Mitochondria buffer NCX-mediated Ca²⁺-entry and limit its diffusion into vascular smooth muscle cells

Abstract

The reverse-mode of the Na⁺/Ca²⁺-exchanger (NCX) mediates Ca²⁺-entry in agonist-stimulated vascular smooth muscle (VSM) and plays a central role in salt-sensitive hypertension. We investigated buffering of Ca²⁺-entry by peripheral mitochondria upon NCX reversal in rat aortic smooth muscle cells (RASMC). [Ca²⁺] was measured in mitochondria ([Ca²⁺]_MT) and the sub-plasmalemmal space ([Ca²⁺]_subPM) with targeted aequorins and in the bulk cytosol ([Ca²⁺]_i) with fura-2. Substitution of extracellular Na⁺ by N-methyl-d-glucamine transiently increased [Ca²⁺]_MT (∼2 μM) and [Ca²⁺]_subPM (∼1.3 μM), which then decreased to sustained plateaus. In contrast, Na⁺-substitution caused a delayed and tonic increase in [Ca²⁺]_i (<100 nM). Inhibition of Ca²⁺-uptake by the sarcoplasmic reticulum (SR) (30 μM cyclopiazonic acid) or mitochondria (2 μM FCCP or 2 μM ruthenium red) enhanced the elevation of [Ca²⁺]_subPM. These treatments also abolished the delay in the [Ca²⁺]_i response to 0Na⁺ and increased its amplitude. Extracellular ATP (1 mM) caused a peak and plateau in [Ca²⁺]_i, and only the plateau was inhibited by KB-R7943 (10 μM), a selective blocker of reverse-mode NCX. Evidence for ATP-mediated NCX-reversal was also found in changes in [Na⁺]_i. Mitochondria normally exhibited a transient elevation of [Ca²⁺] in response to ATP, but inhibiting the mitochondrial NCX with CGP-37157 (10 μM) unmasked an agonist-induced increase in mitochondrial Ca²⁺-flux. This flux was blocked by KB-R7943. In summary, mitochondria and the sarcoplasmic reticulum co-operate to buffer changes in [Ca²⁺]_i due to agonist-induced NCX reversal.

Introduction

The Na⁺/Ca²⁺-exchanger (NCX) plays an important role in vascular smooth muscle (VSM) Ca²⁺ homeostasis. The NCX has been extensively characterized as a Ca²⁺-extrusion mechanism [1], [2], and a growing body of evidence indicates that Ca²⁺-entry via reverse-mode NCX (revNCX) contributes to agonist-mediated stimulation and increased vascular tone in salt-sensitive hypertension [3], [4], [5], [6], [7]. The link between hypertension, elevated plasma [Na⁺] and altered Na⁺/Ca²⁺-exchange was suggested by Blaustein almost 30 years ago [8], [9], but early mechanistic studies failed to provide a consistent correlation between increases in extracellular [Na⁺] and cytosolic [Ca²⁺] ([Ca²⁺]_i). Only later was it discovered that in several types of VSM the NCX communicates more efficiently with the sarcoplasmic reticulum (SR) than the bulk cytosol [1], [10]. This localized Ca²⁺-transport between the extracellular space and the SR is highly dependent on junctional membrane complexes between the plasmalemma (PM) and SR (separated by ∼20 nm) that cover 10–15% of the inner leaflet of the PM [10], [11], [12], [13]. At these junctions, the PM is enriched with NCX and alpha-2 Na⁺/Ka⁺-ATPase, and the SR is enriched with calsequestrin [14], [15]. This arrangement of proteins is thought to provide localized regulation of [Na⁺] in the sub-plasmalemmal cytosol and thus NCX-activity, which regulates SR Ca²⁺ levels [16].

Electron microscopy has shown that 99% of smooth muscle mitochondria are closely associated with SR [17] and that mitochondria can often be found neighbouring PM-SR junctions [12]. Mitochondria are thought to regulate Ca²⁺-release from closely associated SR [18], [19] and in doing so regulate the amplitude and frequency of SR-driven Ca²⁺ oscillations in VSM [20]. Here, we investigated whether mitochondria, presumably those neighbouring the PM-SR junctions, might contribute to the localized regulation of changes in cytosolic [Ca²⁺] mediated by the NCX. In related studies mitochondrial uptake of NCX-mediated Ca²⁺-entry was shown to regulate the activity of bovine cardiac NCX expressed in Chinese hamster ovarian (CHO) cells [21], [22].

The activation of mitochondrial Ca²⁺-uptake and subsequent elevation of mitochondrial [Ca²⁺] ([Ca²⁺]_MT) to levels beyond the average [Ca²⁺]_i is thought to require the generation of supra-micromolar [Ca²⁺] in the cytosolic microdomain between the mitochondria and closely apposed Ca²⁺ sources on the PM or SR [19], [23]. In aortic smooth muscle cells, mitochondria are closely coupled with both inositol-1,4,5 trisphosphate receptors and ryanodine receptors [23], [24], and we have recently shown that the NCX regulates the Ca²⁺ content of the SR and thereby indirectly influences mitochondrial Ca²⁺-uptake [24]. In our current investigation, we have used targeted aequorins and fura-2 to study parallel changes of [Ca²⁺] in mitochondria, the sub-plasmalemmal cytosol and the bulk cytosol. This investigation revealed that the NCX can directly stimulate mitochondrial Ca²⁺-uptake. Reversal of the NCX caused an increase of [Ca²⁺] in the cytosolic domain immediately beneath the PM ([Ca²⁺]_subPM) that was accessible to both SR and mitochondria. This buffering of the NCX-mediated Ca²⁺-entry by the SR and mitochondria appeared to prevent the elevated [Ca²⁺]_subPM from diffusing into the bulk cytosol. Furthermore, NCX reversal appeared to contribute to the agonist-mediated Ca²⁺-entry that was taken up by mitochondria, while elevation of [Ca²⁺]_MT was minimized by an increase in mitochondrial Ca²⁺-extrusion.