UTP-preferring P2 receptor mediates inhibition of sodium transport in porcine thyroid epithelial cells

Abstract

1. The effects of adenosine 5'-triphosphate (ATP), uridine 5'-triphosphate (UTP) and analogues on forskolin-stimulated absorption of Na+ by porcine thyroid epithelial cells were analysed in cultures grown as confluent monolayers on permeable supports in Transwell Ussing chambers. 2. 85% of the forskolin (10 microM)-stimulated short-circuit current was inhibited by phenamil (1 microM), which is a selective antagonist for epithelial type Na+ channels. 3. Phenamil-sensitive current was inhibited in a dose dependent manner by nucleotides added to the apical compartment of Ussing chambers. In contrast, the phenamil-resistant current, previously shown to represent anion secretion, was unaffected by nucleotides. 4. The order of potency (with EC50 values given in microM) was UTP (0.08)>>ATP (6.3)=uridine 5'-diphosphate (UDP) (6. 6)>2methyl-thio-adenosine-5'-triphosphate (2MeSATP) (84.5)>adenosine 5'-diphosphate (ADP) (147.8)>alpha,beta-methylene ATP (>150)>>adenosine (>1000). 5. P2 receptors mediating inhibition of sodium absorption were present on the apical membrane of the cells since addition of UTP (1-1000 microM) to the basal compartment of the Ussing chambers had little effect while subsequent addition to the apical compartment produced a normal response. 6. Cibachron blue (Reactive blue 2) (1-100 microM), an antagonist at some P2 receptor subtypes, inhibited phenamil sensitive current in a dose dependent manner with half maximal inhibition occurring at 14.25 microM. 7. Suramin (100 microM), pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (PPADS) (100 microM) and pyridoxal 5'-phosphate (P5P) (100 microM) showed only slight competitive antagonism against the response to UTP. 8 These results indicate that a UTP-preferring P2 receptor located on the apical membrane of thyroid epithelial cells mediates inhibition of Na+ absorption.

Figure 1

Response of the short-circuit current in a porcine thyroid epithelial cell monolayer cultured on a permeable support in a Transwell Ussing chamber to addition of forskolin (10 μM), multiple additions of control medium (arrows), followed by the sodium channel antagonist, phenamil (1 μM). Data shown are a representative experiment from six replications.

Figure 2

Response of the short-circuit current in a porcine thyroid epithelial cell monolayer cultured on a permeable support in a Transwell Ussing chamber to addition of forskolin (10 μM), followed after settling time by cumulative additions of adenosine triphosphate (ATP), and finally the sodium channel antagonist, phenamil (1 μM). Data shown are a representative experiment from seven replications.

Figure 3

Inhibition of forskolin stimulated (10 μM) short-circuit current in porcine thyroid epithelial cell monolayers cultured on permeable supports in Transwell Ussing chambers by purines and pyrimidines. Data shown are cumulative dose response curves of the depression of short-circuit current expressed as a per cent of the total phenamil sensitive current (1 μM) (see Figure 2); means±s.e.mean (n=5–10). Compounds used were uridine 5′-triphosphate (UTP); uridine 5′-diphosphate (UDP); adenosine 5′-triphosphate (ATP); 2-methyl-thio-adenosine-5′-triphosphate (2MeSATP); adenosine 5′-diphosphate (ADP); α,β-methylene adenosine triphosphate (α,β-MeATP).

Figure 4

Inhibition of forskolin stimulated (10 μM) short-circuit current in porcine thyroid epithelial cell monolayers by uridine 5′-triphosphate (UTP) added to the basal, and then apical, compartments of Transwell Ussing chambers. Data shown are cumulative dose response curves of the depression of short-circuit current expressed as a per cent of the total phenamil sensitive current (1 μM) (see Figure 2). After completion of the cumulative dose response curve for UTP added to the basal compartment 1000 μM UTP remained in the basal compartment while doses of UTP commencing at 0.1 μM were added to the apical compartment. Data are means±s.e.mean (n=6).

Figure 5

Effect on forskolin stimulated (10 μM) short-circuit current in porcine thyroid epithelial cell monolayers cultured on permeable supports in Transwell Ussing chambers of putative antagonists of purinergic receptors. Data shown are cumulative dose response curves of the depression of short-circuit current expressed as a per cent of the total phenamil sensitive current (1 μM) (see Figure 2). Compounds used were Suramin, Cibachron blue (Reactive Blue 2) (CIB), pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS); pyridoxal 5′-phosphate (P5P). Data are means±s.e.mean (n=4–6).

Figure 6

Effect of the purinergic receptor antagonists suramin (100 μM), pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (100 μM) and pyridoxal 5′-phosphate (P5P) (100 μM) on the response to UTP of the forskolin stimulated (10 μM) short-circuit current in porcine thyroid epithelial cell monolayers cultured on permeable supports in Transwell Ussing chambers. Data shown are cumulative dose response curves of the depression of short-circuit current expressed as a per cent of the total phenamil sensitive current (1 μM) (see Figure 2). Data are means±s.e.mean (n=4–5 for UTP+antagonists, or n=10 for UTP control).