Prevention of a hypoxic Ca(2+)(i) response by SERCA inhibitors in cerebral arterioles

Abstract

1. The aim of the study was to investigate the mechanism of a novel effect of hypoxia on intracellular Ca(2+) signalling in rabbit cerebral arteriolar smooth muscle cells, an effect that was resistant to the L-type Ca(2+) channel antagonist methoxyverapamil (D600). 2.[Ca(2+)](i) of smooth muscle cells in intact arteriolar fragments was measured using the Ca(2+)-indicator dye fura-PE3. Hypoxia (PO(2) 10 - 20 mmHg) lowered basal [Ca(2+)](i) but did not inhibit Ca(2+) entry pathways measured by Mn(2+)-quenching of fura-PE3. 3. The effect of hypoxia was completely prevented by thapsigargin or cyclopiazonic acid, selective inhibitors of sarcoplasmic reticulum Ca(2+) ATPase (SERCA). Since these inhibitors do not block Ca(2+) extrusion or uptake via the plasma membrane, the data indicate that the effect of hypoxia depends on a functional sarcoplasmic reticulum. 4. Because actions of nitric oxide (NO) on vascular smooth muscle are also prevented by SERCA inhibitors it was explored whether the effect of hypoxia occurred via modulation of endogenous NO release. Residual NOS-I and NOS-III were detected by immunostaining, and there were NO-dependent effects of NOS inhibitors on Ca(2+)(i)-signalling. Nevertheless, inhibition of endogenous NO production did not prevent the effect of hypoxia on [Ca(2+)](i). 5. The experiments reveal a novel nitric oxide-independent effect of hypoxia that is prevented by SERCA inhibitors.

Figure 1

Hypoxia lowers [Ca²⁺]_i in rabbit pial arteriole smooth muscle cells. (A) Fura-PE3 loading of an enzymatically isolated pial arteriole fragment. The dye was excited by 380 nm light and emission collected at 510 nm. The superimposed white circles are example ‘regions of interest' (ROIs) used for data collection from smooth muscle cells. The scale bar is 10 μm. (B) Carbon fibre O₂ (upper trace) and smooth muscle fura-PE3 Ca²⁺ (lower trace) signals measured simultaneously. Ca²⁺ concentration is represented as the fura-PE3 ratio expressed as a percentage relative to the zero level when bath 1.5 mM Ca²⁺ was replaced with 0.4 mM EGTA (zero-Ca²⁺). Bubbling the bath solution with nitrogen (N₂) lowered PO₂ from 150 to 25 mmHg and reduced the intracellular Ca²⁺ concentration by about 30%. The break in the trace is 8.3 min. (C) Mean±s.e.mean (n=7) fura-PE3 data for experiments as described in (B). The mean minimum PO₂ while bubbling with N₂ was 19.8±2.2 mmHg.

Figure 2

Hypoxia does not inhibit Ca²⁺-influx mechanisms. (A) Effect of 0.5 mM Mn²⁺ (Ca²⁺- and EGTA-free bath solution) on fura-PE3 excited at 360 nm in normoxic bath solution. Fura-PE3 fluorescence is given in arbitrary units. (B) As for (A) except hypoxia was achieved prior to application of Mn²⁺. (C) Mean±s.e.mean data for experiments of the type described in (A) and (B) (n=6 normoxia, and n=5 hypoxia). Fura-PE3 signals were normalized and background (pre-Mn²⁺) quenching rates were subtracted.

Figure 3

SERCA inhibitors prevent the effect of hypoxia. (A) The arteriole was pretreated with thapsigargin (TG, 1 μM) and then exposed to Ca²⁺-free (0.4 mM EGTA) solution before 1.5 mM Ca²⁺ was reintroduced to the bath. Hypoxia (9.5 mmHg) did not affect [Ca²⁺]_i. (B) Mean±s.e.mean hypoxia-induced change (reduction) in fura-PE3 ratio expressed as a percentage relative to the response to Ca²⁺-free bath solution (see Figure 1). Control arterioles were compared with arterioles pretreated with 1 μM TG or 10 μM cyclopiazonic acid (CPA) during the 1-h loading protocol with fura-PE3. The n values for the data sets are given in parentheses. CPA was also present during recordings. **P<0.01. The minimum mean±s.e.mean PO₂ values for TG and CPA groups were 18.4±2.5 and 19.8±1.5 mmHg, compared with 19.8±2.2 mmHg in the controls (n=7). (C) As for Figure 2C except arterioles were pretreated for 1 h with 1 μM TG (n=7 normoxia, and n=6 hypoxia).

Figure 4

Detection of NOS-I and NOS-III in isolated rabbit pial arteriole fragments and smooth muscle cells. Each fluorescence image is overlaid on the bright field image (blue). (A) Isolated arteriolar smooth muscle cells double labelled with anti-α-SMA-Cy3 (red) and anti-NOS-I (green). (B) Isolated arteriolar smooth muscle cells double labelled with anti-α-SMA-Cy3 (red) and anti-NOS-III (green). (C) A long arteriolar fragment labelled with anti-NOS-I (green). (D) A long arteriolar fragment double labelled with anti-VIP (red) and anti-NOS-I (green). Yellow indicates co-localization of VIP and NOS-I. (E) A short arteriolar fragment labelled for NOS-I (green). (F) Partially fragmented long arteriole double labelled with anti-α-SMA-Cy3 (red) and anti-NOS-III (green). The scale bar in (A) is 10 μm and applies to all panels.

Figure 5

Endogenous NO regulates [Ca²⁺]_i but does not play a role in the action of hypoxia. In each panel, Ca²⁺_i signals are expressed as changes in fura-PE3 ratio, mean±s.e.mean values are given, and n values are indicated in parentheses next to the error bars. NOS inhibitors or NO donors were also included during the 1-h fura-PE3 loading protocol. (A) Ca²⁺_i signal observed on changing from Ca²⁺-free to Ca²⁺-containing bath solution compared alternately for control conditions (open) and in the presence of 0.3 mM L-NAME (black). (B) Ca²⁺_i signal observed on changing from Ca²⁺-free to Ca²⁺-containing bath solution and compared alternately for two conditions: On the left for 0.3 mM L-NAME only (black) compared with 0.3 mM L-NAME plus 1 mM L-arginine (single hatch). On the right for 0.3 mM L-NAME only (black) compared with 0.3 mM L-NAME plus 0.3 mM DEA/NONOate (double hatched). (C) Ca²⁺_i signal observed on changing from Ca²⁺-free to Ca²⁺-containing bath solution compared alternately for control conditions (open) and in the presence of 1 μM s-methyl-L-thiocitrulline (black). (D) In the presence of Ca²⁺_o, the effect of hypoxia (minimum PO₂ 9.3 mmHg) on [Ca²⁺]_i in the presence of 0.3 mM L-NAME in the bath.