Keywords

Sodium hypochlorite (NaOCl); Aortic ring; [Ca²⁺]i; Vascular smooth muscle; Signal transduction pathways; Aging

Introduction

Hypochlorite (OCl^-) is one of the most aggressive oxidants of reactive oxygen species which can be produced by the myeloperoxidase (MPO)-H₂O₂-chloride system. During phagocytosis, neutrophils undergo a burst of respiration in which oxygen is reduced to superoxide anion (O₂⁻), which dismutates to form H₂O₂. MPO is discharged from the cytoplasmic granules into the phagosome following particle ingestion. It is thought to utilize H₂O₂ to oxidize halides, which then react with and kill ingested microbes. The initial product of the MPO-H₂O₂-chloride system is hypochlorous acid, and the subsequent formation of chlorine, chloramines, hydroxyl radicals, singlet oxygen, and ozone have been proposed [1-4].

These same toxic agents can be released to the outside of cells, where they may attack normal tissues and cells, and thus contribute to the pathogenesis of disease [2,5]. It is known that hypochlorite plays important roles in both normal and pathological conditions. Reactive oxygen species are continuously generated in vivo, but an excessive level of these species is considered to be responsible for several pathophysiological processes, partly by affecting vascular tone and vessel wall interactions with leukocytes [6]. Under pathological conditions, hypochlorite may be formed at relatively high concentrations, and it has been documented that myeloperoxidase is a component of human atherosclerotic lesions [1]. The MPO-H₂O₂ system can cause chlorination of phospholipids [4].

A large body of studies on blood vessels has shown that reactive oxygen species can modulate vascular tone directly or indirectly leading to vasodilation or vasoconstriction [7-13]. Individual reactive oxygen species may exert different and sometimes complex actions on the tone of blood vessels via different mechanisms [8,10,12,14]. It has been suggested that hypochlorite can produce both relaxant and contractile actions in sheep isolated pulmonary artery rings, which may be dependent upon concentration [15]. However, as for the contractile action of hypochlorite, the available information is relatively scarce and imprecise.

Up to now, the exact mechanism(s) by which NaOCl exerts its vascular effects still remain to be elucidated. It is possible that the NaOCl-induced vascular actions are mediated via second messenger systems. Previous studies from our lab have shown that protein kinase C, mitogen-activated protein kinase, and nonreceptor tyrosine kinases play essential roles in H₂O₂^-, hydroxyl radical- and peroxynitrite-triggered signal transduction pathways in vascular smooth muscle cells [8,9,12,16,17]. It is, however, unclear whether NaOCl acts on vascular tone via similar or different mechanisms. Therefore, the current study was undertaken to investigate the effects of NaOCl on isolated ring segments of rat aorta and to gain insight into the underlying mechanisms of its action, with special attention given to potential signal transduction mediators generated by NaOCl.

Materials and Methods

This is a descriptive study with retrospective data analysis.

General procedures

Male adult Wistar rats (200 g-470 g) were sacrificed by stunning and subsequent decapitation. The aortas were isolated according to previously established methods. The vessels for the ring segments were carefully excised, cleaned, and the tissues cut into 3- to 4 mm segments. For intact tissue preparation, extreme care was taken to avoid damage of endothelial cells. For denuded arteries, the intima of the vessels were removed by gently rubbing against the teeth of a pair of forceps [18]. The tissues were placed in normal Krebs- Ringer bicarbonate (KR buffer) solution containing (in mM): NaCl 118, KCl 4.7, KH₂PO₄ 1.2, MgSO₄ 1.2, CaCl₂ 2.5, dextrose 10 and NaHCO₃ 25 [19,20]. The segments were mounted on stainless-steel pins under 2 g resting isometric tension in organ baths, attached to force transducers (Grass Model FT 03) and connected to Grass Model 7 polygraphs. The organ baths, containing KR buffer, were gassed continuously with 95% O₂ and 5% CO₂ and warmed to 37°C (pH 7.4). Tissues were allowed to equilibrate for at least 90 min before data collection. Incubation media were routinely changed every 15 min as a precaution against interfering metabolites [21]. At the beginning of an experiment, rings were exposed for 30-45 min to 80 mM KCl and this was repeated every 30-45 min, until responses were stable (two to three times). Successful removal of endothelium was assessed by showing that acetylcholine (10^-8 to 10^-6 M) failed to relax the denuded segments. Segments precontracted by 5×10^-7 M phenylephrine did relax 90% in response to acetylcholine in endothelium-intact segments [22,23]. Responses to NaOCl and other drugs were expressed as a percentage of the stable level of contraction induced by 80 mM KCl. All of the experiments and protocols in this study were approved by the Animal Use and Care Committee at our institution.

