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First published online October 5, 2006
Journal of Experimental Biology 209, 4011-4023 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02480

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Hydrogen sulfide as an oxygen sensor/transducer in vertebrate hypoxic vasoconstriction and hypoxic vasodilation

Kenneth R. Olson^1,*, Ryan A. Dombkowski^1,2,, Michael J. Russell^1,, Meredith M. Doellman², Sally K. Head², Nathan L. Whitfield^1,2 and Jane A. Madden^3,4

¹ Indiana University School of Medicine-South Bend, 1234 Notre Dame Avenue, South Bend, IN 46617, USA
² Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA
³ Department of Neurology, The Medical College of Wisconsin, Milwaukee 53226, WI, USA
⁴ Research Service, Zablocki Veterans Affairs Medical Center, Milwaukee, WI 53295, USA

View larger version (15K): [in a new window]   Fig. 1. The response of isolated vessels to hypoxia (N2) or H2S (1 mmol l–1) is temporally and spatially similar, but vessel-specific. (A) Lamprey dorsal aorta (DA), (B) rat thoracic aorta (TA), (C) rat pulmonary artery (PA) pre-contracted with 10–6 mol l–1 norepinephrine (NE), (D) bovine pulmonary artery (PA) pre-contracted with 10–7 mol l–1 U-46619. A slight relaxation of bovine PA often precedes the hypoxic contraction; the H2S contraction appears mono-phasic, but it also is multi-phasic (see Fig. 2). Horizontal lines=10 min; vertical lines=0.5 g tension; W=wash. (1), (2) and (3) in C, indicate stages in the multiphasic response. (A) N2 trace adapted from (Olson et al., 2001), with permission; (C) H2S trace adapted from Dombkowski et al. (Dombkowski et al., 2005), with permission.

View larger version (12K): [in a new window]   Fig. 2. H2S has two dose-dependent effects on pre-contracted (10–7 mol l–1 U-46619) bovine pulmonary arteries. H2S appears to produce a dose-dependent relaxation between 10–8 and 10–5 mol l–1, whereas above 10–5 mol l–1 it produces a dose-dependent constriction. The EC50 for relaxation (5.5±1.8x10–7 mol l–1) is significantly (P0.05) different from the EC50 for contraction (3.7±1.5x10–4 mol l–1). (A) Single trace of cumulative doses; arrowheads indicate log[H2S] (in mol l–1) and time of addition of U-46619; W=wash. (B) Relaxation (filled circles); contraction (open squares; values extrapolated where curves overlap 10–5 mol l–1). Values are means ± s.e.m. of 8 vessels. Stippled rectangle indicates range of H2S reported in the rat plasma (see text for details).

View larger version (14K): [in a new window]   Fig. 3. The effects of hypoxia (N2) and H2S are mutually competitive. (A) Individually, N2 and H2S produce similar contraction of lamprey dorsal aorta (DA; normalized to 80 mmol l–1 KCl contraction=100%) while in the presence of N2, H2S (3x 10–4 mol l–1) relaxes and in the presence of H2S, N2 contractions are significantly (P0.05; N=8 vessels) reduced. (B) In norepinephrine (NE; 10–6 mol l–1) pre-contracted rat thoracic aortas initial exposure to hypoxia (N2; top trace) or H2S (3x10–4 mol l–1; bottom trace) produces a typical relaxation, whereas subsequent application of either H2S (top) or hypoxia (bottom) results in either a slight contraction or no response. (C) In U-46619 (10–6 mol l–1)-contracted bovine pulmonary arteries, 3x10–4 mol l–1 H2S relaxes a pre-existing N2 contraction and N2 relaxes a pre-existing H2S contraction. H2S is lost from continuously aerated baths in C after which normal hypoxic contractions are restored. Values are means ± s.e.m., N=8 vessels; horizontal and vertical scale bars in B and C = 10 min and 0.5 g, respectively.

