Macrophages are a source of extracellular adenosine deaminase-2 during inflammatory responses.

Serum activity of the adenosine deaminase (ADA) isozyme, ADA2, has been reported to be elevated during various disease states. Macrophages have been suggested as the cellular source of extracellular ADA activity because they are one of the only cell types in which intracellular ADA2 activity has been measured, but extracellular secretion has never been demonstrated. Rat primary peritoneal macrophages (PPMs) and peripheral blood monocytes (PBMs) were harvested and incubated for 18 h in RPMI supplemented with horse serum. PPM and PBM lysates were assayed for intracellular ADA activity (ammonia production). In vitro and in vivo extracellular ADA activities were measured in media and rat serum, respectively. Activity of ADA1 was confirmed by selective inhibition with erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA). ADA2 activity was inhibited by 2'-deoxycoformcin only, and was increased at a low pH (6.5). Activity of both ADA isozymes was found in PPMs and PBMs, and their media. In a separate group of rats, peritonitis was induced by ip insertion of 400 mg/kg caecal slurry. PPMs were harvested 24 h later and incubated for 18 h. In PPMs from rats with peritonitis both isozymes were elevated by a similar proportion. In contrast, media from these PPMs had a lower ADA1 and a higher ADA2 activity compared to PPMs from nonseptic rats. This resulted in a greater proportion of ADA2 in media. The isozyme proportions in serum from septic rats more closely resembled that of the PPM media. The response of PBM was small relative to that of PPM. These results suggest that macrophages are a significant source of extracellular ADA isozymes, the activity of which increases during an inflammatory response. Because extracellular isozymes profiles differ from cellular concentrations, the data also suggest differential release of each isozyme from PPMs.

Interdependence of adenosine and nitric oxide in hepato-splanchnic circulation during sepsis.

BACKGROUND: We tested the hypothesis that some of the maintenance of resting, regional hepato-splanchnic perfusion that is mediated by endogenous adenosine (ADO) during sepsis is interdependent with nitric oxide (NO). MATERIALS AND METHODS: Twenty-four hours after sepsis/sham induction, rats were divided into two groups. Group 1 received a 10-min infusion of the ADO antagonist 8-sulfophenyltheophylline (8-SPT; 0.9 mg/kg x min), followed by 10 min of 8-SPT plus L-NAME (0.5 mg/kg x min). Group 2 received L-NAME first followed by 8-SPT in the presence of L-NAME (all groups: n = 6-10). Hemodynamic and regional hepato-splanchnic blood flow measurements were made prior to infusions, 10 min after initiation of each single agent infusion, and again after double agent infusion. RESULTS: Twenty-four hours after sepsis hepato-splanchnic blood flow was significantly elevated, compared to nonseptic rats. Both ADO receptor blockade alone and NOS inhibition alone decreased total hepato-splanchnic blood flow to nonseptic values. Decreases in small intestinal and cecal blood flow accounted for the majority of this decrease, but decreased hepatic arterial perfusion contributed as well. No further alterations were seen when 8-SPT was infused in the presence of L-NAME, nor when L-NAME was infused in the presence of 8-SPT. CONCLUSIONS: These data indicate that there is significant interdependence of endogenous NO and ADO in maintaining resting small bowel, cecal, and hepatic arterial perfusion during sepsis. The lack of responses in other regions of the hepato-splanchnic circulation demonstrate regional specificity of this ADO-NO interdependence.

Central versus peripheral mediation of naloxone's perfusion effects in endotoxic rats.

