Rosuvastatin reduces neointima formation in a rat model of balloon injury.

BACKGROUND: Processes of restenosis, following arterial injury, are complex involving different cell types producing various cytokines and enzymes. Among those enzymes, smooth muscle cell-derived matrix metalloproteinases (MMPs) are thought to take part in cell migration, degrading of extracellular matrix, and neointima formation. MMP-9, also known as gelatinase B, is expressed immediately after vascular injury and its expression and activity can be inhibited by statins. Using an established in vivo model of vascular injury, we investigated the effect of the HMG-CoA reductase inhibitor rosuvastatin on MMP-9 expression and neointima formation. MATERIALS AND METHODS: 14-week old male Sprague Dawley rats underwent balloon injury of the common carotid artery. Half of the animals received rosuvastatin (20 mg/kg body weight/day) via oral gavage, beginning 3 days prior to injury. Gelatinase activity and neointima formation were analyzed 3 days and 14 days after balloon injury, respectively. 14 days after vascular injury, proliferative activity was assessed by staining for Ki67. RESULTS: After 14 days, animals in the rosuvastatin group showed a decrease in total neointima formation (0.194±0.01 mm2 versus 0.124±0.02 mm2, p<0.05) as well as a reduced intima/media ratio (1.26±0.1 versus 0.75±0.09, p<0.05). Balloon injury resulted in increased activity of MMP-9 3 days after intervention for both rosuvastatin treated animals and controls with no significant difference observed between the groups. There was a trend towards a reduction in the number of Ki67-positive cells 14 days after injury. CONCLUSIONS: Rosuvastatin attenuates neointima formation without affecting early MMP-9 activity in a rat model of vascular injury.

Endothelin-1 induces CD40 but not IL-6 in human monocytes via the proinflammatory transcription factor NF-kappaB.

UNLABELLED: The vasoactive peptide endothelin-1 (ET-1) may contribute to the pathogenesis of atherosclerosis and its acute complications. Because inflammation of the vessel wall is a characteristic feature of atherosclerosis, this study investigated the effect of ET-1 on the proinflammatory transcription factor NF-kappaB in monocytes. Monocyte/macrophages are a major source of inflammatory mediators in atheroma and are located in rupture prone plaque areas. In human monocytes ET-1 caused NF-kappaB activation. Specificity of ET-1-induced NF-kappaB activation was ascertained by supershift and competition experiments. This ET-1 effect was blocked by the ET-A-receptor antagonist BQ-123 but not by the ET-B-receptor antagonist BQ-788. PI-1, a specific inhibitor of the IkappaB-alpha-degrading proteasome complex, also prevented NF-kappaB activation. ET-1 stimulated expression of the proinflammatory molecule CD40 but not of the cytokine IL-6 in a NF-kappaB-dependent manner. CONCLUSION: The data demonstrate the ability of ET-1 to activate inflammatory pathways in human monocytes differentially.

Endothelin-1 induces interleukin-6 release via activation of the transcription factor NF-kappaB in human vascular smooth muscle cells.

The potent vasoconstrictor peptide endothelin-1 (ET-1) has been implicated in the pathophysiology of atherosclerosis and its complications. Since inflammation of the vessel wall is a hallmark of atherosclerosis, the purpose of the present study was to investigate the influence of ET-1 on cytokine production in human vascular smooth muscle cells (SMC) as a marker of inflammatory cell activation. ET-1 (100 pM - 1 microM) stimulated interleukin-6 (IL-6) secretion from human vascular SMC in a concentration-dependent manner. The ET-A-receptor antagonist BQ-123 (10 microM), but not the ET-B-receptor antagonist BQ-788, inhibited IL-6 release. ET-1 also transiently increased IL-6 mRNA compatible with regulation of IL-6 release at the pretranslational level. Electrophoretic mobility shift assays demonstrated time- and concentration-dependent activation of the proinflammatory transcription factor nuclear factor-kappaB (NF-kappaB) in ET-1-stimulated human vascular SMC. A decoy oligodeoxynucleotide bearing the NF-kappaB binding site inhibited ET-1-stimulated IL-6 release to a great extent suggesting that this transcription factor plays a key role for cytokine production elicited by ET-1. Moreover, the antioxidant pyrrolidine dithiocarbamate (10 microM) inhibited ET-1-induced IL-6 release indicating involvement of reactive oxygen species in ET-1 signaling. ET-1-stimulated IL-6 secretion was also suppressed by diphenylene iodonium (40 microM), an inhibitor of flavon-containing enzymes such as NADH/NADPH oxidase. The results demonstrate the ability of ET-1 to induce an inflammatory response in human vascular SMC. These observations may contribute to a better understanding of the role of ET-1 in inflammatory activation of the vessel wall during atherogenesis.

