m3 Muscarinic receptor-induced and Gi-mediated heterologous potentiation of phospholipase C stimulation: role of phosphoinositide synthesis.

Agonist activation of thrombin and purinergic receptors endogenously expressed in human embryonic kidney (HEK) cells and of the stably expressed m3 muscarinic acetylcholine receptor (mAChR) induces phospholipase C (PLC) stimulation, with the most pronounced PLC stimulation observed on mAChR activation. These receptor responses were pertussis toxin (PTX) insensitive and nonadditive, suggesting that the receptors share common signaling pathways. Short term (2 min) pretreatment of HEK cells with carbachol (1 mM), but not ATP, followed by agonist washout, caused a long-lasting (> or = 90 min) sensitization of PLC responses. At 30 min after carbachol treatment and washout, mAChR-stimulated PLC activity, measured as formation of either total inositol phosphates or of inositol-1,4,5-trisphosphate, was enhanced by 1.5-2-fold. PLC stimulation by thrombin and purinergic receptors was increased by approximately 3-fold. Furthermore, carbachol pretreatment also enhanced, by approximately 2.5-fold, stimulation of PLC activity on direct activation of G proteins by AIF4- and guanosine-5'-O-(3-thio)-triphosphate in intact and permeabilized cells, respectively. In contrast, PLC activities, measured with exogenous phosphatidylinositol-4,5-bisphosphate [Ptdns(4,5)P2] in HEK cell lysates, were not altered, suggesting that carbachol pretreatment may enhance the cellular level of Ptdlns(4,5)P2. Indeed, the level of Ptdlns(4,5)P2 was found to be increased by approximately 50% in HEK cells 30 min after short term carbachol treatment, whereas the level of phosphatidylinositol was not altered and that of phosphatidylinositol-4-phosphate decreased (by 40-50%). Pretreatment of HEK cells with PTX prevented the m3 mAChR-induced PLC potentiation and reduced the elevation in Ptdlns(4,5)P2 level by approximately 50%. In conclusion, short term agonist activation of m3 mAChRs stably expressed in HEK cells can lead to a longlasting heterologous potentiation of PLC signaling, which processes apparently involve PTX-sensitive G proteins and an enhanced PLC substrate supply.

Restoration of Clostridium difficile toxin-B-inhibited phospholipase D by phosphatidylinositol 4,5-bisphosphate.

Receptor signalling to phospholipase D (PLD) in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor apparently involves Rho proteins. Since phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] has been recognized as an essential cofactor for PLD activity and since activated Rho proteins have been reported to stimulate the synthesis of PtdIns(4,5)P2, we studied whether in HEK cells PLD activity is regulated by PtdIns(4,5)P2 and, in particular, whether PtdIns(4,5)P2 can restore PLD activity inhibited by Clostridium difficile toxin B, which inactivates Rho proteins. Addition of MgATP to permeabilized HEK cells increased basal PLD activity and potentiated PLD stimulation by the stable GTP analogue, guanosine 5'-[gamma-thio]triphosphate (GTP[S]), concomitant with a large increase in PtdIns(4,5)P2. On the other hand, neomycin, which binds to PtdIns(4,5)P2, inhibited basal and GTP[S]-stimulated PLD activities. Addition of PtdIns(4,5)P2 increased PLD activity in HEK cell membranes by 2-3-fold, whereas various other phospholipids were ineffective. Prior treatment of HEK cells with toxin B reduced the level of PtdIns(4,5)P2, measured either in intact cells or in membrane preparations, by about 40%. In membranes of toxin-B-treated cells, basal and GTP[S]-stimulated PLD activities were reduced, when measured with exogenous phosphatidylcholine as enzyme substrate. Inclusion of PtdIns(4,5)P2 with phosphatidylcholine in the substrate vesicles or addition of PtdIns(4,5)P2 fully restored basal and GTP[S]-stimulated PLD activities in membranes of toxin-B-treated cells. In conclusion, the data indicate that PtdIns(4,5)P2 is an essential cofactor for PLD activity in HEK cells and that inhibition of PLD activity by the Rho-inactivating toxin B is apparently caused by depletion of the PLD cofactor, PtdIns(4,5)P2.

Platelet-vessel wall interactions, focal adhesions, and the mechanism of action of endothelial factors.

Endothelial cells produce a variety of vasoactive substances including prostacyclin (PGI2) and endothelium-derived relaxing factor (EDRF/NO) which are potent inhibitors of platelet adhesion/aggregation and vascular smooth muscle cell contraction/proliferation. PGI2 and EDRF elevate cAMP or cGMP, respectively, in vascular cells and other targets. The intracellular effects of cAMP and cGMP in vascular smooth muscle cells and platelets are primarily mediated by the family of cAMP- and cGMP-dependent protein kinases and their substrates. Important effector systems include enzymes, channels and regulatory proteins responsible for the regulation of intracellular Ca++. Other evidence suggests that VASP, a focal adhesion protein phosphorylated in platelets and smooth muscle cells in response to PGI2 and EDRF, is important for the regulation of integrins and cell-matrix interactions.

[A psychiatric survey of a metropolitan district. An analysis of the out-patient-service of the Rheinische Landesklinik Köln-Merheim (author's transl)]. / Psychiatrisch-epidemiologische Patientenstatistik innerhalb einer Grossstadtregion. Eine Untersuchung der Ambulanz der Rheinischen Landesklinik Köln-Merheim.

In the last years there has been an increasing interest in psychiatric surveys as basis of epidemiologic studies and morbidity investigations. Numerous statistics from hospitals, from general psychiarists and general practioners have been referred to and constitute the basis for the 'Enquete zur Lage der Psychiatrie in der Bundesrepublik Deutschland'. The out-patient-service of the Rheinische Landesklinik Köln-Merheim is operating in line with the recommendations of the Enquete committee as one of the first psychiatric establishments in the BRD and as the first in Nordrhein-Westfalen. It consists in a community psychiatric service closely attached to a psychiatric clinic, which is in charge of the overall psychiatric assistance for a municipal district of 500.000 inhabitants. The present study outlines the frequency and categories of psychiatric diagnosis in the psychiatric out-patient-service during a period of 9 months. The case register of ICD underlies the classification of mental diseases. By evaluating a rating scale which had been established for every patient it was possible to demonstrate correlations between diagnosis, age, sex, social status, marital status and nationally. Almost all diagnosis groups demonstrate a prevalence of the female sex except for alcoholism, drug addiction and gerontopsychiatric troubles. Neurotic diseases come at the first place, followed by schizophrenic illnesses. Compared to an evaluation of 1975 there has been an increase of schizophrenia. This fact reveals a modification of concept which puts more stress on after care of ancient in-patients. This group of patients will probably represent the greatest share of out-service patients in future.