Hypoxia inhibits contraction but not calcium channel currents or changes in intracellular calcium in arteriolar muscle cells

Abstract

Objective: We tested the hypothesis that hypoxia inhibits currents through L-type Ca(2+) channels and inhibits norepinephrine-induced rises in intracellular Ca(2+) in cremasteric arteriolar muscle cells, thus accounting for the inhibitory effect of hypoxia on norepinephrine-induced contraction of these cells.

Methods: Single smooth muscle cells were enzymatically isolated from second-order and third-order arterioles from hamster cremaster muscles. The effects of hypoxia (partial pressure of oxygen: 10-15 mm Hg) were examined on Ba(2+) (10 mM) currents through L-type Ca(2+) channels by use of the perforated patch clamp technique. Also, the effect of hypoxia on norepinephrine-induced calcium changes was studied using Fura 2 microfluorimetry.

Results: Hypoxia inhibited the norepinephrine-induced (10 microM) contraction of single arteriolar muscle cells by 32.9 +/- 5.6% (mean +/- SE, n = 4). However, hypoxia had no significant effect on whole-cell currents through L-type Ca(2+) channels: the peak current densities measured at +20 mV were -3.83 +/- 0.40 pA/pF before hypoxia and -3.97 +/- 0.36 pA/pF during hypoxia (n = 15; p > 0.05). In addition, hypoxia did not inhibit Ca(2+) transients in arteriolar muscle cells elicited by 10 microM norepinephrine. Instead, hypoxia increased basal Ca(2+) (13.8 +/- 3.2%) and augmented peak Ca(2+) levels (29.4 +/- 7.3%) and steady-state Ca(2+) levels (15.2 +/- 5.4%) elicited by 10 microM norepinephrine (n = 21; p < 0.05).

Conclusions: These data indicate that hypoxia inhibits norepinephrine-induced contraction of single cremasteric arteriolar muscle cells by a mechanism that involves neither L-type Ca(2+) channels nor norepinephrine-induced Ca(2+) mobilization. Instead, our findings suggest that hypoxia must inhibit norepinephrine-induced contraction by affecting a component of the signaling pathway that lies downstream from the increases in Ca(2+) produced by this neurotransmitter.

Figure 1

Norepinephrine-induced contraction of arteriolar muscle cells is inhibited by hypoxia. The data are given as the mean ± SE. The fraction of cells, expressed as a percentage of the control fraction, that contracted in response to micropipette-applied norepinephrine (10 μM; n = 4). * denotes significant difference from the control value (p < 0.05).

Figure 2

Nifedipine inhibits Ca_L currents in arteriolar muscle cells. The data are given as the mean ± SE. (A) The mean current-value (I-V) relationship (n = 8) for Ca_L channel currents measured at the peak of test pulses in the absence (○), presence (•), and after washout of 1 μM nifedipine (⋄). (B) Raw current traces (top) from a single smooth muscle cell in the presence and absence of nifedipine during a depolarization from −70 mV to +20 mV (bottom).

Figure 3

Hypoxia does not inhibit currents through Ca_L channels in arteriolar muscle cells. All data are given as the mean ± SE. (A) Mean current–voltage relationship (n = 15) of Ca_L channel currents during the activation protocol before (○;) and during hypoxia (•). (B) Normalized inactivation (n = 8) of Ca_L in arteriolar smooth muscle cells before (○) and during hypoxia (•).

Figure 4

Norepinephrine-induced Ca²⁺ transient in an arteriolar muscle cell, as indicated by a representative Fura 2 ratio trace. The Fura 2 ratio changes in response to 60 seconds of norepinephrine were characterized by a pre-norepinephrine baseline, a peak on norepinephrine application, followed by a maintained plateau until norepinephrine removal.

Figure 5

Hypoxia does not inhibit norepinephrine-induced Ca²⁺ in arteriolar muscle cells. All data are given as the mean ± SE. Summary (21 cells) of the baseline, peak, and plateau Fura 2 ratios from arteriolar muscle cells in response to norepinephrine before (white bars), during (black bars), and after recovery (gray bars) from hypoxia. * denotes a significant difference from control value (p < 0.05).