Adenosine Pretreatment Research Articles

Protection Against Ischemic-Reperfusion Injury of Skeletal Muscle: Role of Ischemic Preconditioning and Adenosine Pretreatment

Prolonged tissue ischemia and subsequent reperfusion result in significant tissue injury due to the ischemic-reperfusion syndrome. Although skeletal muscle has significant tolerance to ischemic-reperfusion injury (IRI), compared to other organ systems, IRI of skeletal muscle does occur when there is a prolonged ischemic period. In many reconstructive surgical procedures involving microsurgery and prolonged tissue ischemia time, IRI-induced skeletal-muscle injury is a serious clinical concern. Specifically, there are significant vascular complications (venous thrombosis and arteriolar no-reflow) and loss of transplanted muscle function on reperfusion with prolonged ischemia. Ischemic preconditioning (IPC) or adenosine (ADO) pretreatment applied prior to the ischemic period are known to protect against IRI in cardiac muscle. Recent data from basic science research suggest that IPC or ADO pretreatment may be employed to protect skeletal muscle against IRI. This review summarizes the basic mechanisms and potential clinical relevance of ischemia- and reperfusion-induced skeletal muscle injury and describes how skeletal muscle can be protected against IRI with IPC or ADO pretreatment.

The obligate role of protein kinase C in mediating clinically accessible cardiac preconditioning

Cardiac preconditioning is an adaptation of cardiomyocytes that promotes tolerance to a subsequent ischemic insult. Adenosine receptor signaling is proposed as a mediator of preconditioning, but its mechanism of protection remains unknown. We hypothesized that protection against hypoxia-reoxygenation (H/R) injury could be conferred in a rat ventricle by adenosine-mediated protein kinase C (PKC) activation and that adenosine-mediated cardioprotection could be extended to human ventricular muscle. Isolated rat and human ventricular muscle (VM) strips were subjected to 30 minutes of hypoxia and 60 minutes of reoxygenation (H/R control). The VM was pretreated with 125 mumol/L adenosine, an adenosine antagonist ((p-Sulfophenyl) theophylline [SPT] 50 mumol/L) and adenosine (adenosine + SPT), or with a PKC inhibitor (chelerythrine, 10 mumol/L) and adenosine (adenosine + chelerythrine) before H/R Developed force (DF) and tissue creatine kinase (CK) activity were assessed at end reoxygenation. Human trabeculae were obtained from diseased explanted hearts at cardiac transplantation and were also subjected to H/R injury. Human VM was pretreated with adenosine (125 mumol/L) before H/R injury. Results are expressed as mean +/- standard error of mean. In the rat, adenosine pretreatment conferred protection of DF against H/R injury (adenosine, 62% +/- 6%; H/R control, 27% +/- 2%, p < 0.05). Adenosine + SPT or adenosine + chelerythrine eliminated the functional recovery conferred by adenosine. This recovery of contractile function was associated with greater tissue CK activity (adenosine, 415 +/- 40 units/gm; H/R control, 78 +/- 13 units/gm, p < 0.05). The protective effects of adenosine against H/R were present in the human ventricle and with recovery of DF in adenosine (66% +/- 5%) and H/R control (24% +/- 4%), p < 0.05. Adenosine, a clinically accessible agonist, induces protection against H/R injury through a PKC-mediated mechanism in the rat ventricle. Further, the protection conferred by adenosine against H/R extends to the human ventricle.

Preconditioning with Ischemia or Adenosine Protects Skeletal Muscle from Ischemic Tissue Reperfusion Injury

Prolonged tissue ischemia and subsequent reperfusion results in significant tissue injury due to the ischemic-reperfusion (IR) syndrome. Ischemic preconditioning (IPC) or adenosine (ADO) pretreatment are known to protect IR injury in cardiac muscle. Our aim was to determine whether IPC or ADO pretreatment attenuates and protects against ischemic tissue reperfusion injury in skeletal muscle. Rats were anesthetized and global hindlimb ischemia was induced by 60 min of suprarenal aortic clamping followed by 30 min of reperfusion period. The degree of skeletal muscle dysfunction was determined by decreases in maximum contractile force, and adenosine triphosphate (ATP) and creatine phosphate (CP) levels of extensor digitorum longus (EDL) muscle. The distal tendon of the EDL was attached to a force transducer for maximum isometric force measurement. Samples were taken from the EDL for measurement of ATP and CP levels. The following were protective protocols prior to the IR challenge: (1) four consecutive 5-min periods of ischemia separated by 5-min reperfusion periods (PC/I) or (2) i.v. adenosine infusion (350 μg/kg/min × 10 min, PC/A). Our data suggest that pretreatment with brief periods of ischemia or systemic ADO infusion attenuates ischemic tissue reperfusion injury in skeletal muscle [Table]

