Publications by authors named "Christian Aalkjaer"

134 Publications

ATP induced calcium signaling activity in perivascular cells differ at different vascular branch levels in the porcine retina.

Microvasc Res 2022 Jan 14;139:104256. Epub 2021 Sep 14.

Department of Ophthalmology, Aarhus University Hospital, DK-8200 Aarhus N, Denmark. Electronic address:

Background: The purine adenosine triphosphate (ATP) plays a significant role in retinal blood flow regulation and recent evidence suggests that the vasoactive effect of the compound differs in vessels at different branching level. However, the cellular basis for the regulation of retinal blood flow mediated by ATP has only been scarcely studied.

Methods: Perfused porcine hemiretinas (n = 60) were loaded with the calcium-sensitive fluorophore Oregon Green ex vivo. Spontaneous oscillations in fluorescence were studied in perivascular cells at five different vascular branching levels ranging from the main arteriole to the capillaries, before and after the addition of intra- and extravascular ATP alone or in the presence of a P2-purinergic receptor antagonist.

Results: Intravascular ATP induced an overall significant (p < 0.01) constriction of (mean ± SD) -9.79 ± 13.40% and extravascular ATP an overall significant (p < 0.01) dilatation of (mean ± SD) 19.62 ± 13.47%. Spontaneous oscillations of fluorescence in perivascular cells were significantly more intense around third order arterioles than around vessels at both lower and higher branching levels (p < 0.05 for all comparisons). ATP increased intracellular fluorescence in perivascular cells of first and second order arterioles after extravascular application, and the increase correlated with the accompanying vasodilatation (p < 0.03). Blocking of P2-receptors reduced oscillating fluorescence in pre-capillary arterioles secondary to intravascular ATP (p = 0.03).

Conclusions: Spontaneous oscillations of calcium-sensitive fluorescence in perivascular retinal cells differ at different vascular branching levels. Extravascular ATP increases fluorescence in cells around the larger retinal arterioles exposed to the retinal surface. Future studies should investigate calcium signaling activity in perivascular retinal cells during interventions that simulate retinal pathology such as hypoxia.
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http://dx.doi.org/10.1016/j.mvr.2021.104256DOI Listing
January 2022

Did giraffe cardiovascular evolution solve the problem of heart failure with preserved ejection fraction?

Evol Med Public Health 2021 11;9(1):248-255. Epub 2021 Jun 11.

Zoophysiology, Department of Biology, Aarhus University, Aarhus, Denmark.

The evolved adaptations of other species can be a source of insight for novel biomedical innovation. Limitations of traditional animal models for the study of some pathologies are fueling efforts to find new approaches to biomedical investigation. One emerging approach recognizes the evolved adaptations in other species as possible solutions to human pathology. The giraffe heart, for example, appears resistant to pathology related to heart failure with preserved ejection fraction (HFpEF)-a leading form of hypertension-associated cardiovascular disease in humans. Here, we postulate that the physiological pressure-induced left ventricular thickening in giraffes does not result in the pathological cardiovascular changes observed in humans with hypertension. The mechanisms underlying this cardiovascular adaptation to high blood pressure in the giraffe may be a bioinspired roadmap for preventive and therapeutic strategies for human HFpEF.
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http://dx.doi.org/10.1093/emph/eoab016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8385250PMC
June 2021

Dynein regulates Kv7.4 channel trafficking from the cell membrane.

J Gen Physiol 2021 03;153(3)

Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark.

The dynein motor protein transports proteins away from the cell membrane along the microtubule network. Recently, we found the microtubule network was important for regulating the membrane abundance of voltage-gated Kv7.4 potassium channels in vascular smooth muscle. Here, we aimed to investigate the influence of dynein on the microtubule-dependent internalization of the Kv7.4 channel. Patch-clamp recordings from HEK293B cells showed Kv7.4 currents were increased after inhibiting dynein function with ciliobrevin D or by coexpressing p50/dynamitin, which specifically interferes with dynein motor function. Mutation of a dynein-binding site in the Kv7.4 C terminus increased the Kv7.4 current and prevented p50 interference. Structured illumination microscopy, proximity ligation assays, and coimmunoprecipitation showed colocalization of Kv7.4 and dynein in mesenteric artery myocytes. Ciliobrevin D enhanced mesenteric artery relaxation to activators of Kv7.2-Kv7.5 channels and increased membrane abundance of Kv7.4 protein in isolated smooth muscle cells and HEK293B cells. Ciliobrevin D failed to enhance the negligible S-1-mediated relaxations after morpholino-mediated knockdown of Kv7.4. Mass spectrometry revealed an interaction of dynein with caveolin-1, confirmed using proximity ligation and coimmunoprecipitation assays, which also provided evidence for interaction of caveolin-1 with Kv7.4, confirming that Kv7.4 channels are localized to caveolae in mesenteric artery myocytes. Lastly, cholesterol depletion reduced the interaction of Kv7.4 with caveolin-1 and dynein while increasing the overall membrane expression of Kv7.4, although it attenuated the Kv7.4 current in oocytes and interfered with the action of ciliobrevin D and channel activators in arterial segments. Overall, this study shows that dynein can traffic Kv7.4 channels in vascular smooth muscle in a mechanism dependent on cholesterol-rich caveolae.
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http://dx.doi.org/10.1085/jgp.202012760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7863719PMC
March 2021

Brain capillary pericytes and neurovascular coupling.

Comp Biochem Physiol A Mol Integr Physiol 2021 04 6;254:110893. Epub 2021 Jan 6.

Department of Biomedical Science, University of Copenhagen, DK-2200 Copenhagen N, Denmark; Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark.

The neurovascular coupling ensures that cerebral activity is matched by the relevant blood flow. The control of the blood flow is mediated by capillaries and by the precapillary aterioles. It is the tone of the mural cells, which include pericytes, smooth muscle cells and cells with intermediate phenotypes between pericytes and smooth muscle cells, that determine the the diameter of the blood vessels and consequently the flow. Here we discuss the structure of these blood vessels and the excitationcontraction coupling of the mural cells.
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http://dx.doi.org/10.1016/j.cbpa.2020.110893DOI Listing
April 2021

Sodium bicarbonate cotransporter NBCn1/Slc4a7 affects locomotor activity and hearing in mice.

