Artificial Cells, Blood Substitutes, and Biotechnology, 33: 2736, 2005 Copyright Q Taylor & Francis, Inc.

ISSN: 1073-1199 print/1532-4184 online DOI: 10.1081/BIO-200046643

Hemoglobin Mediated Contraction of the Isolated Rat Thoracic Aorta: Why is Precontraction Necessary?
H. W. Kim and A. G. Greenburg
Brown University and The Miriam Hospital, Providence, Rhode Island, USA

Abstract: A primary mechanism for the hemoglobin (Hb) mediated vascular contraction is believed to be Hb scavenging of endothelial nitric oxide (NO). In the isolated rat thoracic aorta, however, the Hb mediated contraction occurs only after an agonist-induced precontraction. Why? To investigate the question, a rat thoracic aortic ring model was used. Isometric vessel ring tension responses to selected pharmacologic contractile agonists were assessed and compared. The Hb mediated additional contraction occurred in vessel rings precontracted with adrenergic agonists as well as other types of contractile agonists. Even after agonist induced contraction, removal of the vascular endothelium or inhibition of endothelial NO synthase with Nx-nitro-L-arginine methyl ester prevented the Hb mediated additional contraction. Additionally, imposition of passive tension without an agonist pretreatment did not allow Hb mediated contraction. In conclusion, in the isolated rat thoracic aorta, the endothelial NO synthase is minimally active in the basal state but upregulated upon treatment with a contractile agonist. This may explain why the Hb mediated additional contraction occurs only after an agonist induced precontraction.

INTRODUCTION Intravenous administrations of cell-free hemoglobin (Hb) solution to animals and human subjects were shown to cause a transient hypertension [13]. This Hb mediated hypertension was typically characterized by increased systemic vascular resistance without an increase in cardiac output indicating that Hb infusion elicited vasoconstriction. A principal mechanism for the Hb mediated vascular contraction is believed to be Hb
Address correspondence to H. W. Kim, Brown University and The Miriam Hospital, 164 Summit Avenue, Providence, RI 02906, USA. Email: Hae_Kim@ Brown.edu 27

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scavenging of endogenous nitric oxide (NO), a potent vasodilator constitutively released by vascular endothelium [4]. However, in in-vitro experiments with isolated blood vessels of several different species, hemoglobin (Hb), an avid NO scavenger, elicited contraction only after precontraction with a contractile agonist [46]. Further, NO synthase (NOS) inhibitors had no effect on vessels at the basal state but caused further contraction in precontracted vessels [5]. Endothelial nitric oxide synthase (eNOS), an enzyme responsible for synthesis of NO from L-arginine, is generally considered to be constitutively expressed and functionally active in most mammalian blood vessels in the basal state. Why, then, is precontraction necessary for Hb to elicit vascular contraction? To help answer this question, we tested a hypothesis that, in isolated rat thoracic aorta, eNOS activity is quiescent or minimal in the basal state but upregulated upon agonist induced contraction.

MATERIALS AND METHODS Aortic Ring Preparation Male Sprague Dawley rats (250350 g body weight) were anesthetized with methoxy flurane (inhalation anesthetic). Through a midline incision, the heart and lungs were removed en bloc. After a wash in Krebs buffer (in mM, NaCl, 118; KCl, 4.8; CaCl2, 2.5; MgSO4, 1.2; KH2PO4, 1.2; NaHCO3, 24; glucose, 11; and disodium EDTA, 0.03; pH 7.4), the thoracic aorta was carefully excised avoiding endothelial damage and placed in a petri dish containing fresh buffer solution. Following removal of non-vascular tissues, the vessels were cut transversely with sharp surgical scissors into approximately 23 mm ring segments and kept in Krebs buffer until use. The vessel rings were mounted between two opposing stainless hooks, one hook secured to a tissue holder while the other connected to a tension transducer (Grass Model FT03) via a silk suture (30). The vessel ring preparation was placed in a 25 ml experimental tissue bath containing a Krebs buffer maintained at 37C. The buffer was continuously aerated with a 95% O2-5% CO2 gas mixture. The vessels were initially set at imposed tension of 1.52 grams and allowed to relax for 1 hour. Unless otherwise noted, the final vessel ring resting tensions were adjusted to 0.5 g prior to experimental procedure. Tension changes were continuously recorded on a polygraph (Grass Instruments, Quincy, MA). The responsiveness of vessel rings was first assessed by treating with 50 nM norepinephrine (NE); vessel rings that developed less than 0.5 g of contraction were considered nonviable and excluded. The integrity of the endothelium was, then, assessed by treating vessels with 1050 mM