Systemic thrombin inhibition by Hirulog does not alter medial smooth muscle cell proliferation and inflammatory activation after vascular injury in the rabbit.

The study evaluated the role of thrombin in activation of vascular smooth muscle cells early after vascular injury. The direct thrombin inhibitor Hirulog (10 mg/kg SQ tid) or vehicle was administered to rabbits over 3 days following balloon injury to the abdominal aorta and the right iliac artery. Hirulog treatment yielded marked systemic anticoagulation as evidenced by an about 3.5-fold prolongation of quantitative thrombin time one hour after an injection, but with a reduction to almost baseline levels at the end of the dosing interval. After 3 days, proliferating cells in the right iliac artery were enumerated. The expression of intercellular adhesion molecule 1, macrophage-colony stimulating factor, tumor necrosis factor alpha, and interleukin-1beta as markers for inflammatory activation of the vessel wall was examined by immunohistochemistry and graded semiquantitatively. Mitotic indices did not differ between control and Hirulog-treated animals. There was also no difference in the expression of markers of inflammatory activation between both groups. In conclusion, thrombin inhibition by Hirulog administration does not reduce acutely (within 3 days) vascular smooth muscle cell proliferation or inflammatory activation after angioplasty. Thrombin inhibitors may therefore limit restenosis in the rabbit by acting later or via other, unknown pathways. The lack of effect of the thrombin inhibitor on the cellular events during the early phase of the response to balloon injury may explain the failure of such strategies to reduce restenosis in recent clinical trials despite effects towards acute thrombotic complications. Together, these results suggest that acute thrombin generation is not a crucial stimulus for early smooth muscle cell proliferation and inflammatory activation after vascular injury.

Angiotensin induces inflammatory activation of human vascular smooth muscle cells.

Multiple data suggest that the renin-angiotensin system contributes to the pathogenesis of atherosclerosis. The atherogenic effect of the renin-angiotensin system can only in part be explained by the influence of its effector angiotensin II on blood pressure, smooth muscle cell (SMC) growth, or antifibrinolytic activity. Because chronic inflammation of the vessel wall is a hallmark of atherosclerosis, we hypothesized that angiotensin II may elicit inflammatory signals in vascular SMCs. Human vascular SMCs were stimulated with angiotensin. Inflammatory activation was assessed by determination of interleukin-6 (IL-6) release into the culture medium, detection of IL-6 mRNA by RT-PCR, and demonstration of activation of nuclear factor-kappaB in electrophoretic mobility shift assays. Angiotensin II concentration-dependently (1 nmol/L to 1 micromol/L) stimulated IL-6 production by SMCs via activation of the angiotensin II type 1 receptor (demonstrated by the inhibitory action of the receptor antagonist losartan). Angiotensin I increased IL-6 production by SMCs, too. This effect was inhibited by captopril and ramiprilat, suggesting conversion of angiotensin I to angiotensin II by angiotensin-converting enzyme in SMCs. Steady-state mRNA for IL-6 was augmented after stimulation with angiotensin II, suggesting regulation of angiotensin-induced IL-6 release at the pretranslational level. Moreover, the proinflammatory transcription factor nuclear factor-kappaB, which is necessary for transcription of most cytokine genes, was also activated by angiotensin II. Pyrrolidine dithiocarbamate suppressed angiotensin II-induced IL-6 release, a finding compatible with involvement of reactive oxygen species as second messengers in cytokine production mediated by angiotensin. The data demonstrate the ability of angiotensin to elicit an inflammatory response in human vascular SMCs by stimulation of cytokine production and activation of nuclear factor-kappaB. Inflammatory activation of the vessel wall by a dysregulated renin-angiotensin system may contribute to the pathogenesis of atherosclerosis.

