हम एक दृष्टिकोण शुरू करने के लिए मूल्यांकन साइटोसोलिक Ca<sup> 2 +</supपृथक lymphatics में एकाग्रता के लिए अध्ययन Ca<sup> 2 +</sup> निर्भर है और Ca<sup> 2 +</supलसीका चिकनी मांसपेशी संकुचन> sensitizing तंत्र.
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.
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?…
The authors have nothing to disclose.
1. Ringer 5x Stock | |||
Company | Catalog Number | Amount | |
Sodium Chloride | EMD | SX0420-3 | 35 g |
Potassium Chloride | J.T. Baker | 3040 | 1.75 g |
Calcium Chloride | Sigma | C-3881 | 1.47 g |
Magnesium Sulfate | Sigma | M-9397 | 1.44 g |
Sterile Filtered Water | N/A | N/A | Bring to 1 L |
Sterile filter into autoclaved bottles and stores at 4 °C | |||
2. MOPS buffer | |||
Chemical | Company | Catalog Number | Amount |
MOPS | Sigma | M3183 | 125.6 g |
Sterile Filtered Water | N/A | N/A | Bring to 1 L |
Sterile filter into autoclaved bottles and stores at 4 °C | |||
3. Albumin Physiological Salt Solution (APSS) | |||
Chemical | Company | Catalog Number | Amount |
Ringer stock (5x) | N/A | N/A | 200 mL |
Mops Buffer | N/A | N/A | 5 mL |
Sodium Phosphate | Sigma | S-9638 | 0.168 g |
Sodium Pyruvate | Sigma | P5280 | 0.22 g |
EDTA sodium salt | Sigma | ED2SS | 0.0074 g |
Glucose | Sigma | G7528 | 0.901 g |
Albumin, Bovine | USB | 10856 | 10 g |
Sterile Filtered Water | N/A | N/A | Bring to 1 L |
Adjust pH to 7.4 at 37° C, then sterile filter into autoclaved bottles and store at 4 °C. |
Table 1. Specific Reagents Used. Store all at 4 °C.