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 µM 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 µM 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 µM 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 µM to 300 µM) 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 µM) did not induce any changes in [Ca2+]i. (2) Zn-DPBG (50 µM) [Johnson et al., 1995], but not Sn-PP-IX (50 µM) [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 µM) 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.

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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