Mechanisms leading to adenosine-stimulated of microvascular endothelial cells

proliferation

CYNTHIA J. MEININGER AND HARRIS J. GRANGER Microcirculation Research Institute and Department of Medical Physiology, Texas A & M University College of Medicine, College Station, Texas 77843

MEININGER, CYNTHIA J., AND HARRIS J. GRANGER. Ale&anisms leading to adenosine-stimulated proliferation of microvascular endothelial celZs. Am. J. Physiol. ‘258 (Heart Circ. Physiol. 27): Hl98-H206, 1990.-This study investigated the mechanisms by which adenosine stimulates proliferation of microvascular endothelial cells. The metabolic byproducts of adenosine, inosine and hypoxanthine were unable to stimulate proliferation. When adenosine uptake was prevented, the stimulation of proliferation was unchanged, suggesting that uptake of adenosine with subsequent incorporation into the nucleotide pool is not the mechanism for increasing proliferation. Treatment of endothelial cells with adenosine analogues, presumably selective for either the A1 or AS receptor, stimulated proliferation equally. This suggested that adenosine 3’, Y-cyclic monophosphate (CAMP) might not mediate the proliferative response to adenosine. However, radioimmunoassay of cell extracts after treatment with either analogue showed an increase in CAMP. In addition, adenylate cyclase blockade with 2’, 5’dideoxyadenosine prevented the proliferative response brought about by these analogues. These data suggest that the proliferative response to adenosine depends on an increase in CAMP. A 2-h pulse of cholera toxin stimulated endothelial cell proliferation, further supporting a role for CAMP. Pretreatment of endothelial cells with pertussis toxin blocked the stimulation of proliferation, indicating that a Gi or similar G protein is also involved in proliferation. We conclude that the proliferative response to adenosine involves a pertussis toxin-sensitive substrate as well as an increase in CAMP.

adenosine receptors; angiogenesis; G proteins; cyclic monophosphate; pertussis toxin

adenosine

3’, 5’-

or neovascularization, denotes the growth of new capillary vessels from an established microvasculature following stimulation by various physiological or pathological processes. Factors derived from both normal tissues and tumors have been shown to induce angiogenesis (for reviews, see Refs. 1, 11). The mechanism for release and/or activation of these factors is not known. Tissue hypoxia or tissue ischemia is a common feature of many of the conditions in which neovascular growth is observed. For example, the neovascularization associated with tumor growth, diabetic retinopathy, and wound healing are all preceded by the development of poorly perfused tissue (3, 10, 14). All of the tissues involved in these conditions contain angiogenesis-inducing factors, even under conditions of normal blood flow. D’Amore and Thompson (7) postulated that the tissue injury asANGIOGENESIS,

H198

0363-6135/90

$1.50 Copyright

sociated with ischemia results in the release of the mitogens that are sequestered within tissues or cells. Alternatively, alterations in the microenvironment associated with ischemia (e.g., hypoxia, change in pH, release of metabolites) may also affect the angiogenic process. Adenosine is a low-molecular weight metabolite released by hypoxic tissue. Adenosine has long been known as a potent vasodilator (8) but, more recently, was found to stimulate angiogenesis (9, 34) and endothelial cell proliferation (19). This effect of adenosine is caused by binding to specific receptors on the external surface of the endothelial cell (19). Adenosine receptors are coupled to adenylate cyclase via guanosine 5’-triphosphate regulatory proteins (G proteins) and can be subdivided into two classes: A1 (or Ri) receptors coupled to adenylate cyclase in an inhibitory manner and A2 (or R,) receptors coupled to adenylate cyclase in a stimulatory manner. Binding of adenosine to these receptors causes the adenosine 3’, Y-cyclic monophosphate (CAMP) concentration within the cell to rise or fall depending on the type of G protein that couples the receptor with the catalytic subunit of adenylate cyclase. Receptors of the A2 subtype have been demonstrated in cerebral microvessel segments of several species (17,28); however, whether these receptors reside on endothelial cells, vascular smooth muscle cells or both remains to be determined. Adenosine receptors have classically been characterized by their differing affinities for adenosine and its structural analogues (18). The majority of this work was done using brain slices or brain cell membranes. At the A, adenosine receptor the p-substituted adenosine analogue, R-p-phenylisopropyladenosine (R-PIA) is more potent than adenosine or Z-chloroadenosine, which in turn is more potent than the 5’-carboxamides of adenosine, such as 5’ -N-ethylcarboxamide adenosine (NECA). At the A2 adenosine receptor this order of agonist potency is reversed (i.e., NECA is most potent and R-PIA is least potent). More recently, NG-cyclopentyladenosine was found to be even more selective for the A1 receptor (20) The GTP regulatory proteins coupling the adenosine receptor to adenylate cyclase are heterotrimers with subunits designated a, ,6, and y. Differences in the cu-subunits serve to distinguish the various G proteins, whereas the /3- and y-subunits appear very similar between the different receptors. The a-subunits contain a single, high-affinity binding site for GTP and possessthe GTPhydrolyzing activity that is crucial for the action of the

0 1990 the American

Physiological

Society

Downloaded from www.physiology.org/journal/ajpheart at Washington Univ (128.252.067.066) on February 13, 2019.

