A tyrosine kinase regulates propofol-induced modulation of the beta-subunit of the GABA(A) receptor and release of intracellular calcium in cortical rat neurones.

Propofol, an intravenous anaesthetic, has been shown to interact with the beta-subunit of the gamma-amino butyric acid(A) (GABA(A)) receptor and also to cause changes in [Ca2+]i. The GABA(A) receptor, a suggested target for anaesthetics, is known to be regulated by kinases. We have investigated if tyrosine kinase is involved in the intracellular signal system used by propofol to cause anaesthesia. We used primary cell cultured neurones from newborn rats, pre-incubated with or without a tyrosine kinase inhibitor before propofol stimulation. The effect of propofol on tyrosine phosphorylation and changes in [Ca2+]i were investigated. Propofol (3 microg mL(-1), 16.8 microM) increased intracellular calcium levels by 122 +/- 34% (mean +/- SEM) when applied to neurones in calcium free medium. This rise in [Ca2+]i was lowered by 68% when the cells were pre-incubated with the tyrosine kinase inhibitor herbimycin A before exposure to propofol (P < 0.05). Propofol caused an increase (33 +/- 10%) in tyrosine phosphorylation, with maximum at 120 s, of the beta-subunit of the GABA(A)-receptor. This tyrosine phosphorylation was decreased after pre-treatment with herbimycin A (44 +/- 7%, P < 0.05), and was not affected by the absence of exogenous calcium in the medium. Tyrosine kinase participates in the propofol signalling system by inducing the release of calcium from intracellular stores and by modulating the beta-subunit of the GABA(A)-receptor.

Inactivations of p16INK4a-alpha, p16INK4a-beta and p15INK4b genes in 2',3'-dideoxycytidine- and 1,3-butadiene-induced murine lymphomas.

The p16INK4a (alpha and beta form) and p15INK4b genes were analysed for homozygous deletion, hypermethylation and point mutation in B6C3F1 mouse lymphomas induced by 2',3'-dideoxycytidine or 1,3-butadiene. Although the p16INK4a-alpha gene appeared normal in DNA from 2',3'-dideoxycytidine-induced lymphomas, Southern analyses revealed homozygous deletions or rearrangements of the p16INK4a-beta and/or p15INK4b genes in four of 16 tumours. Surprisingly, two of these lymphomas showed exclusive deletions of the p16INK4a EIbeta exon. The p15INK4b promoter region was hypermethylated in two additional 2',3'-dideoxycytidine-induced lymphomas. In contrast, homozygous deletions spanning the p16INK4a and p15INK4b loci were observed in only two of 31 1,3-butadiene-induced tumours. Thus, these cyclin dependent kinase inhibitor genes may play a significant role in chemically induced mouse lymphomas and support the contention of tumour suppressor activity for the p19ARF protein encoded by the p16INK4a-beta gene. Different genetic pathways may be involved in the development of these chemically induced tumours since we have previously shown that mutations in p53 and ras genes are common in 1,3-butadiene- but not 2',3'-dideoxycytidine-induced lymphomas.

Leukotriene D4-induced mobilization of intracellular Ca2+ in epithelial cells is critically dependent on activation of the small GTP-binding protein Rho.

We have previously shown that the leukotriene D4 (LTD4)-induced mobilization of intracellular Ca2+ in epithelial cells is mediated by a G-protein that is distinctly different from the pertussis toxin-sensitive G-protein that regulates the subsequent influx of Ca2+. In the present study, we attempted to gain further knowledge about the mechanisms involved in the LTD4-induced mobilization of intracellular Ca2+ in epithelial cells by investigating the effects of compactin, an inhibitor of the isoprenylation pathway, on this signalling event. In cells preincubated with 10 microM compactin for 48 h, the LTD4-induced mobilization of intracellular Ca2+ was reduced by 75% in comparison with control cells. This reduction was reversed by co-administration of mevalonate (1 mM). The effect of compactin occurred regardless of whether or not Ca2+ was present in the extracellular medium, suggesting that isoprenylation must occur before Ca2+ is released from intracellular stores. In accordance with this, we also found that both the LTD4-induced formation of inositol 1,4,5-trisphosphate and the LTD4-induced phosphorylation of phospholipase C gamma 1 (PLC gamma 1) on tyrosine residues were significantly reduced in compactin-pretreated cells. These results open up the possibility that the activation of PLC gamma 1 is related to a molecule that is sensitive to impaired activity of the isoprenylation pathway, such as a small monomeric G-protein. This idea was supported by the observation that Clostridium botulinum C3 exoenzyme-induced inhibition of Rho proteins abolished the LTD4-induced intracellular mobilization of Ca2+. A regulatory role of Rho proteins in the LTD4-induced activation of PLC gamma 1 is unlikely to be indirectly mediated via an effect on the cytoskeleton, since cytochalasin D had no major effect on the LTD4-induced mobilization of Ca2+. Although the mechanism of interaction remains to be elucidated, the present findings indicate an important role of an isoprenylated protein such as Rho in the LTD4-induced Ca2+ signal.

