Norman Fleming

Different tachykinin receptor subtypes are coupled to the phosphoinositide or cyclic AMP signal transduction pathways in rat submandibular cells

Tachykinins of different classes (NK1, NK2, NK3) caused the concentration‐dependent synthesis of IP3 in rat submandibular acinar cells with the potency rank order of NK1 < NK2 > NK3. Enhancement of IP3 was not affected by pertussis toxin treatment. The reverse rank order was found in the tachykinin inhibition of isoproterenol‐induced cAMP synthesis and this inhibition was abolished by pertussis toxin, an inactivator of the adenylate cyclase Gi regulatory protein. It is suggested that different tachykinin receptor subtypes are preferentially coupled to phospholipase C or adenylate cyclase by separate G regulatory proteins in rat submandibular acinar cells.

Muscarinic, α1 and peptidergic agonists stimulate phosphoinositide hydrolysis and regulate mucin secretion in rat submandibular gland cells

Three classes of agonists, associated with Ca2+-mobilization — α1-adrenergic (methoxamine), muscarinic (carbachol) and peptidergic (substance P, SP) — significantly stimulated the secretion of mucin from enzymatically-dispersed rat submandibular gland acinar cells. The same three secretagogues also caused the hydrolysis of membrane inositol phospholipids, resulting in elevated cellular levels of inositol phosphates, particularly inositol 1,4,5-trisphosphate (IP3). Exogenous IP3 elicited the dose-dependent release of mucin in dispersed cells suggesting that agonist-generated endogenous IP3 may provoke a secretory response. IP3 and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in combination, stimulated an additive secretion of mucin in the model. The potential use of these two agents as specific probes of the IP3- and diacylglycerol-associated legs of the polyphosphoinositide (PPI) breakdown pathway is indicated. Although all three agonists shared a common action in stimulating PPI hydrolysis, their effects on the β-adrenergic mucosecretory response were inconsistent. A brief preincubation of cells with carbachol or SP significantly reduced the subsequent isoproterenol (IPR)-provoked secretion of mucin, whereas methoxamine plus IPR stimulated an additive response. The mechanisms underlying these opposite effects are not known. Failure of IP3 or TPA to modify IPR responses suggests that modulation of the β response may operate at a locus before the generation of diacylglycerol and IP3, possibily at the level of signal transduction. The study indicates a role for Ca2+-mobilizing, agonists in controlling submandibular mucin secretion and provides evidence that receptor-linked phosphoinositide hydrolysis is an early stage in their stimulus-secretion coupling mechanism.

Muscarinic M3 receptors are coupled to two signal transduction pathways in rat submandibular cells

The receptor subtypes involved in muscarinic-induced phosphoinositide hydrolysis and adenylate cyclase inhibition in rat submandibular acinar cells were characterized by comparing the inhibitory potencies of four muscarinic antagonists on the two signal transduction responses. Carbachol-induced phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis was inhibited by all antagonists with a potency rank order of 4-diphenylacetoxy-N-methyl piperidine methobromide (4-DAMP) = atropine much greater than pirenzepine much greater than AF-DX 116 (P less than 0.01). The same rank order was observed in antagonist-reversal of the reduction of cAMP caused by carbachol in the model. These findings suggest that muscarinic effects are mediated by M3 receptors in both the phosphoinositide and adenylate cyclase pathways in the submandibular gland.

Effects of a phorbol ester and diacyglycerols on secretion of mucin and arginine esterase by rat submandibular gland cells

The effects of a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) and a diacylglyceride, 1-oleoyl-2-acetyl-glycerol (OAG) on the secretion of two major exocrine products by dispersed rat submandibular cells were investigated. TPA stimulated the release of acinar cell mucin and ductal cell protease (arginine esterase) in a dose- and time-dependent manner. Mucin secretion was also provoked by OAG, which, however, had no effect on arginine esterase release. The unsaturated diacylglycerol, 1,2-diolein, elicited a greater mucosecretory response than did OAG at the same concentration, while the saturated 1,2-distearin produced a smaller response.Mucin and enzyme secretion caused by TPA or OAG in the rat submandibular model was not inhibited by either of two putative antagonists, the antipsychotic drug, fluphenazine, and the antibiotic, polymyxin B.The involvement of extracellular Ca2+ in TPA-induced secretion was examined by comparing responses of cells maintained in normal or Ca2+-free medium, or in medium containing the ionophore A23187. Although extracellular Ca2+ was not an absolute requirement for a secretory response, the results indicate a synergistic relationship between TPA and Ca2+ in stimulating the release of both mucin and arginine esterase.These results suggest a role for the Ca2+-, phospholipid-dependent enzyme, protein kinase C in the secretory mechanism of mucous and serous cells in the submandibular gland. This is consistent with the proposal that receptor-mediated hydrolysis of membrane phosphoinositides is an initial event in stimulus-response coupling in exocrine cells.

