The role of the opioid-like receptor 1 (ORL1) and its endogenous ligand, nociceptin/orphanin FQ (N/OFQ), in nociception, anxiety, and learning remains to be defined. To allow the rapid identification of agonists and antagonists, a reporter gene assay has been established in which the ORL1 receptor is functionally linked to the cyclic AMP-dependent expression of luciferase. N/OFQ and N/OFQ(1-13)NH(2) inhibited the forskolin-induced luciferase gene expression with IC(50) values of 0.81 +/- 0.5 and 0.87 +/- 0.16 nM, respectively. Buprenorphine was identified as a full agonist at the ORL1 receptor with an IC(50) value of 8.4 +/- 2.8 nM. Fentanyl and 7-benzylidenenaltrexone displayed a weak agonistic activity. The ORL1 antagonist [Phe(1)Psi(CH(2)-NH)Gly(2)]N/OFQ((1-13))NH(2) clearly behaved as an agonist in this assay with an IC(50) value of 85 +/- 47 nM. Thus, there is still a need for antagonistic tool compounds that might help to elucidate the neurophysiological role of N/OFQ.

Induced pluripotent stem cells (iPSCs) have enabled the generation of various difficult-to-access cell types such as human nociceptors. A key challenge associated with human iPSC-derived nociceptors (hiPSCdNs) is their prolonged functional maturation. While numerous studies have addressed the expression of classic neuronal markers and ion channels in hiPSCdNs, the temporal development of key signaling cascades regulating nociceptor activity has remained largely unexplored. In this study, we used an immunocytochemical high-content imaging approach alongside electrophysiological staging to assess metabotropic and ionotropic signaling of large scale–generated hiPSCdNs across 70 days of in vitro differentiation. During this period, the resting membrane potential became more hyperpolarized, while rheobase, action potential peak amplitude, and membrane capacitance increased. After 70 days, hiPSCdNs exhibited robust physiological responses induced by GABA, pH shift, ATP, and capsaicin. Direct activation of protein kinase A type II (PKA-II) through adenylyl cyclase stimulation with forskolin resulted in PKA-II activation at all time points. Depolarization-induced activation of PKA-II emerged after 35 days of differentiation. However, effective inhibition of forskolin-induced PKA-II activation by opioid receptor agonists required 70 days of in vitro differentiation. Our results identify a pronounced time difference between early expression of functionally important ion channels and emergence of regulatory metabotropic sensitizing and desensitizing signaling only at advanced stages of in vitro cultivation, suggesting an independent regulation of ionotropic and metabotropic signaling. These data are relevant for devising future studies into the development and regulation of human nociceptor function and for defining time windows suitable for hiPSCdN-based drug discovery.