Opioid receptor antagonists can act centrally and peripherally. It is unclear if these 2 pathways differentially mediate the perfusion-associated effects of opioid antagonism during endotoxemia. Male, Sprague-Dawley rats (340-390 g) were surgically prepared with left ventricular, tail artery, and jugular vein catheters 24 h before experiments were begun. Conscious, unrestrained rats were challenged with Escherichia coli lipopolysaccharide (LPS; 2 mg/kg/hr over 30 min) infusion. Measurements of regional blood flows were made using radioactive microspheres prior to (baseline), and at 60 and 120 min after LPS infusion. Saline (1 mL/kg bolus + 0.5 mL/kg/h infusion), naloxone (Nlx; 4 mg/kg bolus + 2 mg/kg/h infusion), or naloxone methyl bromide (Nlx-mb; 4.64 mg/kg, bolus + 2.32 mg/kg/h infusion) were administered 40 min after LPS infusion was begun. Nlx-mb does not cross the blood-brain barrier, and was thus used to differentiate central from peripherally mediated responses. At the end of each experiment, blood samples were collected for determination of ET-1 and nitric oxide metabolites (NOx = NO3 + NO2) using enzyme-linked immunosorbent assay (ELISA) and Griess reaction methods, respectively. Endotoxemia produced a significant decrease in cardiac output and an increase in systemic vascular resistance. Treatment with Nlx or Nlx-mb significantly attenuated the endotoxin-induced elevation in systemic vascular resistance and the decrease in cardiac output at 60 min after induction of endotoxemia compared with their respective baseline values. Nlx and Nlx-mb also attenuated the endotoxin-induced increases in hepatic portal and skeletal vascular resistances. These observations suggested that the ameliorative effect of Nlx on endotoxemia-induced regional vascular resistance alterations was mediated via peripheral opioid receptor mechanisms. However, although Nlx attenuated the endotoxin-induced decreases in the blood flow to the stomach and pancreas, Nlx-mb attenuated the endotoxin-induced decreases in the blood flow to the small intestine and cecum, in addition to the pancreas and, to some extent, the stomach. As such, separate central and peripherally mediated actions of opioid receptor antagonism were indicated. Nlx also resulted in an increase in the plasma levels of ET-1 only, whereas Nlx-mb increased the plasma levels of ET-1 and NOx. These observations suggest that separate central and peripheral effects of opioids during endotoxemia play a role in the associated circulatory alterations, and may differentially affect the release and/or synthesis of vasoactive mediators that might be related to their varied hepatosplanchnic vascular response during endotoxemia.

Adenosine and nitric oxide regulate regional vascular resistance via interdependent and independent mechanisms during sepsis.

OBJECTIVE: Adenosine receptor blockade increases regional resting vascular resistance during sepsis. In healthy subjects, part of adenosine's actions are mediated via stimulation of nitric oxide synthase. Because nitric oxide synthase activity is thought to be a major contributor to altered vascular tone in sepsis, we tested the hypothesis that some of the nitric oxide-mediated resting regional resistance during sepsis is secondary to endogenous adenosine stimulation of nitric oxide synthase. DESIGN: Prospective, randomized, controlled experiment. SETTING: Shock-trauma and basic science laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Twenty-four hours after sepsis or sham induction, rats were separated into two groups (n = 6 to 10 in each group). Group 1 received a 10-min infusion of the adenosine antagonist 8-sulfophenyltheophylline (0.9 mg/kg x min) followed by a 10-min infusion of L-nitro-arginine-methyl ester (0.5 mg/kg x min). Group 2 similarly received L-nitro-arginine-methyl ester followed by 8-sulfophenyltheophylline in the presence of L-nitro-arginine-methyl ester. MEASUREMENTS AND MAIN RESULTS: Hemodynamic and blood flow measurements (microspheres) were made before infusions, 10 mins after the administration of each single-agent infusion, and 10 mins after combined-agent infusions were administered. No significant resistance alterations were observed in nonseptic rats. In septic rats, adenosine receptor blockade alone increased hepatosplanchnic and skeletal muscle vascular resistance, but no further increases were seen when L-nitro-arginine-methyl ester was added. Nitric oxide synthase inhibition alone increased hepatosplanchnic and skeletal muscle vascular resistances. When 8-sulfophenyltheophylline was added to the infusion, skeletal muscle vascular resistance increased significantly more than with L-nitro-arginine-methyl ester alone, but there were no further increases in hepatosplanchnic resistance. Renal and adipose vascular resistances increased with L-nitro-arginine-methyl ester infusions, and 8-sulfophenyltheophylline produced no effect. CONCLUSIONS: During sepsis, nitric oxide caused resting vasodilation independent of adenosine in the renal and adipose vasculature. In the hepatosplanchnic circulation, there is reciprocal adenosine-nitric oxide interaction in maintaining resting regional resistance. Skeletal muscle displayed a dual adenosine-mediated (nitric oxide-independent) and nitric oxide-mediated (adenosine receptors required) interaction to regulate resting resistance during sepsis. These data indicate that in the hepatosplanchnic and skeletal muscle vasculature, all of the resting nitric oxide-mediated vasodilation is secondary to endogenous adenosine action, but in adipose and renal vasculature, resting nitric oxide mediated vasodilation is independent of adenosine. Endogenous adenosine also appears to play a significant role in determining resting skeletal muscle resistance that is independent of nitric oxide synthase during sepsis.