Behav Brain Res 2021 03 13;401:113065. Epub 2020 Dec 13.

Department of Biomedicine, Aarhus University, Aarhus, Denmark.

Despite a widespread expression pattern in the central nervous system, the role of the sodium bicarbonate cotransporter NBCn1/Slc4a7 has not been investigated for locomotor activity, emotion and cognition. Here, we addressed the behavioral consequences of NBCn1 knockout and evaluated hearing and vision that are reportedly impaired in an earlier line of NBCn1 knockout mice and may contribute to behavioral changes. In a circular open field, the knockout mice traveled a shorter distance, especially in the periphery of the chamber, than wildtype littermates. The knockout mice also traveled a shorter total distance in a home cage-like open field. Rearing and grooming behaviors were reduced. The knockout and control mice displayed similar time spent and number of open and closed arms in the elevated plus maze test, indicating negligible change in anxiety. In the Morris water maze test, both groups of mice learned the location of an escape platform within comparable time on the training trials and showed similar platform identification on the probe trial. The knockout mice maintained normal visual responses in the optokinetic drum and produced evoked potentials in response to light stimuli. However, these mice failed to produce auditory evoked potentials. qPCR revealed a robust expression of an alternatively transcribed NBCn1 variant in the knockout mouse retina. These results indicate that NBCn1 deletion leads to reduced locomotor activity in mice by affecting their exploratory behaviors or emotionality. The deletion also causes hearing loss, but its effect on vision varies between different lines of knockout mice.
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http://dx.doi.org/10.1016/j.bbr.2020.113065DOI Listing
March 2021

Sympathetic and Sensory-Motor Nerves in Peripheral Small Arteries.

Physiol Rev 2021 04 3;101(2):495-544. Epub 2020 Dec 3.

Deptartment Pharmacology and Personalized Medicine, Maastricht University, Maastricht, The Netherlands.

Small arteries, which play important roles in controlling blood flow, blood pressure, and capillary pressure, are under nervous influence. Their innervation is predominantly sympathetic and sensory motor in nature, and while some arteries are densely innervated, others are only sparsely so. Innervation of small arteries is a key mechanism in regulating vascular resistance. In the second half of the previous century, the physiology and pharmacology of this innervation were very actively investigated. In the past 10-20 yr, the activity in this field was more limited. With this review we highlight what has been learned during recent years with respect to development of small arteries and their innervation, some aspects of excitation-release coupling, interaction between sympathetic and sensory-motor nerves, cross talk between endothelium and vascular nerves, and some aspects of their role in vascular inflammation and hypertension. We also highlight what remains to be investigated to further increase our understanding of this fundamental aspect of vascular physiology.
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http://dx.doi.org/10.1152/physrev.00007.2020DOI Listing
April 2021

A paradoxical increase of force development in saphenous and tail arteries from heterozygous ANO1 knockout mice.

Physiol Rep 2020 11;8(22):e14645

Department of Biomedicine, MEMBRANES, Health, Aarhus University, Aarhus, Denmark.

A Ca -activated Cl channel protein, ANO1, is expressed in vascular smooth muscle cells where Cl current is thought to potentiate contraction by contributing to membrane depolarization. However, there is an inconsistency between previous knockout and knockdown studies on ANO1's role in small arteries. In this study, we assessed cardiovascular function of heterozygous mice with global deletion of exon 7 in the ANO1 gene. We found decreased expression of ANO1 in aorta, saphenous and tail arteries from heterozygous ANO1 knockout mice in comparison with wild type. Accordingly, ANO1 knockdown reduced the Ca -activated Cl current in smooth muscle cells. Consistent with conventional hypothesis, the contractility of aorta from ANO1 heterozygous mice was reduced. Surprisingly, we found an enhanced contractility of tail and saphenous arteries from ANO1 heterozygous mice when stimulated with noradrenaline, vasopressin, and K -induced depolarization. This difference was endothelium-independent. The increased contractility of ANO1 downregulated small arteries was due to increased Ca influx. The expression of L-type Ca channels was not affected but expression of the plasma membrane Ca ATPase 1 and the Piezo1 channel was increased. Expressional analysis of tail arteries further suggested changes of ANO1 knockdown smooth muscle cells toward a pro-contractile phenotype. We did not find any difference between genotypes in blood pressure, heart rate, pressor response, and vasorelaxation in vivo. Our findings in tail and saphenous arteries contrast with the conventional hypothesis and suggest additional roles for ANO1 as a multifunctional protein in the vascular wall that regulates Ca homeostasis and smooth muscle cell phenotype.
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http://dx.doi.org/10.14814/phy2.14645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7695021PMC
November 2020

The Remarkable Cardiovascular System of Giraffes.

Annu Rev Physiol 2021 02 9;83:1-15. Epub 2020 Nov 9.

Department of Biology, Aarhus University, Aarhus C 8000, Denmark; email:

Gravity affects the physiology of many animals, and the effect is, for good reason, most pronounced in tall species. The physiology-in particular, cardiovascular function-of giraffes has therefore captivated the interest of physiologists for centuries. Several studies document high mean arterial blood pressure of giraffes of about 200 mm Hg. This appears necessary to establish a cerebral perfusion pressure on the order of 100 mm Hg at the cranial end of the carotid arteries. Here, we discuss the unique characteristics of blood vessels, the heart, and the kidney of giraffes and how these functional and structural adaptations are related to very high blood pressure. We also discuss how the cerebral circulation of giraffes is established and what we know about how the blood flow and arterial and venous pressures in giraffes change when they stop to drink and subsequently lift their heads 5-6 m in one sweeping movement.
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http://dx.doi.org/10.1146/annurev-physiol-031620-094629DOI Listing
February 2021

Differential Effects of Intra- and Extravascular ATP on the Diameter of Porcine Vessels at Different Branching Levels Ex Vivo.

Invest Ophthalmol Vis Sci 2020 10;61(12)

Department of Ophthalmology, Aarhus University Hospital, DK-8200 Aarhus N, Denmark.