ADENOSINE-STIMULATED

PROLIFERATION

G proteins. This subunit also contains the site for NADdependent ADP-ribosylation catalyzed by bacterial toxins. The purpose of this study was to begin to elucidate the mechanism(s) by which adenosine stimulates the proliferation of microvascular endothelial cells in culture. Because this proliferation is induced by the binding of adenosine to its receptor on the endothelial cell, we investigated the role(s) of the components of this receptor-enzyme complex in the stimulation of proliferation. We used adenosine analogues to study which subclass of adenosine receptor might be mediating the increase in endothelial cell proliferation. We selectively activated or inactivated the a-subunits of the G proteins with bacterial toxins to determine which G proteins play a role in transducing the proliferative signal from the surface receptor to the cell interior. Finally, we looked at the role of CAMP in this proliferative response. MATERIALS

AND

METHODS

Cells. Coronary microvascular endothelial cells were taken from bovine hearts obtained immediately after the death of the animal. Isolation of venular endothelial cells was achieved by a bead attachment technique described previously (26). This method is based on attachment of endothelial cells to microcarrier beads that are tightly lodged in a vessel. By varying the size of the beads perfused, one can select the size of the vessel from which to isolate the endothelial cells. Briefly, the coronary microvasculature was perfused from the aortic ostia with Ca ‘+- and Mg2+-free Dulbecco’s phosphate-buffered saline (DPBS) at 37°C. A sonicated suspension of l&pm polystyrene beads (3M, St. Paul, MN) at a concentration of 8,000 beads/ml in DPBS supplemented with 0.02% EDTA and maintained at 4°C was perfused via small veins connecting the coronary sinus with the venous side of the microcirculation. The endothelial cells became attached to beads that had lodged tightly in vessels. The combination of cold shock and EDTA resulted in dislodging of the attached endothelial cells from the basement membrane but not from the beads. Antegrade perfusion of DPBS (37°C) from the aortic ostia washed bead-cell complexes from the microcirculation, allowing them to be collected at the coronary sinus. These beadcell complexes were recovered by centrifugation (ZOO g, 6 min) and washed free of EDTA by centrifugal washes of balanced salt solution. The bead-cell suspensions were plated in gelatin-coated dishes and grown with Dulbecco’s modified Eagle’s medium (DMEM, K.C. Biological, Lenexa, KA) supplemented with endothelial cell growth factor (ECGF, 100 pg/ml, Biomedical Technologies, Stoughton, MA), 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 pg/ml streptomycin, and 0.25 pg/ml amphotericin B (Fungizone). After the cells neared confluence, the endothelial cell growth factor was replaced with 20% fetal calf serum. Cell lines were passaged by trypsinization using 0.25% trypsin in phosphate-buffered saline containing 0.02% EDTA. Endothelial cell identity was confirmed using fluorescein isothiocyanate (FITC) -conjugated anti-human factor VIIIrelated antigen (Atlantic Antibodies, Scarborough, ME)

OF

ENDOTHELIAL

Hl99

CELLS

and fluorescein-labeled Ulex europaeus Agglutinin I (Vector Laboratories, Burlingame, CA). Growth curues. To measure cell proliferation, endothelial cells (EC) were dispersed by trypsinization and plated in gelatin-coated &well tissue culture trays at a density of 1 X lo4 cells/cm2 in medium containing 10% fetal calf serum. Cells were allowed 2-4 h for attachment. The medium in the wells was aspirated and fresh medium containing the experimental reagents was added to the cells. Cell counts of triplicate wells were made daily. Direct counts of viable cells were obtained by suspending endothelial cells in trypan blue and using a hemacytometer. In experiments utilizing NECA, R-PIA and dideoxyadenosine, cells were seeded at a density of 1 x lo4 cells/ cm2 in medium containing 10% fetal calf serum. Cells were allowed 2-3 h to attach. The medium in the wells was then aspirated, the cells were rinsed with phosphatebuffered saline, and fresh “defined” medium containing the experimental reagents was added to the wells. This defined medium was optimized in our laboratory for coronary microvascular endothelial cells and consisted of DMEM, 1% calf serum, insulin-transferrin-selenium (ITS Premix, Collaborative Research, Bedford, MA), putrescine (1.6 pg/ml, Sigma, St. Louis, MO), linoleic acid-bovine serum albumin (BSA) (10 fig/ml, Sigma), sodium pyruvate (1 mM), L-glutamine (2 mM), and penicillin-streptomycin-Fungizone (Hazleton, Lenexa, KA). Cell counts were made daily as detailed above. CAMP and protein determination. Endothelial cells were seeded in gelatin-coated 35mm tissue culture dishes and grown to confluence. The intracellular level of CAMP was determined by a modification of the method of Rozengurt et al. (25). Briefly, cells were rinsed twice with serum-free DMEM and incubated for 3 h in the same medium containing 1.5 U/ml adenosine deaminase. The cells were again rinsed twice and maintained in serum-free DMEM with or without an adenosine analogue for 2, 5, or 15 min at 37°C. At the end of each incubation period, the medium was rapidly aspirated and the CAMP extracted by adding 400 ~1 of 0.1 N HCI to each dish. After 20 min, the HCl solutions were transferred to tubes and frozen (-20°C) until assayed. The precipitated protein remaining on the dishes was dissolved in 1 ml of 0.1 M NaOH/2% Na2C03 and frozen as above. The CAMP content of the HCl extracts was determined by radioimmunoassay after acetylation (4) using a commercially available kit (New England Nuclear, Boston, MA). Protein content was measured in an enzyme-linked immunosorbent assay (ELISA) reader W max, Molecular Devices, Palo Alto, CA) using a Bradford technique modified for microtiter plates. Briefly, 0.1 ml of diluted protein solution was added to 0.1 ml of 40% (vol/vol) Bio-Rad (Richmond, CA) protein reagent concentrate and the samples were gently mixed. After 10 min, the absorbance was read at 595 nm. Bovine serum albumin was used as the standard for the assay. RESULTS

Inosine and hypoxanthine, products of adenosine, were

the metabolic breakdown tested for their ability to

Downloaded from www.physiology.org/journal/ajpheart at Washington Univ (128.252.067.066) on February 13, 2019.

