Brincidofovir treatment of acyclovir-resistant disseminated varicella zoster virus infection in an immunocompromised host.

Brincidofovir (BCV) is a broad-spectrum antiviral agent active in vitro against double-stranded DNA viruses including herpesviruses, adenoviruses, polyomaviruses, and poxviruses. We report successful BCV use in management of disseminated acyclovir- and cidofovir-resistant varicella zoster virus in an immunocompromised hematopoietic stem cell transplant patient with chronic graft-versus-host disease who was intolerant to foscarnet.

Adenosine-mediated inhibition of platelet aggregation by acadesine. A novel antithrombotic mechanism in vitro and in vivo.

Inhibition of platelet aggregation by acadesine was evaluated both in vitro and ex vivo in human whole blood using impedance aggregometry, as well as in vivo in a canine model of platelet-dependent cyclic coronary flow reductions. In vitro, incubation of acadesine in whole blood inhibited ADP-induced platelet aggregation by 50% at 240 +/- 60 microM. Inhibition of platelet aggregation was time dependent and was prevented by the adenosine kinase inhibitor, 5'-deoxy 5-iodotubercidin, which blocked conversion of acadesine to its 5'-monophosphate, ZMP, and by adenosine deaminase. Acadesine elevated platelet cAMP in whole blood, which was also prevented by adenosine deaminase. In contrast, acadesine had no effect on ADP-induced platelet aggregation or platelet cAMP levels in platelet-rich plasma, but inhibition of aggregation was restored when isolated erythrocytes were incubated with acadesine before reconstitution with platelet-rich plasma. Acadesine (100 mg/kg i.v.) administered to human subjects also inhibited platelet aggregation ex vivo in whole blood. In the canine Folts model of platelet thrombosis, acadesine (0.5 mg/kg per min, i.v.) abolished coronary flow reductions, and this activity was prevented by pretreatment with the adenosine receptor antagonist, 8-sulphophenyltheophylline. These results demonstrate that acadesine exhibits antiplatelet activity in vitro, ex vivo, and in vivo through an adenosine-dependent mechanism. Moreover, the in vitro studies indicate that inhibition of platelet aggregation requires the presence of erythrocytes and metabolism of acadesine to acadesine monophosphate (ZMP).

Accelerated neutrophil apoptosis in the acquired immunodeficiency syndrome.

Neutrophil (PMNL) function defects occur as a consequence of HIV infection. This study examined PMNL apoptosis in patients with the acquired immunodeficiency syndrome (AIDS) to determine if accelerated apoptosis contributes to impaired function. PMNL were isolated from 10 HIV-infected patients with CD4+ lymphocyte counts < 200/mm3 without signs of active infection and 7 healthy volunteers. PMNL were stained with acridine orange and ethidium bromide after 0, 3, 6, and 18 h in culture, and examined for the morphologic changes of apoptosis and viability by fluorescent microscopy. Apoptosis was also demonstrated by electron microscopy, flow cytometry, and DNA gel electrophoresis. Apoptosis was minimal at 0 h, but PMNL from AIDS patients exhibited significantly greater apoptosis than controls at 3 h (22.5+/-11.5 vs. 8.9+/-6.9%, P = 0.015), 6 h (38.1+/-14.2 vs. 18.1+/-4.5%, P = 0.003), and 18 h (71.3+/-19.0 vs. 38.8+/-16.7%, P = 0.002). Viabilities were > or = 88.0% for both groups from 0-6 h, but by 18 h viability was significantly decreased for the HIV group (58.8+/-12.4 vs. 83.5+/-10.4%, P = 0.001) due to an increase in non-viable apoptotic cells. Incubation with serum from AIDS patients had no effect on control PMNL, and incubation with control serum did not reduce the rate of apoptosis of PMNL from AIDS patients. Incubation with granulocyte colony-stimulating factor (G-CSF) in vitro significantly decreased apoptosis for PMNL from AIDS patients. PMNL from patients with AIDS exhibit markedly accelerated apoptosis ex vivo. In vivo, apoptosis and functional impairment of PMNL may contribute to the risk of secondary infections, and cytokine therapy may be of potential clinical benefit in this circumstance.

