]AA in response to the purinergic agonists was still observed; this response was not affected by AACOCF 3 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 AACOCF 3 . It is concluded that the P2X 7 receptor in ductal cells is coupled to kallikrein secretion through a calcium-dependent cPLA 2 and a calcium-independent iPLA 2 .ATP plays an important role as an extracellular agonist that mediates its various effects by acting on specific membrane P2 receptor subtypes (1, 2). P2 receptors comprise receptors of the ligand-gated ion channel type, as well as of the G-proteinlinked superfamily, termed P2X and P2Y, respectively (3). At present, seven genes for P2X receptors have been cloned (4 -8), but their physiological significance has not been fully established yet. One of the most recently cloned P2X receptors (the P2X 7 ) has a pharmacological profile typical of the receptor previously termed P 2Z (9) with the photoactivable analog of ATP, Bz-ATP, 1 the most potent agonist. The P 2Z receptor induces the formation of pores when exposed to concentrations of extracellular ATP in the 100 M to 1 mM range (Refs. 10 -12, but see also Ref. 13). In contrast to other P2X receptors, the P2X 7 receptor has a long COOH-terminal intracellular chain, which is by itself not responsible for the lytic properties of this receptor (14) but probably induces the formation of a second messenger involved in the lysis (12). In summary, the P2X 7 (P 2Z )-receptors share with the other P2X receptors the ability to open a non-selective channel and with P 2Z receptors the induction of cell lysis by repeated applications of the agonist (8).Since the pioneering work of Gallacher (15), ATP has been recognized as a major non-adrenergic non-cholinergic stimulus of saliva secretion. Salivation, like other exocrine secretions, occurs in two steps; (a) acinar cells secrete an isotonic plasmalike fluid, and (b) the electrolyte composition of this primary secretion is modified during its transfer to the mouth by the ductal tree (16). The ducts reabsorb Na ϩ and Cl Ϫ and secrete K ϩ and HCO 3 Ϫ (17). The study of these two phases of the secretory process has been facilitated by the description of an improved technique to separate ducts and acini (18). It could be observed that extracellular ATP increased the intracellular

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 microM concentration range and a further increase at concentrations higher than 100 microM. Among ATP analogs, only benzoyl-ATP stimulated the low affinity component. ATP tau S 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 microM carbachol. By itself, substance P (100 pM-1 microM) 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 microM and substance P (100 pM-1 microM) 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 microM 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 microM 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.

1 The permeabilizing e ect of P2X 7 agonists was tested in rat submandibular acinar cells using the uptake of ethidium bromide as an index. 2 The uptake of ethidium bromide by acini incubated at 378C 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. 3 The response to ATP was dose-dependent (half-maximal concentration around 40 mM) and it was decreased when the temperature was lowered to 258C. 4 Benzoyl-ATP reproduced the response to ATP (half-maximal concentration around 10 mM). UTP or 2-methylthioATP had no e ect. 5 The permeabilization in response to ATP was blocked by oxidized ATP and by magnesium and inhibited by Coomassie blue. 6 ATP increased the activity of a calcium-insensitive phospholipase A 2 (iPLA 2 ). 7 Bromoenol lactone (BEL) inhibited the iPLA 2 stimulated by ATP but potentiated the uptake of ethidium bromide in response to the purinergic agonist. 8 From these results it is concluded that the activation of P2X 7 receptors permeabilizes rat submandibular acinar cells. The pore-forming activity of the receptor might be negatively regulated by the concomitant activation of the iPLA 2 by the receptor.

No abstract

The intracellular pH (pHi) of rat submandibular cells was measured by 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The cells recovered from ammonium (30 mM) prepulse to their resting pHi within 10 min. Ethylisopropylamiloride (EIPA), an inhibitor of the Na+/H+ exchanger, slows the rate of pHi recovery. ATP (1 mM), in the presence of EIPA, increases the rate of recovery 3.7-fold in the absence or presence of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The recovery was blocked by the addition of 5 mM Mg2+ or 10 microM Coomassie blue. The response was elicited by 2'- and 3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate but not by ADP, UTP, adenyl (beta-gamma-methylene)-diphosphonate, 2-methylthioadenosine 5'-triphosphate, or muscarinic or beta-adrenergic agonists. The purinergic response was also observed when the cells were acidified by sodium propionate and could not be mimicked by the depolarization of the plasma membrane. Aluminum fluoride did not reproduce the response to ATP, suggesting that the observed response does not involve a high-molecular-weight GTP-binding protein. It is concluded that the activation of P2z receptors, probably by the opening of nonspecific cation channels, increases the permeability to protons in rat submandibular glands.