Endothelial cells secrete a range of procoagulant, anticoagulant and inflammatory proteins by exocytosis to regulate blood clotting and local immune responses. The mechanisms regulating vesicular exocytosis were studied in human umbilical vein endothelial cells (HUVEC) with high‐resolution membrane capacitance (Cm) measurements. The total whole‐cell Cm and the amplitudes and times of discrete femtoFarad (fF)‐sized Cm steps due to exocytosis and endocytosis were monitored simultaneously. Intracellular calcium concentration [Ca2+]i was elevated by intracellular photolysis of calcium‐DM‐nitrophen to evoke secretion and monitored with the low‐affinity Ca2+ indicator furaptra. Sustained elevation of [Ca2+]i to > 20 μm evoked large, slow increases in Cm of up to 5 pF in 1‐2 min. Exocytotic and endocytotic steps of amplitude 0.5‐110 fF were resolved, and accounted on average for ≈33 % of the total Cm change. A prominent component of Cm steps of 2.5‐9.0 fF was seen and could be attributed to exocytosis of von‐Willebrand‐factor‐containing Weibel‐Palade bodies (WPb), based on the near‐identical distributions of capacitance step amplitudes, with calculated estimates of WPb capacitance from morphometry, and on the absence of 2.5‐9.0 fF Cm steps in cells deficient in WPb. WPb secretion was delayed on average by 23 s after [Ca2+]i elevation, whereas total Cm increased immediately due to the secretion of small, non‐WPb granules. The results show that following a large increase of [Ca2+]i, corresponding to strong stimulation, small vesicular components are immediately available for secretion, whereas the large WPb undergo exocytosis only after a delay. The presence of events of magnitude 9‐110 fF also provides evidence of compound secretion of WPb due to prior fusion of individual granules.

Gap junction channels permit the direct intercellular transfer of ions and small molecules and allow electrotonic coupling within tissues. Porcine aortic endothelial cells were extensively coupled, as assessed by gap junctional transfer of Lucifer yellow and the fluorescent calcium indicators fluo-3 and furaptra, but were not,permeable to rhodamine B isothiocyanate-dextran 10S. The subunit composition of gap junction channels of porcine aortic endothelial cells was characterised using both northern blot analysis and RT-PCR techniques. Messenger RNA encoding connexins 37 and 43, but not 26, 32 or 40, were found in freshly isolated and cultured porcine aortic endothelial cells, Western blots using antipeptide antibodies raised to unique sequences of connexins 37, 40 and 43 showed the presence of connexins 37 and 43, but no connexin 40 was detected. Immunostaining with anticonnexin 43 antibodies showed extensive punctate fluorescent decoration of contacting membranes, whilst antibodies to connexin 37 showed predominantly intracellular staining. Caged InsP(3) was found to readily permeate endothelial gap junctions. These results show that primary cultures of porcine aortic endothelial cells express connexin 37 and 43, and provide strong evidence that the second messenger molecule InsP(3) can permeate porcine endothelial gap junctions.

To understand the complex time course of cytosolic Ca2+ signalling evoked by hormones and neurotransmitters, it is necessary to know the kinetics of steps in the second-messenger cascade, particularly cooperative and inhibitory interactions between components that might give rise to periodic fluctuations. In the case of inositol trisphosphate (InsP3)-evoked Ca2+ release, fast perfusion studies with subcellular fractions or permeabilised cells can be made if sufficient homogeneous tissue is available. Single-cell studies can be made by combining whole-cell patch-clamp techniques and microspectrofluorimetry with flash photolytic release of InsP3 to give quantitative, time-resolved data of Ca2+ release from stores. A technical description is given here of flash photolysis of caged InsP3, and the results of fast perfusion and flash photolytic experiments are reviewed. Studies of kinetics of Ca2+ release have shown that the InsP3 receptor/channel is regulated first by positive and then by negative feedback by free cytosolic Ca2+ concentration, producing a pulse of Ca2+ release having properties that may be important in the spatial propagation of Ca2+ signals within and between cells. The properties of InsP3-evoked Ca2+ release in single cells differ between peripheral tissues, such as the liver, and Purkinje neurones of the cerebellum. Purkinje neurones need 20-50 times higher InsP3 concentrations and release Ca2+ to change the free cytosolic concentration 30 times faster and to higher peak concentrations than in liver. The InsP3 receptors in the two cell types appear to differ in apparent affinity, and the greater Ca2+ efflux from stores in Purkinje cells is probably due to a high receptor density.