Activated human polymorphonuclear leucocytes reduce rabbit papillary muscle function: role of the CD18 glycoprotein adhesion complex.

STUDY OBJECTIVE: The aim was to determine if human polymorphonuclear leucocytes activated by human recombinant C5a (hrC5a) reduce the contractile function of the isolated papillary muscle and if this response depends upon the functional integrity of the CD18 glycoprotein adhesion complex. DESIGN: Human neutrophils with or without pretreatment with monoclonal antibodies to the CD18 adhesion complex were added to organ baths containing isolated papillary muscles of the rabbit and activated with hrC5a. Changes in papillary muscle function were measured. EXPERIMENTAL MATERIAL: 52 right ventricular papillary muscles isolated from rabbit and neutrophils isolated from human whole blood were used. MEASUREMENTS AND MAIN RESULTS: Neither neutrophils nor hrC5a alone reduced papillary muscle function, but activation of neutrophils with hrC5a provoked reduction in myocardial contractility. This correlated with the degree of neutrophil stimulation, assessed by cell aggregation. Pretreatment of neutrophils with antibodies to the CD18 adhesion complex significantly attenuated the neutrophil induced contractile impairment. CONCLUSIONS: Activated human neutrophils can impair contractile function of papillary muscles, which is dependent upon adhesion of the leucocytes to the muscle via the CD18 complex.

Acadesine extends the window of protection afforded by ischaemic preconditioning in conscious rabbits.

OBJECTIVE: Ischaemic preconditioning protects myocardium from infarction if the reperfusion interval between the brief and prolonged ischaemic intervals is less than 1 h. In anaesthetised rabbits acadesine (5-amino-4-imidazolecarboxamide riboside, AICAR), an adenosine enhancer which increases tissue adenosine during ischaemia, prolongs the window of protection to 2 h. The aim of this study was to try to determine the maximum extension of this window of protection, using chronically instrumented, unsedated rabbits. METHODS: Rabbits were instrumented with a balloon occluder around a major branch of the left coronary artery for reversible coronary occlusion. Five to seven days after surgery all animals underwent a 30 min coronary occlusion. Animals were randomised to one of seven groups: (1) No additional treatment (control); (2) Ischaemic preconditioning with 5 min regional ischaemia followed by 10 min reperfusion before the 30 min coronary occlusion; (3) and (4) Ischaemic preconditioning followed by 2 or 4 h of reperfusion before the 30 min occlusion, respectively; (5) Treatment with acadesine (2.5 mg.kg-1.min-1 intravenously for 5 min and then 0.5 mg.kg-1.min-1 beginning 45 min before and continuing until 30 min after release of the 30 min occlusion) without ischaemic preconditioning; (6) and (7) Treatment with the higher dose of acadesine for 5 min beginning 35 min before the 5 min ischaemic period, and then the lower dose continuing until 30 min after release of the 30 min coronary occlusion in rabbits with 4 or 6 h reperfusion intervals, respectively. RESULTS: Rabbits with ischaemic preconditioning with 10 min reperfusion preceding the 30 min coronary occlusion (group 2) had only 5.6(SEM 1.1)% infarction of the ischaemic zone. Ischaemic preconditioning followed by 2 h reperfusion (group 3) offered continued protection [18.2(2.2)% infarction] as compared to control animals [37.7(2.6)% infarction]. However, protection waned if ischaemic preconditioning was followed by 4 h reperfusion (group 4) [36.7(3.0)% infarction]. Additionally, treatment with acadesine alone did not modify infarct size (group 7) [39.5(4.0)%], but acadesine largely restored the protection of ischaemic preconditioning despite a 4 h reperfusion interval (group 5) [20.4(3.0)% infarction, P < 0.01 v control]. However, when reperfusion was extended to 6 h (group 6) acadesine could no longer restore protection [36.2(0.9)% infarction]. CONCLUSIONS: The protection afforded by a 5 min ischaemic preconditioning period lasts from 2 to 4 h in the awake, unsedated rabbit, and acadesine can extend the duration of this window of protection to at least 4 h but not to 6 h.