Pretreatment of human myocardium with adenosine during open heart surgery.

Depressed myocardial performance after cardiac surgery can be contributed to ischemic reperfusion injury (IRI) incurred during and following the cardiopulmonary bypass (CPB). Myocardial preconditioning (PC) achieved by brief ischemia and subsequent reperfusion appears to be a clinically useful method of improved cardiac protection during surgery involving CPB by retarding IRI. Based on animal studies, activation of cardiac adenosine (ADO) receptors prior to the prolonged ischemic period appears to mimic this PC phenomenon. We investigated whether the human myocardial PC can be mimicked with ADO in the setting of the coronary artery bypass graft (CABG). The specific proposed objective of this study was to determine whether ADO infusion just prior to starting the CPB can improve post-CPB myocardial hemodynamics. Patients undergoing elective CABG with poor ventricular function (ejection fraction approximately 30%), and with at least three-vessel disease were selected for this study (n = 7 ADO, and n = 7 control). Our results show that ADO infusion (250-350 micrograms/kg X 10 min) just prior to CPB resulted in an immediately improved postbypass cardiac index (CI) in the OR (CI increase of 41.5% +/- 11.1% for ADO vs 9.7% +/- 6.0% for control, p < 0.05). Forty hours postoperatively in the intensive care unit, ADO patients had improved CI (3.3 +/- 0.2 L/min per m2 for ADO, vs 2.6 +/- 4 L/min per m2 for control, p < 0.05). ADO patients maintained lowered resting heart rate (90 +/- 6 for ADO, vs 108 +/- 4 for control, p < 0.05) 40 hours after surgery. ADO patients also released significantly less CPK during the first 24 hours of the postoperative period. Based on these measurements, ADO pretreated patients had improved ventricular performance postoperatively. It also appears that ADO pretreatment results in lowered postoperative myocardial energy demand and less myocellular injury during CPB. To our knowledge, this is the first study to demonstrate that human myocardium can be hemodynamically improved with ADO pretreatment, and may be protected against IRI incurred during and following the CPB. We believe that a cardiac surgeon may now have the unique opportunity to confer myocardial protection during and after a cardiac surgical procedure.

Adenosine pretreatment for prolonged cardiac storage: an evaluation with St. Thomas' Hospital and University of Wisconsin solutions

Adenosine pretreatment has been shown to be beneficial in several models of ischemia-reperfusion. We wished to evaluate whether adenosine pretreatment is cardioprotective for prolonged cardiac storage and whether the presence of adenosine in the storage media affects the results. Isolated rodent hearts were obtained from Sprague-Dawley rats, mounted on a Langendorff apparatus, instrumented with an intraventricular balloon, and ventricularly paced at 300 beats/min. Four groups of hearts were studied in a 2 × 2 factorial experiment ( n = 8 to 12 per group). Hearts were subjected to normal perfusion or to solution supplemented with adenosine 50 μmol/L for 10 minutes followed by adenosine-free perfusion for 10 minutes. Hearts then were stored for 8 hours at 0º C in either University of Wisconsin solution (adenosine 5 mmol/L) or St. Thomas' Hospital II solution (adenosine free). Adenosine pretreatment increased tissue levels of adenosine triphosphate before storage ( p = 0.04). Nonfunction was less common after storage (1/19 versus 6/20 hearts, p < 0.05), and diastolic function was better preserved in the adenosine groups in the reperfusion phase ( p = 0.01). The beneficial effects of adenosine pretreatment were independent of which storage solution was used. Developed pressure was increased ( p < 0.05) and release of creatine kinase and lactate dehydrogenase was reduced ( p < 0.0001) in hearts treated with University of Wisconsin solution compared with those treated with St. Thomas' Hospital solution. These studies suggest that adenosine pretreatment improves recovery after prolonged hypothermic storage and that the presence of adenosine in the preservation solution does not alter the results. The experiments provide further evidence that extended myocardial protection is better enhanced with University of Wisconsin solution than with St. Thomas' Hospital II solution. (J T HORAC C ARDIOVASC S URG 1995;110:293-301)