Mechanisms of myocardial protection by adenosine-supplemented cardioplegia: differential response of calcium-independent protein kinase C isozymes.

BACKGROUND: Adenosine-supplemented cardioplegia improves myocardial function after cardioplegic arrest. However, the underlying cellular mechanism(s) responsible for adenosine's protective actions remains unclear. We tested the hypothesis that protection by adenosine-supplemented cardioplegia would be associated with selective activation of protein kinase C (PKC) isozymes delta and epsilon. MATERIALS AND METHODS: Isolated rat hearts were perfused (37 degrees C, Krebs-Ringer bicarbonate buffer) for 30 min, after which baseline functional measurements were made. This was followed by 120 min of cold cardioplegic arrest at 4 degrees C with either St. Thomas No. 2 (ST#2), ST#2 + adenosine (100 microM, ADO) or ST#2 + ADO + 8-sulfophenyltheophylline (50 microM, SPT). Hearts were reperfused for 60 min and functional measurements made. Distribution of PKC isoforms was determined (immunoblotting) after 30 min of warm perfusion (No-CDPL) or after 30 min of perfusion followed by 15 min of cardioplegic arrest. RESULTS: ADO prevented myocardial dysfunction after cardioplegic arrest. PKC-delta did not differ in the cytosolic fraction among groups. However, ADO prevented increases in particulate fraction PKC-delta, but elicited a significant increase in the particulate fraction PKC-epsilon, while ST#2 or SPT significantly decreased the cytosolic fraction PKC-epsilon. Both functional and cellular changes associated with ADO were receptor mediated. CONCLUSION: This novel, dual action of adenosine-supplemented cardioplegia on PKC isoforms may be responsible for the associated functional improvements.

Mechanisms of myocardial protection by adenosine-supplemented cardioplegic solution: myofilament and metabolic responses.

OBJECTIVE: Adenosine supplementation of cardioplegic solutions in cardiac operations improves postarrest myocardial recovery after cardioplegic arrest and reperfusion; however, the mechanism of the action of adenosine remains unknown. We tested the hypotheses that adenosine-supplemented cardioplegic solution improves myofibrillar protein cooperative interaction and increases myocardial anaerobic glycolysis. METHODS: The hearts of male Sprague-Dawley rats were randomized to undergo 120 minutes of cardioplegic arrest with 1 of 3 cardioplegic solutions: (1) St Thomas' Hospital No. 2 cardioplegic solution (St Thomas group), (2) St Thomas' Hospital No. 2 cardioplegic solution plus adenosine (100 micromol/L) (adenosine group), and (3) St Thomas' Hospital No. 2 cardioplegic solution plus adenosine (100 micromol/L) plus the nonspecific adenosine receptor antagonist 8-p -sulfophenyltheophylline (50 micromol/L) (sulfophenyltheophylline group). A fourth group of hearts underwent no cardioplegic arrest. RESULTS: Systolic and diastolic functional recovery was improved in the adenosine group compared with that in the other two groups, independent of coronary flow. Adenosine supplementation of cardioplegic solution prevented the decrease in myofibrillar protein cooperative interaction seen after cardioplegic arrest and reperfusion (St Thomas and sulfophenyltheophylline groups). Adenosine-supplemented cardioplegic solution also caused significantly increased anaerobic glycolysis during cardioplegic arrest. These responses were blocked in the sulfophenyltheophylline group. CONCLUSIONS: The changes in myocardial glycolytic activity and myofilament cooperativity coincided with functional recovery in the three cardioplegia groups and may represent mechanisms underlying protection with adenosine-supplemented cardioplegic solution.

Multiple intrasplenic hepatocyte transplantations in the dalmatian dog.

