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At least three types of opioid receptors have been identified in the nervous system. In this paper we report molecular cloning and expression of a rat kappa opioid receptor. PCR was performed on double-stranded cDNA derived from poly(A)+ RNA of the rat striatum with primers similar to those of Libert and co-workers [Libert, Parmentier, Lefort, Dinsart, Van Sande, Maenhaut, Simons, Dumont and Vassart (1989) Science 244, 569-572]. One of the PCR products, which had 65% sequence similarity to the mouse delta opioid receptor, was used to screen a rat striatum cDNA library. Two positive clones were isolated and found to be identical. The clone had a 2.1-kb insert, which was termed RKOR-1. RKOR-1 has an open reading frame of 1140 bp and encodes a 380-amino-acid protein. Hydropathy analysis indicates that RKOR-1 has seven putative transmembrane domains with short intra- and extra-cellular loops. Membranes of Cos-7 cells transfected with RKOR-1 exhibited high specific binding for [3H]diprenorphine ([3H]DIP), a non-selective opioid ligand. Naloxone inhibited [3H]DIP binding with stereospecificity. [3H]DIP binding was potently inhibited by selective kappa opioid ligands, with Ki values in the nanomolar or subnanomolar range, but much less potently inhibited by drugs selective for mu or delta receptors. Thus, RKOR-1 represents an opioid receptor with kappa characteristics.

Objective-Hyperglycemia has been suggested to play a role in the development of vascular disease associated with diabetes. Atypical Ca 2ϩ signaling and gene expression are characteristic of vascular dysfunction; however, little is known regarding the effects of high glucose on Ca 2ϩ -dependent transcription in the vascular wall. Methods and Results-Using confocal immunofluorescence, we show that modest elevation of extracellular glucose (ie, from 2 to 11.5 mmol/L) increased [Ca 2ϩ ] i , leading to nuclear accumulation of nuclear factor of activated T cells (NFAT) in intact cerebral arteries from mouse. This was accompanied by increased NFAT-dependent transcriptional activity. Both the increase in Ca 2ϩ and NFAT activation were prevented by the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. We provide evidence that the potent vasoconstrictors and growth stimulators UTP and UDP mediate glucose-induced NFAT activation via P2Y receptors. NFAT nuclear accumulation was inhibited by the voltage-dependent Ca 2ϩ channel blockers verapamil and nifedipine, the calcineurin inhibitor cyclosporine A, and the novel NFAT blocker A-285222. High glucose also regulated glycogen synthase kinase 3␤ and c-Jun N-terminal kinase activity, yielding decreased kinase activity and reduced export of NFAT from the nucleus, providing additional mechanisms underlying the glucose-induced NFAT activation. Key Words: NFAT Ⅲ high glucose Ⅲ vascular smooth muscle Ⅲ extracellular nucleotides Ⅲ GSK-3 G lucose has been shown to activate nuclear factor of activated T cells (NFAT) in pancreatic ␤-cells, promoting insulin gene transcription. 1 Previous studies have shown that this transcription factor is expressed in native vascular smooth muscle and can be activated in response to vasoconstrictor agonist stimulation. 2 Furthermore, NFAT inhibition has been shown recently to reduce balloon injury-induced neointima formation in rat carotid artery. 3 However, it is unknown whether glucose can activate NFAT in the vasculature. Conclusions-OurThere are 4 well-characterized members of the NFAT family, all of which depend on dephosphorylation by calcineurin to translocate to the nucleus. 4 In immune cells, activation of this signaling pathway leads to production of cytokines and T-cell proliferation. 4 Inhibition of NFAT nuclear translocation is largely responsible for the immunosuppressive actions of cyclosporine A and tacrolimus (FK506), which specifically block calcineurin. 5 Although originally thought to be restricted to T cells, NFAT has since been shown to regulate heart valve development, 6 skeletal muscle differentiation, 7 and vascular development during embryogenesis. 8 NFAT has also been implicated in the pathogenesis of cardiac 9 and skeletal muscle hypertrophy 10 and might be predicted to play a role in smooth muscle hypertrophy associated with, for example, hypertension and atherosclerosis. Indeed, Amberg et al 11 recently demonstrated that sustained angiotensin II stimulation, which in vi...