Salutary effects of exogenous adenosine administration on in vivo myocardial stunning

Augmentation of endogenous adenosine levels is associated with decreased myocardial ischemic-reperfusion injury. The purpose of this study was to determine whether exogenous adenosine administered before ischemia could attenuate postischemic myocardial dysfunction. Regional myocardial stunning was induced by 15 minutes of coronary artery occlusion and 90 minutes of reperfusion in an open-chest canine preparation. Regional ventricular function was assessed by measurement of systolic wall thickening. Control untreated hearts were compared with two groups of hearts treated immediately before ischemia with intracoronary adenosine (5 micrograms/kg per minute and 50 micrograms/kg per minute). A fourth group of hearts was treated for the first 30 minutes of reperfusion with adenosine (50 micrograms/kg per minute). Preischemic adenosine administration increased coronary flow sixfold to sevenfold without altering regional function, mean arterial pressure, or left ventricular end-diastolic pressure. Both adenosine pre-treatments attenuated stunning compared with results in control animals (14.7% +/- 5.1% and 21.6% +/- 7.3% of preischemic systolic wall thickness versus -14.0% +/- 10%). Adenosine treatment during reperfusion transiently increased function in parallel with increased coronary blood flow, but after termination of the infusion regional function was not different from that in control stunned hearts (-5.0% +/- 13.1% of preischemic systemic wall thickness). These results indicate that adenosine pretreatment is associated with attenuation of stunning, an effect that can be produced at doses that do not alter systemic hemodynamics.

932-90 Transient Adenosine Infusion and Washout Before Ischemia Protects the Heart Against Metabolic Damage During Ischemia and Reperfusion, but Does not Attenuate Stunning in Pigs – Comparison with Ischemic Preconditioning

Recent studies indicate that ischemic preconditioning (IP) may be mediated by adenosine (ADO) receptor stimulation. This study compared the effects of adenosine pretreatment on myocardial metabolism and function with those of IP. Control hearts (C) underwent 15 min LAD occlusion (occl) followed by 120 min reperfusion (R). ADO (200/μg/kg/min) was infused into left atrium for 15 min starting at 20 min before LAD occl. IP was elicited by two cycles of 5 min occl & 5 min R. Tissue levels of ATP. creatine phosphate (CP) and pH in the area at risk were measured by 31 P-MRS, and %segment shortening (%SS) by sonomicrometry ADO infusion decreased blood pressure (–26%) and heart rate (–13%), and increased three times regional myocardial blood flow (RMBF). However, within 5 min after stopping ADO infusion, hemodynamic parameters returned to baseline. During sustained ischemia and reperfusion, there were no differences in hemodynamic parameters or RMBF among three groups. 15 min occl 120 min R 120 min R Groups ATP CP pH ATP CP %SS C(n = 10) 64 ± 7% 7 ± 6% 6.3 ± 0.2 71 ± 7% 105 ± 12% 39 ± 16% ADO (n = 10) 76 ± 6% * 9 ± 6% # 6.5 ± 0.1 * 90 ± 8% * 101 ± 6% # 29 ± 6% IP(n = 10) 74 ± 9% * 18 ± 6% * 6.6 ± 0.1 * 91 ± 90% 126 ± 7% 32 ± 16% ATP, CP and %SS values; % of baseline * p &lt; 0.05 vs C # p &lt; 0.05 vs IP ADO pretreatment and IP had similar effects on ATP and pH, but not on CP. In spite of the protective effects on myocardial metabolism during ischemia and reperfusion, neither of the interventions improved %SS after ischemia.

Inhibition of TPA and 12(S)-HETE-stimulated tumor cell adhesion by prostacyclin and its stable analogs: rationale for their antimetastatic effects.