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acetylcholine (Ach). If vessel rings relaxed less than 10% of pretreatment values, functional endothelium was considered absent.

Assay Hemoglobin Solution Highly purified stroma-free Hb reconstituted in a Ringers lactate solution (6.4 .2 gHb=dl) (Hemosol, Inc., Etobicoke, Canada) was used. The Hb solution was aliquoted in 1 ml vials and stored frozen 80C until use. On the day of experiment Hb solution was thawed and diluted as needed with the Krebs buffer before use. Total Hb content and percent of oxyHb and metHb were evaluated with a IL282 Co-Oximeter (Instrument Laboratories, Lexington, MA). Only Hb solution with less than 10% metHb content was used.

Drugs and Chemicals Commercial pharmaceutical preparations of norepinephrine (NE; levarterenol bitartarate, Winthrop Laboratories, New York, NY) and phenylephrine hydrochloride (PhE; Schein Pharmaceutical, Port Washington, NY) was used. Nx-nitro-L-arginine methyl ester (NAME) and other chemicals were purchased from Sigma Chemical Co.(Saint Louis, MO).

Inducible NOS (iNOS) Gene Expression Selected vessel rings were assayed for iNOS gene expression by reverse transcription-polymerase chain reaction (RT-PCR). Briefly, total RNA was prepared from 100300 mg tissue samples by homogenizationextraction, phase separation, and precipitation using TRI1 reagent (MRC, Cincinnati, OH), chloroform, and isopropyl alcohol, respectively. Two mg of purified total RNA from each organ was reverse transcribed into the first-strand cDNA using MuLV reverse transcriptase (Perkin Elmer, Foster City, CA) according to the manufacturers protocol. PCR was conducted with 20 mL RT product in a reaction tube containing 50 mL PCR buffer, 0.05 U=L AmpliTaq1 DNA polymerase (Perkin Elmer), 1.5 mM MgCl2, 125 mM NTPs, and 0.05 mM each of either iNOS (Biognostic; Gottingen, Germany) or glyeraldehyde-3-phosphatedehydrogenase (G3PDH, control gene) primers (Clonetech, Palo Alto, CA). The PCR was performed on a thermal cycler (MJ Research, Watertown, MA) using following thermal cycling protocol; an initial melting for 5 min at 95C, 35 cycles of 1 min at 95C, 2 min at 57C, and 3 min at 72C followed by a 7 minute of final extension. The final PCR products