Arachidonic Acid Stimulates Intracellular Calcium Mobilization and Regulates Protein Synthesis, ATP Levels, and Mucin Secretion in Submandibular Gland Cells

Earlier observations that arachidonic acid inhibited the synthesis of membrane inositol phospholipids in rat submandibular acinar cells prompted the present study on whether the fatty acid may also regulate other key physiological processes in the model. Arachidonate, at concentrations above 10 pmol/L, inhibited up to 97% protein synthesis in acinar cells. The acid also lowered cellular ATP levels to 25% of control values by a ouabaininsensitive mechanism. In endoplasmic reticulum-calcium studies in permeabilized cells, arachidonic acid stimulated the mobilization of up to 73% loaded ER- 45Ca2+ to the cytosol, a much greater response than those caused by other calcium translocators, thapsigargin or inositol 1,4,5-trisphosphate. Additionally, arachidonate provoked the release of over 80% of total cell 45Ca2+ to the extracellular space in intact cells and stimulated mucin secretion in the submandibular model. The inhibitory effect of arachidonic acid on protein synthesis was duplicated by carbachol, thapsigargin, and BAPTA/AM, three agents that cause net efflux of ER-Ca2+ by different mechanisms. Furthermore, comparable with the arachidonate effect on ATP, carbachol and thapsigargin also significantly reduced cellular levels of the nucleotide. It is concluded that arachidonic acid acts as a regulator of central synthetic/secretory processes in mucous acinar cells of rat submandibular gland and suggested that at least some of its effects may be secondary to its calcium-mobilizing action.

Regulation of the cAMP signal transduction pathway by protein kinase C in rat submandibular cells

Treatment of rat submandibular acinar cell extracts with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) caused the dose-dependent activation of protein kinase C (PKC), assessed by the phosphorylation of a novel and highly specific substrate. This effect was duplicated by a diacylglycerol, but not by the 4α-phorbol ester 4α-phorbol 12,13-didecanoate. The TPA elevation of PKC was blocked by the PKC inhibitors H-7 and sangivamycin. In intact cells, TPA caused the translocation of PKC from cytosol to membrane, consistent with its known mode of activation. The β-adrenergic agonist, isoproterenol, stimulated cAMP levels which were significantly reduced by preactivation of PKC. This inhibitory PKC effect was reversed by H-7. When cAMP was stimulated at the post-receptor level, however, by forskolin, NaF or GTP[γS], PKC did not inhibit, but rather enhanced the cyclic nucleotide response. Since PKC phosphorylated an endogenous protein of 55 kDa, the size of the β1 receptor, these findings indicate that, as in other cell types, PKC can desensitize adenylate cyclase by direct phosphorylation of the β receptor, but potentiate the cAMP response by a post-receptor mechanism. In mucin secretion studies in the model, TPA alone caused the cAMP-independent release of up to 44% total mucin, which was much less than additive with the isoproterenol response. When the cAMP-mucosecretory response was stimulated at the adenylate cyclase level by forskolin, however, the TPA + forskolin effects were additive. These findings on the modulation of cAMP by PKC indicate cross-talk regulation in the phosphoinositide-cAMP signal transduction pathways in submandibular acinar cells.

ALUMINUM FLUORIDE INHIBITION OF CABBAGE PHOSPHOLIPASE D BY A PHOSPHATE-MIMICKING MECHANISM

Aluminum fluoride (AlF4 −) inhibited phospholipase D (PLD) purified from cabbage in both PIP2‐dependent and PIP2‐independent assays, consistent with its previously observed effect on mammalian PLD. The possibility that AlF4 − may exert this effect through its known phosphate‐mimicking property was examined. Inorganic phosphate, as well as two phosphate analogs, beryllium fluoride and orthovanadate, also inhibited cabbage PLD. Enzyme kinetic studies confirmed that PLD followed Hill kinetics, characteristic for allosteric enzymes, with an apparent Hill coefficient (n app) of 3.8, indicating positive cooperativity among multiple substrate‐binding sites and suggesting possible functional oligomerization of the enzyme. AlF4 − modification of PLD kinetics was consistent with a competitive mode of enzyme inhibition. It is therefore proposed that AlF4 −, and other phosphate analogs, inhibits plant PLD by competing with a substrate phosphate group for a substrate‐binding site, thereby preventing the formation of an enzyme‐phosphatidyl intermediate. This may be a conserved feature of PLD superfamily enzymes.

