The proU locus, which encodes a high-affinity betaine transport system, and the kdp operon, which encodes a potassium transport system, are the principal osmoresponsive genes in Escherichia coli and Salmonella typhimurium. The kdp operon is known to be induced in response to changes in cell turgor. We have investigated the control of proU expression and shown that it differs from that of kdp in a number of fundamental ways. Rather than responding to changes in turgor, proU expression is principally determined by the intracellular accumulation of potassium ions. Potassium and betaine were shown to play distinct osmoprotective roles. Potassium serves as the principal osmoprotectant and is accumulated in response to low-level osmotic stress to restore turgor. As external osmolarity is increased to a level at which the corresponding increase in internal potassium concentrations is potentially deleterious to enzyme function, betaine (when available) is accumulated in preference to potassium. The different mechanisms ofproU and kdp regulation reflect the different physiological roles of these two osmoprotectants.

The mechanism by which N-ethylmaleimide (NEM) elicits potassium efflux from Escherichia coli has been investigated. The critical factor is the formation of specific glutathione metabolites that activate transport systems encoded by the kefB and kefC gene products. Formation of N-ethyl-succinimido-S-glutathione (ESG) leads to the activation of potassium efflux via these transport systems. The addition of dithiothreitol and other reducing agents to cells reverses this process by causing the breakdown of ESG and thus removing the activator of the systems. Chlorodinitrobenzene, p-chloromercuribenzoate and phenylmaleimide provoke similar effects to NEM. lodoacetate, which leads to the formation of S-carboxymethyl-glutathione, does not activate the systems but does prevent the action of NEM. It is concluded that the KefB and KefC systems are gated by glutathione metabolites and that the degree to which they are activated is dependent upon the nature of the substituent on the sulphydryl group.

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The temporal and dose-response relationships of platelet-activating-factor (PAF)-induced changes in the concentrations of cytosolic Ca2+ ([Ca2+]i), Ins(1,4,5)P3 and 1,2-diacylglycerol (DAG) were examined. In addition, phosphorylation of protein kinase C (PKC) substrate (40-47 kDa protein) was determined. In high-dose PAF-activated platelets, all three signal molecules increased rapidly and transiently, with the peak Ins(1,4,5)P3 concentration preceding maximal elevation of [Ca2+]i by 5 s. In low-dose PAF-activated platelets there were large increases in [Ca2+]i and dense-granule release, without any increase in Ins(1,4,5)P3 and DAG or 40-47 kDa protein phosphorylation. Staurosporine, a non-specific PKC inhibitor, produced enhanced elevations in the concentrations of Ins(1,4,5)P3, DAG and thromboxane B2, and the duration of the Ca2+ signal in platelets stimulated with a high dose, but not a low dose, of PAF. These results suggest there are both phospholipase C-dependent and -independent changes in Ca2+ homoeostasis. Endogenously activated PKC regulates the formation of signal molecules.