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Invited Symposium: Carbon Monoxide and Cardiovascular Function






Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References




Discussion
Board

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Direct Effect of Zinc Protoporphyrin-IX on Calcium Homeostasis of Vascular Smooth Muscle Cells


Contact Person: Lingyun Wu (Wuli@ERE.UMontreal.CA)


Introduction

Metalloporphyrins, especially zinc protoporphyrin XI (Zn-PP-IX), have been widely used as the tool in studying the biological effects of endogenously generated carbon monoxide (CO). The rationale of using metalloporphyrins in CO study is that certain metalloporphyrins can inhibit heme oxygenase (HO) in a relatively specific manner. The inhibition of HO would decrease the degradation of heme-containing molecules, thus leading to a reduced production of CO and bilirubin. Among the most often used metalloporphyrins Zn-PP-IX has been used to selectively inhibit both HO-1 and HO-2 [Martasek et al., 1988]. However, the non-specific effects of Zn-PP-IX has been demonstrated. For instance, Zn-PP-IX may, independent of the inhibition of HO, interact with various membrane receptors [Ny et al., 1995], inhibit soluble guanylyl cyclase and nitric oxide synthase [Christodoulides et al., 1995; Ignarro et al., 1984; Luo & Vincent, 1994; Meffert et al., 1994], and inhibit voltage-gated calcium channel current [Linden et al, 1993] in different preparations. On the other hand, the role of CO as a biological signalling gaseous molecule has been indicated in many cases mainly based on the effects of Zn-PP-IX. If Zn-PP-IX is not a specific HO inhibitor, many of the reported Zn-PP-IX-induced biological changes may not be completely ascribed to the speculated endogenous CO. In the present study, we examined whether Zn-PP-IX could modulate the intracellular free calcium concentrations, [Ca2+]i, in cultured vascular smooth muscle cells from rat tail artery, and whether this effect was due to a inhibited HO activity. Other related metalloporphyrins were also studied to corroborate our observations on Zn-PP-IX. The direct effect exogenous CO on [Ca2+]i was also determined.

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Materials and Methods

Single smooth muscle cells (SMCs) were isolated from rat tail arteries and identified as described previously [Wang et al., 1998]. Briefly, tail arteries were isolated from male Sprague- Dawley rats (150-200 g). The vessel was cut open longitudinally and enzymatically digested with collagenase/dispase, elastase and collagenase for different periods of time. The tissues were then triturated and isolated cells were plated in 35 mm Petri dishes and cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum in a CO2 incubator at 37oC. SMCs in primary culture from 16 to 48 hours were loaded with 5 M acetoxymethylester of Fura-2. Fluorescence measurements were carried out using an epifluorescence microscope attached to a dual-excitation spectrofluorometer (Spex Fluorolog II) with excitation wavelengths set at 340 (F340) and 380 nm (F380) and an emission wavelength at 505 nm. The relative changes in [Ca2+]i were estimated in individual cells from the ratio of the emitted Fura-2 fluorescence, referred to as R340/380 [Wang et al., 1998]. SMCs were continuously superfused with a standard Earle's solution composed of (mM): 121 NaCl, 5.4 KCl, 1.8 CaCl2, 0.8 MgSO4, 6 NaHCO3, 1 NaH2PO4, 5.5 glucose, and 25 N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffered at pH 7.3. Zn-PP-IX was from Aldrich (Milwaukee); Sn-PP-IX and zinc deuteroporphyrin 2,4-bis glycol (Zn-DPBG) were from Porphyrin Products (Logan, UT). Unless otherwise specified, all metalloporphyrin-containing solutions were prepared and stored in the darkness, and Fura-2 experiments were carried out in the dark room. The data were expressed as means S.E.M.. Statistical significance was analyzed using unpaired Student's t test or the Newman-Keuls variance analysis where applicable. The difference was considered to be significant when p < 0.05.

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Results

In our initial experiments, Zn-PP-IX at 100 M induced a transient increase in [Ca2+]i in cultured rat tail artery SMCs (n=6). This effect was independent of the presence or absence of extracellular calcium. However, blockade of intracellular calcium releasing pool with thapsigargin completely abolished the Zn-PP-IX induced increase in [Ca2+]i. Therefore, it appeared that Zn-PP-IX transiently released intracellular calcium from vascular smooth muscle cells. The transient increase in [Ca2+]i induced by Zn-PP-IX would trigger many calcium-dependent cellular events, such as the activation of calmodulin-dependent constitutive nitric oxide synthase. Whether this effect of Zn-PP- IX was mediated by a decreased activity of HO was further investigated. It has been reported that at concentrations lower than 50 M Zn-PP-IX and Sn-PP-IX had no effect on NO synthase or soluble guanylyl cyclase [Zakhary et al., 1996]. Therefore, it was suggested that at this low concentration range these metalloporphyrins might specifically inhibit HO. Interestingly, it was found in our experiments that Zn-PP-IX concentration-dependently (10 M to 300 M) increased [Ca2+]i. This effect of Zn-PP-IX was unlikely mediated by HO activities based on the following observations. (1) Bathing the vascular SMCs with the CO-containing Earle's solution (100 M) did not induce any changes in [Ca2+]i. (2) Zn-DPBG (50 M) [Johnson et al., 1995], but not Sn-PP-IX (50 M) [Morita et al., 1995; Ny et al., 1995], induced a similar transient increase in [Ca2+]i in these vascular smooth muscle cells. (3) Light-treated Zn-PP-IX (100 M) still induced a transient increase in [Ca2+]i in vascular SMCs. It has been known that Zn-PP-IX is photosensitive and light-exposed metalloporphyrins lost their ability to inhibit HO [Greenbaum & Kappas, 1991]. In another study by Zygmunt et al. [1994] the HO-independent vasoactive effect of Zn-PP-IX has been demonstrated under different light exposure conditions. The effect of Zn-PP-IX on [Ca2+]i was also not due to the autofluorescence of the metalloporphyrin because the autofluorescence and background fluorescence had been subtracted and the effects of Zn-PP-IX could be suppressed after thapsigargin treatment. Moreover, the induced increase in [Ca2+]i was transient although the cells were continuously superfused with the Zn-PP-IX containing bath solutions.

