Pharmacological Reports
Copyright © 2006
by Institute of Pharmacology
Polish Academy of Sciences
2006, 58, 884–889
ISSN 1734-1140
Cyclic AMP generating system in human
microvascular endothelium is highly responsive
to adrenaline
Magdalena Namiecinska1, Anna Wiktorowska-Owczarek2,
Agnieszka Loboda3, Józef Dulak3, Jerzy Z. Nowak1,2
Center of Medical Biology, Polish Academy of Sciences, Lodowa 106, PL 93-232 £ódŸ, Poland
Department of Pharmacology, Medical University of £ódŸ, ¯eligowskiego 7/9, PL 90-752 £ódŸ, Poland
Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology,
Jagiellonian University, Gronostajowa 7, PL 30-387 Kraków, Poland
!
Correspondence: Jerzy Z. Nowak,
e-mail: [email protected]
Abstract:
We have tested cultured human microvascular endothelial cells (HMEC-1) for their ability to synthesize cyclic adenosine
3’,5’-monophosphate (cAMP) in the absence or presence of various drugs. The accumulation of cAMP was only slightly affected by
the addition of a phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) to the incubation medium. A direct stimulator of
adenylyl cyclase forskolin and adrenergic drugs, such as adrenaline and noradrenaline, strongly increased cAMP accumulation in
IBMX-treated HMEC-1 cells, whereas some other drugs known to stimulate the nucleotide synthesis in different cell/tissues were
inactive (dopamine, histamine). Adrenaline was significantly more potent than noradrenaline. The effect of adrenaline on cyclic
AMP production was reproduced by a selective b-adrenoceptor agonist isoprenaline, antagonized by b-blocker propranolol, and was
not influenced by both a1- and a2-selective antagonists, prazosin and yohimbine, respectively. Adrenaline did not significantly
affect the ability of HMEC-1 cells to produce vascular endothelial growth factor (VEGF) or interleukin-8 (IL-8), the major
angiogenic mediators. The results indicate that under basal (non-stimulated) conditions, the cAMP generating system of HMEC-1
cells maintained in culture remains rather quiescent, yet it can strongly respond to the hormone adrenaline acting on b-adrenergic
receptors.
Key words:
human microvascular endothelium, HMEC-1, cyclic AMP, isoprenaline, adrenaline, noradrenaline, forskolin, b-adrenergic
receptor, VEGF, IL-8
Introduction
Cyclic adenosine 3’,5’-monophosphate (cAMP) is an
important signaling molecule operating in different
cell types of various species. Its effective concentra-
884
Pharmacological Reports, 2006, 58, 884–889
tion, resulting from the local activity of the cyclic nucleotide synthesizing and inactivating enzymes,
adenylyl cyclase (AC) and phosphodiesterase (PDE),
respectively, varies widely not only among different
tissues and species, but also among similar type of
cells within the same organism, as is the case for e.g.,
Human endothelial cells are responsive to adrenaline
Magdalena Namiecinska et al.
endothelial cells originating from different blood vessels [5, 7, 11, 19, 20].
Stimulation of many receptors may positively or
negatively affect the activity of the cyclic AMP generating system, and one signaling molecule, depending
on receptor type with which it interacts, may either
stimulate or inhibit the production of cAMP, contributing to the final biological response of a given cell
[23]. Endothelial cells possess receptor-driven cyclic
AMP system(s) [5, 19, 20]; they actively participate in
various physiological events, including transport,
metabolic, synthetic or secretory processes, whose
repertoire and/or intensity may differ depending on
both the size (large, small, capillary) and type (artery,
vein) of a vessel [4, 32].
The aim of this work was twofold: 1. to study the
ability of human microvascular endothelial cells
(HMEC-1) maintained in culture to synthesize cAMP
under basal and stimulated conditions (including catecholamines), and 2. to see whether adrenaline modulates the expression of vascular endothelial growth
factor (VEGF) or IL-8 in HMEC-1 cells. We report
here that the cAMP generating system of HMEC-1
cells, being weakly active under basal conditions, can
be largely stimulated by adrenaline used at physiological concentrations. In contrast to the cAMP response,
adrenaline affected neither VEGF nor IL-8 levels.
