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; Dette arbejde blev støttet af NIH give P20RR018766 og en bevilling fra ABMRF / Foundation for Alkoholforskning.</p

1. Dyr <ol><li> Alle procedurer blev godkendt af Institutional Animal Care og bruge udvalget ved Louisiana State University Health Sciences Center, og blev udført i overensstemmelse med retningslinjerne fra National Institute of Health (NIH publikation nr. 85-12, revideret 1996). Mand Sprague-Dawley rotter (Charles River Laboratories, 270-350 g krop wt) blev anbragt i en kontrolleret temperatur (22 ° C) og kontrolleret belysning (00:12 H lys mørke cyklus) miljø. Efter ankomsten blev rotterne indgivet til en uges a...

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En ny kombination af metoder, der var ansat til at studere iboende pumpning af lymfekar. Evnen til samtidigt at måle ændringer i [Ca 2 +] i og diameter i pumpe lymfevejene vil give mulighed for studier af de relative bidrag af Ca 2 +-afhængige og Ca 2 +-sensibiliserende signalveje i den samlede ordning, der gælder for lymfe kontraktile cyklus. Det lymfe kontraktile cyklus består af phasic sammentrækninger ovenpå tone. Dataene viser, at hver ...

<table border="1"><tbody><tr><td> 1. Ringer 5x Stock </td></tr><tr><td Kemisk </td><td> Firma </td><td> Katalognummer </td><td> Beløb </td></tr><tr><td> Sodium Chloride </td><td> EMD </td><td> SX0420-3 </td><td> 35 g </td></tr><tr><td> Kaliumklorid </td><td> JT Baker </td><td> 3040 </td><td> 1,75 g </td></tr><tr><td> Calcium Chloride </td><td> Sigma </td><td> C-3881 </td><td> 1,47 g </td></tr><tr><td> Magnesium Sulfate </td><td> Sigma </td><td> M-9397 </td><td> 1,44 g </td></tr><tr><td> Sterile filtreret vand </td><td> N / A </td><td> N / A </td><td> Bring til 1 L </td></tr><tr><td> Steril filter i autoklaveres flasker og gemmer ved 4 ° C </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 2. MOPS buffer </td><td></td><td></td><td></td></tr><tr><td> Kemisk </td><td> Firma </td&gt; <td> Katalognummer </td><td> Beløb </td></tr><tr><td> MOPS </td><td> Sigma </td><td> M3183 </td><td> 125,6 g </td></tr><tr><td> Sterile filtreret vand </td><td> N / A </td><td> N / A </td><td> Bring til 1 L </td></tr><tr><td> Steril filter i autoklaveres flasker og gemmer ved 4 ° C </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 3. Albumin Fysiologiske Salt Solution (APSS) </td></tr><tr><td> Kemisk </td><td> Firma </td><td> Katalognummer </td><td> Beløb </td></tr><tr><td> Ringer materiel (5x) </td><td> N / A </td><td> N / A </td><td> 200 ml </td></tr><tr><td> Mops Buffer </td><td> N / A </td><td> N / A </td><td> 5 ml </td></tr><tr><td> Natrium Fosfat </td><td> Sigma </td><td> S-9638 </td><td> 0,168 g </td></tr><tr><td> Sodium Pyruvat </td><td> Sigma </td><td> P5280 </td><td> 0,22 g </td></tr><tr><td> EDTA natrium salt </td><td> Sigma </td><td> ED2SS </td><td> 0,0074 g </td></tr><tr><td> Glucose </td><td> Sigma </td><td> G7528 </td><td> 0,901 g </td></tr><tr><td> Albumin, Bovin </td><td> USB </td><td> 10856 </td><td> 10 g </td></tr><tr><td> Sterile filtreret vand </td><td> N / A </td><td> N / A </td><td> Bring til 1 L </td></tr><tr><td> Justér pH til 7,4 ved 37 ° C, så sterilt filter i autoklaveres flasker og opbevares ved 4 ° C. </td></tr></tbody></table> Tabel 1. Specifikke reagenser, der anvendes. Opbevar alle ved 4 ° C.

