PMC

Distribution of Ca²⁺ flashes during and following EFS (10 Hz, 1 s). Ca²⁺ flashes typically show two episodes of activity, one during EFS, and the second peaking between 2–8 s following EFS (A, control). Inhibition of purinergic receptors (α,β-meATP–suramin, 10 μm each) nearly abolishes Ca²⁺ flashes during EFS, but prolongs the second episode of Ca²⁺ flashes following EFS. Ca²⁺ flashes were obtained from paired experiments (4 preparations) in control and after inhibition of P2X1Rs (A). Similarly, when P2X1^−/− were compared to WT, Ca²⁺ flashes during EFS were absent; however, the Ca²⁺ flashes following EFS were greatly increased (B). Ca²⁺ flashes were summed from 5 preparations (wild type) and 6 preparations (P2X1^−/−).

(A) Isolated mitochondria labeled with MitoSOX red. (B) Time-lapse images and (C) typical trace of superoxide flashes visualized by MitoSOX red. Different types of traces of superoxide flashes (D) low variation slope traces, (E) high variation slope traces, (F) multi-event traces. (G) Comparison of superoxide flashes frequency in mitochondria isolated from petal and style of M. denudata. (H) Comparison of superoxide flashes frequency in mitochondria isolated from petal and receptacle of N. nucifera. Scale bar: 5 µm.

BK promotes superoxide flash generation in YAC128 MEFs. A, representative mt-cpYFP images showing superoxide flashes in mitochondria of WT and YAC128 MEFs. Arrowheads depict individual mitochondria with superoxide flashes. Insets are enlarged views showing mitochondria undergoing superoxide flashes. B, representative mitochondrial superoxide flash traces (mt-cpYFP fluorescence with excitation at 488 nm, ΔF/F₀) in WT and YAC128 MEFs without or with 2.5 μm BK stimulation. C and D, statistical analysis of superoxide flashes in WT and YAC128 MEFs in response to 2.5 μm BK. C, in resting cells, the amplitude of spontaneous mitochondrial superoxide flashes in YAC128 (n = 37) MEFs was significantly higher than in WT MEFs (n = 21) (**, p < 0.01). In response to BK, both WT (n = 21) and YAC128 MEFs (n = 17) showed significantly higher amplitudes of superoxide flashes when compared with spontaneous flashes in resting MEFs (**, p < 0.01), and BK caused significantly higher superoxide flashes in YAC128 MEFs (n = 17) than in WT (n = 21) MEFs (**, p < 0.01). Flashes occurring 8 min before BK application and 8 min after BK application were averaged for superoxide flash amplitude analysis. D, in resting cells, spontaneous mitochondrial superoxide flashes occurred at a similar frequency in WT and YAC128 MEFs (p > 0.05). In response to BK, superoxide flashes in YAC128 MEFs occurred at a significantly higher frequency at 2–4 min after BK treatment when compared with resting cells (**, p < 0.01) and with WT MEFs (**, p < 0.01). Data are shown as the mean ± S.E.

Adjusted means in average IMT by hot flash frequency among women in daily hot flash group.
Quartile (Q) 1: ≤ 4 physiologic hot flashes, ≤ 1 self-reported hot flashes; Q2: 5–9 physiologic hot flashes, 2–3 self-reported hot flashes; Q3: 10–14 physiologic hot flashes, 4–5 self-reported hot flashes; Q4: ≥ 15 physiologic hot flashes, ≥ 6 self-reported hot flashes; waking hot flashes Adjusted for age, race, body mass index, education, high density lipoprotein cholesterol, low density lipoprotein cholesterol, triglycerides, systolic blood pressure, diastolic blood pressure, homeostatic model assessment, blood pressure-lowering medications, lipid-lowering medications, diabetes medications
IMT = intima media thickness
*p<.05; **p<.01; ***p<.001 relative to Q1