Nucleotide-, chemotactic peptide- and phorbol ester-induced exocytosis in HL-60 leukemic cells

Undifferentiated and differentiated HL-60 leukemic cells possess nucleotide receptors which functionally couple to phospholipase C via pertussis toxin-sensitive guanine nucleotide-binding proteins (G-proteins). We investigated the role of extracellular nucleotides in the regulation of beta-glucuronidase release in HL-60 cells. In dibutyryl cyclic AMP (Bt2cAMP)-differentiated HL-60 cells, the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), the phosphorothioate analogue of ATP, adenosine 5'-O-[3-thio]triphosphate (ATP[gamma S]), and UTP increased cytosolic Ca2+ from 100 nM up to 1.2 microM with EC50 values of 4 nM, 1 microM and 100 nM, respectively. In these cells, ATP[gamma S] induced exocytosis with an EC50 of 4 microM and an effectiveness amounting to 50-70% of that of fMet-Leu-Phe. ATP, ITP, UTP, CTP, and uridine 5'-O-[2-thio]diphosphate activated exocytosis as well. Phorbol myristate acetate (PMA) induced exocytosis with an EC50 of 115 ng/ml and an effectiveness similar to that of ATP[gamma S]. Cytochalasin B (CB) differently potentiated exocytosis induced by ATP[gamma S], fMet-Leu-Phe and PMA. Treatment of Bt2cAMP-differentiated HL-60 cells with pertussis toxin (500 ng/ml) for 24 h resulted in ADP-ribosylation of more than 97.5% of the G-proteins. Under these conditions, pertussis toxin almost completely inhibited the increase in cytosolic Ca2+ and beta-glucuronidase release induced by fMet-Leu-Phe but only partially inhibited the effects of ATP[gamma S] and UTP. fMet-Leu-Phe at a non-stimulatory concentration (1 nM) potentiated ATP[gamma S]-induced beta-glucuronidase release in the presence but not in the absence of CB. In contrast, ATP[gamma S] and fMet-Leu-Phe synergistically activated superoxide formation in the absence of CB. PMA potentiated superoxide formation induced by ATP[gamma S] or fMet-Leu-Phe and did not affect exocytosis induced by ATP[gamma S] or fMet-Leu-Phe. In undifferentiated HL-60 cells, fMet-Leu-Phe, ATP[gamma S], UTP and PMA did not induce beta-glucuronidase release. fMet-Leu-Phe did not increase cytosolic Ca2+ in undifferentiated HL-60 cells, whereas ATP[gamma S] and UTP were similarly potent and effective as in Bt2cAMP-differentiated cells. In differentiated HL-60 cells, fMet-Leu-Phe induced aggregation, and ATP[gamma S] induced a transient shape change. Our results show (I) that exocytosis in HL-60 cells does not obligatorily depend on CB. (II) Purine and pyrimidine nucleotides activate exocytosis via pertussis toxin-sensitive and -insensitive signal transduction pathways.(ABSTRACT TRUNCATED AT 400 WORDS

Cyclosporin H is a potent and selective formyl peptide receptor antagonist. Comparison with N-t-butoxycarbonyl-L-phenylalanyl-L-leucyl-L-phenylalanyl-L- leucyl-L-phenylalanine and cyclosporins A, B, C, D, and E

The cyclic undecapeptide, cyclosporin (Cs) H, is a potent inhibitor of FMLP-induced superoxide anion (O2-) formation in human neutrophils. We studied the effects of CsH in comparison with those of N-t-butoxycarbonyl-L-phenylalanyl-L-leucyl-L-phenylalanyl-L-leucyl-L- phenylalanine (BocPLPLP), a well known formyl peptide receptor antagonist, and of other Cs on activation of N6,2'-O-dibutyryl adenosine 3:5'-monophosphate-differentiated HL-60 cells and human erythroleukemia cells (HEL cells). CsH inhibited FMLP binding in HL-60 membranes with a Ki (inhibition constant) of 0.10 microM. CsH inhibited activation by FMLP of high affinity GTPase (the enzymatic activity of alpha-subunits of heterotrimeric regulatory guanine nucleotide-binding proteins) in HL-60 membranes with a Ki of 0.79 microM. CsH inhibited the stimulatory effects of FMLP on cytosolic Ca2+ concentration ([Ca2+]i), O2- formation, and beta-glucuronidase release with Ki values of 0.08, 0.24, and 0.45 microM, respectively. BocPLPLP was 14-fold less potent than CsH in inhibiting FMLP binding and 4- to 6-fold less potent than CsH in inhibiting FMLP-induced GTP hydrolysis, rises in [Ca2+]i, O2- formation, and beta-glucuronidase release. CsA reduced FMLP-induced O2- formation by 20%, but CsB, CsC, CsD, and CsE did not. CsA, CsB, CsC, CsD, and CsE did not affect FMLP-induced rises in [Ca2+]i. BocPLPLP inhibited leukotriene B4-induced rises in [Ca2+]i with a Ki of 0.33 microM, whereas CsH showed no inhibitory effect. CsH and BocPLPLP did not inhibit the rises in [Ca2+]i induced by several other stimuli in HL-60 cells and HEL cells. Our results show that 1) CsH is a more potent formyl peptide receptor antagonist than BocPLPLP; 2) unlike BocPLPLP, CsH is selective; and 3) N-methyl-D-valine which is present at position 11 of the amino acid sequence of CsH but not of other Cs is crucial for FMLP antagonism