BACKGROUND: Hepatocyte transplantation is an attractive potential treatment for liver-based inborn errors of metabolism and for fulminant hepatic failure. Dalmatian dogs have a metabolic error that results in hyperuricosuria. This report focuses on the effect of multiple, sequential intrasplenic transplants of fresh and cryopreserved hepatocytes in dalmatians. METHODS: Dalmatians underwent intrasplenic hepatocyte transplantation with hepatocytes taken from healthy mongrels. Dalmatian urinary uric acid excretion was measured preoperatively, and this served as the control value. Three hepatocyte transplantations were performed at 30-day intervals--the first with freshly isolated cells, and both the second and the third with cryopreserved hepatocytes from the same donor. Urinary uric acid excretion was measured postoperatively twice per week. RESULTS: The urinary uric acid excretion decreased an average of 54% after the first hepatocyte transplantation. The effect was transient and lasted an average of 22 days (range, 19-50 days). Subsequent intrasplenic hepatocyte transplantation with cryopreserved hepatocytes resulted in similar decreases in urinary uric acid excretion. Each transplant resulted in a significant decrease in urinary uric acid excretion when compared with baseline values (P = < .001). CONCLUSIONS: Sequential intrasplenic hepatocyte transplantation is feasible in this model. This method provided a significant, but transient, correction in urinary uric acid excretion that was similar with either fresh or cryopreserved hepatocytes. A substantial biologic effect provided by cryopreserved hepatocytes has important implications in clinical hepatocyte transplantation.

Elevated coronary endothelin-1 but not nitric oxide in diabetics during CABG.

BACKGROUND: After coronary artery bypass grafting procedures, a higher incidence of morbidity and mortality has been reported in diabetic patients. We tested whether coronary artery bypass grafting in diabetics affects the endothelin-1 and nitric oxide coronary effluent profile during reperfusion. METHODS: Twenty-one consecutive patients (9 with type II diabetes mellitus, 12 non-diabetics) underwent coronary artery bypass grafting by one surgeon. The two groups did not differ in preoperative ejection fraction, Parsonnet score, number of vessels bypassed, or cross-clamp time. Each patient was treated in the same intraoperative manner with single atrial, aortic, and antegrade and retrograde cardioplegia (CPL) cannulas. Cold CPL arrest was by antegrade and retrograde infusion of modified Buckberg CPL solution. Warm CPL solution was infused before reperfusion. Coronary sinus blood samples were obtained for estimation of endothelin-1 and nitrite plus nitrate before CPL arrest and at 1 and 15 minutes after each of 2 reperfusion periods. RESULTS: In diabetics, endothelin-1 was significantly increased at all reperfusion times as compared with non-diabetics. Nitrite plus nitrate levels were significantly higher in patients with diabetes than in those without, but did not change with time in either of the groups. CONCLUSIONS: Reperfusion after CPL during coronary artery bypass grafting procedure can trigger the release of endothelin-1 in patients with diabetes mellitus. This may favor increased vascular tone or positive inotropic responses after coronary artery bypass grafting and may contribute to significant cardiovascular consequences in diabetic patients.

Cardiopulmonary bypass increases coronary IL-8 in diabetic patients without evidence of reperfusion injury.

BACKGROUND: Endothelin-1 (ET-1) has been shown to be a potent agonist for monocyte production of the neutrophil chemotactic cytokine interleukin-8 (IL-8). We have shown that diabetic patients demonstrate elevated coronary ET-1 after coronary artery bypass grafting (CABG). We hypothesized that these same diabetic patients would manifest elevated coronary IL-8 and conjugated diene concentrations (an index of reperfusion injury). METHODS: Sixteen patients [9 nondiabetics and 7 type II diabetics] underwent nonemergent CABG. The two groups did not differ significantly in preoperative ejection fraction, number of vessels bypassed, or cross-clamp time. Coronary sinus samples were obtained prior to cardioplegic arrest (baseline) and at 1 and 15 min after reperfusion periods A and B (A, reperfusion of native coronaries + LIMA; B, reperfusion of saphenous vein grafts in addition to native coronary system + LIMA). Plasma samples were analyzed for IL-8 (ELISA) and conjugated dienes (spectrophotometry). RESULTS: Initially after reperfusion, IL-8 in both groups was significantly lower than precardioplegia values. In reperfusion B, only the diabetic group demonstrated a significant increase in IL-8 concentrations at 1 and 15 min compared to nondiabetics. Conjugated diene levels were significantly higher in diabetics at each time point than nondiabetics. CONCLUSIONS: This study demonstrates an early decrease in IL-8 in both groups, most likely related to depressed production secondary to hypothermia. The subsequent elevation in IL-8 only in the diabetic group was seen without concomitant conjugated diene elevation. While no evidence of reperfusion injury was demonstrated in this time frame, the elevation of IL-8 in diabetics after CABG may contribute to later infiltration and associated oxidative damage.

Sepsis produces depression of testosterone and steroidogenic acute regulatory (StAR) protein.

