Yoram Oron

Stable cholinergic-muscarinic inhibition of rat parotid adenylate cyclase

1. Introduction There are numerous reports describing cY-adrener- gic and cholinergic inhibition of CAMP accumulation in various tissues [ l-101 . The rat parotid gland pro- vides an excellent model system for the elucidation of inter-receptor interactions. The stimulation of a-adrenergic, /3-adrenergic and cholinergic-muscarinic receptors has been well characterized pharmacol- ogically, biochemically and morphologically and has been integrated into an overall scheme in [ 11 ,121. Cholinergic-muscarinic and a-adrenergic inhibition of CAMP accumulation, stimulated by activation of /3-adrenergic receptors in the rat parotid has been reported [13-IS]. Recent data from our laboratory demonstrated a different mechanism of action for the cholinergic and cu-adrenergic effect and also indicated adenylate cyclase as the target enzyme [ 16,171 . In the present communication we report a stable inhibi- tion of parotid adenylate cyclase in homogenates and washed membrane preparations following the ex- posure of tissue slices to carbamylcholine. 2. Experimental methods Male Wistar rats (120-200 g) were used through- out. Rats were fasted overnight prior to sacrifice. The

Concanavalin A-stimulated Ca2+ uptake in rat splenocytes.

SummaryCommercially available concanavalin A binds Ca2+ with high apparent affinity. In order to dissociate concanavalin A stimulated Ca2+ uptake (defined as an increased association of 45Ca2+ with cells) in rat splenocytes and Ca2+ binding to cell-bound concanavalin A, conditions were developed to remove more than 75% of the bound concanavalin A. Under these conditions concanavalin A treated cells showed a considerable increase in 45Ca2+ uptake over control. The concanavalin A stimulated uptake of 45Ca2+ occurred within minutes, and required concentrations of concanavalin A which promoted [3H]thymidine uptake into these cells. Succinyl concanavalin A was less potent in promoting Ca2+ uptake than concanavalin A. Sodium periodate inhibited Ca2+ uptake at concentrations which promoted 3H-thymidine incorporation into splenocytes.It is concluded that con canavalin A promotes Ca2+ uptake which is not due to binding of 45Ca2+ to concanavalin A. Although the concanavalin A-promoted Ca2+ uptake occurs at lectin concentrations that cause lymphocyte proliferation as measured by 3H-thymidine incorporation, the role of Ca2+ in this event remains unclear.

Kaposi's Sarcoma-Associated Herpesvirus-G Protein-Coupled Receptor-Expressing Endothelial Cells Exhibit Reduced Migration and Stimulated Chemotaxis by Chemokine Inverse Agonists

A constitutively active G protein-coupled receptor (GPCR) encoded by Kaposis sarcoma-associated herpesvirus (human herpesvirus-8) (KSHV) is expressed in endothelial (spindle) cells of Kaposis sarcoma lesions. In this study, we report novel effects of basal signaling by this receptor and of inverse agonist chemokines on migration of KSHV-GPCR-expressing mouse lung endothelial cells. We show that basal signaling by KSHV-GPCR inhibits migration of endothelial cells in two systems, movement through porous filters and in vitro wound closure. Naturally occurring chemokines, interferon γ-inducible protein-10 and stromal-derived factor-1, which act as inverse agonists at KSHV-GPCR, abrogate the inhibition of migration and stimulate directed migration (or chemotaxis) of these cells. Thus, the expression of KSHV-GPCR may allow infected endothelial cells in situ to remain in a localized environment or to directionally migrate along a gradient of specific chemokines that are inverse agonists at KSHV-GPCR.

A modified rapid filtration assay of glycogen synthase.

The filter paper assay of glycogen synthase according to Thomas et al.(J. A. Thomas, K. K. Schlender, and J. Larner, 1968, Anal. Biochem.25, 486–489) has been modified to obtain results in a significantly shorter period of time with increased sensitivity and no loss in accuracy. The modified method is based on filtration on glass-fiber filter disks using a multiple filtration apparatus. The assay was examined on glycogen synthase activity present in muscle extracts and was found to be superior to the original assay procedure as regards reproducibility and time required for processing samples. The new method has been used in our laboratory for over 6 months.

Proteinase-activated receptors differentially modulate in vitro invasion of human pancreatic adenocarcinoma PANC-1 cells in correlation with changes in the expression of CDC42 protein.