Purpose: Adenosine triphosphate (ATP) is involved in the diameter regulation of retinal vessels. The compound has been shown to induce both constriction and dilatation, but the detailed mechanisms underlying these effects and the site of action of the compound are not known in detail. Therefore, the purpose of the present study was to investigate whether the vasoactive effects of ATP on retinal vessels depend on intra- and extravascular application, and to study whether the effects differ at different vascular branching levels.

Methods: Diameter changes in arterioles, pre-capillary arterioles, and capillaries were studied in perfused porcine hemiretinas (n = 48) ex vivo after intra- and extravascular application of the nondegradable ATP analogue ATP-γ-S or ATP in the presence or not of antagonists to the CD73/ecto-5'-nucleotidase (AOPCP), the P2-purinergic receptor (PPADS), the A3-adenosine receptor (MRS1523), and the synthesis of cyclooxygenase products (ibuprofen).

Results: Intravascular ATP-induced constriction and extravascular ATP-induced dilatation of retinal arterioles, pre-capillary arterioles and capillaries, and dilatation was inhibited by ibuprofen. Both constriction and dilatation of arterioles were inhibited by antagonizing ATP degradation. Furthermore, constriction at all three branching levels was antagonized by blocking the A3 purinoceptor, whereas constriction in arterioles and pre-capillary arterioles was antagonized by blocking the P2 purinoceptor.

Conclusions: ATP affects the diameter of retinal arterioles, pre-capillary arterioles, and capillaries through different pathways, and the effects depend on whether the compound is administered intravascularly or extravascularly. This may form the basis for selective interventions on retinal vascular disease with differential involvement of vessels at different branching levels.
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http://dx.doi.org/10.1167/iovs.61.12.8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7552936PMC
October 2020

Increased Alcohol Consumption in Mice Lacking Sodium Bicarbonate Transporter NBCn1.

Sci Rep 2020 07 3;10(1):11017. Epub 2020 Jul 3.

Department of Physiology, Emory University School of Medicine, Atlanta, GA, 30322, USA.

The previous reports on an addiction vulnerability marker in the human SLC4A7 gene encoding the Na/HCO transporter NBCn1 suggest that this pH-regulating protein may affect alcohol-related behavior and response. Here, we examined alcohol consumption and sensitivity to the sedative effects of alcohol in male NBCn1 knockout mice. These mice displayed lower pH in neurons than wildtype controls, determined by intracellular pH in hippocampal neuronal cultures. Neurons from knockout mice had a higher action potential threshold and a more depolarized membrane potential, thus reducing membrane excitability. In a two-bottle free choice procedure, knockout mice consumed more alcohol than controls and consistently increased alcohol consumption after repeated alcohol deprivation periods. Quinine and sucrose preference was similar between genotypes. Knockout mice showed increased propensity for alcohol-induced conditioned place preference. In loss of righting reflex assessment, knockout mice revealed increased sensitivity to alcohol-induced sedation and developed tolerance to the sedation after repeated alcohol administrations. Furthermore, chronic alcohol consumption caused NBCn1 downregulation in the hippocampus and striatum of mice and humans. These results demonstrate an important role of NBCn1 in regulation of alcohol consumption and sensitivity to alcohol-induced sedation.
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http://dx.doi.org/10.1038/s41598-020-67291-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7335059PMC
July 2020

A sex-specific, COX-derived/thromboxane receptor activator causes depolarization and vasoconstriction in male mice mesenteric resistance arteries.

Basic Clin Pharmacol Toxicol 2020 Aug 4;127(2):152-159. Epub 2020 May 4.

Department of Biomedicine, Aarhus University, Aarhus C, Denmark.

We investigated whether sex differences exist in cyclooxygenase-dependent effects on membrane potential and relaxation in mice mesenteric resistance arteries. Mesenteric small arteries of 9- to 12-week-old, male and female, wild-type mice, db/+ mice and diabetic db/db mice were mounted in myographs for measurements of isobaric diameter and smooth muscle cell membrane potential. Acetylcholine caused smaller dilation of arteries from male db/+ mice compared to arteries from female db/+ mice. In the presence of the NO synthase inhibitor N -nitro-L-arginine methyl ester (L-NAME), acetylcholine-induced dilation of arteries from males increased in the presence of indomethacin and the COX-1-specific inhibitor FR122047. The presence of indomethacin was also associated with a more negative membrane potential in arteries from males. In arteries from db/db mice, no sex differences were seen. In arteries from male but not female wild-type mice, the thromboxane receptor blocker SQ29,548 increased relaxation to acetylcholine. In contrast to arteries from female mice, COX (most likely COX-1)-derived prostanoids and activation of thromboxane receptors counteract acetylcholine vasodilatation probably through increased smooth muscle depolarization in arteries from male mice. In mice with diabetes and pronounced endothelial dysfunction, inhibition of COX did not enhance acetylcholine vasodilatation.
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http://dx.doi.org/10.1111/bcpt.13413DOI Listing
August 2020

Abnormal neurovascular coupling as a cause of excess cerebral vasodilation in familial migraine.

Cardiovasc Res 2020 10;116(12):2009-2020

Department of Biomedicine, Aarhus University, Høegh-Guldbergs Gade 10, Aarhus 8000, Denmark.

Aims: Acute migraine attack in familial hemiplegic migraine type 2 (FHM2) patients is characterized by sequential hypo- and hyperperfusion. FHM2 is associated with mutations in the Na, K-ATPase α2 isoform. Heterozygous mice bearing one of these mutations (α2+/G301R mice) were shown to have elevated cerebrovascular tone and, thus, hypoperfusion that might lead to elevated concentrations of local metabolites. We hypothesize that these α2+/G301R mice also have increased cerebrovascular hyperaemic responses to these local metabolites leading to hyperperfusion in the affected part of the brain.