View larger version (11K): [in a new window]   Fig. 4. H2S is produced by homogenates of bovine pulmonary artery and vein and lamprey dorsal aorta. H2S production by pulmonary arteries is inhibited by the CBS inhibitor, amino-oxyacetate (AOA; 1 mmol l–1), the pyridoxyl 5'-phosphate-dependent enzyme inhibitor, hydroxylamine (HA; 1 mmol l–1), but not by the CSE inhibitor D,L-propargylglycine (PPG; 10 mmol l–1); HA also inhibits H2S production in veins. Bovine vessels were pooled from two animals, lamprey aortas were pooled from six fish. Total sulfide (H2S and HS–) was measured in triplicate with ion-selective electrodes after alkaline conversion to S2–. Values are means ± s.e.m.; *significantly different from respective control (P0.05).

View larger version (26K): [in a new window]   Fig. 5. Constrictory and dilatory responses of isolated vessels to hypoxia (stippled bars) is partially or completely prevented by inhibition of H2S synthesis (black bars). (A) Hypoxic vasoconstriction in lamprey dorsal aorta (DA; N=8) is unaffected by three consecutive bouts of N2 exposure (left bars), whereas 1 mmol l–1 of the pyridoxyl 5'-phosphate-dependent enzyme inhibitor, hydroxylamine (HA), reduces the N2 response by over 80%. (B) Hypoxic vasodilation of norepinephrine (NE; 10–5 mol l–1) pre-contracted rat thoracic aorta (TA) is nearly completely blocked by PPG (N=6). (C) In NE pre-contracted rat pulmonary arteries (PA), both the hypoxic phase 1 contraction (1) and phase 2 relaxation (2) are partially inhibited by the CSE inhibitor, ß-cyanoalanine (BCA; 5 mmol l–1) or a combination of 1 mmol l–1 AOA, 10 mmol l–1 BCA and 10 mmol l–1 PPG (N=8 each group). (D) Hypoxic vasoconstriction in 10–7 µmol l–1 U-46619 pre-contracted (contraction not shown) bovine pulmonary arteries is unaffected by the CSE inhibitor D,L-propargylglycine (PPG; 10 mmol l–1), partly blocked by the CBS inhibitor amino-oxyacetate (AOA; 1 mmol l–1) and converted to slight relaxation by HA and a strong relaxation by a combination of the three inhibitors (N=6 each group). Values are means ± s.e.m.; *significantly different from respective control (P0.05).

View larger version (20K): [in a new window]   Fig. 6. Contribution of the H2S precursor, cysteine, to hypoxic responses. (A) Addition of cysteine to lamprey dorsal aorta (DA) significantly and specifically increases the magnitude of a hypoxic contraction. In the absence of exogenous cysteine, a hypoxic contraction (N2) develops as much force as a reference 80 mmol l–1 KCl contraction (broken line). Addition of cysteine (Cys; 1 mmol l–1) produces a slight, transient contraction, doubles the strength of the hypoxic contraction (N2+Cys; P0.05), but does not affect a second KCl contraction. Addition of glycine (Gly; 1 mmol l–1) also produced a slight contraction but did not affect either hypoxic (N2+GLY) or KCl (KCl+Gly) contractions. (B) Rat thoracic aortas (TA) were exposed to hypoxia for 15 h in the absence (Con) or presence of cysteine (Cys), returned to normoxia, pre-contracted with U-46619, and exposed twice to hypoxia. Incubation with cysteine significantly (P0.05) reduced the magnitude of the first hypoxic relaxation but enabled the vessels to respond to re-oxygenation and a second hypoxia. (C) Bovine pulmonary arteries (PA) were exposed to hypoxia for 15 h in the absence (Con) or presence of cysteine (Cys), returned to normoxia, pre-contracted with U-46619, and exposed twice to hypoxia. Incubation with cysteine increased the magnitude of the initial hypoxic contraction and the second hypoxic contraction was sustained longer.