Extracellular calcium (Ca²⁺) and intracellular Ca²⁺ experiments

For the extracellular Ca²⁺-free experiments, the rat aortic ring segments were equilibrated in Ca²⁺-free KR buffer containing 0.2 mM EGTA for at least 90 min before initiation of the experiments [24]. For intracellular Ca²⁺-buffered experiments, the acetyl methyl ester of bis (o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM) (a membrane permeable Ca²⁺ chelator) was added to the bath medium, at a final bath concentration of 10 μM BAPTA-AM [24]. After obtaining stable conditions for the ring segments, experiments were begun [15,12,24].

Reagents

The following pharmacological agents were purchased from Sigma (St. Louis, MO): NaOCl, bisindolylmaleimide I HCl, acetylcholine HCl, phenylephrine HCl, N,N-tetraacetic acid (EGTA), indomethacin, propranolol HCl, naloxone HCl, atropine sulfate, H₂O₂, phentolamine methanesulfonate, staurosporine, and genistein. 1, 2-Bis-(O-aminophenoxy) ethane-N, N, N', N'- tetraacetic acid acetoxymethyl ester (BAPTA/AM) was purchased from Molecular Probes (Eugene, OR). Dimethyl sulfoxide (DMSO), Gö6976 (12-(2-Cyanoethyl)-6,7,12,13- tetrahydro-13-methyl-5-oxo-5H-indolo{2,3-a}pyrrolo{3,4-c} carbazole), PD-098059 {2-(2-Amino-3-methoxyphenyl)-4H-1- benzopyran-4-one} and wortmannin were purchased from CALBIOCHEM (La Jolla, CA, USA). All other organic and inorganic chemicals were obtained from Fisher Scientific (Fair Lawn, NJ, USA) and were of the highest purity.

Results

Rat age, body weight and NaOCl-induced vasoconstriction of intact aortic rings

To examine for a possible role of age and body weight in NaOCl-induced vasoconstriction of intact aortic rings, experiments were carried out in rats of different ages and weights. As shown in Figure 1, we observed a stronger contractile effect of NaOCl on aortic segments with increasing age and weight of the rats. These results suggest that the contractile responses of the vessels to NaOCl are associated with both age and body weight. This was not observed for KClinduced contractions.

Figure 1: Contractile concentration-effect curves to NaOCl on rat aortic rings in the presence of endothelium. The data were obtained in rats of increasing age and weight. Values are expressed as means ± S.E. as percentage of the tension developed by 80 mM KCl; Group 200 - <275 g, n=3; 275 - <425 g, n=21; ≥425 g, n=4. *p<0.05 compared to each other.

Contractile effects of NaOCl on rat aortic rings

In the absence of any vasoactive agent, NaOCl alone induced contractile effects on rat aortic rings. Figure 2 shows a typical tracing of the vasoconstrictor effects of NaOCl on rat aortic rings with and without endothelium. As shown in Figures 2 and 3, NaOCl elicits concentration-dependent contractions of most rat aortic rings at concentrations of from 75 μM to 5 mM. However, reproducible threshold contractile responses were obtained up to 100 μM NaOCl. In addition, a comparison of the concentration-response curves with and without endothelium demonstrates that most of the contractile effects exerted by NaOCl on rat aortic rings are endothelium-independent (Figure 3). NaOCl-induced contractions of intact rat aortic rings appeared to be slightly stronger than those on denuded rat aortic rings at the highest concentration, but no paired points are significantly different from one another. It should be noted, here, that the vasoconstrictor effects of NaOCl on rat aortic rings were neither reversible nor reproducible on the same ring. Although not shown, preliminary experiments using the trypan blue exclusion assays indicate that contractions of hypochlorite greater than 100μM cause cytotoxicity to primary aortic smooth muscle cells in culture.

Figure 1: Contractile responses to NaOCl (mM) in isolated intact E(+) and endothelium-denuded E(−) rat aortic rings.

Figure 3: Contractile concentration-effect curves to NaOCl on rat aortic rings in the absence or presence of endothelium. Values are expressed as means ± S.E. No paired points are significantly different from one another at p>0.05 (paired t-test), n=5. E(+) = tissue with endothelial cells, E(−) = tissue without endothelial cells.