Methods And Results: Neurovascular coupling was compared in α2+/G301R and matching wild-type (WT) mice using Laser Speckle Contrast Imaging. In brain slices, parenchymal arteriole diameter and intracellular calcium changes in neuronal tissue, astrocytic endfeet, and smooth muscle cells in response to neuronal excitation were assessed. Wall tension and smooth muscle membrane potential were measured in isolated middle cerebral arteries. Quantitative polymerase chain reaction, western blot, and immunohistochemistry were used to assess the molecular background underlying the functional changes. Whisker stimulation induced larger increase in blood perfusion, i.e. hyperaemic response, of the somatosensory cortex of α2+/G301R than WT mice. Neuronal excitation was associated with larger parenchymal arteriole dilation in brain slices from α2+/G301R than WT mice. These hyperaemic responses in vivo and ex vivo were inhibited by BaCl2, suggesting involvement of inward-rectifying K+ channels (Kir). Relaxation to elevated bath K+ was larger in arteries from α2+/G301R compared to WT mice. This difference was endothelium-dependent. Endothelial Kir2.1 channel expression was higher in arteries from α2+/G301R mice. No sex difference in functional responses and Kir2.1 expression was found.

Conclusion: This study suggests that an abnormally high cerebrovascular hyperaemic response in α2+/G301R mice is a result of increased endothelial Kir2.1 channel expression. This may be initiated by vasospasm-induced accumulation of local metabolites and underlie the hyperperfusion seen in FHM2 patients during migraine attack.
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http://dx.doi.org/10.1093/cvr/cvz306DOI Listing
October 2020

Perivascular Adipose Tissue Contributes to the Modulation of Vascular Tone in vivo.

J Vasc Res 2019 24;56(6):320-332. Epub 2019 Sep 24.

Department of Biomedicine, Aarhus University, Aarhus, Denmark,

Background: Perivascular adipose tissue (PVAT) reduces vascular tone in isolated arteries in vitro, however there are no studies of PVAT effects on vascular tone in vivo. In vitro adipocyte β3-adrenoceptors play a role in PVAT function via secretion of the vasodilator adiponectin.

Objective: We have investigated the effects of PVAT on vessel diameter in vivo, and the contributions of β3-adrenoceptors and adiponectin.

Method: In anaesthetised rats, sections of the intact mesenteric bed were visualised and the diameter of arteries was recorded. Arteries were stimulated with electrical field stimulation (EFS), noradrenaline (NA), arginine-vasopressin (AVP), and acetylcholine (Ach).

Results: We report that in vivo, stimulation of PVAT with EFS, NA, and AVP evokes a local anti-constrictive effect on the artery, whilst PVAT exerts a pro-contractile effect on arteries subjected to Ach. The anti-constrictive effect of PVAT stimulated with EFS and NA was significantly reduced using β3-adrenoceptor inhibition, and activation of β3-adrenoceptors potentiated the anti-constrictive effect of vessels stimulated with EFS, NA, and AVP. The β3-adrenoceptor agonist had no effect on mesenteric arteries with PVAT removed. A blocking peptide for adiponectin receptor 1 polyclonal antibody reduced the PVAT anti-constrictive effect in arteries stimulated with EFS and NA, indicating that adiponectin may be the anti-constrictive factor released upon β3-adrenoceptor activation.

Conclusions: These results clearly demonstrate that PVAT plays a paracrine role in regulating local vascular tone in vivo, and therefore may contribute to the modulation of blood pressure. This effect is mediated via adipocyte β3-adrenoceptors, which may trigger release of the vasodilator adiponectin.
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http://dx.doi.org/10.1159/000502689DOI Listing
January 2020

[Ca ] changes in sympathetic varicosities and Schwann cells in rat mesenteric arteries-Relation to noradrenaline release and contraction.

Acta Physiol (Oxf) 2019 08 26;226(4):e13279. Epub 2019 Apr 26.

Department of Biomedicine, University of Aarhus, Aarhus C, Denmark.

Aim: This study aimed to assess intracellular Ca dynamics in nerve cells and Schwann cells in isolated rat resistance arteries and determine how these dynamics modify noradrenaline release from the nerves and consequent force development.

Methods: Ca in nerves was assessed with confocal imaging, noradrenaline release with amperometry and artery tone with wire myography. Ca in axons was assessed after loading with Oregon Green 488 BAPTA-1 dextran. In other experiments, arteries were incubated with Calcium Green-1-AM which loads both axons and Schwann cells.

Results: Schwann cells but not axons responded with a Ca increase to ATP. Electrical field stimulation of nerves caused a frequency-dependent increase in varicose [Ca ] ([Ca ] ). ω-conotoxin-GVIA (100 nmol/L) reduced the [Ca ] transient to 2 and 16 Hz by 60% and 27%, respectively; in contrast ω-conotoxin GVIA inhibited more than 80% of the noradrenaline release and force development at 2 and 16 Hz. The K channel blocker, 4-aminopyridine (10 µmol/L), increased [Ca ] , noradrenaline release and force development both in the absence and presence of ω-conotoxin-GVIA. Yohimbine (1 µmol/L) increased both [Ca ] and noradrenaline release but reduced force development. Acetylcholine (10 µmol/L) caused atropine-sensitive inhibition of [Ca ] , noradrenaline release and force. In the presence of ω-conotoxin-GVIA, acetylcholine caused a further inhibition of all parameters.

Conclusion: Modification of [Ca ] in arterial sympathetic axons and Schwann cells was assessed separately. K 3.1 channels may be important regulators of [Ca ] , noradrenaline release and force development. Presynaptic adrenoceptor and muscarinic receptor activation modify transmitter release through modification of [Ca ] .
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http://dx.doi.org/10.1111/apha.13279DOI Listing
August 2019

Rat mesenteric small artery neurogenic dilatation is predominantly mediated by β -adrenoceptors in vivo.

J Physiol 2019 04 21;597(7):1819-1831. Epub 2019 Feb 21.

Department of Biomedicine, Membranes, University of Aarhus, Aarhus, Denmark.

Key Points: The prevailing dogma about neurogenic regulation of vascular tone consists of major vasodilatation caused by CGRP (and possibly substance P) released from sensory-motor nerves and vasoconstriction caused by noradrenaline, ATP and neuropeptode Y release from sympathetic nerves. Most studies on perivascular nerve-mediated vasodilatation are made in vitro. In the present study, we provide evidence indicating that in vivo electrical perivascular nerve stimulation in rat mesenteric small arteries causes a large β1-adrenoceptor-mediated vasodilatation, which contrasts with a smaller vasodilatation caused by endogenous CGRP that is only visible after inhibition of Y1 NPY receptors.

