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Abstract

Tachylcinin receptors (NK1, NK2 and NK3) and transient receptor potential vanilloid
channels (TRPV1 and TRPV2) are widely distributed throughout the central nervous
system (CNS) and peripheral tissues of mammals. They play central roles in
nociception, cardiovascular and respiratory function. In addition, tachykinin
receptors and TRPV1 appear to play vital roles in neurogenic inflammation and in
the modulation of the innate immune system through PBMCs and monocytes.
This thesis represents a 10 year research journey that documents: 1) the development
of an extensive range of molecular biological techniques for the analysis of
tachykinin receptor and TRPV expression; 2) the effects of capsaicin administration
on tachykinin receptors and TRPV expression in CNS and peripheral tissue of rats,
and 3) the expression and potential function of TRPV1 in human peripheral blood
mononuclear cells (PBMC), naïve monocytes and the human monocytic, THP-1 cell
line.
In the rat studies, quantitation of tachykinin receptor and TRPV1 expression in both
CNS and peripheral tissues, including PBMC, provided some interesting fmdings.
The mRNA expression of cFOS as a marker of FOS mediated transcription activity
was also quantified as an early measure of cellular transcription levels. Overall,
cFOS was the most highly expressed gene in all tissues studied with expression
levels >1 x 106 copies mRNA transcript/106 copies GAPDH.
In CNS tissues, NK3 expression was consistently higher than NK1 and NK2
expression, which is supported by later published observations based on gene chip
data. NK2 expression was very low in all CNS tissues studied, particularly the brain
stem which was in agreement with autoradiographic mapping of NIC2 receptors using [ 125 I]-NKA in our laboratory. In general, the expression profile of tachykinin
receptors in CNS tissue was NK3>NK1>NK2. In the peripheral tissues studied, the
expression profiles differed markedly, though as a general rule they agreed with
previously published radioligand binding data.
Systemic capsaicin administration produced changes in tachykinin receptor mRNA
expression in specific tissues. There was no effect on tachykinin receptor mRNA
expression in the cerebral cortex or brain stem in response to capsaicin (data which
agreed with our initial pilot study). However, in the cervical spinal cord of
capsaicinised rats, NK1 and NK3 mRNA transcripts were significantly lower
compared to vehicle controls indicating a significant down-regulation, whilst NK2
and TRPV1 message were significantly higher. Surprisingly, up-regulation of cFOS
expression was not observed with the significant changes in receptor expression in
the cervical spinal cord. In peripheral tissues, capsaicin administration significantly
down-regulated NK1 transcription in the urinary bladder This finding corresponds
well with previous data showing a decrease of NK1 receptor radioligand binding in
the urinary bladder in response to capsaicin. The most marked changes in receptor
expression levels were seen in PBMC where, although initial receptor expression
was low there were significant changes in cF0S, NK1, NK2 and NK3 mRNA
expression in response to capsaicin. Thus, stimulation of TRPV1—expressing neurons
by capsaicin in both the CNS and peripheral tissues leads to quite dramatic changes
in the mRNA expression of tachylcinin receptors. Collectively, this series of
experiments showed that modern molecular techniques could be used to identify
quantitative changes in pharmacological receptor transcript following specific
interventions.
In the human studies, both TRPV1 and TRPV2 expression was demonstrated in
PBMC and isolated naïve monocytes for the first time. The mRNA expression of
both TRPVI and TRPV2 was much greater in PBMC (>90% lymphocytes)
compared to isolated naïve monocytes, whilst TRPV2 expression was significantly
higher than TRPV1 expression. TRPV1 mRNA and protein (measured by Western
blot) was also highly expressed by monocytic THP-1 cells. In PBMC, naive
monocytes and THP-1 cells, low concentrations of capsaicin dose-dependently
increased cell metabolic activity. This effect occurred immediately and was
maintained for at least 1 hour, on exposure to capsaicin concentrations that had
previously been shown to cause cell death. The capsaicin-induced increase in
metabolic activity was abolished by prior exposure to the TRPVI antagonist, 5'-iodoresiniferatoxin,
indicating a TRPV I mediated mechanism. In addition to stimulating
metabolic activity, capsaicin was also shown to dramatically modify mitogeninduced
inflammatory cytokine release from THP-1 cells. Both phytohaemagglutinin
A (PHA) and lipopolysaccharide (LPS) stimulated the release of IL-1I3, IL-6, TNF-a
and MCP-1 from THP-1 cells. Capsaicin pretreatment significantly reduced PHAsimulated
release of all four cytokines, an effect which concentration-dependent. In
contrast, capsaicin pretreatment significantly increased LPS-stimulated IL-6 and
TNF-a secretion. Thus, prior exposure of cells to capsaicin, at levels that would
theoretically equate to those present after a high dose chilli meal, resulted in
significant reductions in PHA-stimulated inflammatory cytokine release. The overall
anti-inflammatory effects of capsaicin reported here may partly explain the apparent
protective role of TRPV1 in atherosclerosis and diabetes. Thus, this series of
experiments identified TRPV expression on naïve monocytes, and validated THP-I
cells as a model system for the study of TRPV1 message, protein and function
monocytes.
In conclusion, the studies described in this thesis demonstrate a number of important
features of tachykinin receptor and TRPV expression in rat and human cells. In the
rat brain stem, there is no change in mRNA expression of the tachykinin receptors in
response to the systemic administration of capsaicin. This suggests that the
modulation of respiratory function identified in previous studies in this laboratory
may not have been due to changes in genetic expression but some other dynamic
regulatory process (although the capsaicin treatment regimens were different).
Significant and surprising changes in cFOS and tachykinin receptor expression were
seen in whole blood suggesting an immunological aspect to tachykinin functions. For
the first time, TRPV 1 and 2 expression profiles of PBMC (>90% lymphocytes) and
naïve monocytes were compared and shown to be substantially different. Moreover,
a (THP-1) cell culture system for the study of TRPV1 function in monocytes was
established and revealed that capsaicin not only produces a novel metabolic response
in monocytes but may also have significant immunomodulatory effects by
dramatically altering mitogen-stimulated cytolcine release.

Item Type:	Thesis - PhD
Authors/Creators:	Kunde, Dale Andrew
Copyright Holders:	The Author
Copyright Information:	Copyright 2010 the Author - The University is continuing to endeavour to trace the copyright owner(s) and in the meantime this item has been reproduced here in good faith. We would be pleased to hear from the copyright owner(s).
Additional Information:	Thesis (PhD)--University of Tasmania, 2010. Includes bibliographical references
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