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Discussion and Conclusion

Zn-PP-IX induced a transient increase in cultured rat tail artery SMCs in a HO activity- independent fashion. This effect of Zn-PP-IX is concentration-dependent and extracellular calcium independent. Zn-DPBG, but not Sn-PP-IX, had a similar effect as Zn-PP-IX on [Ca2+]i in these vascular SMCs. Our results suggested that the conclusions on the biological functions of endogenous CO based solely on the effect of Zn-PP-IX, even at relatively low concentrations, should be cautiously re-evaluated.

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References

  1. Christodoulides N, Durante W, Kroll MH, Schafer AI: Vascular smooth muscle cell heme oxygenases generates guanylyl cyclase-stimulatory carbon monoxide. Circulation. 91:2306-2309, 1995.
  2. Greenbaum NL, Kappas A: Comparative photoactivity of tin and zinc porphyrin inhibitors of heme oxygenase: pronounced photolability of the zinc compounds. Photochem. Photobiol. 54:183-192, 1991.
  3. Ignarro LJ, Ballot B, Wood KS: Regulation of soluble guaylate cyclase activity by porphyrins and metalloporphyrins. J. Bio. Chem. 259: 6201-6207, 1984.
  4. Johnson RA, Lavesa M, Askari B, Abraham NG, Nasjletti A: A heme oxygenase product, presumably carbon monoxide, mediates a vasodepressor function in rats. Hypertension 25:166-169, 1995.
  5. Linden DJ, Narasimhan K, Gurfel D: Protoporphyrins modulate voltage-gated Ca current in AtT-20 pituitary cells. J. Neurophysiol. 70: 2673-2677, 1993.
  6. Luo D, Vincent SR: Matalloporphyrins inhibit nitric oxide-dependent cGMP formation in vivo. Eur. J. Pharmacol. 267: 263-267, 1994.
  7. Martasek P, Solangi K, Goodman AI, Levere RD, Chernick RJ, Abraham NG: Properties of human kidney heme oxygenase: inhibition by synthetic heme analogues and metalloporphyrins. Biochem. Biophys. Res. Commun. 157:480-487, 1988.
  8. Meffert MK, Haley JE, Schumann EM, Schulman H, Madison DV: Inhibition of hippocampal heme oxygenase, nitric oxide synthase and long-term potentiation by metalloporphyrins. Neuron 13: 1225-1233, 1994.
  9. Morita T, Perrella MA, Lee ME, Kourembanas S: Smooth muscle cell-derived carbon monoxide is a regulator of vascular cGMP. Proc. Natl. Acad. Sci. USA.. 92:1475-1479, 1995.
  10. Ny L, Andersson K-E, Grundemar L: Inhibition by zinc protoporphyrin-IX of receptor-mediated relaxation of the rat aorta in a manner distinct from inhibition of haem oxygenase. Br. J. Pharmacol. 115: 186-190, 1995.
  11. Wang R, Liu Y, Sauv R, Anand-Srivastava MB: Diabetes-related abnormal calcium mobilization in smooth muscle cells are induced by hyperosmolality. Mol. Cell. Biochem. 183: 79-85, 1998.
  12. Zakhary R, Gaine SP, Dinerman JL, Martial R, Flavahan NA, Snyder SH. Heme oxygenase-2: endothelial and neuronal localization and role in endothelium-dependent relaxation. Proc. Natl. Acad. Sci. USA. 93: 795-798, 1996.
  13. Zygmunt PM, Hogestatt ED, Grundemar L: Light-dependent effects of zinc protoporphyrin IX on endothelium-dependent relaxation resistant to N omega-nitro-L-arginine. Acta Physiol. Scand1. 52:137-143, 1994.

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Wu, L; Wang, R; (1998). Direct Effect of Zinc Protoporphyrin-IX on Calcium Homeostasis of Vascular Smooth Muscle Cells. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/wang/wu0210/index.html
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