Abstract: Mesenteric arteries are densely innervated and the nerves are important regulators of vascular tone and hence blood pressure and blood flow. Perivascular sensory-motor nerves have been shown to cause vasodilatation in vitro. However, less is known about their function in vivo. Male Wistar rats (10-12 weeks old; n = 72) were anaesthetized with ketamine (3 mg kg ) and xylazine (0.75 mg kg ) or pentobarbital (60 mg kg ). After a laparotomy, a section of second-order mesenteric artery was visualized in an organ bath after minimal removal of perivascular adipose tissue. The effects of electrical field stimulation (EFS) and drugs on artery diameter and blood flow were recorded with intravital microscopy and laser speckle imaging. EFS caused vasodilatation in arteries constricted with 1 μm U46619 in the presence of 140 μm suramin and 1 μm prazosin. The vasodilatation was inhibited by 1 μm tetrodotoxin and 5 μm guanethidine, although not by the 1 μm of the CGRP receptor antagonist BIBN4096bs. In the presence of 0.3 μm Y1 receptor antagonist BIBP3226, BIBN4096bs partly inhibited the vasodilatation. Atenolol at a concentration 1 μm inhibited the vasodilatation, whereas 0.1 μm of the β -adrenoceptor selective antagonist ICI-118,551 had no effect. Increasing the extracellular [K ] to 20 mm caused vasodilatation but was converted to vasoconstriction in the presence of 1 μm BIBN4096bs, and constriction to 30 mm potassium was potentiated by BIBN4096bs. Atenolol but not BIBN4096bs increased contraction to EFS in the absence of suramin and prazosin. In mesenteric small arteries of anaesthetized rats, EFS failed to stimulate major dilatation via sensory-motor nerves but induced sympathetic β -adrenoceptor-mediated dilatation.
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http://dx.doi.org/10.1113/JP277368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441891PMC
April 2019

The vasodilating effect of glucose differs among vessels at different branching level in the porcine retina ex vivo.

Exp Eye Res 2019 02 10;179:150-156. Epub 2018 Nov 10.

Department of Ophthalmology, Aarhus University Hospital, DK-8000, Aarhus C, Denmark.

Diabetic retinopathy is characterized by retinal lesions related to disturbances in retinal blood flow. The metabolic dysregulation in diabetes involves hyperglycemia which in both clinical and experimental studies has been shown to induce dilatation of larger retinal vessels, which has been suggested to be mediated by nitric oxide (NO). However, the effects of glucose on the diameter of smaller retinal vessels that are the site of development of diabetic retinopathy are unknown. Diameter changes in porcine retinal arterioles, pre-capillary arterioles and capillaries were studied ex vivo during acute changes in intraluminal glucose concentrations that mimicked changes in plasma glucose in diabetic patients. The experiments were repeated during blocking of NO-synthesis. Intravascular application of 2 mM glucose dilated arterioles and capillaries significantly, while 20 mM glucose dilated precapillary arterioles significantly. Intravascular application of 20 mM glucose dilated precapillary arterioles previously exposed to 2 mM glucose, while no significant diameter changes were observed after application of 2 mM glucose in vessels previously exposed to 20 mM glucose. No diameter changes were observed after application of 5.5 mM glucose in vessels previously exposed to both 2 mM and 20 mM glucose in either order. There was no significant difference between the diameter responses in the absence and presence of NO-synthesis blocker. Glucose induced dilatation of porcine precapillary arterioles ex vivo differs from the response in larger arterioles and capillaries, and the response is unaffected by the blocking of NO-synthesis. This may have implications for understanding the pathophysiology of diseases in the retinal microcirculation, such as diabetic retinopathy.
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http://dx.doi.org/10.1016/j.exer.2018.11.006DOI Listing
February 2019

The Na,K-ATPase-Dependent Src Kinase Signaling Changes with Mesenteric Artery Diameter.

Int J Mol Sci 2018 Aug 23;19(9). Epub 2018 Aug 23.

Department of Biomedicine, Health, Aarhus University, DK-8000 Aarhus C, Denmark.

Inhibition of the Na,K-ATPase by ouabain potentiates vascular tone and agonist-induced contraction. These effects of ouabain varies between different reports. In this study, we assessed whether the pro-contractile effect of ouabain changes with arterial diameter and the molecular mechanism behind it. Rat mesenteric small arteries of different diameters (150⁻350 µm) were studied for noradrenaline-induced changes of isometric force and intracellular Ca in smooth muscle cells. These functional changes were correlated to total Src kinase and Src phosphorylation assessed immunohistochemically. High-affinity ouabain-binding sites were semi-quantified with fluorescent ouabain. We found that potentiation of noradrenaline-sensitivity by ouabain correlates positively with an increase in arterial diameter. This was not due to differences in intracellular Ca responses but due to sensitization of smooth muscle cell contractile machinery to Ca. This was associated with ouabain-induced Src activation, which increases with increasing arterial diameter. Total Src expression was similar in arteries of different diameters but the density of high-affinity ouabain binding sites increased with increasing arterial diameters. We suggested that ouabain binding induces more Src kinase activity in mesenteric small arteries with larger diameter leading to enhanced sensitization of the contractile machinery to Ca.
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http://dx.doi.org/10.3390/ijms19092489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6164810PMC
August 2018

Smooth muscle Ca sensitization causes hypercontractility of middle cerebral arteries in mice bearing the familial hemiplegic migraine type 2 associated mutation.

J Cereb Blood Flow Metab 2019 08 7;39(8):1570-1587. Epub 2018 Mar 7.