Carbohydrate- and CD18-dependent neutrophil adhesion to cardiac myocytes: effects of adenosine.

OBJECTIVE: Adenosine inhibits neutrophil adhesion and injury to isolated cardiac myocytes. In the present study, the contribution of selectin and CD18 interactions to neutrophil-myocyte adhesion and their sensitivity to adenosine were assessed. METHODS: Activated human neutrophils and canine myocytes were incubated with inhibitors of CD18 or selectin binding, adenosine, or combinations of both for 30-50 min at 37 degrees C. Neutrophils were pretreated with 0.1 microM fMLP for 10 min to study L-selectin-independent adhesion. Adhesion was measured by phase contrast microscopy. RESULTS: Anti-L-selectin mAb and the selectin-blocking carbohydrates sialyl Lewisx or mannose-6-phosphate, as well as anti-CD18 or anti-ICAM-1 mAbs, inhibited cell adhesion (by 84-99%, P < 0.05). CD11a, but not CD11b, was responsible for most of the CD18-mediated binding. An L-selectin-independent interaction between neutrophils and cardiac myocytes was observed that was delayed (peak adhesion at 40-50 min, rather than 30 min), but still inhibited by anti-CD18 mAb (by 65 +/- 11%, P < 0.05) and carbohydrates (by 87-97%, each P < 0.05). Adenosine (100 nM) inhibited this late CD18-dependent/L-selectin-independent phase of adhesion (by 61 +/- 14%, P < 0.05). The combination of adenosine and anti-CD18 mAb was additive such that adhesion was completely blocked (P < 0.05, compared to either agent alone). Inhibition of adhesion by adenosine was prevented by the A2 antagonist, DMPX (100 nM), and mimicked by the A2 agonist, CGS-21680 (10 nM) or the adenosine regulating agents, acadesine (100 microM) or GP531 (10 microM). CONCLUSION: Neutrophil-myocyte adhesion involved both L-selectin-dependent and L-selectin-independent carbohydrate binding as well as CD11a/CD18. Inhibition of adhesion by adenosine interferes with L-selectin-independent carbohydrate binding and possibly CD18.

Acadesine extends the window of protection afforded by ischaemic preconditioning.

OBJECTIVE: The aim was to test whether acadesine (5-amino-4-imidazolecarboxamide riboside, AICAR), an adenosine regulating agent which increases tissue adenosine during ischaemia, could prolong the window of protection from ischaemic preconditioning. METHODS: A branch of the left coronary artery of a rabbit heart was occluded for 30 min and reperfused for 180 min to induce infarction. Infarct size was determined with triphenyl tetrazolium staining. Prior to the 30 min ischaemia, rabbits were subjected to one of the following seven protocols: (1) No treatment (controls). (2) Preconditioning with 5 min of regional ischaemia followed by 2 h of reperfusion. (3) Treatment with acadesine (2.5 mg.kg-1.min-1 intravenously for 5 min starting 155 min prior to 30 min ischaemia followed by 210 min infusion of 0.5 mg.kg-1.min-1. (4) Treatment with acadesine (same schedule as in group 3) plus preconditioning as in group 2. (5) Treatment with acadesine for a shorter period (acadesine 2.5 mg.kg-1.min-1 for 5 min starting 30 min prior to preconditioning followed by 0.5 mg.kg-1.min-1 for only 60 min) plus preconditioning as in group 2. (6) Treatment with preconditioning followed by adenosine receptor blockade with 8-(p-sulphophenyl)theophylline (SPT) 10 mg.kg-1 intravenously immediately after and again 15 min after preconditioning. (7) Treatment with short infusion of acadesine plus preconditioning plus SPT. RESULTS: Preconditioning followed by 2 h of reperfusion offered little protection against infarction [28.6(SEM 2.7)% of the ischaemic zone infarcted] as compared to control [38.7(3.1)% infarction]. Treatment with acadesine alone did not modify the infarct size [37.8(3.5)%], but both of the acadesine plus preconditioning groups showed a significant limitation of infarct size with 13.9(3.1)% infarction in group 4 and 12.7(2.2)% infarction in group 5 (both p < 0.01 v control). Although SPT alone did not modify the infarct size [26.8(3.3)%], SPT blocked the protective effect of acadesine [25.3(2.9)%, p < 0.05 v group 5]. CONCLUSION: Acadesine can delay the natural decay of preconditioning. This delay appeared to be mediated by adenosine and may have therapeutic potential.

