Abstract
In the microcirculation blood flow is highly regulated dependent on the metabolic activity of the tissues. Among several mechanisms, mechanisms involved in the coupling of changes in tissue oxygen tension due to changes in the metabolic activity of the tissue play an important role. In the systemic artery system hypoxia causes dilatation of arteries and arterioles (hypoxic vasodilatation), whereas hyperoxia causes constriction (hyperoxic vasoconstriction).
The aim of this ph.d. study is to examine: I) mechanisms involved in hypoxic vasodilatation and hyperoxic vasoconstriction, and II) whether localized hypoxia or hyperoxia is able to induce a conducted vasomotor response in mouse cremaster arterioles in situ.
In anesthetized C57BL/6J male mice, cremaster arterioles (~30 μm) where examined during superfusion with a Krebs’ buffer equilibrated with gas mixtures containing 0% (low oxygen superfusate) or 21% O2(high oxygen superfusate), 5% CO2 andN2 balance. Low oxygen superfusates (PO2 ~15 mmHg) caused dilatation of the arterioles and high oxygen superfusates (PO2 ~160 mmHg) caused vasoconstriction. The role of ATP-sensitive K+ channels (KATP channels) was tested. Application of 10 μM glibenclamide (inhibitor of KATP channels) in the superfusate abolished both vasodilatation and constriction to low and high oxygen superfusate, indicating that KATP channels are involved in both hypoxic vasodilatation and hyperoxic vasoconstriction. Red blood cells (RBCs) have been proposed to release ATP and/or NO in response to hypoxia, which acts on the vascular wall causing vasodilatation. In cremaster arterioles devoid of RBCs, achieved by buffer perfusion via a cannula in the abdominal aorta of the animals, the cremaster arterioles showed the same degree of dilatation and constriction to low and high oxygen as in the intact blood-perfused arteriole. This indicates that RBCs are not essential for hypoxic vasodilatation. In addition several potential pathways were evaluated. Application of DPCPX (inhibitor of adenosine A1 and A2 receptors) and L-NAME (inhibitor of NO-synthase) did not affect vasomotor responses to low or high oxygen superfusate. This indicates that adenosine and NO- release are not involved in the regulation of blood flow induced by changes in tissue oxygen tension. The roles of prostaglandins and 20-HETE were also tested. While application of indomethacin (inhibitor of cyclooxygenase) in the superfusate caused a temporary inhibition of vasodilatation during low oxygen superfusate, the arterioles quickly regained their response to low and high oygen superfusates. This suggests that prostaglandins are not involved in hypoxic vasodilatationin mouse cremaster arterioles. Conversely, application of DDMS (inhibitor of 20-HETE production) inhibited vasoconstriction to high oxygen superfusates, indicating that 20-HETE is involved in hyperoxic vasoconstriction.
Using a custom-built gas exchange chamber with a small aperture covered by a gas-permeable membrane, we were able to induce highly localized oxygen changes to parts of the cremaster microcirculation. We showed that induction of localized low oxygen and high oxygen caused a local vasodilatation and constriction, which was conducted along the arteriole to a site, located 1000 μm upstream (distant site). Functional transmural damage to a short segment of the arteriole located midway (500 μm) between the local site and distant site, utilizing a method called “light dye treatment”, abolished the conducted response, but not the local response. This indicates that changes in oxygen tension are able to induce a conducted vasomotor response.
In a separate study we examined if low or high oxygen caused any change in the intracellular Ca2+ concentration of isolated mouse cremaster arterioles using fluorescent microscopy. Cremaster arterioles were isolated by gentle microdissection, loaded with Fura-PE3 (calcium fluorescent dye) and placed in a small chamber which was perfused with low or high oxygen Krebs’ buffer. While there was a significant increase in the intracellular Ca2+ concentration during application of 75 mM KCl or 100 μM phenylephrine, no change in the intracellular Ca2+ concentration was seen during change from low to high oxygen perfusate. Possible explanations for these findings could be: a) that the oxygen sensing mechanism was damaged during the dissection procedure, b) changes in oxygen tension does not cause changes in the intracellular Ca2+ concentration, but rather changes the Ca2+ sensitivity of contractive apparatus and c) that the arterioles were not perfused and pressurized, which rendered them unresponsive to changes in oxygen tension. To examine the last possibility, mouse mesenteric arteries (diameter ~200 μm) were isolated and mounted in a pressure myography. During intraluminal pressure of 40 mmHg, change from low to high oxygen caused no change in vessel diameter, however when intraluminal pressure was raised to 80-120 mmHg, change in oxygen tension caused change in vessel diameter. This indicates that the vessels need to be pressurized in order to be able to sense and react to changes in the oxygen tension.
