5‐Hydroxytryptamine stimulation of phospholipase D activity in the rabbit isolated mesenteric artery

The involvement of phospholipase D (PLD) in the 5‐hydroxytryptamine 5‐HT 1B /5‐HT 1D ‐signalling pathway was assessed in the rabbit isolated mesenteric artery. RT–PCR analysis of mesenteric smooth muscle cells revealed a strong signal corresponding to mRNA transcript for the 5‐HT 1B receptor. The PCR fragment corresponded to the known sequence for the 5‐HT 1B receptor. No signal corresponding to 5‐HT 1D mRNA was detected. Neither 5‐HT (3 μ M ) nor KCl (45 m M ) individually stimulated any significant increase in the smooth muscle concentration of [ 33 P]‐PtdBut to reflect PLD activity. However, in the presence of KCl (45 m M ), 5‐HT evoked a concentration‐dependent increase in [ 33 P]‐PtdBut, to a maximum of 84% with 5‐HT (3 μ M ). [ 33 P]‐PtdBut accumulation evoked by 5‐HT in the presence of KCl was abolished in nominally calcium‐free Krebs‐Henseleit Buffer (KHB) or with the selective protein kinase C inhibitor, Ro‐31 8220 (10 μ M , 20 min). 5‐HT (3 μ M ) in the presence of KCl (45 m M ) failed to increase either the accumulation of [ 33 P]‐phosphatidic acid in the presence of butanol, or total [ 3 H]‐inositol phosphates ([ 3 H]‐InsP) in the presence of LiCl (10 m M ). 5‐HT (0.1–1 μ M ) abolished forskolin (1 μ M ) stimulated increases in cyclic AMP (15 fold increase), an action which was pertussis toxin‐sensitive. Therefore, in the presence of raised extracellular potassium 5‐HT can stimulate PLD via 5‐HT 1B receptors in the rabbit mesenteric artery. This action requires extracellular calcium and the activation of protein kinase C. These characteristics are identical to the profile for 5‐HT 1B /5‐HT 1D ‐receptor evoked contraction in vascular smooth muscle cells, suggesting a role for PLD in this response to 5‐HT.British Journal of Pharmacology (1999) 126 , 1601–1608; doi: 10.1038/sj.bjp.0702484