Impaired endothelium-dependent relaxations in rabbits subjected to aortic coarctation hypertension.

Rabbits were rendered hypertensive by suprarenal coarctation of the abdominal aorta. Seven days later, endothelium-dependent and endothelium-independent vascular relaxations were examined in vascular rings taken from hypertensive (thoracic aorta, carotid artery) and normotensive (abdominal aorta) regions. Relaxation of phenylephrine-contracted rings in response to endothelium-dependent agonists (acetylcholine, A23187) was impaired, compared with that in sham-operated and intact controls, in regions exposed to the elevated blood pressure (i.e., above the coarctation). Responses to acetylcholine and A23187 in the abdominal aorta, below the coarctation, were not altered. The diminished endothelium-dependent responses in the thoracic aorta were not affected by pretreatment with the cyclooxygenase inhibitor indomethacin. In contrast to acetylcholine and A23187, responses to the endothelium-independent agonist nitroprusside were not attenuated in vessels from hypertensive regions, indicating that the defect occurred in the endothelium. The EC50 for acetylcholine-induced relaxations of thoracic aorta correlated significantly with mean arterial pressure above the coarctation, indicating that the extent to which endothelium-dependent relaxation is impaired is in proportion to the degree of blood pressure elevation. This study suggests that the diminished relaxations by endothelium-dependent agonists is a local response to the elevation of blood pressure and is not due to a circulating factor.

Thromboxane synthase inhibition enhances action of converting enzyme inhibitors.

Mean arterial blood pressure was measured over a 24-hour period from the femoral artery of conscious, unrestrained spontaneously hypertensive rats. Oral administration of the angiotensin converting enzyme inhibitor CGS 16617 significantly lowered mean arterial pressure. In contrast, both the thromboxane synthase inhibitor CGS 12970 and the thromboxane receptor antagonist BM 13505 lacked an antihypertensive action in the spontaneously hypertensive rat. When administered concurrently, the thromboxane synthase inhibitor CGS 12970 potentiated the antihypertensive action of the angiotensin converting enzyme inhibitor CGS 16617. This effect was not observed with the thromboxane receptor antagonist BM 13505. In addition to CGS 16617, CGS 12970 also potentiated the hypotensive effect of two structurally dissimilar angiotensin converting enzyme inhibitors, benazapril HCL and captopril. Indomethacin blocked the thromboxane synthase inhibition-induced potentiation of the antihypertensive action of angiotensin converting enzyme inhibitors. The thromboxane synthase inhibitor CGS 12970 had no effect on the hypotension induced by hydralazine, indicating that the hypotension is not a nonspecific action related to the fall in blood pressure. These results may suggest that converting enzyme inhibition augments the levels and actions of a hormone that stimulates prostaglandin formation. It is well established that thromboxane synthase inhibitors eliminate the formation of the vasoconstrictor thromboxane A2 and allow reorientation of eicosanoid production toward the formation of vasodilating prostaglandins, which could enhance the antihypertensive action of angiotensin converting enzyme inhibitors.

Presence of cytochrome P-450-dependent monooxygenase in intimal cells of the hog aorta.

Cytochrome P-450-dependent mixed function oxidase activity is present in vascular tissue; however, as far as we could determine, the distribution of monooxygenase activity across the blood vessel wall has not previously been assessed. The aryl-hydrocarbon hydroxylase activity was examined by metabolism of benzo[a]pyrene in microsomes prepared from intimal and smooth muscle cell scrapings of the hog thoracic aorta. Microsomes of intimal cells comprising 95% endothelial cells showed an approximately 2.5-fold increase in aryl-hydrocarbon hydroxylase activity compared with that in microsomes prepared from medial smooth muscle cells. Michaelis-Mentin kinetics for the intimal enzyme yielded an apparent Km value of 11.11 microM and an apparent Vmax of 3-OH benzo[a]pyrene of 40 pmol/mg protein/10 min. Aryl-hydrocarbon hydroxylase activity was dependent on nicotinamide adenine dinucleotide phosphate and was inhibited by 7,8 benzoflavone, SKF 525A, and carbon monoxide. The localization of cytochrome P-450-dependent mixed function oxidase primarily to the intimal surface of the aorta may indicate a role for this enzyme system in vasoregulation and the pathogenesis of atherosclerosis.

