Relative Roles of Heme-Irons and Globin-Thiols in the Genesis of Acellular Hemoglobin Mediated Vasoconstriction

Figures & data

Figure 1. Vascular responses to native and cysteines masked Hbs. a) In rat thoracic aortic rings precontracted with 100 nM NE, treatment with 33 μM Ach produced a signifcant relaxation, indicating presence of intact functional endothelium. In these vessel rings, subsequent treatment with 0.4 μM oxyhemoglobin (Fe⁺²) elicited a signifcant contraction. A representative tracing is shown. b) In similarly treated rat aortic rings, treatment with 0.4 μM NEM-HbO₂ (oxyhemoglobin with (βCys93 masked by NEM treatment) also caused a signifcant contraction. c) In these vessel rings, both HbO2 and NEM-HbO2 produced comparable contractions (58.1 ±7.0% and 50.7±9.5% of baseline values, respectively). N=7-8 vessels each.

^**significantly different from respective pretreatment value (P<0.01)

NS: not signifcantly different between the two groups (P>0.05)

Figure 2. Vascular responses to Hb⁺CN, an Hb with ferric heme-irons, and NEM- Hb⁺CN, an Hb with both ferric heme-irons and masked (βCys93 cysteines. a) In rat thoracic aortic rings precontracted with 100 nM NE, treatment with 33 μM Ach produced signifcant relaxation indicating presence of intact functional endothelium. In these vessel rings, treatment with 0.4 μM Hb⁺CN did not cause any signifcant contraction. But subsequent treatment with 0.4 μM HbO₂ (Fe⁺²) elicited a signifcant contraction. A representative tracing is shown. b) In similarly treated rat aortic rings, treatment with 0.4 μM NEM- Hb⁺CN did not cause a signifcant contraction, either. But subsequent treatment with 0.4 μM HbO₂ again elicited a signifcant contraction. c) and d) In these vessel rings, neither Hb⁺CN nor NEM- Hb⁺CN produced signifcant contractions (− 3.7±4.6% and 12.0±4.5% of baseline values, respectively). N=7 vessels each.

^**signifcantly different from respective pretreatment value (P<0.01)

NS: not signifcantly different from respective pretreatment value (P>0.05)

Figure 3. Vascular responses to HbNO, an Hb with nitrosylated heme-irons, and NEM-HbNO, an Hb with both nitrosylated heme-irons and masked (βCys93 cysteines. a) In rat thoracic aortic rings precontracted with 100 nM NE, treatment with 33 μM Ach produced signifcant relaxation, indicating presence of intact functional endothelium (tracings not shown). In these vessel rings, treatment with 2 μM HbNO did not cause any signifcant contraction. But subsequent treatment with 0.4 μM HbO₂ elicited a signifcant contraction. A representative tracing is shown. b) In similarly treated rat aortic rings, treatment with 0.4 μM NEM-HbNO, Hb with both nitrosylated hem-iron and masked cysteines, did not cause a signifcant contraction, either. But, again, subsequent treatment with 0.4 μM HbO₂ elicited a signifcant contraction. A representative tracing is shown. c) and d) In these vessel rings, both HbNO and NEM-HbNO produced no signifcant contractions (—5.5 ±6.0% and 3.2± 1.7% of baseline values, respectively). N=7 and 3 vessels each, respectively.

Figure 4. Vascular responses to ferrous (Fe⁺²) and ferric (Fe⁺³) sperm whale myoglobins. a) In rat thoracic aortic rings precontracted with 50 nM NE, treatment with 2 μM ferric sperm whale myoglobin (metMb) did not cause any signifcant contraction. But subsequent treatment with 2 μM oxymyoglobin (MbO₂) elicited a signifcant contraction. A representative tracing is shown. b) In these vessel rings, 2 μM MbO₂ and metMb produced vessel tensions of 55 ±13.2% and —0.8±3.1% of baseline values, respectively). N=10 and 6 vessels each, respectively.

Figure 5. Vascular responses to ferrous (Fe⁺²) and ferric (Fe⁺³) equine heart cytochrome-C. a) In rat thoracic aortic rings precontracted with 50 nM NE, treatment with 2 μM equine heart ferrous or ferric cytochrome C (Cyt-C) did not cause any signifcant contraction. But subsequent treatment with 2 μM oxyhemoglobin (Hb O₂) or oxymyoglobin (MbO₂) elicited a signifcant contraction (tracings not shown). b) In these vessel rings, neither 2 μM ferrous Cyt-C nor 2 μM ferric Cyt-C produced signifcant contractions (− 6.8 ±4.3% and — 3.0±3.2% of baseline values, respectively). N = 10 and 6 vessels each, respectively.