Name: measurement of cytosolic ca2+ in isolated contractile lymphatics
Uploaded: 2011-12-08T13:00:00
Description: Watch this Scientific Journal Video about Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics at JoVE.com

<p class="jove_content"&gt; यह काम NIH अनुदान P20RR018766 और शराब अनुसंधान के लिए ABMRF / फाउंडेशन से अनुदान द्वारा समर्थित किया गया था.</p

1. पशु <ol><li> सभी प्रक्रियाओं संस्थागत पशु देखभाल और लुइसियाना राज्य विश्वविद्यालय के स्वास्थ्य विज्ञान केंद्र में प्रयोग करें समिति द्वारा अनुमोदित किया गया और गया राष्ट्रीय स्वास्थ्य संस्थान (एनआईए...

<ol>
	<li>Chakraborty, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic+system:+a+vital+link+between+metabolic+syndrome+and+inflammation.">Lymphatic system: a vital link between metabolic syndrome and inflammation.</a> Ann. N. Y. Acad. Sci. 1207, 94-94 (2010).</li><li>Zawieja, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic+biology+and+the+microcirculation:+past,+present+and+future.">Lymphatic biology and the microcirculation: past, present and future.</a> Microcirculation. 12, 141-141 (2005).</li><li>Benoit, J. N., Zawieja, D. C., Goodman, A. H., Granger, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+intact+mesenteric+lymphatic+pump+and+its+responsiveness+to+acute+edemagenic+stress.">Characterization of intact mesenteric lymphatic pump and its responsiveness to acute edemagenic stress.</a> Am. J. Physiol. 257, H2059-H2059 (1989).</li><li>Davis, M. J., Davis, A. M., Ku, C. W., Gashev, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myogenic+constriction+and+dilation+of+isolated+lymphatic+vessels.">Myogenic constriction and dilation of isolated lymphatic vessels.</a> Am. J. Physiol. Heart. Circ. Physiol. 296, H293-H293 (2009).</li><li>Dougherty, P. J., Davis, M. J., Zawieja, D. C., Muthuchamy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+sensitivity+and+cooperativity+of+permeabilized+rat+mesenteric+lymphatics.">Calcium sensitivity and cooperativity of permeabilized rat mesenteric lymphatics.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 294, R1524-R1524 (2008).</li><li>Fay, F. S., Shlevin, H. H., Granger, W. C., Taylor, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aequorin+luminescence+during+activation+of+single+isolated+smooth+muscle+cells.">Aequorin luminescence during activation of single isolated smooth muscle cells.</a> Nature. 280, 506-506 (1979).</li><li>Wang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+myosin+light+chain+phosphorylation+decreases+rat+mesenteric+lymphatic+contractile+activity.">Inhibition of myosin light chain phosphorylation decreases rat mesenteric lymphatic contractile activity.</a> Am. J. Physiol. Heart. Circ. Physiol. 297, 726-726 (2009).</li><li>Karaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+movements,+distribution,+and+functions+in+smooth+muscle.">Calcium movements, distribution, and functions in smooth muscle.</a> Pharmacol. Rev. 49, 157-157 (1997).</li><li>Ratz, P. H., Berg, K. M., Urban, N. H., Miner, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+smooth+muscle+calcium+sensitivity:+KCl+as+a+calcium-sensitizing+stimulus.">Regulation of smooth muscle calcium sensitivity: KCl as a calcium-sensitizing stimulus.</a> Am. J. Physiol. Cell. Physiol. 288, C769-C769 (2005).</li><li>Somlyo, A. P., Somlyo, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++sensitivity+of+smooth+muscle+and+nonmuscle+myosin+II:+modulated+by+G+proteins,+kinases,+and+myosin+phosphatase.">Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase.</a> Physiol. Rev. 83, 1325-1325 (2003).</li><li>Souza-Smith, F. M., Kurtz, K. M., Molina, P. E., Breslin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+mesenteric+collecting+lymphatic+pump+function+following+acute+alcohol+intoxication.">Adaptation of mesenteric collecting lymphatic pump function following acute alcohol intoxication.</a> Microcirculation. 17, 514-514 (2010).</li><li>Breslin, J. W., Yuan, S. Y., Wu, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-C+alters+barrier+function+of+cultured+lymphatic+endothelial+cells+through+a+VEGFR-3-dependent+mechanism.">VEGF-C alters barrier function of cultured lymphatic endothelial cells through a VEGFR-3-dependent mechanism.</a> Lymphat. Res. Biol. 5, 105-105 (2007).</li><li>Shirasawa, Y., Benoit, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretch-induced+calcium+sensitization+of+rat+lymphatic+smooth+muscle.">Stretch-induced calcium sensitization of rat lymphatic smooth muscle.</a> Am. J. Physiol. Heart. Circ. Physiol. 285, H2573-H2573 (2003).</li><li>Imtiaz, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pacemaking+through+Ca2++stores+interacting+as+coupled+oscillators+via+membrane+depolarization.">Pacemaking through Ca2+ stores interacting as coupled oscillators via membrane depolarization.</a> Biophys. J. 92, 3843-3843 (2007).</li><li>Muller, J. M., Davis, M. J., Kuo, L., Chilian, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+coronary+endothelial+cell+Ca2++concentration+during+shear+stress-+and+agonist-induced+vasodilation.">Changes in coronary endothelial cell Ca2+ concentration during shear stress- and agonist-induced vasodilation.</a> Am. J. Physiol. 276, 1706-1706 (1999).</li><li>Ferrusi, I., Zhao, J., van Helden, D., von der Weid, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclopiazonic+acid+decreases+spontaneous+transient+depolarizations+in+guinea+pig+mesenteric+lymphatic+vessels+in+endothelium-dependent+and+-independent.">Cyclopiazonic acid decreases spontaneous transient depolarizations in guinea pig mesenteric lymphatic vessels in endothelium-dependent and -independent.</a> 286, H2287-H2287 (2004).</li></ol>