HZ00

ADENOSINE-STIMULATED

PROLIFERATION

stimulate the proliferation of coronary venular endothelial cells. In an attempt to mimic the in vitro response seen with adenosine (0.5 and 5.0 ,uM) (19), inosine and hypoxanthine were added to endothelial cells at equivalent doses. Table 1 summarizes data from several experiments. The data have been normalized to compensate for the variability between experiments. The control cell counts are set at 100%. The counts for cells grown in the presence of adenosine, inosine, or hypoxanthine are recorded as a percentage of the control cell count. Only the group treated with adenosine showed a significant increase in proliferation over the control group. Inosine and hypoxanthine were unable to stimulate the proliferation of endothelial cells at the concentrations tested. To test whether the uptake of adenosine contributed to the increased proliferation, endothelial cells were cultured with adenosine alone or with adenosine plus an adenosine uptake inhibitor. Figure 1 shows one representative experiment utilizing dipyridamole. Adenosine was able to stimulate proliferation of endothelial cells. When dipyridamole (1 ,uM) was added to the culture medium this proliferation was no longer seen. This would suggest that uptake was necessary to stimulate proliferTABLE

1. Effect of adenosine metabolites Day

Control 5.0 pM 0.5 ,uM 5.0 pM 0.5 ,uM 5.0 ,uM

1

100 llOk8 102k6 98k7 103t6 10624

ADO IN0 IN0 HYP HYP

Day 2

100 176t6* 102t6 102k4 109t7 106t9

Day 3

100 159-+4* 99t4 94-1-4 102t4 9623

Day

4

100 132t4* lOOrt3 992x3 lOOt2 lOOk2

Endothelial cell proliferation is presented as the mean % of the nontreated control cultures t SE of the mean (n = 9). Endothelial cells were grown in 24-well trays and stimulated with either adenosine (ADO), inosine (INO), or hypoxanthine (HYP). Control cells received medium with fetal calf serum only. * P < 0.05.

OF

ENDOTHELIAL

CELLS

ation. However, when compared with control cells, cells with dipyridamole alone in the culture medium grew at a slower rate. This suggested that dipyridamole blocked the action of adenosine indirectly by blocking the normal proliferation of the cells. When adenosine uptake was inhibited by nitrobenzylthioinosine [NBTI (1 PM)], adenosine was still able to stimulate the proliferation of endothelial cells (Fig. 2). Control cells grown with NBTI only were not different from control cells grown in fetal calf serum only, indicating that NBTI had no detrimental effect on the growth of the cells. As an alternative to uptake inhibitors, a large molecular weight form of adenosine was utilized to test whether or not uptake of adenosine was necessary to stimulate proliferation of endothelial cells. Polyadenylic acid (Poly A), a long-chain polymer of adenylic acid molecules, was added to cultures of endothelial cells. The large molecular weight of this compound precludes its being taken up by the cells via facilitated transfer. Only the adenosine moiety at the 3’ end of the molecule possessesall the adenosine-like structural characteristics required to bind to an adenosine receptor on the endothelial cell surface (21). Figure 3 shows one representative experiment. Poly A stimulated proliferation of endothelial cells. The effect of Poly A was dose dependent with 0.5 nM showing the peak response. Doses greater or lower than 0.5 nM exhibited less proliferation. It should be noted that a sample of Poly A is a mixture of molecules of molecular weight X0? This mixture was arbitrarily assigned a molecular weight of 106. Therefore, the concentrations indicated here do not correspond to ones used for adenosine. However, the data presented suggests that uptake of adenosine with subsequent incorporation of adenosine into the adenine nucleotide pool is not the mechanism 2x105 1

A w

-+-

I I

CONTROL DIPYRIDAMOLE ADENOSINE

T

CONTROL NBTI ADO NBTI + A

.---A---.

. . . . . . . e...

Y 4........+.......

DAYS

IN CULTURE

1. Effect of dipyridamole (DIPYR) on stimulation of proliferation of coronary venular endothelial cells by adenosine(AD0). Endothelial cells were grown in 24-well trays in medium with fetal calf serum only (control), 1 ,uM DIPYR, 5 PM ADO, or 1 PM DIPYR + 5.0 pM ADO (DIPYR + ADO). Mean endothelial cells counts for one representative experiment are shown. Cell counts are shown as means t SE of triplicate wells. FIG.

I I

I

I

I

I

1

0

1

2

3

4

5

DAYS

IN CULTURE

2. Effect of nitrobenzylthioinosine (NBTI) on stimulation of proliferation of coronary venular endothelial cells by adenosine (ADO). Endothelial cells were grown in 24-well trays in medium with fetal calf serum only (control), 1 pM NBTI, 5.0 PM ADO, or 1 PM NBTI + 5.0 ,uM ADO. Mean endothelial cells counts for one representative experiment are shown. Cell counts are shown as means t SE of triplicate wells. FIG.

Downloaded from www.physiology.org/journal/ajpheart at Washington Univ (128.252.067.066) on February 13, 2019.