G regulatory proteins and muscarinic receptor signal transduction in mucous acini of rat submandibular gland

The involvement of G regulatory proteins in muscarinic receptor signal transduction was examined in electrically permeabilized rat submandibular acinar cells. The guanine nucleotide analog, GTP gamma S, caused the dose dependent hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate to release IP3. This response was insensitive to pertussis toxin treatment and was duplicated by NaF but not by GDP beta S. Enhanced IP3 synthesis was observed with a combination of GTP gamma S and carbachol. Exogenous IP3, as well as carbachol and GTP gamma S, provoked the release of sequestered 45Ca2+ from non-mitochondrial stores. In intact cells, carbachol significantly reduced the level of cyclic AMP induced by the beta-adrenergic agonist, isoproterenol, to 69% of its normal value. Pertussis toxin abolished this inhibitory action of carbachol on cyclic nucleotide levels. These results suggest that muscarinic receptors are coupled to two separate G regulatory proteins in submandibular mucous acini-the pertussis toxin-insensitive Gp of the phosphoinositide transduction pathway associated with elevated cytosolic calcium levels, and the pertussis toxin-sensitive Gi inhibitory protein of the adenylate cyclase complex.

Aluminum fluoride inhibits phospholipase D activation by a GTP-binding protein-independent mechanism

Aluminum fluoride (AlF4 −) inhibited guanine nucleotide‐activated phospholipase D (PLD) in rat submandibular gland cell‐free lysates in a concentration‐dependent response. This effect was consistent in permeabilized cells with endogenous phospholipid PLD substrates. Inhibition was not caused by either fluoride or aluminum alone and was reversed by aluminum chelation. Inhibition of PLD by aluminum fluoride was not mediated by cAMP, phosphatases 1, 2A or 2B, or phosphatidate phosphohydrolase. AlF4 − had a similar inhibitory effect on rArf‐stimulated PLD, but did not block the translocation of Arf from cytosol to membranes, indicating a post‐GTP‐binding‐protein site of action. Oleate‐sensitive PLD, which is not guanine nucleotide‐dependent, was also inhibited by AlF4 −, supporting a G protein‐independent mechanism of action. A submandibular Golgi‐enriched membrane preparation had high PLD activity which was also potently inhibited by AlF4 −, leading to speculation that the known fluoride inhibition of Golgi vesicle transport may be PLD‐mediated. It is proposed that aluminum fluoride inhibits different forms of PLD by a mechanism that is independent of GTP‐binding proteins and that acts via a membrane‐associated target which may be the enzyme itself.

Muscarinic regulation of phospholipase D and its role in arachidonic acid release in rat submandibular acinar cells

The characteristics of muscarinic cholinergic-induced phospholipase D (PLD) activation, and the involvement of the enzyme in the release of arachidonic acid were examined in rat submandibular acinar cells. Carbachol produced a dose-related activation of PLD to around fivefold control values at 100 μM agonist concentration. This was associated with the appearance of free choline, phosphatidic acid and arachidonic acid, indicating that the PLD substrate was phosphatidylcholine. The response to carbachol was inhibited by 60% by U73122, a blocker of a phospholipase C (PLC) specific to phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], suggesting that the cleavage of phosphatidylcholine by PLD was, at least in part, secondary to agonist-coupled hydrolysis of PtdIns(4,5)P2 by PLC. Consistent with this, PLD was also activated to levels comparable to those induced by carbachol, by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), and the Ca2+ mobilizer, thapsigargin, two agents that respectively mimic the activation of protein kinase C (PKC) by diacylglycerol and the elevation of cytosolic Ca2+by inositol 1,4,5-triphosphate [Ins(1,4,5)P3] in the phosphoinositide effect. The cell-permeant Ca2+chelator 1,2-bis-(O-aminophenoxy)-ethane-N, N,N′,N′-tetraacetic acid, tetraacetoxymethyl ester (BAPTA/AM) abolished the thapsigargin-induced activation of PLD and inhibited the responses of PLD to carbachol and TPA by 60%. The PKC inhibitor, Ro-31-8220, also inhibited the activation of PLD by carbacol and TPA to a level of approximately double control values, but had no effect on the thapsigargin-induced elevation of PLD. A role for both the PKC-associated and Ca2+-mobilizing arms of the PtdIns(4,5)P2-PLC pathway in PLD regulation is thus suggested. Pretreatment of cells with the phosphatidate phosphohydrolase blocker, propranolol, significantly enhanced the carbachol-induced elevation of phosphatidic acid, but decreased agonist-stimulated production of diacylglycerol and arachidonic acid, indicating that phosphatidlycholine was the likely source of arachidonic acid. We therefore propose that, in submandibular mucous acinar cells, muscarinic activation of the PtdIns(4,5)P2-PLC pathway regulates phosphatidylcholine-specific PLD through both the PKC- and Ca2+-mobilizing arms of the phosphoinositide response, and that diacylglycerol, derived from phosphatidylcholine via phosphatidic acid, is a source of free arachidonic acid.