Induction of apoptosis by thrombin in the cultured neurons of dorsal motor nucleus of the vagus.

BACKGROUND: A previous study demonstrated the presence of protease-activated receptor (PAR) 1 and 2 in the dorsal motor nucleus of vagus (DMV). The aim of this study is to characterize the effect of thrombin on the apoptosis of DMV neurons. METHODS: The dorsal motor nucleus of vagus neurons were isolated from neonatal rat brainstems using micro-dissection and enzymatic digestion and cultured. Apoptosis of DMV neurons were examined in cultured neurons. Apoptotic neuron was examined by TUNEL and ELISA. Data were analyzed using anova and Student's t-test. KEY RESULTS: Exposure of cultured DMV neurons to thrombin (0.1 to 10 U mL(-1)) for 24 h significantly increased apoptosis. Pretreatment of DMV neurons with hirudin attenuated the apoptotic effect of thrombin. Similar induction of apoptosis was observed for the PAR1 receptor agonist SFLLR, but not for the PAR3 agonist TFRGAP, nor for the PAR4 agonist YAPGKF. Protease-activated receptors 1 receptor antagonist Mpr(Cha) abolished the apoptotic effect of thrombin, while YPGKF, a specific antagonist for PAR4, demonstrated no effect. After administration of thrombin, phosphorylation of JNK and P38 occurred as early as 15 min, and remained elevated for up to 45 min. Pretreatment of DMV neurons with SP600125, a specific inhibitor for JNK, or SB203580, a specific inhibitor for P38, significantly inhibited apoptosis induced by thrombin. CONCLUSIONS & INFERENCES: Thrombin induces apoptosis in DMV neurons through a mechanism involving the JNK and P38 signaling pathways.

Functional protease-activated receptors in the dorsal motor nucleus of the vagus.

BACKGROUND: Protease-activated receptors (PARs), a family member of G-protein coupled receptors, are present and functionally active in a wide variety of cells. The object of this study was to demonstrate the presence and function of PAR-1 and PAR-2 in the dorsal motor nucleus of the vagus (DMV). METHODS: DMNV neurons were isolated from neonatal rat brainstems using micro-dissection and enzymatic digestion. Neurons were cultured in Neurobasal medium A containing 2% B27 supplement. Intracellular calcium concentration ([Ca(2+)](i)) was measured using fura-2 based microspectrometry. Expression of PARs was detected by RT-PCR and immunofluorescent staining. KEY RESULT: Thrombin and PAR-1 agonist peptide activate PAR-1 with a maximum change in [Ca(2+)](i) expressed as DeltaF/F0 of 229 +/- 14% and 137 +/- 7%, respectively. Trypsin and PAR-2 agonist peptide activate PAR-2 with a maximum DeltaF/F0 change of 258 +/- 12% and 242 +/- 10%, respectively. Inhibition of phospholipase C (PLC) by U73312 (1 microm) decreased the maximal change in DeltaF/F0 induced by PAR-1 activation from 140 +/- 17% to 21 +/- 3%, while the PAR-2-mediated maximal change in DeltaF/F0 decreased from 185 +/- 21% to 19 +/- 6%. Blockade of IP3 receptor with 2APB inhibited the maximal change in DeltaF/F0 due to PAR-1 and PAR-2 activation by 72 +/- 13% and 71 +/- 20% respectively. PAR-1 immnuoreactivity was present in DMV neurons. Increase in transcripts for PAR-1 and PAR-2 were detected in DMV tissues derived from IBD rats relative to control animals. CONCLUSIONS & INFERENCES: Our results indicate that PAR-1 and PAR-2 are present in the DMV neurons, and their activation leads to increases in intracellular calcium via signal transduction mechanism that involves activation of PLC and the production of IP3.

Arcuate nucleus transcriptome profiling identifies ankyrin repeat and suppressor of cytokine signalling box-containing protein 4 as a gene regulated by fasting in central nervous system feeding circuits.

The arcuate nucleus of the hypothalamus is a primary site for sensing blood borne nutrients and hormonal messengers that reflect caloric status. To identify novel energy homeostatic genes, we examined RNA extracts from the microdissected arcuate nucleus of fed and 48-h fasted rats using oligonucleotide microarrays. The relative abundance of 118 mRNA transcripts was increased and 203 mRNA transcripts was decreased during fasting. One of the down-regulated mRNAs was ankyrin-repeat and suppressor of cytokine signalling box-containing protein 4 (Asb-4). The predicted structure of Asb-4 protein suggested that it might encode an intracellular regulatory protein, and therefore its mRNA expression was investigated further. Reverse transcription quantitative polymerase chain reaction was used to validate down-regulation of Asb-4 mRNA in the arcuate nucleus of the fasted Sprague-Dawley rat (relative expression of Asb-4 mRNA: fed = 4.66 +/- 0.26; fasted = 3.96 +/- 0.23; n = 4, P < 0.01). Down-regulation was also demonstrated in the obese fa/fa Zucker rat, another model of energy disequilibrium (relative expression of Asb-4 mRNA: lean Zucker = 3.91 +/- 0.32; fa/fa = 2.93 +/- 0.26; n = 5, P < 0.001). In situ hybridisation shows that Asb-4 mRNA is expressed in brain areas linked to energy homeostasis, including the arcuate nucleus, paraventricular nucleus, dorsomedial nucleus, lateral hypothalamus and posterodorsal medial amygdaloid area. Double in situ hybridisation revealed that Asb-4 mRNA colocalises with key energy homeostatic neurones. In the fed state, Asb-4 mRNA is expressed by 95.6% of pro-opiomelanocortin (POMC) neurones and 46.4% of neuropeptide Y (NPY) neurones. By contrast, in the fasted state, the percentage of POMC neurones expressing Asb-4 mRNA drops to 73.2% (P < 0.001). Moreover, the density of Asb-4 mRNA per fasted POMC neurone is markedly decreased. Conversely, expression of Asb-4 mRNA by NPY neurones in the fasted state is modestly increased to 52.7% (P < 0.05). Based on its differential expression, neuroanatomical distribution and colocalisation, we hypothesise that Asb-4 is a gene involved in energy homeostasis.

Tissue distribution of recombinant human tumor necrosis factor alpha derivative in mice.

AIM: To study the tissue distribution and its mechanism of a new recombinant tumor necrosis factor alpha derivative (rhTNF alpha Da) in mice. METHODS: 125I-rhTNF alpha Da was prepared by Iodogen method. Tissue distribution of 125I-rhTNF alpha Da in mice was studied by determining radioactivity of tetrachloroacetic acid (TCA)- precipitable fraction in tissues. The isolated heart-lung perfusion study using 125I-rhTNF alpha Da perfusate was carried out to study the distribution characteristics of 125I-rhTNF alpha Da in lung. RESULTS: Except for thyroid, AUC of the TCA-precipitable 125I-rhTNF alpha Da in tissues was highest in lung, which was 12.2-fold of that in serum, while concentrations in other tissues were all lower than that in serum. Perfusion study in vitro revealed that the concentration of radio-labeled peptide in lung was higher than that in perfusate. On the contrary, level in heart was much lower than that in perfusate. The overall distribution of 125I-rhTNF alpha Da in lungs showed rapidly equilibratory, dose-dependent, saturable, competitive, and highly affinitive, with Kd 47.6 pmol.L-1 and Bmax 348 fmol.g-1 (lung tissue). CONCLUSION: The specific distribution of rhTNF alpha Da in lungs was its distinctive characteristics.