We have investigated the regulatory role of PGI2 and its stable analogs, i.e., iloprost and cicaprost, on 12(S)-HETE- and TPA-enhanced tumor cell integrin expression and adhesion. Walker 256 carcinosarcoma cells express alpha IIb beta 3 integrin receptors, which mediate their adhesion to endothelium, subendothelial matrix and fibronectin. Adhesion is enhanced by treatment with exogenous 12(S)-HETE but not 12(R)-HETE or other lipoxygenase-derived hydroxy fatty acids, as well as by TPA. Both 12(S)-HETE and TPA enhanced alpha IIb beta 3 expression on W256 cells. PGI2 iloprost and cicaprost inhibited both 12(S)-HETE- and TPA-enhanced adhesion to endothelium and subendothelial matrix as well as alpha IIb beta 3 expression on W256 cells. The mechanism responsible for the effect of PGI2 was explored. Prostacyclin treatment of W256 cells resulted in an enhanced production of cAMP in a time- and dose-dependent manner. Pre-treatment of tumor cells with increasing concentrations of adenosine resulted in a dose-dependent decrease in the PGI2 effect on TPA or 12(S)-HETE-enhanced adhesion, suggesting that the PGI2 effect is mediated through PKA. Dibutyryl cAMP also blocked the 12(S)-HETE- or TPA-enhanced adhesion, and adenosine pre-treatment did not result in an inhibition of the dibutyryl cAMP effect. Collectively, our results suggest that the cyclooxygenase metabolite PGI2 can antagonize the lipoxygenase metabolite 12(S)-HETE- and TPA-enhanced alpha IIb beta 3 expression and tumor cell adhesion via activation of adenylate cyclase and elevation of intracellular levels of cAMP.

Increased Acetylcholine Content Induced by Adenosine in a Sympathetic Ganglion and Its Subsequent Mobilization by Electrical Stimulation

The present study was initiated to examine the effects of ATP on acetylcholine (ACh) synthesis. The exposure of superior cervical ganglia to ATP increased ACh stores by 25%, but this effect was also evident with ADP, AMP, and adenosine, but not with beta gamma-methylene ATP, a nonhdydrolyzable analogue of ATP, or with inosine, the deaminated product of adenosine. Thus, we attribute the enhanced ACh content caused by ATP to the presence of adenosine derived from its hydrolysis by 5'-nucleotidase. The adenosine-induced increase of tissue ACh was not the consequence of an adenosine-induced decrease of ACh release. The extra ACh remained in the tissue for more than 15 min after the removal of adenosine, but it was not apparent when ganglia were exposed to adenosine in a Ca(2+)-free medium. Incorporation of radiolabelled choline into [3H]ACh was also enhanced in the presence of adenosine, suggesting an extracellular source of precursor. Moreover, the synthesis of radiolabelled forms of phosphorylcholine and phospholipid was not reduced in adenosine's presence, suggesting that the extra ACh was not likely derived from choline destined for phospholipid synthesis. Aminophylline did not prevent the adenosine effect to increase ACh content; this effect was blocked by dipyridamole, but not by nitrobenzylthioinosine (NBTI). In addition, two benzodiazepine stereoisomers known to inhibit stereoselectively the NBTI-resistant nucleoside transporter displayed a similar stereoselective ability to block the effect of adenosine. Together, these results argue that adenosine is transported through an NBTI-resistant nucleoside transporter to exert an effect on ACh synthesis. The extra ACh accumulated as a result of adenosine's action was releasable during subsequent preganglionic nerve stimulation, but not in the presence of vesamicol, a vesicular ACh transporter inhibitor. We conclude that the mobilization of ACh is enhanced as a result of adenosine pretreatment.

