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The enzymatic pathway of MMP2 is involved in synaptic remodeling induced by axotomy of rat superior cervical ganglion neurons
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Abstracts / Journal of Physiology - Paris 99 (2006) 245–278
heterologous regulation between these receptors appears. It can be comprehended as ‘‘fine tuning’’ of signal brought by neurotransmitters (Selbie and Hill, 1998). Recently, we have demonstrated heterologous regulation of this pair of receptors in many cases in the heart tissue (Myslivecˇek and Trojan, 2003). Now, we wondered to know: (1) if this pair of receptors could adapt to targeted disruption of CRH (corticotropine releasing hormone) gene, (2) if there are some sex differences in receptor expression, (3) if there could be difference in the effect on a-adrenoceptors and badrenoceptors due to different affinity to noradrenaline (increased in CRH KO), and (4) what are the effects of immobilisation stress in CRH KO mice in comparison to their wild type (WT) counterparts. We investigated the densities of muscarinic (MR) and of a1- (AAR), total number of b-adrenoceptors (BAR), b1- and b2-adrenoceptors in the lungs of CRH KO. Lung tissue can serve as a model of tissue in which the effects of sympathetic neurotransmiters (i.e., adrenaline or noradrenaline) are antagonised by the effects of parasympathetic neurotransmitters. Compounds [3H]quinuclidinyl benzilate, [3H]CGP 12177 and [3H]prazosin were utilised as specific markers of the muscarinic and adrenergic receptors, respectively. The respective subtypes were identified using specific antagonists (CGP 20712A, ICI 118.551). The amount of receptor binding sites in CRH KO mice were decreased in comparison to their wild type counterparts. This effect was more prominent in males than in females and AAR were mainly affected. The stress brings about profound decrease in MR, BAR and AAR both in WT and KO females while lesser effect was seen in males. The changes in total BAR densities brought by stress were caused by b2-adrenoceptors, the b1-adrenoceptors were not changed or increased (in females, after seventh immobilization). On the other hand, the changes in the densities of MR and AAR brought by stress were more noticeable in WT animals. We can therefore conclude that: (1) targeted disruption of CRH gene is able to change the densities of MR, AAR and BAR in the lung tissue; (2) stress reaction differs in CRH KO animals in comparison to their WT counterparts; (3) there are sex differences (both in receptor expression and reaction to stress). Our results showed that receptors adapt to the changed environmental condition through deep changes in the level of receptor binding sites. Supported by grant of SP 51/028 08 00/028 08, EU Centre of Excellence Grant ICA1-CT-2000-70008 and by NATO LST.EAP.CLG.980745. doi:10.1016/j.jphysparis.2005.12.060 Myslivecˇek, J., Trojan, S., 2003. Regulation of b-adrenoceptors and muscarinic receptors in the heart. Gen. Physiol. Biophy. 221, 3–14. Selbie, L.A., Hill, S.J., 1998. G protein-coupled-receptor cross-talk: the fine-tuning of multiple receptor-signaling pathways. Trends Pharmacol. Sci. 19, 87–93.