The hypothesis that induction of chronic peritoneal sepsis would produce depression of serum testosterone due to a decrease in Leydig cell steroidogenic acute regulatory (StAR) protein or P450c17 steroidogenic enzyme was tested. Male Sprague-Dawley rats (350-400 g) were randomized to septic and nonseptic groups. Sepsis was induced with a cecal slurry (200 mg/kg in 5 mL of 5% dextrose in water (D5W); intraperitoneal) while nonseptic rats received only sterile D5W. Animals (n = 6, in each group) were killed by CO2 asphyxiation and blood samples were collected by direct cardiac puncture at 24 h after induction of sepsis/sham sepsis. The serum concentration of corticosterone, progesterone, estradiol, and testosterone was determined using radioimmunoassay. Western blot analysis was utilized to quantify Leydig cell StAR protein and P450c17 enzyme. Sepsis produced a significant decrease in the serum concentration of testosterone, a down-regulation of StAR protein, and an increase in serum estradiol 24 h after induction of sepsis (as compared with the nonseptic group). Protein levels of P450c17 in Leydig cells and serum concentrations of progesterone and corticosterone 24 h after induction of sham sepsis or sepsis were not different. It is concluded that the decreases in serum testosterone after 24 h of chronic peritoneal sepsis correlated with reductions in StAR protein.

End-diastolic pressure-volume relationship in sepsis: relative contributions of compliance and equilibrium chamber volume differ.

BACKGROUND: Compliance is a complex parameter to measure both clinically and in the laboratory. Investigations in recent years have interpreted changes in the end-diastolic pressure-volume relationship (EDPVR) as changes in compliance. However, without considering the equilibrium chamber volume (LV volume when transmural pressure = 0), changes in the EDPVR may not reflect changes in left ventricular chamber compliance. In the present study, chamber compliance was differentiated from equilibrium chamber volume to determine their respective contributions to the EDPVR in a clinically relevant animal model of chronic sepsis. MATERIALS AND METHODS: Rats were made septic by intraperitoneal injection of a cecal slurry (200 mg/kg; 5 ml/kg of 5% dextrose in water). At 1, 3, and 7 days post-sepsis induction, hearts were perfused on an isolated heart apparatus. A latex balloon was placed in the left ventricle to record peak systolic and end-diastolic pressures at various volumes, and the starting volume in the balloon was recorded. Systolic performance was evaluated by calculating the developed pressure (systolic pressure minus end-diastolic pressure) and peak dP/dt at end-diastolic pressures of 5 and 10 mm Hg. RESULTS: Developed pressure and peak dP/dt were impaired 3 days after sepsis induction and continued to be so through Day 7 of sepsis. The diastolic data were fitted to an exponential equation, the results of which indicated a strong leftward shift in the EDPVR through 7 days of sepsis with a concomitant decrease in equilibrium chamber volume. The LV chamber compliance decreased at 1 day after sepsis induction, as indicated by significant changes in regression coefficients for the curves, transiently returned toward control by Day 3, but decreased again by 7 days of sepsis. CONCLUSIONS: Our data indicate that early in sepsis, compliance changes contribute to a left-shifting EDPVR, but at later times in the course of the disease, unstressed chamber volume becomes an important determinant of the left shift. The decrease in compliance (suggesting diastolic dysfunction) occurred prior to systolic impairment, which may have valuable prognostic implications for septic patients.

Velopharyngeal changes after maxillary advancement in cleft patients with distraction osteogenesis using a rigid external distraction device: a 1-year cephalometric follow-up.