Materials and Methods
Chemicals
The following substances used were: MCDB 131 medium, fetal bovine serum, penicillin-streptomycin soution (5,000 units/ml penicillin and 5,000 µg/ml
streptomycin sulfate in physiological saline),
phosphate-buffered saline (PBS) (pH = 7.4) and
trypsin-EDTA (0.25% trypsin, 1mM EDTA-4 Na)
were purchased from Invitrogen (Carlsbad, California). Epinephrine (adrenaline), L-arterenol (noradrenaline), 3-isobutyl-1-methylxanthine (IBMX), isoprenaline, dopamine, histamine, forskolin, propranolol, prazosin and yohimbine were purchased from
Sigma (St. Louis, MO, USA) and radioactive compounds: 2,8-[3H]adenine (specific activity 24.40
Ci/mmol) was from PerkinElmer Life Sciences, Inc.
(Boston, MA, USA) and [14C]cAMP (specific activity
56 mCi/mmol) was from Moravek Biochemicals
(Brea, CA, USA).
Cell culture
HMEC-1 (human microvascular endothelial cells)
were kindly provided by Dr. F. Candal from Center
for Disease Control and Prevention (Atlanta). The
cells were used between passages 32–41 and cultured
in 25 cm3 flasks in MCDB 131 medium supplemented
with 10% fetal bovine serum, 10 ng/ml of epidermal
growth factor, 1 µg/ml of hydrocortisone and penicillin-streptomycin solution, in humidified atmosphere
of 95% O2 and 5% CO2 at 37°C. Every third day cells
were harvested in trypsin-EDTA (0.25% trypsin,
1mM EDTA) solution.
Assay of cAMP formation
Cells used in experiments were seeded in 12-well
plates at a density of 250,000 cells/well in 500 µl of
culture medium and cultured overnight. Next day, culture medium was removed, fresh serum-free culture
medium was added and cells were incubated in the
presence of [3H]adenine for 2 h at 37°C. Next, the
medium was removed, cells were rinsed two times
with pre-warmed phosphate-buffered saline (PBS)
and serum-free culture was added. Then, cells were
preincubated for 20 min at 37°C in the presence of
3-isobutyl-1-methylxanthine (IBMX; 0.1 mM). After
this preincubation period, cells were exposed to appropriate agonists for further 15 min. Antagonists,
when tested, were applied 15 min before an agonist.
The reaction was stopped by adding 500 µl of an icecold 10% trichloroacetic acid. The resulting mixture
was then transferred into test tubes, centrifuged and
the formed cAMP was quantified in a supernatant
fraction.
The formation of [3H] cAMP in [3H]adenineprelabeled cells was assayed according to Shimizu et
al. [31]. The formed [3H]cyclic AMP was isolated by
sequential Dowex-alumina column chromatography
according to Salomon et al. [27]. The results were individually corrected for percentage recovery with the
aid of [14C]cAMP added to each column system prior
to the nucleotide extraction. The accumulation of
cAMP during a 15-min stimulation period was assessed as a percentage of the conversion of [3H]adenine to [3H]cAMP. Details of the whole procedure
were described earlier [34].
Pharmacological Reports, 2006, 58, 884–889
885
VEGF and IL-8 concentrations in cell culture media
were determined by a commercially available ELISA
kits according to the vendor’s protocols (R&D System, Abingdon, UK).
Data analysis
All data are expressed as the mean ± SEM values. For
statistical evaluation of the results, analysis of variance (ANOVA) was used followed by the post-hoc
Student-Newman-Keuls test.
Results and Discussion
In order to check the ability of HMEC-1 cells to synthesize cAMP under basal, non-stimulated conditions,
we compared the accumulation of [3H]cAMP in
[3H]adenine-prelabeled cells in the absence or presence of a PDE inhibitor IBMX (0.1 mM) after 60 and
180 min. The obtained time-dependent results without
IBMX are: 0.30 and 0.25% conversion, and with
IBMX: 0.38 and 0.35% conversion, showed only a
slight increase in the nucleotide accumulation with
time (127 and 140% of respective control taken as
100%), which suggests that under our experimental
conditions the basal activity of the cAMP generating
system in HMEC-1 cells was rather low.
Earlier studies have shown a highly differentiated
basal activity of the cAMP generating system operating in different endothelial cells, such as, e.g., human
umbilical vein endothelial cells (HUVEC), human
adipose microvascular endothelial cells (HAMVEC),
pig pulmonary artery endothelial cells (PPAEC), bovine aortic endothelial cells (BAEC) [20, 21]. Although the mean levels of cyclic AMP were similar in
HUVEC, BAEC and PPAEC (9.4–12.5 pmol/mg of
protein), those in HAMVEC were significantly higher
(26 pmol/mg of protein). Yet, a completely different
picture has emerged when the various cell types were
incubated with IBMX where the basal cAMP levels
were found elevated 2- to 9-fold depending on the cell
type (BAEC >> HUVEC ³ HAMVEC > PPAEC).