Angiotensin II activates the proinflammatory transcription factor nuclear factor-kappaB in human monocytes.

The renin-angiotensin system may contribute to the pathogenesis of atherosclerosis. A common feature of all stages of atherosclerosis is inflammation of the vessel wall. The transcription factor nuclear factor-kappaB (NF-kappaB) participates in most signaling pathways involved in inflammation. This study therefore examined the effect of angiotensin (ANG) II on NF-kappaB activation in monocytic cells, a major cellular component of human atheroma, by electrophoretic mobility shift assay. ANG II, like TNFalpha, caused rapid activation of NF-kappaB in human mononuclear cells isolated from peripheral blood by Ficoll density gradient. This ANG II effect was blocked by the angiotensin AT1 receptor antagonist losartan. Specificity of ANG II-induced NF-kappaB activation was ascertained by supershift and competition experiments. Moreover, ANG II stimulated NF-kappaB activation in human monocytes, but not in lymphocytes from the same preparation. Together, the data demonstrate the ability of the vasoactive peptide ANG II to activate inflammatory pathways in human monocytes.

Thrombin promotes activation of matrix metalloproteinase-2 produced by cultured vascular smooth muscle cells.

Thrombin generated at sites of vascular injury not only participates in the coagulation cascade but can signal other events related to development and complication of atherosclerotic plaques. We investigated here a novel non-thrombotic action of thrombin: the possibility that this protease influences the expression or activation of matrix metalloproteinases (MMPs) produced by vascular smooth muscle cells (SMCs). Matrix-degrading proteinases likely contribute to several aspects of vascular lesion development. Vascular SMCs constitutively elaborate the zymogen form of gelatinase A (MMP-2), found in cell supernatants complexed with its inhibitor, the tissue inhibitor of metalloproteinases (TIMP)-2. When activated, MMP-2 digests collagens and elastin and may thus promote cell migration and vascular remodeling. Analysis of culture supernatants harvested from either human or rabbit vascular SMCs by gelatin zymography revealed that compared with supernatants of unstimulated SMCs, media conditioned by thrombin-stimulated cells contained increased amounts of proteolytically processed MMP-2, suggesting activation of this MMP. Further experiments tested whether thrombin directly activates MMP-2. In cell-free experiments, when added to medium harvested from unstimulated SMCs, alpha-thrombin increased in a dose- and time-dependent manner the amount of proteolytically processed MMP-2, as shown by zymography and by Western blotting with specific antibodies. Thrombin cleaved pro-MMP-2 within 4 hours, even when the gelatinase was bound with its inhibitor, TIMP-2. Thrombin treatment rendered culture media of unstimulated SMCs able to degrade collagen type IV, consistent with generation of active MMP-2. Addition of inhibitors of either thrombin or MMPs decreased this type IV collagenolytic activity, but thrombin in the absence of SMC-conditioned medium containing pro-MMP-2 exhibited only minimal collagenolysis. Our results suggest that at sites of vascular injury, thrombin may activate locally produced MMP-2 and thereby facilitate cell migration and proliferation. In the case of complicated atherosclerotic plaques, episodes of intraplaque hemorrhage or plaque disruption with thrombosis may promote plaque instability by increasing local matrix-degrading activity.