Release of a neutrophil-derived vasoconstrictor agent which augments platelet-induced contractions of blood vessels in vitro.

1. The effects of neutrophil-derived products on vascular tone in vitro were examined by adding purified rabbit neutrophils to siliconized organ baths containing rings of rabbit thoracic aorta. 2. Neutrophil-derived products induced a concentration-dependent contraction of the blood vessels generating 3.9 +/- 0.2 g of tension at a cell concentration of 2 X 10(6) ml-1. This contractile response was not dependent on an intact endothelium and was not ameliorated by treatment of the neutrophils with inhibitors of lipoxygenase or cyclo-oxygenase enzymes, by free radical scavengers or by the use of end organ antagonists to angiotensin II, desArg9-bradykinin, histamine, catecholamines or acetylcholine. 3. Neutrophils stimulated with phorbol myristate acetate, formyl methionyl-leucyl-phenylalanine or calcium ionophore A23187 released a contractile factor into the supernatant which produced qualitatively similar contractions compared to those elicited by incubation with intact unstimulated neutrophils. 4. Ten to twenty times more platelets were required to evoke equivalent contractions to those observed with neutrophils. However, neutrophil supernates significantly augmented platelet-mediated contractions (P less than 0.001). 5. Contractions elicited by neutrophils and supernates derived from activated neutrophils were partially antagonized by 5-hydroxytryptamine (5-HT) receptor antagonists, methysergide and ketanserin. However, the inhibition of exogenous 5-HT-induced contractions on rabbit aorta and rat stomach strips by both antagonists was greater than the inhibition of contractions produced by neutrophils and neutrophil-derived supernates. 6. Extraction of the biologically active material from supernatants of activated neutrophils into acetone, but not chloroform-methanol or ethyl acetate, suggests the contractile factor may be a protein/peptide. Partial purification on a Sephadex G100 column yields a contractile factor with a molecular weight of less than 4,000 daltons. 7. This factor may augment vascular tone, either directly or by interactions with platelets, in pathophysiological states associated with neutrophil activation and accumulation.

Prostacyclin can either increase or decrease heart rate depending on the basal state.

1 The influence of the basal heart rate on the change in rate induced by prostacyclin (PGI2) was investigated in beagles anaesthetized with chloralose. 2 In male dogs with a low basal heart rate (less than 100 beats/min) PGI2, in doses up to 0.5 microgram/kg intravenously, induced hypotension and tachycardia. 3 In contrast, PGI2-induced hypotension was accompanied by bradycardia when either the basal heart rate was increased (greater than 130 beats/min) with isoprenaline or nitroprusside, or the dose of PGI2 was increased. 4 Female beagles were less sensitive than males to the stimulation of a reflex bradycardia by PGI2. 5 The influence of prostaglandin E2 (PGE2) and bradykinin on heart rate was also found to depend upon the basal state in some dogs. 6 Bilateral vagotomy reversed the bradycardia provoked by PGI2, PGE2 and bradykinin. 7 Thus, PGI2-induced bradycardia in dependent on both the dose and the basal heart rate. Similarly the effects of PGE2 and bradykinin on heart rate also depend upon the basal state in some dogs. Moreover, there is a correlation between the ability of all three agonists to induce bradycardia, suggesting a common mechanism of action.

Protection against injury during ischemia and reperfusion by acadesine derivatives GP-1-468 and GP-1-668. Studies in the transplanted rat heart.

