European Journal of Pharmacology, 214 (1992) 269-272 © 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
5-Hydroxytl3,ptamine stimulates 4 S e a 2 + uptake by human umbilical vein endothelial cells in culture: mediation by 5-HT 2 receptor subtypes J a s w i n d e r K. Gill, G e r r a r d S t a n s b y
a, N e e l i m a
Shukla a George Hamilton a n d J a m i e Y. J e r e m y
M a n u e l A. B a r r a d a s
Department of Chemical Pathology and Human Metabolism, aAcademic Department of Surgery, Royal Free Hospital and School of Medicine, Pond Street, London NW3 2QG, U.K. Received 3 December 1991, revised MS received 18 February 1992, accepted 25 February 1992
In cultured human umbilical vein endothelial cells, 5-HT and a-methyl 5-HT stimulated [45Ca2+] uptake in concentration-dependent manner, whereas the 5-HT l agonists, m-CPP (1-(3-chlorophenyl)piperazine and 2-MPP (1-(2-methoxyphenyl)piperazine), were without effect. In turn, 5-HT-stimulated [45Ca2+] uptake was inhibited in concentration-dependent manners by the 5-HT receptor antagonists ketanserin (5-HTz), LY 53,857 (5-HT2) and methiothepine (5-HT1/2) and to a lesser degeree by MDL 72222 (5-HT3) and BRL 43694 (5-HT 3) whereas (+)-propranolol (5-HT 1) was without effect. These data indicate that 5-HT stimulates Ca 2+ uptake by endothelial cells via activation of a 5-HT 2 receptor subtype. 5-HT was without effect on de novo prostacyclin (PGI 2) synthesis over the concentration of 5-HT that elicited [45Ca2+] uptake. Since 5-HT did not stimulate PGI 2, an event associated with an increase in levels of intracellular Ca 2+, it is postulated that the uptake of 45Ca2+ reflects changes of Ca 2+ at the level of the plasma membrane rather than on intracellular changes. 5-HT-stimulated Ca 2+ uptake may be of relevance to endothelium-dependent relaxation, vascular permeability and endothelial repair and proliferation. 5-HT (5-hydroxytryptamine, serotonin); Ca2+; Endothelium; Prostacyclin
1. Introduction The vascular endothelium has many functions: control of vascular permeability, control of vascular contractility, haemostasis, control of growth and repair and movement of white blood cells across the blood vessel wall (De Clerck et al., 1984; Furchgott, 1983). In turn, serotonin (5-hydroxytryptamine; 5-HT), a major platelet release substance, increases vascular permeability (De Clerck et al, 1981; 1985) and elicits dilation via the release of an endothelial factor (Cocks and Angus, 1983). There is evidence that both permeability and endothelium-dependent relaxation are Ca2+-medi ated (DeClerck et al., 1981; Furchgott, 1983; Luckhoff et al., 1988). In order to further clarify the role of Ca 2÷ in mediating the actions of 5-HT on endothelial function, the effect of 5-HT o n [45Ca2+] uptake by cultured human endothelial cells was studied. The 5-HT receptor subtype involved (5-HT1, 5-HT 2 and 5-HT 3) was investigated using a range of agonists and antagonists
Correspondence to: J.Y. Jeremy, Department of Chemical Pathology and Human Metabolism, Royal Free Hospital and School of Medicine, Pond Street, London NW3 2QG, U.K.
to 5-HT receptors. In order to monitor a known Ca 2+sensitive system in these cells, the effect of 5-HT on the synthesis of prostacyclin (PGI2; Luckhoff et al., 1988) was concomitantly studied.
