Dysfunctional TRPM8 signalling in the vascular response to environmental cold in ageing
- Section of Vascular Biology and Inflammation, School of Cardiovascular Medicine and Sciences, BHF Centre of Research Excellence, King’s College London, United Kingdom
- Cancer Research UK, Cambridge Institute, University of Cambridge, United Kingdom
- Skin Research Institute, Agency of Science, Technology, and Research (A*STAR), Singapore
Figures
Figure 1 with 1 supplement
Cold-induced vascular response is impaired with ageing.
(a) Diagram illustrates the experimental setup of cold-induced vascular response protocol; FLPI from top measures the blood flow in the hindpaw of the anaesthetised mouse when on a heating mat in …
Figure 1—figure supplement 1
Analysis of cold-induced blood flow in the mouse paw.
(a) The blood flow graph from cold treatment was used to calculate the maximum vasoconstriction, area under the curve (AUC) analysis of vasoconstriction and recovery of blood flow from cold …
Figure 2 with 1 supplement
Blood flow, skin temperature and tissue oxygen saturation with cold treatment in ageing.
(a) The mean ± s.e.m. blood flow trace of the vascular response with cold (4 °C) water treatment (n = 8). (b) % change in hindpaw blood flow from baseline to 0–2 min following cold treatment …
Figure 2—figure supplement 1
Oxidative stress with ageing.
(a) The representative western blot analysis of 3-nitrotyrosine (3-NT), which is a biomarker of oxidative stress produced by reactive nitrogen species, in hind paw skin of naïve young and aged mice. …
Figure 3
Behavioural analysis with cold plate in young and aged mice.
(a–c) Time of first response of mice to cold plate set at 4 °C, 10 °C, and 20 °C. (d-f) The total number of responses from mice during the cold plate experiment at 4 °C, 10 °C, and 20 °C. (g–h) Time …
Figure 4
TRPA1 and TRPM8 are involved in cold-induced vascular response.
Vascular responses with cold (4 °C) water treatment in mice pre-treated with combined TRPA1 antagonist A967079 (100 mg kg–1) and TRPM8 antagonist AMTB (10 mg kg–1), or vehicle control (Veh - 10 % …
Figure 5 with 1 supplement
TRPA1 and TRPM8 activity deteriorates with ageing (a) Graph shows the % mean ± s.e.m.
of blood flow change from baseline in response to topical application of menthol (10%) and vehicle (Veh - 10 % DMSO in ethanol) in ear of young and aged mice. (b) % maximum change in ear blood flow …
Figure 5—figure supplement 1
Agonist induced blood flow response in mouse ear.
(a) The representative image from FLPI shows the blood flow response induced by topical application of vehicle (left ear) and 10 % cinnamaldehyde (right ear) in the anaesthetised mouse. (b) The …
Figure 6 with 1 supplement
Dysfunctional sympathetic signalling in ageing (a) Graph shows the mean ± s.e.m.
blood flow in hindpaw with intraplantar injection of noradrenaline (1.25 ng/µl in saline in 20 µl) in young and aged mice (n = 8). (b) % maximum change in blood flow from baseline induced by …
Figure 6—figure supplement 1
Phospho-tyrosine hydroxylase with ageing.
(a) The representative western blot analysis of phospho-tyrosine hydroxylase in hind paw skin of naïve young and aged mice. The densitometric analysis is normalised to total tyrosine hydroxylase. …
Figure 7 with 2 supplements
Sympathetic-sensory signalling and influence of ageing.
(a–b) RT-PCR CT analysis shows the expression and fold change of Trpa1 and Trpm8 in young and aged SG normalised to three housekeeping genes collected from the cervical and thoracic paravertebral …
Figure 7—figure supplement 1
Sympathetic-sensory signalling in young and aged mice.
(a–c) RT-PCR CT analysis shows fold change of α2a adrenoceptor, α2c adrenoceptor and Trpv1 in young and aged mice normalised to three housekeeping genes in (DRG). Results are shown as mean ± s.e.m. …
Figure 7—figure supplement 2
Characterisation of sympathetic markers.
(a) RT-PCR CT analysis shows fold change of the sympathetic nerve marker Dopamine β-hydroxylase (Dbh) in DRG and SG normalised to three housekeeping genes. (b) The western blot analysis of the …
Figure 8
Proposed cold-induced vasoconstriction signalling pathway in young and aged mice.
The local cold exposure produces rapid vasoconstriction which is significantly blunted in the aged mice (see blood flow graph at top centre). Cold water (4 °C) exposure to hindpaw activates the cold …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Other | CD1 | Charles River | 022; RRID:IMSR_CRL:22 | Mouse (Mus musculus) |
| Antibody | Anti- TRPM8 (Rabbit polyclonal) | Alomone Labs | Cat# ACC-049, RRID:AB_2040254 | “(1:500)” |
| Antibody | Anti-alpha2c adrenergic receptor (Rabbit polyclonal) | Novus Biologicals | Cat# NB100-93554, RRID:AB_1236641 | “(1:1000)” |
| Antibody | Anti-tyrosine hydroxylase (phospho ser41) (Rabbit polyclonal) | Novus Biologicals | Cat# NB300-173, RRID:AB_2201648 | “(1:1000)” |
| Antibody | Anti-tyrosine hydroxylase (Rabbit polyclonal) | Novus Biologicals | Cat# NB300-109, RRID:AB_10077691 | “(1:1000)” |
| Antibody | Anti-p21 (Mouse monoclonal) | Santa Cruz Biotechnology | Cat# sc-6246, RRID:AB_628073 | “(1:1000)” |
| Antibody | Anti- GAPDH (Rabbit polyclonal) | Thermo Fisher Scientific | Cat# PA1-987, RRID:AB_2107311 | “(1:2000)” |
| Antibody | Anti- β- actin(Mouse monoclonal) | Sigma-Aldrich | Cat# A5441, RRID:AB_476744 | “(1:2000)” |
| Antibody | Anti-α- tubulin(Rat monoclonal) | Millipore | Cat# MAB1864, RRID:AB_2210391 | “(1:2000)” |
Additional files
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Supplementary file 1
List of primer sequences.
- https://cdn.elifesciences.org/articles/70153/elife-70153-supp1-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/70153/elife-70153-transrepform1-v2.docx
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Source data 1
Orginal blots.
- https://cdn.elifesciences.org/articles/70153/elife-70153-supp2-v2.docx
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Source data 2
Raw source files.
Figure 1c: FLPI blood flow data; Figure 2a: VMS Blood flow data; Figure 2d: Skin Temperature; Figure 2g: Oxygen Saturation; Figure 4a: BF data with antagonist; Figure 4b: BF data A967; Figure 4c: BF data AMTB; Figure 5a: BF - Menthol; Figure 5d: BF - CA; Figure 5g: BF – Capsaicin; Figure 6a: BF – NA; Figure 6c: BF - Medetomidine; Figure 7d: Calcium imaging data; Figure 7f: Calcium imaging data.
- https://cdn.elifesciences.org/articles/70153/elife-70153-supp3-v2.xlsx