ADENOSINE-STIMULATED 1.6 X lo5

1

-

PROLIFERATION

CONTROL

2x104 0

I

I

I

1

1

2

3

4

DAYS IN CULTURE 3. Effect of polyadenylic coronary venular endothelial cells. well trays in medium with fetal various concentrations of Poly A. one representative experiment are means k SE of triplicate wells. FIG.

acid (Poly A) on proliferation of Endothelial cells were grown in 24calf serum only (control) or with Mean endothelial cells counts for shown. Cell counts are shown as

1x105

1x104 1

2

3

4

5

DAYS 4. Effect of venular endothelial with fetal calf serum cell counts for one are shown as means FIG.

adenosine analogues on proliferation of coronary cells. Endothelial cells were grown in 24well trays only or an adenosine analogue. Mean endothelial representative experiment are shown. Cell counts t SE of triplicate wells.

for bringing about increased proliferation of endothelial cells. Since the identity of adenosine receptors on endothelial cells has not been clearly established, we investigated the ability of some adenosine analogues to stimulate endothelial cell proliferation. NG-cyclopentyladenosine (CPA) and 5 ’ -N- (cyclopropyl) -carboxamidoadenosine (NCPCA), with selectivity in the brain for A1 and A2 receptors, respectively, were added to the culture medium. Figure 4 shows three sets of growth curves for these analogues. A dose-dependent stimulation of endothelial cell proliferation could be seen with either analogue. However, in the concentration range of 5-500 nM, both adenosine receptor agonists were equipotent in

OF

ENDOTHELIAL

HZ01

CELLS

terms of stimulating proliferation of endothelial cells. Two other analogues were tested under “defined” conditions: R-PIA and NECA. Table 2 summarizes data from several experiments. These analogues were also equipotent in stimulating endothelial cell proliferation, and the proliferative response was equivalent to that seen with adenosine. Functional data concerning receptor effects on intracellular CAMP levels have been obtained with model cell types exhibiting only one type of adenosine receptor (i.e., platelets and adipocytes). The Al adenosine receptor inhibits adenylate cyclase causing CAMP levels in the cell to go down. The A2 receptor activates adenylate cyclase causing CAMP levels in the cell to rise. If the analogues used in our study were truly selective for one receptor over the other, and if CAMP was mediating the response to adenosine, one would have expected to see increased proliferation with one analogue and not the other. Since the data shown above suggested otherwise, we tested the role of CAMP in the proliferative response to adenosine in another way. The adenosine analogue 2’,5’-dideoxyadenosine (DDA) blocks the catalytic action of adenylate cyclase without binding to surface receptors (6). By using DDA, one can distinguish between events causally related to changes in CAMP from events concomitant with but not causally related to changes in CAMP. Figure 5 shows one representative experiment. The addition of DDA (25 PM) to the culture medium blocked the stimulation of proliferation brought about by adenosine, NECA, or R-PIA (Fig. 5, B-D ). The growth curve of cells with DDA alone was not significantly different from the growth curve for control cells (Fig. 5A). These data would suggest that CAMP does indeed play a role in the proliferative response to adenosine or an adenosine analogue. The level of CAMP within cells treated with these analogues was measured directly by utilizing a radioimmunoassay. Figure 6 illustrates that an increase in CAMP was detected in cells treated with either analogue. Thus an increase in CAMP in endothelial cells in response to adenosine may be positively coupled to the proliferative response of these cells. The mechanism by which the adenosine receptor communicates with the adenylate cyclase enzyme is via a GTP binding protein (or G protein). The identity of the G protein determines whether CAMP will rise or fall in the cell. The inhibitory G protein, or Gi, inhibits adenylate cyclase and thus causes CAMP levels to fall. This G protein can be inactivated by NAD-dependent ADP-ribosylation catalyzed by pertussis toxin. ADP-ribosylation by pertussis toxin TABLE

2. Effect of adenosine analogues Day

Control 5.0 pM ADO 5.0 pM NECA 5.0 pM R-PIA

1

Day

100

100

125t6* 136t9* 123t6*

136tl4* 129t5* 13lt7*

2

Day

3

100 122IM* 133-c-4* 12lt2*

Day 4

100 133t9* 136t6* 133&g*

Endothelial cell proliferation is presented as the mean % of the nontreated control cultures t SE of the mean (n = 9). Endothelial cells were grown in 24-well trays under “defined” conditions with either adenosine (ADO), 5’-N-ethylcarboxamide adenosine (NECA), or RN6-phenylisopropyladenosine (R-PIA). * P < 0.05.

Downloaded from www.physiology.org/journal/ajpheart at Washington Univ (128.252.067.066) on February 13, 2019.

H202

ADENOSINE-STIMULATED

PROLIFERATION

OF

ENDOTHELIAL

CELLS

A __f_ CONTROL DDA

A - _ _ -A- - _ _

CONTROL ADO ADO’DDA

1

s---: ---f---

1x104

! 0

e ;;

CONTROL NECA NECA’DDA

I 1

1 3

i 4

t 5

0

1

1

1

2

3

4

5

CONTROL R-PIA RPIA’DDA

e--*-e-

1 2

1

1

DAYS IN CULTURE

DAYS IN CULTURE

FIG. 5. Effect of 2, Y-dideoxyadenosine (DDA) on stimulation of coronary venular endothelial cell proliferation by adenosine (ADO, 5 PM) or ADO analogues (5 PM). Endothelial cells were seeded in 24-well trays with medium containing 10% fetal calf serum and allowed 2-3 h to attach. All wells were rinsed and “defined medium” containing ADO or an ADO analogue was added. Matched cultures were grown under same conditions along with DDA (25 FM). Mean endothelial cells counts for one representative experiment are shown. Cell counts are shown as means t SE of triplicate wells.