BACKGROUND: Acadesine (AICAr: 5-amino-4-imidazole carboxamide riboside) has been shown to afford sustained protection against injury during ischemia and reperfusion. The present studies used the heterotopically transplanted rat heart to assess the protective properties of two new acadesine analogs: GP-1-468 and GP-1-668. METHODS AND RESULTS: Hearts were excised, arrested with a 2-minute infusion of cardioplegic solution, and subjected to 4 hours of global ischemia (20 degrees C) with cardioplegic reinfusion for 2 minutes every 30 minutes. The hearts were then transplanted (1 hour of additional ischemia) into the abdomens of recipient rats and reperfused in situ for 30 minutes or 24 hours. The hearts were then excised, perfused aerobically for 20 minutes, and contractile function was assessed. GP-1-468 or GP-1-668 was administered to donor rats (20 mg/kg intravenously, 30 minutes before excision). They were also added to the cardioplegic solution (10 mumol/L for GP-1-468, 5 mumol/L for GP-1-343, the active metabolite of GP-1-668) and were also given to recipient rats (20 mg/kg intravenously, 30 minutes before transplantation, so that the drugs were present during reperfusion). Nine groups of hearts were studied. Three groups of studies were carried out (n = 24 transplants for each group). The first group of hearts was reperfused for 30 minutes, the second group was reperfused for 24 hours, and the third group was transplanted but not reperfused; instead, they were frozen at the end of 5 hours of ischemia and taken for metabolite analysis. Within each group were three subgroups (n = 8 per group) receiving GP-1-468, GP-1-668, or saline solution. In the 30-minute reperfusion group the recoveries of left ventricular developed pressure were 88 +/- 4, 87 +/- 7, and 50 +/- 9 mm Hg, respectively (p < 0.05 versus saline-treated controls); left ventricular volumes (recorded at 12 mm Hg) were 112 +/- 20, 132 +/- 28, and 41 +/- 9 microliters, respectively (p < 0.05 versus saline-treated controls), and coronary flows were 13.1 +/- 0.7, 13.4 +/- 1.0, and 9.9 +/- 0.5 ml/min, respectively (p < 0.05 versus saline-treated controls). In addition to improving functional recovery, the two analogs increased the tissue content of adenosine at the end of the ischemic period (5.4 +/- 0.6 and 7.3 +/- 0.5 mumol/gm dry weight, respectively, versus 2.7 +/- 0.4 mumol/gm dry weight in the saline-treated controls; p < 0.05); however, they did not influence adenosine triphosphate or its catabolites. In the 24-hour reperfusion group the corresponding values were 77 +/- 6 and 88 +/- 6 versus 35 +/- 4 mm Hg for left ventricular developed pressure (p < 0.05), 111 +/- 9 and 121 +/- 11 versus 41 +/- 8 microliters for left ventricular volume (p < 0.05), and 13.7 +/- 0.7 and 13.0 +/- 0.6 versus 11.7 +/- 0.7 ml/min for coronary flow (no significant difference). Thus both analogs afforded an early and comparable degree of protection of contractile function that was sustained even after 24 hours of reperfusion. CONCLUSIONS: Both GP-1-468 and GP-1-668 increase the rate and extent of early postischemic recovery, and this protection is sustained for at least 24 hours. These beneficial actions were associated with an increase of the tissue content of adenosine during ischemia, but they appeared to be independent of the status of the high-energy metabolism.

Activated neutrophils release mediators that may contribute to myocardial injury and dysfunction associated with ischemia and reperfusion.

Neutrophils accumulate in the ischemic myocardium and exacerbate postischemic cardiac dysfunction and injury. The formation of lipoxygenase metabolites of AA, derived either directly from the neutrophils or by interactions with other blood elements or cells, may promote neutrophil-mediated injury. Recognition of the roles played by neutrophils and AA metabolites in reperfusion injury may lead to the development of new therapies that can be used in conjunction with thrombolytic drugs to reduce the complications associated with restoring blood flow to the ischemic heart.

Modulation of vascular tone by 12(R)-, but not 12(S)-, hydroxyeicosatetraenoic acid.

