Effect of cyclosporine on oxidative phosphorylation and adenylate energy charge of regenerating rat liver

1 Co-administration of the calcineurin inhibitor cyclosporine and the mTOR inhibitors sirolimus or everolimus increases the efficacy of immunosuppression after organ transplantation. However, clinical studies showed enhancement of cyclosporine toxicity. To characterize the biochemical mechanisms involved, we assessed the time-dependent effects of cyclosporine in combination with mTOR inhibitors on energy production (ex vivo 31 P-MRS), glucose metabolism (ex vivo 13 C-MRS), and reactive oxygen species (ROS) formation (using the fluorescent agent 2 0 ,7 0 -dichlorofluorescein diacetate) in perfused rat brain slices. 2 Cyclosporine alone inhibited energy production (ATP: 7579%), the Krebs cycle (4-13 C-glutamate from 1-13 C-glucose: 61727%), and oxidative phosphorylation (NAD þ : 62725%) after 4 h of perfusion. After 10 h, activation of anaerobic glycolysis (3-13 C-lactate: 140717%) compensated for inhibition of mitochondrial energy production and lowered the intracellular pH. ROS formation was increased after 4 h (285755% of untreated control), but not after 10 h. mTOR inhibitors alone inhibited lactate production. When combined with cyclosporine, sirolimus enhanced cyclosporineinduced inhibition of energy metabolism (ATP: 6479%) and ROS formation (367746%). Most importantly, sirolimus inhibited cytosolic glycolysis and therefore compensation for cyclosporineinduced ATP reduction after 10 h. In contrast to sirolimus, everolimus antagonized cyclosporineinduced inhibition of mitochondrial energy metabolism (ATP: 9177%) and ROS formation (170749%). The antioxidant tocopherol antagonized all cyclosporine effects on cell metabolism. 3 Cyclosporine time-dependently inhibited mitochondrial metabolism and increased ROS, followed by compensation involving anaerobic glycolysis. Everolimus antagonized cyclosporine-induced mitochondrial dysfunction, whereas sirolimus inhibited compensatory anaerobic glycolysis, thus enhancing cyclosporine's negative effects. ROS play the key role in mediating the negative effects of cyclosporine on cell energy metabolism.

Section: Discussionmentioning

confidence: 99%

Alterations in glucose metabolism by cyclosporine in rat brain slices link to oxidative stress: interactions with mTOR inhibitors

Christians

Gottschalk

Miljuš

et al. 2004

“…Our study gave additional insights into the mechanisms underlying the reduction of high‐energy phosphate concentrations by cyclosporine. Previously described mechanisms involved in the cyclosporine‐mediated reduction of cellular ATP concentrations include increased radical formation ( Uemoto et al ., 1989 ; Wang & Salahudeen, 1994 ; Wolf et al ., 1997 ) and mitochondrial Ca 2+ ‐overload ( Salducci et al ., 1996 ). The accumulation of oxaloacetate and lactate indicated that cyclosporine significantly inhibited the Krebs cycle at its entrance, possibly by inhibiting pyruvate dehydrogenase.…”

Section: Discussionmentioning

confidence: 99%

Sirolimus, but not the structurally related RAD (everolimus), enhances the negative effects of cyclosporine on mitochondrial metabolism in the rat brain