Thrombin potently stimulates cytokine production in human vascular smooth muscle cells but not in mononuclear phagocytes.

Thrombosis frequently occurs during atherogenesis and in response to vascular injury. Accumulating evidence supports a role for inflammation in the same situation. The present study therefore sought links between thrombosis and inflammation by determining whether thrombin, which is present in active form at sites of thrombosis, can elicit inflammatory functions of human monocytes and vascular smooth muscle cells (SMCs), two major constituents of advanced atheroma. Human alpha-thrombin (EC50, approximately equal to 500 pmol/L) potently induced interleukin (IL)-6 release from SMCs. The tethered-ligand thrombin receptor appeared to mediate this effect. Furthermore, alpha-thrombin also rapidly increased levels of mRNA encoding IL-6 and monocyte chemotactic protein-1 (MCP-1) in SMCs. In contrast, only alpha-thrombin concentrations of > or = 100 nmol/L could stimulate release of IL-6 or tumor necrosis factor-alpha (TNF alpha) in peripheral blood monocytes or monocyte-derived macrophages. Lipid loading of macrophages did not augment thrombin responsiveness. Likewise, only alpha-thrombin concentrations of > or = 100 nmol/L increased levels of IL-6, IL-1 beta, MCP-1, or TNF alpha mRNA in monocytes. Differential responses of SMCs and monocytes to thrombin extended to early agonist-mediated increases in [Ca2+]i. SMCs and endothelial cells, but not monocytes, contained abundant mRNA encoding the thrombin receptor and displayed cell surface thrombin receptor expression detected with a novel monoclonal antibody. Thus, the level of thrombin receptors appeared to account for the differential thrombin susceptibility of SMCs and monocytes. These data suggest that SMCs may be more sensitive than monocytes/macrophages to thrombin activation in human atheroma. Cytokines produced by thrombin-activated SMCs may contribute to ongoing inflammation in atheroma complicated by thrombosis or subjected to angioplasty.

Macrophage foam cells from experimental atheroma constitutively produce matrix-degrading proteinases.

Monocyte-derived foam cells figure prominently in rupture-prone regions of atherosclerotic plaques. Peripheral blood monocytes in culture can produce certain enzymes that degrade extracellular matrix, known as matrix metalloproteinases (MMPs). Lipid-laden macrophages may thus contribute to weakening of extracellular matrix of rupture-prone atherosclerotic plaques. However, the spectrum and regulation of MMP production by foam cells remain unknown. To investigate this issue, we isolated lipid-laden macrophages from rabbit aortic lesions produced by a combination of hypercholesterolemia and balloon injury. Freshly isolated aortic macrophage foam cells, identified using cell-specific antibodies, contained immunoreactive stromelysin and interstitial collagenase, whereas alveolar macrophages isolated from the lungs of same rabbits did not. Macrophages from both tissue sources released gelatinolytic activity consistent with the 92-kDa gelatinase. In vitro, lipid-laden aortic macrophages, but not alveolar macrophages, synthesized de novo and released immunoprecipitable stromelysin and collagenase, with or without stimulation by phorbol ester or bacterial lipopolysaccharide. These stimuli caused foam cells to release additional gelatinolytic activity that migrated faster than a purified preparation of 92-kDa gelatinase in substrate-containing polyacrylamide gels, indicating activation of the 92-kDa gelatinase or induction of the 72-kDa gelatinase. Our results show that lipid-laden macrophages elaborate MMPs capable of degrading the major constituents of vascular extracellular matrix even without further stimulation. Therefore, these cells may contribute to remodeling of the extracellular matrix during atherogenesis and to the disruption of plaques often responsible for acute clinical manifestations of atherosclerosis.

Interrelation between atherosclerosis and restenosis.

Atherosclerosis and restenosis following PTCA share many pathophysiologic features although they differ in the inciting stimuli and in their time-course. Both are characterized by an inflammatory reaction of the vessel wall whose modulation may offer therapeutic approaches for either entity.