तरीकों का एक उपन्यास संयोजन लसीका वाहिकाओं के आंतरिक पंप अध्ययन करने के लिए नियोजित किया गया था. के लिए एक साथ में बदलाव को मापने की क्षमता [2 Ca +] और मैं पम्पिंग lymphatics में व्यास 2 Ca के रिश्तेद?...

<table border="1"><tbody><tr><td> 1. घंटी 5x शेयर </td></tr><tr><td रासायनिक </td><td> कंपनी </td><td> सूचि संख्या </td><td> राशि </td></tr><tr><td> सोडियम क्लोराइड </td><td> ईएमडी </td><td> SX0420 - 3 </td><td> 35 छ </td></tr><tr><td> पोटेशियम क्लोराइड </td><td> संयुक्त बेकर </td><td> 3040 </td><td> 1.75 छ </td></tr><tr><td> कैल्सियम क्लोराइड </td><td> सिग्मा </td><td> सी 3881 </td><td> 1.47 छ </td></tr><tr><td> मैग्नीशियम सल्फेट </td><td> सिग्मा </td><td> M-+९,३९७ </td><td> 1.44 छ </td></tr><tr><td> बाँझ छानने का पानी </td><td> N / A </td><td> N / A </td><td> 1 एल को लाओ </td></tr><tr><td> 4 autoclaved बोतलें और दुकानों में बाँझ फिल्टर डिग्री सेल्सियस </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 2. MOPS बफर </td><td></td><td></td><td></td></tr><tr><td> रासायनिक </td><td> कंपनी </p&gt; <td> सूचि संख्या </td><td> राशि </td></tr><tr><td> MOPS </td><td> सिग्मा </td><td> M3183 </td><td> 125.6 छ </td></tr><tr><td> बाँझ छानने का पानी </td><td> N / A </td><td> N / A </td><td> 1 एल को लाओ </td></tr><tr><td> 4 autoclaved बोतलें और दुकानों में बाँझ फिल्टर डिग्री सेल्सियस </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 3. Albumin शारीरिक नमक समाधान (APSS) </td></tr><tr><td> रासायनिक </td><td> कंपनी </td><td> सूचि संख्या </td><td> राशि </td></tr><tr><td> घंटी शेयर (5x) </td><td> N / A </td><td> N / A </td><td> 200 एमएल </td></tr><tr><td> Mops बफर </td><td> N / A </td><td> N / A </td><td> 5 एमएल </td></tr><tr><td> सोडियम फास्फेट </td><td> सिग्मा </td><td> S-9638 </td><td> .168 छ </td></tr><tr><td> सोडियम पाइरूवेट </td><td> सिग्मा </td><td> P5280 </td><td> 0.22 छ </td></tr><tr><td> EDTA सोडियम सालटी </td><td> सिग्मा </td><td> ED2SS </td><td> ०.००७४ जी </td></tr><tr><td> ग्लूकोज </td><td> सिग्मा </td><td> G7528 </td><td> .901 छ </td></tr><tr><td> Albumin गोजातीय </td><td> यूएसबी </td><td> 10856 </td><td> 10 छ </td></tr><tr><td> बाँझ छानने का पानी </td><td> N / A </td><td> N / A </td><td> 1 एल को लाओ </td></tr><tr><td> 7,4 करने के लिए 37 में पीएच समायोजित डिग्री सेल्सियस, तो 4 बजे autoclaved बोतलें और दुकान में बाँझ फिल्टर डिग्री सेल्सियस </td></tr></tbody></table> तालिका 1 विशिष्ट अभिकर्मकों प्रयुक्त.. 4 में सभी स्टोर डिग्री सेल्सियस