2. Materials and methods 2.1. [45Ca2+] uptake by cultured human umbilical endothelial cells H u m a n umbilical endothelial cells were prepared by the method of Jaffe et al. (1973). Following initial culture in medium 199 containing 20% fetal calf serum cells were trypsinised and seeded into 96-well tissue culture plates and further cultured in medium 199 containing 20% fetal calf serum. When confluent, the human umbilical endothelial cells were washed twice with H E P E S buffer A (composition in mM: 140 NaCI, 2.7 KC1, 1 E D T A , 0.001 PGE1, 3.8 HEPES, 0.1% glucose, 0.1% bovine serum albumin; adjusted to p H 7.6 with Tris base). To each well, 200 /xl Ca2+-free H E P E S buffer B (mM: 140 NaCI, 2.7 KC1, 8 N a z H P O 4, 1.5 KH2PO4, 0.1 MgCI 2, 1 glucose; adjusted to p H 7.6 with Tris base) containing 0.75/xCi [45Ca2+] and vary-
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- l o g E5 HT-]
I 9 -log
( M )
Fig. 1. (A) Effect of 5-HT (e), a-methyl 5-HT ( 0 ) , m-CPP (©) and 2-MPP ( v ) on 4SCa2+ uptake by cultured human vascular endothelial cells. Each point represents mean+S.E., n = 6. (B) Effect of 5-HT receptor antagonists on 5-HT-stimulated 45Ca2+ uptake by cultured human vascular endothelial cells. LY 53, 857 ( • ), ketanserin (e), methiothepine (0), BRL 43694 ( • ), MDL 72222 (©), ( _+)-propranolol ( • ). Each point represents mean +S.E., n = 6.
the actual quantities of [45Ca2+] taken up in response to 5-HT. In six experiments basal uptake was calculated as 0.62 + 0.04 pmol 45Ca2+. well- 1. min- 1 (mean + S.E.). Data on antagonists are expressed as % inhibition of 5-HT stimulated values (arbitrarily expressed as 0%); 100% inhibition thus represent basal values.
ing concentrations of 5-HT or other 5-HT agonists were added and the culture plates incubated at 37°C for 90 s (Gill et al., 1991). Following incubation, the wells were rapidly washed twice with buffer A. The cells were treated with Triton X-100 to disrupt the cells and radioactivity counted. In the antagonist studies cells were pro-incubated with antagonists for 15 min prior to the simultaneous addition of a stimulatory dose (3 × 10 - 7 M) of 5-HT and [45Ca2+]. The cells were then further incubated and processed as above for agonist studies. In a separate study, the effect of 5-HT on PGI 2 synthesis by human umbilical endothelial ceils was investigated. Confluent cells, in HEPES buffer B, were treated with 5-HT for either 90 s or 1 h at 37°C. Following incubation, aliquots of supernatant were taken for estimation of 6-oxo-PGFl~ (the spontaneous, stable hydrolysate of PGI2), as previously described (Stansby et al., 1991).
2.3. Drugs Serotonin (5-hydroxytryptamine, 5-HT) and ( + ) propranolol were purchased from Sigma Chemical Co. (Poole, Dorset, UK). MDC 72222 was a kind gift of Merrel Dow Pharmaceuticals (Staines, Middlesex, UK), BRL 43699 of Smith Kline and Beecham (Betchworth, Surrey, UK) and methiothepine maleate of Roche Pharmaceuticals (Welwyn Garden City, Herts, UK). a-Methyl 5-HT maleate, 1-(3-chlorophenyl)piperazine dihydrochloride (m-CPP), 1-(2-methoxyphenyl)piperazine hydrochloride (2-MPP) and LY 53,857 maleate were purchased from RBI Research Biochemicals Inc. (Natick, MA, USA). Medium 199 and fetal calf serum were purchased from Gibco Biocult (Paisley Scotland, UK).
2.2. Calculation of resufts For the 5-HT-stimulated [45Ca2+] uptake data, basal uptake (unstimulated) values (arbitrarily expressed as zero) were subtracted from stimulated values to give
TABLE 1 Effect of 5-HT on PGI2 synthesis by endothelial cells (pg 6-oxo-PGFl=. well-l, unit t i m e - i _+S.D., n = 4). Zero values represent undetectable quantities of 6-oxo-PGF1,, ( < 10 pg. well-l). - log [5-HT] (M) pg 6-oxo-PGFl,~ .well - i. 90 s - l pg 6-oxo-PGFt,, .well 1.1h - l
271 3. Results 5 - H T and to a lesser extent, a-methyl 5-HT, stimulated [45Ca2+] uptake by h u m a n umbilical endothelial cells in c o n c e n t r a t i o n - d e p e n d e n t manners, whereas the 5-HT~ agonists, m - C P P and 2 - M P P were without effect (fig. 1A). In turn, 5-HT-stimulated [45Ca2+] uptake was inhibited in c o n c e n t r a t i o n - d e p e n d e n t m a n n e r s by the 5 - H T ~ / 5 - H T 2 receptor antagonists, ketanserin, L Y 53,857 and m e t h i o t h e p i n e and to a lesser degree by M D L 72222 (5-HT 3 antagonist) and B R L 43699 (5-HT 3 antagonist) (fig. 1B). ( + ) - P r o p r a n o l o l (5-HT 1 antagonist) was without effect on [45Ca2+] uptake (fig. 1B). 5 - H T (up to 10 /xM) was without effect on de novo P G I 2 synthesis, either at 90 s or 1 h following addition of 5 - H T (table 1).
