Phosphorylation of 3 particulate proteins in rat pancreatic acini in response to vasoactive intestinal peptide (VIP), secretin and cholecystokinin (CCK-8)

Rat pancreatic acini were preincubated with 0.4 mM 32Pi for 45 min at 37 degrees C, then exposed for 15 min to VIP, secretin or CCK-8. The incubation was terminated with a stop solution and a fraction rich in mitochondria and zymogen granules was separated from a microsome-rich fraction by differential centrifugation. After heating in the presence of SDS, beta-mercaptoethanol was added and the pattern of equivalent amounts of 32P-labelled proteins was examined by autoradiography of SDS-PAGE gels. VIP, secretin, and CCK-8 stimulated the phosphorylation of a Mr=33 K microsomal protein and that of two proteins of Mr=21 K and Mr=25 K mostly present in a fraction rich in mitochondria and zymogen granules. Stimulations were dose-dependent, the highest stimulant concentrations tested allowing 2- to 3-fold increases of phosphorylation over basal. When 1 nM CCK-8 was used simultaneously with 1 microM VIP, the cyclic AMP levels attained and the pattern of protein phosphorylation were similar to those obtained with VIP alone, and there was a potentiation of amylase secretion; when a supra-maximal 0.1 microM CCK-8 concentration was added, the VIP-induced elevation in cyclic AMP levels and the phosphorylation of the Mr=21 K and Mr=25 K proteins were partially antagonized, and no potentiation any more of secretion occurred. To conclude the in vitro phosphorylation of three particulate proteins (Mr=33 K, 25 K, and 21 K) was similarly increased in rat pancreatic acini in response to secretin and VIP (acting through cyclic AMP) and to CCK-8 (acting mostly through Ca2+).     

Ivermectin-dependent release of IL-1beta in response to ATP by peritoneal macrophages from P2X<Subscript>7</Subscript>-KO mice

The response to ATP of peritoneal macrophages from wild-type (WT) and P2X₇-invalidated (KO) mice was tested. Low concentrations (1–100 μM) of ATP transiently increased the intracellular concentration of calcium ([Ca²⁺]_i) in cells from both mice. The inhibition of the polyphosphoinositide-specific phospholipase C with U73122 inhibited this response especially in WT mice suggesting that the responses coupled to P2Y receptors were potentiated by the expression of P2X₇ receptors. One millimolar ATP provoked a sustained increase in the [Ca²⁺]_i only in WT mice. The response to 10 μM ATP was potentiated and prolonged by ivermectin in both mice. One millimolar ATP increased the influx of extracellular calcium, decreased the intracellular concentration of potassium ([K⁺]_i) and stimulated the secretion of interleukin-1β (IL-1β) only in cells from WT mice. Ten micromolar ATP in combination with 3 μM ivermectin reproduced these responses both in WT and KO mice. The secretion of IL-1β was also increased by nigericin in WT mice and the secretory effect of a combination of ivermectin with ATP in KO mice was suppressed in a medium containing a high concentration of potassium. In WT mice, 150 μM BzATP stimulated the uptake of YOPRO-1. Incubation of macrophages from WT and KO mice with 10 μM ATP resulted in a small increase of YOPRO-1 uptake, which was potentiated by addition of 3 μM ivermectin. The uptake of this dye was unaffected by pannexin-1 blockers. In conclusion, prolonged stimulation of P2X₄ receptors by a combination of low concentrations of ATP plus ivermectin produced a sustained activation of the non-selective cation channel coupled to this receptor. The ensuing variations of the [K⁺]_i triggered the secretion of IL-1β. Pore formation was also triggered by activation of P2X₄ receptors. Higher concentrations of ATP elicited similar responses after binding to P2X₇ receptors. The expression of the P2X₇ receptors was also coupled to a better response to P2Y receptors.     