Modulatory effects of adenosine on Porichthys luminescence

AbstractThe modulatory effects of exogenous applications of adenosine on luminous organs (photophores) of the fish Porichthys were studied. Pretreatment of photophores with adenosine (10−7 to 10−3 M) potentiated both phases of adrenaline induced luminescence. The first peak of light (Lmax1) showed a 3‐fold increase following 10−7 M adenosine treatment; a maximal increase of 12‐fold was observed with 10−3 M adenosine. The second peak of light (Lmax2) exhibited a similar dose‐response curve, but the maximal increase was attained with 10−4 M adenosine. The potentiating effect of 1 uM adenosine on light emission triggered by adrenaline increased for adrenaline concentrations ranging from 10−7 to 10−4 M for Lmax1, but it remained stable for Lmax2. All 4 P1‐purinergic agonists tested at the final concentration of 10−4 M induced a potentiation of adrenaline luminescence with the following rank of potency: L‐PIA &gt; NECA &gt; CADO ≥ CHA = ADO for Lmax1 and NECA &gt; CADO &gt; ADO &gt; CHA &gt; L‐PIA for Lmax2. The potentiating effect of adenosine was antagonized with the following rank of potency by the P‐1 purinergic antagonists: CPT &gt; DPMX ≥ AMINO &gt; THEO for Lmax1 and CPT &gt; AMINO &gt; THEO ≥ DPMX for Lmax2. According to the classification of purinergic receptors, our results suggest that P1‐purinergic receptors might be implicated in the adenosine potentiation of Porichthys adrenaline luminescence. Experiments also showed that nor‐adrenaline luminescence is inhibited by 1 uM adenosine pretreatment. Further work will be necessary to elucidate the mechanism involved in the modulatory roles played by the purinergic receptors on adrenaline and noradrenaline luminescence in Porichthys photophores. © 1993 Wiley‐Liss, Inc.

Adenosine alters glucose use during ischemia and reperfusion in isolated rat hearts.

Adenosine possesses marked cardioprotective properties, but the mechanisms for this beneficial effect are unclear. The objective of this study was to determine the effect of adenosine given before ischemia or at reperfusion on mechanical function, glucose oxidation, glycolysis, and metabolite levels in isolated, paced (280 beats per minute) working rat hearts. Hearts were perfused with Krebs-Henseleit buffer containing 11 mM glucose, 1.2 mM palmitate, and 500 microU.mL-1 insulin at an 11.5 mm Hg left atrial preload and 80 mm Hg aortic afterload. Adenosine (100 microM) pretreatment or adenosine (100 microM) at reperfusion markedly increased the recovery of mechanical function (from 44% to 81% and 96%, respectively) after 60 minutes of low-flow ischemia (coronary flow, 0.5 mL.min-1). Glucose oxidation (mumol.min-1 x g dry wt-1) was inhibited during ischemia (from 0.44 +/- 0.04 to 0.12 +/- 0.01), and this was not altered by adenosine (100 microM). During reperfusion, glucose oxidation recovered (to 0.38 +/- 0.02) and adenosine (100 microM), given at reperfusion, further increased glucose oxidation (to 0.52 +/- 0.06). The rate of glycolysis (mumol.min-1 x g dry wt-1), which was unaffected by ischemia per se, was inhibited by adenosine pretreatment (from 4.7 +/- 0.3 to 2.6 +/- 0.3). During reperfusion, glycolysis was also inhibited by adenosine relative to control (3.9 +/- 0.8) either when present during ischemia (2.6 +/- 0.6) or during reperfusion (1.4 +/- 0.4). These effects of adenosine on glucose metabolism reduced the calculated rate of H+ production attributable to glucose metabolism during the ischemic and reperfusion periods. Tissue lactate levels (mumol.g dry wt-1), which increased during ischemia (from 9.3 +/- 1.1 to 87.4 +/- 10.3) and then declined during reperfusion (to 26.2 +/- 3.7), were depressed further by adenosine pretreatment (to 19.7 +/- 4.1) and by adenosine at reperfusion (to 13.6 +/- 2.1). ATP levels (mumol.g dry wt-1), which were depressed by ischemia (from 18.1 +/- 1.1 to 10.6 +/- 1.3) and tended to be further depressed during reperfusion (to 7.1 +/- 0.7), were increased by adenosine pretreatment (to 14.1 +/- 1.2) and by adenosine at reperfusion (to 15.6 +/- 2.4). The effects of adenosine on glucose metabolism that would tend to decrease cellular acidosis and hence, Ca2+ overload, may explain the beneficial effects of adenosine on mechanical function observed in these hearts during reperfusion after ischemia.

