Stephan Nees

Evidence for a cell surface adenosine receptor on coronary myocytes and atrial muscle cells

SummaryWe have studied the site of action of adenosine with the aid of a large molecular weight adenosine derivative which is confined to the extracellular space. A stable protein-AMP-conjugate was formed using 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide to covalently couple AMP by P-N linkage to lysine residues of a carbonic anhydrase preparation. The conjugate was characterized by disc-SDS-electrophoresis and exhibited a mean molecular weight of about 30000. When infused into the coronary arteries of isolated guinea pig hearts the protein-AMP-conjugate induced vasodilation which was similar in magnitude and time course to that elicited by free AMP or adenosine. The dilatory response of the coronaries was caused by the protein-AMP-conjugate itself and not by free AMP or by adenosine liberated from the conjugate. This conclusion is based on the facts that: i) The electrophoretic mobility of the conjugate remained unchanged after its passage through the heart. ii) Addition of 5′-nucleotidase and adenosine deaminase to the protein-AMP-conjugate prior to its infusion into the coronaries did not alter the vasoactive effects. iii) Perfusion of the isolated hearts with equipotent concentrations of14C-AMP,14C-adenosine or protein-14C-AMP-conjugate resulted in a significant incorporation of radioactivity into cardiac adenine nucleotides only in the case of labeled AMP and adenosine. Besides its effects on the coronaries, the protein-AMP-conjugate also rapidly abolished the calcium-dependent action potential in the atrial muscle. Since the biological effects observed are most likely caused by the adenosine moiety of AMP our results provide evidence that AMP as well as adenosine act via a receptor site on the surface of coronary myocytes and atrial muscle cells.

Cardiac ventricular β2-adrenoceptors in guinea-pigs and rats are localized on the coronary endothelium

SummaryIn mammalian heart tissue β2 are known to coexist with β1. In the present study, evidence that β2 in guinea-pig and rat ventricles are primarily localized on the coronary endothelium is provided by competition binding studies with the subtype-selective β-adrenoceptor antagonists ICI 89.406 (β1) and ICI 118.551 (β2) on four different plasma membrane preparations. (1) Following density gradient centrifugation of cardiac ventricular microsomes from rats or guinea-pigs, endothelial plasma membranes migrated at slightly higher density than the sarcolemmal membranes, as verified by endothelial (angiotensin converting enzyme) and sarcolemmal markers (adenylate cyclase, [3H] ouabain binding). At the activity peak of angiotensin converting enzyme, the relative amount of β2 in guinea-pigs and rats was 25% and 65%, respectively. (2) On sarcolemmal membranes corresponding to the activity peak of adenylate, cyclase, β-adrenoceptors consisted of the β1 exclusively (guinea-pig), or to at least 90% (rat). (3) Cultures of coronary endothelial cells derived from guinea-pigs revealed only β2. (4) Isolated guinea-pig cardiomyocytes contained only β1, a finding recently established in rat myocytes as well.

Functional evidence for the presence of adenosine A2-receptors in cultured coronary endothelial cells.

SummaryAdenosine and the adenosine receptor agonists, R- and S-N6-phenylisopropyladenosine (R- and S-PIA) and 5′-N-ethylcarboxamidoadenosine (NECA), enhanced [3H]cAMP accumulation in [3H]adenine-labelled cultured endothelial cells isolated from the microvasculature of guinea pig hearts. As shown by their concentration-response curves, NECA was a more potent agonist than R-PIA or adenosine. Their respective concentrations at half-maximal stimulation of [3H]cAMP accumulation were 0.7 μM, 10.5 μM and 12.6 μM, indicating a 15- to 18-fold potency difference between NECA and the other agonists. The increased [3H]cAMP accumulation elicited by 10−5 M NECA was inhibited by the xanthine derivative 8-phenyltheophylline, 3-isobutyl-l-methylxanthine, theophylline or caffeine. These findings provide functional evidence for the presence of adenosine receptors of the A2-type in microvascular coronary endothelial cells in culture. The functional significance of these receptors remains to be established, but they may be involved in the regulation of vascular permeability.