Suppression of neointimal thickening and smooth muscle cell proliferation after arterial injury in the rat by inhibitors of Na(+)-H+ exchange.

Replication of vascular smooth muscle cells is a key event in the pathogenesis of restenosis following angioplasty. Little is known about early biochemical events involved in the proliferation of smooth muscle cells following arterial injury. In the present study, the effect of Na(+)-H+ exchange inhibitors on neointima formation after balloon injury of the rat carotid artery was investigated. Neointima formation was quantified 14 days after injury by morphometric measurement of cross-sectional neointimal area and by fluorometric determination of DNA content. The specific Na(+)-H+ exchange inhibitor 3-methylsulfonyl-4-piperidino-benzoyl guanidine mesylate (Hoe 694) dose-dependently reduced neointimal area and DNA content, the latter finding indicating a true antiproliferative effect. The structurally different Na(+)-H+ exchange blocker 5-(N-ethyl-N-isopropyl)amiloride hydrochloride had comparable inhibitory effects on neointimal area and DNA content, whereas 5-methylsulfonyl-2-piperidino-benzoyl guanidine hydrochloride, a position isomer of Hoe 694 lacking Na(+)-H+ exchange blocking properties, did not suppress neointima formation. The effect of Na(+)-H+ exchange blockers on neointima formation depended on the duration of drug application. Maximal suppression was achieved only when Hoe 694 was applied throughout the entire experiment for 14 days. This inhibitory effect of Na(+)-H+ exchange blocker application for the first 2 weeks following injury lasted for 2 months. In conclusion, the results of the present study reveal a potential role of Na(+)-H+ exchange for smooth muscle cell proliferation in vascular disease.

Effect of angiotensin converting enzyme inhibitors on cardiac noradrenaline release.

Angiotensin II facilitates sympathetic transmitter release in the heart. Thus, angiotensin converting enzyme inhibitors may be assumed to suppress noradrenaline release in the heart. To test this hypothesis endogenous noradrenaline release was induced either by electrical stimulation of the stellate ganglion or by global ischaemia in rat hearts. Noradrenaline was determined by high performance liquid chromatography in coronary venous overflow. Endogenous angiotensin formation was blocked by the angiotensin converting enzyme inhibitors captopril and ramiprilat and the activity of the endogenous renin-angiotensin system was modulated by variations in nutritional sodium load prior to the experiments. Both angiotensin converting enzyme inhibitors significantly reduced noradrenaline release evoked by nerve stimulation when the animals had been fed a low sodium diet. Following a high sodium diet, however, captopril and ramiprilat had no effect on stimulation-induced noradrenaline release. Global ischaemia (20 min) resulted in noradrenaline overflow from sympathetic nerves, independent of nerve stimulation. This ischaemia-induced noradrenaline release was not influenced by either converting enzyme inhibitor. In conclusion, the results suggest a sodium-dependent endogenous angiotensin formation in the heart which facilitates physiological noradrenaline release and is sensitive to angiotensin converting enzyme inhibitors. In contrast, noradrenaline release in ischaemia is not affected by converting enzyme blockers, due to a different, non-exocytotic release mechanism of noradrenaline during myocardial ischaemia.

Effect of digitalis glycosides on norepinephrine release in the heart. Dual mechanism of action.