causes an impaired ability to interact with the adenosine receptor. When endothelial cells were pretreated with pertussis toxin in a concentration range of l-500 rig/ml, they lost the ability to increase proliferation in response to adenosine (Fig. 7). Pertussis toxin pretreatment alone did not affect control cell growth (data not shown). This result would suggest that a Gi or similar protein is involved in the proliferative response to adenosine. A stimulatory G protein, or G,, activates adenylate cyclase causing CAMP levels in the cell to rise. This G protein can be ADP ribosylated by cholera toxin, resulting in an activation of adenylate cyclase. When endothelial cells were pretreated with cholera toxin for 2 h, they were stimulated to proliferate with or without the presence of adenosine (Fig. 8). This would support the hypothesis that a rise in CAMP is a necessary signal to initiate cell proliferation. DISCUSSION

Adenosine is removed from the circulation by uptake into endothelial cells, chiefly by carrier-mediated uptake. Once inside an endothelial cell, two processing pathways can be distinguished (21). One is a high-affinity process, K M = 1 pM, and presumably reflects the action of aden-

The other is a low-affinity process, KM = measures the activity of adenosine deaminase. Adenosine deaminase converts adenosine to inosine, which is further converted into hypoxanthine. Unlike adenosine, these metabolic breakdown products were unable to stimulate microvascular endothelial cell proliferation. Thus, adenosine does not mediate the proliferative response via interaction of breakdown products with the cell surface or interior. In our system adenosine was added at a concentration of 5 PM. One would expect that at this concentration the adenosine would be preferentially incorporated into nucleotides. When uptake was blocked by using dipyridamole, the growth of the endothelial cells was decreased below that of the control cells. This was most likely because of the fact that dipyridamole is not selective for adenosine uptake but blocks thymidine uptake as well (31). A more specific adenosine transport inhibitor, nitrobenzylthioinosine, did not block the stimulation of proliferation brought about by adenosine. This would suggest that the uptake of adenosine and subsequent incorporation into the nucleotide pool is not the mechanism for stimulating proliferation. Earlier work in our laboratory using the adenosine osine kinase.

450 PM, and probably

Downloaded from www.physiology.org/journal/ajpheart at Washington Univ (128.252.067.066) on February 13, 2019.

ADENOSINE-STIMULATED

PROLIFERATION

OF

ENDOTHELIAL

CELLS

H203

120000

ST E 2 100000

u h v . m

‘3 iii 0,

Control Cholera Toxin Adenosine Chol. Toxin +Adenosine

80000

K W ii

=

60000

.

i

W

u

40000 _I_fi_

0

-

NECA R-PIA

5

10

15

TIME

DAYS IN CULTURE 8. Effect of cholera toxin on stimulation of proliferation of coronary venular endothelial cells by adenosine. Endothelial cells were pretreated for 2 h with either control medium (control) or medium containing 1 rig/ml cholera toxin. Control and cholera toxin-pretreated cells were then incubated with 5.0 PM adenosine, and a growth curve was generated. Mean endothelial cells counts for one representative experiment are shown. Cell counts are shown as means t SE of triplicate wells. FIG.

FIG. 6. Effect of adenosine analogues on intracellular concentration of CAMP in coronary venular endothelial cells. Endothelial cells were treated for 2,5, or 15 min with serum-free culture medium only [control (not shown)], 5.0 FM 5’-N-ethylcarboxamide adenosine (NECA) or 5.0 PM P-R- phenylisopropyladenosine (R-PIA). Data are presented as percent of control cell response.

-u

,+

I

I

0

DAYS

TROL TREA #TED 1 NG/ML PT + ADO 500 NG/ML PT + ADO

I

I

2

3

1

IN CULTURE

7. Effect of pertussis toxin on stimulation of proliferation of coronary venular endothelial cells by adenosine (ADO). Endothelial cells were pretreated for 2 h with either control medium (control) or medium containing l-500 rig/ml pertussis toxin (PT) (intermediate concentrations not shown). Control and PT-treated cells were then incubated with 5.0 FM ADO, and a growth curve was generated. Mean endothelial cells counts for one representative experiment are shown. Cell counts are shown as means -+- SE of triplicate wells. FIG.

receptor blocker S-phenyltheophylline suggestedthat the stimulatory response to adenosine was receptor mediated (19). Adenosine receptors are found on the external surface of the cell membrane. This conclusion is based on two types of data. First, in the presence of adenosine transport inhibitors, the physiological effects of adenosine persist or are enhanced (32). Second, adenosine linked to various macromolecules that are not easily transported across the cell membrane produces the same

results as does free adenosine (13, 23, 27). In our system, adenosine “linked” to a chain of adenylic acid molecules was able to mimic the effect of free adenosine. Thus, the proliferative reponse to adenosine involves the binding of adenosine to an extracellular receptor without a requirement for adenosine uptake. Adenosine receptors have historically been defined in functional terms on the basis of inhibition or stimulation of adenylate cyclase. However, when discrepancies between receptor, activity of adenylate cyclase, levels of CAMP, and physiological responses were noted, direct radioligand-binding studies were initiated. As a consequence, the classification of adenosine receptors based on pharmacological criteria has also been reported for several cell types in the literature. We used the selective adenosine analogues, NCPCA, CPA, NECA, and R-PIA to elucidate which type of adenosine receptor might be mediating the proliferative response to adenosine in venular endothelial cells. At doses equivalent to those used with adenosine, these analogues caused an equal stimulation of proliferation. No selectivity was evident at the concentration of analogues used (i.e., 5-500 nM). Binding studies in the literature utilize membrane fractions treated with nanomolar concentrations of adenosine analogues. Even at these concentrations it was noted that the potencies at A, and AZ receptors were nearly equivalent. In brain homogenates, NECA can bind with high affinity to both A, and A2 receptors (33). Therefore, in our system the apparent “nonselectivity” is not surprising, since micromolar concentrations were utilized in many of the experiments. However, even at a concentration of 5 nM (Fig. 4) no selectivity could be demonstrated. Radioimmunoassay of extracts of cells treated with NECA or R-PIA demonstrated an increase

Downloaded from www.physiology.org/journal/ajpheart at Washington Univ (128.252.067.066) on February 13, 2019.