Endothelium-mediated coronary dilatation by adenosine does not depend on endothelial adenylate cyclase activation: studies in isolated guinea pig hearts

Adenosine, applied into the coronary system of guinea pigs at up to 10−6 M, elicits dilatation solely via an endothelium-mediated process. We investigated the role of coronary A2 receptors in this dilation, since the coronary endothelium possesses adenosine A2-receptors with a stimulatory action on the adenylate cyclase. In situ, A2 receptor stimulation can be assessed by prelabeling the coronary endothelial adenine nucleotide pool with3H-adenosine and subsequently determining the rate of release of radiolabeled cAMP induced by A2 agonists. Thus, perfusion of isolated hearts with 5′-N-ethylcarboxamidoadenosine (NECA) dose-dependently increased coronary flow and the release of3H-cAMP from the endothelium. In the presence of 50 μM 2′, 5′-dideoxyadenosine (ddA), a P-site agonist which inhibits the catalytic activity of adenylate cyclase, coronary flow increases induced by both adenosine and NECA wre unaffected. In contrast, ddA reduced the release of labeled cAMP in response to NECA by about 60%. In cultured endothelial cells, ddA likewise inhibited cAMP accumulation due to NECA by about 70%. Moreover, ddA antagonized the adenylate cyclase mediated flow response due to the PGI2 analogue, iloprost, as well as the positive chronotropic and motropic actions of isoproterenol. The dissociation elicited by ddA between the coronary flow response and the release of cAMP strongly indicates that the endothelial A2 receptors which are linked to adenylate cyclase are not causally involved in endothelium-dependent coronary dilatation induced by adenosine.

Pericytes in the macrovascular intima: possible physiological and pathogenetic impact

The frequently observed de-endothelialization of venous coronary bypass grafts prepared using standard methods exposes subendothelial prothrombotic cells to blood components, thus endangering patients by inducing acute thromboembolic infarction or long-term proliferative stenosis. Our aim was to gain deeper histological and physiological insight into these relations. An intricate network of subendothelial cells, characterized by histological features specific for true pericytes, was detected even in healthy vessels and forms, coupled to the luminal endothelium, a second leaflet of the macrovascular intima. These cells, and particularly those in the venous intima, express enormous concentrations of tissue factor and can recruit additional amounts of up to the 25-fold concentration within 1 h during preincubation with serum (intimal pericytes of venous origin activate 30.71 +/- 4.07 pmol coagulation factor x.min(-1).10(-6) cells; n = 15). Moreover, decoupled from the endothelium, they proliferate rapidly (generation time, 15 +/- 2.1 h, n = 8). Central regions of atherosclerotic plaques, as well as of those of restenosed areas of coronary vein grafts, consist almost completely of these cells. In stark contrast with the prothrombogenicity of the intimal pericytes, intact luminal endothelium recruits high concentrations of thrombomodulin (CD 141) specifically within its intercellular junctions, activates Protein C rapidly (42 +/- 5.1 pmol/min.10(6) venous endothelial cells at thrombin saturation; n = 15), can thus actively prevent coagulatory processes, and never expresses histologically detectable and functionally active tissue factor. Given this strongly prothrombotic potential of the intimal pericytes and their overshooting growth behavior in endothelium-denuded vascular regions, they may play important roles in the development of atherosclerosis, thrombosis, and saphenous vein graft disease.