The vascular activity of the two stereoisomers of 12-hydroxyeicosatetraenoic acid (12-HETE) was assessed on rabbit thoracic aortic rings. In vessels contracted in K+-free media, 12(R)-HETE inhibited the relaxations produced by the addition of KCl, in a concentration-dependent manner, while 12(S)-HETE had little effect. 12(R)-HETE, but not the 12(S) isomer, potentiated phenylephrine-induced contractions of aortic rings in normal buffer. Thus 12(R)-HETE can modulate vascular tone, perhaps by inhibition of Na+,K+-ATPase activity.

Prostacyclin mediates the potentiated hypotensive effect of bradykinin following captopril treatment.

The effect of angiotensin-converting enzyme inhibition by captopril on the release of a prostacyclin-like substance by bradykinin, angiotensin I and angiotensin II was studied by means of the blood-bathed bioassay technique of Vane. Administration of captopril abolished the release of prostacyclin-like substance induced by angiotensin I, potentiated the release provoked by bradykinin and did not alter that due to angiotensin II. Potentiation of the bradykinin-induced renal vasodilatation with captopril could be completely reversed by indomethacin, which also abolished the kinin-induced release of prostacyclin-like substance. Potentiation of the bradykinin-induced hypotension was markedly attenuated but not completely reversed by cyclo-oxygenase inhibition. It is suggested that following converting inhibition increased production of prostacyclin by elevated kinin levels may contribute to the antihypertensive action of angiotensin-converting enzyme inhibitors.

The interactions of platelet-derived mediators on isolated canine coronary arteries.

The interactions of 5-hydroxytryptamine (5-HT) and a thromboxane A2 mimetic, U44069, were investigated on rings of canine coronary arteries. 5-HT (10(-8) - 10(-5) M) and U44069 (10(-9) - 10(-6) M) produce coronary vasoconstriction which is potentiated by indomethacin (3 X 10(-6) M). In the presence of 10(-9) M 5-HT, U44069-induced vasoconstriction is enhanced. Similarly, 10(-9) M U44069 enhances the effects of 5-HT. In both cases this potentiation is greatest in the presence of indomethacin. Synergism between the platelet-derived mediators could contribute to coronary vasospasm and myocardial ischaemia.

Cardioprotection with a novel adenosine regulating agent mediated by intravascular adenosine.

Adenosine is cardioprotective in models of myocardial stunning and infarction, but the precise compartment within the heart in which adenosine elicits its cardioprotective effects has not been determined. The goals of the present study were to (i) investigate the effects of a novel adenosine regulating agent, GP531 (5-amino-1-beta-n-(5-benzylamino-5-deoxyribofuranosyl) imidazole-4-carboxamide), on post-ischemic myocardial function, and (ii) examine the contribution of endogenous adenosine in the intravascular and interstitial compartments in mediating the beneficial effects. Pigs were instrumented for measurement of myocardial segment shortening, and for sampling of coronary venous blood and myocardial interstitial fluid for determination of adenosine concentration. Myocardial dysfunction was induced by 4 x 8 min coronary occlusions, and recovery of regional function was monitored for 2 h. In control pigs, function recovered to 24 +/- 2% of baseline after 2 h. Treatment with GP531 improved functional recovery to 55 +/- 3%. GP531-mediated cardioprotection was prevented by adenosine receptor blockade with 8-sulfophenyltheophylline (23 +/- 2%). GP531 did not affect basal adenosine levels, but caused a 2-fold greater increase in vascular adenosine concentration with ischemia (54.6 +/- 10.6 vs. 28.1 +/- 8.0 microM in controls. P < 0.05). In contrast, the interstitial adenosine concentration was not significantly different in treated vs. untreated control pigs (9.4 +/- 3.9 vs. 15.0 +/- 1.8 microM in controls). These data indicate that (1) GP531 improves recovery of myocardial function following ischemia reperfusion injury via an adenosine receptor-dependent mechanism, and (2) the cardioprotection is associated with increased intravascular, but not interstitial, adenosine concentration during ischemia. Therefore, we conclude that cardioprotection elicited by GP531-enhanced endogenous adenosine is dependent on an intravascular site of action.

Dual activation of adenosine A1 and A3 receptors mediates preconditioning of isolated cardiac myocytes.

