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Institutionen för fysik, kemi och biologi
Examensarbete
TRP channels and regulation of blood flow in the brood
patch of Zebra finches (Taeniopygia guttata)
Malin Silverå Ejneby
Examensarbetet utfört vid IFM
2010-06-01
LITH-IFM-G-EX--10/2339—SE
Linköpings universitet Institutionen för fysik, kemi och biologi
581 83 Linköping
Nyckelord
Keyword
4α-PDD, Brood patch, Carvacrol, TRPV3, TRPV4, Zebra finch
Sammanfattning
Abstract
During the breeding season Zebra finch, Taeniopygia guttata, females develops a brood patch on
the ventral surface which facilitates heat exchange between the incubating bird and the egg. The
brood patch has to be sensitive to changes in temperature, so that the eggs can be kept at an
optimal temperature for embryo development. If the egg temperature drops it has to be re-warmed.
Mild cooling of the brood patch has been shown to cause cold induced vasodilation, but the
responsible mechanism for this is not known. In this study we investigated if known
thermoreceptors, TRPV3 and TRPV4, could be involved in the alteration of blood flow. To
activate TRPV3 and TRPV4 two agonists, carvacrol and 4α-PDD respectively, were applied on
the brood patch. Changes in skin temperature and vascularity were then examined. The results
obtained did not reveal any changes in the vascularity. Temperature changes in the skin that could
be caused by an alteration in blood flow did not significantly change either. Still, a role of these
channels in the brood patch cannot be excluded.
Titel
Title
TRP channels and regulation of blood flow in the brood patch of Zebra finches (Taeniopygia
guttata)
Författare
Author
Malin Silverå Ejneby
ISBN
__________________________________________________
ISRN
__________________________________________________
Serietitel och serienummer ISSN
Title of series, numbering
LITH-IFM-G-Ex—10/2339--SE
URL för elektronisk version
Rapporttyp
Report category
Licentiatavhandling
x Examensarbete
C-uppsats
D-uppsats
Övrig rapport
_______________
Språk
Language
Svenska/Swedish
x Engelska/English
________________
Datum
Date
2010-06-01
Avdelning, Institution
Division, Department
Biology, IFM
Content
1 Abstract ........................................................................................................................................... 1
2 Abbreviations .................................................................................................................................. 1
3 Introduction ..................................................................................................................................... 1
4 Method ............................................................................................................................................ 2
4.1 Animal handling ...................................................................................................................... 2
4.2 Nest monitoring ....................................................................................................................... 2
4.3 Brood patch scoring ................................................................................................................ 2
4.4 Agonists ................................................................................................................................... 3
4.5 Experimental procedure .......................................................................................................... 3
4.6 Recordings ............................................................................................................................... 4
4.7 Statistic .................................................................................................................................... 4
5 Results ............................................................................................................................................. 4
5.1 Brood patch scoring ................................................................................................................ 4
5.2 Temperatures ........................................................................................................................... 4
5.3 Vascularity .............................................................................................................................. 6
5.4 Statistics .................................................................................................................................. 7
6 Discussion ....................................................................................................................................... 7
6.1 TRPV3 ..................................................................................................................................... 7
6.1.1. Physiological role ............................................................................................................ 7
6.1.2. Agonists ........................................................................................................................... 8
6.1.3. Vasoactive role ................................................................................................................ 8
6.2 TRPV4 ..................................................................................................................................... 9
6.2.1 Physiological role ............................................................................................................ 9
6.2.2 Agonist ............................................................................................................................ 9
6.2.3. Vasoactive effects .......................................................................................................... 10
7 Acknowledgment ........................................................................................................................... 10
8 References ..................................................................................................................................... 11
Appendix 1 ............................................................................................................................................ 14
Appendix 2 ............................................................................................................................................ 15
1
1 Abstract
During the breeding season Zebra finch, Taeniopygia guttata, females develops a brood patch
on the ventral surface which facilitates heat exchange between the incubating bird and the egg.