Effects of extra-and intracellular calcium on NaOCl-induced vasoconstriction of intact rat aortic rings

The relationship between NaOCl-induced contraction and extra- and intracellular Ca²⁺ was next examined in this study. As shown in Figures 4 and 5 removal of either extracellular Ca²⁺ from the bathing medium, or use of buffered intracellular Ca²⁺ by preincubation of the tissues in 10 μM BAPTA-AM, results in significant attenuation of NaOCl-induced contractions. As shown in Figure 6, incubation in [Ca²⁺]o-free medium together with use of BAPTA-AM, produced significant and almost complete inhibition of the contractile effects of NaOCl. These results suggest the NaOCl-induced contractions on rat aortic rings are dependent upon both extracellular- and intracellular calcium.

Figure 4: Contractile effects to NaOCl on rat aortic rings with endothelium, obtained in the absence or presence of extracellular Ca²⁺. Values are expressed as means ± S.E. as percentage of the tension developed by 80 mM KCl; n=4 each. *p<0.05 compared to control (paired t-test).

Figure 5: Contractile effects to NaOCl on rat aortic rings with endothelium, obtained in the presence or absence of BAPTA-AM. Values are expressed as means ± S.E. as percentage of the tension developed by 80 mM KCl; n=7 each. *p<0.05 compared to control (paired t-test).

Figure 6: Contractile effects to NaOCl on rat aortic rings with endothelium, obtained in the presence or absence of extracellular Ca²⁺ plus BAPTA-AM. Values are expressed as means ± S.E. as percentage of the tension developed by 80 mM KCl; n=3 each. *p<0.01 compared to control (paired ttest).

Effects of protein kinase C (PKC) antagonists on NaOCl-induced vasoconstriction of intact rat aortic rings

Figure 7 shows that pretreatment of intact rat aortic rings with staurosporine, bisindolylmaleimide I HCl (both potent antagonists of PKC), or Gö6976 (a PKCα-and PKCβ1-selective antagonist), for 20 min, completely inhibited NaOCl-induced vasoconstrictions. These three PKC antagonists were all dissolved in DMSO. The DSMO concentrations applied in these experiments were <1 μM for staurosporine and bisindolylmaleimide I HCl, and around 7.5 μM for Gö6976. In control experiments, equivalent amounts of DMSO were employed and exerted no effects on NaOCl-induced vasoconstriction (n=4 each, data not shown).

Figure 7: Contractile effects to NaOCl on rat aortic rings with endothelium, obtained in the presence of staurosporine, bisindolylmaleimide I HCl and Gö6976. Values are expressed as means ± S.E. as percentage of the tension developed by 80 mM KCl; n=4 each. The abbreviations used are: Sta, Staurosporine; Bis, Bisindolylmaleimide I HCl; Gö, Gö6976. *p<0.01, compared to control (paired t-test).

Effects of PD-098059, genistein and wortmannin on NaOCl-induced vasoconstriction of intact rat aortic rings

Figure 8 illustrates that pretreatment of intact rat aortic rings with PD-098059 (an antagonist of mitogen-activated protein kinase), genistein (an antagonist of protein tyrosine kinase) or wortmannin {an antagonist of phosphatidylinositol 3-kinases (PI₃Ks)} for 20 min completely inhibited NaOClinduced vasoconstrictions of rat aortic rings. The DSMO concentrations applied in these experiments were <1 μM. In control experiments, equivalent amounts of DMSO were employed and exerted no effects on NaOCl-induced vasoconstriction (n=4 each, data not shown).

Figure 8: Contractile effects to NaOCl on rat aortic rings with endothelium, obtained in the presence of PD-098059, genistein and wortmannin. Values are expressed as means ± S.E. as percentage of the tension developed by 80 mM KCl; n=4 each. The abbreviations used are: PD, PD-098059; Gen, genistein; Wort, wortmannin. *p<0.01 compared to control (paired t-test).

Variety of pharmacologic antagonists fail to attenuate or inhibit the contractile action of NaOCl on rat aortic rings

Incubation of intact rat aortic rings with a wide variety of specific amine, opiate and prostanoid antagonists (i.e., 10^-6 M diphenhydramine, 10^-5 M cimetidine, 10^-6 M phentolamine, 10^-6 M methysergide, 10^-5 M propranolol, 10^-6 M atropine, 10^-5 M naloxone and, 10^-5 M indomethacin) failed to either attenuate or interfere with contractions induced by NaOCl (data not shown, n=4-6 each).