Thus, the observed here a relatively low basal activity
of the cAMP generating system in HMEC-1 cells does
886
Pharmacological Reports, 2006, 58, 884–889
not seem to be a feature unique for this type of endothelial cells.
In contrast to a rather weak basal activity of the
cAMP generating system of HMEC-1 cells, the addition of forskolin, a direct activator of adenylyl cyclase, or catecholamines, such as noradrenaline, and –
especially – adrenaline to the IBMX-enriched incubation medium, strongly stimulated the nucleotide accumulation (Fig. 1). Adrenaline added to the medium
without IBMX also significantly stimulated cAMP
production, and the obtained results of experiments
carried out in parallel with and without IBMX for
100 µM hormone were (in % conversion): without
IBMX – control 0.39 ± 0.03(9), adrenaline 2.51 ± 0.16(9),
with IBMX – control 0.43 ± 0.10(7), adrenaline 7.01
± 0.19(12).
As shown in Figure 1, noradrenaline also substantially stimulated cyclic AMP synthesis in HMEC-1
cells, it appeared, however, to be significantly less potent than adrenaline; for example, at 10 and 100 µM
their respective effects were (in % conversion): control: 0.30 ± 0.02(25); 10 µM: 1.44 ± 0.12(12), with
min-max values of 0.74–1.89 vs. 7.42 ± 0.25(6), with
14
C
10 µM
cAMP formation, % conversion
ELISA assays
12
100 µM
10
8
6
4
2
0
C
Iso
Adr
NA
C
For
Effects of adrenaline (Adr), noradrenaline (NA), isoprenaline
(Iso) and forskolin (For) on cyclic AMP synthesis in HMEC-1 cells.
Bars represent the means (± SEM of 6–25 experiments). *** p < 0.001;
C – control values
Fig. 1.
min-max values of 6.68–8.47; 100 µM: 3.44 ±
0.31(22), min-max values of 2.14–4.60% conversion;
vs. 8.43 ± 0.39(18), min-max values of 4.85–10.27%
conversion. A similar picture has been observed in
bovine endothelial cells (BAEC) where noradrenaline
was at least one order of magnitude weaker than
adrenaline in stimulating adenylyl cyclase activity
[28]. Unfortunately, in neither study (BAEC,
Human endothelial cells are responsive to adrenaline
Magdalena Namiecinska et al.
HMEC-1) the concentration curves reached their plateaus, which makes impossible to compare the respective maximal (or near maximal) effects of the two
catecholamines.
The cAMP-enhancing effect of the studied catecholamines in HMEC-1 cells was mediated through
b-adrenergic receptors, as b-adrenoceptor blocker
propranolol (10 µM) prevented the action of adrenaline, whereas a1- and a2-selective blockers, prazosin
and yohimbine, respectively, applied at 10 µM dose
had no effect (Fig. 2). In addition, as shown in Figure
1, a selective b-agonist isoprenaline reproduced the
effects of adrenaline.
cAMP formation, % conversion
12
10
8
C
Adr 100 µM
6
4
Adr+Prop 10 µM
Adr+Praz 10 µM
Adr+Yoh 10 µM
2
recently been confirmed by the results of Loboda et
al. [17, 18] which demonstrated the induction of
VEGF expression by both hypoxia (1% O2) and cobalt chloride (CoCl2), a hypoxia mimicking agent
[16]. The mentioned data considered together
prompted us to check whether HMEC-1 cells will express VEGF when exposed to adrenaline, the hormone endowed with a potent cAMP-enhancing effect
just in these cells (present data). An additional argument supporting such an idea comes from the data
that noradrenaline, acting via the cAMP-dependent
mechanism, did stimulate VEGF-mRNA expression
in rodent brown adipocytes [1,10] and the b-adrenoceptor agonist isoprenaline did so in aortic smooth
muscle cells [24].
However, in our hands (Fig. 3), adrenaline (10 and
100 µM) incubated with HMEC-1 cells for 3, 6, 12
and 24 h did not significantly modify VEGF levels,
although CoCl2 (250 µM) as expected stimulated the
cytokine synthesis, and the effect was particularly impressive after 24 h (508% of control value; Fig. 3).
Adrenaline (100 µM; 24 h) also had no significant effect on IL-8 production (results not shown).