Serkova¹,

Jacobsen²,

Niemann³

et al. 2001

1 Clinical studies have shown enhancement of cyclosporine toxicity when co-administered with the immunosuppressant sirolimus. We evaluated the biochemical mechanisms underlying the sirolimus/ cyclosporine interaction on rat brain metabolism using magnetic resonance spectroscopy (MRS) and compared the e ects of sirolimus with those of the structurally related RAD. 2 Two-week-old rats (25 g) were allocated to the following treatment groups (all n=6): I. control, II. cyclosporine (10 mg kg 71 d 71 ), III. sirolimus (3 mg kg 71 d 71 ), IV. RAD (3 mg kg 71 d 71 ), V. cyclosporine+sirolimus and VI. cyclosporine+RAD. Drugs were administered by oral gavage for 6 days. Twelve hours after the last dose, metabolic changes were assessed in brain tissue extracts using multinuclear MRS. 3 Cyclosporine signi®cantly inhibited mitochondrial glucose metabolism (glutamate: 78+6% of control; GABA: 67+12%; NAD + : 76+3%; P50.05), but increased lactate production. Sirolimus and RAD inhibited cytosolic glucose metabolism via lactate production (sirolimus: 81+3% of control, RAD: 69+2%; P50.02). Sirolimus enhanced cyclosporine-induced inhibition of mitochondrial glucose metabolism (glutamate: 60+4%; GABA: 59+8%; NAD + : 45+5%; P50.02 versus cyclosporine alone). Lactate production was signi®cantly reduced. In contrast, RAD antagonized the e ects of cyclosporine (glutamate, GABA, and NAD + , not signi®cantly di erent from controls). 4 The results can partially be explained by pharmacokinetic interactions: co-administration increased the distribution of cyclosporine and sirolimus into brain tissue, while co-administration with RAD decreased cyclosporine brain tissue concentrations. In addition RAD, but not sirolimus, distributed into brain mitochondria. 5 The combination of cyclosporine/RAD compares favourably to cyclosporine/sirolimus in regards to their e ects on brain high-energy metabolism and tissue distribution in the rat.

“…Cyclosporine perfusate concentrations of 100 μg l −1 corresponded to the lower limit of the blood concentration range targeted in transplant patients (Oellerich et al ., 1995). Various previous studies (Gabe et al ., 1998; Henke et al ., 1992; Massicot et al ., 1997; Riuz‐Cabello et al ., 1994; Uemoto et al ., 1989) demonstrated that cyclosporine specifically inhibits mitochondrial energy production and decreases ATP level in target organs of its toxicity such as kidney, liver and intestine in vitro as well as in vivo . Impairment of energy metabolism in the brain results in excitotoxic neuronal death in various neurodegenerative illnesses (Beal, 1992).…”

Section: Discussionmentioning

confidence: 99%

The novel immunosuppressant SDZ‐RAD protects rat brain slices from cyclosporine‐induced reduction of high‐energy phosphates

Serkova

Litt

Leibfritz

et al. 2000

1 SDZ-RAD, 40-O-(2-hydroxyethyl)-rapamycin, is a novel macrolide immunosuppressant. Because of its synergistic interaction, SDZ-RAD is under clinical investigation as immunosuppressant in combination with cyclosporine after organ transplantation. Neurotoxicity is a critical side-e ect of cyclosporine. 2 We studied the e ect of SDZ-RAD and its combination with cyclosporine on high-energy phosphates, phosphocreatine (PCr) and nucleoside triphosphates (NTP), in brain slices using 31 Pmagnetic resonance spectroscopy (MRS). 3 Cyclosporine signi®cantly reduced high-energy phosphates after 2 h in a dose-dependent manner (100 mg l 71 : 93+3% of control (NTP), 91+3% (PCr); 500 mg l 71 : 84+2% (NTP), 73+2 (PCr); 5000 mg l 71 : 68+3% (NTP), 55+5% (PCr); n=6; P50.02). 4 In contrast, after perfusion for 2 h, SDZ-RAD (500 mg l 71 and 5000 mg l 71 ) signi®cantly increased high-energy phosphate concentrations in the brain slices (P50.02). Even at the lowest concentration, SDZ-RAD protected brain energy metabolism against cyclosporine toxicity: 100 mg l 71 SDZ-RAD+5000 mg l 71 cyclosporine: 86+3% (NTP), 83+7% (PCr), n = 3, P50.03 compared to cyclosporine alone. 5 As evaluated using an algorithm based on Loewe isobolograms, the e ects of SDZ-RAD/ cyclosporine combinations on brain energy reduction were antagonistic. Both drugs were found in mitochondria using h.p.l.c-MS analysis. 6 We conclude that cyclosporine inhibits mitochondrial high-energy phosphate metabolism, which can be antagonized by SDZ-RAD.