Mitochondria modulate ameloblast Ca2+ signaling.

The role of mitochondria in enamel, the most mineralized tissue in the body, is poorly defined. Enamel is formed by ameloblast cells in two main sequential stages known as secretory and maturation. Defining the physiological features of each stage is essential to understand mineralization. Here, we analyzed functional features of mitochondria in rat primary secretory and maturation-stage ameloblasts focusing on their role in Ca2+ signaling. Quantification of the Ca2+ stored in the mitochondria by trifluoromethoxy carbonylcyanide phenylhydrazone stimulation was comparable in both stages. The release of endoplasmic reticulum Ca2+ pools by adenosine triphosphate in rhod2AM-loaded cells showed similar mitochondrial Ca2+ (m Ca2+ ) uptake. However, m Ca2+ extrusion via Na+ -Li+ -Ca2+ exchanger was more prominent in maturation. To address if m Ca2+ uptake via the mitochondrial Ca2+ uniporter (MCU) played a role in cytosolic Ca2+ (c Ca2+ ) buffering, we stimulated Ca2+ influx via the store-operated Ca2+ entry (SOCE) and blocked MCU with the inhibitor Ru265. This inhibitor was first tested using the enamel cell line LS8 cells. Ru265 prevented c Ca2+ clearance in permeabilized LS8 cells like ruthenium red, and it did not affect ΔΨm in intact cells. In primary ameloblasts, SOCE stimulation elicited a significantly higher m Ca2+ uptake in maturation ameloblasts. The uptake of Ca2+ into the mitochondria was dramatically decreased in the presence of Ru265. Combined, these results suggest an increased mitochondrial Ca2+ handling in maturation but only upon stimulation of Ca2+ influx via SOCE. These functional studies provide insights not only on the role of mitochondria in ameloblast Ca2+ physiology, but also advance the concept that SOCE and m Ca2+ uptake are complementary processes in biological mineralization.

Chronic treatment with red wine modulates the purinergic neurotransmission and decreases blood pressure in hypertensive SHR and diabetic-STZ rats.

It is known that red wine has cardioprotective properties. However, its influence is unknown about purinergic system. Therefore, we study the influence of the treatment with red wine or ethanol in purinergic neurotransmission. We used Wistar Kyoto rats (WKY), diabetic streptozotocin-induced WKY and spontaneously hypertensive rats (SHR), treated with red wine (12.5%) or ethanol (12.5%). The cardiovascular function stimulated with purinergic agonists and systolic blood pressure (SBP) was assessed. In atria of diabetics and SHRs, the P1 receptor response was decreased, unlike the P2 receptor response was increased. Likewise, in aorta the affinity to adenosine (ADO) was decreased from SHRs and diabetics. Furthermore, the P2X function was increased just SHRs. All these alterations were improved after treatment with red wine, resulting in reduction of SBP from diabetics and SHRs, but not when treated with ethanol. This study has important implications, because it is shown that consumption of red wine can improve cardiovascular system by purinergic neurotransmission.

Fluoride exposure alters Ca2+ signaling and mitochondrial function in enamel cells.

Fluoride ions are highly reactive, and their incorporation in forming dental enamel at low concentrations promotes mineralization. In contrast, excessive fluoride intake causes dental fluorosis, visually recognizable enamel defects that can increase the risk of caries. To investigate the molecular bases of dental fluorosis, we analyzed the effects of fluoride exposure in enamel cells to assess its impact on Ca2+ signaling. Primary enamel cells and an enamel cell line (LS8) exposed to fluoride showed decreased internal Ca2+ stores and store-operated Ca2+ entry (SOCE). RNA-sequencing analysis revealed changes in gene expression suggestive of endoplasmic reticulum (ER) stress in fluoride-treated LS8 cells. Fluoride exposure did not alter Ca2+ homeostasis or increase the expression of ER stress-associated genes in HEK-293 cells. In enamel cells, fluoride exposure affected the functioning of the ER-localized Ca2+ channel IP3R and the activity of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) pump during Ca2+ refilling of the ER. Fluoride negatively affected mitochondrial respiration, elicited mitochondrial membrane depolarization, and disrupted mitochondrial morphology. Together, these data provide a potential mechanism underlying dental fluorosis.

Altered mitochondrial function, capacitative calcium entry and contractions in the aorta of hypertensive rats.

OBJECTIVE: It has been suggested that Ca entry through store-operated Ca channels (SOCs) is regulated by a dynamic interplay between the endoplasmic reticulum Ca stores and the mitochondria. These relationships drive the activation and inactivation of SOCs, yet it remains unclear whether this regulation of SOCs by mitochondria is altered in the aorta of spontaneously hypertensive rats (SHRs). METHODS: We performed a thorough study of the mitochondrial membrane potential, the ability of mitochondria to deal with cytosolic Ca, capacitative Ca entry (CCE), and stromal interaction molecule 1 (STIM1) and calcium release-activated calcium modulator 1 (orai1) protein expression, as well as the contractile capacity of aortic rings, in normotensive Wistar Kyoto rats (WKYs) and SHRs. RESULTS: Changes were observed in aortic tissue and cultured vascular smooth muscle cells isolated from SHRs relative to WKYs, including more depolarized mitochondria, stronger CCE upon the addition of Ca, larger cytosolic Ca transients (cytosolic Ca concentration) or aortic ring contraction elicited by endoplasmic reticulum depletion and a significant increase in STIM1 protein expression but not of orai1. CONCLUSION: These results suggest that the impaired Ca buffering capacity of partially depolarized mitochondria dysregulates CCE, leading to overfilling of the endoplasmic reticulum Ca store through enhanced STIM1/orai1 interactions and an increase in aorta contractions in SHRs. Thus, understanding the implications of the alterations to STIM1/orai1, and their relationship to mitochondria, may aid drug development and therapeutic strategies to treat hypertension, as well as its long-term sequelae in poorly controlled patients.