The effect of ouabain on exocytotic and nonexocytotic norepinephrine release was investigated in perfused rat and guinea pig hearts. The overflow of endogenous norepinephrine and its neuronal metabolite 3,4-dihydroxyphenylethyleneglycol (DOPEG) was determined by high-pressure liquid chromatography. DOPEG served as the indicator of free axoplasmic norepinephrine concentrations. The overflow of the norepinephrine cotransmitter neuropeptide Y (NPY) was determined by radioimmunoassay and NPY was used as marker for exocytotic release. Electrical stimulation of the left stellate ganglion resulted in exocytotic norepinephrine release in rat and guinea pig hearts. Ouabain caused an increase in stimulation-induced norepinephrine overflow from rat and guinea pig hearts by 40%. However, overflow of NPY was decreased by 40%, indicating a reduced exocytosis rate. Ouabain increased both norepinephrine and NPY overflow, suggesting enhancement of exocytosis, when neuronal catecholamine uptake (uptake1) was blocked by desipramine or when presynaptic alpha 2-adrenoceptors were inhibited by yohimbine. The results demonstrate an interaction of ouabain with both calcium-dependent exocytosis and uptake1 of norepinephrine. Under calcium-free conditions, ouabain or potassium-free perfusate resulted in norepinephrine release from hearts when the axoplasmic norepinephrine concentration was elevated by the reserpinelike agent Ro 4-1284. This release was independent from neural activity, not accompanied by NPY overflow, and suppressed by the uptake1 blocker desipramine. These findings are in keeping with carrier-mediated nonexocytotic norepinephrine release that is caused by reversal of the transport direction of the uptake1 carrier. During myocardial ischemia nonexocytotic norepinephrine release was accelerated and enhanced by inhibition of Na+,K(+)-ATPase before ischemia. This study demonstrates the potential of digitalis glycosides to interact both with transmitter exocytosis and with the neuronal catecholamine transport system by Na+,K(+)-ATPase inhibition. Interaction with the catecholamine transport system involves both inhibition of norepinephrine inward transport and induction of norepinephrine outward transport, resulting in nonexocytotic norepinephrine release.

Effect of gallopamil on myocardial ischaemia during percutaneous transluminal coronary angioplasty.

This report summarises selected preliminary results from an ongoing study designed to investigate the effect of the calcium antagonist gallopamil on myocardial ischaemia during percutaneous transluminal coronary angioplasty (PTCA). To date, 12 adult males with coronary artery disease and significant proximal stenosis of the left anterior descending coronary artery (LAD) have been randomly assigned to gallopamil or placebo under double-blind conditions. Patients with recent myocardial infarction, apparent collateralisation of the LAD, myocardial failure, sinoatrial or atrioventricular block, severe hepatic disease or renal failure were excluded from the study. PTCA was performed using at least 2 balloon inflations, each of 2 minutes' duration. Gallopamil 0.4 mg or placebo (normal saline) were administered during the 10-minute interval between the 2 inflations. Blood samples were taken simultaneously from the coronary sinus and the femoral artery before and immediately after each inflation. Lactate concentration and the relative amount of activated neutrophils were selected for trend analysis. Furthermore, ECG changes were analysed by calculating the sum of the absolute ST-segment deviations (80 msec after J point, maximal T deviation) of leads I, II, III, V2, V4 and V6. In the presence of gallopamil, the degree of ST-segment/T-wave changes induced by balloon inflation was reduced. Additionally, gallopamil attenuated myocardial lactate release and appeared to prevent the increase in activated neutrophils observed during control inflations. These preliminary results suggest a beneficial effect from intracoronary administration of gallopamil during PTCA, achieved by attenuation of the ischaemic reaction during coronary occlusion.

Role of calcium channels and protein kinase C for release of norepinephrine and neuropeptide Y.