Endothelial cells synthesize prostacyclin in response to a wide variety of vasoactive stimuli. The transduction pathway is dependent on elevation of intracellular Ca2+ and specifically desensitizes cells to the stimulus used, thus providing tight temporal regulation of the release of this potent vasodilator and inhibitor of platelet aggregation.

Single pig aortic endothelial cells in culture loaded with the Ca(2+)-sensitive fluorescent dye Indo-1 were stimulated with ATP (0.1-100 microM) or bradykinin (0.1-5.0 nM). Spiking or oscillations of [Ca2+]i were seen in approx. 50% of cells stimulated with either agonist. Non-spiking or transient responses in which [Ca2+]i returned to pre-stimulation levels rapidly 9120-250 s), or sustained responses in which [Ca2+]i remained elevated for many minutes, were seen in a further 20% of cells in each case, stimulated with either agonist. There was a marked variation between individual cells in the latency, magnitude, frequency and overall pattern of oscillations induced by ATP and bradykinin, although the patterns of response to bradykinin were less variable. In cells where repetitive spikes were seen, a relation between concentration of ATP and the latency of the response and the frequency of spiking was evident. Effects of removal of extracellular Ca2+, elevation of extracellular K+ concentration (35 or 70 mM) or exposure to phorbol 12,13-dibutyrate or 1,2-dioctanoyl-sn-glycerol were tested on the spiking Ca2+ responses. Each of these procedures reversibly slowed or prevented Ca2+ spiking evoked by ATP or bradykinin. In contrast, the inactive phorbol ester 4 alpha-phorbol didecanoate had no effect on Ca2+ spiking evoked by these hormones. Our results thus indicate that the responses of single cells to ATP or bradykinin exhibit marked heterogeneity, and suggest that secretory events driven by extracellular Ca2+ may be regulated by repetitive spikes or oscillations of Ca2+.

Annals of the New York Academy of SciencesVolume 603, Issue 1 p. 267-273 Effects of Extracellular ATP on the Release of Vasoactive Mediators from Endothelium JEREMY D. PEARSON, JEREMY D. PEARSON Section of Vascular Biology Clinical Research Centre Medical Research Council Harrow, Middlesex HA1 3UJ, EnglandSearch for more papers by this authorTHOMAS D. CARTER, THOMAS D. CARTER Section of Vascular Biology Clinical Research Centre Medical Research Council Harrow, Middlesex HA1 3UJ, EnglandSearch for more papers by this author JEREMY D. PEARSON, JEREMY D. PEARSON Section of Vascular Biology Clinical Research Centre Medical Research Council Harrow, Middlesex HA1 3UJ, EnglandSearch for more papers by this authorTHOMAS D. CARTER, THOMAS D. CARTER Section of Vascular Biology Clinical Research Centre Medical Research Council Harrow, Middlesex HA1 3UJ, EnglandSearch for more papers by this author First published: December 1990 https://doi.org/10.1111/j.1749-6632.1990.tb37678.xCitations: 7 National Institute for Medical Research, Mill Hill, London, England. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat References 1 De QMey, J. G. & P. M. Vanhoutte. 1981 Role of the intima in cholinergic and purinergic relaxation of isolated canine femoral arteries. J. Physiol. 316: 347–355. 10.1113/jphysiol.1981.sp013792 PubMedWeb of Science®Google Scholar 2 Palmer, R. M., A. G. Ferrige & S. Moncada. 1987 Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327: 524–526. 10.1038/327524a0 CASPubMedWeb of Science®Google Scholar 3 Gordon, J. L. & W. Martin. 