The regulation of leukotriene D4-induced calcium influx in human epithelial cells involves protein tyrosine phosphorylation.

Leukotriene D4 (LTD4) has been found to induce calcium signalling in the intestinal epithelial cell line Int 407, and this action involves the activation of both different GTP-binding proteins (G-proteins) and phospholipase C of the gamma-subtype (PLC-gamma). With this knowledge as the incentive, we investigated the possible regulatory role of protein tyrosine kinase activities in the calcium signalling system of the LTD4 receptor. The tyrosine kinase inhibitors genistein and herbimycin. A both reduced the LTD4-induced calcium signal by 70% when Int 407 cells were stimulated in the presence of extracellular calcium, but had no effect on the signal when the cells were stimulated in a calcium-free medium. In accordance with these findings, pretreatment with a tyrosine kinase inhibitor also blocked thapsigargin-induced cellular influx of calcium. These inhibitors had no effect on the intracellular mobilisation of calcium, which was supported by the findings that LTD4 was able to induce an increase in the tyrosine phosphorylation of PLC-gamma even when one of the tyrosine kinase inhibitors was present. Of possible interest regarding the effect of genistein on LTD4-induced calcium influx is that two major tyrosine phosphorylated protein bands were detected in immunoprecipitates obtained with PLC-gamma antibodies from LTD4-stimulated cells. These proteins, which associate with PLC-gamma, have estimated molecular weights of 84 and 97 kD. Preincubation with genistein completely abolished the LTD4-induced increase in tyrosine phosphorylation of the major 97 kD band, whereas the 84 kD protein band, like the PLC-gamma band, still exhibited an increased phosphorylation of tyrosine residues in response to LTD4. Neither this effect nor any of the other effects of genistein were induced when cells were preincubated with daidzein, an inactive analogue of genistein. The present results suggest that LTD4-induced calcium signalling in epithelial cells involves not only tyrosine phosphorylation of PLC-gamma, but also a tyrosine kinase-dependent step which occurs downstream of PLC-gamma activation and is directly implicated in the regulation of agonist-mediated calcium influx.

A human epithelial cell line, intestine 407, can produce 5-hydroxyeicosatetraenoic acid and leukotriene B4.

The present study was carried out to further characterize the role of non-inflammatory cells in the inflammatory process. More specifically, we have investigated whether human epithelial cells can generate inflammatory lipid mediators via activation of the 5-lipoxygenase pathway. The cells were stimulated with the calcium ionophore A23187 (5 microM) for different periods of time, after which the production of eicosanoids was determined by gradient reverse-phase high performance liquid chromatography (RP-HPLC) and rapid spectral detection, permitting continuous ultraviolet spectroscopy. In both non-prelabeled cells and cells prelabeled with [1-14C]arachidonic acid, cell stimulation for 30 min or more resulted in the production of two important 5-lipoxygenase products: 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4 (LTB4). Stimulation for 15 min or less, however, led solely to the formation of 5-HETE. The identities of 5-HETE and LTB4 were confirmed by HPLC retention times and UV spectra, as well as by gas chromatography-mass spectrometry for 5-HETE and radioimmunoassay for LTB4. It can therefore be concluded that human epithelial cells in general can produce important inflammatory mediators, which suggests that epithelial cells may play a more active role in the inflammatory process than is normally assumed.