The effect of maxillary advancement on speech may have benefits on articulation improvement but compromises velopharyngeal (VP) closure by increasing the nasopharyngeal distance. The purpose of this study was to evaluate the static VP anatomic changes on lateral cephalograms in patients who underwent maxillary advancement through distraction osteogenesis (DO) with a rigid external distraction device and to correlate these changes with clinical speech data. Twenty-two patients (5 female and 17 male) underwent maxillary advancement through DO utilizing a rigid external distraction device (age, 5.2 to 25.7 years) with various diagnoses, including 13 unilateral cleft lip and palate (CLP) patients, 5 bilateral CLP patients, 1 isolated cleft palate patient, 2 facial cleft patients, and 1 patient with craniosynostosis. Lateral cephalograms of preoperative, immediate postdistraction, and 1-year postdistraction were obtained for analysis. Speech evaluation was performed preoperatively, immediate postdistraction, and then at 6-month intervals, and included assessment of air pressure flow, hypernasality, and articulation. With an average amount of 8.9 mm maxillary forward advancement, 14% of patients (3 of 21) presented deterioration in hypernasality. However, 57% of patients (12 of 21) demonstrated improvement in articulation. The cephalometric analysis demonstrated an increase in nasopharyngeal depth by 8.5 mm (1:1 ratio with bony movement) and velar angle by 14.1 deg. The length of the soft palate remained unchanged. The need ratio (intersection of palatal plane and posterior pharyngeal wall-posterior nasal spine/posterior nasal spine--tip of uvula) worsens after distraction. The deterioration of hypernasality was related to the amount of forward distraction, especially in patients without a preexisting pharyngeal flap (PF). Speech evaluation is an important aspect concerning treatment planning for maxillary distraction. The increase in nasopharyngeal depth may compromise VP closure. The increase in velar angle was considered to be part of the compensation in the VP mechanism. An adverse effect of a preexisting PF on maxillary distraction was not observed; however, it prevented postoperative hypernasality.

Depressed myofilament co-operativity associated with post-cardioplegic myocardial depression.

The mechanism underlying myocardial depression after procedures involving cardioplegia are unknown. We tested the hypothesis that such depression was associated with altered myofilament interactions, using isolated hearts perfused with warm (37 degreesC), oxygenated (95% O2/5% CO2) Krebs-Ringer's bicarbonate (KRB) buffer. A latex balloon was inserted into the left ventricle (LV) to monitor LV function. All hearts underwent a 30-min equilibration period. One group of hearts (CPL+RPR) were arrested with St Thomas #2 cardioplegic solution (4 degreesC; 3 ml followed by 1 ml every 15 min) for 120 min, followed by reperfusion with warm, oxygenated KRB. A second group underwent cardioplegic arrest with no reperfusion (CPL). A third group underwent 60 min of warm, oxygenated perfusion with KRB beyond the equilibration period (60 MIN). The last group only underwent the equilibration period (EQUIL). LV function was assessed at the end of equilibration, and at 30 and 60 min of reperfusion (or 30 and 60 min additional perfusion in the 60 MIN group). All hearts were frozen at the end of the temporal protocol for each group, and stored at -70 degreesC for later measurement of Ca2+-stimulated Mg2+ ATPase activity after isolation of myofibrils. CPL+RPR hearts demonstrated significant depression of systolic pressure and elevation diastolic pressure at fixed volumes, compared to baseline and 60 MIN group values. There were no significant changes in the amount of constituent myofilament proteins, as assessed by densinometric analyses of Western blots. There were also no changes in the minimal or maximal ATPase activities, nor in the pCa50, indicating no effect of cardioplegic arrest on myofilament sensitivity to calcium. However, all hearts that underwent cardioplegic arrest were found to have significantly lower Hill coefficients (1.85+/-0.09 and 1.85+/-0.13 v 2.31+/-0.13 and 2.34+/-0. 14 in CPL+RPR and CPL v 60 MIN and EQUIL hearts, respectively), suggesting decreased co-operativity of the actomyosin interaction. Such a decrease in co-operativity would contribute to both the systolic and diastolic alterations associated with myocardial depression after cardioplegic arrest. These changes were associated with the cardioplegic event, and appeared to be independent of reperfusion.

Diabetic patients produce an increase in coronary sinus endothelin 1 after coronary artery bypass grafting.

Diabetes is associated with altered vascular responses, and diabetic patients demonstrate increased morbidity and mortality after coronary artery bypass grafting (CABG). We tested whether endothelin (ET)-1 levels in this patient population differed from those in nondiabetic subjects after CABG. Of 14 consecutive patients who underwent CABG by the same surgeon, 7 had type 2 diabetes and 7 were nondiabetic. The two groups did not differ significantly in preoperative ejection fraction, number of vessels bypassed, cross-clamp time, or Parsonnet's score. Coronary sinus blood samples were obtained before cardioplegic arrest and then obtained at 1 and 15 min after each of two reperfusion periods: reperfusion A (native coronary perfusion plus the left internal mammary artery), reperfusion B (saphenous vein graft perfusion). ET-1 was significantly increased at all reperfusion time points in diabetic patients compared with nondiabetic patients. In diabetic patients, reperfusion after CABG can trigger the release of ET-1, which may be a contributing factor in the increased cardiac morbidity seen in this patient population.