1983 Endothelium-dependent relaxation of the pig aorta: Relationship to stimulation of 86Rb efflux from isolated endothelial cells. Br. J. Pharmacol. 79: 537–541. Google Scholar 4 Martin, W., N. J. Cusack, J. S. Carleton & J. L. Gordon. 1985 Specificity of the P2-purinoceptor that mediates endothelium-dependent relaxation of the pig aorta. Eur. J. Pharmacol. 134: 295–299. Google Scholar 5 Burnstock, G. & C. Kennedy. 1985. Is there a basis for distinguishing two types of P2-purinoceptor? Gen. Pharmacol. 16: 433–440. 10.1016/0306-3623(85)90001-1 CASPubMedWeb of Science®Google Scholar 6 Pearson, J. D., L. L. Slakey & J. L. Gordon. 1983 Stimulation of prostacyclin production through purinoceptors on cultured porcine aortic endothelial cells. Biochem. J. 214: 273–276. 10.1042/bj2140273 CASPubMedWeb of Science®Google Scholar 7 Needham, L., N. J. Cusack, J. D. Pearson & J. L. Gordon. 1987 Characteristics of the P2-purinoceptor that mediates endothelial prostacyclin production. Eur. J. Pharmacol. 134: 199–209. CASPubMedWeb of Science®Google Scholar 8 Pearson, J. D. & T. D. Carter. 1989. Transduction of purinoceptor-mediated endothelial cell responses. In Vascular Endothelium: Receptors and Transduction Mechanisms. J. D. Catravas &. C. N. Gillis &. U. S. Ryan, Eds: 189–195. Plenum. New York , NY . 10.1007/978-1-4684-8532-5_19 Web of Science®Google Scholar 9 Pearson, J. D. & J. L. Gordon. 1989 P2-purinoceptors in the blood vessel wall. Biochem. Pharmacol. 38: 4157–4163. 10.1016/0006-2952(89)90509-1 CASPubMedWeb of Science®Google Scholar 10 Pirotton, S., E. Rapse, D. Demolle, L. Erneux & J. M. Boeynaems. 1987 Involvement of inositol 1,4,5-trisphosphate and calcium in the actions of adenine nucleofides on aortic endothelial cells. J. Biol. Chem. 262: 17461–17467. CASPubMedWeb of Science®Google Scholar 11 Luckhoff, A. & R. Busse. 1986 Increased free calcium in endothelial cells under stimulation with adenine nucleotides. J. Cell. Physiol. 126: 414–420. 10.1002/jcp.1041260312 CASPubMedWeb of Science®Google Scholar 12 Hallam, T. J. & J. D. Pearson. 1986 Exogenous ATP raises cytoplasmic free calcium in fura-2-loaded piglet aortic endothelial cells. FEBS Lett. 176: 139–143. Web of Science®Google Scholar 13 Carter, T. D., T. J. Hallam, N. J. Cusack & J. D. Pearson. 1988 Regulation of P2Y-purinoceptor-mediated prostacyclin release from human endothelial cells by cytoplasmic calcium concentration. Br. J. Pharmacol. 94: 1181–1190. Google Scholar 14 Hallam, T. J., R. Jacob & J. E. Merritt. 1988 Evidence that agonists stimulate bivalentcation influx into human endothelial cells. Biochem. J. 255: 179–184. 10.1042/bj2550179 CASPubMedWeb of Science®Google Scholar 15 Carter, T. D., T. J. Hallam & J. D. Pearson. 1989 P2-purinoceptor-stimulated mobilization of intracellular calcium precedes divalent cation influx in human endothelial cells. Br. J. Haematol. 73: 437. Web of Science®Google Scholar 16 Toothill, V. J., L. Needham, J. L. Gordon & J. D. Pearson. 1988 Desensitization of agonist-stimulated prostacyclin release in human umbilical vein endothelial cells. Eur. J. Pharmacol. 157: 189–196. CASPubMedWeb of Science®Google Scholar 17 Berridge, M. J. & R. F. Irvine. 1989 Inositol phosphates and cell signalling. Nature 341: 197–205. 10.1038/341197a0 CASPubMedWeb of Science®Google Scholar 18 Carter, T. D., T. J. Hallam & J. D. Pearson. 1989 Protein kinase C activation alters the sensitivity of agonist-stimulated endothelial cell prostacyclin production to intracellular ionised calcium. Biochem. J. 262: 431–437. 