Agonist-evoked calcium entry in vascular smooth muscle cells requires IP₃ receptor-mediated activation of TRPC1

Abstract

Transient receptor potential canonical (TRPC) proteins have been proposed to function as plasma membrane Ca²⁺ channels activated by store depletion and/or by receptor stimulation. However, their role in the increase in cytosolic Ca²⁺ activated by contractile agonists in vascular smooth muscle is not yet elucidated. The present study was designed to investigate the functional and molecular properties of the Ca²⁺ entry pathway activated by endothelin-1 in primary cultured aortic smooth muscle cells. Measurement of the Ca²⁺ signal in fura-2-loaded cells allowed to characterize endothelin-1-evoked Ca²⁺ entry, which was resistant to dihydropyridine, and was blocked by 2-aminoethoxydiphenylborate (2-APB) and micromolar concentration of Gd³⁺. It was not activated by store depletion, but was inhibited by the endothelin ET_A receptor antagonist BQ-123, and by heparin. On the opposite, thapsigargin-induced store depletion activated a Ca²⁺ entry pathway that was not affected by 2-APB, BQ-123 or heparin, and was less sensitive to Gd³⁺ than was endothelin-1-evoked Ca²⁺ entry. Investigation of the gene expression of TRPC isoforms by real-time RT-PCR revealed that TRPC1 was the most abundant. In cells transfected with TRPC1 small interfering RNA sequence, TRPC1 mRNA and protein expression were decreased by 72±3% and 86 ± 2%, respectively, while TRPC6 expression was unaffected. In TRPC1 knockdown cells, both endothelin-1-evoked Ca²⁺ entry and store-operated Ca²⁺ entry evoked by thapsigargin were blunted. These results indicate that in aortic smooth muscle cells, TRPC1 is not only involved in Ca²⁺ entry activated by store depletion but also in receptor-operated Ca²⁺ entry, which requires inositol (1,4,5) triphosphate receptor activation.

Introduction

Intracellular calcium is considered as an important messenger in the control of vascular smooth muscle contractile tone. Activator Ca²⁺ can be released from intracellular stores or can enter the cell from the extracellular space through several types of ion channels. In vascular smooth muscle cell, two main classes of plasmalemmal Ca²⁺ channels have been identified based on the voltage sensitivity of the gating process. Voltage-operated Ca²⁺ channels (VOC) are highly selective for Ca²⁺. They have been widely studied, and their molecular structures have been identified (Catterall, 2000). The prominent role played by a second group of Ca²⁺ channels that are not activated by membrane depolarisation, has been recognised more recently. Most of them have non-selective cation conductance. They are resistant to dihydropyridines, the well-known inhibitors of L-type VOC, and no reliable selective antagonist is available (Albert and Large, 2006, Beech et al., 2004, Inoue et al., 2003).

The mechanisms of activation and molecular composition of non-selective cation channels have not yet been conclusively identified. In most cells, depletion of intracellular Ca²⁺ stores is pivotal for Ca²⁺ entry commonly termed capacitive Ca²⁺ entry (Berridge, 1995, Putney et al., 2001). Thapsigargin and cyclopiazonic acid are often used to study this pathway because they selectively inhibit the active uptake of Ca²⁺ into the sarco-endoplasmic reticulum via Ca²⁺-ATPase pump, allowing the stores to be passively depleted, without receptor activation. The capacitative pathway is not however, the only pathway through which Ca²⁺ can enter cells in response to receptor activation. Non-selective cation channels are also activated in response to a signal generated by a range of intracellular messengers released after the binding of an agonist to its receptor including diacylglycerol (Albert and Large, 2006, Helliwell and Large, 1997), arachidonic acid (Munaron et al., 1997, Shuttleworth et al., 2004), inositol (1,4,5) triphosphate (IP₃) (Dong et al., 1995). The later channels have been called “receptor-operated channels or ROC”, in contrast to “store-operated channels or SOC”. However, whether this classification reflects distinct molecular entities is not yet determined.

The canonical transient receptor potential TRPC proteins have been proposed to form non-selective cation channels and considerable research has been carried out on the seven members of this family. RT-PCR analysis has revealed the expression of TRPC1, 4, 5, and 6 mRNA in vascular smooth muscle, including cerebral arteries (Bergdahl et al., 2005), aorta (Xu and Beech, 2001), mesenteric resistance artery (Hill et al., 2006), and pulmonary artery (McDaniel et al., 2001, Sweeney et al., 2002, Wang et al., 2004, Yu et al., 2003).