Role of increased cytosolic free calcium concentration in myocardial ischemic injury.

Increases in cytosolic free calcium concentration ([Ca2+]I) may play an important role in myocardial ischemic injury. An early effect of the rise in [Ca2+]I may be impaired postischemic contractile function if the ischemic myocardium is reperfused during the reversible phase of ischemic injury; furthermore, if the rise in [Ca2+]I is prolonged, a cascade of events may be initiated which ultimately results in lethal injury. With the development of methods for measuring [Ca2+]I, it has become possible to evaluate directly the role of increased [Ca2+]I in myocardial ischemic injury. Although it has been possible to show that inhibition of the transport processes which contribute to the early rise in [Ca2+]I attenuates stunning and the rise in [Ca2+]I concurrently, if increased [Ca2+]I plays an important role in ischemic injury, then it should be possible to show that interventions which alter the timecourse of ischemic injury also alter the timecourse of the rise in [Ca2+]I in a parallel manner. Recently, considerable effort has been expended to investigate the mechanisms underlying the preconditioning phenomenon, whereby repetitive brief periods of ischemia prior to a sustained period of ischemia protects the myocardium from injury during the sustained period of ischemia, and this has stimulated additional work to understand the possible involvement of adenosine as a mediator of preconditioning as well as to understand the protective effects of adenosine. Measurements of [Ca2+]I using 19F NMR of 5FBAPTA-loaded hearts have shown that preconditioning attenuates the rise in [Ca2+]I during 30 min of ischemia and reduces stunning during reflow. Adenosine pretreatment mimics the effects of preconditioning on the rise in [Ca2+]I and on stunning, but adenosine receptor antagonists do not eliminate the protective effects of preconditioning, although some adenosine antagonists also block hexose transport and under these conditions, the ability of preconditioning to attenuate the rise in [Ca2+]I is abolished and there is a corresponding loss of the protective effect of preconditioning on stunning. Although it has been suggested that the beneficial effect of preconditioning on infarct size can be eliminated by pretreatment with glibenclamide, in the isolated rat heart glibenclamide does not affect the attenuation of the rise in [Ca2+]I induced by preconditioning and does not affect stunning. All of these studies show a consistent relationship between the magnitude of the rise in [Ca2+]I during ischemia and the degree of stunning during reperfusion. The data suggest that increased [Ca2+]I plays a very important role in myocardial ischemic injury.

Myocardial protective effects of adenosine. Infarct size reduction with pretreatment and continued receptor stimulation during ischemia.

We hypothesized that 1) endogenous adenosine released during ischemia conferred an inherent cardioprotection, and 2) a pretreatment dose of adenosine before ischemia would provide additional protection independent of hemodynamic effects. Thirty-six anesthetized New Zealand White rabbits underwent 30 minutes of regional ischemia produced by coronary occlusion followed by 2 hours of reperfusion. The adenosine group (ADO, n = 9) received a 5-minute pretreatment infusion of 140 micrograms/kg/min of adenosine before ischemia. A control group (SAL, n = 9) received saline before ischemia. To separate the effects of adenosine used as a pretreatment versus the effects during ischemia, a third group (ADO+SPT, n = 9) received adenosine as pretreatment followed by 10 mg/kg 8-p-sulfophenyl theophylline (8-SPT), an A1/A2-receptor antagonist given before ischemia, thus allowing pretreatment with adenosine but antagonizing its effects during ischemia. To preclude any protection from endogenous adenosine released during ischemia, the fourth group (SAL+SPT, n = 9) received saline as pretreatment and 8-SPT before ischemia. Area of necrosis within the area at risk (infarct size) was determined with tetrazolium and Evans blue stains, and transmural blood flow was measured using radioactive microspheres. Collateral blood flow in the area at risk was similar in all groups, as was the size of the area at risk. Infarct size was reduced by adenosine pretreatment (ADO, 8.4 +/- 7.2%) in contrast to saline vehicle (SAL, 27.8 +/- 6.3%; p less than 0.05 versus ADO). alpha 1/alpha 2-Receptor blockade after adenosine pretreatment abolished the ischemic protection provided by pretreatment adenosine (ADO+SPT, 42.7 +/- 8.3%; p less than 0.05 versus ADO). Finally, receptor blockade of endogenously released adenosine without adenosine pretreatment increased infarct size by 24% over the nonpretreated saline group (SAL+SPT, 51.5 +/- 9.0%; p less than 0.05 versus SAL). We conclude that 1) endogenous adenosine building up during ischemia is cardioprotective, and 2) pretreatment with adenosine confers cardioprotection independent of hemodynamic effects. Whether pretreatment effects of adenosine subsequently modulate the effects of endogenous adenosine (through alterations in receptor population or sensitivity) or endogenous and exogenous adenosine represent additive compartments is unclear.

