[Plasma lipids profile of non treated HIV infected adults in Ouagadougou (Burkina Faso)]. / Profil lipidique au cours de l'infection par le VIH à Ouagadougou - Burkina Faso: intérêt des marqueurs lipidiques dans le suivi de l'évolution de l'infection à VIH.

Many authors reported metabolic perturbations in connection with HIV infection. The aim of this studies was to determinate plasma lipids profile in non treated HIV infected adults in Ouagadougou (Burkina Faso). The results obtain with 187 HIV infected patients showed a significative (p < 10(-6)) high level of LDL, triglycerid, atherogenic indice; HDL was decreased. The atherogenic risk is increased with lymphocytes CD4 depletion. Plasma lipids levels must be consider in the choice of antiretroviral treatment.

Modulation by propranolol of the uptake of ethidium bromide by rat submandibular acinar cells exposed to a P2X(7) agonist or to maitotoxin.

We have compared the formation of pores in rat submandibular acinar cells in response to 2',3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (Bz-ATP) and maitotoxin. Bz-ATP (100 microM) permeabilized the cells to ethidium bromide. The uptake of ethidium increased to 29+/-1% of maximal uptake in 10 min. DL-Propranolol (300 microM) inhibited the Bz-ATP-induced uptake of ethidium bromide by 40% without affecting the P2X(7)-gated cation channel. The inhibitory effect of DL-propranolol on the formation of pores by Bz-ATP was reproduced by D-propranolol, an optical isomer with very poor beta-blocking activity. Tenidap, an antiinflammatory drug, enhanced the permeabilization in response to Bz-ATP. Propanolol inhibited the response to tenidap plus Bz-ATP. The effect of propranolol was reproduced by labetolol, a beta-adrenergic antagonist with membrane-stabilizing properties, but not by atenolol, which blocks beta-adrenergic receptors but has no effect on the stability of the membrane. In the presence of extracellular calcium, maitotoxin also increased the uptake of ethidium bromide. Tenidap had no effect on this response, which was delayed by propranolol. In conclusion, we have shown that propranolol, in a range of 10-300 microM, inhibits the pore-forming activity of the P2X(7) receptor without affecting the opening of the cation channel coupled to this receptor. This inhibition is not related to its beta-adrenergic blocking activity but rather to its membrane-stabilizing properties. Propranolol also delays the uptake of ethidium bromide in response to maitotoxin. This is in agreement with the current view that P2X(7) agonists and maitotoxin share a common pore.

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

The permeabilizing effect of P2X(7) 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 degrees 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 microM) and it was decreased when the temperature was lowered to 25 degrees C. Benzoyl-ATP reproduced the response to ATP (half-maximal concentration around 10 microM). 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 A(2) (iPLA(2)). Bromoenol lactone (BEL) inhibited the iPLA(2) 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 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.

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 micromol/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 NH4+ 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 NH4+. 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 NH4+ was independent of the presence of calcium in the medium. Isoproterenol increased the uptake of NH4+ by the cotransporter. Conversely, 1 mmol/l extracellular ATP blocked the basal uptake of NH4+ 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 ADPbetaS. ATP only slightly inhibited the increase of cyclic AMP (-22%) by isoproterenol but fully blocked the stimulation of the cotransporter by the beta-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.

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.

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 microM). 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 microM), without activation of phospholipase C. The purinergic antagonists oxidized ATP, suramin, and Coomassie Blue partly inhibited the response to 1 mM ATP and 100 microM Bz-ATP; the response was also blocked by the addition of Mg2+ or Ni2+. Expression of P2X7 receptor 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.

Activation of the Na+-K+(NH4+)-2Cl(-)- cotransporter from rat submandibular glands in response to VIP.

A cellular suspension from rat submandibular glands was prepared with collagenase. The intracellular pH (pHi) was estimated with 2',7'-bis-(2-carboxy-ethyl)-5(6)-carboxyfluorescein (BCECF). After exposure to NH4Cl, the pHi transiently increased (diffusion of NH3) and then dropped (influx of NH4+). Isoproterenol increased 2.5-fold the rate of NH4+ influx; bumetanide, an inhibitor of the Na+-K+-2Cl(-)-cotransporter blocked the response to isoproterenol, confirming that the beta-adrenergic agonist stimulated the cotransporter. Forskolin (1 micromol/L) mimicked the response to isoproterenol. VIP (1 nmol/L(-1) micromol/L) also increased the activity of the cotransporter. Cyclic AMP rather than calcium was the mediator of this activation since 1) carbachol which increased the [Ca2+]i fivefold increased the uptake of NH4+ by only 50%; 2) only high concentrations of VIP significantly increased the [Ca2+]i; 3) incubation in the presence of EGTA had no effect on the response to VIP; 4) low concentrations (nmol/L) of the neuropeptide increased the intracellular level of cAMP; and 5) the stimulation of the cotransporter by VIP, forskolin, and isoproterenol was inhibited by H8, an inhibitor of cAMP-dependent protein kinase. It is concluded that the Na+-K+-2Cl(-)-cotransporter of rat submandibular glands is activated by isoproterenol, forskolin, and neuropeptides of the VIP family by a mechanism involving cAMP-dependent processes. The activation of the cotransporter by VIP could partly explain the potentiating effect of VIP on the response to sialagogues like substance P or muscarinic agonists.

Differential sensitivity to nickel and SK&F96365 of second messenger-operated and receptor-operated calcium channels in rat submandibular ductal cells.

The intracellular concentration of calcium ([Ca2+]i) of rat submandibular ductal cells was measured with the intracellular fluorescent dye Fura-2. Carbachol (100 microM) and ATP (1 mM) both increased the [Ca2+]i. The late response to ATP was blocked by 0.5 mM Ni2+. This concentration of Ni2+ also blocked the increase of the [Ca2+]i and the uptake of manganese and calcium in response to 2'- and 3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP, 100 microM), a specific agonist of P2X receptors from salivary glands. The increase of the [Ca2+]i in response to 2-methylthioadenosine 5'-triphosphate (2-MeSATP, 100 microM) a specific P2Y agonist in salivary glands or to a muscarinic agonist (carbachol) was not affected by 0.5 mM Ni2+. Only higher concentrations of Ni2+ (in the millimolar range) inhibited the uptake of extracellular calcium in response to carbachol. SK&F96365, a blocker of store-operated calcium channels, inhibited the uptake of extracellular calcium in response to carbachol without affecting the response to BzATP. It is concluded that at low concentrations (below 0.5 mM), Ni2+ inhibits the non-specific cation channel coupled to P2X receptors. The uptake of extracellular calcium by store-operated calcium channels is inhibited by higher concentrations of Ni2+ and by SK&F96365.

Study of nonspecific cation channel coupled to P2z purinergic receptors using an acid load technique.

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.