Eduardo Alzola

Activation by P2X7 agonists of two phospholipases A2 (PLA2) in ductal cells of rat submandibular gland. Coupling of the calcium-independent PLA2 with kallikrein secretion.

Isolated ductal cells of rat submandibular gland phospholipid pools were labeled with [3H]arachidonic acid (AA). The tracer was incorporated preferentially to phosphatidylcholine (46% of the lipidic fraction). Extracellular ATP induced the release of [3H]AA to the extracellular medium in a time- and dose-dependent manner (EC50 = 220 μm). Among other agents tested, only 2′,3′-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (Bz-ATP) was able to mimic the effect of ATP (EC50 = 15 μm), without activation of phospholipase C. The purinergic antagonists oxidized ATP, suramin, and Coomassie Blue partly inhibited the response to 1 mm ATP and 100 μmBz-ATP; the response was also blocked by the addition of Mg2+ or Ni2+. Expression of P2X7receptor mRNA in these cells was confirmed by reverse transcription-polymerase chain reaction. In the presence of extracellular calcium, the phospholipase A2 inhibitor 2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid (a nonspecific inhibitor), arachidonyl trifluoromethylketone (AACOCF3, an inhibitor of the calcium-dependent cytosolic PLA2(cPLA2)), and bromoenol lactone (an inhibitor of the calcium-independent PLA2 (iPLA2)) inhibited the release of [3H]AA induced by ATP and Bz-ATP. In the absence of extracellular calcium, the release of [3H]AA in response to the purinergic agonists was still observed; this response was not affected by AACOCF3 and completely blocked by bromoenol lactone. ATP and Bz-ATP stimulated a calcium-independent secretion of kallikrein, which could be blocked by BEL but which was enhanced by AACOCF3. It is concluded that the P2X7 receptor in ductal cells is coupled to kallikrein secretion through a calcium-dependent cPLA2 and a calcium-independent iPLA2.

Bromoenol lactone enhances the permeabilization of rat submandibular acinar cells by P2X7 agonists

The permeabilizing effect of P2X7 agonists was tested in rat submandibular acinar cells using the uptake of ethidium bromide as an index. The uptake of ethidium bromide by acini incubated at 37°C in the presence of 1 mM ATP increased with time and reached after 5 min about 10% of maximal uptake measured in the presence of digitonin. The response to ATP was dose‐dependent (half‐maximal concentration around 40 μM) and it was decreased when the temperature was lowered to 25°C. Benzoyl‐ATP reproduced the response to ATP (half‐maximal concentration around 10 μM). UTP or 2‐methylthioATP had no effect. The permeabilization in response to ATP was blocked by oxidized ATP and by magnesium and inhibited by Coomassie blue. ATP increased the activity of a calcium‐insensitive phospholipase A2 (iPLA2). Bromoenol lactone (BEL) inhibited the iPLA2 stimulated by ATP but potentiated the uptake of ethidium bromide in response to the purinergic agonist. From these results it is concluded that the activation of P2X7 receptors permeabilizes rat submandibular acinar cells. The pore‐forming activity of the receptor might be negatively regulated by the concomitant activation of the iPLA2 by the receptor.

Low affinity purinergic receptor modulates the response of rat submandibular glands to carbachol and substance P.

The effect of extracellular ATP on the intracellular calcium concentration ([Ca2+]i) in rat submandibular glands was tested. The dose‐response curve for ATP was biphasic with a first increase in the 1–30 μM concentration range and a further increase at concentrations higher than 100 μM. Among ATP analogs, only benzoyl‐ATP stimulated the low affinity component. ATPτS blocked this response. All the other analogs tested reproduced the high‐affinity low capacity response. Magnesium and Coomassie blue selectively blocked the low affinity component. High concentrations of ATP blocked the increase of the intracellular calcium concentration [Ca2+]i in response to 100 μM carbachol. By itself, substance P (100 pM‐1 μM) increased the [Ca2+]i. One mM ATP potentiated the response to concentrations of substance P higher than 10 nM. This potentiation was reversed by extracellular magnesium. Carbachol 100 μM and substance P (100 pM‐1 μM) increased the release of inositol trisphosphate (IP3) from polyphosphoinositides (polyPI). Activation of the low affinity ATP receptors did not activate the polyPI‐specific phospholipase C but inhibited its activation by 100 μM carbachol (−50%) and by 100 nM substance P (−60% at 1 nM substance P and −40% at 100 nM substance P). Substance P induced a strong homologous desensitization: a preincubation with 1 nM substance P nearly completely abolished the response to 1 μM substance P. When the cells were exposed to ATP before the second addition of substance P, the purinergic agonist partially restored the response to the tachykinin without totally reversing the desensitization. It is concluded that two types of purinergic receptors coexist in rat submandibular glands; a high‐affinity, low capacity receptor which remains pharmacologically and functionally undefined and a low affinity site, high capacity receptor of the P2Z type coupled to a non‐selective cation channel. The occupancy of these low affinity sites blocks the increase of the [Ca2+]i in response to a muscarinic agonist and the activation of polyPI‐specific phospholipase C by carbachol and substance P. It potentiates the effect of high concentrations of substance P on the [Ca2+]i.

Regulation by P2 agonists of the intracellular calcium concentration in epithelial cells freshly isolated from rat trachea.

Epithelial cells were isolated from rat trachea by incubation of the organ in a calcium-free medium. The intracellular concentration of calcium ([Ca(2+)](i)) was measured with the calcium-sensitive fluorescent dye fura2. In resting conditions, the cells maintained a low [Ca(2+)](i) in spite of the presence of millimolar concentration of calcium in the incubation medium. These cells had retained intracellular stores of calcium which were emptied after exposure of the cells to thapsigargin, an inhibitor of intracellular calcium ATPases. Substance P (125 nM) transiently increased 2.5-fold the [Ca(2+)](i). ATP (1 mM) doubled the [Ca(2+)](i) after a few seconds and further induced a sustained increase of the [Ca(2+)](i). Coomassie blue fully blocked the response to ATP and extracellular magnesium only inhibited the delayed response to ATP. Among purinergic analogs, only benzoyl-ATP (Bz-ATP), an agonist on P2X ionotropic purinergic receptors, reproduced the response to ATP. UTP and 2-methylthioATP (two agonists on P2Y metabotropic purinergic receptors) transiently increased the [Ca(2+)](i). Thapsigargin, ATP and Bz-ATP increased the uptake of extracellular calcium. RT-PCR analysis revealed that two metabotropic receptors (P2Y(1) and P2Y(2)) and two ionotropic receptors (P2X(4) and P2X(7)) were expressed by the cells present in the suspension. It is concluded that purinergic agonists can modulate the response of rat tracheal epithelial cells by several mechanisms. The activation of metabotropic receptors should mobilize intracellular IP(3)-sensitive calcium pools. The activation of the ionotropic receptors should not only open a non-specific cation channel leading to the entry of calcium but should also induce the formation of pores in cells expressing the P2X(7) receptors, which could be deleterious to these cells.