The role of calcium for the release of norepinephrine (NE, determined by high-pressure liquid chromatography) and neuropeptide Y (NPY, determined by radioimmunoassay) was investigated in guinea pig perfused hearts with intact sympathetic innervation. In the presence of extracellular calcium (1.85 mM), electrical stimulation of the left stellate ganglion (12 Hz, 1 min) induced a closely related release of NE and NPY with the molar ratio of approximately 400-600 (NE) to 1 (NPY). The stimulation-evoked overflow of both transmitters was dependent from the extracellular calcium concentration and was almost completely suppressed by calcium-free perfusion. The corelease of both transmitters was not affected by the L-type calcium channel blocker felodipine (1-10 microM). However, the overflow of NE and NPY was markedly attenuated by the unselective calcium antagonist flunarizine (1-10 microM) and completely prevented by the neuronal (N-type) calcium channel blockers omega-conotoxin (1-100 nM) and cadmium chloride (10-100 microM), indicating a key role for N-type calcium channels in the exocytotic release of transmitters from cardiac sympathetic nerve fibers. Possibly due to unspecific actions, such as interference with sodium channels or uptake1-blocking properties, the phenylalkylamines verapamil (0.01-10 microM) and gallopamil (1-10 microM) reduced NPY overflow with only a minor effect on NE overflow. The stimulation-induced transmitter release was increased up to twofold by activation of protein kinase C (phorbol 12-myristate 13-acetate, 3 nM-3 microM) and completely suppressed by inhibition of protein kinase C (polymyxin B, 100 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction between the release of adenosine and noradrenaline during sympathetic stimulation: a feed-back mechanism in rat heart.

Interactions between the release of adenosine and noradrenaline were studied during sympathetic stimulation in rat heart perfused in situ. Cardiac sympathetic nerves were activated by electrical stimulation of the left cervicothoracic ganglion, and endogenous noradrenaline and adenosine were measured in the effluent from the heart. Following the onset of a continuous stimulation (6 min) a rise of heart rate was observed which was accompanied by the release of noradrenaline and adenosine. Specific blockade of adenosine receptors by 8-phenyltheophylline enhanced the stimulation induced release of noradrenaline suggesting an effective suppression of the noradrenaline release by endogenous adenosine. Heart rate and the release of adenosine were reduced by the beta 1-adrenergic antagonist bisoprolol, while noradrenaline overflow increased. These results are compatible with the concept of a negative feed-back regulation of noradrenaline release by endogenous adenosine from the stimulated cardiomyocytes. In order to characterize the subtype of the presynaptic adenosine receptors involved, the inhibitory potency on stimulus induced noradrenaline release of metabolically stable adenosine agonists was tested. The order of potency (Cyclohexyladenosine greater than or equal to R-phenylisopropyl-adenosine greater than N-ethylcarboxamidoadenosine greater than S-phenylisopropyl-adenosine) suggests an adenosine A1-receptor mediated presynaptic inhibition of noradrenaline release.

Adenosine inhibits exocytotic release of endogenous noradrenaline in rat heart: a protective mechanism in early myocardial ischemia.

The effects of exogenous and endogenous adenosine on exocytotic noradrenaline release were studied in rat hearts perfused in situ. Exocytotic release of endogenous noradrenaline (determined by high pressure liquid chromatography) was induced by electrical stimulation of the left cervicothoracic ganglion. Exogenous adenosine significantly reduced noradrenaline overflow from the heart. This suppression of noradrenaline overflow was not influenced by desipramine, indicating a mechanism independent from noradrenaline reuptake. The A1 subtype specific agonists cyclohexyladenosine and R-phenylisopropyladenosine had inhibitory effects at lower concentrations than adenosine and S-phenylisopropyladenosine, suggesting the relevance of presynaptic inhibitory adenosine receptors of the A1 subtype. Short ischemic periods of 3 minutes resulted in a marked coronary venous overflow of adenosine during reperfusion. This was accompanied by an inhibition of noradrenaline release evoked by nerve stimulation during ischemia. The adenosine antagonists theophylline and 8-phenyltheophylline prevented this suppression of noradrenaline release. Blockade of oxidative phosphorylation by cyanide in combination with glucose-free perfusion induced an increased formation of endogenous adenosine and suppression of stimulation-evoked noradrenaline overflow. Again, in the presence of the adenosine antagonists theophylline or 8-phenyltheophylline, this suppression was abolished. These results indicate that adenosine is a potent inhibitor of exocytotic noradrenaline release in the heart with relevance during conditions of increased endogenous adenosine formation such as myocardial ischemia.