Isolation, bulk cultivation, and characterization of coronary microvascular pericytes: the second most frequent myocardial cell type in vitro

Densely arranged pericytes engird the endothelial tube of all coronary microvessels. Since the experimental access to these abundant cells in situ is difficult, a prerequisite for broader investigation is the availability of sufficient numbers of fully differentiated pericytes in homogenous culture. To reach this goal, we applied strictly standardized cell isolation techniques, optimized culture methods and specific histological staining. Approximately 1,000-fold enriched pericytes were proteolytically detached from highly purified coronary microvascular networks (density gradient centrifugation) of eight mammalian species including human. Addition of species-autologous fetal or neonatal serum (10-20% vol/vol) was a precondition for longer term survival of homogenous pericyte cultures. This ensured optimal growth (doubling time <14 h) and full expression of pericyte-specific markers. In 3-mo, 10(10) pericytes (15 g) could be cultivated from 1 bovine heart. Pericytes could be stored in liquid N(2), recultured, and passaged repeatedly without loss of typical features. In cocultures with EC or vascular smooth muscle cells, pericytes transferred fluorescent calcein to each other and to EC via their antler-like extensions, organized angiogenetic sprouting of vessels, and rapidly activated coagulation factors X and II via tissue factor and prothrombinase. The interconnected pericytes of the coronary system are functionally closely correlated with the vascular endothelium and may play key roles in the adjustment of local blood flow, the regulation of angiogenic processes, and the induction of procoagulatory processes. Their successful bulk cultivation enables direct experimental access under defined in vitro conditions and the isolation of pericyte specific antigens for the production of specific antibodies.

Focus on cardiac pericytes

The wall of myocardial terminal vessels, consisting of a continuous endothelial tube with an adventitial coat of pericytes in their extracellular matrix, constitutes a remarkably tight barrier to solute transport between the blood and the parenchyma. This constructional principle of precapillary arterioles, capillaries and postcapillary venules extends both up- and downstream into the arterial and venous limbs, where the original microvessel tube widens and becomes the innermost layer—the intima—of all the larger coronary vessels. In the myocardium’s smallest functional units and in the intima of the coronaries, the pericytes play key roles by virtue of both their central histological localization and their physiological functions. Recognition and integration of these properties has led to new pathogenetic models for diverse heart diseases and suggests that current therapeutic concepts need to be revised.

Formation of Adenosine by Vascular Endothelium: a Homeostatic and Antithrombogenic Mechanism?

Under physiological conditions, concentrations of adenosine in arterial and venous blood are very similar, although the nucleoside is rapidly metabolized by blood cells and the vascular endothelium. In order to characterize the possible regulatory role of endothelial cells in the homeostasis of adenosine in the blood, studies concerning metabolism of adenosine and adenine nucleotides were carried out on cultured endothelial cells of various origin and on different vessel preparations.

Adenosine Receptors at the Coronary Endothelium: Functional Implications

Cultured coronary endothelial cells (CEC) are characterized by an active metabolism of adenosine (AR) and adenine nucleotides (AN). Extracellularly applied AN (AMP, ADP or, ATP) are rapidly dephosphorylated by a cascade of nucleotidases at the cell surface. AR, formed by these processes or directly added into the incubation medium, is avidly taken up: small amounts are preferentially phosphorylated, larger amounts are degraded mainly to uric acid. With a specially developed dual-chamber perfusion system the ability of AR and AN to penetrate across confluent endothelial layers was determined. Regardless whether these vasoactive compounds were applied at the apical or basal side of the cell layer, at concentrations <10−6 M only vasoinactive degradatives were detected at the contralateral side.

Radioimmunoassay for adenosine in biological samples.

A sensitive and specific radioimmunoassay for adenosine has been developed. Antibodies directed against adenosine (titer 1∶400–1∶700) were obtained by immunizing rabbits with adenosine, conjugated via its vicinal hydroxyl groups to bovine serum albumin (periodate oxidation). Interfering adenosine deaminase activity was removed from the antisera by treatment with DEAW-cellulose. Free and antibody bound3H-adenosine was separated by either the “second antibody” precipitation method or by a simple filtration step. The sensitivity and assay range for adenosine was 1–100 pmoles per assay tube. Structurally related purine compounds (adenine nucleotides, adenine) crossreacted with adenosine binding and were removed by a single chromatographic step. Analysis of the adenosine content in normoxic guinea pig hearts yielded 2.53 nmoles/g, a value which was confirmed by spectrophotometric analysis.