Effect of NG-nitro-L-arginine methyl ester on testicular blood flow and serum steroid hormones during sepsis.

Production of nitric oxide (NO) via NO synthase (NOS) has been implicated in the regulation of steroidogenesis in normal physiology and septic pathophysiology. The hypothesis that blockade of NOS by NG-nitro-L-arginine methyl ester (L-NAME) would affect testicular blood flow and circulating levels of steroid reproductive hormones was tested. Male Sprague-Dawley rats (350-450 g) were randomized to septic and nonseptic groups. Sepsis was induced with an intraperitoneal (i.p.) injection of a cecal slurry (200 mg/kg in 5 mL 5% dextrose in water (D5W)) in rats, while nonseptic rats received only sterile D5W. The rats (n = 6 per group) were catheterized in the jugular vein, left ventricle (via right carotid artery), and tail artery to determine blood flow and systemic hemodynamics and to collect blood at 24 h after induction of sepsis/sham sepsis. After baseline (24 h post-cecal slurry challenge) measurement, L-NAME (.50 mg/ kg x min) was infused through the jugular vein for 10 min, blood flow was determined using a radioactive microsphere technique, and blood samples were collected. The serum concentrations of corticosterone, progesterone, and testosterone were determined using radioimmunoassay. Plasma concentrations of NO byproducts (NOx) were determined using the Greiss reaction. After 24 h, heart rate, testicular blood flow, and NOx levels were significantly increased, whereas the serum concentration of testosterone was significantly decreased in the septic group as compared with the nonseptic group. However, serum concentrations of progesterone and corticosterone at 24 h after induction of sham-sepsis or sepsis were not statistically different. Infusion of L-NAME significantly reduced the testicular blood flow and serum NOx levels in septic rats as compared with their baseline values. The administration of L-NAME significantly increased the concentration of testosterone in nonseptic and septic rats as compared with their respective basal values. However, testosterone levels in septic rats were still significantly lower than in nonseptic rats. The results of this study indicate that the synthesis of NO through NO synthase may play a role in the regulation of testicular blood flow and the serum levels of testosterone, associated with chronic peritoneal sepsis in the rat.

Rapid determination of glomerular filtration rate by single-bolus inulin: a comparison of estimation analyses.

Rapid measurement of glomerular filtration rate (GFR) by an inulin single-bolus technique would be useful, but its accuracy has been questioned. We hypothesized that reported inaccuracies reflect the use of inappropriate mathematical models. GFR was measured in 14 intact and 5 unilaterally nephrectomized conscious male Sprague-Dawley rats (mean weight 368 +/- 12 g) by both single-bolus (25 mg/kg) and constant-infusion techniques (0.693 mg . kg-1 . min-1). The temporal decline in plasma inulin concentration was analyzed through biexponential curve fitting, which accounted for renal inulin loss before complete vascular and interstitial mixing. We compared our mathematical model based on empirical rationale with those of other investigators whose studies suggest inaccuracy of single-bolus methods. Our mathematical model yielded GFR values by single bolus that agreed with those obtained by constant infusion [slope = 0.94 +/- 0.16 (SE); y intercept = 0.23 +/- 0.64; r = 0.82]. In comparison to the data obtained by constant inulin infusion, this method yielded a very small bias of -0.0041 +/- 0.19 ml/min. Two previously reported models yielded unsatisfactory values (slope = 1. 46 +/- 0.34, y intercept = 0.47 +/- 1.5, r = 0.72; and slope = 0.17 +/- 1.26, y intercept = 17.15 +/- 5.14, r = 0.03). The biases obtained by using these methods were -2.21 +/- 0.42 and -13.90 +/- 1. 44 ml/min, respectively. The data indicate that when appropriate mathematical models are used, inulin clearance after single-bolus delivery can be used to measure GFR equivalent to that obtained by constant infusion of inulin. Attempts to use methods of analysis for simplicity or expediency can result in unacceptable measurements relative to the clinical range of values seen.

Cardiac myofilament protein function is altered during sepsis.