Several reports have proposed that TRPC1 forms SOC in vascular smooth muscle cell, as in several other cell types (Beech, 2005). TRPC1 specific antibody inhibits store-operated Ca²⁺ influx in human, mouse and rabbit arterial smooth muscle cells (Xu and Beech, 2001). TRPC1 antisense oligonucleotides inhibit SOC in pulmonary artery cell line (Sweeney et al., 2002), while in rat cerebral artery in organ culture, increase in TRPC1 expression is associated with upregulated SOC activity (Bergdahl et al., 2005). Similarly, overexpression of TRPC1 in pulmonary artery increases capacitative Ca²⁺ entry (Kunichika et al., 2004). In pulmonary artery smooth muscle cell as in the stable A7r5 cell line, (small interfering) siRNA-mediated knockdown of TRPC1 selectively inhibits thapsigargin-activated Ca²⁺ entry (Brueggemann et al., 2006, Lin et al., 2004).

On the other hand, the TRPC3/6/7 subfamily is generally proposed to form ROC. TRPC6 is activated by angiotensin II in rabbit mesenteric artery (Saleh et al., 2006), and by α1-adrenergic agonist in rabbit portal vein (Inoue et al., 2001). In A7r5 cell line, Ca²⁺ current activated by Arg-vasopressin shares kinetic and pharmacological properties with heterologously expressed TRPC6 channel protein (Jung et al., 2002), and is suppressed by dominant-negative mutant TRPC6 (Maruyama et al., 2006). In rabbit ear artery, noradrenaline activates TRPC3 channels. TRPC3 is also activated by UTP in cerebral arteries (Reading et al., 2005) or, in association with TRPC7, by endothelin-1 in rabbit coronary artery (Peppiatt-Wildman et al., 2007).

The mechanism(s) of activation of Ca²⁺ entry induced by store depletion or receptor activation in vascular smooth muscle cells and their relative contribution to Ca²⁺ signalling are still not defined. One difficulty probably arises from the differences between vascular beds, that could be related to the expression of different TRPC isoforms. Moreover, many studies reported in literature were performed in overexpression systems, and their conclusions cannot be translated to native cells since the composition of the channels and by consequence their properties, depend on the relative expression of the TRPC proteins (Putney, 2004, Vazquez et al., 2003).

Discrepancy in the responses to vasopressin in A7r5 cells is a good example of the conflicting results that are found in literature: vasopressin has been reported to activate non-capacitative Ca²⁺ entry (Broad et al., 1999, Jung et al., 2002), to activate capacitative Ca²⁺ entry (Brueggemann et al., 2005, Brueggemann et al., 2006) or to reciprocally activate capacitative and non-capacitative Ca²⁺ entry (Moneer et al., 2005, Moneer and Taylor, 2002). These puzzling results are suggested to be related to different expression in TRPC subtypes in variant cell lines: non-capacitative entry is observed in cells expressing TRPC6 (Jung et al., 2002), while TRPC1 is involved in capacitative Ca²⁺ entry (Brueggemann et al., 2006, Moneer et al., 2005).

The present study was designed to characterize the Ca²⁺ entry pathway activated by contractile agonist in rat aortic SMC and to investigate the role of TRPC1 in the response to agonist. The potent vasoconstrictor endothelin-1 was used to stimulate aortic cells. Endothelin-1 binds to endothelin ET_A receptor on vascular smooth muscle cells (VSMC) (Oonuma et al., 2000) and induces an increase in intracellular Ca²⁺ responsible for the development of contraction through the activation of non-selective cation channels (Miwa et al., 2005). Receptor-activated Ca²⁺ entry evoked by endothelin-1 was compared to Ca²⁺ entry activated by store depletion evoked by treatment of the cells with thapsigargin. Results indicated that, in aortic smooth muscle cells, endothelin-1 activates a Ca²⁺ entry pathway that is distinct from SOC and requires IP₃ receptor activation. Inhibition of TRPC1 expression in cells transfected with TRPC1 small interfering RNA sequence abolished endothelin-1 activated Ca²⁺ entry, suggesting that TRPC1 is an indispensable element of ROC in vascular smooth muscle.