The brood patch has to be sensitive to changes in temperature, so that the eggs can be kept at
an optimal temperature for embryo development. If the egg temperature drops it has to be re-
warmed. Mild cooling of the brood patch has been shown to cause cold induced vasodilation,
but the responsible mechanism for this is not known. In this study we investigated if known
thermoreceptors, TRPV3 and TRPV4, could be involved in the alteration of blood flow. To
activate TRPV3 and TRPV4 two agonists, carvacrol and 4α-PDD respectively, were applied
on the brood patch. Changes in skin temperature and vascularity were then examined. The
results obtained did not reveal any changes in the vascularity. Temperature changes in the skin
that could be caused by an alteration in blood flow did not significantly change either. Still, a
role of these channels in the brood patch cannot be excluded.
2 Abbreviations
4α- PDD – 4α-phorbol 12,13-didecanoate
AVA – Arteriovenous anastomoses
CIVD – Cold induced vasodilation
EDHF – Endothelium derived hyperpolarizing factor
NO – Nitric oxide
TRP – Transient receptor potential
TRPV- Transient receptor potential vanilloid
3 Introduction
During the breeding season many birds develop a brood patch on the ventral surface (Lea and
Klandorf, 2002). This is the case for Zebra finch females, Taeniopygia guttata, but not males
because they do not develop a brood patch (Zann and Rossetto, 1991). The formation of the
brood patch includes morphological changes such as 1) de-feathering 2) an increase in
vascularity 3) edema, which can be seen as puffiness and wrinkles (Kern and Coruzzi, 1979;
Lea and Klandorf, 2002). The brood patch facilitates heat exchange between the incubating
bird and the eggs, which is important because if the egg temperature drops to values that
would suppress embryo development, the egg has to be re-warmed (Brummermann and
Reinertsen, 1991). Normally, local cooling of the skin respond with vasoconstriction due to a
reflex which include input from an afferent pathway, integration of the information in central
nervous system and an efferent pathway that target organs to control heat transfer (Daanen,
2003). Vasoconstriction is important to reduce heat loss in cold ambient temperatures
(Daanen, 2003), but in the brood patch of Bantam hens it has been shown that in response to
cooling, the blood flow increases (Midtgård et al, 1985). This response is known as cold
induced vasodilation (CIVD). Arteriovenous anastomoses (AVAs) are medium sized blood
vessels in which blood can be passed from arterioles to venules without going through
capillaries (Daanen, 2003; Lea and Klandorf, 2002) and they are suggested to have an
important thermoregulatory role in the brood patch (Midtgård, 1985). AVAs has a thick
muscular wall which is under control of the sympathetic nervous system and when these
vessels are open large amount of blood can be delivered (Daanen, 2003). The mechanism
responsible for CIVD is not yet known but the initial response seems to be a neural reflex
probably targeting AVAs, followed by a local mechanism largely independent of neurons
(Peltonen and Pyörnilä, 2004).
2
In this study we hypothesized that known thermoreceptors will alter blood flow in the brood
patch when they are stimulated, in vivo. Only Zebra finch females were chosen to participate
in the study because they have more blood vessels compared with males. If there were any
visible responses in vascularity it should be extra visible in these subjects. There are nine TRP
(transient receptor potential) cation channels activated by different temperatures and six of
these are thought to have a role in temperature sensation. TRPV1 are activated by
temperatures > 42˚C, TRPV2 > 52˚C, TRPV3 > 33˚C, TRPV4 >25-35˚C, TRPA1 < 17˚C and
TRPM8 < 25˚C (Bandell et al, 2007). Transient receptor potential vanilloid, member 3 and 4
(TRPV3, TRPV4) are activated in the temperatures expected to play a major role for thermo-
sensitivity in the brood patch during incubation and therefore these two are chosen to be
further investigated. TRPV3 is expressed predominately in keratinocytes and neural tissues
(Vogt-Eisele et al, 2007) and it have also been found in the skin of Jungle fowls (Jafari, 2009).
TRPV4 is expressed in tissues such as kidney, brain, liver, heart, fat, vascular endothelium,
hypothalamus and keratinocytes (Patapoutian et al, 2003) but in the study by Jafari (2009) the
expression of TRPV4 in the skin of Jungle fowls was more uncertain though.