0
Although the b-adrenoceptor-mediated stimulation
of cAMP production has been reported in different endothelial cells [2, 19–21, 28, 30], such an effect in
HMEC-1, an immortalized human dermal microvascular endothelial cell line, has not been studied till
now. On the other hand, it has recently been shown
that in HMEC-1 cell line adrenaline and noradrenaline, acting via b-adrenoceptors, stimulated IL-6 induction [12]. Interestingly, several recent papers have
focused on the AC-cAMP signaling pathway in
HMEC-1 cells, showing its activity upon stimulation
by, e.g., forskolin [33] or adenosine [8, 15]. Of special
interest are the latter results, as the authors showed
that microvascular HMEC-1 cells preferentially express A2B, while macrovascular HUVEC cells – A2A
adenosine receptors, both being positively linked to
cAMP formation; yet, only HMEC-1 cells simultaneously responded with an increased expression of IL-8,
bFGF and VEGF [8, 15]. The ability of HMEC-1 cells
to express the major angiogenic mediator VEGF has
200
3h
180
***
6h
160
VEGF [pg/ml]
Fig. 2. Effects of adrenergic receptor antagonists propranolol (Prop),
prazosin (Praz), and yohimbine (Yoh) on 100 µM adrenaline-evoked
stimulation of cyclic AMP accumulation in HMEC-1 cells. Bars represent the means (± SEM of 6–22 experiments). *** p < 0.001; C – control value
12 h
140
24 h
120
100
80
*
60
40
20
0
C
Adr 10
Adr 100
CoCl2
Fig. 3. Effects of adrenaline (Adr, 10 and 100 µM) and cobalt chloride
(CoCl , 250 µM) on VEGF levels in HMEC-1 cells. Bars represent the
means (± SEM of 3 experiments). * p < 0.05; *** p < 0.001
A biological significance of the cAMP response of
HMEC-1 cells to b-adrenoceptor agonists, especially
to adrenaline – a powerful stimulator of the nucleotide
synthesis, remains at present unknown. Earlier findings have shown on porcine aortic endothelial cells
(PAEC) [13, 14] and HUVEC cells [9] that stimulation of cAMP synthesis evoked by isoprenaline or
forskolin led to nitric oxide (NO) generation; Ferro et
al. [9] have also shown that isoprenaline relaxed umbilical vein rings, which would indicate that activaPharmacological Reports, 2006, 58, 884–889
887
tion of the b-adrenoceptor-dependent cAMP cascade
eventually give rise to vasorelaxation. Recent data
have demonstrated that adrenaline via the cAMPPKA dependent cascade stimulated the release of von
Willebrand factor in macrovascular HUVEC cells
[25, 26], yet such an activity of the hormone has not
been reported for the microvascular HMEC-1 cells.
The above-cited observations linking catecholamines
(adrenaline, noradrenaline) and cAMP in different endothelial cells, including HMEC-1 cells, suggest that
the spectrum of biological events regulated by adrenaline and/or noradrenaline is much broader than previously thought, and more study is needed to substantiate the profile of physiological (induced by endogenously released catecholamines, especially adrenaline)
and pharmacological (induced by exogenously applied b-adrenoceptor agonists, including native catecholamines) effects resulting from adrenoceptor activation.
In addition to adrenaline, noradrenaline and isoprenaline, we also studied the effects of dopamine and
histamine on cAMP in HMEC-1 cells for two reasons,
firstly, these agents are well-known stimulators of the
nucleotide production in many body cells and/or tissues [7, 19, 22, 23, 29], and secondly, some reports
described their effects also in endothelial cells [3, 6,
20]. In our hands, these two agents used at 100 µM
concentration had no significant stimulatory activity
on the cAMP generating system in HMEC-1 cells (results not shown). However, in other studies, histamine
(10–100 µM) has been reported to be active in BAEC
and HUVEC model systems [20] and inactive in
HMEC cells, although in these cells histamine did
stimulate phospholipase C-linked signaling cascade
through H1-type receptors [6], whereas dopamine
stimulated cAMP production in human cerebromicrovascular endothelium [3]. These data further indicate
that different endothelial cells may express different
sets of receptors for a number of hormones and factors which may influence various aspects of the endothelium physiology.
In conclusion, we have shown in immortalized human dermal microvascular endothelial cells (HMEC-1)
that the basal activity of the cAMP generating system
of these cells is very low, yet the system can be
strongly stimulated by b-adrenoceptor agonists, of
which the hormone adrenaline (which physiologically
occurs and sometimes distinctly fluctuates in the
blood) appeared to be a very effective stimulator.