measurement of cytosolic ca2+ in isolated contractile lymphatics

Lymfekar omfatter et multifunktionelt transport-system, der sikrer flydende homeostase, leverer lipider til den centrale cirkulation, og fungerer som et overvågningssystem for potentielt skadelige antigener, optimering slimhinde immunitet og adaptive immunrespons 1. Lymfeknuder er dannet af interstitiel væske, der kommer ind blind-ended indledende lymfevejene, og derefter transporteres mod et tryk gradient i større indsamling lymfevejene. Hver indsamle lymfe består af en række segmenter kaldet lymphangions, adskilt af bicuspid ventiler, der forhindrer tilbageløb. Hver lymphangion besidder en kontraktile cyklus, der driver lymfeknuder mod et tryk gradient mod det centrale cirkulation 2. Denne phasic kontraktile mønster svarer til hjertets cyklus, med et systolisk og diastolisk faser, og med en lavere sammentrækning frekvens 4. Desuden genererer lymfe glat muskeltonus og viser myogenic konstriktion og dilatation jegn reaktion på stigninger og fald i luminale pres, henholdsvis 5. En hybrid af molekylære mekanismer, der understøtter både phasic og tonic kontraktilitet af lymfevejene er således foreslået. Sammentrækning af glat muskulatur er generelt reguleret af cytosole Ca 2 + koncentration ([Ca 2 +] i) plus følsomhed for Ca 2 +, af de kontraktile elementer i reaktion på ændringer i miljøet omkring cellen 6. [Ca 2 +] Jeg er bestemmes af kombinationen af den frie bevægelighed for Ca 2 + gennem plasmamembranen ligand eller spændingsfølsomme Ca 2 + kanaler og frigivelse og optagelse af Ca 2 + fra interne butikker. Cytosole Ca 2 + binder sig til calmodulin og aktiverer enzymer såsom myosin lyskæde (MLC) kinase (MLCK), som igen phosphorylates MLC fører til actin-myosin-medieret kontraktion 8. Men følsomheden af ​​denne vej til Ca <sup&gt; 2 + kan reguleres af MLC fosfatase (MLCP) 9. MLCP virksomhed er omfattet af Rho-kinase (ROCK) og myosin fosfatase-hæmmer protein CPI-17. Her præsenterer vi en metode til at vurdere ændringer i [Ca 2 +] i over tid i isolerede, perfunderet lymfevejene for at studere Ca 2 +-afhængige og Ca 2 +-sensibiliserende mekanismer af lymfatisk glatte muskelsammentrækning. Brug af isolerede rotte mesenteriallymfeknuderne indsamle lymfevejene vi studerede stretch-inducerede ændringer i [Ca 2 +] i og kontraktile aktivitet. Den isolerede lymfe-modellen har den fordel, tryk, flow, og den kemiske sammensætning af badet løsningen kan blive nøje kontrolleret. [Ca 2 +] Jeg blev bestemt ved at indlæse lymfevejene med ratiometrisk, Ca 2 +-bindende dye Fura-2. Disse undersøgelser vil give en ny tilgang til det bredere problem med at studere de forskellige molekylære mekanismer, der regulerer phasicsammentrækninger versus tonic konstriktion i lymfe glatte muskulatur.

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

Vi introducerer en tilgang til at evaluere cytosol Ca<sup&gt; 2 +</sup&gt; Koncentration i isolerede lymfevejene at studere Ca<sup&gt; 2 +</sup&gt;-Afhængige og Ca<sup&gt; 2 +</sup&gt;-Sensibiliserende mekanismer af lymfatisk glatte muskelsammentrækning.

Måling af cytosole Ca 2 + I isolerede kontraktile lymfekar

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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Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

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