4 Method
4.1 Animal handling
In the start of the study there were 31 zebra finches, 10 males, 14 females and 7 fledglings.
During the period of the study 1 female died and 9 hatchlings fledged which gives a total
number of 39 zebra finches. The birds were kept in a cage (2.17 x 2.16 m wide and 2.00 m
high) build with a wooden frame, covered with chicken wire within a greenhouse. The cage
contained 10 nests with an inner size of (9 x 9 x 9 cm) and had an entrance with a diameter of
4 cm. The floor in the cage was covered with soil and in the middle of the study some
rhubarb, Rheum rhabarbum, was planted in the cage to stimulate natural living. The birds
were exposed to natural daylight but with a minimum of 12 h light. Temperature were
fluctuating between 20- 30 ˚C but were not allowed to fall under 19 ˚C. The birds were given
a mix with boiled eggs, eggshell and sprouted seeds form alfalfa, Medicágo sativa, and mung
beans, Vigna radiata, twice a week. They also had free access to water and mixed seeds (Fink
blandning, Imazo AB, Sweden). Cocos and string fibers were supplied at the floor as nest
building material.
4.2 Nest monitoring
The nests were checked once a day (Monday - Friday) to determine nest status. Building
material amount, number of eggs and number of hatchlings were checked. To confirm which
bird attending which nest and separate the broody from the non-broody birds they all have a
microchip implanted subcutaneously on their backs between the wings. A microchip-reader
(Portable transceiver system, FS 2001 F-ISO, Newport) was placed around the nest and
microchip readings were stored in a file every 15 seconds if any bird attended the nest. New
microchip detection was stored even if 15 seconds had not passed.
4.3 Brood patch scoring
Birds were caught and brood patch development was assessed according to de-feathering,
vascularity and edema in the beginning of the study. The birds were held in the hand kept on
their backs. De-feathering was scored first, then water soluble gel (Klick, RFSU AB, Sweden)
diluted in water 1:1 was applied on the brood patch with a cotton tipped applicator to get a
more visible view. Afterward edema seen as wrinkles and puffiness was scored. USB digital
microscope (Dino-lite pro, AM-413ZT, AnMo Electronics Corporation, Taiwan) was used to
3
easier score vascularity. Information obtained was used to develop a brood patch scoring
protocol for later experiments (Table 1).
Table 1. Brood patch scoring protocol. Protocol was established by examine brood patch of birds. Used as a
protocol for scoring the brood patch in later experiments.
Score De-feathering Edema Vascularity
1 ~20 down feathers Normal tight skin Big vessel visible in the middle
of the thorax
2 Feathers on both abdomen
and thorax but less than a
total of 20 down feathers
Big vessel more visible in the
middle of the thorax and
branching seen
3 Less down feathers on
abdomen
Loose skin One other vessel visible, total
number of two
4 Less down feathers on thorax Two other vessels visible, total
number of three
5 No feathers on abdomen and
thorax
Loose fluid filled skin Vessels on abdomen shaped
like a three
4.4 Agonists
All substance was purchased from Sigma Aldrich including carvacrol and 4α-PDD (4α-
phorbol 12,13-didecanoate). Carvacrol was prepared in solution of 8 mM in ethanol (79%).
4α-PDD was prepared in a stock solution of 1.5 mM in ethanol (100%), stored at -30˚C.
Further dilutions of 4α-PDD were accomplished in water.