Contribution of two ionotropic purinergic receptors to ATP responses in submandibular gland ductal cells

The effect of extracellular ATP on salivary gland function was compared in wild-type (WT) and P2X(7) knockout (KO) mice. The increase in the intracellular concentration of calcium ([Ca(2+)](i)) in response to carbachol was similar in submandibular ductal cells of WT and KO mice. ATP and its analog, benzoyl-ATP, induced a sustained increase in the [Ca(2+)](i) in WT animals. In KO mice, ATP slightly and transiently increased the [Ca(2+)](i) and benzoyl-ATP had no effect. The response to ATP of WT but not KO mice was blocked by KN-62, Coomassie blue and magnesium. The small response of ATP observed in KO mice was completely blocked in the absence of extracellular calcium, unchanged by U73122 and potentiated by ivermectin indicating the probable involvement of a P2X(4) receptor. A RT-PCR and a Western blot confirmed the presence of these receptors in ducts of both WT and KO mice. ATP increased the permeability of the cells to ethidium bromide and stimulated a phospholipase A(2) activity in WT but not KO mice. Mice submandibular gland cells secreted IL-1beta but this secretion was not modified by ATP and was similar in both groups of animals. The volume of saliva provoked by pilocarpine and the concentration of proteins, sodium and chloride in this saliva was similar in both groups of animals. The concentration of potassium was higher in KO mice. We can conclude that the major purinergic receptors expressed in mice submandibular ductal cells are P2X(7) receptors but that P2X(4) receptors are also involved in some ATP effects.     

Cholesterol depletion perturbs calcium handling by rat submandibular glands

The sensitivity to cholesterol depletion of calcium handling by rat submandibular glands was investigated. The glands were digested with collagenase. After homogenization, the lysate was extracted at 4 degrees C with 0.5% Triton X-100 and the extract was submitted to an ultracentrifugation in a sucrose discontinuous gradient. A population of detergent-resistant membranes (DRM) was collected at the 5%-35% interface. The DRM had a higher content of cholesterol, saturated and long-chain fatty acids. Caveolin-1 and alpha(q/11) were located in these membranes. They were more ordered than vesicles from total cellular lysate as determined by anisotropy measurement. They disappeared after cholesterol extraction with methyl-beta-cyclodextrin (MCD). Exposure of the cellular suspension with MCD nearly abolished the response to carbachol, epinephrine, and substance P and inhibited the activation of phospholipase C (PLC) by these agonists and by sodium fluoride. MCD did not affect the mobilization of intracellular pools of calcium by thapsigargin. It increased the uptake of extracellular calcium or barium and did not inhibit the uptake of calcium after depletion of the intracellular stores of this ion. From these results, it is concluded that Triton X-100 can extract a fraction of membrane resistant to detergents. Treatment of the cells with MCD disrupts these membranes. The coupling between the heterotrimeric GTP-binding protein G(q/11) and poly-phosphoinositide-specific PLC is affected by disruption of these membrane fractions. At the opposite, the store-operated calcium channel (SOCC) is not affected by DRM-disruption.     

Effect of pluronic acid F-127 on the toxicity towards eukaryotic cells of CSA-13, a cationic steroid analogue of antimicrobial peptides

Aims: CSA‐13 is an antimicrobial cationic steroid with some toxicity against eukaryotic cells. The purpose of this work was to test whether pluronic acid F‐127 could interfere with the toxicity of CSA‐13 on human umbilical vein endothelial (HUVEC) without modifying its bactericidal activity against Pseudomonas aeruginosa. Methods and Results: The addition of pluronic acid F‐127 slightly decreased the number of dead cells after exposure to CSA‐13. Pluronic acid F‐127 blocked the permeabilizing effect of CSA‐13 on the plasma membrane of HUVEC (uptake of ethidium bromide, release of lactate dehydrogenase) without modifying its toxic effect on their mitochondrial function (MTT test, uptake of tetramethyl rhodamine ethyl ester). Conclusion: Pluronic acid F‐127 decreased the toxicity of CSA‐13 against eukaryotic cells without completely protecting them from mitochondrial damage at high concentrations of the drug. Significance and Impact of the Study: This work establishes that studies on the toxic effects of synthetic antimicrobials on eukaryotic cells should not only focus on the permeability of the plasma membrane but also on the integrity of the mitochondria.     

