Publisher Summary This chapter discusses the role of glutathione (GSH) and glutathione s-transferases in metabolism of chemical carcinogens and other electrophilic agents. GSH is a tripeptide (I) that is present in nearly all living cells and is the most abundant sulfhydryl compound present in animal tissues, mainly in the cytosol. The chapter illustrates the wide range of electrophilic agents, including several known mutagens and carcinogens, which conjugate with GSH, a process usually catalyzed by the GSH S-transferases. This conjugation is probably a protective mechanism and is the initial stage in mercapturic acid biosynthesis for the elimination of foreign compounds from the body. GSH S-transferases provide protection not only by catalyzing the conjugation of a potential toxicant with GSH, but also by preferentially binding, even covalently, that toxicant. The reactive electrophiles that conjugate with GSH also bind to DNA, RNA, and protein and identification of GSH conjugates provide information on the nature of these biologically active intermediates or even their immediate precursors. Thus, the knowledge of the way in which mutagens and carcinogens are metabolized is essential to a better understanding of their mode of action and of the processes for their detoxication.

The role of glutathione and glutathione S-transferases in the metabolism of chemical carcinogens and other electrophilic agents.

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Ventricular myocytes were isolated by means of collagenase and hyaluronidase. Hearts of rats or guinea pigs were perfused in a retrograde manner; bovine ventricular tissue was incubated in from of tissue chunks.




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In “low Ca medium” (aCa=1 μM), the myocytes beat spontaneously. Resting potentials of −4 mV and input resistances of 1 MΩ, associated with a fast decay of aKi, indicated “hyperpermeability” of the cell membrane. Within 2 h, the cells aged: they granulated (swelling of mitochondria), contracted, rounded up and finally disaggregated. When the superfusate was changed to Tyrode solution (3.6 mM CaCl2), 90–98% of those cells responded with the “Ca paradox”. Initially, aCai increased from 0.2 μM to >10 μM, and the sarcomere length (SL) shortened to <1.5 μm. Within the following 3 min aCai renormalized but the contracture was sustained. After another 5 min, the SL shortened to <1.2 μm, but a delay of 1–2 min passed before aCai started to increase towards aCa0. The 2–10% Ca tolerant cells beat spontaneously. Their resting potentials were between −10 and −70 mV, their input resistances between 4 and 20 MΩ. Repolarization by anodal current flow could restore the sodium spike but not the plateau of the action potential.




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Freshly prepared myocytes were pre-incubated in a KB medium composed of 85 mM KCl, 30 mM K2HPO4, 5 mM MgSO4, 5 mM Na2ATP, 5 mM pyruvate, 5 mM succinate, 5 mM β-OH-butyrate, 5 mM creatine, adjusted with KOH to pH 7.2 and with EGTA to pCa 7.5. The minimum time of preincubation was 1 h, the maximum 5 days when the cells were stored at 5°C. The conditioning with the KB medium improved both Ca tolerance and electrophysiology significantly. About 70% of the rod shaped myocytes were Ca tolerant. These cells had resting potentials between −75 and −90 mV, the membrane resistances were around 7 kΩ×cm2. The action potentials showed well pronounced plateaus and lasted for 125 ms (rat), 300 ms (guinea pig) or 400 ms (bovine). Their shape showed the typical species related pecularities.




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We discuss that both ageing and Ca intolerance of the un-conditioned cells result from Ca overload through the hyperpermeable membrane, and that Ca overload causes ATP depletion via mitochondrial disfunction. The constituents of the KB medium may act to prevent these processes.

Calcium tolerant ventricular myocytes prepared by preincubation in a "KB medium".

This review is aimed at providing readers with a comprehensive reference article about the distribution and function of P2 receptors in all the organs, tissues, and cells in the body. Each section provides an account of the early history of purinergic signaling in the organ/cell up to 1994, then summarizes subsequent evidence for the presence of P2X and P2Y receptor subtype mRNA and proteins as well as functional data, all fully referenced. A section is included describing the plasticity of expression of P2 receptors during development and aging as well as in various pathophysiological conditions. Finally, there is some discussion of possible future developments in the purinergic signaling field.

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Cellular distribution and functions of P2 receptor subtypes in different systems.

https://www.cell.com/biophysj/pdf/S0006-3495(02)75621-X.pdf

Two-Photon Fluorescence Spectroscopy and Microscopy of NAD(P)H and Flavoprotein

Adenosine is released from the myocardium in response to a decrease in the oxygen supply/demand ratio, as is seen in myocardial ischemia; its protective role is manifested by coronary and collateral vessel vasodilation that increase oxygen supply and by multiple effects that act in concert to decrease myocardial oxygen demand (i.e., negative inotropism, chronotropism, and dromotropism). During periods of oxygen deprivation, adenosine enhances energy production via increased glycolytic flux and can act as a substrate for purine salvage to restore cellular energy charge during reperfusion. Adenosine limits the degree of vascular injury during ischemia and reperfusion by inhibition of oxygen radical release from activated neutrophils, thereby preventing endothelial cell damage, and by inhibition of platelet aggregation. These effects help to preserve endothelial cell function and microvascular perfusion. Long-term exposure to adenosine may also induce coronary angiogenesis.

Jocelyn W. Dow

Papers

Review: Isolated cardiac myocytes: I Preparation of adult myocytes and their homology with the intact tissue

A possible protective role for sulphydryl compounds in acute alcoholic liver injury

Review: Isolated cardiac myocytes: II Functional aspects of mature cells

Accumulation and salvage of adenosine and inosine by isolated mature cardiac myocytes.

Uptake of bepridil into isolated ventricular myocytes. Retention by actin.