4.5 Experimental procedure
A plastic-bag was filled with isoflurane (7.7%) supplied from a gas vaporizer (DrägerWerk
AB, Lübeck) and the bird was placed inside. Once the bird was anaesthetized it was placed on
a heat pad (OBH Nordica, S8-S) which could be adjusted at two levels to keep the body
temperature stable. The head was put in an open whole-head mask connected to plastic tubes
from the gas vaporizer, supplying isoflurane (2.3%). A temperature probe was inserted in the
cloaca to measure core temperature and ventilation events were logged in a recording system
(Power lab 4/25T, ADInstruments) to obtain breathing frequency. Temperature and ventilation
were allowed to stabilize before experiments began. If ventilation was irregular or lower than
~50 breaths per minute isoflurane was lowered to 1.5%. The core temperature was kept as
stable as possible by adjusting heat pad temperature and by putting heat blankets (Klini drape,
Mölmlicke healthcare) over the bird. The heat blankets had a cut hole in the middle, so that
the brood patch could be exposed. Brood patch scoring and other experimental protocols such
as cold probe stimulation were carried out before the application of TRP agonists. TRPV3 and
TRPV4 ion channels were stimulated with their agonists carvacrol (8 mM) (Earley et al, 2010;
Vogt-Eisele et al, 2007; Xu et al, 2006; Ueda et al, 2009) and 4α-PDD (10 μM) (Birder et al,
2007 ; Fian et al, 2007; Vriens et al, 2004; Watanabe et al, 2002) respectively. 20 μl carvacrol
or 4α-PDD were applied with a pipette on the brood patch and a cotton- tipped applicator with
parafilm on was used to distribute it on the abdomen of the brood patch. Both agonists were
applied for 4 min. A cotton tipped applicator was then used to remove and absorb the liquid
4
on brood patch. After the experimental protocol was completed, approximately after two
hours, the rectal probe was removed and isoflurane was turned off. The bird was then allowed
to recover in a smaller cage for at least 30 minutes before it was returned to the big aviary in
the greenhouse.
4.6 Recordings
Temperature on the brood patch was measured with an infrared thermometer (68 Infrared
thermometer, Fluke Corporation, Everett) and a infrared camera (InfraCAM, FLIR systems,
Danderyd). Core temperature values used in graphs and statistics were documented at the
same time brood patch temperature was measured with the infrared thermometer. Visible
changes in vascularity were documented by USB digital microscope (Dino-lite pro, AM-
413ZT, AnMo Electronics Corporation, Taiwan). A digital camera (Panasonic, NR DMC-
FZ18, Matsushita electric industrial co. Ltd, Japan) was used to save pictures of the brood
patch if later needed for localization of other brood patch pictures with USB microscope or
infrared camera. All recordings were carried out before the agonists were applied,
immediately after a 4 min application and then every second minute for 10 min.
4.7 Statistic
Data analyses of the results were performed using a general linear model in SPSS statistics
17.0 (SPSS inc.,Chicago, USA). Time was used as the repeated measurement factor and
treatment as the between subjects factor. P-values used for core temperature and infrared
thermometer are the ones corresponding to a linear model and p-values for infrared camera are
the ones corresponding to a quadratic model. Choice of the appropriate model was done
visually. Paired t-test performed in Excel 2007 (Microsoft Corporation, Kisa, Sweden) was
used to detect changes in time for individual treatments.
5 Results
5.1 Brood patch scoring
Zebra finch females were selected based on nest status, nest attendance, and brood patch
score. The brood patch scorings for all subjects used are present in Table 2.
Table 2. Brood patch scorings. Each subjects brood patch score and average for all subjects used in experiment.
Subject 1 2 3 4 5 6 7 8 9 Average
De-feathering 4 4 4 5 3 4 4 4 5 4.1±0.6
Edema 3 1 1 3 1 3 1 1 5 2.1±1.4
Vascularity 4 2 5 5 2 5 5 3 5 4±1.3
5.2 Temperatures
Core temperature measured with a rectal probe during the experiment (Figure 1) was tried to
be kept as stable as possible by regulating heat pad temperature and by putting heat blankets
over the bird if needed. This would minimize thermoregulatory responses due to changes in
core temperature. Still there were a significant changed with time (p <0.05) but no significant
change due to the treatment. These results suggest that changes in core temperature were not
due to the activation of agonists but instead an effect of heat pad adjusting or heat blankets put
over the bird.
5
Figure 1. Core temperature recordings measured with rectal probe during experiment. Core temperature
changes over time were significant (p <0.05) but there was no significant differences due to the treatments.
Data shown as average and standard deviation.
Brood patch temperature measured with infrared thermometer (Figure 2) does not show any
significant change with time or treatment suggesting that there is no effect due to the agonists
or the applied liquid. However compared with temperatures obtained with the infrared camera
(shown below) these temperatures are lower. This could probably be due to the distance
between the infrared thermometer and brood patch while measuring. A closer distance than
the optimal could border the area where temperature is measured. This would result in that a
larger area than the brood patch itself contribute to the obtained temperature values.
