Oxyhemoglobin stimulation of endothelin production in cultured endothelial cells

Abstract

Oxyhemoglobin and endothelin have both been linked to the development of the severe and sustained cerebral vasospasm associated with subarachnoid hemorrhage. The effects of oxyhemoglobin on endothelin biosynthesis in cultured endothelial cells were evaluated. Oxyhemoglobin (0.01 to 100 microM) produced concentration-dependent increases in immunoreactive endothelin levels in bovine pulmonary artery endothelial cell-conditioned medium. The median effective concentration for oxyhemoglobin-induced increases in immunoreactive endothelin levels was approximately 0.5 microM, and the maximum stimulation of immunoreactive endothelin levels was approximately 5.5-fold over basal conditions. In addition to directly stimulating basal production of immunoreactive endothelin, oxyhemoglobin significantly augmented immunoreactive endothelin production following platelet-mediated stimulation of endothelin production. An l-arginine analog inhibitor of nitric oxide synthase, L-NG-monomethyl arginine (L-NMMA, 200 microM), did not significantly affect basal immunoreactive endothelin levels. However, L-NMMA significantly augmented platelet-induced immunoreactive endothelin production. Methylene blue (10 microM), an inhibitor of soluble guanylate cyclase, did not significantly affect basal immunoreactive endothelin levels, nor did it significantly affect the platelet-mediated stimulation of immunoreactive endothelin production in cultured endothelial cells. The present results reveal that oxyhemoglobin can directly stimulate endothelin biosynthesis in cultured endothelial cells. This newly identified property of oxyhemoglobin suggests a potential mechanism for the sustained and severe cerebral vasospasm associated with subarachnoid hemorrhage.

Source and cause of endothelin-1 release to cerebrospinal fluid after subarachnoid hemorrhage

“…[9,24,40] On the contrary, we found that production of ET-1 by the cells was inhibited by exposure to oxyhemoglobin or methemoglobin. The discrepancy between our results and those of others [9,24,40] may be related to their use of a commercially available bovine double-crystallized hemoglobin (Sigma Chemical Co., St. Louis, MO), which is contaminated with hemin [27] and endotoxins [35] and which may be responsible for the increased production of ET-1 by the cells.…”

“…[9,11,14,19,24,33,37,40,[47][48][49] This controversial [5,19] hypothesis is supported by a delayed onset of long-lasting spasm of the intracranial vessels produced by intracisternal administration of ET-1, [3,47] by an increase in ET-1 levels in CSF after SAH in humans, [11,14,48,49] and by a decrease in the incidence of vasospasm after use of ET-1 receptor antagonists. [8,23,34,38,57] Astrocytes, neurons, and pituitary cells produce ET-1.…”

“…[8,23,34,38,57] Astrocytes, neurons, and pituitary cells produce ET-1. [10,17,26,29,56] However, ET-1 is also released by endothelial and smooth-muscle cells when they are stimulated by oxyhemoglobin [9,24,40] and by astrocytes when they are stimulated by thrombin. [10] Thus, both the source and the mechanism of ET-1 presence in the subarachnoid space after SAH appear to be unclear.…”

“…[31,46] One of the potential oxyhemoglobin-related mechanisms of vasospasm is the release of endothelin-1 (ET-1), [3,33,37] a potent endothelium-derived vasoconstricting agent, [21,55] into the cerebrospinal fluid (CSF) after SAH. [3,11,14,15,19,21,25,33,[47][48][49] Not only are astrocytes, neurons, and pituitary cells a normal source of extraluminal ET-1, [10,17,26,29,56] but endothelial [9,24,40] and smooth-muscle [24] cells also release ET-1 when stimulated by oxyhemoglobin [9,24,40] or thrombin. [10 ] Because an increase in the levels of ET-1 in CSF has been described in patients with cerebral vasospasm and delayed ischemic neurological deficits after SAH, [11,14,48,49] the production of ET-1 may also be induced by ischemia.…”

Molecular biological considerations in cerebral vasospasm following aneurysmal subarachnoid hemorrhage

“…Oxyhemoglobin lends itself to consideration as a pathogen in this context: as the major intracellular protein of the RBC it is liberated after lysis of this cell, and levels of oxyhemoglobin appear to peak in the cerebrospinal fluid (CSF) at 3 to 4 days. [4] Oxyhemoglobin has been demonstrated in vitro to activate the ET-1 gene and increase levels of ET-1 mRNA; [19,34,54] it is also eventually enzymatically depleted in the CSF. [60] [38,43] and to cytokines such as interleukin-1 as noted previously.…”