Naima Chaib

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.

Study on the activation of phospholipases A2 by purinergic agonists in rat submandibular ductal cells

Extracellular ATP and benzoyl-ATP (Bz-ATP) increased the release of [3H]arachidonic acid ([3H]AA) from prelabeled rat submandibular gland (RSMG) ductal cells respectively two- and threefold. Both agonists also increased the release of [3H]AA from acini but at a lower level (+50% and +100% respectively). Carbachol had no significant effect on either cellular population. In ductal cells phorbol myristate acetate, an activator of protein kinase C, slightly increased the basal release of [3H]AA but did not affect the release of [3H]AA in response to ATP. Staurosporine, an inhibitor of protein kinases, inhibited the response to the purines. The removal of calcium from the extracellular medium decreased the response to ATP and Bz-ATP. Only barium could partly substitute for calcium to restore the purinergic response. Zinc inhibited the release of [3H]AA. Permeabilization of the cells with streptolysin O (SLO) activated the calcium-independent phospholipase A2 activity (iPLA2). The iPLA2, not the calcium-dependent PLA2 (cPLA2), released [3H]oleic acid ([3H]OA) from RSMG ductal cells. It is concluded that RSMG ducts have a higher PLA2 activity when compared to acini. This activity is accounted for by iPLA2 and cPLA2. Both enzymes are activated by P2X agonists by a staurosporine-sensitive mechanism. Cells permeabilized with SLO or membranes from Escherichia coli as a substrate are not good models to study the regulation of these enzymes. In intact RSMG ductal cells the two activities can be distinguished by rather specific inhibitors, by different ionic conditions and also by the fatty acid used to label the cells.

Multiple effects of trichloroethanol on calcium handling in rat submandibular acinar cells

The effect of trichloroethanol (TCEt), the active metabolite of chloral hydrate, on the intracellular concentration of calcium ([Ca2+]i) was investigated in rat submandibular glands (RSMG) acini loaded with fura‐2. TCEt (1–10 mM) increased the [Ca2+]i independently of the presence of calcium in the extracellular medium. Dichloroethanol (DCEt) and monochloroethanol (MCEt) reproduced the stimulatory effect of TCEt but at much higher concentrations (about 6 fold higher for DCEt and 20 fold higher for MCEt). TCEt mobilized an intracellular pool of calcium, which was depleted by a pretreatment with thapsigargin, an inhibitor of the sarcoplasmic and endoplasmic reticulum calcium‐dependent ATPases, but not with FCCP, an uncoupler of mitochondria. TCEt 10 mM inhibited by 50% the thapsigargin‐sensitive microsomal Ca2+‐ATPase. DCEt 10 mM and MCEt 10 mM inhibited the ATPase by 20 and 10%, respectively. TCEt inhibited the increase of the [Ca2+]i and the production of inositol phosphates in response to carbachol, epinephrine and substance P. TCEt inhibited the uptake of calcium mediated by the store‐operated calcium channel (SOCC). ATP and Bz‐ATP increased the [Ca2+]i in RSMG acini and this effect was blocked by extracellular magnesium, by Coomassie blue and by oxydized ATP (oATP). TCEt potentiated the increase of the [Ca2+]i and of the uptake of extracellular calcium in response to ATP and Bz‐ATP. TCEt had no effect on the uptake of barium and of ethidium bromide in response to purinergic agonists. These results suggest that TCEt, at sedative concentrations, exerts various effects on the calcium regulation: (1) it mobilizes a thapsigargin‐sensitive intracellular pool of calcium in RSMG acini; (2) it inhibits the uptake of calcium via the SOCC; (3) it inhibits the activation by G protein‐coupled receptors of a polyphosphoinositide‐specific phospholipase C. It does not interfere with the activation of the ionotropic P2X receptors. The use of chloral hydrate should be avoided in studies exploring the in vivo responses to sialagogues.

Regulation of the Na+-K+(NH4+)-2Cl- cotransporter of rat submandibular glands.

A cellular suspension from rat submandibular glands was exposed to different concentrations of NH4Cl, and the variations of the intracellular concentration of calcium ([Ca2+]i) and the intracellular pH (pHi) were measured using fura‐2 and 2′,7′‐bis‐(2‐carboxy‐ethyl)‐5(6)‐carboxyfluorescein. More than 5 mmol/l NH4Cl significantly increased the [Ca2+]i without affecting the response to 100 µmol/l carbachol. When exposed to 1 and 5 mmol/l NH4Cl, the cells acidified immediately. At 30 mmol/l, NH4Cl first alkalinized the cells and the pHi subsequently dropped. This drop reflects the uptake of NH  +4 ions that dissociate to NH3 and H+ in the cytosol. These protons are exchanged for extracellular sodium by the Na+/H+ exchanger because the presence of an inhibitor of the exchanger in the medium increased the acidification induced by 1 mmol/l NH4Cl. Ouabain partly blocked the uptake of NH  +4 . In the combined presence of ouabain and bumetanide (an inhibitor of the Na+‐K+‐2Cl− cotransporter), 1 mmol/l NH4Cl alkalinized the cells. The contribution of the Na/K ATPase and the Na+‐K+‐2Cl− cotransporter in the uptake of NH  +4 was independent of the presence of calcium in the medium. Isoproterenol increased the uptake of NH  +4 by the cotransporter. Conversely, 1 mmol/l extracellular ATP blocked the basal uptake of NH  +4 by the cotransporter. This inhibition was reversed by extracellular magnesium or Coomassie Blue. It was mimicked by benzoyl‐ATP but not by CTP, GTP, UTP, ADP, or ADPβS. ATP only slightly inhibited the increase of cyclic AMP (−22%) by isoproterenol but fully blocked the stimulation of the cotransporter by the β‐adrenergic agonist. ATP increased the release of 3H‐arachidonic acid from prelabeled cells but SK&F 96365, an imidazole‐based cytochrome P450 inhibitor, did not affect the inhibition by ATP. It is concluded that the activation of a purinoceptor inhibits the basal and the cyclic AMP‐stimulated activity of the Na+‐K+‐2Cl− cotransporter. J. Cell. Physiol. 180:422–430, 1999.