Figure 2. Temperature measured with an infrared thermometer on the abdomen where the different agonists
were applied. There were no significant differences depending on time or treatment. Data shown as average and
standard deviation.
6
Temperature measured with an infrared camera (Figure 3) shows a change in temperature with
time (p<0.05) which seems to be more due to the liquid applied than the agonists because no
significant differences in temperature between treatments were detected. Human skin has an
emissivity of ~98 % which allows the thermal camera to measure temperature with a relatively
high accuracy, but when applying a medium on the skin the emissivity will change (Williams,
2009). In the infrared camera pictures for the controls and 4α-PDD colder spots on the brood
patch can be seen after the liquid was removed but not for carvacrol (example seen in
appendix 1). These differences could probably be explained by difference in emissivity caused
by the liquids since carvacrol was prepared in a solution with a larger concentration of alcohol
or the fact that it was absorbed by the skin easier.
Figure 3. Temperature measured with an infrared camera on the brood patch. The results show that the
temperature change with time (p<0.05) but there were no significant differences between the treatments. Data is
shown as average and standard deviation.
5.3 Vascularity
USB microscope pictures were scored based on visible changes in vascularity. Pictures before
were compared with pictures after 4 min of application of the agonist. Scores were defined as
described in Table 3.
Table 3. USB microscope picture scores. Scores were assed based on changes in the vascularity seen in the
picture after 4 minutes compared with the before pictures. Scores were defined as described above.
Score Definition
2 Clear change due to vasodilatation
1 Small change that could be due to vasodilatation
0 No visible change
-1 Small change that could be due to vasoconstriction
-2 Clear change due to vasoconstriction
Neither of the treatment reached a score large enough to be assessed as a change due to
vasoconstriction or vasodilatation as seen in figure 4.
7
Figure 4. USB microscope prictures scoring.. USB pictures after 4 minutes of treatmens was scored based on
changes in vascularity. Results show that there is no treatment that reaches a score that could suggest that there
are any changes in vascularity. Data shown as mean and standard deviation.
5.4 Statistics
The statistic results obtained with a general linear model in SPSS are seen appendix 2.
6 Discussion
Zebra finches egg temperature is kept between 36-38 C˚ (Zann and Rossetto, 1991). This
temperature range is optimal for embryo development and if the eggs are exposed to an
extended time of cold, it will cause abnormalities in embryo development (Brummermann and
Reinertsen, 1991). Therefore, temperature sensing on brood patch is important so that an
appropriate response could be induced if the egg has to be re-warmed. In bantham hens it has
been shown that temperatures between 25- 40 C˚ will cause CIVD. As mentioned previously,
CIVD is suggested to be initiated by a neural reflex followed by a local response largely
independent of neurons (Peltonen and Pyörnilä, 2004). In this study we hypothesized that
known thermoreceptors present in the skin (Jafari, 2009) were involved in changes of the
blood flow. Two receptors were chosen to be investigated, TRPV3 and TRPV4, because of
their activation in given temperature range responsible for CIVD. Even if no obvious changes
in vascularity and temperature could be obtained small changes cannot be excluded. Today,
the knowledge about TRPV3 and TRPV4 is an emerging field and below known physiological
roles that can be important in brood patch, available agonists and the obtained results for these
channels will be discussed.