4. Discussion Since 5 - H T and a-methyl 5 - H T (a 5 - H T 1 c / 5 - H T 2 receptor agonist), but not m - C P P or 2 - M P P (agonists at the 5 - H T l c receptor but antagonists at the 5 - H T 2 receptor; Hoyer, 1988; C o n n and Sanders-Bush, 1987) stimulated the uptake of 45Ca2+ by h u m a n umbilical endothelial cells, the present data are indicative of a specific effect of 5 - H T on 5 - H T 2 receptor subtypes. This is consolidated by the lack of effect of the 5-HT1 receptor antagonist, ( + ) - p r o p r a n o l o l on 5-HT-stimulated 45Ca 2+ uptake, whereas the 5 - H T l c / 5 - H T 2 antagonists, ketanserin, L Y 53,857 and methiothepine were p o t e n t inhibitors of 5-HT-stimulated 45Ca2+ uptake. In the present study, 5 - H T did not stimulate P G I z synthesis in h u m a n umbilical endothelial ceils, even after an incubation of 1 h. Since agonist-stimulated P G I 2 synthesis in cultured endothelial cells is accompanied by increases in intracellular Ca 2+, which in turn is p u r p o r t e d to stimulate P G I 2 synthesis via activation of phospholipase A 2 (Luckhoff et al., 1988; Hallam et al., 1988), we can only surmise that 5 - H T does not elicit changes in intracellular Ca ~+ (at least not e n o u g h to stimulate P G I z synthesis). W e are also unaware of any studies that have shown 5 - H T to increase intracellular Ca 2 + in cultured endothelial cells using Ca 2+-sensitive dyes such as Fura, Q u i n 2 or aequorin. In contrast to P G I 2, L u c k h o f f et al. (1988) have established that agonist-stimulated E D R F release from cultured bovine endothelial ceils is directly controlled by extracellular Ca 2+ and not by changes of intracellular Ca 2+. In turn, 5 - H T has b e e n shown to possess both vasodilator activity via stimulation of E D R F release and vasoconstrictor activity by direct action on vascular smooth muscle tissue (Cocks and Angus, 1983). T h e present data are therefore consistent with mediation of 5 - H T stimulated E D R F release by Ca 2+ influx. A l t h o u g h it
has b e e n suggested that 5-HT-stimulated E D R F release is m e d i a t e d by '5-HTl-like' not by 5 - H T 2 receptor subtypes (Left et al., 1987), o t h e r studies have indicated E D R F to be 5 - H T 2 r e c e p t o r - m e d i a t e d (Imaizumi et al., 1984). T h e relationship between 5-HT, E D R F and Ca 2+ in h u m a n endothelial cells remains to be clarified. 5 - H T also increases vascular permeability, an effect thought to be due to a combination of endothelial cell gap formation and an increase in blood flow subsequent to arteriolar dilation (DeClerck et al., 1981, 1984, 1985). In turn, both endothelial cell gap formation and increase in permeability are d e p e n d e n t on extracellular Ca 2+ and are inhibited by ketanserin (De Clerck et al., 1981, 1984, 1985). 5 - H T has also b e e n shown to stimulate bovine endothelial cell proliferation, an event a c c o m p a n i e d by an uptake of 45Ca2+ ( D ' A m o r e and Shepro, 1977). Since 5 - H T (and other mitogens) is released by platelets and platelets adhere to sites of vascular injury, this was p r o p o s e d as a m e c h a n i s m by which endothelial repair is initiated. In this context, protein kinase C (PKC), a Ca2+-activated enzyme that moves into the plasma m e m b r a n e from the cytosol is recognised as a key mediator of cell division (Nishizuka, 1984). In turn, 5 - H T (via 5 - H T 2 receptor subtypes) stimulates phosphoinositide turnover in vascular ceils ( R o t h et al., 1984), a process which generates diacyl glycerol, an activator of P K C (Nishizuka, 1984). Thus, w h e t h e r the 5-HT-stimulated Ca 2+ uptake is a c o m p o n e n t of the P K C activation sequence warrants further investigation.
