Regulation of Cell Proliferation by the Guanosine-Adenosine Mechanism: Role of Adenosine Receptors

Abstract

A recent study (American Journal of Physiology-Cell Physiology 304: C406-C421, 2013) suggests that extracellular guanosine increases extracellular adenosine by modifying the disposition of extracellular adenosine ("guanosine-adenosine mechanism") and that the guanosine-adenosine mechanism is not mediated by classical adenosine transport systems (SLC28 and SLC29 families) nor by classical adenosine-metabolizing enzymes. The present investigation had two aims: 1) to test the hypothesis that the "guanosine-adenosine mechanism" affects cell proliferation; and 2) to determine whether the transporters SLC19A1, SLC19A2, SLC19A3 or SLC22A2 (known to carrier guanosine analogues) might be responsible for the guanosine-adenosine mechanism. In the absence of added adenosine, guanosine had little effect on the proliferation of coronary artery vascular smooth muscle cells (vascular conduit cells) or preglomerular vascular smooth muscle cells (vascular resistance cells). However, in the presence of added adenosine (3 or 10 μmol/L), guanosine (10 to 100 μmol/L) decreased proliferation of both cell types, thus resulting in a highly significant (p<0.000001) interaction between guanosine and adenosine on cell proliferation. The guanosine-adenosine interaction on cell proliferation was abolished by 1,3-dipropyl-8-(p-sulfophenyl)xanthine (adenosine receptor antagonist). Guanosine (30 μmol/L) increased extracellular levels of adenosine when adenosine (3 μmol/L) was added to the medium. This effect was not reproduced by high concentrations of methotrexate (100 μmol/L), thiamine (1000 μmol/L), chloroquine (1000 μmol/L) or acyclovir (10,000 μmol/L), archetypal substrates for SLC19A1, SLC19A2, SLC19A3, and SLC22A2, respectively; and guanosine still increased adenosine levels in the presence of these compounds.

Conclusion: The guanosine-adenosine mechanism affects cell proliferation and is not mediated by SLC19A1, SLC19A2, SLC19A3 or SLC22A2.

Keywords: SLC19 family; SLC22A2; adenosine; cell proliferation; guanosine.

Figure 1

Effects of guanosine on [³H]-thymidine incorporation in human coronary artery vascular smooth muscle cells in the absence and presence of adenosine. Values represent means and SEMs. Two-factor ANOVA indicated a significant interaction between guanosine and adenosine on [³H]-thymidine incorporation (P < 0.000001). Letter “a” indicates a significant inhibitory response to guanosine at the indicated concentration of adenosine, and “b” indicates that the inhibitory response to guanosine is significantly greater in the presence of adenosine.

Figure 2

Effects of guanosine on [³H]-thymidine incorporation in preglomerular vascular smooth muscle cells isolated from normotensive Wistar-Kyoto rats (WKY) in the absence and presence of adenosine. Values represent means and SEMs. Two-factor ANOVA indicated a significant interaction between guanosine and adenosine on [³H]-thymidine incorporation (P < 0.000001). Letter “a” indicates a significant inhibitory response to guanosine at the indicated concentration of adenosine, and “b” indicates that the inhibitory response to guanosine is significantly greater in the presence of adenosine.

Figure 3

Effects of guanosine on [³H]-thymidine incorporation in preglomerular vascular smooth muscle cells isolated from spontaneously hypertensive rats (SHR) in the absence and presence of adenosine. Values represent means and SEMs. Two-factor ANOVA indicated a significant interaction between guanosine and adenosine on [³H]-thymidine incorporation (P < 0.000001). Letter “a” indicates a significant inhibitory response to guanosine at the indicated concentration of adenosine, and “b” indicates that the inhibitory response to guanosine is significantly greater in the presence of adenosine.

Figure 4

Effects of adenosine on [³H]-thymidine incorporation in human coronary artery vascular smooth muscle cells in the absence (left panel) and presence (right panel) of guanosine, both without (no DPSPX; top graph) and with (pretreated with DPSPX; bottom graph) 1,3-dipropyl-8-(p-sulfophenyl)xanthine (DPSPX; 100 μmol/L; adenosine receptor antagonist). Values represent means and SEMs. Two-factor ANOVA indicated a significant interaction between guanosine and adenosine on [³H]-thymidine incorporation in the “no DPSPX” group (P < 0.000001) but not in the “pretreated with DPSPX” group. Letter “a” indicates significantly different from corresponding group without adenosine, and “b” indicates significantly different from corresponding “no guanosine” group.

Figure 5

Effects of adenosine on [³H]-thymidine incorporation in preglomerular vascular smooth muscle cells isolated from normotensive Wistar-Kyoto rats (WKY) in the absence (left panel) and presence (right panel) of guanosine, both without (no DPSPX; top graph) and with (pretreated with DPSPX; bottom graph) 1,3-dipropyl-8-(p-sulfophenyl)xanthine (DPSPX; 100 μmol/L; adenosine receptor antagonist). Values represent means and SEMs. Two-factor ANOVA indicated a significant interaction between guanosine and adenosine on [³H]-thymidine incorporation in the “no DPSPX” group (P < 0.000001) but not in the “pretreated with DPSPX” group. Letter “a” indicates significantly different from corresponding group without adenosine, and “b” indicates significantly different from corresponding “no guanosine” group.

Figure 6

Effects of adenosine on [³H]-thymidine incorporation in preglomerular vascular smooth muscle cells isolated from spontaneously hypertensive rats (SHR) in the absence (left panel) and presence (right panel) of guanosine, both without (no DPSPX; top graph) and with (pretreated with DPSPX; bottom graph) 1,3-dipropyl-8-(p-sulfophenyl)xanthine (DPSPX; 100 μmol/L; adenosine receptor antagonist). Values represent means and SEMs. Two-factor ANOVA indicated a significant interaction between guanosine and adenosine on [³H]-thymidine incorporation in the “no DPSPX” group (P = 0.0002) but not in the “pretreated with DPSPX” group. Letter “a” indicates significantly different from corresponding group without adenosine, and “b” indicates significantly different from corresponding “no guanosine” group.

Figure 7

Rat preglomerular vascular smooth muscle cells isolated from spontaneously hypertensive rats were incubated with adenosine (3 μmol/L) for 1 h in the absence or presence of guanosine (30 μmol/L) and either without or with: (A) methotrexate (0.1 mmol/L; SLC19A1 inhibitor); (B) thiamine (1 mmol/L; SLC19A2 inhibitor); (C) chloroquine (1 mmol/L; SLC19A3 inhibitor); or (D) acyclovir (10 mmol/L; SLC22A2 inhibitor). The medium was assayed for adenosine by mass spectrometry. Values represent means and SEMs. Two-factor ANOVA indicated a significant interaction between methotrexate and guanosine (P = 0.0038) and thiamine and guanosine (P = 0.0175), but not between chloroquine and guanosine or acyclovir and guanosine. Letter “a” indicates significantly different from corresponding “no guanosine” group, “b” indicates significantly different from corresponding “guanosine” group.