Purine and pyrimidine nucleotides potentiate activation of NADPH oxidase and degranulation by chemotactic peptides and induce aggregation of human neutrophils via G proteins

Whereas the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), induced NADPH-oxidase-catalyzed superoxide (O2-) formation in human neutrophils, purine and pyrimidine nucleotides per se did not stimulate NADPH oxidase but enhanced O2- formation induced by submaximally and maximally stimulatory concentrations of fMet-Leu-Phe up to fivefold. On the other hand, FMet-Leu-Phe primed neutrophils to generate O2- upon exposure to nucleotides. At a concentration of 100 microM, purine nucleotides enhanced O2- formation in the effectiveness order adenosine 5'-O-[3-thio]triphosphate (ATP[gamma S]) greater than ITP greater than guanosine 5'-O-[3-thio]triphosphate (GTP[gamma S]) greater than ATP = adenosine 5'-O-[2-thio]triphosphate (Sp-diastereomer) = GTP = guanosine 5'-O-[2-thio]diphosphate (GDP[beta S] = ADP greater than adenosine 5'-[beta, gamma-imido]triphosphate = adenosine 5'-O-[2-thio]triphosphate] (Rp-diastereomer). Pyrimidine nucleotides stimulated fMet-Leu-Phe-induced O2- formation in the effectiveness order uridine 5'-O-[3-thio]triphosphate (UTP[gamma S]) = UTP greater than CTP. Uracil (UDP[beta S]) = uridine 5'-O[2-thio]triphosphate (Rp-diastereomer) (Rp)-UTP[beta S]) = UTP greater than CTP. Uracil nucleotides were similarly effective potentiators of O2- formation as the corresponding adenine nucleotides. GDP[beta S] and UDP[beta S] synergistically enhanced the stimulatory effects of ATP[gamma S], GTP[gamma S] and UTP[gamma S]. Purine and pyrimidine nucleotides did not induce degranulation in neutrophils but potentiated fMet-Leu-Phe-induced release of beta-glucuronidase with similar nucleotide specificities as for O2- formation. In contrast, nucleotides per se induced aggregation of neutrophils. Treatment with pertussis toxin prevented aggregation induced by both nucleotides and fMet-Leu-Phe. Our results suggest that purine and pyrimidine nucleotides act via nucleotide receptors, the nucleotide specificity of which is different from nucleotide receptors in other cell types. Neutrophil nucleotide receptors are coupled to guanine-nucleotide-binding proteins. As nucleotides are released from cells under physiological and pathological conditions, they may play roles as intercellular signal molecules in neutrophil activation

Similar apparent constitutive activity of human histamine H_2-receptor fused to long and short splice variants of Gsa,

Fusion proteins allow for the analysis of receptor/G protein coupling under defined conditions. The beta 2-adrenoceptor (beta 2AR) fused to the long splice variant of Gsalpha (Gsalpha L) exhibits a higher apparent constitutive activity than the beta 2-adrenoceptor fused to the short splice variant of Gsalpha (Gsalpha S). Experimentally, this results in higher efficacy and potency of partial agonists and in higher efficacy of inverse agonists at the beta 2AR fused to Gsalpha L relative to the beta 2AR fused to Gsalpha S, indicating that the agonist-free beta 2AR and the beta 2AR occupied by partial agonists promote GDP dissociation from Gsalpha L more efficiently than from Gsalpha S. In fact, the GDP affinity of Gsalpha S fused to the beta 2AR is higher than the GDP affinity of Gsalpha L fused to the beta 2AR. We asked the question whether the histamine H2-receptor (H2R) exhibits similar coupling to Gsalpha splice variants as the beta 2AR. To address this question, we studied H2R-Gsalpha fusion proteins expressed in Sf9 cells. In contrast to beta 2AR-Gsalpha fusion proteins, the potencies and efficacies of partial agonists and the efficacies of inverse agonists were similar at the H2R fused to Gsalpha L and Gsalpha S as assessed by guanosine-5'-O-(3-thio)triphosphate binding and/or steady-state GTPase activity. However, the time course analysis of guanosine-5'-O-(3-thio)triphosphate binding indicated that Gsalpha S fused to the H2R possesses a higher GDP-affinity than Gsalpha L fused to the H2R. Our data show that the H2R fused to Gsalpha L and Gsalpha S possesses similar constitutive activity and is insensitive to differences in GDP affinity of Gsalpha splice variants. Thus, GDP affinity of G proteins does not generally determine constitutive activity of receptors