10.1042/bj2620431 CASPubMedWeb of Science®Google Scholar 19 Carter, T. D., J. S. Newton, R. Jacob & J. D. Pearson. 1989. Desensitization of ATP-mediated elevations in cytosolic [Ca2+] and prostacyclin production in human endothelial cells: A protein kinase C-independent mechanism. Biochem. J.: submitted for publication. PubMedWeb of Science®Google Scholar 20 Gonsalez, F. A., E. Rozengurt & L. E. Heppel. 1989 Extracellular ATP induces the release of calcium from intracellular stores without the activation of protein kinase C in Swiss 3T6 mouse fibroblasts. Proc. Natl. Acad. Sci. USA 86: 4530–4534. 10.1073/pnas.86.12.4530 PubMedWeb of Science®Google Scholar 21 Kelm, M., M. Feelisch, R. Spahr, H.-M. Piper, E. Noacke & J. Schrader. 1988 Quantitative and kinetic characterization of nitric oxide and EDRF released from cultured endothelial cells. Biochem. Biophys. Res. Commun. 154: 236–244. 10.1016/0006-291X(88)90675-4 CASPubMedWeb of Science®Google Scholar 22 Lewis, M. J. & A. H. Henderson. 1987 A phorbol ester inhibits the release of endothelium-derived relaxing factor. Eur. J. Pharmacol. 137: 167–171. CASPubMedWeb of Science®Google Scholar 23 Sage, S. O. & T. J. Rink. 1987 The kinetics of changes in intracellular calcium concentrations in fura-2-loaded human platelets. J. Biol. Chem. 262: 16364–16369. CASPubMedWeb of Science®Google Scholar 24 Benham, C. D. & R. W. Tsien. 1987 A novel receptor-operated, Ca2+-permeable channel activated by ATP in smooth muscle. Nature 328: 275–278. 10.1038/328275a0 CASPubMedWeb of Science®Google Scholar 25 Sage, S. O., J. E. Merritt, T. J. Hallam & T. J. Rink. 1989 Receptor-mediated calcium entry in fura-2-loaded human platelets stimulated with ADP and thrombin. Biochem. J. 258: 923–926. 10.1042/bj2580923 CASPubMedWeb of Science®Google Scholar 26 Boeynaems, J. M. & J. D. Pearson. 1990 P2-purinoceptors on vascular endothelial cells: Physiological significance and transduction mechanisms. Trends Pharmacol. Sci.. 11: 34–37. 10.1016/0165-6147(90)90039-B CASPubMedWeb of Science®Google Scholar Citing Literature Volume603, Issue1Biological Actions of Extracellular ATPDecember 1990Pages 267-273 ReferencesRelatedInformation

Single human umbilical-vein endothelial cells in culture loaded with the Ca2(+)-sensitive dye fura-2 exhibited characteristic increases in cytosolic Ca2+ concentrations [(Ca2+]i) in response to extracellular ATP. The rapid decline of [Ca2+]i to prestimulated levels in the continued presence of ATP, with in most cells no sustained or oscillatory increase in [Ca2+]i, indicated desensitization. This was agonist-specific, and contrasted with the [Ca2+]i response to histamine, though each agonist mobilized Ca2+ from the same internal store. In populations of cells, when desensitization was variably induced by a second challenge with ATP after different times, desensitization of the initial peak [Ca2+]i was directly related to desensitization of prostacyclin release. This was not affected by treatment with the protein kinase C inhibitor staurosporine, under conditions where a similar degree of desensitization of peak [Ca2+]i induced by phorbol 12-myristate 13-acetate was blocked. Sequential addition of ATP to cell populations cumulatively desensitized the peak elevation of [Ca2+]i, but did not block the second, sustained, phase of the response. We conclude that desensitization of prostacyclin synthesis by ATP is likely to be due to uncoupling of the P2Y purinoceptor from phosphoinositidase C, but does not involve protein kinase C activation.

