Background and Purpose-Sphingosine 1-phosphate (S1P) is a platelet-derived bioactive lipid that exerts a variety of biological responses, including vasocontraction. To understand the involvement of S1P in cerebral vasospasm, we investigated the effect of S1P on vasocontraction of the canine basilar artery in vitro and in vivo. Methods-We recorded isometric tension in basilar arterial rings from dogs in vitro and estimated time-course changes in the diameter of canine basilar arteries and the S1P concentration in cerebrospinal fluid (CSF) by angiography and radioreceptor assays, respectively, after administering S1P into the cisterna magna. Changes in the supernatant S1P concentration during clot formation were monitored by using the in vitro subarachnoid hemorrhage model, in which blood is mixed with CSF. Results-At concentrations ranging between 100 nmol/L and 10 mol/L, S1P induced a dose-dependent contraction of the basilar artery in vitro. This effect was significantly inhibited by Y-27632, a highly selective Rho-kinase inhibitor. The administration of S1P into the CSF induced a 60% to 70% decrease in the arterial diameter within 15 minutes, and vasocontraction continued for 2 days thereafter. The concentration of S1P in the supernatant during clot formation in vitro reached Ϸ300 nmol/L. Conclusions-S1P induces vasocontraction in the canine basilar artery in vitro and in vivo, possibly through a mechanism involving activation of the Rho/Rho-kinase pathway. Thus, S1P might be considered as a novel spasmogenic substance involved in cerebral vasospasm after subarachnoid hemorrhage.

ROSs have direct vasocontractile effects on the canine basilar artery in vitro, but different ROSs have different contractile characteristics. Such contractions might be related to the pathophysiology of cerebral vasospasm. MCI-186 had a clear and selective inhibitory effect against *OH-induced contraction in vitro. Comparison of different radical scavengers may be important in pharmacological assessment, especially targeted on cerebral vasospasm.

Several spasmogenic mediators released after SAH may cause vasospasm through Rho-kinase-mediated increase in calcium sensitization. Rho-kinase inhibitors, including Y-27632, may be effective for the prevention of cerebral vasospasm after SAH.

PRL-releasing peptide receptor (PrRPR) mRNA was expressed in pituitary adenomas but was not detected in patients treated with bromocriptine, a specific agonist of dopamine 2 (D2) receptor. Although medical treatment with bromocriptine is effective for patients with pituitary adenomas, little is known about the molecular mechanisms of gene regulation mediated by D2 receptors. The cloned human PrRPR gene spanned approximately 2.0 kb and contained two exons and one intron. Two functional polyadenylation signals located at 510 and 714 bp downstream from the stop codon. A primer extension analysis demonstrated two major transcriptional start sites at 139 and 140 bp upstream from the translational start site and an additional minor site at -161. The promoter region contained several putative binding sites for transcriptional factors including pituitary-specific transcription factor (Pit 1), activator protein 1 (AP-1), and specificity protein (Sp1), but no typical TATA or CAAT box. This promoter showed the strong activity in the pituitary-derived GH4C1 cells, and the region between -697 and -596 bp was responsible for the stimulation both by forskolin and overexpression of cAMP response element binding protein (CREB). These stimulations were significantly suppressed by incubation with bromocriptine in a dose- and time-dependent manner, and the mutant CREB (S133A) completely abolished the inhibitory events of bromocriptine. However, EMSA studies demonstrated that CREB did not bind to this region, to which an approximately 60-kDa protein was strongly bound, and that antibodies against CREB, c-Fos, and Sp1 did not supershift this complex. Furthermore, the amount of this unknown protein was apparently reduced by treatment with bromocriptine. A series of mutation analyses demonstrated that the specific sequence, 5'-cccacatcat-3', was required for both the binding to the 60-kDa protein and the repression by bromocriptine. Therefore, the transcriptional repression of the PrRPR gene by bromocriptine required CREB but was independent of direct binding of CREB to the gene and that the sequence -663 -- -672, 5'-cccacatcat-3', bound to the 60-kDa protein appeared to be critical for this event.

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