1 Department of Biomedicine, Aarhus University, Aarhus, Denmark.

Familial hemiplegic migraine type 2 (FHM2) is associated with inherited point-mutations in the Na,K-ATPase α2 isoform, including G301R mutation. We hypothesized that this mutation affects specific aspects of vascular function, and thus compared cerebral and systemic arteries from heterozygote mice bearing the G301R mutation (Atp1a2) with wild type (WT). Middle cerebral (MCA) and mesenteric small artery (MSA) function was compared in an isometric myograph. Cerebral blood flow was assessed with Laser speckle analysis. Intracellular Ca and membrane potential were measured simultaneously. Protein expression was semi-quantified by immunohistochemistry. Protein phosphorylation was analysed by Western blot. MSA from Atp1a2 and WT showed similar contractile responses. The Atp1a2 MCA constricted stronger to U46619, endothelin and potassium compared to WT. This was associated with an increased depolarization, although the Ca change was smaller than in WT. The enhanced constriction of Atp1a2 MCA was associated with increased cSrc activation, stronger sensitization to [Ca] and increased MYPT1 phosphorylation. These differences were abolished by cSrc inhibition. Atp1a2 mice had reduced resting blood flow through MCA in comparison with WT mice FHM2-associated mutation leads to elevated contractility of MCA due to sensitization of the contractile machinery to Ca, which is mediated via Na,K-ATPase/Src-kinase/MYPT1 signalling.
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http://dx.doi.org/10.1177/0271678X18761712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6681533PMC
August 2019

Variable Contribution of TMEM16A to Tone in Murine Arterial Vasculature.

Basic Clin Pharmacol Toxicol 2018 Jul 30;123(1):30-41. Epub 2018 Mar 30.

Department of Biomedicine, Aarhus University, Aarhus, Denmark.

TMEM16A is essential for Ca -activated Cl conductance in vascular smooth muscle. The importance of TMEM16A for agonist-induced vascular constriction and blood pressure control is, however, under debate. Previous studies suggested that TMEM16A might have a complex cellular function beyond being essential for the Ca -activated Cl conductance, for example modulation of Ca channel expression. Mice with constitutive, smooth muscle-specific expression of siRNA directed against Tmem16a (transgenic mice, TG) were generated. Isometric constrictions of isolated aorta, mesenteric, femoral and tail arteries from TG mice were compared with wild-types. Protein expression was analysed by Western blots. Blood pressure and heart rate were studied telemetrically. Significant TMEM16A down-regulation was seen in aorta and tail arteries, while no changes were detected in mesenteric and femoral arteries. Contractile responses of mesenteric and femoral arteries from TG and wild-type mice were not different. Aorta from TG mice showed reduced agonist-induced constriction, while their responses to elevated K were unchanged. Tail arteries from TG mice also constricted less to adrenergic stimulation than wild-types. Surprisingly, tail arteries from TG mice constricted less to elevated K too and were more sensitive to nifedipine-induced relaxation. Consistently, TMEM16A down-regulation in tail arteries was associated with reduction in CACNA1C protein (i.e. vascular L-type Ca channel) expression. No differences in blood pressure and heart rate between the groups were seen. This study suggests a complex contribution of TMEM16A in vascular function. We suggest that TMEM16A modulates arterial contractility, at least in part, indirectly via regulation of CACNA1C expression.
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http://dx.doi.org/10.1111/bcpt.12984DOI Listing
July 2018

Microtubule Regulation of Kv7 Channels Orchestrates cAMP-Mediated Vasorelaxations in Rat Arterial Smooth Muscle.

Hypertension 2018 02 26;71(2):336-345. Epub 2017 Dec 26.

From the Department of Biomedical Sciences, Ion Channels Group, University of Copenhagen, Denmark (J.L., M.M.K., P.R.L., C.A., T.A.J.); and Department of Biomedicine, Aarhus University, Denmark (M.M.K., C.A.).

Microtubules can regulate GPCR (G protein-coupled receptor) signaling in various cell types. In vascular smooth muscle, activation of the β-adrenoceptor leads to production of cAMP to mediate a vasorelaxation. Little is known about the role of microtubules in smooth muscle, and given the importance of this pathway in vascular smooth muscle cells, we investigated the role of microtubule stability on β-adrenoceptor signaling in rat renal and mesenteric arteries. In isometric tension experiments, incubation with the microtubule inhibitors colchicine and nocodazole enhanced isoprenaline-mediated relaxations of renal and mesenteric arteries that the microtubule stabilizer, paclitaxel, prevented. Sharp microelectrode experiments showed that colchicine treatment caused increased hyperpolarization of mesenteric artery segments in response to isoprenaline. Application of the Kv7 channel blocker, XE991, attenuated the effect of colchicine on isoprenaline relaxations, whereas iberiotoxin-a BKCa channel blocker-had no effect. In addition, colchicine improved the relaxations to the Kv7.2 to 7.5 activator, S-1, in both renal and mesenteric artery segments compared with dimethyl sulfoxide incubation. We determined that increased mesenteric artery myocytes treated with colchicine showed increased Kv7.4 membrane expression, but Western blot analysis showed no change in total Kv7.4 protein. This study is the first to show microtubule disruption improves the β-adrenoceptor-mediated relaxations of mesenteric and renal arteries and determine this enhancement to be because of increased membrane expression of the Kv7 voltage-gated potassium channels.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.117.10152DOI Listing
February 2018

The ins and outs of acid-base transport in skeletal muscle.

J Gen Physiol 2018 01 13;150(1):3-6. Epub 2017 Dec 13.

Department of Biomedicine, Aarhus University, Aarhus, Denmark.

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http://dx.doi.org/10.1085/jgp.201711955DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5749118PMC
January 2018

Loss-of-activity-mutation in the cardiac chloride-bicarbonate exchanger AE3 causes short QT syndrome.

Nat Commun 2017 11 22;8(1):1696. Epub 2017 Nov 22.

Department of Cardiology, Aarhus University Hospital, Palle Juul-Jensens Boulevard 99, DK-8200, Aarhus N, Denmark.

