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1 The full therapeutic potential of the main immunosuppressive drug, cyclosporin A (CsA), is limited because of its side effects, namely nephrotoxicity and hypertension. Several lines of evidence suggest that the origin of both side effects could be CsA-induced vasoconstriction. However Cyclosporin A (CsA) is currently the main immunosuppressive drug used during transplantation and autoimmune disorders. The mechanism of its immunosuppressive effect has recently been elucidated (McKeon, 1991;Schreiber & Crabtree, 1992;Swanson et al., 1992). The formation of a tertiary complex between CsA, the rotamase cyclophilin, and the protein phosphatase, calcineurin, inhibits the activity of the latter. This leads to decreased interleukin-2 (IL-2) production by lymphocyte T cells resulting in inhibition of their activation and proliferation (Cohen et al., 1984;Kunz & Hall, 1993). However, clinical use of CsA is limited due to its nephrotoxicity and ability to induce hypertension (Kahan, 1989;Mason, 1990;Textor et al., 1994). Most studies in animals as well as in CsA-treated patients suggest that CsA-induced vasoconstriction is the underlying cause of both side effects. However, the molecular mechanism of this effect is still an object of some controversy: (1) An increased sympathetic activity has been noted both in experimental models as well as in man (Moss et al., 1985;Scherrer et al., 1990;Morgan et al., 1991;Chiu et al., 1992;Lyson et al., 1993;1994). (2) Stimulation of the renin-angiotensin system has been described in hypertensive transplant recipients under CsA treatment (Julien et al., 1993) as well as in animals (Muller-Schweinitzer, 1988 British Journal of Pharmacology (1996) 118, 885-892 A. Lo Russo et al Cyclosporins and Ca2, signalling noradrenaline and AVP-induced contraction on isolated mesenteric arteries. We have also extended our previous findings to other vasoconstrictor hormones using cultured VSMC. In addition, to determine the possible role of cyclophilin or calcineurin inhibition in CsA-induced calcium potentiation, we have investigated the effect of some derivatives of CsA devoid of immunosuppressive activity. MethodsDiameter measurement of mesenteric arteriesMale Wistar Kyoto rats (200 to 300 g) were anaesthetized by i.p. injection of a solution of pentobarbitone (200 mg kg-' body weight) and were decapitated. Resistance arteries of ca. 200 uM diameter were prepared from the mesenteric bed using segments of the third order branch. They were cleaned of adherent tissue in ice-cold Krebs-Ringer solution (KRS: see below), mounted at both ends onto glass cannulae in a chamber (Living Systems Instrumentation, Burlington, VT, U.S.A.) filled with KRS and placed on a microscope stage. The vessels were continuously superfused with oxygenated KRS at 370C.A steady pressure of 40 mmHg was created in the vessel with a pump coupled to a feed-back pressure system. Vasoconstrictor drugs were applied via the superfusate in a low [Ca2+] KRS (see Chemicals and buffers) to avoid contractions due to calcium entry throu...

1 In Duchenne muscular dystrophy (DMD) dysregulation of cytosolic calcium appears to be involved in the degeneration of skeletal muscle fibres. Therefore, we have studied the regulation of the free cytosolic calcium concentration ([Ca2+]) under specific stress conditions in cultured myotubes isolated from the hind limbs of wild-type (C57BL10) and dystrophin-deficient mutant mdx mice. [Ca2+] [Ca2+]c when the cells were exposed to the hypo-osmotic buffer (100 mOsm).5 Incubation of the cell culture for 3-5 days from the onset of induction of myotube formation with the membrane permeable protease inhibitor, calpeptin (50 gM) abolished the rise in [Ca2+]c in mdx myotubes upon exposure to hypo-osmotic shock.6 Treatment of the cell culture for 3-5 days with ac-methylprednisolone (PDN, 10 guM) attenuated the rise in [Ca2+]c following hypo-osmotic stress for both normal and mdx myotubes by about 50%.7 The results described here suggest an increased permeability of mdx myotubes to Ca2+ under specific stress conditions. The ameliorating effect of PDN on [Ca2+]c could explain, at least partly, the beneficial effect of this drug on DMD patients.

1 Glucocorticoids, namely a-methylprednisolone (PDN) and de¯azacort, are the only drugs reported to have a bene®cial e ect on the degenerative course of Duchenne muscular dystrophy (DMD). Increased cytosolic calcium concentrations ([Ca 2+ ] c ) have been implicated as one of the pathological events responsible for the degeneration of dystrophic skeletal muscles. In previous studies, we have demonstrated that PDN treatment of both normal and dystrophic murine skeletal muscle cells was able to normalize elevated [Ca 2+ ] c and improved myogenesis. Here we have investigated the mechanism underlying the e ects of glucocorticoids on cellular Ca 2+ in¯ux into C2C12 skeletal muscle cells. 2 Long-term incubation of C2C12 myocytes with PDN was necessary to observe a reduction of 45 Ca 2+ in¯ux. PDN was most e ective in inhibiting 45 Ca 2+ uptake when added for 4 days (at the time of fusion of myoblasts into myotubes) and to a lesser extent, when added after fusion. It was ine ective when added to C2C12 cells at the myoblast stage. Short PDN incubation times, at the time of fusion were insu cient to elicit a response. 3 Several steroids were tested for their ability to inhibit 45 Ca 2+ in¯ux in C2C12 myocytes. All four glucocorticoids examined were able to reduce Ca 2+ in¯ux, dexamethasone being the most potent (IC 50 3.14+0.34610 78 M). Mineralocorticoids (aldosterone and 11-deoxycorticosterone) were also able to reduce Ca 2+ in¯ux. 4 The vitamin E-derived lazaroid U-83836E and the glucocorticoid-derived lazaroid U-74389G also elicited a decrease in Ca 2+ in¯ux, but higher concentrations were necessary. Because both glucocorticoids and lazaroids display antioxidant properties, but U-83836E is devoid of glucocorticoid activity, the reduction in Ca 2+ in¯ux was suspected to be triggered via an antioxidant mechanism. 5 To test this hypothesis, we assessed the action of several antioxidants, such as vitamin E, vitamin C, 2-tert.-butyl-4-methoxyphenol (BHA), 2,6-di-tert.-butyl-4-methyl-phenol (BHT) and nordihydroguaiaretic acid (NDGA), on 45 Ca 2+ in¯ux. None of these agents had an e ect on 45 Ca 2+ in¯ux. In addition, several oxidants were tested (either acutely or chronically) for their ability to elicit 45 Ca 2+ in¯ux in C2C12 myocytes and were found to be inactive. 6 The involvement of the glucocorticoid receptor on the modulation of Ca 2+ in¯ux was investigated. The glucocorticoid receptor antagonist mifepristone (code name RU38486, 10 76 M) caused a shift of two orders of magnitude of the PDN response. However, neither actinomycin D nor cycloheximide a ected the response to PDN. 7 Results with the phospholipase A 2 inhibitor, manoalide, suggest that glucocorticoid-induced protein synthesis (e.g. enhanced stimulation of lipocortin) does not play a role in the reduction of calcium in¯ux. 8 Our results suggest that steroids elicit a decrease in calcium in¯ux in C2C12 skeletal muscle cells. This decrease is not due to an antioxidant mechanism or to a mechanism which requires gene expression. Since mineralocorticoids and U...