Cultured coronary endothelial cells and the coronary endothelium of isolated perfused guinea-pig hearts are characterized by a very active adenosine and adenine nucleotide metabolism. Adenosine applied to the endothelium at low concentrations is avidly metabolized and preferentially incorporated into different nucleotide pools--only a minor amount is degraded to uric acid. Physiologically, the coronary endothelium therefore functions as an impermeable metabolic barrier for interstitially or intravascularly accumulating adenosine. Only at concentrations greater than or equal to 10(-6) M adenosine can pass the endothelial barrier. As a consequence, the vasodilatory action of adenosine formed in or administered into the coronary system cannot be induced by a direct association of the nucleoside with the putative adenosine receptor of the arteriolar smooth muscle cells, but must be mediated by the endothelium. High molecular weight derivatives of adenosine, clearly confined to the coronary system, can also induce a coronary dilation. The endothelium-mediated smooth muscle relaxation is therefore obviously due to triggering of an extracellular adenosine receptor at the luminal surface of the endothelium. Since this process is accompanied by a rapid and pronounced activation of the adenylate cyclase system, the endothelial receptor conforms to an A2-type. According to our results it is necessary to reconsider qualitative and quantitative facets of the adenosine hypothesis of metabolic regulation of coronary blood flow, which--in its original formulation--exclusively centers on the cardiomyocyte metabolism. With respect to the vasoactivity of adenosine one obviously has to distinguish between its action from the interstitial space directly via the myocyte receptors of the vessel wall, and/or its action from the intracoronary space via the newly detected endothelial A2-receptor. More information is needed to determine the extent to which both receptor populations actually participate in the metabolic regulation of coronary flow under physiological and pathophysiological conditions.
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.
SUMMARY1. Coronary endothelial cells were isolated from adult guinea-pig hearts (Nees, Gerbes & Gerlach, 1981) and the electrical properties of primary cultures were studied using the tight-seal whole-cell recording mode of the patch clamp technique.2. On the third or fourth day in culture whole-cell clamp records from single coronary endothelial cells were obtained at 37 'C. The resting potential was -33 + 6 mV (n = 10). The membrane time constant determined with rectangular current pulses was 68 + 22 ms (n = 10).3. In voltage clamp experiments, no time-dependent membrane conductance changes were found in the range -80 to + 40 mV. The current-voltage relation was linear in normal physiological salt solution containing 5-4 mM-K+. The input resistance was 1P7 +0 4 GQ. When the external K+ concentration was increased to 116 mm the cells depolarized to about -3 mV and the clamp currents showed marked inward rectification.4. Between days four and seven in culture the endothelial cells formed confluent monolayers which showed the characteristic 'cobblestone' morphology. The input resistance of cells in a monolayer was 8 + 3 MQ, i.e. a factor of 200 lower than that found in single cells. It was concluded that the cells in the confluent monolayer are coupled electrically by gap junctions.5. Exposure of coronary endothelial cells to K+-free solution for 5 min produced a depolarization of about 8 mV. Upon readmission of normal external K+ the cells transiently hyperpolarized by up to 20 mV. This transient hyperpolarization decayed with a time constant of 1-9 + 0-3 min.6. The transient hyperpolarization could be abolished by application of 2 x 10-4 Mdihydro-ouabain (DHO). Application of DHO in the steady state produced a depolarization of 8+1 mV. From these findings it was concluded that coronary endothelial cells possess an electrogenic sodium pump which contributes about -8 mV to the resting potential.7. From the passive electrical properties of single cells and the morphological data t Present address:
Microvascular cells derived from rat hindlimb muscles demonstrated endothelial characteristics. These cells accumulated reduced vitamin C by means of Na+-dependent ascorbate transporters, which are distinct from hexose carriers. The high endothelial ascorbate concentration at steady-state is consistent with the role of ascorbate as a major antioxidant in the skeletal muscle microvasculature.
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