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 responses. 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 circulation. This phasic contractile pattern is analogous to the cardiac cycle, with systolic and diastolic phases, and with a lower contraction frequency. In addition, lymphatic smooth muscle generates tone and displays myogenic constriction and dilation in response to increases and decreases in luminal pressure, respectively. 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 Ca(2+) concentration ([Ca(2+)](i)) plus sensitivity to Ca(2+) of the contractile elements in response to changes in the environment surrounding the cell. [Ca(2+)](i) is determined by the combination of the movement of Ca(2+) through plasma membrane ligand or voltage gated Ca(2+) channels and the release and uptake of Ca(2+) from internal stores. Cytosolic Ca(2+) binds to calmodulin and activates enzymes such as myosin light chain (MLC) kinase (MLCK), which in turn phosphorylates MLC leading to actin-myosin-mediated contraction. However, the sensitivity of this pathway to Ca(2+) can be regulated by the MLC phosphatase (MLCP). 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 [Ca(2+)](i) over time in isolated, perfused lymphatics in order to study Ca(2+)-dependent and Ca(2+)-sensitizing mechanisms of lymphatic smooth muscle contraction. Using isolated rat mesenteric collecting lymphatics we studied stretch-induced changes in [Ca(2+)](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. [Ca(2+)](i) was determined by loading lymphatics with the ratiometric, Ca(2+)-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.
Department of Physiology, School of Medicine, Louisiana State University Health Sciences Center, USA.
Flavia M
FM
Souza-Smith
Kristine M
KM
Kurtz
Jerome W
JW
Breslin
D000818:Animals; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry* / Q000378:metabolism; D042601:Lymphatic Vessels / Q000737:chemistry* / Q000378:metabolism; D008297:Male; D009119:Muscle Contraction; D009130:Muscle, Smooth / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D002118:Calcium