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It has been shown that infusion of ATP-MgCl2 proved beneficial in the treatment of shock; however, it is not known whether this effect is due to improvement in the microcirculation or direct provision of energy or a combination of the above or other effects. To elucidate the mechanism of the salutary effect of ATP-MgCl2, we have now examined the in vitro uptake of ATP by liver and kidney of animals in shock. Rats were bled to a mean arterial pressure of 40 Torr and so maintained for 2 hrs. After the rats were killed, liver and kidney were removed and slices of tissue (0.3-0.5 mm thick) were incubated for 1 h in 1.0 ml of Krebs-HCO3 buffer containing 10 mM glucose, 5 mM MgCl2, and 5 mM [8-14C]ATP or 5 mM [8-14C]ADP, or 5 mM [8-14C]AMP, or 5 mM [8-14C]adenosine in 95% O2-5% CO2 and then homogenized. Tissue and medium samples were subjected to electrophoresis to separate and measure the various nucleotides. The uptake of [14C]ATP but not that of [14C]ADP or [14C]adenosine by liver and kidney slices from animals in shock was 2.5 times greater than the corresponding uptake by control slices. Thus, the beneficial effect of ATP-MgCl2 in shock could be due to provision of energy directly to tissue in which ATP levels were lowered.

Isolated rat kidneys were subjected to 45, 60, and 90 min of normothermic ischemia. Kidneys reperfused with a saline solution after ischemia had very low initial whole kidney inulin clearances (Cin), urine flow rates (V), renal perfusate flow rates (RPF), and fractional absorption of sodium values that remained depressed throughout the 110 min of reperfusion. Kidneys subjected to 45 min of warm ischemia and reperfused with a solution containing 0.3 mM ATP-MgCl2 had higher initial Cin and attained complete recovery of Cin by 75 min of perfusion. Fractional absorption of sodium and water were also improved, and RPF, V, and tissue ATP levels returned to control values after 110 min of perfusion. Kidneys subjected to 60 min of ischemia and reperfused with a solution containing 0.3 mM ATP-MgCl2 also had higher initial Cin values and attained 50% recovery of Cin after 110 min of perfusion. RPF also improved, and V reached control levels. Kidneys subjected to 90 min ischemia showed no improvement in renal function even with 2 mM ATP-MgCl2. Because, even in isolated kidneys, administration of ATP-MgCl2 after 45 and 60 min of ischemia results in an early recovery of renal function, the beneficial effects of ATP-MgCl2 are exerted directly on the kidney and are not mediated via systemic effects.

Gluconeogenic and oxidative capabilities with lactate as a substrate were studied in perfused livers isolated from rats in late sepsis. Glucose release in the presence of 5 mM lactate was significantly depressed in livers from septic rats. When gluconeogenesis was stimulated by phenylephrine, livers from septic rats exhibited both a decreased sensitivity and lower maximal rate of glucose release when compared with livers from sham-operated rats. Oxygen consumption (VO2) by perfused livers from septic rats was also depressed under the above conditions. The addition of lysine in concentrations greater than 0.5 mM restored glucose production in livers from septic rats to a rate not different from sham-operated controls but did not restore VO2. However, inclusion of lysine (5 mM) in the perfusate was not able to restore sensitivity to stimulation by phenylephrine in livers from septic rats. Although hepatic ATP levels were depressed in sepsis, the decrease was not sufficient to explain the decreased rates of glucose production. We conclude from these results that primary cellular defects in gluconeogenic and oxidative capabilities occur during sepsis that are independent of inadequate perfusion.