Distinct interaction of human and guinea pig histamine H_2-Receptor with guanidine-type agonists,

It is unknown why the potencies and efficacies of long-chained guanidine-type histamine H2-receptor (H2R) agonists are lower at the H2R of human neutrophils than at the H2R of the guinea pig atrium. To elucidate these differences, we analyzed fusion proteins of the human H2R (hH2R) and guinea pig H2R (gpH2R), respectively, and the short splice variant of Gsalpha (Gsalpha S) expressed in Sf9 cells. The potencies and efficacies of small H2R agonists in the GTPase assay and the potencies of antagonists at inhibiting histamine-stimulated GTP hydrolysis by hH2R-Gsalpha S and gpH2R-Gsalpha S were similar. In contrast, the potencies and efficacies of guanidines were lower at hH2R-Gsalpha S than at gpH2R-Gsalpha S. Guanidines bound to hH2R-Gsalpha S with lower affinity than to gpH2R-Gsalpha S, and high-affinity binding of guanidines at gpH2R-Gsalpha S was more resistant to disruption by GTPgamma S than binding at hH2R-Gsalpha S. Molecular modeling suggested that the nonconserved Asp-271 in transmembrane domain 7 of gpH2R (Ala-271 in hH2R) confers high potency to guanidines. This hypothesis was confirmed by Ala-271right-arrowAsp-271 mutation in hH2R-Gsalpha S. Intriguingly, the efficacies of guanidines at the Ala-271right-arrowAsp-271 mutant and at hH2R/gpH2R chimeras were lower than at gpH2R. Our model suggests that a Tyr-17/Asp-271 H-bond, present only in gpH2R-Gsalpha S but not the other constructs studied, stabilizes the active guanidine-H2R state. Collectively, our data show 1) distinct interaction of H2R species isoforms with guanidines, 2) that a single amino acid in transmembrane domain 7 critically determines guanidine potency, and 3) that an interaction between transmembrane domains 1 and 7 is important for guanidine efficacy

Unerwünschte Arzneimittelwirkungen in der Gerontopsychiatrie

Zusammenfassung Ziel der Studie war es zu uberprufen, ob die Verordnung potenziell inadaquater Wirkstoffe" (PIW) mit einem erhohten Risiko fur unerwunschte Arzneimittelwirkungen (UAW) einhergeht. Methodik 304 stationare gerontopsychiatrische Patienten wurden in die Studie eingeschlossen. Anhand der klinischen Daten und Verlaufsdokumentationen wurden Hinweise auf UAW identifiziert. Der Kausalzusammenhang der UAW und der verordneten Wirkstoffe wurde von Experten bewertet. Ergebnisse Vor der stationaren Aufnahme erhielten ca. 30% aller Patienten >= 1 PIW, bei Entlassung waren es 22%. Risikofaktoren fur die Verordnung >= 1 PIW waren zunehmende Anzahl verordneter Wirkstoffe und Schizophrenie. Protektiv wirkten daher die Faktoren Demenz und zunehmendes Lebensalter. Die Verordnung von >= 1 PIW fuhrte zu einem 5-fach erhohten Risiko fur das Auftreten von >= 1 UAW; das Risiko fur UAW stieg mit der Zahl der PIW. Die Verordnung von >= 1 PIW ging mit einem 4-fach erhohten Risiko fur das Auftreten schwerwiegender UAW einher. Bei Einnahme von >= 2 PIW war das Risiko hierfur sogar um ein 10-Faches erhoht. Schlussfolgerungen Die PRISCUS-Liste bildet wesentliche Risiken fur das Auftreten von UAW in der Gerontopsychiatrie ab. Abstract Objective The purpose of this study was to examine the extent to which "potentially inappropriate drugs" (PID) are associated with an increased risk for adverse drug reactions (ADR). Methods Data from 304 geriatric psychiatric inpatients was collected. Medical documentation was used to find indications of ADRs. Causal relationship between the ADR and the prescribed drugs was assessed by experts. Results Almost 30% of patients received >= 1 PID before admission to hospital, in comparison to 22% at discharge. Increasing number of total prescriptions and the diagnosis of schizophrenia resulted in an increased risk for receiving >= 1 PID. Higher age and dementia were protective factors. Patients receiving >= 1 PID had a 5-fold increased risk of experiencing >= 1 ADR. Risk for an ADR increased with number of PID prescriptions. Patients treated with >= 1 PID had a 4-fold increased risk of experiencing severe ADRs. Risk for severe ADRs was 10-fold higher in patients treated with >= 2 PIDs. Conclusion The PRISCUS list predicts significant risk factors for the occurrence of ADRs in the geriatric psychiatric setting