Discussion

Reactive oxygen species are known to be involved in the progression of various cardiovascular diseases. One source of reactive oxygen species is activated neutrophils, which can release superoxide anion radicals and hydrogen peroxide by membrane-bound NADPH oxidases. These reactive oxygen species not only destroy bacteria, but may also affect mammalian tissues. In addition, hydrogen peroxide serves as a substrate for myeloperoxidase, an enzyme that is released by activated neutrophils during inflammatory processes, as seen, for instance, in reperfusion injury and atherosclerosis [24]. Myeloperoxidase catalyzes the oxidation of chloride by hydrogen peroxide, yielding hypochlorite, an extremely potent oxidant [24], and the local concentration of HOCl potentially exceeds 100 μM/l under pathological conditions in vivo [16,25-27].

The hypochlorite concentrations in our studies could be considered as biologically/pathologically-relevant to some diseases such as atherosclerosis and inflammation in vivo.

The present study demonstrates that NaOCl produces vasoconstriction of isolated rat aortic rings in a concentrationdependent manner. The fact that almost full contractile actions on the rat aortic rings are observed, after removal of the endothelium, indicates that most, if not all, of the NaOClinduced contractile are independent of endothelial cellderived mediators. Our findings indicate that NaOCl exerts a stronger contractile action on aortic segments obtained from older and heavier rats. Such data suggests that aortic smooth muscle cells become more and more sensitive to the contractile (and possibly) destructive actions of OCl- with age. This could support the notion that the aging process predisposes the vasculature to pathological actions of oxidants, such as hypertension, atherogenesis, heart diseases, inflammatory conditions, and strokes. The current work also demonstrates that the NaOCl-produced contractions are not reproducible and elicit tachyphylaxis in any one ring (with or without endothelium). What is of singular interest, here, is that even challenge sometimes with threshold levels of NaOCl results in tachyphylaxis. This indicates that the contractile actions of NaOCl are not reversible and probably lead to permanent vascular damage at low levels of hypochlorite. This vascular damage may, in part, be due to apoptotic phenomena as preliminary studies in our lab indicate that concentrations of NaOCl >100 μM cause apoptosis (e.g., activation of several caspase enzymes) in primary cultured rat aortic smooth muscle cells and cerebral vascular smooth muscle cells, similar to that we reported for peroxynitrite, H₂O₂ and hydroxyl radicals [17,18]. This is currently under investigation in our lab.

At present, the underlying mechanisms whereby NaOCl triggers vasoconstriction are poorly understood. The current work was designed to better define the effects of NaOCl on rat aortic rings and to probe the relationships between contractility of vessels induced by generation of OCl^-, Ca²⁺ and possible signaling pathways. It was also of interest to design the current study in a way to be able to compare potential mechanisms of action of NaOCl, H₂O₂ and hydroxyl radicals on rat aortic smooth muscle.

It has been well documented that cytosolic calcium increases during contraction of vascular smooth muscles either as a consequence of influx of Ca²⁺ from the extracellular space or release of Ca²⁺ from intracellular stores. Conversely, relaxation is usually accompanied by a decrease in the cytosolic Ca²⁺ level in smooth muscle cells [28]. In this study, removal of extracellular Ca²⁺ as well as buffering intracellular Ca²⁺ with the membrane-permeable Ca²⁺ chelator BAPTA-AM both inhibited most of the contractile actions of NaOCl on rat aortic rings. This suggests that the passage of extracellular calcium ions across the vascular smooth membranes as well as a release of intracellular free calcium ions from intracellular stores probably play essential roles in NaOCl-induced contractile effects. This is in agreement with a previous conclusion that hydroxyl radicals can enhance the voltagedependent influx of Ca²⁺ in rabbit lingual smooth muscle cells [29] and compares favorably to the actions of H₂O₂ on rat aortic rings [12] and cerebral arterial smooth muscle cells [14] as well as hydroxyl radicals in rat aortic rings [16].