888
Pharmacological Reports, 2006, 58, 884–889
A functional meaning of this adrenaline-induced endothelial response remains to be established.
Acknowledgments:
This study was partly supported by funds from Medical University
of £ódŸ (grant 502-11-245) and by Center of Medical Biology, PAS,
£ódŸ, and the funds from Jagiellonian University.
References:
1. Asano A, Morimatsu M, Nikami H, Yoshida T, Saito M:
Adrenergic activation of vascular endothelial growth factor mRNA expression in rat brown adipose tissue: implication in cold-induced angiogenesis. Biochem J, 1997,
328, 179–183.
2. Bacic F, McCarron RM, Uematsu S, Spatz M: Adrenergic receptors coupled to adenylate cyclase in human
cerebromicrovascular endothelium. Metab Brain Dis,
1992, 7, 125–137.
3. Bacic F, Uematsu S, McCarron R, Spatz M: Dopaminergic receptors linked to adenylate cyclase in human cerebramicrovascular endothelium. J Neurochem, 1991, 57,
1774–1780.
4. Born GVR, Schwartz CJ: Vascular Endothelium: Physiology, Pathology and Therapeutics. Schatthauer Verlag,
Stuttgart, 1997.
5. Bounassisi V, Venter JC: Hormone and neurotransmitter
receptors in an established vascular endothelial cell line.
Proc Natl Acad Sci USA, 1976, 73, 1612–1616.
6. Bull HA, Cohen J, Dowd PM: Responses of human
microvascular endothelial cells to histamine and their
modulation by interleukin 1 and substance P. J Invest
Dermatol, 1991, 97, 787–792.
7. Daly JW (Ed.): Cyclic Nucleotides in the Nervous System. Raven Press, New York, 1977.
8. Feoktistov I, Goldstein AE, Ryzhov S, Zeng D, Belardinelli L, Voyno-Yasenetskaya T, Biaggioni I: Differential
expression of adenosine receptors in human endothelium
cells. Role of A2B receptors in angiogenic factor regulation. Circ Res, 2002, 90, 531–538.
9. Ferro A, Queen LR, Priest RM, Ritter JM, Poston L,
Ward JPT: Activation of nitric oxide synthase by
b2-adrenoceptors in human umbilical vein endothelium
in vitro. Br J Pharmacol, 1999, 126, 1872–1880.
10. Frederiksson JM, Lindquist JM, Bronnikov GE, Nedergaard J: Norepinephrine induces vascular endothelial
growth factor gene expression in brown adipocytes
through a b-adrenoceptor/cAMP/protein kinase A pathway involving Src but independently of Erk1/2. J Biol
Chem, 2000, 275, 13802–13811.
11. Gerritsen ME: Functional heterogeneity of vascular endothelial cells. Biochem Pharmacol, 1987, 36, 2701–2711.
12. Gornikiewicz A, Sautner T, Brostjan C, Schmierer B,
Fugger R, Roth E, Muhlbacher F, Bergmann M: Catecholamines up-regulate lipopolysaccharide-induced IL-6
Human endothelial cells are responsive to adrenaline
Magdalena Namiecinska et al.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
production in human microvascular endothelial cells.
FASEB J, 2000, 14, 1093–1100.
Graier WF, Groschner K, Schmidt K, Kukovetz WR: Increases in endothelial cyclic AMP levels amplify
agonist-induced formation of endothelium-derived relaxing factor (EDRF). Biochem J, 1992, 288, 345–349.
Graier WF, Kukovetz WR, Groschner K: Cyclic AMP
enhances agonist-induced Ca2+ entry into endothelial
cells by activation of potassium channels and membrane
hyperpolarization. Biochem J, 1993, 291, 263–267.
Grant MB, Tarnuzzer RW, Caballero S, Ozeck MJ, Davis
MI, Spoerri PE, Feoktistov I et al.: Adenosine receptor activation induces vascular endothelial growth factor in human retinal endothelial cells. Circ Res, 1999, 85, 699–706.
Grasselli F, Basini G, Bussolati S, Bianco F: Cobalt chloride, a hypoxia-mimicking agent, modulates redox status
and functional parameters of cultured swine granulose
cells. Reprod Fer Dev, 2005, 17, 715–720.
