STIM and Orai proteins: players in sexual differences in hypertension-associated vascular dysfunction?

Abstract

Sex-associated differences in hypertension have been repeatedly observed in epidemiological studies. However, the mechanisms conferring vascular protection to females are not totally elucidated. Sex-related differences in intracellular Ca²⁺ handling or, more specifically, in mechanisms that regulate Ca²⁺ entry into vascular smooth muscle cells have been identified as players in sex-related differences in hypertension-associated vascular dysfunction. Recently, new signaling components that regulate Ca²⁺ influx, in conditions of intracellular store depletion, were discovered: stromal interaction molecule 1 (STIM1), which works as an intracellular Ca²⁺ sensor; and Orai1, which is a component of the Ca⁺² release-activated Ca⁺² (CRAC) channels. Together, these proteins reconstitute store-operated Ca²⁺ channel function. Disturbances in STIM1/Orai1 signaling have been implicated in pathophysiological conditions, including hypertension. In this review we analyze evidence for sex-related differences in Ca²⁺ handling and propose a new hypothesis where sex-related differences in STIM/Orai signaling may contribute to hypertension-associated vascular differences between male and female subjects.

1.0 - Introduction

Defective regulation of intracellular calcium (Ca²⁺) is a hallmark of hypertension-associated vascular dysfunction and plays a key role in the augmented vascular reactivity, characteristic of clinical and experimental hypertension. The recent discovery of new signaling components linking intracellular Ca⁺² stores to plasma membrane Ca²⁺ entry brought a new insight into the understanding of Ca²⁺ homeostasis. Stromal interaction molecule 1 (STIM1) is the Ca²⁺ sensor protein that triggers Ca²⁺ influx in response to Ca²⁺ store depletion, whereas Orai is an essential component of CRAC (Ca²⁺ release-activated Ca²⁺) channels. Although research on STIM1/Orai signaling is entering an exponential phase of growth, the role of these proteins in vascular dysfunction is unknown.

Mechanisms contributing to hypertension and its associated end-organ dysfunction are differentially regulated in males and females. Critical sex differences are observed in the intrinsic vascular mechanisms that regulate total peripheral resistance, namely that gonadally intact females display less vascular dysfunction associated with experimental hypertension [1–6], compared to gonadally intact males. Although the existence of sex differences in vascular Ca²⁺ handling is well established [7–10], no studies have addressed differences in vascular STIM1/Orai signaling in male and female hypertensive animals.

Given the importance of STIM1/Orai signaling in intracellular Ca²⁺ homeostasis, it seems plausible that increased activation of STIM1/Orai contributes to increased vascular contraction in the vasculature of hypertensive animals.

Here we will review evidence supporting these hypotheses. First, we will address the participation of STIM1 and Orai1 on Ca⁺² handling mechanisms during physiological condition as well as in hypertension. Then, we will focus on how alteration of STIM1/Orai signaling contributes to differences in Ca⁺² handling, and how this impairment may contribute to sex-related differences in vascular function in hypertension.

2.0 - STIM1-Orai1 pathway represents a key Ca⁺² handling mechanism

Nearly all cell types depend on cytoplasmatic Ca⁺² signals to trigger specific responses [11]. Upon stimulation, most excitable cells display a biphasic increase in cytosolic Ca²⁺ concentrations. The initial transient increase is due to Ca²⁺ release from intracellular stores, e.g. inositol trisphosphate (IP₃)-mediated release of ER Ca²⁺, whereas the subsequent prolonged increase requires extracellular Ca²⁺ influx through various pathways. Upon depletion of Ca²⁺ from the ER, Ca²⁺ channels are activated in the plasma-membrane to refill internal Ca²⁺ stores. This mechanism, by which the ER acts as a capacitor, lead to the term store-operated Ca²⁺ entry (SOCE) [12, 13]. SOCE carries a highly Ca²⁺–selective, non-voltage-gated, inwardly rectifying current termed CRAC (Ca²⁺ release-activated Ca²⁺) current, or I_CRAC [13, 14].

The discovery of new signaling components linking intracellular Ca⁺² stores to plasma membrane Ca²⁺ entry brought a new insight into the understanding of Ca²⁺ homeostasis. Stromal interaction molecule 1 (STIM1) was identified as a Ca²⁺ sensor essential for Ca²⁺–store depletion-triggered Ca²⁺ influx [15, 16]. Roos and colleagues (2005) showed that knockdown of STIM in Drosophila S2 cells significantly reduced thapsigargin-dependent Ca²⁺ entry and completely suppressed I_CRAC [15]. In addition to being an ER Ca²⁺ sensor, STIM1 functions within the plasma membrane to control operation of the Ca²⁺ entry channel itself [17] and STIM1 migrates from the Ca²⁺ store to the plasma membrane, in conditions of store depletion [18].

It was later demonstrated that Orai1 is an essential pore subunit of the CRAC channel [19]. Accordingly, upon depletion of ER Ca²⁺ stores, STIM1 and Orai move in a coordinated fashion to form closely apposed clusters in the ER and plasma membrane [20], creating the elementary unit of SOCE [21]. In addition, the interaction between STIM1 and Orai1 is greatly enhanced after thapsigargin treatment, which acts as selective inhibitor of the ER Ca²⁺ ATPase resulting in depletion of ER Ca²⁺ stores [22].