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were analyzed on a 1.3% agarose gel premixed with ethidium bromide. The gel was imaged using a digital camera. Statistical Analysis Data are presented as mean 1 standard deviation (SD). Statistical significance was determined by Students t-tests at p 0.05 level. RESULTS AND DISCUSSION Role of Endothelial No Synthase in Hb Mediated Contraction Typically, in aortic rings with intact endothelium without NE induced precontraction, Hb dose as high as 4 mM did not elicit a notable contraction (Figure 1). In contrast, in vessel rings precontracted with 1050 nM NE, Hb as low as 0.2 mM elicited a notable additional contraction. In vessel rings denuded of the endothelium by a gentle intimal rubbing, Hb concentration 4 mM failed to elicit contraction even after NE induced precontraction. In these vessel rings, treatment with NAME had no effect. In the vessel rings with intact endothelium, pretreatment with 2 mM NAME prevented the Hb induced additional contraction. In endothelium intact vessel rings with approximately 0.5 g resting tension, 50 nM NE significantly increased vessel ring tension to 1.79 0.43 g (P < 0.01, N 6). In these vessel rings, 2 mM Hb elicited 0.7 0.34 g additional contraction (P < 0.05) (Figure 2). If vessel rings were pretreated with 2 mM NAME, the same dose of NE caused a slightly greater increase in vessel ring tension (2.07 0.78 g, P < 0.01, N 6) than values without NAME pretreatment. In these vessel rings, 2 mM Hb did not elicit significant additional contractions. In vessel rings devoid of functional endothelium, 50 nM NE still elicited a significant vessel tension increase to 1.52 0.55 g (P < 0.01, N 6). However, the Hb mediated additional contraction did not occur. Similar results were obtained when the vessel rings were treated with phenylephrine, an alpha specific adrenergic agonist, in lieu of NE. Passive Tension and Hb Mediated Additional Contraction In aortic rings with intact endothelium, increasing NE doses had a positive correlation with tension increases. For NE doses 5 nM-3 mM, vessel tension increased linearly with NE doses reaching a plateau at around 0.1 mM (Figure 3). In these vessel rings, 2 mM Hb elicited generally significant additional contractions than those with NE alone (P < 0.01, N 6

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Figure 1. Typical responses of rat aortic rings with or without the endothelium to norepinephrine (NE) and subsequent Hb treatment. In vessel rings with intact endothelium (E), 1050 nM NE produced a notable contraction. Subsequent treatment with 0.24 mM Hb elicited an additional contraction. In contrast, in vessel rings denuded of the endothelium ( E), Hb mediated additional contraction did not occur despite precontraction with NE. When vessel rings with intact endothelium were treated with 0.5 mM Nx-nitro-L-arginine methyl ester (NAME; a NOS inhibitor), 4 mM Hb had no effect.

each). In contrast, imposition of passive tension comparable to a level developed by 50 nM NE did not allow any notable additional contraction following treatment with 4 mM Hb (Figure 4). The magnitude of Hb mediated additional contraction in NE precontracted vessel rings was significantly greater than that of vessel rings with PT (P < 0.01, N 6). Inducible NOS Gene Expression and Hb Mediated Contraction The possibility that the Hb mediated additional contraction was due to increased level of NO release through activation of aortic smooth muscle iNOS was explored. In all the aortic samples tested before and after

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Figure 2. Hb mediated additional contraction in NE precontracted rat aortic rings with or without functional endothelium. In endothelium intact vessel rings pretreated with 50 nM NE, 2 mM Hb elicited a significant additional contraction (P < 0.05, N 6). In vessel rings without functional endothelium, 50 nM NE produced similar precontractions. However, 2 mM Hb did not elicit a significant additional contraction. (P > 0.05, N 6). When vessel rings with intact endothelium were pretreated with 2 mM NAME, the NE induced contraction responses were not altered but 2 mM Hb did not elicit significant additional contractions. (P > 0.05, N 6). The Hb mediated additional tension increases in endothelium intact vessel rings were significantly greater than those of vessels with denuded endothelium or pretreated with NAME (P < 0.01, N 6 each).

NE=Hb treatment, No notable band for iNOS RT-PCR product was detected (Figure 5). This result indicates that NO present in the vessel ring was not derived from iNOS activation.

Non-adrenergic Agonist Induced Contraction The Hb mediated additional contraction appears to occur not only with alpha adrenergic contraction but also with several other types of contractile agonists including prostaglandin F2a (arachidonic acid metabolite), potassium chloride (depolarizing agent), arginine vasopressor (vasoactive peptide), or serotonin (phenolic amine) [7]. For these agonists, removal of the endothelium or pretreatment of vessel rings with NAME had no significant effect on the contraction responses with these agonists but prevented the Hb mediated additional contractions.

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Figure 3. Aortic ring tension responses to 0.3 nM3 mM norepinephrine (NE) in the absence or presence of 2 mM Hb. At NE doses greater than 5 nM, vessel tension increased linearly with NE doses reaching a plateau at around 0.1 mM. At these NE doses, presence of 2 mM Hb allowed generally significantly greater contractions than NE alone ( P < 0.05).

