Different phospholipase-C-coupled receptors differentially regulate capacitative and non-capacitative Ca2+ entry in A7r5 cells

Abstract

Several receptors, including those for AVP (Arg8-vasopressin) and 5-HT (5-hydroxytryptamine), share an ability to stimulate PLC (phospholipase C) and so production of IP3 (inositol 1,4,5-trisphosphate) and DAG (diacylglycerol) in A7r5 vascular smooth muscle cells. Our previous analysis of the effects of AVP on Ca2+ entry [Moneer, Dyer and Taylor (2003) Biochem. J. 370, 439-448] showed that arachidonic acid released from DAG stimulated NO synthase. NO then stimulated an NCCE (non-capacitative Ca2+ entry) pathway, and, via cGMP and protein kinase G, it inhibited CCE (capacitative Ca2+ entry). This reciprocal regulation ensured that, in the presence of AVP, all Ca2+ entry occurred via NCCE to be followed by a transient activation of CCE only when AVP was removed [Moneer and Taylor (2002) Biochem. J. 362, 13-21]. We confirm that, in the presence of AVP, all Ca2+ entry occurs via NCCE, but 5-HT, despite activating PLC and evoking release of Ca2+ from intracellular stores, stimulates Ca2+ entry only via CCE. We conclude that two PLC-coupled receptors differentially regulate CCE and NCCE. We also address evidence that, in some A7r5 cells lines, AVP fails either to stimulate NCCE or inhibit CCE [Brueggemann, Markun, Barakat, Chen and Byron (2005) Biochem. J. 388, 237-244]. Quantitative PCR analysis suggests that these cells predominantly express TRPC1 (transient receptor potential canonical 1), whereas cells in which AVP reciprocally regulates CCE and NCCE express a greater variety of TRPC subtypes (TRPC1=6>2>3).

Role of Endogenous TRPC6 Channels in Ca2+ Signal Generation in A7r5 Smooth Muscle Cells

“…Despite the quantitative uncertainty of Western analysis, assessment of TRPC protein levels rather than mRNA levels is obviously a more reliable indicator of the presence and relative levels of endogenous TRPC channels. In a paper published after completion of this work, Moneer et al (36) revealed significant variations in the expression of TRPC channels in different strains of A7r5 cells. In certain strains, significant levels of TRPC3 message could be detected, although no information on protein levels was given.…”

Stimulation of arachidonic acid release by vasopressin in A7r5 vascular smooth muscle cells mediated by Ca2+‐stimulated phospholipase A2

“…AA appears also to differ in its effectiveness according to how it is produced. We have shown earlier that release of AA by DAG lipase from DAG produced by AVP-activated PLC causes reciprocal regulation of CCE and NCCE [6,7], but the much larger amounts of AA produced by cPLA 2 (Fig. 1) appear not to significantly influence Ca 2+ entry (Fig.…”

“…A7r5 vascular smooth muscle cells, although their relative roles may differ under different conditions [4][5][6][7][8][9], influenced by both the pattern of expression of TrpC proteins [7,10] and the nature of the stimulus [7]. It is, however, clear that in A7r5 cells, as in many other cells [11][12][13], arachidonic acid (AA) contributes to the coordinate regulation of CCE and NCCE [5,6,14].…”

“…AA, verapamil and AVP were from Sigma. Other materials are listed in previous publications [6][7][8].…”

“…The medium bathing the cells (1 ml) was then removed (for determination of 3 H-AA content) and replaced (1 ml) at intervals of 1-min. All experiments were performed at 20°C; the same temperature was used for analyses of AVP-evoked Ca 2+ signals [4][5][6][7][8]. At the end of each experiment, Triton-X-100 (1%) was added to lyze the cells, and the medium was removed to determine its 3 H-AA content.…”