Ischemic preconditioning reduces post-ischemic myocardial injury by activating myocellular adenosine A1 receptors. Adenosine A3 receptors have also been implicated but there is no evidence for A3 receptors in cardiac myocytes. The aim of this study was to develop a model of preconditioning in isolated cardiac myocytes to evaluate the role of the adenosine A1 and A3 receptors in preconditioning-induced protection from ischemic injury. Reverse transcription polymerase chain reaction (PCR) was also employed to establish the presence of adenosine A3 receptors in these cells. In the preconditioning studies, ischemic injury was simulated by exposing isolated rabbit myocytes (placed in the cell chamber and paced at l Hz) to buffer containing (in mM) 2'-deoxyglucose (20), NaCN (1), Na (+)-lactate (20), KCl (10) at pH 6.6 (37 degrees C). Changes of diastolic and systolic cell length were monitored with an optical-video edge imaging system, and hypercontracture was assessed as an index of irreversible cell injury. Preconditioning (2 min brief ischemia and 15 min reperfusion) significantly reduced cell injury resulting from a subsequent prolonged ischemia (10 min) and reperfusion (15 min), as indicated by a reduction in the incidence of cell hypercontracture from 67 +/- 6% to 29 +/- 5% (P < 0.001). Preconditioning-induced cardioprotection was only partially blocked by a maximally effective concentration (100 nM) of the adenosine A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (cell hypercontracture = 43 +/- 3%, P < 0.05 vs. control) but completely blocked by either the combination of DPCPX (100 nM) with the adenosine A1/A3 receptor antagonist DPCPX +8-(4-carboxyethylphenyl)-1,3-dipropylxanthine (BWA1433; 1 microM) or the non-selective adenosine receptor antagonist, 8-(p-sulfophenyl)theophylline (8-SPT; 100 microM) (cell hypercontracture = 64 +/- 4%, 59 +/- 5%, respectively; P = NS vs. control). In non-hypercontractured myocytes, preconditioning also substantially enhanced the recovery of the contractile amplitude and, similarly, this effect was only partially blocked by DPCPX but completely blocked by either the combination of DPCPX with BWA1433, or 8-SPT. These studies suggest that preconditioning protects isolated cardiac myocytes from ischemic injury independent of other cell types, and that maximal preconditioning-induced cardioprotection requires activation of both adenosine A1 and A3 receptors. Reverse transcription-PCR using primers for the rabbit receptor provide evidence for the presence of adenosine A3 receptors in these cells.

Biotransformation and cardiovascular effects of arachidonic acid in the dog.

The biotransformation and cardiovascular effects of arachidonic acid (AA) were studied in the circulation of anaesthetized dogs. Arterial blood was continuously bioassayed for arachidonate metabolites using the blood-bathed organ technique of Vane. AA (5-10 microgram/ml) infused into an incubation coil of flowing blood was converted into a labile substance which contracted the vascular tissues (rabbit aorta, RbA; rabbit coeliac and mesenteric arteries, RbCA and RbMA; bovine coronary artery, BCA) and the gastrointestinal smooth muscle strips (rat stomach strip, RSS; rat colon, RC). These effects could be mimicked by exogenously generated thromboxane A2 (TXA2). Conversion of AA was inhibited by indomethacin and the selective thromboxane synthetase inhibitor, imidazole (100 microgram/ml). The half-life of TXA2 in blood was 30-47 sec, a similar value to that found in aqueous solutions at 37 degrees C. PGH2 was also converted in blood to other product(s) which contracted RSS and RC, relaxed RbCA and RbMA but had little effect on RbA. Intravenous infusion of AA (50-800 microgram kg-1 min-1) caused effects on the bioassay tissues which could be mimicked by prostacyclin. The AA infusion also induced falls in pulmonary and systemic arterial pressures and bradycardia. All effects were abolished by indomethacin (5 mg/kg) or aspirin (200 mg/kg). Radioimmunoassay confirmed that the major product of intravenously infused AA was 6-oxo-PGF1alpha, the chemical degradation product of prostacyclin. Thus, although AA is transformed to the vasoconstrictor TXA2 when incubated for sufficient time with blood alone, on rapid pulmonary transit it is transformed into a prostacyclin-like substance.