alanine in position 2 of the N-terminal sequence are cieaved. However, X-Pro-Pro- . . . structures, e. g. as in bradykinin, are not attacked. 1 mM bis-(6nitrophenyI)phosphate or 1 mM diisopropylfluorophosphate completely inactivate the peptidase within 30 min at 30°C (pH 8). The peptidase is also completely inhibited by 1 mM Zn²⁺ and by other heavy metals.