Physiological Concentrations of Zinc Have Dual Effects on P2X Myenteric Receptors of Guinea Pig

Abstract

We, hereby, characterize the pharmacological effects of physiological concentrations of Zinc on native myenteric P2X receptors from guinea-pig small intestine and on P2X2 isoforms present in most myenteric neurons. This is the first study describing opposite effects of Zinc on these P2X receptors. It was not possible to determine whether both effects were concentration dependent, yet the inhibitory effect was mediated by competitive antagonism and was concentration dependent. The potentiating effect appears to be mediated by allosteric changes induced by Zinc on P2X myenteric channels, which is more frequently observed in myenteric neurons with low zinc concentrations. In P2X2-1 and P2X2-2 variants, the inhibitory effect is more common than in P2X myenteric channels. However, in the variants, the potentiatory effect is of equal magnitude as the inhibitory effect. Inhibitory and potentiatory effects are likely mediated by different binding sites that appear to be present on both P2X2 variants. In conclusion, in myenteric native P2X receptors, Zinc has quantitatively different pharmacological effects compared to those observed on homomeric channels: P2X2-1 and P2X2-2. Potentiatory and inhibitory Zinc effects upon these receptors are mediated by two different binding sites. All our data suggest that myenteric P2X receptors have a more complex pharmacology than those of the recombinant P2X2 receptors, which is likely related to other subunits known to be expressed in myenteric neurons. Because these dual effects occur at Zinc physiological concentrations, we suggest that they could be involved in physiological and pathological processes.

Keywords: ATP; Gastrointestinal tract; Myenteric neurons; P2X native receptors; P2X2 recombinant receptors; Zinc.

Fig. 1

Zinc has potentiatory and inhibitory actions on guinea-pig myenteric P2X receptors. Sequential application of ATP–ATP + Zinc-ATP induces opposite effects in different neurons (a, b). Similarly, the pretreatment of Zinc during 15 to 60 s can also have opposite effects in different neurons (c, d). I_ATP were induced by 10 µM ATP and the same concentration of Zinc was applied. Cells were held at a holding potential of − 60 mV. Proportion of myenteric neurons that responded to Zinc (e), low concentrations induced most frequently a potentiation of the current induced by ATP but at 30 µM it induced current inhibition. Change magnitude (f) produced by different concentrations of Zinc on the ATP current

Fig. 2

Zinc has potentiatory and inhibitory actions in the same guinea-pig myenteric neurons with varying ATP concentrations. a pretreatment of Zinc inhibited the ATP—induced (100 µM) current in a reversible fashion. However, in this same neuron, activation of I_ATP with lower agonist concentration (10 µM) resulted in Zinc-mediated potentiation of the current (b). Each cell (n = 6) were held at a holding potential of − 60 mV

Fig. 3

Zinc inhibition of myenteric P2X receptors was remounted by increasing ATP concentration. a sets of I_ATP from two different myenteric neurons before, during, and after the washed out of Zinc (30 µM). Notice that inhibition was larger when currents were elicited by 100 than with 300 µM ATP. b ATP Concentration–response curves in the presence and the absence of Zinc (30 µM). Inhibition of myenteric P2X receptors was abolished by increasing ATP concentration, suggesting that Zinc effect is mediated by a competitive antagonism. Each symbol is the average of 4–7 neurons. Cells were held at a holding potential of − 60 mV

Fig. 4

Zinc inhibitory effect on P2X myenteric receptors is concentration dependent when they are activated by 100 µM ATP. Inhibition was the only effect of Zinc when a higher concentration of ATP was used (n = 20). Each symbol is the average of 4–6 neurons. Cells were held at a holding potential of − 60 mV

Fig. 5

Zinc inhibited recombinant P2X2 receptors when high concentrations of ATP are used. Concentration–response curves for the inhibitory effects of Zinc on I_ATP mediated by P2X2-1a (100 µM ATP) or P2X2-2b (300 µM ATP) receptors. Zinc was applied 2 min before ATP. Estimated IC₅₀ was 250 ± 34 µM (P2X2-1a; n = 4–5) and 485 ± 30 µM (P2X2-2b; n = 3–5). Effect of Zinc was reverted within 3 min of its removal. Complete inhibition (100%) of I_ATP was assumed for fitting these data. Cells were held at a holding potential of − 60 mV

Fig. 6

Zinc effects on homomeric P2X2-1a and P2X2-2b receptors are quantitatively different than those found in native myenteric P2X receptors. Representative traces of ATP induced-currents recorded from Xenopus laevis oocytes expressing P2X2-2b (a, b) or P2X2-1a (c, d). Horizontal bars above traces indicate ATP application (10 µM). e and g, frequency distribution of the percentage of neurons showing inhibition and potentiation by Zinc. Low Zinc concentrations induced inhibition more frequently than in neurons (see Fig. 1). f and h, magnitude of the change produced by different concentrations of Zinc on ATP-induced currents. Cells were held at a holding potential of − 60 mV