measurement of cytosolic ca2+ in isolated contractile lymphatics

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a surveillance system for potentially harmful antigens, optimizing mucosal immunity and adaptive immune responses1. Lymph is formed from interstitial fluid that enters blind-ended initial lymphatics, and then is transported against a pressure gradient in larger collecting lymphatics. Each collecting lymphatic is made up of a series of segments called lymphangions, separated by bicuspid valves that prevent backflow. Each lymphangion possesses a contractile cycle that propels lymph against a pressure gradient toward the central circulation2. This phasic contractile pattern is analogous to the cardiac cycle, with systolic and diastolic phases, and with a lower contraction frequency4. In addition, lymphatic smooth muscle generates tone and displays myogenic constriction and dilation in response to increases and decreases in luminal pressure, respectively5. A hybrid of molecular mechanisms that support both the phasic and tonic contractility of lymphatics are thus proposed.
Contraction of smooth muscle is generally regulated by the cytosolic Ca2+ concentration ([Ca2+]i) plus sensitivity to Ca2+, of the contractile elements in response to changes in the environment surrounding the cell6. [Ca2+]i is determined by the combination of the movement of Ca2+ through plasma membrane ligand or voltage gated Ca2+ channels and the release and uptake of Ca2+ from internal stores. Cytosolic Ca2+ binds to calmodulin and activates enzymes such as myosin light chain (MLC) kinase (MLCK), which in turn phosphorylates MLC leading to actin-myosin-mediated contraction8. However, the sensitivity of this pathway to Ca2+ can be regulated by the MLC phosphatase (MLCP)9. MLCP activity is regulated by Rho kinase (ROCK) and the myosin phosphatase inhibitor protein CPI-17.
Here, we present a method to evaluate changes in [Ca2+]i over time in isolated, perfused lymphatics in order to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction. Using isolated rat mesenteric collecting lymphatics we studied stretch-induced changes in [Ca2+]i and contractile activity. The isolated lymphatic model offers the advantage that pressure, flow, and the chemical composition of the bath solution can be tightly controlled. [Ca2+]i was determined by loading lymphatics with the ratiometric, Ca2+-binding dye Fura-2. These studies will provide a new approach to the broader problem of studying the different molecular mechanisms that regulate phasic contractions versus tonic constriction in lymphatic smooth muscle.

Watch this Scientific Journal Video about Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics at JoVE.com

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

हम एक दृष्टिकोण शुरू करने के लिए मूल्यांकन साइटोसोलिक Ca<sup&gt; 2 +</supपृथक lymphatics में एकाग्रता के लिए अध्ययन Ca<sup&gt; 2 +</sup&gt; निर्भर है और Ca<sup&gt; 2 +</supलसीका चिकनी मांसपेशी संकुचन&gt; sensitizing तंत्र.

साइटोसोलिक Ca के मापन 2 + पृथक सिकुड़ा lymphatics में

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a ...

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Immunology and Infection

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

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