Release and extracellular metabolism of ATP by ecto-nucleotidase eNTPDase 1-3 in hypothalamic and pituitary cells

Abstract

Hypothalamic and pituitary cells express G protein-coupled adenosine and P2Y receptors and cation-conducting P2X receptor-channels, suggesting that extracellular ATP and other nucleotides may function as autocrine and/or paracrine signaling factors in these cells. Consistent with this hypothesis, we show that cultured normal and immortalized pituitary and hypothalamic cells release ATP under resting conditions. RT-PCR analysis also revealed the presence of transcripts for ecto-nucleotidase eNTPDase 1-3 in these cells. These enzymes were functional as documented by degradation of endogenously released and exogenously added ATP. Blocking the activity of eNTPDases by ARL67156 led to an increase in ATP release in perifused pituitary cells and inhibition of degradation of extracellularly added ATP. Furthermore, the addition of apyrase, a soluble ecto-nucleotidase, and the expression of recombinant mouse eNTPDase-2, enhanced degradation of both endogenously released and exogenously added ATP. The released ATP by resting hypothalamic cells was sufficient to activate and desensitize high-affinity recombinant P2X receptors, whereas facilitation of ATP metabolism by the addition of apyrase protected their desensitization. These results indicate that colocalization of ATP release sites and ecto-nucleotidase activity at the plasma membrane of hypothalamic and pituitary cells provides an effective mechanism for the operation of nucleotides as extracellular signaling molecules.

Figure 1

Constitutive release and metabolism of ATP by immortalized hypothalamic and pituitary cells in static cultures. (a) Dependence of ATP release on density of cells. ATP concentrations in medium from hypothalamic GT1-7 cells cultured in 6-well plates at density of 1 × 10⁵ and 5 × 10⁵ cells/well. As controls, media from wells without cells were used. Dishes were cultured for 24 h, washed twice and kept in 1 ml ATP-free Krebs-Ringer buffer for indicated times at room temperature. At the end of incubation, media were collected and immediately tested for ATP concentration. (b) Cell-type specificity of ATP release and degradation. Cells were cultured at density of 5 × 10⁵ per well. (c) Time-course of PRL release by GH₃ pituitary cells when cultured in experimental conditions identical to that shown in panel (b). In this and following figures with cells in static cultures, data points are means ± SEM from three experiments, each performed in sextuplicate determinations. Results for GT1-7 cells in (a) and (b) were obtained from two different experiments.

Figure 2

Constitutive PRL (a) and ATP (b) release by perifused hypothalamic and pituitary cell lines. Prior to experiments, cells (5 × 10⁶) attached on beads were transferred into 0.5-ml chamber and perifused with ATP-free KrebsYRinger medium for 2.5 h to establish a stable baseline. During experiments, samples were collected every minute and immediately tested for ATP and PRL concentrations. Chambers loaded with beads without cells were used as controls. In this and following figures with perifused pituitary cells, data points are means ± SEM from three independent experiments.

Figure 3

Expression of eNTPDase 1–3 transcripts in hypothalamic and pituitary tissues and cells. For negative controls, PCR was conducted performing first-strand cDNA synthesis without RT (–). In both panels, DNA markers are shown in the last right lanes. (a) Mouse cell lines. (b) Rat cells and tissues. GAPDH primers were used as internal control to monitor the quality of RNA preparation. For details, see Materials and methods.

Figure 4

Cell-type specificity of extracellular ATP metabolism. Chamber without or with cells (5 × 10⁶ per column) were perifused with Krebs-Ringer buffer at flow rate of 0.8 ml/min. Samples were collected every minute and immediately tested for ATP concentration. Horizontal bars above traces indicate the duration of ATP application.

Figure 5

Effects of ARL67156 on ATP release and metabolism in perifused pituitary and GH₃ cells. (a) Increase in endogenous ATP levels in pituitary cells perifused with ARL67156. (b) Concentration-dependent effects of ARL67156 on degradation of exogenously added ATP in pituitary cells. (c) Effects of ARL67156 on ATP metabolism in perifused GH₃ cells. Gray areas indicate the duration of ARL67156 treatment and horizontal bars indicate the duration of ATP application.

Figure 6

Facilitation of ATP metabolism by over-expression of mouse ecto-ATPase eNTPDase-2 (a) and addition of 1 µg/ml apyrase (b) in αT3-1 and GT1-7 cells. Culture medium was washed and replaced with ATP-free or ATP-containing Krebs-Ringer buffer and cells were incubated for 5 min. Numbers below the bars indicate amounts of ATP added to the buffer. Asterisks indicate significant differences between pairs (P < 0.01). For details see Materials and methods.

Figure 7

Desensitization of recombinant rat P2XRs expressed in GT1-7 cells by endogenous ATP. Left column: The lack of effects of exogenously added ATP on activation of P2X₃R (a) and chimeric receptors (b and c), but not P2X₂Rs (d and e), in cells cultured in medium without apyrase, a soluble ecto-ATPase, for 60 min. Central column: Patterns of ATP-induced [Ca²⁺]_i signaling by parental receptors and their chimeras in cells incubated in medium without apyrase for 15 min. Right column: Patterns of ATP-induced [Ca²⁺]_i signaling by parental and chimeric receptors in cells incubated in medium containing apyrase. In dishes with parental receptors the enzyme was present for 1 h, whereas in those with cells expressing chimeras apyrase was present at least 4 h. Traces shown are mean values from at least 10 EGFP-positive cells in one from three to five dishes. For details on expression of P2XRs, see Materials and methods.