The extracellular cardiac purine metabolome in sepsis.

The total cardiac purine metabolome includes all of the adenine and guanine nucleoside and nucleosides and related molecules involved throughout the intracellular and extracellular compartments and various cell types in the heart. In considering purines as molecules involved in autocrine and paracrine communication, effective interstitial concentrations of the nucleoside adenosine, or purine metabolites, are of greatest interest. These molecules arise from the complex interactions between cardiac-specific cell types, including fibroblasts and myocytes, and noncardiac cells, such as tissue-resident macrophages and other immune cells that have vascular access. In the interstitial environment, adenosine can regulate vascular resistance, contractile function, and immunochemical interactions. The breakdown of purines can produce reactive oxygen species that also influence autocrine and paracrine interactions. A central enzyme in this paradigm, adenosine deaminase, is a pivotal molecule in regulating the balance between pro-inflammatory and anti-inflammatory signaling cascades. A new role for adenosine deaminase as an allosteric regulator of relevant membrane proteins has yet to be explored in the heart.

Adding bupivacaine to high-potassium cardioplegia improves function and reduces cellular damage of rat isolated hearts after prolonged, cold storage.

BACKGROUND: Bupivacaine retards myocardial acidosis during ischemia. The authors measured function of rat isolated hearts after prolonged storage to determine whether bupivacaine improves cardiac protection compared with standard cardioplegia alone. METHODS: After measuring cardiac function on a Langendorff apparatus, hearts were perfused with cardioplegia alone (controls), cardioplegia containing 500 microm bupivacaine, or cardioplegia containing 2 mm lidocaine; were stored at 4 degrees C for 12 h; and were then reperfused. Heart rate and left ventricular developed pressures were measured for 60 min. Maximum positive rate of change in ventricular pressure, oxygen consumption, and lactate dehydrogenase release were also measured. RESULTS: All bupivacaine-treated, four of five lidocaine-treated, and no control hearts beat throughout the 60-min recovery period. Mean values of heart rate, left ventricular developed pressure, maximum positive rate of change in ventricular pressure, rate-pressure product, and efficiency in bupivacaine-treated hearts exceeded those of the control group (P < 0.001 at 60 min for all). Mean values of the lidocaine group were intermediate. Oxygen consumption of the control group exceeded the other groups early in recovery, but not at later times. Lactate dehydrogenase release from the bupivacaine group was less than that from the control group (P < 0.001) but did not differ from baseline. CONCLUSIONS: Adding bupivacaine to a depolarizing cardioplegia solution reduces cell damage and improves cardiac function after prolonged storage. Metabolic inhibition may contribute to this phenomenon, which is not entirely explained by sodium channel blockade.

Advances in understanding adenosine as a plurisystem modulator in sepsis and the systemic inflammatory response syndrome (SIRS).

Adenosine is a ubiquitous molecule that influences every physiological system studied thus far. In this review, we consider the influence of this purine nucleoside on some of the physiological systems affected during sepsis and SIRS. In the control of perfusion and cardiac output distribution, endogenous adenosine appears to play an important role in regulating perfusion in various vascular beds. Some of this control is mediated by stimulation of adenylyl cyclase, while part occurs by stimulating the production of nitric oxide. In the heart, adenosine may act as an inhibitory modulator of TNF-alpha expression. With regard to innate immune responses the effects of adenosine vary considerably, and are complex. However, the dominant responses relevant to SIRS indicate attenuation of inflammatory responses. Many of the effects of adenosine may also involve modulating oxyradical-mediated response. This occurs via increased oxyradical production via adenosine degradation (xanthine oxidase pathway), or limiting inflammatory oxyradical generation. Attempts to exploit the beneficial responses to adenosine have met with some success, and are considered here.

Adenosine attenuates C-terminal but not N-terminal proteolysis of cTnI during cardioplegic arrest.