The aim of this ph.d. study is to examine: I) mechanisms involved in hypoxic vasodilatation and hyperoxic vasoconstriction, and II) whether localized hypoxia or hyperoxia is able to induce a conducted vasomotor response in mouse cremaster arterioles in situ.
In anesthetized C57BL/6J male mice, cremaster arterioles (~30 μm) where examined during superfusion with a Krebs’ buffer equilibrated with gas mixtures containing 0% (low oxygen superfusate) or 21% O2(high oxygen superfusate), 5% CO2 andN2 balance. Low oxygen superfusates (PO2 ~15 mmHg) caused dilatation of the arterioles and high oxygen superfusates (PO2 ~160 mmHg) caused vasoconstriction. The role of ATP-sensitive K+ channels (KATP channels) was tested. Application of 10 μM glibenclamide (inhibitor of KATP channels) in the superfusate abolished both vasodilatation and constriction to low and high oxygen superfusate, indicating that KATP channels are involved in both hypoxic vasodilatation and hyperoxic vasoconstriction. Red blood cells (RBCs) have been proposed to release ATP and/or NO in response to hypoxia, which acts on the vascular wall causing vasodilatation. In cremaster arterioles devoid of RBCs, achieved by buffer perfusion via a cannula in the abdominal aorta of the animals, the cremaster arterioles showed the same degree of dilatation and constriction to low and high oxygen as in the intact blood-perfused arteriole. This indicates that RBCs are not essential for hypoxic vasodilatation. In addition several potential pathways were evaluated. Application of DPCPX (inhibitor of adenosine A1 and A2 receptors) and L-NAME (inhibitor of NO-synthase) did not affect vasomotor responses to low or high oxygen superfusate. This indicates that adenosine and NO- release are not involved in the regulation of blood flow induced by changes in tissue oxygen tension. The roles of prostaglandins and 20-HETE were also tested. While application of indomethacin (inhibitor of cyclooxygenase) in the superfusate caused a temporary inhibition of vasodilatation during low oxygen superfusate, the arterioles quickly regained their response to low and high oygen superfusates. This suggests that prostaglandins are not involved in hypoxic vasodilatationin mouse cremaster arterioles. Conversely, application of DDMS (inhibitor of 20-HETE production) inhibited vasoconstriction to high oxygen superfusates, indicating that 20-HETE is involved in hyperoxic vasoconstriction.
Using a custom-built gas exchange chamber with a small aperture covered by a gas-permeable membrane, we were able to induce highly localized oxygen changes to parts of the cremaster microcirculation. We showed that induction of localized low oxygen and high oxygen caused a local vasodilatation and constriction, which was conducted along the arteriole to a site, located 1000 μm upstream (distant site). Functional transmural damage to a short segment of the arteriole located midway (500 μm) between the local site and distant site, utilizing a method called “light dye treatment”, abolished the conducted response, but not the local response. This indicates that changes in oxygen tension are able to induce a conducted vasomotor response.
In a separate study we examined if low or high oxygen caused any change in the intracellular Ca2+ concentration of isolated mouse cremaster arterioles using fluorescent microscopy. Cremaster arterioles were isolated by gentle microdissection, loaded with Fura-PE3 (calcium fluorescent dye) and placed in a small chamber which was perfused with low or high oxygen Krebs’ buffer. While there was a significant increase in the intracellular Ca2+ concentration during application of 75 mM KCl or 100 μM phenylephrine, no change in the intracellular Ca2+ concentration was seen during change from low to high oxygen perfusate. Possible explanations for these findings could be: a) that the oxygen sensing mechanism was damaged during the dissection procedure, b) changes in oxygen tension does not cause changes in the intracellular Ca2+ concentration, but rather changes the Ca2+ sensitivity of contractive apparatus and c) that the arterioles were not perfused and pressurized, which rendered them unresponsive to changes in oxygen tension. To examine the last possibility, mouse mesenteric arteries (diameter ~200 μm) were isolated and mounted in a pressure myography. During intraluminal pressure of 40 mmHg, change from low to high oxygen caused no change in vessel diameter, however when intraluminal pressure was raised to 80-120 mmHg, change in oxygen tension caused change in vessel diameter. This indicates that the vessels need to be pressurized in order to be able to sense and react to changes in the oxygen tension.
| Originalsprog | Engelsk |
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| Status | Udgivet - 8 dec. 2010 |
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