Adenosine-sensitive phosphoinositide turnover in a newly established renal cell line

To aid in characterizing adenosine receptors in renal cells, primary cultures of rabbit cortical collecting tubule (RCCT) cells were infected with an adenovirus 12-simian virus 40 hybrid, resulting in a continuous cell line. The cells, designated RCCT-28A, retained their epithelial morphology and reacted with a monoclonal antibody specific for rabbit collecting tubule. Adenosine 3',5'-cyclic monophosphate (cAMP) accumulation was stimulated by vasopressin (AVP), isoproterenol, prostaglandin E2 (PGE2), calcitonin, parathyroid hormone, and a potent adenosine A1- and A2-receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA). A more selective adenosine A1-receptor agonist, N6-cyclohexyl adenosine (CHA) inhibited basal and AVP-stimulated cAMP accumulation. Cytosolic free calcium was transiently elevated by bradykinin, PGE2, NECA, and CHA. To examine the mechanism by which adenosine analogues increase intracellular free calcium, phosphoinositide (PI) turnover was assessed in the 28A cells after labeling with myo-[3H]inositol. NECA and CHA increased [3H]inositol phosphate formation with an approximate half-maximal effective concentration of 0.1 microM for both analogues. The increase in PI turnover was blocked by the selective adenosine A1-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine and pretreatment of the 28A cells with pertussis toxin. These results suggest that adenosine analogues increase cytosolic free calcium by stimulating PI turnover.

An experimental evaluation of nucleotide enhancement techniques for kidney transplantation

The effect of various nucleotide-enhancing agents on renal function and intracellular nucleotide levels was evaluated in a canine autotransplant model. Thirty-five dogs (18–28 kg) underwent left nephrectomy and 30 min of warm ischemia followed by Collins C-4 flush and 24 hr of cold-storage preservation. Heterotopic autotransplantation and immediate contralateral nephrectomy was then performed. Seven equal groups were evaluated: group A—controls, group B—adenosine pretreatment (1.0 g), group C—dipyridamole pretreatment (10 mg), group D—adenosine (1.0 g), and dipyridamole (10 mg) pretreatment, group E—adenosine (200 mg) and EHNA (2.5 mg/kg) pretreatment, group F—adenosine (200 mg) and EHNA (2.5 mg/kg) in the Collins C-4 flush, and group G—adenosine (200 mg) and EHNA (2.5 mg/kg) at the time of autotransplantation. All kidneys underwent cortical biopsies at the end of preservation and 1 hr after restoration of blood flow for determinations of AMP, ADP, and ATP. In the pretreatment groups (groups B through E) there was 60% graft survival whereas the controls (group A) and the groups treated after ischemia (groups F and G) had 0, 0, and 20% graft survival, respectively. In groups B and E, ATP levels were greater than controls after preservation and 1 hr after restoration of blood flow. Group C AMP and ADP levels and group D energy charge were greater than controls in the post-transplantation biopsies. Administration of adenosine and EHNA after ischemia was not associated with increased intracellular nucleotide levels. One hour post-transplantation biopsies demonstrated greater ability to regenerate cortical nucleotides in the surviving animals but no absolute value could be identified as a predictor of viability. In conclusion, pretreatment with adenosine, dipyridamole, and EHNA alone and in combination is beneficial in ischemically injured kidneys undergoing cold-storage preservation.