References Cocks, T.M. and J.A. Angus, 1983, Endothelium-dependent relaxation of coronary arteries by noradrenaline and serotonin, Nature 305, 627. Conn, PJ. and E. Sanders-Bush, 1987, Relative efficacies of piperazines at the phosphoinositide hydrolysis-linked serotonergic (5HT 2 and 5-HT k) receptors, J. Pharmacol. Exp. Ther. 242, 552. D'Amore, P. and D. Shepro, 1977, Stimulation of growth and calcium influx in cultured bovine aortic endothelial cells by platelets and vasoactive substances, J. Cell. Physiol. 92, 177. De Clerck, F., M. De Brabander, H. Neels and V.V. de Velde, 1981, Direct evidence for the contractile capacity of endothelial cells, Thromb. Res. 23, 505. De Clerck, F., J.M. Van Neuten and R.S. Reneman, 1984, Platelet vessel wall interactions: implication of 5-hydroxytryptamine, Agents Action 15, 612. De Clerck, F., L. Van Goy, J. Beetens and R.S. Reneman, 1985, Platlet-mediated vascular permeability in the rat: a predominant role for 5-hydroxytryptamine, Thromb. Res. 38, 321. Furchgott, R.F. 1983, Role of endothelium in response of vascular smooth muscle, Circ. Res. 53, 557. Gill, J., P. Dandona and J.Y. Jeremy, 1991, Activation of atypical /3 and a-adrenoceptors, dibutyryl cAMP and iloprost stimulate [45Ca2+] uptake by human platelets, Platelets 2, 93. Hallam, T.J., J.D. Pearson and L.A. Needham, 1988, Thrombinstimulated elevation of human endothelial cell cytoplasmic free
272 calcium concentration causes prostacylin production, Biochem. J. 251,243. Hoyer, D., 1988, Molecular pharmacology and biology of 5HTIc receptors, Trends Pharmacol. Sci. 9, 89. Imaizumi, Y., M. Baba, Y. Imaizumi and M. Watanabe, 1984, Involvement of endothelium in the relaxation of isolated chick jugular vein by 5-hydroxytryptamine, Eur. J. Pharmacol. 97, 335. Jaffe, E.A., R.L. Nachman, C.G. Becker and C.R. Minick, 1973, Culture of endothelial cells derived from umbilical veins. Identification by morphological and immunological criteria, J. Clin. Invest. 52, 2745. Leff, P., G.R. Martin and J.M. Morse, 1987, Differential classification of vascular smooth muscle and endothelial cell 5-HT receptors by use of tryptamine analogues, Br. J. Pharmacol. 91,321.
Luckhoff, A., U. Pohl, A. Mulsch and R. Busse, 1988, Differential role of extra- and intracellular calcium in the release of EDRF and prostacylin from cultured endothelial cells, Br. J. Pharmacol. 95, 189. Nishizuka, Y., 1984, The role of protein kinase C in cell surface signal transduction and tumour promotion, Nature 308, 693. Roth, B.L., T. Nakaki, D. Chuang and E. Costa, 1984, Aortic recognition sites for serotonin (5-HT) are coupled to phospholipase C and modulate phosphatidyl inositol turnover, Neuropharmacology 23, 1223. Stansby, G., N. Shukla, G. Hamilton and J.Y. Jeremy, 1991, Comparison of prostanoid synthesis in cultured human vascular endothelial cells derived from omentum and umbilical vein, Eur. J. Vasc. Surg. 5, 501.