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

Bandell, M., Macpherson, L.J and Patapoutian, A (2007) From chills to chilis: mechanisms for

thermosensation and chemesthesis via thermoTRPs. Current opinion in Neurobiology. 17, 490 - 497

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Appendix 1

A) Control

Before After 4 min

B) 4α-PDD

Before After 4 min

C) Carvacrol

Before After 4 min

15

Appendix 2

Table 1 Results from one way ANOVA with repeated measurements.

ANOVA p-value Time Interaction Treatment

Infrared thermometer 0,38 0,655 0,942

Core temperature 0,083 0,953 0,99

Infrared camera 0,031 0,172 0,153