BACKGROUND: Specific site proteolysis and loss of troponin I (TnI) during myocardial ischemic events can contribute to myocardial dysfunction. Adenosine supplementation of cardioplegic solutions results in improved functional preservation of the heart. We investigated the effect of adenosine on N-terminal and C-terminal proteolysis of TnI in the heart. MATERIALS AND METHODS: Hearts from male Sprague-Dawley rats were isolated and perfused at a constant pressure. Cardioplegic arrest (St. Thomas #2 +/- 100 microm adenosine) was induced and hearts frozen at various times during the arrest. Antibodies directed against specific regions of TnI were used to visualize TnI in whole heart homogentates, as well as from cellular fractions, using western blot analysis. RESULTS: Cardioplegic arrest alone resulted in early N-terminal proteolysis of TnI, followed by later loss of sequences from the C-terminal end of the molecule. In addition, secondary protein bands that were immunoreactive to amino acid sequences centrally located on the TnI molecule were observed. There was also evidence of dissociation of TnI from the other myofibrillar proteins. The supplementation of cardioplegic solution with adenosine significantly attenuated the late C-terminal proteolytic degradation of TnI and its apparent dissociation from myofibrils proteins but had no effect on the early N-terminal proteolysis associated with cardioplegic arrest. CONCLUSIONS: These data may provide an explanation for partial protection against postarrest myocardial dysfunction provided by adenosine.

Subcellular distribution of protein kinase C isozymes during cardioplegic arrest.

BACKGROUND: On the basis of the hypothesis that cardioplegia-associated myocardial depression was due to activation of protein kinase C, we examined whether specific protein kinase C isozymes would translocate to a cellular fraction containing myofilaments. METHODS: Isolated rat hearts were perfused with Krebs-Ringer bicarbonate buffer for 30 minutes and arrested with 4 degrees C St Thomas No. 2 cardioplegic solution for 0 to 120 minutes (n = 5 per group). The 3 fractions of the left ventricle tissue represented the myofibrillar/nuclear fraction (P1), membranes (P2), and cytosol (supernatant). The distributions of protein kinase C isozymes alpha, delta, epsilon, and eta were examined after separation by electrophoresis, immunoblotting/chemiluminescence, and densitometry. RESULTS: A significant increase in protein kinase C-delta in the P1 fraction was detected after 5 minutes of cardioplegic arrest and remained increased for 60 minutes. Increases in P1 protein kinase C-alpha and -epsilon were seen transiently at 5 minutes, and protein kinase C-epsilon demonstrated a secondary increase in P1 at 30 to 60 minutes. There was also a significant relative increase in protein kinase C-alpha and protein kinase C-delta in the P2 fraction after 60 minutes of cardioplegia. CONCLUSIONS: These data are consistent with our hypothesis that activation of protein kinase C isozymes is associated with altered myofilament function after cardioplegic arrest.

Therapeutic potential for transient inhibition of adenosine deaminase in systemic inflammatory response syndrome.

OBJECTIVE: We sought to determine the potential usefulness of 2'-deoxycoformycin (pentostatin), an inhibitor of adenosine deaminase, as a postinsult, or prophylactic treatment for systemic inflammatory response syndrome resulting from fecal peritonitis. DESIGN: Prospective, randomized, controlled experiment. SETTING: Small animal basic science laboratory. SUBJECTS: Male Spague-Dawley rats, weighing 300 to 350 g. INTERVENTIONS: Rats with fecal peritonitis (intraperitoneal cecal slurry) were treated with 1 mg/kg pentostatin intraperitoneally 24 hrs before, or intravenously when signs of illness presented (2 hrs after induction of peritonitis). Signs of illness included tachycardia, tachypnea, and leukopenia. All rats received 50 mL/kg 0.9% saline resuscitative fluid at 2 hrs. MEASUREMENTS AND MAIN RESULTS: Survival to day 6 was 100% in nonseptic sham rats, but 33% in untreated septic rats. In rats given pentostatin either 2 hrs after the insult, or 24 hrs before the insult, 6-day survival improved to 81% and 78%, respectively. Histology revealed diffuse peritonitis, and evidence of systemic inflammatory response syndrome, including local and distant site vascular damage and leukocyte activation. These responses to the septic challenge were abrogated by pentostatin treatment. Return of significant amount of tissue adenosine deaminase activity by 24 hrs and later recovery of white blood cell counts argue against any potential for inappropriate immunosuppression by pentostatin. CONCLUSIONS: These data indicate that the novel use of pentostatin to prevent systemic inflammatory response syndrome secondary to fecal peritonitis shows uncommon promise as a therapeutic tool. All indices of systemic inflammatory response syndrome were abrogated and survival improved when pentostatin was not given until after signs of the illness became manifest. Because protection was afforded with treatment 24 hrs in advance of the inciting insult, pentostatin also has the unique potential for use as a true prophylactic agent.