Cellular Ca2+ monitored by aequorin in adenosine-mediated smooth muscle relaxation

We studied the effect of adenosine on cytoplasmic ionized calcium-force relationships in vascular smooth muscle (VSM) and determined the dose dependence of the observed effects. The bioluminescent protein aequorin was used as an index of cytoplasmic ionized calcium and was chemically loaded into ferret portal vein strips. The VSM strips were contracted with 33 mM potassium (K+), 5 X 10(-6) M phenylephrine (PE), or electrical stimulation. Force and aequorin light, i.e., cytoplasmic ionized calcium, were simultaneously recorded. Adenosine pretreatment (3.7 X 10(-6) M) reduced both force and light responses in contractures with K+, PE, or electrical stimulation. In contrast, the addition of adenosine during PE or K+ contractions decreased force without a change in light. Dose-response curves for the effects of adenosine on K+ contractures indicated that at low doses adenosine decreases force and cytoplasmic ionized calcium but that at high concentrations (greater than 3.7 X 10(-6) M) adenosine increases light and apparently relaxes VSM by desensitizing the myofilaments to cytoplasmic ionized calcium.

Regulation of depolarization-dependent release of neurotransmitters by adenosine: cyclic AMP-dependent enhancement of release from PC12 cells.

We have used pheochromocytoma cells, clone PC12, as a model system for studying the effects of adenosine on neurosecretion. Exposure of the cells to adenosine or 2-chloroadenosine caused immediate activation of adenylate cyclase, increases in cellular cyclic AMP content, and inhibition of SAM-dependent phospholipid N-methylation and protein carboxymethylation. However, the effects on methylation were only observed with concentrations of adenosine 100 times greater than those that elevated cyclic AMP. Exposure of the cells to adenosine and 2-chloroadenosine did not alter the release of [3H]norepinephrine [(3H]NE) in the absence of depolarization. However, depolarization-dependent release of [3H]NE was markedly elevated by short (1-20 min) pretreatments with adenosine or 2-chloroadenosine. The enhancement of release was observed irrespective of the nature of the depolarizing stimulus (elevated K+, carbamylcholine, or veratridine). Release of [3H]acetylcholine in response to elevated K+ also was increased by adenosine pretreatment. These effects of adenosine and 2-chloroadenosine on neurotransmitter release closely paralleled elevation of cellular cyclic AMP but not inhibition of methylation. Taken together, the results show that adenosine, probably acting through adenosine receptors coupled to stimulation of adenylate cyclase, is able to modulate the neurosecretory process in PC12 cells. Furthermore, the enhancement of release occurred even though the extent of depolarization (measured as 86Rb+ flux through the acetylcholine receptor channel) and the amount of 45Ca2+ which entered upon depolarization were unchanged. Therefore, the enhancement of release produced by elevated cyclic AMP appeared to reflect increased efficiency of the stimulus-secretion coupling process.

Aggregation of platelets in the mesenteric microcirculation of the rat induced by α- toxin (phospholipase C) of Clostridium perfringens

We have investigated the effects of purified α-toxin (phospholipase C) of Clostridium perfringens ( Yamakawa and Ohsaka, 1977) on the behavior of cellular components in the microcirculation, using cinematography on a microscopic level and electron microscopy. We demonstrated that after topical application of α-toxin to the mesentery of the rat, rolling of leucocytes along the vessel wall and sticking of leucocytes to the vessel wall occurred in venules but not in arterioles. Some of the leucocytes remained attached to the vessel wall. Thrombi were formed frequently in venules and capillaries, and at a later stage, in arterioles. With time, thrombi increased in number and size, leading eventually to stasis of the blood stream. Thrombi were also observed frequently in the mesenteric microcirculation when toxin was injected into the jugular vein of the rat. The experiments with adenosine pretreatment followed by topical application of α-toxin to the mesentery suggested that the formation of thrombi induced by this toxin does not involve the mediation of ADP. Electron-microscopic examination confirmed the formation of thrombi consisting solely of platelets. It was concluded that thrombosis must be involved as an early step in the pathogenesis of necrosis caused by α-toxin. The death of the animals injected intravenously with α-toxin may be due, at least in part, to thrombosis. It is possible that thrombosis induced by α-toxin may be one of the factors involved in the causation of toxemia often manifested in the late stage of gas gangrene.