6.1 TRPV3
6.1.1. Physiological role
Transient receptor potential vanilloid, member 3 (TRPV3) is an ion channel selective for Ca2+
and Na2+
(10:1) (Early et al, 2010) and it is expressed predominantly in keratinocytes (Peier et
al, 2002). In humans TRPV3 has also been found in tongue, spinal cord, dorsal root ganglion
(DRG), brain, trimegal ganglion (Sherkheli et al 2009) and in rats it has been found in low
levels in vascular endothelium of cerebral and cerebellar arteries (Earley et al, 2010). TRPV3
8
is activated by temperatures above 33 ˚C (Dhaka et al, 2006) and has been shown to
participate in thermosensation. TRPV3 expressed in skin keratinocytes mediate release of
ATP due to heating and blockage of purinoreceptors, activated by ATP, located on sensory
neurons has been shown to disrupt the communication between keratinocytes and sensory
neurons (Mandadi et al, 2009). These results suggest that ATP act as a signal molecule for
thermal information between keratinocytes and purinergic nerves innervating the skin
(Mandadi et al, 2009). It is also supported by results obtained with mice lacking
purinoreceptor, P2X3, which shows an impaired response to innocuous heat, 34-38 ˚C, with a
consentient trend toward noxious heat (Shimizu et al 2005). And mice lacking TRPV3 also
have an impaired sensation to innocuous and noxious heat (Moqrich et al 2005). In addition to
temperature sensation TRPV3 has been suggested to participate in regulation of local blood
flow (Lee and Caterina, 2005). Carvacrol, a TRPV3 agonist, has been shown to induce
arterial relaxation in rat cerebral and cerebellar arteries, in vitro, (Early et al, 2010). The
mechanism responsible for vasodilation caused by TRPV3 agonist carvacrol is due to an
increased intracellular [Ca2+
] in the endothelial cells which activate Ca2+
dependent K+
channels. In turn this results in a hyperpolarization of the endothelial cell membrane, which
spreads to the vascular smooth muscles and cause vasodilation (Early et al, 2010). But even if
these results suggest that TRPV3 expressed in endothelium may control local blood flow the
expression level is low. Therefore other mechanism involving TRPV3 and regulation of local
blood flow can be suggested. Lee and Caterina (2005) suggested an involvement for NO or
other signaling molecules release by keratinocytes, induced by activation of TRPV3 and
TRPV4.
6.1.2. Agonists
Today, there are no selective agonist available for TRPV3 but there are several compounds
that activate TRPV3 such as camphor, thymol, menthol, eugenol, vanillin, incensole acetate,
2-APB (2-aminoethoxydiphenylborate) (Vriens et al 2009), dihydrocaveol, 1,8-cineol and
carvacrol (Sherkheli et al 2009). Carvacrol is a monoterpenoid phenol found in oregano
(Early et al, 2010). It has been shown that carvacrol activate TRPV3 and stimulate Ca2+
influx
in skin epithelial cells, tongue epithelial cells (Xu et al 2006) vascular endothelium (Earley et
al, 2010) colonic epithelial cells (Ueda et al 2009) and corneal epithelial cells (Yamada et al,
2010). The effect of carvacrol is also potentiated at physiological temperatures, 37 ˚C,
compared with 22 ˚C (Xu et al 2006). Although, carvacrol activates both TRPV3 and TRPA1
(Xu et al 2006) the compound was still chosen to be used in this study. TRPA1 activity
rapidly desensitizes in response to continuously presence of carvacrol and TRPV3 is
sensitized (Xu et al 2006). These suggest that any changes in vascularity should be due to
TRPV3 activity. Carvacrol was also chosen because it is lipophilic and can target TRPV3 in
keratinocytes (Xu et al 2006).
6.1.3. Vasoactive role
The results obtained in this study did not reveal any changes in blood flow or temperature
caused by the TRPV3 agonist carvacrol. However, smaller changes could exist. Isoflurane
used to anaesthesia the birds has been shown to alter the blood flow and cause vasodilation
(Negoro et al, 2007) which could make changes in the local blood flow due to carvacrol more
difficult to detect. Temperature changes in brood patch due to the treatment could not be
detected, but the temperature changes in the brood patch measured with thermal camera
showed a large individual variation. If this variation is caused by differences in response to
carvacrol or a decrease in emissivity has to be further investigated. Anyway, there was no
9
pattern of the variation due to incubation status or brood patch score which otherwise could
have been suggested to play a role.
