Molecular and pharmacological characterization of zebrafish 'contractile' and 'inhibitory' prostanoid receptors.

Prostanoids comprising prostaglandins (PGs) and thromboxanes (TXs) have been shown to play physiological and pathological roles in zebrafish. However, the molecular basis of zebrafish prostanoid receptors has not been established. Here, we demonstrate that there exist at least five 'contractile' (Ca(2+)-mobilizing) and one 'inhibitory' (Gi-coupled) prostanoid receptors in zebrafish; five 'contractile' receptors consisting of two PGE2 receptors (EP1a and EP1b), two PGF2α receptors (FP1 and FP2), and one TXA2 receptor TP, and one 'inhibitory' receptor, the PGE2 receptor EP3. [(3)H]PGE2 specifically bound to the membranes of cells expressing zebrafish EP1a, EP1b and EP3 with a Kd of 4.8, 1.8 and 13.6nM, respectively, and [(3)H]PGF2α specifically bound to the membranes of cells expressing zebrafish FP1 and FP2, with a Kd of 6.5 and 1.6nM, respectively. U-46619, a stable agonist for human and mouse TP receptors, significantly increased the specific binding of [(35)S]GTPγS to membranes expressing the zebrafish TP receptor. Upon agonist stimulation, all six receptors showed an increase in intracellular Ca(2+) levels, although the increase was very weak in EP1b, and pertussis toxin abolished only the EP3-mediated response. Zebrafish EP3 receptor also suppressed forskolin-induced cAMP formation in a pertussis toxin-sensitive manner. In association with the low structural conservation with mammalian receptors, most agonists and antagonists specific for mammalian EP1, EP3 and TP failed to work on each corresponding zebrafish receptor. This work provides further insights into the diverse prostanoid actions mediated by their receptors in zebrafish.

Molecular and pharmacological characterization of zebrafish 'relaxant' prostanoid receptors.

Prostanoids comprising prostaglandins (PGs) and thromboxanes have been shown to play physiological and pathological roles in zebrafish. However, the molecular basis of zebrafish prostanoid receptors has not been characterized to date. Here, we demonstrate that there exist at least six 'relaxant' (Gs-coupled) prostanoid receptors in zebrafish; one PGI2 receptor IP and five PGE2 receptors comprising two EP2 (EP2a and EP2b), and three EP4 receptors (EP4a, EP4b and EP4c). In contrast, we failed to find a zebrafish PGD2 receptor with any structure and/or character similarities to the mammalian DP1 receptor. [(3)H]iloprost, a stable IP radioligand, specifically bound to the membrane of cells expressing zebrafish IP with a Kd of 42nM, and [(3)H]PGE2 specifically bound to the membranes of cells expressing zebrafish EP2a, EP2b, EP4a, EP4b and EP4c with a Kd of 6.9, 6.0, 1.4, 3.3 and 1.2nM, respectively. Upon agonist stimulation, the 'relaxant' prostanoid receptors showed intracellular cAMP accumulation. The responsiveness of these zebrafish receptors to subtype-specific agonists correlated with their structural conservation to the corresponding receptor in mammals. RT-PCR analysis revealed that the six zebrafish prostanoid receptors show unique tissue distribution patterns; each receptor gene may hence be under unique transcriptional regulation. This work provides further insights into the diverse functions of prostanoids in zebrafish.

New predictive model of the touchdown times in a high level 110 m hurdles.

The present study aimed to establish a more robust, reliable statistical model of touchdown times based on the data of elite 110 m hurdlers to precisely predict performance based on touchdown times. We obtained 151 data (race time: 13.65 ± 0.33 s, range of race time: 12.91 s- 14.47 s) from several previous studies. Regression equations were developed to predict each touchdown time (times from the start signal to the instants of the leading leg landing after clearing 1st to 10th hurdles) from the race time. To avoid overtraining for each regression equation, data were split into training and testing data sets in accordance with a leave-one-out cross-validation. From the results of cross-validation, the agreement and generalization were compared between the present study model and the existing model. As a result, the proposed predictive equations showed a good agreement and generalization (R2 = 0.527-0.981, MSE = 0.0015-0.0028, MAE = 0.019-0.033) compared to that of existing equations (R2 = 0.481-0.979, MSE = 0.0017-0.0039, MAE = 0.034-0.063). Therefore, it can be assumed that the proposed predictive equations are a more robust, reliable model than the existing model. The touchdown times needed for coaches and elite hurdlers to set their target records will be accurately understood using the model of this study. Therefore, this study model would help to improve training interventions and race evaluations.

Kinematic Factors Associated with Hitting Hurdles During the Initial Phase of a 110-m Hurdle Race.

This study aimed to clarify the kinematic factors for the cause and effect of hitting hurdles during the initial phase of a 110-m hurdle run. Nine experienced male hurdlers participated in this study (body height: 1.74 ± 0.04 m, body mass: 67.4 ± 5.9 kg, age: 20.2 ± 1.4 years, personal best: 15.21 ± 0.47 s, seasonal best: 15.33 ± 0.55 s). Hurdlers undertook 12 trials of the initial phase of hurdling from the start to the second hurdle landing. Dual-sided sagittal plane motion was obtained from images from two high-speed cameras operating at 120 Hz. One 'hit' trial which had the largest horizontal displacement of markers fixed on the hurdle and one 'non-hit' trial which had the fastest time of hurdle clearance were extracted for each participant. Kinematic variables were compared between the two trials. Significantly lower height of the whole-body centre of mass at the take-off was found as a possible cause of hitting hurdles, caused by insufficient swing-up of the lead leg thigh. In contrast to conventional understanding, take-off velocity, take-off distance and the take-off angle were comparable between the 'hit' trial and 'non-hit' trial. Regarding the effect of hitting hurdles, it was observed that running velocity during hurdling was not substantially reduced. However, several characteristic movements were identified that might induce inefficient motion to re-accelerate running velocity during the following landing steps.

Essential role of prostaglandin E2 and the EP3 receptor in lymphatic vessel development during zebrafish embryogenesis.

Lymphatic endothelial cells arise from the venous endothelial cells in embryonic lymphatic development. However, the molecular mechanisms remain to be elucidated. We here report that prostaglandin (PG) E2 plays essential roles in the embryonic lymphatic development through the EP3 receptor, one of the PGE2 receptors. Knockdown of the EP3 receptor or inhibition of cyclooxygenases (COX; rate-limiting enzymes for PG synthesis) impaired lymphatic development by perturbing lymphatic specification during zebrafish development. These impairments by COX inhibition were recovered by treatment with sulprostone (EP1/3 agonist). Knockdown of the EP3 receptor further demonstrated its requirement in the expression of sex determining region Y-box 18 (sox18) and nuclear receptor subfamily 2, group F, member 2 (nr2f2), essential factors of the lymphatic specification. The EP3 receptor was expressed in the posterior cardinal vein (region of embryonic lymphatic development) and the adjacent intermediate cell mass (ICM) during the lymphatic specification. COX1 was expressed in the region more upstream of the posterior cardinal vein relative to the EP3 receptor, and the COX1-selective inhibitor impaired the lymphatic specification. On the other hand, two COX2 subtypes did not show distinct sites of expression around the region of expression of the EP3 receptor. Finally, we generated EP3-deficient zebrafish, which also showed defect in lymphatic specification and development. Thus, we demonstrated that COX1-derived PGE2-EP3 pathway is required for embryonic lymphatic development by upregulating the expression of key factors for the lymphatic specification.