The enzymatic pathway of MMP2 is involved in synaptic remodeling induced by axotomy of rat superior cervical ganglion neurons Lucia Leone a, Maria Egle De Stefano a, Arianna Del Signore a, Tamara Corina Petrucci b, Paola Paggi a a
Dip. Biologia Cellulare e dello Sviluppo, Universita` ‘‘La Sapienza’’, Italy b Istituto Superiore di Sanita`, Roma, Italy E-mail address: [email protected] (P. Paggi) We previously demonstrated an increase in the expression and activation of metalloproteinase2 (MMP2) in rat superior cervical ganglion (SCG) after ganglionic neuron axotomy. To clarify the enzymatic pathway leading to MMP2 activation, we investigated the expression pattern of both the membrane-type 1 MMP (MT1-MMP) and TIMP2, the main MMP2 activator and inhibitor, respectively. We used Western blot to analyze the expression levels of MT1MMP and TIMP2 in SCG extracts at 15, 30, 60 min, and 1, 3 and 5 d post-axotomy. The level of MT1-MMP begins to increase at 30 min post-axotomy and remains higher than that of the control till 1 d after injury. On the other hand, TIMP2 level shows a first increase at 30 min, followed by a second one starting at 1 d after axotomy. The increase in MT1-MMP and TIMP2 levels suggests an activation cascade for MMP2 similar to that described in other experimental systems (Toth et al., 2000). RT-PCR analysis (15 min, 1, 6 h, and 1, 3 and 5 d post-axotomy) reveals that the level of MMP2 mRNA is not significantly affected by postganglionic nerve crush, while MT1-MMP and TIMP2 mRNAs gradually increase reaching a peak at 1 d. Recent data indicate b-dystroglycan (b-DG), a protein implicated in synaptic contact stabilization, as a possible MMPs substrate in the central nervous system. We investigated whether DG was modified in injured SCG. Western blot reveals an increase in the 30 kDa b-DG degradation product as early as 1 d after axotomy. In conclusion, our data indicate that both MT1MMP and TIMP2, together with MMP2, are involved in intraganglionic synapses remodeling after SCG neuron axotomy and that DG may be one of MMP2 substrate. doi:10.1016/j.jphysparis.2005.12.061 Toth, M., Bernardo, M.M., Gervasi, D.C., Soloway, P.D., Wang, Z., Bigg, H.F., Overall, C.M., DeClerck, Y.A., Tschesche, H., Cher, M.L., Brown, S., Mobashery, S., Fridman, R., 2000. Tissue inhibitor of metalloproteinase (TIMP)-2 acts synergistically with synthetic matrix metalloproteinase (MMP) inhibitors but not with TIMP-4 to enhance the (membrane type 1)-MMP-dependent activation of pro-MMP-2. J. Biol. Chem. 275 (52), 41415–41423.
Molecular determinants of allosteric modulators of the calcium-sensing receptor Christophe Pe´trel a, Albane Kessler b, Philippe Dauban b, Robert H. Dodd b, Didier Rognan c, Martial Ruat a
Abstracts / Journal of Physiology - Paris 99 (2006) 245–278
heterologous regulation between these receptors appears. It can be comprehended as ‘‘fine tuning’’ of signal brought by neurotransmitters (Selbie and Hill, 1998). Recently, we have demonstrated heterologous regulation of this pair of receptors in many cases in the heart tissue (Myslivecˇek and Trojan, 2003). Now, we wondered to know: (1) if this pair of receptors could adapt to targeted disruption of CRH (corticotropine releasing hormone) gene, (2) if there are some sex differences in receptor expression, (3) if there could be difference in the effect on a-adrenoceptors and badrenoceptors due to different affinity to noradrenaline (increased in CRH KO), and (4) what are the effects of immobilisation stress in CRH KO mice in comparison to their wild type (WT) counterparts. We investigated the densities of muscarinic (MR) and of a1- (AAR), total number of b-adrenoceptors (BAR), b1- and b2-adrenoceptors in the lungs of CRH KO. Lung tissue can serve as a model of tissue in which the effects of sympathetic neurotransmiters (i.e., adrenaline or noradrenaline) are antagonised by the effects of parasympathetic neurotransmitters. Compounds [3H]quinuclidinyl benzilate, [3H]CGP 12177 and [3H]prazosin were utilised as specific markers of the muscarinic and adrenergic receptors, respectively. The respective subtypes were identified using specific antagonists (CGP 20712A, ICI 118.551). The amount of receptor binding sites in CRH KO mice were decreased in comparison to their wild type counterparts. This effect was more prominent in males than in females and AAR were mainly affected. The stress brings about profound decrease in MR, BAR and AAR both in WT and KO females while lesser effect was seen in males. The changes in total BAR densities brought by stress were caused by b2-adrenoceptors, the b1-adrenoceptors were not changed or increased (in females, after seventh immobilization). On the other hand, the changes in the densities of MR and AAR brought by stress were more noticeable in WT animals. We can therefore conclude that: (1) targeted disruption of CRH gene is able to change the densities of MR, AAR and BAR in the lung tissue; (2) stress reaction differs in CRH KO animals in comparison to their WT counterparts; (3) there are sex differences (both in receptor expression and reaction to stress). Our results showed that receptors adapt to the changed environmental condition through deep changes in the level of receptor binding sites. Supported by grant of SP 51/028 08 00/028 08, EU Centre of Excellence Grant ICA1-CT-2000-70008 and by NATO LST.EAP.CLG.980745. doi:10.1016/j.jphysparis.2005.12.060 Myslivecˇek, J., Trojan, S., 2003. Regulation of b-adrenoceptors and muscarinic receptors in the heart. Gen. Physiol. Biophy. 221, 3–14. Selbie, L.A., Hill, S.J., 1998. G protein-coupled-receptor cross-talk: the fine-tuning of multiple receptor-signaling pathways. Trends Pharmacol. Sci. 19, 87–93.
