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...

The use of the immunosuppressant cyclosporin A (CsA) is frequently associated with hypertension. Drug-induced local vasoconstriction appears to be responsible for this effect. Using fura-2 and 45Ca2+ efflux techniques, we have examined variations in the cytosolic calcium concentration ([Ca2+]c) in rat aortic smooth muscle cells and have shown that increases in [Ca2+]c after [Arg8]vasopressin, serotonin, endothelin-1 or angiotensin II stimulation were potentiated after preincubation of cells with CsA. This effect was independent of cyclophilin or calcineurin inhibition by CsA. Measurements of inositol phosphates (InsPn) after agonist stimulation showed that CsA also potentiated their formation. As for 45Ca2+ efflux this effect was not related to cyclophilin or calcineurin inhibition. Direct stimulation of G proteins with aluminium tetrafluoride induced an increase in InsPn formation and 45Ca2+ efflux. Neither of these responses was potentiated by CsA. These results indicate that CsA acts on a target upstream of G protein activation, possibly at the receptor level, resulting in a potentiation of InsPn formation and subsequent calcium increase.

1 The use of the immunosuppressive drug cyclosporin A (CsA) is limited by two major side e ects, nephrotoxicity and hypertension, which are caused by drug-induced local vasoconstriction. We have recently shown that CsA potentiates the contraction of isolated resistance arteries to vasoconstrictor hormones and increases the calcium response to these agents in vascular smooth muscle cells (VSMC). The goal of the present study was to investigate further the molecular mechanism(s) involved in these e ects. H]-AVP on VSMC showed that CsA increased the number of AVP receptors by about two fold without a ecting receptor a nity. Actinomycin D completely blocked this increase. 5 These results demonstrate for the ®rst time that incubation of VSMC with CsA increases the expression of AVP receptors, resulting in a potentiation of InsP formation and calcium response upon stimulation with AVP. This e ect of CsA is likely to occur with other vasoconstrictor hormone receptors as well and could be a key mechanism in the induction of vasoconstriction, and subsequent drug-induced nephrotoxicity and hypertension.

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