Loboda A, Jazwa A, Jozkowicz A, Molema G, Dulak J:
Angiogenic transcriptome of human microvascular endothelial cells: effect of hypoxia, modulation by atorvastatin. Vascul Pharmacol, 2006, 44, 206–214.
Loboda A, Jazwa B, Wegiel B, Jozkowicz A, Dulak J:
Heme oxygenase-1-dependent and –independent regulation of angiogenic expression: effect of cobalt protoporphyrin and cobalt chloride on VEGF and IL-8 synthesis in human microvascular endothelial cells. Cell Mol
Biol, 2005, 51, 347–355.
Manolopoulos VG, Fenton JW, Lelkes PI: The thrombin
receptor in adrenal medullary microvascular endothelial
cells is negatively coupled to adenylyl cyclase through
a Gi protein. Biochim Biophys Acta, 1997, 1356, 321–332.
Manolopoulos VG, Liu J, Unsworth BR, Lelkes PI:
Adenylyl cyclase isoforms are differentially expressed in
primary cultures of endothelial cells and whole tissue homogenates from various rat tissues. Biochem Biophys
Res Commun, 1995, 208, 323–331.
Monolopoulos VG, Samet MM, Lelkes PI: Regulation of
the adenylyl cyclase signaling system in various types of cultured endothelial cells. J Cell Biochem, 1995, 57, 590–598.
Nowak JZ: Histamine in the retina and some other components of the visual system. Prog Retinal Res, 1993, 12,
41–74.
Nowak JZ, Sek B, Schorderet M: Bidirectional regulation of cAMP generating system by dopamine-D1 and
D2-receptors in the rat retina. J Neural Transm, 1990, 81,
235–240.
Pueyo ME, Chen Y, D’Angelo G, Michel JB: Regulation
of vascular growth factor expression by cAMP in rat aor-
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
tic smooth muscle cells. Exp Cell Res, 1998, 238,
354–358.
Rondaij MG, Bierings R, Kragt A, Gijzen KA, Sellink E,
van Mourik JA, Fernandez-Borja M et al.: Dyneindynactin complex mediates protein kinase A-dependent
clustering of Weibl-Palade bodies in endothelial cells.
Arterioscler Thromb Vasc Biol, 2006, 26, 49–55.
Rondaij MG, Sellink E, Gijzen KA, Klooster JP, Hordijk
PL, von Mourik JA, Voorberg J: Small GTP-binding protein Ral is involved in cAMP-mediated release of von
Willebrand factor from endothelial cells. Arterioscler
Thromb Vasc Biol, 2004, 24, 1315–1320.
Salomon Y, Londos C, Rodbell M: A highly sensitive
adenylate cyclase assay. Anal Biochem, 1974, 58, 541–544.
Schafer AI, Gimbrone MA, Handin RI: Endothelial cell
adenylate cyclase: activation by catecholamines and
prostaglandin I2. Biochem Biophys Res Commun, 1980,
96, 1640–1647.
Schorderet M, Nowak JZ: Retinal dopamine D1 and D2
receptors: characterization by binding or pharmacological studies and physiological functions. Cell Mol Neurobiol, 1990, 10, 303–325.
Sexl V, Mancusi G, Baumgartner-Parzer S, Schutz W,
Freissmuth M: Stimulation of human umbilical vein endothelial cell proliferation by A2-adenosine and b2-adrenoceptors. Br J Pharmacol, 1995, 114, 1577–1586.
Shimizu H, Daly JW, Creveling CR: A radioisotopic
method for measuring the formation of adenosine 3’,5’cyclic monophosphate in incubated slices of brain.
J Neurochem, 1969, 16, 1609–1619.
Vane JR. The Croonian Lecture 1993: The endothelium:
maestro of the blood circulation. Philos Trans R Soc
Lond B Biol Sci, 1994, 343, 225–246.
Vanhauwe JF, Thomas TO, Minshall RD, Tiruppathi C,
Li A, Gilchrist A, Yoon EJ et al: Thrombin receptors activate Go proteins in endothelial cells to regulate intracellular calcium and cell shape changes. J Biol Chem, 2002,
277, 34143–34149.
Zawilska JB, Gendek-Kubiak H, Woldan-Tambor A,
Wiktorowska-Owczarek A, Nowak JZ: Histamineinduced cyclic AMP formation in the chick hypothalamus: interaction with vasoactive intestinal peptide. Pharmacol Rep, 2005, 57, 188–194.
Received:
October 27, 2006, in revised form: November 28, 2006.
Pharmacological Reports, 2006, 58, 884–889
889