The enzymatic pathway of MMP2 is involved in synaptic remodeling induced by axotomy of rat superior cervical ganglion neurons Lucia Leone a, Maria Egle De Stefano a, Arianna Del Signore a, Tamara Corina Petrucci b, Paola Paggi a a
Dip. Biologia Cellulare e dello Sviluppo, Universita` ‘‘La Sapienza’’, Italy b Istituto Superiore di Sanita`, Roma, Italy E-mail address: [email protected] (P. Paggi) We previously demonstrated an increase in the expression and activation of metalloproteinase2 (MMP2) in rat superior cervical ganglion (SCG) after ganglionic neuron axotomy. To clarify the enzymatic pathway leading to MMP2 activation, we investigated the expression pattern of both the membrane-type 1 MMP (MT1-MMP) and TIMP2, the main MMP2 activator and inhibitor, respectively. We used Western blot to analyze the expression levels of MT1MMP and TIMP2 in SCG extracts at 15, 30, 60 min, and 1, 3 and 5 d post-axotomy. The level of MT1-MMP begins to increase at 30 min post-axotomy and remains higher than that of the control till 1 d after injury. On the other hand, TIMP2 level shows a first increase at 30 min, followed by a second one starting at 1 d after axotomy. The increase in MT1-MMP and TIMP2 levels suggests an activation cascade for MMP2 similar to that described in other experimental systems (Toth et al., 2000). RT-PCR analysis (15 min, 1, 6 h, and 1, 3 and 5 d post-axotomy) reveals that the level of MMP2 mRNA is not significantly affected by postganglionic nerve crush, while MT1-MMP and TIMP2 mRNAs gradually increase reaching a peak at 1 d. Recent data indicate b-dystroglycan (b-DG), a protein implicated in synaptic contact stabilization, as a possible MMPs substrate in the central nervous system. We investigated whether DG was modified in injured SCG. Western blot reveals an increase in the 30 kDa b-DG degradation product as early as 1 d after axotomy. In conclusion, our data indicate that both MT1MMP and TIMP2, together with MMP2, are involved in intraganglionic synapses remodeling after SCG neuron axotomy and that DG may be one of MMP2 substrate. doi:10.1016/j.jphysparis.2005.12.061 Toth, M., Bernardo, M.M., Gervasi, D.C., Soloway, P.D., Wang, Z., Bigg, H.F., Overall, C.M., DeClerck, Y.A., Tschesche, H., Cher, M.L., Brown, S., Mobashery, S., Fridman, R., 2000. Tissue inhibitor of metalloproteinase (TIMP)-2 acts synergistically with synthetic matrix metalloproteinase (MMP) inhibitors but not with TIMP-4 to enhance the (membrane type 1)-MMP-dependent activation of pro-MMP-2. J. Biol. Chem. 275 (52), 41415–41423.
Molecular determinants of allosteric modulators of the calcium-sensing receptor Christophe Pe´trel a, Albane Kessler b, Philippe Dauban b, Robert H. Dodd b, Didier Rognan c, Martial Ruat a