H204

ADENOSINE-STIMULATED

PROLIFERATION

in CAMP with either analogue, reiterating the lack of specificity. The fact that the level of CAMP rose with either analogue and that proliferation was stimulated by both analogues to the same degree suggests that the change in CAMP might be involved in the response to adenosine. When the catalytic subunit of adenylate cyclase was blocked with DDA, the proliferative response was also blocked. It would appear that the rise in CAMP is positively coupled to the proliferative response to adenosine or an adenosine analogue. Leitman et al. (16) reported that agents that raise intracellular levels of CAMP (i.e., phosphodiesterase inhibitors, forskolin, and CAMP analogues) inhibited endothelial cell proliferation. However, it should be noted that although these pharmacological agents raise CAMP levels, they do so in an irreversible manner. As a result, CAMP levels rise and remain high. The preponderance of evidence suggesting a role for CAMP as a positive regulator of cell proliferation comes from observations of transient CAMP surges before DNA replication (for review, see Ref. 2). A brief surge of CAMP production in cells is a necessary event in the onset of DNA synthesis, whereas a sustained CAMP signal is inhibitory on continuation of DNA replication. The effect of adenosine analogues added to our endothelial cell cultures is a transient rise in CAMP. G proteins serve as regulatory elements interposed between the adenosine receptor and the adenylate cyclase enzyme, i.e., serve as a mechanism by which the cell surface receptor communicates with its effector. Information flows from the extracellularly oriented receptor to G protein to intracellular effector. Adenylate cyclase is stimulated or inhibited depending on the identity of the G protein coupling the receptor to the enzyme. Presently available evidence suggests that stimulation of adenylate cyclase by G, is induced by the dissociation of G, into a, and ,@, followed by association of activated, GTP-bound as with the adenylate cyclase catalytic subunit (13, 29). The activation process appears to be terminated by hydrolysis of cu,-bound GTP to GDP and reassociation of a- and &subunits. With regard to adenylate cyclase inhibition by Gi, it is presently unclear which of the subunits of Gi causes the inhibition of the adenylate cyclase and how this inhibition is induced. One mechanism that has been proposed is the following. The reaction is triggered by a receptor-induced dissociation of Gi into ai- and ,&y-subunits. The released @y-subunit inactivates G, (cu,), resulting in a reduction in adenylate cyclase activity (13, 29). The role of the G proteins in the proliferative response to adenosine was investigated using bacterial toxins that

CHOLERA

CYCLASE

Fj

PERTUSSIS

OF

ENDOTHELIAL

CELLS

selectively ADP-ribosylate the a-subunits of these proteins. ADP-ribosylation of Gi (ai) by pertussis toxin results in an impaired ability of the G protein to interact with the receptor (15, 30). This is probably because of a conformational change in the Gi protein such that selective coupling to receptors is prevented (30). When the cells were pretreated with pertussis toxin for 2 h, they lost the ability to respond to adenosine. Thus a Gi or similar protein must be involved in the proliferative response to adenosine. The proliferative response to adenosine requires that adenosine bind to the adenosine receptor on the surface of the endothelial cell. The data presented here indicates that CAMP levels are raised in response to adenosine analogue treatment. In this regard, the adenosine receptor would be expected to raise CAMP via the G, protein. Cholera toxin pretreatment of these cells caused a stimulation of proliferation in and of itself. The addition of adenosine after toxin treatment potentiated the response slightly, but this was only evident if the cells were kept in serum-free conditions for 48 h before cholera toxin treatment. This would suggest that the activation of G, with the subsequent rise in the CAMP level within the cell is a general signal to the cell to initiate cell division. By raising CAMP in the cell, adenosine may be able to stimulate endothelial cell proliferation. Indeed, a rise in CAMP is one of the early signals in the mitogenic response of cells (24). However, the ability to raise cellular CAMP alone is not sufficient to stimulate cell division. Rozengurt (24) proposed that a proliferative agent must activate protein kinase C and enhance ionic fluxes in addition to increasing CAMP. Activation of protein kinase C is brought about by hydrolysis of phosphoinositide-4, Sbisphosphate catalyzed by phospholipase C. It has been reported in some cell types that pertussis toxin can block phospholipase C action (5). Pertussis toxin in our system may block a G protein coupling the adenosine receptor with phospholipase C. These venular endothelial cells exhibit a rise in intracellular calcium when loaded with indo-l and stimulated with adenosine in calcium-free buffer (Meininger et al., 19a). The calcium response is abolished if the cells are pretreated with pertussis toxin. Nees et al. (22) described a purinergic receptor on the surface of guinea pig coronary endothelial cells that used phospholipase C as its signaling system and might be involved in regulation of coronary flow. This receptor was designated a P1 + 2 receptor, because it responded to adenosine as well as the adenine nucleotides AMP, ADP, and ATP. These data suggest that adenosine may be able to activate the phospholipase C pathway FIG. 9. Possible mechanism for proliferative effect of adenosine (ADO). ADO binds to a surface receptor “linked” to adenylate cyclase and phospholipase C via a G protein(s). This G protein is stimulated by cholera toxin but inhibited by pertussis toxin. Activation of both adenylate cyclase and phospholipase C pathways leads to cell proliferation.

Downloaded from www.physiology.org/journal/ajpheart at Washington Univ (128.252.067.066) on February 13, 2019.