Patients with short QT syndrome (SQTS) may present with syncope, ventricular fibrillation or sudden cardiac death. Six SQTS susceptibility genes, encoding cation channels, explain <25% of SQTS cases. Here we identify a missense mutation in the anion exchanger (AE3)-encoding SLC4A3 gene in two unrelated families with SQTS. The mutation causes reduced surface expression of AE3 and reduced membrane bicarbonate transport. Slc4a3 knockdown in zebrafish causes increased cardiac pH, short QTc, and reduced systolic duration, which is rescued by wildtype but not mutated SLC4A3. Mechanistic analyses suggest that an increase in pH and decrease in [Cl] shortened the action potential duration. However, other mechanisms may also play a role. Altered anion transport represents a mechanism for development of arrhythmia and may provide new therapeutic possibilities.
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http://dx.doi.org/10.1038/s41467-017-01630-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5700076PMC
November 2017

4-Aminopyridine: a pan voltage-gated potassium channel inhibitor that enhances K 7.4 currents and inhibits noradrenaline-mediated contraction of rat mesenteric small arteries.

Br J Pharmacol 2018 02 5;175(3):501-516. Epub 2018 Jan 5.

Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Background And Purpose: K 7.4 and K 7.5 channels are regulators of vascular tone. 4-Aminopyridine (4-AP) is considered a broad inhibitor of voltage-gated potassium (K ) channels, with little inhibitory effect on K 7 family members at mmol concentrations. However, the effect of 4-AP on K 7 channels has not been systematically studied. The aim of this study was to investigate the pharmacological activity of 4-AP on K 7.4 and K 7.5 channels and characterize the effect of 4-AP on rat resistance arteries.

Experimental Approach: Voltage clamp experiments were performed on Xenopus laevis oocytes injected with cRNA encoding KCNQ4 or KCNQ5, HEK cells expressing K 7.4 channels and on rat, freshly isolated mesenteric artery smooth muscle cells. The effect of 4-AP on tension, membrane potential, intracellular calcium and pH was assessed in rat mesenteric artery segments.

Key Results: 4-AP increased the K 7.4-mediated current in oocytes and HEK cells but did not affect K 7.5 current. 4-AP also enhanced native mesenteric artery myocyte K current at sub-mmol concentrations. When applied to NA-preconstricted mesenteric artery segments, 4-AP hyperpolarized the membrane, decreased [Ca ] and caused concentration-dependent relaxations that were independent of 4-AP-mediated changes in intracellular pH. Application of the K 7 channel blocker XE991 and BK channel blocker iberiotoxin attenuated 4-AP-mediated relaxation. 4-AP also inhibited the NA-mediated signal transduction to elicit a relaxation.

Conclusions And Implications: These data show that 4-AP is able to relax NA-preconstricted rat mesenteric arteries by enhancing the activity of K 7.4 and BK channels and attenuating NA-mediated signalling.
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http://dx.doi.org/10.1111/bph.14097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773965PMC
February 2018

Reply from Vladimir V. Matchkov and Christian Aalkjaer.

J Physiol 2017 11;595(21):6785-6787

Aarhus University, Ole Worms Alle bygn.4, 1160, Aarhus, 8000, Denmark.

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http://dx.doi.org/10.1113/JP275145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5663824PMC
November 2017

The crystal structure of the regulatory domain of the human sodium-driven chloride/bicarbonate exchanger.

Sci Rep 2017 09 21;7(1):12131. Epub 2017 Sep 21.

Norwegian Centre for Molecular Medicine, Nordic EMBL Partnership University of Oslo, Gaustadalléen 21, 0349, Oslo, Norway.

The sodium-driven chloride/bicarbonate exchanger (NDCBE) is essential for maintaining homeostatic pH in neurons. The crystal structure at 2.8 Å resolution of the regulatory N-terminal domain of human NDCBE represents the first crystal structure of an electroneutral sodium-bicarbonate cotransporter. The crystal structure forms an equivalent dimeric interface as observed for the cytoplasmic domain of Band 3, and thus establishes that the consensus motif VTVLP is the key minimal dimerization motif. The VTVLP motif is highly conserved and likely to be the physiologically relevant interface for all other members of the SLC4 family. A novel conserved Zn-binding motif present in the N-terminal domain of NDCBE is identified and characterized in vitro. Cellular studies confirm the Zn dependent transport of two electroneutral bicarbonate transporters, NCBE and NBCn1. The Zn site is mapped to a cluster of histidines close to the conserved ETARWLKFEE motif and likely plays a role in the regulation of this important motif. The combined structural and bioinformatics analysis provides a model that predicts with additional confidence the physiologically relevant interface between the cytoplasmic domain and the transmembrane domain.
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http://dx.doi.org/10.1038/s41598-017-12409-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608694PMC
September 2017

ACCURACY OF NONINVASIVE ANESTHETIC MONITORING IN THE ANESTHETIZED GIRAFFE (GIRAFFA CAMELOPARDALIS).

J Zoo Wildl Med 2017 09;48(3):609-615

This study evaluated the accuracy of pulse oximetry, capnography, and oscillometric blood pressure during general anesthesia in giraffes (Giraffa camelopardalis). Thirty-two giraffes anesthetized for physiologic experiments were instrumented with a pulse oximeter transmittance probe positioned on the tongue and a capnograph sampling line placed at the oral end of the endotracheal tube. A human size 10 blood pressure cuff was placed around the base of the tail, and an indwelling arterial catheter in the auricular artery continuously measured blood pressure. Giraffes were intermittently ventilated using a Hudson demand valve throughout the procedures. Arterial blood for blood gas analysis was collected at multiple time points. Relationships between oxygen saturation as determined by pulse oximetry and arterial oxygen saturation, between arterial carbon dioxide partial pressure and end-tidal carbon dioxide, and between oscillometric pressure and invasive arterial blood pressure were assessed, and the accuracy of pulse oximetry, capnography, and oscillometric blood pressure monitoring evaluated using Bland-Altman analysis. All three noninvasive methods provided relatively poor estimates of the reference values. Receiver operating characteristic curve fitting was used to determine cut-off values for hypoxia, hypocapnia, hypercapnia, and hypotension for dichotomous decision-making. Applying these cut-off values, there was reasonable sensitivity for detection of hypocapnia, hypercapnia, and hypotension, but not for hypoxemia. Noninvasive anesthetic monitoring should be interpreted with caution in giraffes and, ideally, invasive monitoring should be employed.
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http://dx.doi.org/10.1638/2016-0276.1DOI Listing
September 2017

PDE1A inhibition elicits cGMP-dependent relaxation of rat mesenteric arteries.