2.1 - STIM1-Orai1 pathway alterations and hypertension

Since a rise in intracellular free Ca²⁺ concentration is the principal process that initiates contraction of vascular smooth muscle cells (VSMCs) [23], the maintenance of the steady-state Ca²⁺ is critically important to maintain vascular tone and, consequently, total peripheral resistance [24]. Defective regulation of intracellular Ca²⁺ plays a major role in the augmented vascular reactivity [25]. Augmented Ca²⁺ levels in VSMCs from hypertensive animals can be attributed to various mechanisms, including increased Ca²⁺ release from intracellular stores [26]; reduced Ca²⁺ uptake by the SR [27]; impaired function of Ca²⁺-binding proteins [28]; decreased Ca²⁺ extrusion mechanisms in the plasma membrane [26]; and increased Ca²⁺ influx [29, 30].

Here we will focus on how increased Ca²⁺ influx through STIM1 and Orai1 pathway may contribute to augmented vascular reactivity in hypertension. We first demonstrated that aortas from stroke-prone spontaneously hypertensive rats (SHRSP), displayed increased force-development during Ca²⁺ loading, upon depletion of intracellular Ca²⁺ stores [31]. The SR Ca²⁺ store is larger in aortas from SHRSP due to an enhanced influx of Ca²⁺ across the sarcolemma rather than an impaired recycling of the cation by the SR Ca²⁺-ATPase [32].

It was shown that depletion of ER Ca²⁺ stores induces greater SOCE activation in vascular myocytes from SHRSP, compared to that in control Wistar Kyoto (WKY - Figure 1) rats [30]. This is associated with augmented vascular contractile responses to Ca²⁺, which is blocked by molecular (neutralizing antibodies against STIM1 and Orai1) and pharmacological (2-APB and Gd³⁺) inhibition of STIM1/Orai signaling. In addition, vascular expression of STIM1 (Figure 2) and Orai proteins is increased in SHRSP versus WKY. Thus, augmented STIM1/Orai signaling may represent a mechanism leading to impaired control of intracellular Ca⁺² in hypertension.

Fig.1. SHRSP aortas display augmented spontaneous tone after depletion of Ca²⁺ stores.

Inhibition of the SR Ca²⁺-ATPase with thapsigargin augmented contractile responses to Ca²⁺ with greater effects in SHRSP. Representative traces.

Fig. 2. SHRSP displays increased vascular expression of STIM-1.

Immunocytochemistry performed in rat aorta vascular smooth muscle cells. Cells were incubated with primary antibodies rabbit anti-STIM1 and counterstained with a Cy3-conjugated anti-mouse IgG secondary antibody. Cells were incubated with 4′,6-diamidino-2-phenyindole (DAPI, D-9564) to detect nuclei.

3.0 - Sex differences in hypertension

Blood pressure is higher in men than in age-matched women and there is a lower incidence of hypertension in pre-menopausal women than men [33–36]. Although gender-associated differences during hypertension have been repeatedly observed in epidemiological studies, the mechanisms for gender differences in blood pressure control are not totally elucidated.

Additionally, because Ca⁺² triggers VSMC function and its regulation is highly controlled, differences in Ca⁺² handling mechanisms have been proposed to explain sex-related differences in vascular function during hypertension [37] as will be discussed.

3.1 - Sex differences in hypertension and Ca²⁺ handling by vascular myocytes

Although sex-associated differences in hypertension are well established, with important differences in the neural, renal and vascular mechanisms associated with blood pressure homeostasis ^[49–53], the mechanisms that determine differences in blood pressure control in males and females are not totally elucidated. Considering that sex-related differences in mechanisms that regulate Ca²⁺ entry and storage in VSMCs have been identified, differential Ca⁺² handling in VSMCs from males and females may explain sex-related differences in vascular function in hypertension [37]. Accordingly, in aorta from both normotensive and female SHR, Ca²⁺ influx upon contractile stimuli is decreased, compared to that in male SHR [10]. In addition, VSMCs from female rats display reduced Ca²⁺ entry and reduced depolarization-induced Ca²⁺ levels, compared to those in male [8]. Differences in intracellular Ca²⁺ increases in VSMC from male and female SHR are abolished in the absence of extracellular Ca²⁺ [37–39].

Since augmented STIM1/Orai function may represent one mechanism that contributes to abnormal Ca⁺² in VSMC, we hypothesize that vascular protection in hypertensive females reflects an attenuated signaling through STIM1/Orai in vascular myocytes from hypertensive females (figure 3).

Fig. 3. Putative mechanism by which augmented STIM-1/Orai1 signaling contributes to abnormal Ca²⁺ homeostasis in vascular myocytes.

Compared to female, arteries from male subjects display increased activation of STIM1/Orai1. In conditions of depletion or decrease of intracellular Ca²⁺ stores, STIM1 is activated, resulting in the dimerization of Orai1, and the formation of CRAC channels. Ca²⁺ influx, which is increased in VSMCs from hypertensive males compared to hypertensive females, is stimulated via STIM1 and CRAC channels (Orai1). Therefore, arteries from hypertensive male subjects display increased Ca²⁺ influx. SR Ca²⁺ ATPase is activated and the intracellular Ca²⁺stores are refilled. However, the SR in VSMCs of female and male hypertensive displays similar buffering capacity. Consequently, arteries from hypertensive male subjects show increased free cytosolic Ca²⁺, which can lead to increased vascular contraction.

In agreement with this hypothesis, we showed that upon store depletion, force development during Ca²⁺ loading period is augmented in aortas from male SHRSP, compared to female SHRSP. Interestingly, pharmacological blockade of CRAC channel is able to abolish sex-differences in spontaneous contractions during Ca²⁺ loading period [40]. Additionally, after store depletion, neutralizing antibodies against STIM1 and Orai1 abolish sex-differences in spontaneous contractions.

It is possible that augmented STIM1/Orai signaling represents a mechanism for increased activation of Ca²⁺-dependent signaling pathways in arteries from hypertensive animals. Identification of mechanisms leading to sex differences in hypertension may uncover a regulatory mechanism that may be used to confer cardiovascular protection.