Temperature effects on morphological integrity and Ca²⁺signaling in freshly isolated murine feed artery endothelial cell tubes

Abstract

To study Ca(2+) signaling in the endothelium of murine feed arteries, we determined the in vitro stability of endothelial cell (EC) tubes freshly isolated from abdominal muscle feed arteries of male and female C57BL/6 mice (5-9 mo, 25-35 g). We tested the hypothesis that intracellular Ca(2+) concentration ([Ca(2+)](i)) responses to muscarinic receptor activation would increase with temperature. Intact EC tubes (length: 1-2 mm, width: 65-80 μm) were isolated using gentle enzymatic digestion with trituration to remove smooth muscle cells. A freshly isolated EC tube was secured in a chamber and superfused at 24 (room temperature), 32, or 37°C. Using fura-2 dye, [Ca(2+)](i) was monitored (ratio of fluorescence at 340- to 380-nm wavelength) at rest and in response to bolus doses of ACh (20 nmol to 200 μmol). The morphological integrity of EC tubes was preserved at 24 and 32°C. Based on the Ca(2+) K(d) values we determined for fura-2 (174 nM at 24°C and 146 nM at 32°C), resting [Ca(2+)](i) remained stable for 180 min at both 24 and 32°C (27 ± 4 and 34 ± 2 nM, respectively), with peak responses to ACh (20 μmol) increasing from ∼220 nM at 24°C to ∼500 nM at 32°C (P < 0.05). There was no difference in responses to ACh between EC tubes from male versus female mice. When EC tubes were maintained at 37°C (typical in vivo temperature), resting [Ca(2+)](i) increased by ∼30% within 15 min, and gaps formed between individual ECs as they retracted and extruded dye, precluding further study. We conclude that EC tubes enable Ca(2+) signaling to be evaluated in the freshly isolated endothelium of murine feed arteries. While Ca(2+) responses are enhanced by approximately twofold at 32 versus 24°C, the instability of EC tubes at 37°C precludes their study at typical body temperature.

Calcium and electrical signaling in arterial endothelial tubes: New insights into cellular physiology and cardiovascular function

“…As an alternative to the study of single isolated ECs and/or cell culture, use of intact endothelial “tubes” (see Figure 1A) freshly isolated from hamster cremaster arterioles was first developed and published by the Jackson laboratory (68). The Segal laboratory applied further refinements of the endothelial tube preparation of rodent skeletal muscle arteries and arterioles by streamlining the enzyme digestion process (e.g., omitting elastase treatment step while maintaining use of a papain, dithioerythritol, and collagenase cocktail) (23), optimizing experimental temperature (69), loading of Ca 2+ dyes and confocal microscopy applications (69, 70), and mechanical stability for intracellular V m measurements (40). As a result, Ca 2+ and electrical signals characteristic of the intact endothelium have been resolved in depth for the first time (39, 40, 70).…”

Tuning Electrical Conduction Along Endothelial Tubes of Resistance Arteries Through Ca 2+ -Activated K + Channels

“…To avoid such confounding influences, we have freshly isolated EC tubes from feed arteries of mouse abdominal skeletal muscle. 29, 30 Intact EC tubes from these resistance vessels can exceed 3 mm in length, enabling the efficacy of electrical conduction along the endothelium to be evaluated. Using sharp intracellular microelectrodes, current microinjection into a single EC alters membrane potential (V m ) independent of agonists or ion channel activation.…”

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

“…While our preparations from the SEA have proven stable and healthy at ambient room temperature (~24 °C) and for several hours at 32 °C, morphological and functional degradation occur in less than an hour at 37 °C 9 . A second important limitation of the endothelial tube is the loss of myoendothelial junctions and their inherent signaling domains that are integral to EC function in the intact vessel wall [14][15][16] .…”

“…This procedure was adapted from one originally developed to isolate endothelial tubes from arterioles of the hamster cremaster muscle 8 . Utilizing minor variations of the techniques presented here, we have isolated endothelial tubes from a variety of vascular beds, including: feed arteries of the hamster retractor muscle and cheek pouch arterioles 10 , mouse superior epigastric artery 6,7,9 , mouse mesenteric and cerebral arteries and lymphatic microvessels (unpublished observations; references are included for isolating mesenteric vessels 12 and cerebral vessels 13 ). Isolating endothelial tubes from new vascular beds may require modifications to the original protocol.…”

“…The key features of this method are that intact lengths (1-3 mm) of microvascular endothelium are isolated, secured against a glass coverslip in a flow chamber in which they are superfused with PSS, and imaged for Ca 2+ signaling [7][8][9] or impaled for electrical signaling 6,8 . Within the flow chamber, the upper half of a tube is exposed to the superfusion solution and responds to experimental interventions, while the bottom half (in contact with the coverslip) is protected and remains quiescent.…”