Regulation of phospholipase D by P2X7 receptors in submandibular ductal cells

ATP (1 mM) increased the phospholipase D (PLD) activity of rat submandibular gland (RSMG) ductal cells in a concentration-dependent and calcium-sensitive manner. The response to ATP was reproduced by benzoylbenzoyl-ATP (Bz-ATP, 100 microM) and also partly by adenosine 5'-(gamma-thio)triphosphate (ATPgammaS, 1 mM). A similar stimulation was observed in control mice (P2X7R+/+ mice) but not in mice lacking the P2X7 receptors (P2X7R-/- mice). Oxidized ATP and Coomassie blue or the addition of magnesium or nickel to the incubation medium inhibited the response to ATP. The stimulation of PLD by purinergic agonist was inhibited by about 50% by calphostin C and chelerythrine, two protein kinase C (PKC) inhibitors. The stimulation of PLD by Bz-ATP and by o-tetradecanoylphorbol 13-acetate (TPA), a phorbol ester which activates PKC, were not additive.From these results we can conclude that the activation of P2X7 receptors in RSMG ductal cells is coupled to the activation of a PLD. This activation is partly mediated by protein kinase C.     

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 (pH_i) was estimated with 2′,7′-bis-(2-carboxy-ethyl)-5(6)-carboxyfluorescein (BCECF). After exposure to NH₄Cl, the pH_i 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 μmol/L) mimicked the response to isoproterenol. VIP (1 nmol/L-1 μmol/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.¹     

Evidence that helodermin, a newly extracted peptide from Gila monster venom, is a member of the secretin/VIP/PHI family of peptides with an original pattern of biological properties

Helodermin, a newly isolated peptide from the venom of Gila monster (Heloderma suspectum) was shown to stimulate the adenylate cyclase activity of rat pancreatic membranes as efficiently as secretin and VIP. It also increased cyclic AMP levels and inhibited [125I]VIP binding in rat pancreatic acini. Finally, helodermin activated adenylate cyclase in membranes from rat heart, rat brain, and human heart, showing properties analogous yet distinct from those of secretin, VIP and PHI.     

Adenylate cyclase stimulation by VIP in rat and human parotid membranes

Adenylate cyclase activity was stimulated by vasoactive intestinal peptide (VIP) in rat parotid membranes, in the presence of 100 microM guanosine triphosphate (GTP). The threshold concentration of VIP was 300 nM and the activity doubled at the maximal VIP concentration tested (30 microM). The relative potency of peptides of the VIP family was: VIP greater than peptide histidine isoleucinamide (PHI) greater than secretin. The beta-adrenergic agent isoproterenol was a more efficient activator of rat parotid adenylate cyclase and its stimulatory effect, like that of VIP, depended on the presence of GTP. The effects of VIP and isoproterenol were both potentiated by 10 microM forskolin. By comparison with rat parotid preparations, membranes from a human parotid gland responded similarly to the VIP family of peptides (VIP greater than PHI greater than secretin). In both rat and human parotid membranes, two proteins (Mr 44 kDa and 53 kDa) of the alpha-subunit of Ns (the guanyl nucleotide-binding stimulatory protein) were labelled by ADP-ribosylation, in the presence of cholera toxin. Taken together, these results indicate that VIP receptors, when coupled to Ns, were able to activate the adenylate cyclase system in rat and human parotid membranes.     

Insulin inhibition of alpha-adrenergic actions in liver.

The effects of insulin on alpha-agonist (phenylephrine)- and [Arg8]vasopressin-induced Ca2+ and glucose release and mitochondrial Ca2+ fluxes in isolated perfused rat livers were examined. Insulin (6 nM) inhibited the ability of phenylephrine (1 and 0.5 microM) to elicit Ca2+ and glucose release, whereas it was without effect on vasopressin (10 and 2.5 nM) actions. Correspondingly, insulin inhibited the action of phenylephrine to induce a stable increase in mitochondrial Ca2+ uptake, but it did not affect the alteration caused by vasopressin. Phenylephrine and vasopressin caused transient increases in hepatocyte respiration. Insulin inhibited the effect of phenylephrine on this parameter, but not that of vasopressin. Insulin added alone did not alter any of the above parameters. It is concluded from these data that insulin does not alter cellular Ca2+ fluxes and respiration themselves, but selectively inhibits alpha-adrenergic stimulation of these processes. It is proposed that insulin acts either to inhibit binding of alpha-agonists to their specific plasma-membrane receptors or to alter generation and/or degradation of the putative alpha-adrenergic 'second messenger'. If this latter possibility is the case, then the alpha-adrenergic 'second messenger' must be different from the 'second messenger' of vasopressin.