ADENOSINE-STIMULATED

PROLIFERATION

proposed to be involved in the proliferation of cells as well as increase CAMP. The ADP-ribosylation of Gi (ai) by pertussis toxin impairs its ability to interact with the adenosine receptor and therefore prevents the transduction of the inhibitory signal to the adenylate cyclase system. Therefore, pertussis toxin should indirectly raise CAMP. We have argued that CAMP is positively coupled to proliferation of cells, and yet pertussis toxin blocked the stimulation of proliferation by adenosine. A possible linkage of the adenosine receptor with phospholipase C by a pertussis toxin-sensitive G protein might explain this observation. A cell requires the protein kinase C/ionic flux responses initiated by phospholipase C in addition to the CAMP signal. If the G protein transducing the signal from the adenosine receptor to phospholipase C is inactivated by pertussis toxin, this second set of proliferative signals would not occur. An increase in CAMP alone would not be capable of initiating cell division. The action of cholera toxin must also be examined using the same argument. Cholera toxin treatment raises intracellular levels of CAMP. Cholera toxin treatment also stimulates the proliferation of endothelial cells in the absence of adenosine. If a rise in intracellular CAMP alone is not sufficient to stimulate cell division, then how does cholera toxin mediate its proliferative response? One possible interpretation of the data involves a G protein that is a substrate for both cholera toxin and pertussis toxin (Fig. 9). This G protein would be “shared” between the adenylate cyclase and the phospholipase C pathways. Activation of this G protein by adenosine and/ or cholera toxin would lead to a stimulation of adenylate cyclase as well as phospholipase C. Inactivation of this G protein by pertussis toxin would prevent the interaction of the G protein and the receptor, thus preventing the activation of protein kinase C/ionic fluxes necessary for proliferation. It would appear that the action of this G protein is critical for transducing a signal from the cell surface to the replicative machinery inside the cell. In conclusion, the data presented here indicate that adenosine and adenosine analogues stimulate microvascular endothelial cell proliferation by activating a surface receptor which, in turn, activates adenylate cyclase via a cholera toxin-sensitive G protein. The resulting rise in CAMP within the endothelial cell serves as a signal to the cell to initiate cell division. In addition, a pertussis toxin-sensitive pathway also appears to be involved in the proliferative response of the microvascular endothelial cell to adenosine. The nature of the pertuss is toxinsensitive pathway remains to be elucidated. This study was supported by National Heart, Institute Grants HL-21498-11 and HL-07782-01. Address reprint requests to C. J. Meininger. Received

22 February

1989; accepted

in final

form

Lung,

and

21 July

1989.

Blood

REFERENCES 1. AUERBACH, R. Angiogenesis-inducing factors: a review. Lymphokines 4: 69-88, 1981. 2. BOYNTON, A. L., AND J. F. WHITFIELD. The role of CAMP in cell proliferation: a critical assessment of the evidence. In: Advances in Cyclic Nucleotide Research, edited by P. Greengard and G. A. Robinson. New York: Raven, 1983, vol. 15, p. 193-294. 3. BRESNICK, G. H., R. ENGERMAN, M. D. DAVIS, G. DE VENECIA,