Objectives Proteinase-activated receptor-1 (PAR-1) and PAR-2 have been associated with increased invasiveness and metastasis in human malignancies. The role of PAR-3 has been less investigated. We examined the role of PARs in a human pancreatic adenocarcinoma PANC-1 cell line phenotype in vitro. Methods We knocked down PAR-1, PAR-2, or PAR-3, whereas empty vector-infected cells served as controls. Specific peptide agonists of PARs were used to stimulate the receptors. In vitro assays of colony formation, migration, and invasion were used to characterize the phenotypes, and Western analysis was used to follow cell division control protein 42 homolog (CDC42) expression. Results PAR-1 and PAR-2 knockdowns (KDs) were markedly less, whereas PAR-3 KDs were robustly more migratory and invasive than the controls. Stimulation of PAR-1 or PAR-2 by their peptide agonists increased, whereas PAR-3 agonist reduced the invasion of the control cells. Knockdowns of all three PARs exhibited changes in the expression of CDC42, which correlated with the changes in their invasion. Conversely, stimulation of vector-control cells with PAR-1 or PAR-2 agonists enhanced, whereas PAR-3 agonist reduced the expression of CDC42. In the respective KD cells, the effects of the agonists were abrogated. Conclusion The expression and/or activation of PARs is linked to the invasiveness of PANC-1 cells in vitro, probably via modulation of the expression of CDC42.

Insulin action in intact mouse diaphragm II.

SummaryThe incubation of intact mouse diaphragms with insulin caused a dose and time dependent increase in the independent activity of glycogen synthase in tissue extracts. 2-deoxyglucose (2–10 mm) alone markedly stimulated the conversion of glycogen synthase to the independent activity under conditions in which tissue ATP concentrations were not affected. The incubation of diaphragms with both insulin and 2-deoxyglucose resulted in a greater than additive effect. Insulin stimulated the uptake of 2-deoxyglucose into mouse diaphragms, accumulating as 2-deoxyglucose-6-phosphate. The accumulation of 2-deoxyglucose-6-phosphate correlated well with the increase in the independent activity of glycogen synthase and with the activation of glycogen synthase phosphatase in tissue extracts. The uptake of 3-0-methyl glucose was also markedly stimulated by insulin, without affecting the activity of glycogen synthase. Both glucose-6-phosphate and 2-deoxyglucose-6-phosphate stimulated the activation of endogenous glycogen synthase phosphatase activity in muscle homogenates. We conclude that insulin, in addition to its effects in the absence of exogenous sugars, increases the independent activity of glycogen synthase through increased sugar transport resulting in increased concentrations of sugar-phosphates which promote the activity of glycogen synthase phosphatase.

Serum Deprivation Induces Glucose Response and Intercellular Coupling in Human Pancreatic Adenocarcinoma Panc-1 Cells

Objective This study aimed to investigate whether the previously described differentiating islet-like aggregates of human pancreatic adenocarcinoma cells (PANC-1) develop glucose response and exhibit intercellular communication. Methods Fura 2–loaded PANC-1 cells in serum-free medium were assayed for changes in cytosolic free calcium ([Ca]i) induced by depolarization, tolbutamide inhibition of K(ATP) channels, or glucose. Dye transfer, assayed by confocal microscopy or by FACS, was used to detect intercellular communication. Changes in messenger RNA (mRNA) expression of genes of interest were assessed by quantitative real-time polymerase chain reaction. Proliferation was assayed by the MTT method. Results Serum-deprived PANC-1 cell aggregates developed [Ca]i response to KCl, tolbutamide, or glucose. These responses were accompanied by 5-fold increase in glucokinase mRNA level and, to a lesser extent, of mRNAs for K(ATP) and L-type calcium channels, as well as increase in mRNA levels of glucagon and somatostatin. Trypsin, a proteinase-activated receptor 2 agonist previously shown to enhance aggregation, modestly improved [Ca]i response to glucose. Glucose-induced coordinated [Ca]i oscillations and dye transfer demonstrated the emergence of intercellular communication. Conclusions These findings suggest that PANC-1 cells, a pancreatic adenocarcinoma cell line, can be induced to express a differentiated phenotype in which cells exhibit response to glucose and form a functional syncytium similar to those observed in pancreatic islets.

Nuclear glycogen synthase--an artifact of preparation.

Nuclear deposits of glycogen, are usually related to pathological conditions. These include human hepatocytes following prednisolone treatment [ 11, in liver biopsies obtained in cases of hepatitis [2,3], in Novikoff hepatoma cells [4] and in Erlich ascites [5-81 where glycogen synthase was reported in purified nuclei [9] and the accumulation of nuclear glycogen demonstrated by electron microscopy and autoradiography [lo]. In a limited number of reports an abnormal deposition of glycogen in the nucleus was linked with diabetes [ 111. We have examined the activity of glycogen synthase in liver nuclei of normal and diabetic rats. Here we demonstrate low levels of nuclei-associated activity of glycogen synthase. The electron-microscopic data indicate that glycogen synthase activity associated with liver nuclei is most probably an artifact of preparation.