Upregulation of vasopressin V_1A receptor mRNA and protein in vascular smooth muscle cells following cyclosporin A treatment

Abstract

1 The major side eects of the immunosuppressive drug cyclosporin A (CsA) are hypertension and nephrotoxicity. It is likely that both are caused by local vasoconstriction. 2 We have shown previously that 20 h treatment of rat vascular smooth muscle cells (VSMC) with therapeutically relevant CsA concentrations increased the cellular response to [Arg 8 ]vasopressin (AVP) by increasing about 2 fold the number of vasopressin receptors.3 Displacement experiments using a speci®c antagonist of the vasopressin V 1A receptor (V 1A R) showed that the vasopressin binding sites present in VSMC were exclusively receptors of the V 1A subtype. 4 Receptor internalization studies revealed that CsA (10 76 M) did not signi®cantly alter AVP receptor tracking. 5 V 1A R mRNA was increased by CsA, as measured by quantitative polymerase chain reaction. Time-course studies indicated that the increase in mRNA preceded cell surface expression of the receptor, as measured by hormone binding. 6 A direct eect of CsA on the V 1A R promoter was investigated using VSMC transfected with a V 1A R promoter-luciferase reporter construct. Surprisingly, CsA did not increase, but rather slightly reduced V 1A R promoter activity. This eect was independent of the cyclophilin-calcineurin pathway. 7 Measurement of V 1A R mRNA decay in the presence of the transcription inhibitor actinomycin D revealed that CsA increased the half-life of V 1A R mRNA about 2 fold. 8 In conclusion, CsA increased the response of VSMC to AVP by upregulating V 1A R expression through stabilization of its mRNA. This could be a key mechanism in enhanced vascular responsiveness induced by CsA, causing both hypertension and, via renal vasoconstriction, reduced glomerular ®ltration.

Vasopressin Type 1A Receptor Up-regulation by Cyclosporin A in Vascular Smooth Muscle Cells Is Mediated by Superoxide

“…In addition, we have shown that the effect of CsA on receptor protein expression was preceded by an increase in V 1A receptor mRNA levels starting at 2 h and peaking at 6 -10 h after CsA addition (13). These data suggest that shorter times of CsA exposure may be sufficient to induce V 1A receptor up-regulation.…”

“…At least for vasopressin and angiotensin II, the expression of their respective cell surface receptors was shown to be increased by CsA in rat and human aortic smooth muscle cells (10 -12). Recently, we have shown that CsA up-regulated the vasopressin type 1A (V 1A ) receptor via an increase in the corresponding mRNA levels in RASMC (13). Up-regulation of vasoconstrictor hormone receptors may be responsible for the enhanced vasoconstriction under in vivo conditions, thus leading, via an increase in peripheral resistance, to hypertension and to a decrease in glomerular filtration (4 -6).…”

“…Real-time Quantitative PCR Analysis-Real-time quantitative PCR analysis was performed with a PerkinElmer Life Sciences 7700 Sequence Detector (PerkinElmer Life Sciences) as illustrated (13). Results were normalized for GAPDH expression for each sample.…”

“…[ 3 H]AVP Binding-Binding experiments were carried out as described (13). Results are expressed in percent of control to normalize for inter-experimental variations.…”

“…The RNA integrity was assessed by electrophoresis in non-denaturing 1.2% agarose gels stained with ethidium bromide. DNase treatment and reverse transcription were executed as described (13).…”