A chimeric protein (rscu-PA-40-kDa/Hir), consisting of the C-terminal amino acids 53 -65 of hirudin (Hir), fused via a 14-amino-acid linker sequence to the C-terminal of a 40-kDa fragment (Ser47-Leu411) of recombinant (r) single-chain (sc) urokinase-type plasminogen activator (rscu-PA), was produced by expression of the corresponding chimeric cDNA in Escherichia coli cells. The thrombin inhibitory potential of purified rscu-PA-40-kDdHir was confirmed by complete inhibition of the coagulant activity of thrombin at 20-30-fold molar excess of the chimera, and by the resistance of rscu-PA-40-kDa/Hir to proteolytic cleavage by thrombin. rscu-PA-40-kDa/Hir prolonged the thrombin time of normal human plasma in a dose-dependent way (reduction of the apparent thrombin concentration to 50% with 95 nM chimeric protein as compared to 4.7 nM hirudin), and inhibited thrombin-mediated platelet aggregation (reduction of the apparent thrombin concentration to 50% with 40 nM chimeric protein).The chimera had a specific activity on fibrin films of 57000 IU/mg as compared to 95000 IU/mg for rscu-PA. The urokinase-like amidolytic activity of the single-chain protein was only 220 IU/mg but increased to 169000 IU/mg after treatment with plasmin, which resulted in quantitative conversion to a two-chain (tc) derivative (rtcu-PA-40-kDa/Hir). Corresponding values for rscu-PA were 270 and 226 000 IU/mg. The catalytic efficiencies for plasmin-mediated conversion to two-chain molecules were comparable for rscu-PA-40-kDaMr and rscu-PA (0.63 and 0.65 pM-' . s-', respectively). The plasminogenactivating potential of the single-chain chimera was comparable to that of rscu-PA ; the catalytic efficiencies for plasminogen activation by their two-chain counterparts were also similar (0.55 and 0.73 pM-I . s-', respectively). In 2 h, 50% lysis of 'z51-fibrin-labeled clots prepared from platelet-poor human plasma and immersed in normal plasma was obtained with 1.3 pg/ml rscu-PA-40-kDa/Hir and with 0.67 pg/ml rscu-PA, with corresponding residual fibrinogen levels of 74% and 87%, respectively. In the absence of fibrin, 50% fibrinogenolysis in 2 h i n normal human plasma required 2.1 pgiml rscu-PA, but 7.9 pgiml rscu-PA-40-kDa/Hir.Thus, the chimera rscu-PA-40-kDUHir has maintained the specific fibrinolytic and plasminogen activating activity of rscu-PA as well as its fibrinolytic potency in plasma, whereas it displayed a similar or somewhat better fibrin specificity. In addition, the fibrinolytically active concentration in a plasma medium is severalfold lower than the concentration required for thrombin inhibition, which may limit systemic anticoagulant activity. Therefore, further evaluation of the thrombolytic and antithrombotic potential of such chimeric molecules seems to be warranted.Keywurds: urokinase-type plasminogen activator; hirudin; thrombolytic agent; antithrombotic agent.Clinical experience, mainly in the treatment of patients with acute myocardial infarction, has revealed that the presently Correspondence to H. R.

The blood clotting enzyme thrombin plays a central role in the aetiology of occlusive disorders such as stroke and acute myocardial infarction. During fibrinolytic therapy with plasminogen activators, thrombin is neutralized by anticoagulative drugs. In order to combine plasminogen-activating and thrombin-inhibitory activities we constructed chimeric derivatives of recombinant single-chain, urokinase-type plasminogen activator (rscu-PA) which comprise the kringle and protease domain of rscu-PA fused via a linker sequence to a thrombin-inhibitory domain. The inhibitory domain contains a sequence element directed to the active site of thrombin and a sequence taken from either hirudin or the human thrombin receptor both binding to the fibrinogen recognition site of thrombin. Analysing different sets of point mutants showed that the linker between the protease domain and the active site-directed sequence is contributing significantly to the thrombin-inhibitory potential. Kinetic analysis of thrombin inhibition revealed that most of the chimeras tested competitively inhibit the thrombin-mediated cleavage of a peptide substrate in a concentration-dependent manner; however, in two examples the insertion of one glycine residue into the active site directed-sequence abolished the blockade of the active site. This supports the conclusion that the chimeras with high thrombin-inhibitory potential interact with the active site and the fibrinogen recognition site of thrombin.

In order to design plasminogen activators with improved thrombolytic properties, bifunctional proteins with both plasminogen-activating and anticoagulative activity were constructed by fusing a thrombin-inhibitory moiety itself comprises four elements: linker 1, a motif directed to thrombin's active site, linker 2 and a fragment of hirudin which binds to the fibrinogen-recognition site of thrombin. In order to improve further the anticoagulative activity, the thrombin-inhibitory domain was modified by substituting linker 2. Introduction of a linker (FLLRNP) from the human thrombin receptor conferred about a 10-fold increase in anticoagulative activity in protein M37 compared with the parent molecule M23 carrying an aliphatic linker. The increase in anticoagulative activity was also reflected in the shift of the Ki value from 159 +/- 20 nM for M23 to 2.0 +/- 0.5 nM for M37. The increased thrombin-inhibitory activity of M37 may be due to the presence of an arginine in the linker from the thrombin receptor which may interact with one of two glutamic acid residues located at the exit of the thrombin substrate binding pocket. This explanation is supported by the observation that another chimera (M35) carrying a linker sequence with two acidic residues has relatively weak thrombin-inhibitory activity. The thrombin-inhibitory activity of M37 may be strong enough to substitute anticoagulative co-medication during fibrinolytic treatment.