Br J Pharmacol 2017 Nov 15;174(22):4186-4198. Epub 2017 Oct 15.

Division of Synaptic Transmission, H. Lundbeck A/S, Valby, Denmark.

Background And Purpose: PDE1, a subfamily of cyclic nucleotide PDEs consisting of three isoforms, PDE1A, PDE1B and PDE1C, has been implicated in the regulation of vascular tone. The PDE1 isoform(s) responsible for tone regulation is unknown. This study used isoform-preferring PDE1 inhibitors, Lu AF58027, Lu AF64196, Lu AF66896 and Lu AF67897, to investigate the relative contribution of PDE1 isoforms to regulation of vascular tone.

Experimental Approach: In rat mesenteric arteries, expression and localization of Pde1 isoforms were determined by quantitative PCR and in situ hybridization, and physiological impact of PDE1 inhibition was evaluated by isometric tension recordings.

Key Results: In rat mesenteric arteries, Pde1a mRNA expression was higher than Pde1b and Pde1c. In situ hybridization revealed localization of Pde1a to vascular smooth muscle cells (VSMCs) and only minor appearance of Pde1b and Pde1c. The potency of the PDE1 inhibitors at eliciting relaxation showed excellent correlation with their potency at inhibiting PDE1A. Thus, Lu AF58027 was the most potent at inhibiting PDE1A and was also the most potent at eliciting relaxation in mesenteric arteries. Inhibition of NOS with l-NAME, soluble GC with ODQ or PKG with Rp-8-Br-PET-cGMP all attenuated the inhibitory effect of PDE1 on relaxation, whereas PKA inhibition with H89 had no effect.

Conclusions And Implications: Pde1a is the dominant PDE1 isoform present in VSMCs, and relaxation mediated by PDE1A inhibition is predominantly driven by enhanced cGMP signalling. These results imply that isoform-selective PDE1 inhibitors are powerful investigative tools allowing examination of physiological and pathological roles of PDE1 isoforms.
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http://dx.doi.org/10.1111/bph.14034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5659990PMC
November 2017

Down-regulation of K2.3 channels causes erectile dysfunction in mice.

Sci Rep 2017 06 19;7(1):3839. Epub 2017 Jun 19.

Department of Biomedicine, Pulmonary and Cardiovascular Pharmacology, Aarhus University, Aarhus, Denmark.

Modulation of endothelial calcium-activated K channels has been proposed as an approach to restore arterial endothelial cell function in disease. We hypothesized that small-conductance calcium-activated K channels (K2.3 or SK3) contributes to erectile function. The research was performed in transgenic mice with overexpression (K2.3 ) or down-regulation (K2.3 ) of the K2.3 channels and wild-type C57BL/6-mice (WT). QPCR revealed that K2.3 and K1.1 channels were the most abundant in mouse corpus cavernosum. K2.3 channels were found by immunoreactivity and electron microscopy in the apical-lateral membrane of endothelial cells in the corpus cavernosum. Norepinephrine contraction was enhanced in the corpus cavernosum of K2.3 versus K2.3 mice, while acetylcholine relaxation was only reduced at 0.3 µM and relaxations in response to the nitric oxide donor sodium nitroprusside were unaltered. An opener of K2 channels, NS309 induced concentration-dependent relaxations of corpus cavernosum. Mean arterial pressure was lower in K2.3 mice compared with WT and K2.3 mice. In anesthetized mice, cavernous nerve stimulation augmented in frequency/voltage dependent manner erectile function being lower in K2.3 mice at low frequencies. Our findings suggest that down-regulation of K2.3 channels contributes to erectile dysfunction, and that pharmacological activation of K2.3 channels may have the potential to restore erectile function.
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http://dx.doi.org/10.1038/s41598-017-04188-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5476588PMC
June 2017

Intravital investigation of rat mesenteric small artery tone and blood flow.

J Physiol 2017 08 30;595(15):5037-5053. Epub 2017 Jun 30.

Department of Biomedicine, Membranes, Aarhus University, Aarhus, Denmark.

Key Points: Substantial information on rat mesenteric small artery physiology and pharmacology based on in vitro experiments is available. Little is known about the relevance of this for artery function in vivo. We here present an intravital model where rat mesenteric small artery diameters are studied under isolated and controlled conditions in situ with simultaneous measurement of blood flow. The responses of the isolated arteries vary with the anaesthetic used, and they are quantitatively but not qualitatively different from the responses seen in vitro.

Abstract: Functional characteristics of rat mesenteric small arteries (internal diameter ∼150-200 μm) have been extensively studied in vitro using isometric and isobaric myographs. In vivo, precapillary arterioles (internal diameter < 50 μm) have been studied, but only a few studies have investigated the function of mesenteric small arteries. We here present a novel approach for intravital studies of rat mesenteric small artery segments (∼5 mm long) isolated in a chamber. The agonist-induced changes in arterial diameter and blood flow were studied using video imaging and laser speckle analysis in rats anaesthetized by isoflurane, pentobarbital, ketamine-xylazine, or by a combination of fentanyl, fluanison and midazolam (rodent mixture). The arteries had spontaneous tone. Noradrenaline added to the chamber constricted the artery in the chamber but not the downstream arteries in the intestinal wall. The constriction was smaller when rats were anaesthetized by rodent mixture in comparison with other anaesthetics, where responses were qualitatively similar to those reported in vitro. The contraction was associated with reduction of blood flow, but no flow reduction was seen in the downstream arteries in the intestinal wall. The magnitude of different endothelium-dependent relaxation pathways was dependent on the anaesthesia. Vasomotion was present under all forms of anaesthesia with characteristics similar to in vitro. We have established an intravital method for studying the tone and flow in rat mesenteric arteries. The reactivity of the arteries was qualitatively similar to the responses previously obtained under in vitro conditions, but the choice of anaesthetic affects the magnitude of responses.
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http://dx.doi.org/10.1113/JP274604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5538206PMC
August 2017
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