Adenosine deaminase inhibition attenuates microvascular dysfunction and improves survival in sepsis.

The ability of increased endogenous adenosine to mitigate microvascular derangements in sepsis was studied. Pentostatin (2'-deoxycoformycin), an inhibitor of adenosine deaminase, was administered to mice immediately after induction of sepsis by cecal ligation and puncture. Intravital video microscopy of cremasteric postcapillary venules was performed. Leukocyte rolling and adhesion were significantly increased in septic mice compared with control mice. Treatment of septic mice with pentostatin significantly decreased leukocyte rolling and adhesion (6.02 +/- 0.09 versus 1.72 +/- 0.12 rolling cells/min, 2.07 +/- 0.04 versus 0.62 +/- 0.05 adherent cells/100 microm per minute; p < 0.001). Albumin leakage (ratio) was significantly attenuated in septic animals treated with pentostatin (0.42 +/- 0.05 versus 0.21 +/- 0.04; p < 0.01). Circulating levels of interleukin-6, tumor necrosis factor-alpha, and soluble tumor necrosis factor type II receptor were decreased in septic mice treated with pentostatin. Survival was significantly improved at 48 hours in mice treated with pentostatin. These results suggest an important role for adenosine in modulating both leukocyte-dependent and -independent mechanisms of endothelial injury in sepsis. Exploiting the advantageous action of endogenous adenosine represents a potentially useful and novel therapeutic approach for the treatment of sepsis.

Inhibiting adenosine deaminase modulates the systemic inflammatory response syndrome in endotoxemia and sepsis.

By pharmacological manipulation of endogenous adenosine, using chemically distinct methods, we tested the hypothesis that endogenous adenosine tempers proinflammatory cytokine responses and oxyradical-mediated tissue damage during endotoxemia and sepsis. Rats were pretreated with varying doses of pentostatin (PNT; adenosine deaminase inhibitor) or 8-sulfophenyltheophylline (8-SPT; adenosine receptor antagonist) and then received either E. coli endotoxin (lipopolysaccharide; 0.01 or 2.0 mg/kg) or a slurry of cecal matter in 5% dextrose in water (200 mg/kg). Resultant levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-10 were measured in serum and in liver and spleen. Untreated, 2 mg/kg lipopolysaccharide elevated serum TNF-alpha, IL-1beta, and IL-10. PNT dose dependently attenuated, without ablating, the elevation in serum TNF-alpha and IL-1beta and raised liver and spleen IL-10. PNT also attenuated elevation of TNF-alpha in serum, liver, and spleen at 4 and 24 h after sepsis induction, and 8-SPT resulted in higher proinflammatory cytokines. Modulating endogenous adenosine was also effective in exacerbated (8-SPT) or diminished (PNT) tissue peroxidation. Survival from sepsis was also improved when PNT was used as a posttreatment. These data indicate that endogenous adenosine is an important modulatory component of systemic inflammatory response syndromes. These data also indicate that inhibition of adenosine deaminase may be a novel and viable therapeutic approach to managing the systemic inflammatory response syndrome without ablating important physiological functions.