6.2 TRPV4
6.2.1 Physiological role
Transient receptor potential vanilloid, member 4 (TRPV4) is expressed in a broad range of
tissues such as heart, fat, liver, kidney, inner ear, hypothalamus, peripheral nervous system,
trachea and skin kerationocytes (Dhaka et al, 2006). TRPV4 is activated by temperatures >25-
35 ˚C (Bandell et al, 2007) and it is desensitized upon sustained or repeated heat application
(Chung et al, 2004). TRPV4 like TRPV3 is expressed in keratinocytes and heat has been
shown to activate TRPV4 in these cells too (Chung et al, 2003). In mice lacking TRPV4 it has
been shown that thermosensation to innocuous warmth and moderately hot temperatures is
altered. The TRPV4 knockout mice showed a prolonged withdraw latency during acute tail
heating, in hot water (46-47 ˚C). It also showed a larger preference for slightly higher
temperatures than wildtype mice on a floor with a temperature gradient ranging from 1- 49 ˚C.
In the same study it was also shown that wildtype mice were incapable of distinguish between
30˚C and 34˚C while mice lacking TRPV4 had a strong preference for 34˚C (Lee et al, 2005).
Taken together, these results suggest that TRPV4, like TRPV3, is responsible for
thermosensation. The role for TRPV4 in local blood flow has also been investigated. In
contrast to TRPV3, TRPV4 is highly expressed in endothelial cells ranging from aorta to
capillaries (Everaerts et al, 2009). It has been shown that activation of TRPV4 with 4α-PDD
in cerebral arteries results in an increased influx of Ca2+
and an increase in endothelium
derived hyperpolarizing factor (EDHF), resulting in vasodilation (Marrelli et al, 2007).
Further, in mice lacking TRPV4 there has been shown to be a decrease in EDHF- and NO-
mediated vasodilation while the endothelium-independent vasodilation remained (Saliez et al,
2008), suggesting an important role for TRPV4 expressed in endothelial cells. In addition to
thermosensation and regulation of blood flow TRPV4 is involved in urine bladder contraction.
Activation of TRPV4 in urothelial cells causes an increase in Ca2+
influx and release of ATP
from these cells. In the same study intravesicular administration of 4α-PDD altered urine
bladder contractility in rats when they were awake and inhibition of purinoreceptor P2X3
abolished this alteration. Therefore it seems that TRPV4 is involved in a neural reflex
controlling urinary bladder contractions, by mediating release of ATP from urothelial cells
(Birder et al, 2007). It may not appear relevant for TRPV4s role in the brood patch but it
shows that release of ATP due to the channel activity may have an important physiological
role for mediating neuron reflexes, and could probably do that even in the skin.
6.2.2 Agonist
TRPV4 was first discovered as an osmosensitive channel, activated by hypotonic cell swelling
but it could also be activated by other physical stimuli and chemical ligands (Vriens et al,
2009). It is activated by arachidonic acid, 5,6- epoxyeicosatrienoic acid (5,6-EET),
bisandrographolide A (BAA) (Vriens et al, 2009), and the synthetic phorbol ester 4α-PDD
(Vriens et al, 2009; Wantanabe et al, 2002; Fian et al, 2007; Birder et al, 2007). It has been
found that a tyrosin-serin motif on TRPV4 is important for its activation by heat and 4α-PDD.
A mutation in these sites caused a large decrease in channel activity. However, the response
for cell-swelling, arachidonic acid and 5,6-EET were not altered by these mutation (Vriens et
al, 2004). Today, 4α-PDD is the most selective agonist for TRPV4 but activation of 4α-PDD
is slow and diffusion in the cell might be rate limited (Vriens et al, 2009) and therefore all
substance applied on the brood patch were left for four minutes.
10
6.2.3. Vasoactive effects
Changes in vascularity could not be detected in these experiments. The lack of visible changes
could be explained as above for TRPV3 but it could also be due to the fact that diffusion of
4α-PDD in the cell is slow, suggesting that longer application of agonist is needed for
activation of TRPV4. Any significant temperature changes with the thermal camera due to 4α-
PDD could not be detected. However, comparing the graphs obtained with 4α-PDD and the
controls may suggest that temperature decreases more in the controls after 4 min application.
But as for carvacrol a large individual variation could be seen.
7 Acknowledgment
I want to acknowledge my supervisor, Dr Jordi Altimiras, for being enthusiastic, supportive
and helpful during the project. I also want to thanks Anna Södergren and Sofia Klubb for their
important role during this project and also for their motivating and supportive role during
these years at Linköpings universitet.
11
8 References
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