Effects of eugenol on respiratory burst generation in newborn rat brainstem-spinal cord preparations.

Eugenol is contained in several plants including clove and is used as an analgesic drug. In the peripheral and central nervous systems, this compound modulates neuronal activity through action on voltage-gated ionic channels and/or transient receptor potential channels. However, it is unknown whether eugenol exerts any effects on the respiratory center neurons in the medulla. We examined the effects of eugenol on respiratory rhythm generation in the brainstem-spinal cord preparation from newborn rat (P0-P3). The preparations were superfused by artificial cerebrospinal fluid at 25-26 °C, and inspiratory C4 ventral root activity was monitored. Membrane potentials of respiratory neurons were recorded in the parafacial region of the rostral ventrolateral medulla. Bath application of eugenol (0.5-1 mM) decreased respiratory rhythm accompanied by strong inhibition of the burst activity of pre-inspiratory neurons. After washout, respiratory rhythm partly recovered, but the inspiratory burst duration was extremely shortened, and this continued for more than 60 min after washout. The shortening of C4 inspiratory burst by eugenol was not reversed by capsazepine (TRPV1 antagonist) or HC-030031 (TRPA1 antagonist), whereas the depression was partially blocked by GABAA antagonist bicuculline and glycine antagonist strychnine or GABAB antagonist phaclofen. A spike train of action potentials in respiratory neurons induced by depolarizing current pulse was depressed by application of eugenol. Eugenol decreased the negative slope conductance of pre-inspiratory neurons, suggesting blockade of persistent Na+ current. These results suggest that changes in both membrane excitability and synaptic connections are involved in the shortening of respiratory neuron bursts by eugenol.

Adhesion Control of Branched Catecholic Polymers by Acid Stimulation.

Biomimetic material design is a useful method for producing new functional materials. In recent years, catecholic polymers inspired from the adhesion mechanism of marine organisms have attracted attention. Here, we demonstrated the preparation of catecholic polymers by reversible addition-fragmentation chain transfer (RAFT) polymerization of an acetonide-protected catecholic monomer, that is, N-(2-(2,2-dimethylbenzo-1,3-dioxol-5-yl)ethyl)-acrylamide (DDEA). By selecting the specific RAFT reagents, well-defined branched PDDEA and linear PDDEA were obtained. These PDDEA samples showed stronger adhesion strength after deprotection by acid stimulation compared with that before deprotection. In addition, we demonstrated the adhesion control of synthetic polymers by photoirradiation in the presence of photoacid generators, which decompose under light and release an acid.

Effects of a TRPV1 agonist capsaicin on respiratory rhythm generation in brainstem-spinal cord preparation from newborn rats.

The heat-sensitive transient receptor potential vanilloid 1 (TRPV1) channels are expressed in the peripheral and central nervous systems. However, there is no report on how the activation of TRPV1 causes the modulation of neuronal activity in the medullary respiratory center. We examined effects of capsaicin, a specific agonist of TRPV1 channels, on respiratory rhythm generation in brainstem-spinal cord preparation from newborn rats. Capsaicin induced a biphasic response in the respiratory rhythm (a transient decrease followed by an increase in the C4 rate). The second-phase excitatory effect (but not the initial inhibitory effect) in the biphasic response was partly blocked by capsazepine or AMG9810 (TRPV1 antagonists). Capsaicin caused strong desensitization. After its washout, the strength of C4 burst inspiratory activity was augmented once per four to five respiratory cycles. The preinspiratory and inspiratory neurons showed tonic firings due to membrane depolarization during the initial inhibitory phase. In the presence of TTX, capsaicin increased the fluctuation of the membrane potential of the CO2-sensitive preinspiratory neurons in the parafacial respiratory group (pFRG), accompanied by slight depolarization. The C4 inspiratory activity did not stop, even 60-90 min after the application of 50/100 µM capsaicin. Voltage-sensitive dye imaging demonstrated that the spatiotemporal pattern of the respiratory rhythm generating networks after application of capsaicin (50 µM, 70-90 min) was highly similar to the control. A histochemical analysis using TRPV1 channel protein antibodies and mRNA demonstrated that the TRPV1 channel-positive cells were widely distributed in the reticular formation of the medulla, including the pFRG. Our results showed that the application of capsaicin in the medulla has various influences on the respiratory center: transient inhibitory and subsequent excitatory effects on the respiratory rhythm and periodical augmentation of the inspiratory burst pattern. The effects of capsaicin were partially blocked by TRPV1 antagonists but could be also induced at least partially via the non-specific action. Our results also suggested a minor contribution of the TRPV1 channels to central chemoreception.

A bioluminescent enzyme immunoassay for prostaglandin E(2) using Cypridina luciferase.

Prostaglandin E(2) is one of the major cyclooxygenase metabolites of arachidonic acid. We developed a competitive immunosorbent assay for prostaglandin E(2) utilizing a bioluminescent enzyme Cypridina luciferase. The prostaglandin E(2) amount could be quantified over the concentration ranging from 7.8 to 500 pg/mL. The amount of unlabeled prostaglandin E(2) required to displace 50% of the maximal binding of Cypridina luciferase-labeled prostaglandin E(2) (B/B(0)) was approximately 35 pg/mL. The results show a great potential of Cypridina luciferase as a new labeling enzyme for enzyme-linked immunosorbent assay.