Angiotensin II Disrupts Neurovascular Coupling by Potentiating Calcium Increases in Astrocytic Endfeet

Figures

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  Figure 1. Ang II attenuates CBF responses to whisker stimulation and mGluR activation in the somatosensory cortex. A, Thirty‐minute perfusion with Ang II (50 nmol/L) attenuates CBF increases in response to whisker stimulations (n=5–6) and to the mGluR agonist, t‐ACPD (5 minutes, 25 µmol/L; n=4–6). B, Traces of averaged resting CBF acquired before and during Ang II (50 nmol/L) superfusion. C, Traces of averaged CBF responses induced by whisker stimulation (left panel) or t‐ACPD (right panel) superfusion in the presence or absence of Ang II were acquired at 1 Hz using laser Doppler flowmetry. SD is represented by the lighter tone shade surrounding each curve. (**P<0.01; 2‐way ANOVA followed by Bonferroni correction). Ang II indicates angiotensin II; CBF, cerebral blood flow; mGluR, metabotropic glutamate receptor; SD, standard deviation; and t‐ACPD, 1S, 3R‐1‐aminocyclopentane‐trans‐1,3‐dicarboxylic acid1S.

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  Figure 2. Ang II promotes constriction over dilation of the somatosensory cortex parenchymal arteries in response to t‐ACPD in acute brain slices. A, Differences expressed in percent change between the vascular responses to t‐ACPD (50 µmol/L) before (resting) and after 20 minutes of incubation with the vehicle (artificial cerebrospinal fluid), Ang II (100 nmol/L), or Ang II in the presence of the AT1 antagonist, candesartan (10 µmol/L). Candesartan was added 5 minutes before Ang II. B, Representative pictures of resting vascular state and maximum vascular response to t‐ACPD after 20 minutes of incubation with the vehicle or Ang II. Images are obtained from infrared differential interference contrast infrared differential interference contrast imaging. The lumen of parenchymal arteries is outlined by red lines. The diameter was calculated from the average of 20 successive images at resting state and maximum vascular response to t‐ACPD (scale bar=20 µm). C, Time‐course traces of luminal diameter changes in response to t‐ACPD after 20 minutes of incubation with the vehicle (black line) or Ang II (red line). Vasodilatation to t‐ACPD in the presence of the vehicle is converted into…

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  Figure 3. Ang II amplifies Ca2+ increases in astrocytic endfeet in response to t‐ACPD in acute brain slices. A, Ang II (100 nmol/L) significantly increases the amplitude of astrocytic endfeet Ca2+ response to t‐ACPD (50 µmol/L), measured as fractional fluorescence (F1/F0). B, Representative images showing astrocytic endfoot Ca2+ increases in response to t‐ACPD before and after 20 minutes of incubation with Ang II or its vehicle. [Ca2+] i in astrocytic endfeet surrounding a parenchymal arteriole in brain slice is pseudocolor‐mapped (based on fluo‐4 fluorescence) (Pseudocolors legend unit corresponds to nmol/L of Ca2+; scale bar=10 µm). The white arrows show Ca2+ spots in analyzed astrocytic endfeet. The lumen of the artery is outlined by white lines. (**P<0.01; 2‐tailed unpaired t test; n=9–10). Ang II indicates angiotensin II; and t‐ACPD, 1S, 3R‐1‐aminocyclopentane‐trans‐1,3‐dicarboxylic acid.

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  Figure 4. In acute brain slices, Ang II increases resting [Ca2+] i and t‐ACPD‐induced Ca2+ rises in astrocytic endfeet. A, Estimated [Ca2+] i from the fluo‐4 signal and calculated using Maravall’s formula at resting state and in response to t‐ACPD (50 µmol/L) in astrocytic endfeet incubated with the vehicle, Ang II (100 nmol/L), or Ang II+candesartan (Can, 10 µmol/L). Can was added 5 minutes before Ang II incubation (n=4–5). B, Average of the estimated Ca2+ levels of all experiments for each time point in response to t‐ACPD, suggesting a potentiated response in the Ang II group as compared with the vehicle and the Ang II+Can groups. SD is shown by the lighter tone shade surrounding each curve. C, AUC of Ca2+ increases in response to t‐ACPD after 20 minutes of incubation with vehicle, Ang II, or Ang II+Can (n=4–5). D, The CV in percentage of the resting spontaneous Ca2+ oscillations in the presence of the vehicle or Ang II in cortical astrocytes (n=4). E, Traces of averaged resting [Ca2+] i acquired in the presence of the vehicle or Ang II in cortical astrocytes. Shaded areas represent SD (*P<0.05, **P<0.01, ***P<0.001; 1‐way ANOVA followed by Bonferroni correction for multiple comparisons…

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  Figure 5. Ang II does not modulate the vascular response to Ca2+ increases controlled by photolysis or Ca2+ chelation in acute brain slices. A, Example of simultaneous recording of changes in arteriolar diameter (upper panels) and astrocytic endfoot Ca2+ increases (lower panels) before (resting) and after 2‐photon Ca2+ uncaging (excitation volume <3 μm3) for ≈0.5 s in acute brain slices incubated with Ang II (100 nmol/L) or its vehicle. Upper panels: Images of parenchymal arteries obtained from infrared differential interference contrast imaging. Lower panels: Pseudocolor‐mapped [Ca2+] i (based on fluo‐4 fluorescence) representing [Ca2+] i in astrocytic endfeet surrounding a parenchymal arteriole in acute brain slice (Pseudocolors legend unit corresponds to nmol/L of Ca2+; scale bar=10 µm). Dashed white lines in the upper panels and arrows in the lower panels show an astrocyte endfoot abutting a parenchymal arteriole in acute brain slice loaded with the caged Ca2+, DMNP‐EDTA (10 μmol/L, 1 h). The lumen of parenchymal arteries is outlined by red lines in the upper panels and white lines in the lower panels. B, Time course traces of changes in endfoot Ca2+ (red) and arteriole diameter…

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  Figure 6. IP3Rs and TRPV4 channels mediate Ang II action on astrocytic endfoot Ca2+ levels in acute brain slices. A, Astrocytic endfeet Ca2+ increases in response to t‐ACPD, measured as F1/F0 in brain slices perfused with vehicle or in the presence of the sarcoplasmic reticulum (SR)/ER Ca2+ ATPase (SERCA) inhibitor, CPA (30 µmol/L) or the partial IP3Rs inhibitor, XC (10 µmol/L; n=5–6). B, Astrocytic endfeet Ca2+ increases in response to t‐ACPD, measured as F1/F0 in brain slices perfused with Ang II (100 nmol/L) alone or in the presence of CPA 30 µmol/L or XC 10 µmol/L (n=4–6). C, Estimated [Ca2+] i at resting state and in response to t‐ACPD in astrocytic endfeet with the vehicle or HC (10 µmol/L; n=4–5). D, Estimated [Ca2+] i at resting state and in response to t‐ACPD in astrocytic endfeet in the presence of Ang II (50 nmol/L) or with HC 10 µmol/L (n=5–8) in different groups of brain slices. (*P<0.05, **P<0.01; A through B, 1‐way ANOVA followed by a Bonferroni correction for multiple comparisons; D, 2‐way ANOVA followed by Bonferroni correction for multiple comparisons). Ang II indicates angiotensin II; CPA, cyclopiazonic acid; HC, HC067047; IP3Rs, inositol 1,4,5‐trisphosphate receptor;…