Mourad Metioui

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.

Regulation by thapsigargin and carbachol of the intracellular calcium concentration in rat submandibular glands.

1. Carbachol and thapsigargin both increased the intracellular calcium concentration in rat submandibular cells in the presence and in the absence of extracellular calcium. Depletion of intracellular calcium pools with thapsigargin prevented the response to carbachol. 2. The two agents also increased the influx of calcium. The muscarinic agonist stimulated the efflux of calcium outside the cell. 3. From these results it is concluded that submandibular cells possess several intracellular calcium pools sensitive to thapsigargin, among which some are sensitive to IP3. Depletion of these pools increase the uptake of extracellular calcium.

Are salivary glands cell lines in culture a good model for purinergic receptors in salivary glands

A major obstacle in studying the physiological and biochemical processes of salivary secretion is the lack of a good ductal cell line model. HSY, an immortalised cell line originating from human parotid gland intercalated ducts, provides a possible model for purinergic mechanisms in ductal cells. Unlike the biphasic dose response to ATP of isolated submandibular ductal cells, the rise in [Ca2+]i in HSY cells shows single Michaelis-Menten kinetics with an apparent Ka of 0.8 microM. Pre-incubation with thapsigargin inhibited the ATP induced [Ca2+]i rise. Both ATP (10 microM) and carbachol (100 microM) increased IP3 production. Intercalated duct cells may differentiate into acinar or ductal cells in response to appropriate stimuli from extracellular matrix We therefore attempted to induce a duct-like phenotype in the striated duct-derived HSY cells by growing them on microcarrier beads coated with type I collagen. In Ca-containing medium cells grown on all substrates showed similar responses to ATP. In contrast, in Ca-free medium, [Ca2+]i rose only slightly in cells grown on beads relative to those on glass. This probably resulted from reduced IP3 production. Carbachol also induced a much smaller increase in [Ca2+]i and less IP3 production in cells grown on Cytodex-3. The HSY response to purinergic stimuli by an increase in [Ca2+]i and IP3 means that they can be used to study the metabotropic purinergic pathway. The impairment in the HSY responses grown on Cytodex-3 can be used to probe phosposinositol signal transduction in salivary cells.

Inhibitory effect of caffeine on the response to carbachol in rat pancreatic acini

1. Caffeine did not affect by itself the amylase release, nor intracellular calcium concentration in rat pancreatic acini. 2. Concentration of caffeine higher than 1 mM inhibited the amylase released in response to the muscarinic agonist carbamylcholine. This inhibition was also seen on the intracellular calcium elevation: 20 mM caffeine inhibited by 80% the calcium elevation in response to 100 microM carbachol. 3. Caffeine also prevented the specific binding of [3H]-N-methylscopolamine ([3H]-NMS) on rat pancreatic muscarinic receptors. 4. We conclude that caffeine behaves as a muscarinic antagonist.

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.

Mepacrine inhibits amylase secretion mediated by a guanylnucleotide binding protein.

1. In intact rat pancreatic acini, 10 nM bombesin, 100 nM phorbol ester TPA and 10 mM fluoroaluminate increased amylase secretion 5-, 3- and 4-fold respectively. 2. Mepacrine dose-dependently inhibited the response to bombesin and fluoroaluminate but did not affect the response to TPA. 3. In permeabilized acini, mepacrine inhibited the secretory response to GTP gamma S without modifying the response to TPA.