Male Sprague-Dawley rats (350-500 g) were made septic by intraperitoneal injection of 200 mg/kg cecal material in 5% dextrose in water (D5W; 5 ml/kg). Control rats (n = 11) received D5W. Preparations were studied on days 1 (n = 7), 3 (n = 7), and 7 (n = 8) of sepsis. In isolated hearts, ventricular function was depressed on days 3 and 7 of sepsis. Densitometric analysis of myofilament proteins from septic rats separated by SDS-PAGE showed no differences in relative amounts of actin, troponin, tropomyosin and myosin light chains compared to control. Myofilament function, assessed by measuring ATPase activities, was altered during sepsis. CA(2+)-independent Mg-ATPase activity was elevated on days 1 and 3 of sepsis, returning toward control by day 7. Maximal ATPase activity was unchanged on day 1, but was increased on days 3 and 7 sepsis. Myofibrillar myosin K(EDTA)-, Ca(2+)-, and Mg(2+)-ATPase activities were not altered, nor were there any apparent changes in myosin heavy chain isoform populations. Our data are the first to demonstrate alterations in minimal and maximal ATPase activities and myofilament CA(2+)-sensitivity during chronic peritoneal sepsis. These alterations may contribute to observed changes in ventricular function.

Adenosine receptor antagonism affects regional resting vascular resistance during rat peritoneal sepsis.

BACKGROUND: To identify vascular beds where endogenous adenosine plays a significant role as a mediator of resting perfusion alterations associated with sepsis, we tested the hypothesis that adenosine receptor blockade would cause differential regional increases in vascular resistance during intraperitoneal (ip) sepsis in the rat. MATERIALS AND METHODS: Rats (250-350 g) were catheterized and randomized to septic or nonseptic groups. Sepsis was induced with an ip injection of cecal slurry (150 mg/kg in D5W; 5 ml/kg), and baseline hemodynamics, cardiac output (CO), and blood flows (microspheres) were measured 24 h later. Animals then received the adenosine receptor antagonist 8-phenyltheophylline (8-PTH; 10 mM, 1.5 ml/kg), its vehicle (1.5 ml/kg), or normal saline (1.5 ml/kg), iv, and measurements were repeated. RESULTS: Septic animals treated with 8-PTH had a significant increase in skeletal muscle, hepatic portal, and cerebral vascular resistance with concomitant decreases in CO when compared with vehicle at 1 min. No significant resistance changes were observed in the renal, adipose, or coronary vasculatures. Adenosine receptor blockade caused a significant increase in +dP/dt and -dP/dt during sepsis, indicating that the reduced CO was not secondary to myocardial depression. CONCLUSIONS: These data suggest that adenosine receptor-mediated actions during sepsis affect vascular beds selectively and indicate a significant role for adenosine in resting perfusion redistribution in sepsis.

Splanchnic vascular control during sepsis and endotoxemia.

Endotoxemia and sepsis often result in circulatory derangements which manifest as perfusion maldistributions. It has been widely accepted that the splanchnic circulation decreases in perfusion during advanced septic or endotoxemic states. Impaired perfusion of splanchnic organs may result not only in organ dysfunction but also exacerbations of polymicrobial bacteremia due to intestinal mucosal leakage. Consequently, evaluation of the splanchnic mechanisms of vasoregulation and how perfusion is maintained is vital to any topic concerning the management of the septic patient.

Evidence that adenosine contributes to maintenance of hepatosplanchnic blood flow during peritoneal sepsis in rats.

Sepsis induced derangements in hepatosplanchnic perfusion can contribute to organ damage and death. Adenosine, a common and potent metabolic vasodilator, has not been evaluated as a mechanism for maintenance of blood flow during sepsis. We tested the hypothesis that adenosine receptor blockade would cause a decrease in hepatosplanchnic blood flow during intraperitoneal (i.p.) sepsis in the rat. Rats (250-350 g) were catheterized for hemodynamic and blood flow measurements with tracer microspheres. Sepsis was induced with an i.p. injection of cecal material (150 mg/kg in D5W; 5 mL/kg), and baseline measurements were taken 24 h later. Animals then received either the adenosine receptor antagonist 8-PTH (10 mM, 1.5 mL/kg), its vehicle (1.5 mL/kg) or normal saline (1.5 mL/kg), intravenously, and measurements were repeated 1 and 10 min later. There was a significant increase in hepatosplanchnic portal resistance in septic animals given 8-PTH, with no change in mean arterial blood pressure (MAP) or heart rate. Regionally, there was a significant decrease in gastric, small intestinal, cecal, and pancreatic blood flow when compared with vehicle. Adenosine receptor blockade caused a significant reduction in hepatosplanchnic blood flow during sepsis, suggesting that maintenance of splanchnic blood flow during sepsis involves receptor mediated adenosine actions.