Figure 4. Imposition of passive tension (PT) to a level produced by 50 nM NE did not allow any notable additional contraction following treatment with 4 mM Hb. The magnitude of Hb mediated additional contraction in NE precontracted vessel rings was significantly greater than that of vessel rings with PT (P < 0.01, N 6).

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Figure 5. RT-PCR assay results for iNOS gene expression in the rat thoracic aortic rings. Lane 1: Molecular weight marker (New England Bio Labs, 100 bp DNA ladder); Lanes 23: RT-PCR products from 2 normal rat aorta samples. Absence of a 278 bp band indicates lack of iNOS gene expression; Lanes 45: Rat iNOS cDNA band (278 bps; positive controls); Lanes 68: Glycerol aldehyde-3phosphate dehydrogenase (GAPDH) cDNA (983 bps; control gene expression).

Possible Mechanism for the Hb Mediated Additional Contraction The Hb mediated additional contraction was not limited to one specific type of contractile agonist. This suggests that the condition=consequence quence caused as a result of smooth muscle contraction may be an important factor for the Hb mediated contraction to occur rather than direct causal effect of a specific precontractile agonist. In addition, the Hb mediated additional contractions occurred only in the presence of intact endothelium and were inhibitable by NAME pretreatment. These results suggest that agonist induced aortic ring contraction may activate= upregulate endothelial NO release. How could vascular contraction augment endothelial NO release? Several possible mechanisms could be considered, including direct stimulation of eNOS by contractile agonists and NO release by peri-vascular adrenergic nerve endings [8]. However, this mechanism does not explain since the contraction coupled NO release also appears to occur with other agonists [7]. The contraction coupled endothelial NO release may be mediated through chemical messenger(s) released from the contracting vascular smooth muscle to activate eNOS. For example, during vascular contraction, a rise in sarcoplasmic Ca may trigger myoendothelial Ca conductance activating the Ca-calmodulin mediated eNOS pathway. The

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contraction coupled endothelial NO release observed did not appear to depend on the Ca-calmodulin pathway since it was not inhibited by calmidazolium, a calmodulin antagonist. Calmidazolium inhibits acetylcholine mediated vascular relaxation, a Ca-calmodulin dependent process [9]. Interestingly, a Ca-calmodulin pathway is reported not involved in flow=shear stress or isometric tension induced endothelial NO release [9,10]. Blocking intercellular gap junction channels was shown to impair the Hb mediated contraction suggesting that gap junction channels may play a role in contraction coupled endothelial NO release [7]. The gap junction channels have been reported to serve as conduits for Ca and other intercellular messenger trafficking [1114]. Interestingly, the agonist induced contraction coupled NO release did not involve eNOS phophorylation since a nonspecific PK inhibitor staurosporine or a tyrosine specific kinase inhibitor did not alter the Hb mediated additional contractions in norepinephrine precontracted rat aortic vessel rings [7]. Because flow induced endothelial NO release is reported to occur through eNOS phosphorylation [9,10], the contraction coupled NO release may occur through a yet unknown pathway. Alternatively, the contraction coupled NO release may be mediated through mechanical strain exerted on the endothelial cytoskeletal elements during smooth muscle contraction. Deformation of endothelial cytoskeletal elements such as F-actin filaments, microtubules, and other elements has been shown to induce endothelial NO release [1215]. In isolated rat thoracic aorta precontracted with various contractile agonists, Hb elicits additional contractions. The Hb mediated additional contraction requires the presence of a functional endothelium and is inhibited by NOS inhibitor. These results indicate that agonist induced vascular contraction elicits endothelial NO release. This contraction induced NO release does not appears to depend on Ca-calmodulin or protein kinase dependent pathways. In rat thoracic aorta and, perhaps in other vessels, the endothelial NOS may be minimally active in the basal state but upregulated upon agonist induced contraction. This may be why, in isolated rat thoracic aorta, Hb mediated additional contraction requires agonist induced precontraction. REFERENCES
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