Multiple cellular signal pathways are known to participate in mechanisms of vasoconstriction, such as activation of protein kinase C (PKC), protein tyrosine kinase, mitogen-activated protein kinase (MAPK), and PI₃Ks [28]. Vascular smooth muscle cells after exposure to NaOCl may result in release of a variety of vasoactive substances that in turn modulate vascular tone. To assess the involvement of such possible substances in NaOCl-induced contraction, several specific pharmacological inhibitors as well signaling pathway inhibitors were employed in the present study. A variety of pharamacologic amine antagonists, an opiate antagonist and a Cox-I inhibitor failed to inhibit the contractile action of NaOCl, suggesting that endogenous formation or release of catecholamines, histamine, serotonin, acetylcholine, opiates, or prostanoids do not play any role in NaOCl-induced contractile actions.

PKC, a family of Ca²⁺-sensitive and Ca²⁺-insensitive phospholipid-dependent protein kinases, that are present in high concentrations in vascular smooth muscle, have been shown to play important roles in cellular signal transduction [20,28,30,31]. However, up until the present study, no experimental evidence has appeared on the possible role of PKC in NaOCl-induced contractile effects on rat aortic rings. In the present work, NaOCl-induced vasoconstrictions were found to be completely inhibited in the presence of two potent PKC antagonists (bisindolylmaleimide I and staurosporine) and one selective PKC_α and PKC_â1 antagonist (Gö6976). This suggests that PKC isoforms, probably PKC_α (possibly PKC_β1 as well) may be a pivotal signaling mechanism by which NaOCl acts on the aortic smooth muscle cells to promote vasoconstriction. This is, thus, similar to that found for H₂O₂ in rat aortic rings and cerebral arteries [12,14] and hydroxyl radicals in rat aortic rings [16].

It has been reported that PI₃Ks are a group of enzymes that act directly to provide biochemical links between a novel phosphatidylinositol pathway and nonreceptor tyrosine kinases that appear to modulate Ca²⁺ channels through activity of these PI₃Ks [32]. Up to now, there has been no information regarding PI₃Ks and NaOCl-induced vasoconstriction on any vessel type. In this paper, we report that one potent antagonist of PI₃Ks (wortmannin) completely attenuated NaOCl-induced contractions, suggesting the possible involvement of products of PI₃Ks in such responses. Recently, H₂O₂-induced contractions [12,14,33] and hydroxyl radical-induced contractions [16] were found to be somewhat dependent on PI₃Ks as well.

Previous studies have shown that protein tyrosine phosphorylation is an essential component in signal transduction pathways in smooth muscle cells [34,35]. Our present data also demonstrates that genistein, an inhibitor of protein tyrosine kinase [35], which is an enzyme for catalyzing protein tyrosine phosphorylation, completely suppressed the NaOCl-induced vasoconstriction, which indicates the likely involvement of protein tyrosine phosphorylation in contractions caused by NaOCl. This conclusion is supported by other investigators who have demonstrated that oxidative stress can promote protein tyrosine phosphorylation in smooth muscle cells [12,13,16,36].

Several studies have been reported which indicate MAPK responds to diverse stimuli, including reactive oxygen species, and can transduce signals from the cell membrane to the nucleus [13,36-41]. Activation of MAPK plays an important role in modulating vascular tone [13,41]. In this investigation, the specific MAPK antagonist, PD-098059 [37-43], significantly prevented NaOCl-induced contractions in rat aortic rings. This suggests that activation of MAPK is probably an important pathway in NaOCl-mediated action on rat aortic smooth muscle cells.

Conclusion

In summary, the data from the present investigation suggest that NaOCl induces contractile actions on rat aortic rings in concentrations of from 75 μM to 5 mM, but not relaxation on rat aortic rings under resting tension. These NaOCl-induced contractions are mediated via the activation of several wellknown signal transduction pathways, and elevation of intracellular free Ca²⁺ levels, and are independent of the endothelium. Production of, and contact, of at least rat aorta with this most highly-reactive oxygen species, even in low concentration, appears to result in irreversible damage of the aortic smooth muscle cells. Such findings are thus quite different from those observed for H₂O₂, O₂.⁻, or peroxynitrite [8,10,12,14,37-43] but resemble those obtained for hydroxyl radicals [16] and requires further investigation. Lastly, if our new findings hold for the peripheral microvasculature, they could be suggestive of a very important role for generation of hypochlorite in regulation of the microvasculature and its pathophysiology, particularly in inflammatory processes, atherogenesis, coronary heart diseases and the aging process.

Acknowledgement

The work described herein was supported in part by a NIH Research Grants (National Heart, Lung and Blood Institute; National Institute on Alcoholism and Alcohol Abuse. National Institute on Mental Health) to B.M.A. and B.T.A.

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