OF

ENDOTHELIAL

CELLS

HZ05

AND F. L. MYERS. Patterns of ischemia in diabetic retinopathy. Trans. Am. Acad. Ophthalmol. Otolarygol. 81: OP694-709, 1976. 4. BROOKER, G., J. F. HARPER, W. L. TERASAKI, AND R. D. MOYLAN. Radioimmunoassay of cyclic AMP and cyclic GMP. Adv. Cyclic Nucleotide Res. 10: l-33, 1979. 5. CASEY, P. J., AND A. G. GILMAN. G protein involvement in receptor-effector coupling. J. Biol. Chem. 263: 2577-2580, 1988. 6. DALY, J. (Editor). Accumulation of cyclic nucleotides. In: Cyclic Nucleotides in the Nervous System. New York: Plenum, 1977, p. 97-209. 7. D’AMORE, P. A., AND R. W. THOMPSON. Mechanisms of angiogenesis. Ann. Rev. Physiol. 49: 453-464, 1987. 8. DRURY, A. N., AND A. SZENT-GYORGY. The physiological activity of adenine compounds with especial reference to their action upon the mammalian heart. J. Physiol. Lond. 68: 213-237, 1929. 9. DUSSEAU, J. W., P. M. HUTCHINS, AND D. S. MALBASA. Stimulation of angiogenesis by adenosine on the chick chorioallantoic membrane. Circ. Res. 59: 163-170, 1986. 10. FOLKMAN, J. Tumor angiogenesis. Adu. Cancer Res. 19: 331-358, 1974. 11. FOLKMAN, J., AND M. KLAGSBRUN. Angiogenic factors. Science Wash. DC 235: 442-447,1987. 12. GHAI, G., AND S. F. MUSTAFA. Adenosine receptors in blood vessels. Direct evidence. In: Physiology and Pharmacology of Adenosine Derivatives, edited by J. Daly, Y. Kurota, J. W. Phillips, H. Shimizu, and M. Ui. New York: Raven, 1983, p. 71-76. 13. GILMAN, A. G. G proteins and dual control of adenylate cyclase. Cell 36: 577-579, 1984. 14. KNIGHTON, D. R., T. K. HUNT, H. SCHEUENSTUHL, B. J. HALLIDAY, Z. WERB, AND J. CARR. Oxygen tension regulates the expresion of angiogenesis factors by macrophages. Science Wash. DC 221: 1283-1285,1983. 15. KUROSE, H., T. KATADA, T. HAGA, K. HAGA, A. ICHIYAMA, AND M. UI. Functional interaction of purified muscarinic receptors with purified inhibitory guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles. J. Biol. Chem. 261: 6423-6428, 1986. 16. LEITMAN, D. C., R. R. FISCUS, AND F. MURAD. Forskolin, phosphodiesterase inhibitors, and CAMP analogs inhibit proliferation of cultured bovine aortic endothelial cells. J. Cell. Physiol. 127: 237-243,1986. 17. LI, Y.-O, AND B. B. FREDHOLM. Adenosine analogues stimulate CAMP formation in rabbit cerebral microvessels via adenosine ASreceptors. Acta Physiol. Stand. 124: 253-259, 1985. 18. LONDOS, C., D. M. F. COOPER, AND J. WOLFF. Subclasses of external adenosine receptors. Proc. Natl. Acad. Sci. USA 77: 25512554,198O. 19. MEININGER, C. J., M. E. SCHELLING, AND H. J. GRANGER. Adenosine and hypoxia stimulate proliferation and migration of endothelial cells. Am. J. Physiol. 255 (Heart Circ. Physiol. 24): H554H562,1988. ,,,.MEININGER, C. J., C. L. MONTGOMERY, AND H. J. GRANGER. Intracellular changes in endothelial cell calcium in response to adenosine (Abstract). Circulation 80, Suppl. 2: 11-448, 1989. 20. Moos, W. H., D. S. SZOTEK, AND R. F. BRUNS. w-cycloalkyladenosines. Potent Al-selective adenosine agonists. J. Med. Chem. 28: 1383-1384,1985. 21. NEES, S., M. BOCK, V. HERZOG, B. F. BECKER, C. DES ROSIERS, AND E. GERLACH. The adenine nucleotide metabolism of the coronary endothelium: implications for the regulation of coronary flow by adenosine. In: Adenosine: Receptors and Modulation of Cell Function, edited by V. Stefanovich, K. Rudolphi, and P. Schubert. Oxford, UK: IRL, 1985, p. 419-436. 22. NEES, S., C. DES ROSIERS, AND M. BOCK. Adenosine receptors at the coronary endothelium: functional implications. In: Topics and Perspectives in Adenosine Research, edited by E. Gerlach and B. F. Becker. Berlin: Springer-Verlag, 1987, p. 454-467. 23. OLSSON, R. A., C. C. DAVIS, AND E. M. KHOURI. Coronary vasoactivity of adenosine covalently linked to polylysine. Life Sci 21: 1343-1350,1977. 24. ROZENGURT, E. Early signals in the mitogenic response. Science Wash. DC 234: 161-166,1986. 25. ROZENGURT, E., P. STROOBANT, M. D. WATERFIELD, T. F. DEUEL, AND M. KEEHAN. Platelet-derived growth factor elicits cyclic AMP

Downloaded from www.physiology.org/journal/ajpheart at Washington Univ (128.252.067.066) on February 13, 2019.

HZ06

26.

27.

28.

29.

ADENOSINE-STIMULATED

PROLIFERATION

accumulation in Swiss 3T3 cells: role of prostaglandin production. Cell 34: 265-272, 1983. SCHELLING, M. E., C. J. MEININGER, J. R. HAWKER, AND H. J. GRANGER. Venular endothelial cells from bovine heart. Am. J. Physiol. 254 (Heart Circ. Physiol. 23): H1211-H1217, 1988. SCHRADER, J., S. NEES, AND E. GERLACH. Evidence for a cell surface adenosine receptor on coronary myocytes and atria1 muscle cells. Studies with an adenosine derivative of high molecular weight. Pfluegers Arch. 369: 251-257, 1977. SCHUTZ, W., G. STEURER, AND E. TUISL. Functional identification of adenylate cyclase-coupled adenosine receptors in rat brain microvessels. Eur. J. Pharmacol. 85: 177-184, 1982. SMIGEL, M., T. KATADA, J. K. NORTHUP, G. M. BOKOCH, M. UI, AND A. G. GILMAN. Mechanisms of guanine nucleotide-mediated regulation of adenylate cyclase activity. Adv. Cyclic Nucleo. Prot. Phosphor. Res. 17: l-18, 1984.

30.

31.

32. 33.

34.

OF

ENDOTHELIAL

CELLS

UI, M. Islet-activating

protein, pertussis toxin: a probe for functions of the inhibitory guanine nucleotide regulatory component of adenylate cyclase. Trends Pharmacol. Sci. 5: 277-299, 1984. WIDSTROM, O., C. BUSCH, G. TORNLING, B. S. NILSSON, AND G. UNGE. Dipyridamole suppresses uptake of thymidine in human and bovine cells in vitro. Agents Actions 11: 287-289, 1981. WOLFF, J., C. LONDOS, AND D. M. F. COOPER. Adenosine receptors and the regulation of adenylate cyclase. Adv. Cyclic Nucleotide Res. 14: 199-214, 1981. YEUNG, S. -M. H., AND R. D. GREEN. [3H]-5’-N-ethylcarboxamide adenosine binds to both R, and Ri adenosine receptors in rat striatum. Naunyn-Schmiedeberg’s Arch. Pharmacol. 325: 218-225, 1984. ZIADA, A. M., 0. HUDLICKA, K. R. TYLER, AND A. J. WRIGHT. The effect of long term vasodilatation on capillary growth and performante in rabbit heart and skeletal muscle. Cardiovasc. Res. 18: 724732, 1984.

Downloaded from www.physiology.org/journal/ajpheart at Washington Univ (128.252.067.066) on February 13, 2019.