Activation of Muscarinic Receptors in Porcine Airway Smooth Muscle Elicits a Transient Increase in Phospholipase D Activity

Abstract

Phospholipase D (PLD) is a phosphodiesterase that catalyses hydrolysis of phosphatidylcholine to produce phosphatidic acid and choline. In the presence of ethanol, PLD also catalyses the formation of phosphatidylethanol, which is a unique characteristic of this enzyme. Muscarinic receptor-induced changes in the activity of PLD were investigated in porcine tracheal smooth muscle by measuring the formation of [³H]phosphatidic acid ([³H]PA) and [³H]phosphatidylethanol ([³H]PEth) after labeling the muscle strips with [³H]palmitic acid. The cholinergic receptor agonist acetylcholine (Ach) significantly but transiently increased formation of both [³H]PA and [³H]PEth in a concentration-dependent manner (>105–400% vs. controls in the presence of 10^–6 to 10^–4 M Ach) when pretreated with 100 mM ethanol. The Ach receptor-mediated increase in PLD activity was inhibited by atropine (10^–6 M), indicating that activation of PLD occurred via muscarinic receptors. Activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) increased PLD activity that was effectively blocked by the PKC inhibitors calphostin C (10^–8 to 10^–6 M) and GFX (10^–8 to 10^–6 M). Ach-induced increases in PLD activity were also significantly, but incompletely, inhibited by both GFX and calphostin C. From the present data, we conclude that in tracheal smooth muscle, muscarinic acetylcholine receptor-induced PLD activation is transient in nature and coupled to these receptors via PKC. However, PKC activation is not solely responsible for Ach-induced activation of PLD in porcine tracheal smooth muscle.

Loss of receptor regulation by a phospholipase D1 mutant unresponsive to protein kinase C

“…Carbachol stimulation of the m1 and m3 muscarinic receptors results in immediate activation and translocation of PKC, Rho and ARF (Rumenapp et al, 1995;Keller et al, 1997), and stimulation of endogenous PLD (BoyanoAdanez et al, 1997). m1 muscarinic receptor stimulation of PLD has been reported to be abrogated by inhibitors of PKC (BoyanoAdanez et al, 1997;Mamoon et al, 1999), although as discussed above, these experiments do not address the direct stimulation of PLD1 by PKC. Moreover, one of the most widely used PKC catalytic inhibitors, bisindolylmaleimide, actually binds directly to muscarinic receptors and inhibits them (Lazareno et al, 1998).…”

“…However, PLD1 is stimulated through interaction with the regulatory domain of PKC, the stimulation does not involve kinase activity, and it is not blocked by these inhibitors in vitro S.M.Hammond and A.J.Morris, manuscript submitted); thus, the in vivo inhibition is clearly mediated through unknown indirect effects on cellular pathways (reviewed in Houle and Bourgoin, 1999). Inhibition of PKC activation through the regulatory domain inhibitor calphostin is also quite potent at blocking PLD stimulation (Min et al, 1998;Mamoon et al, 1999). However, calphostin is now appreciated to be an extremely potent direct inhibitor of PLD1 and PLD2 (S.M.…”

Protein kinase C isoenzymes: a review of their structure, regulation and role in regulating airways smooth muscle tone and mitogenesis

“…Several agonists that act through G-protein-coupled receptors including ACh and bradykinin activate PLD in [ 3 H]-palmitic acid-labelled porcine and guinea-pig tracheal smooth muscle respectively in the presence of a primary alcohol as indicated by a signi®cant increase in the formation of [ 3 H]-phosphatidylalcohol (Pyne & Pyne, 1993a,b;Mamoon et al, 1999). Such an eect leads to the formation of phosphatidic acid and, ultimately, DAG by the action of phosphatidic acid phosphohydrolase.…”