Local elementary purinergic-induced Ca2+ transients: from optical mapping of nerve activity to local Ca2+ signaling networks

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Abstract

The autonomic nervous system regulates smooth muscle contractility through both sympathetic and parasympathetic influences. In some tissues, such as the urinary bladder, parasympathetic influences predominate and nerves communicate to detrusor smooth muscle through the release of acetylcholine (ACh). In other tissues, such as the vas deferens and mesenteric arterial circulation, the primary autonomic influence is sympathetic, and norepinephrine (NE) is the predominant neurotransmitter. NE and ACh act on smooth muscle cells through Gq-coupled α-adrenergic and muscarinic receptors, respectively, which signal through PLC to elevate diacylglycerol and inositol trisphosphate (IP3), which in turn, activate PKC and IP3 receptors (IP3Rs) in the SR. IP3-mediated Ca2+ release from the SR of vascular smooth muscle cells gives rise to Ca2+ waves (Iino and Tsukioka, 1994; Jaggar and Nelson, 2000; Wray et al., 2005; Kim et al., 2008), which are propagating elevations in Ca2+ that are thought to contribute to vascular smooth muscle contraction (Mauban et al., 2001; Zang et al., 2006). The consequences of G protein–coupled signaling events manifest after a characteristic lag, reflecting the temporal dynamics of multiple sequential and parallel molecular linkages. Although NE and ACh are the prototypical transmitters released by autonomic nerves, it has long been known that ATP is coreleased with NE at sympathetic nerve–muscular junctions and with ACh at parasympathetic nerve–muscular junctions. Coreleased ATP acts on P2X receptor channels in the plasma membrane of smooth muscle cells. Because P2X receptors are ion channels, once activated, their effects are experienced almost immediately by the cell. This rapid time course is in contrast to the more delayed influence of the G protein–coupled adrenergic and muscarinic receptors. P2X receptors represent a family of seven receptors (P2X1–7) that belong to the transmitter-gated ion channel superfamily, which also includes nicotinic-like receptors and glutamate-like receptors (for review see Khakh, 2001). Each P2X receptor subunit possesses intracellular N and C termini and two membrane-spanning domains linked by a large extracellular domain (for review see Khakh, 2001; North, 2002). P2X receptors are thought to consist of three subunits (Aschrafi et al., 2004), which is also the simplest stoichiometry among ionotropic receptors. At least three ATP molecules bind to the extracellular domain of P2X channels (Jiang et al., 2003). Upon binding ATP, P2X receptors undergo conformational changes that result in the opening of the pore within milliseconds, although the underlying molecular details have not yet been elucidated. P2X receptors are nonselective cation channels that exhibit a permeability to Ca2+ approximately equal to that of sodium (Na+) (Schneider et al., 1991). Thus, activation of P2X receptors by ATP released at nerve–muscle junctions causes a rapid local influx of Na+ and Ca2+ (Lamont and Wier, 2002; Lamont et al., 2006). Although most of the excitatory junction current (EJC) associated with P2X activation is carried by the more abundant (∼70-fold) Na+ ions, the influx of Ca2+ is quite substantial. In fact, the fractional Ca2+ currents mediated by the rat (∼12.4%) and human (∼11%) P2X1 isoforms are not significantly different from that of the NMDA channel (∼14%) (Egan and Khakh, 2004), long considered the gold standard for high-level, ligand-gated Ca2+ entry. The current mediated by Na+ and Ca2+ influx creates an excitatory junction potential (EJP) that contributes directly to the increase in postjunctional excitability associated with autonomic stimulation. The P2X1 receptor is the predominant P2X receptor isoform expressed in smooth muscle. It was originally cloned from the vas deferens (Valera et al., 1994), and immunocytochemical studies in mice have shown that P2X1 expression in the urinary bladder is restricted to detrusor smooth muscle (Vial and Evans, 2000). The most compelling evidence for the prominence of the P2X1 isoform in smooth muscle comes from studies using P2X1 receptor knockout (P2X1R-KO) mice. These studies have shown that ATP-evoked EJCs and EJPs are absent in the vas deferens from P2X1R-KO mice (Mulryan et al., 2000). Similarly, these mice lack nerve-evoked purinergic contractile responses in bladders (Vial and Evans, 2000) and mesenteric arteries (Vial and Evans, 2002; Lamont et al., 2006).

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