Agonist-stimulated release of prostacyclin (PGI2) from endothelial cells requires elevation of the concentration of intracellular ionized calcium ([Ca2+]i) above a threshold value, and raised [Ca2+]i provides a sufficient transduction signal to account for the extent of PGI2 production. However, chronic activation of protein kinase C has been reported separately to potentiate PGI2 release, but to depress agonist-induced elevations of [Ca2+]i. We show here that pretreatment with phorbol 12-myristate 13-acetate (PMA) dose-dependently induces PGI2 release over many minutes after a significant lag period without any change in [Ca2+]i. In addition, PMA potentiates the transient release of PGI2 in response to agonists in a complex manner depending on the time of pre-incubation and the concentrations of both PMA and agonist. Concomitant measurement of [Ca2+]i and PGI2 release demonstrates that PMA pretreatment dose-dependently inhibits both the peak [Ca2+]i transient and the subsequent steady-state elevation of [Ca2+]i in response to agonists. Determination of the quantitative [Ca2+]i/PGI2 dose/response relationship, when PGI2 release is driven purely by elevating [Ca2+]i with ionomycin, demonstrates that PMA also enhances the Ca2+-sensitivity of PGI2 release. The observed effects of PMA on PGI2 release can be explained quantitatively by its abilities to lower the threshold [Ca2+]i required for PGI2 synthesis and to depress the peak [Ca2+]i evoked by agonist. We propose that these effects are due respectively to actions of PMA on phospholipase A2 and on a G-protein (Gp) that couples activated receptors to phospholipase C.

ATP and ATP analogues induced prostacyclin (PGI2) secretion from human cultured umbilical vein endothelial cells. The threshold active concentration for ATP was ≤ 1 μm. The rank order of potency of analogues was 2‐chloroadenosine 5′‐triphosphate (2‐C1ATP) > 2‐methylthioadenosine 5′‐triphospate (2‐MeSATP) > ATP > ADP, while adenosine 5′‐(α,βfanethylene)triphosphonate, AMP and adenosine were inactive, indicating the presence of P2y‐purinoceptors. In contrast to their actions on P2y‐receptors in guinea‐pig taenia coli, isopolar analogues of 2‐methylthioadenosine 5′‐(β,γ‐methylene)triphosphonate were less effective than ATP. ATP and ATP analogues increased intracellular free calcium ions, [Ca2+]i, giving a rapid transient peak due predominantly to release from intracellular stores, followed by a maintained steady‐state elevated level due to influx. The dose‐response curves for peak [Ca2+]i induced by ATP, 2‐C1ATP and 2‐MeSATP were very similar to those for PGI2 production. Elevations of [Ca2+]i, above a threshold value of 0.8–1 μm, were necessary for PGI2 production in response to P2y‐receptor activation. The dose relationships between PGI2 release and peak [Ca2+]i were equivalent whether [Ca2+]i was raised by ionomycin or via P2y‐receptor activation by ATP or 2‐ClATP, indicating that elevations of [Ca2+]i provide the major, if not the exclusive intracellular pathway for P2y‐purinoceptor‐mediated PGI2 synthesis.