The aims of this study were to determine 1) the effects of anoxia and reoxygenation on electrolytes and ATP content of isolated hepatocytes and 2) whether exogenous ATP-MgCl2 has any beneficial effects on the cellular alterations that are produced during the reoxygenation period. After 90 min anoxia, intracellular Na and inorganic phosphate (Pi) increased, K and ATP decreased, whereas Ca and Mg did not change. After 60 min of reoxygenation, intracellular Mg and Na levels decreased, Ca increased, and Pi levels returned to normal, but ATP levels remained low. These results suggest that the plasma membrane is relatively impermeable to divalent ions such as Ca2+, Mg2+, and HPO2-4 during anoxia but becomes permeable to them after reoxygenation. When anoxic cells were treated with ATP-MgCl2 during reoxygenation, intracellular ATP and Mg levels increased, but accumulation of Ca and Pi was also observed. Thus enhancement of Mg transport and specific stimulation of Ca-Pi sequestration occurs with ATP-MgCl2 treatment. Na and K levels exhibited biphasic reciprocal dose-response changes to ATP-MgCl2 treatment; i.e., 0.1 mM ATP-MgCl2 (low-dose) treatment increased K and decreased Na, whereas 1 mM ATP-MgCl2 (high-dose) treatment decreased K and increased Na. These results indicate that although addition of a single high-dose ATP-MgCl2 was deleterious over a period of 60 min, low-dose ATP-MgCl2 accelerates the recovery of postanoxic cellular electrolyte homeostasis.

In this study we investigated in vivo changes in hepatic cellular electrolytes and resting transmembrane potentials (Em) during hemorrhagic shock. Hepatic Na-K transport and cell volume regulation were assessed in vitro. Rats were bled and the ensuing hypotension (40 mmHg) was maintained by returning 25-30% (intermediate-shock, IS) or 55-60% (late-shock, LS) of the shed blood. We resuscitated IS rats by reinfusion of all of the remaining shed blood and Ringer's lactate solution. Hepatic cellular Na and Cl increased and K decreased progressively with shock. Resuscitation of IS rats restored cell K and Cl but not Na to preshock levels. Em decreased from the control average value of -40 (mV) to -31 in IS and -19 in LS. Em was partially restored (-36 mV) after resuscitation. We evaluated changes in relative membrane permeability to Na and K (PNa/PK) with shock by assuming Em either to be a Na-K exchange diffusion potential or due to an unequally coupled movement of Na and K. These evaluations show a lack of effect of shock (IS, with or without resuscitation) on PNa/PK. Our observations are compatible with failure of an electrogenic Na pump in shock. This may be related to loss of hepatic cell volume regulation in shock.

Hepatic ischemia followed by reflow results in a myriad of metabolic and circulatory derangements that may eventually result in liver failure and death. In the present experiments we have used the technique of intravital fluorescence microscopy with fluoroscein isothiocyanate conjugated to bovine serum albumin as the intravascular fluorochrome to study the effects of ischemia and reperfusion on the hepatic microcirculation in vivo. Total hepatic ischemia was produced for 90 min to the left and median lobes of pentobarbital-anesthetized rats. After ischemia, reflow was allowed for 2 h. Three groups were studied: sham-ischemia controls and rats treated with either 1 ml saline or 12.5 mumol ATP-MgCl2 in 1-ml volume at the beginning of reflow. Although control rats exhibited stable microcirculation throughout the experiment, in saline-treated rats the number of perfused centrilobular areas and perfused sinusoids per unit area on the surface of the liver was decreased to approximately 50 and 40% of sham-ischemia controls, respectively. However, in rats treated with ATP-MgCl2 the density of perfused centrilobular areas and perfused sinusoids was 86 and 80% of sham-ischemia controls, respectively. From these results we conclude that intravital fluorescence microscopy is a potentially valuable method for the study of the hepatic microcirculation in vivo. Moreover, the results with ATP-MgCl2 treatment indicate that its effect on the microcirculation is a major factor in its beneficial effects on hepatic function after ischemia and reflow.

ATP-MgCl2 X administration had been shown to accelerate the recovery of renal function following warm ischemia. However, since the major breakdown product of ATP is adenosine, the relative contribution of ATP vs. adenosine in improving renal function following ischemia remains to be determined. To study this, kidneys were subjected to 45 min of normothermic ischemia and then perfused at 100 mmHg with oxygenated Krebs-HCO3 buffer containing albumin, [3H]inulin, substrates, and either 0.3 mM ATP-MgCl2 or adenosine-MgCl2 for 110 min. Perfusate and timed urine samples were collected and analyzed for radioactivity and [Na+]. The functional parameters indicated that although adenosine-MgCl2 treatment provided a transient improvement, it failed to provide a sustained improvement in renal function or attain control values compared with ATP-MgCl2 treatment. Thus, the salutary effects of ATP-MgCl2 following warm ischemia in the kidney are not mediated by adenosine.