Invited Symposium: Carbon Monoxide and Cardiovascular Function
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.
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 ÁM 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.
In our initial experiments, Zn-PP-IX at 100 ÁM 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 ÁM 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 ÁM to 300 ÁM) 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 ÁM) did not induce any changes in [Ca2+]i. (2) Zn-DPBG (50 ÁM) [Johnson et al., 1995], but not Sn-PP-IX (50 ÁM) [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 ÁM) 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.  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.
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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|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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