Journal of the New Zealand Medical Association, 15-July-2005, Vol 118 No 1218
Proceedings of the 178th meeting of the Otago Medical School Research Society, 7 July 2005
Possible modulation of serotonin receptor 2A by filamin-C. Erin Cawston, A Fitches, D Markie, R Olds. Department of Pathology, Dunedin School of Medicine, University of Otago, Dunedin.
Serotonin receptor 2A (5-HT2A) has many biological roles. In the peripheral nervous system it has been shown to mediate contraction of vascular and extra-vascular smooth muscle and is involved in platelet aggregation. In the central nervous system, clinical studies have shown that 5-HT2A may be differentially regulated in schizophrenia and other psychiatric disorders, but the direction and magnitude of these changes remains controversial. It has also been shown that classic hallucinogens and atypical antipsychotic drugs act principally through 5-HT2A to modulate human perception and cognition. We hypothesised that intracellular proteins that interact with the cytoplasmic C-terminal of 5-HT2A may modulate the function of the receptor.
The C-terminal cytoplasmic region of murine 5-HT2A was used to find binding partners from a 7 day mouse embryo cDNA expression library. This mating strategy screened the C-terminal of 5-HT2A (amino acids 384-471) by a yeast two-hybrid approach. BLAST searches of sequence databases showed similarity to murine filamin-C for seven of the interacting clones. The interaction between the human homologues of 5-HT2A (aa387-471) and filamin-C (aa2141-2705) was confirmed using the yeast two-hybrid system and by an in vitro capture assay. The interaction is biologically plausible, as co-expression of both 5-HT2A and filamin-C was identified in two of eight neural cell lines. The 5-HT2A-binding domain of filamin-C has been further localised to repeats 20 and 21 and appears to be enhanced by the presence of a unique sequence, found only in filamin-C and not in the closely related filamins A or B.
Filamin proteins have been shown to act as scaffolding proteins, therefore we suggest the interaction between filamin and 5-HT2A may provide a mechanism for internalisation or recycling of the receptor, so influencing the sensitivity of the cell.
The interaction between the delta epithelial sodium channel (δENaC) and its downregulator Murr1. Tina Chang, FJ McDonald. Department of Physiology, Otago School of Medical Sciences, University of Otago, Dunedin.
The epithelial Na+ channel (ENaC) is an important regulator of salt and water balance in the body, and hence blood pressure. The classic ENaC consists of three subunits, α, β and γ. A fourth ENaC subunit, named δ, is highly expressed in “non-traditional” epithelial tissues, such as brain, testis, ovary and pancreas, and the δENaC channel is activated by acidic extracellular pH. However, the physiological role of δENaC remains uncertain. A copper-toxicosis related protein called Murr1 was identified as a binding partner of δENaC, and it inhibits δENaC channel activity through direct interaction. The present study aims to explore the interaction between δENaC and Murr1 by investigating the sites of protein-protein interaction and subcellular locations.
Deletion constructs for both δENaC and Murr1 were prepared, and a glutathione S-transferase pull down assay and Western blot analysis were used to identify the sites of protein interactions. The Murr1 binding site was narrowed down to amino acids 592-615 of δENaC, and the δENaC binding site in Murr1 was located between amino acids 140-150. Immunocytochemical studies and subcellular fractionation revealed that FLAG-tagged δENaC (δENaCFLAG) is located in the cytosol, in association with vesicular compartments, and HA-tagged Murr1 (Murr1HA) is located around the nucleus in COS-7, HEK293 and MDCK cell lines. When coexpressed, Murr1HA colocalised with δENaCFLAG, suggesting regulation of δENaC by Murr1 occurs in the cytosol.
In summary, the binding site for Murr1 was narrowed down to a 24 amino acid region in the δENaC C-terminal domain, whereas the binding site for δENaC was narrowed down to a 10 amino acid region in Murr1. The cytosolic colocalisation of δENaCFLAG and Murr1HA suggests that Murr1 may be a regulator of δENaC intracellular trafficking.
Supported by a University of Otago Research Grant and the Marsden Fund
Clomethiazole spares mitochondrial dysfunction during hypoxia-ischaemia-induced hippocampal diaschisis. Andrew N Clarkson, DM Jackson, DS Kerr, IA Sammut. Department of Pharmacology and Toxicology, Otago School of Medical Sciences, University of Otago, Dunedin.
Transhemispheric diaschisis describes remote changes that occur contralateral to an ischaemic insult. We have shown that cerebral hypoxia-ischaemia (HI) results in lasting suppression of hippocampal CA1 region activity, contralateral to a carotid artery occlusion in post-natal day 26 rats. Mitochondria have been shown to be specifically damaged by oxidative stress arising during an ischaemic insult, resulting in diminished respiratory function and ATP production.
The present study assessed mitochondrial dysfunction in rat hippocampal tissue homogenates, contralateral and ipsilateral to carotid artery occlusion. Assessment of mitochondrial impairment was carried out using oxygen electrode-derived respiratory measurements and mitochondrial electron transport chain enzyme kinetics (enzyme complexes I-V). Mitochondrial dysfunction was also assessed following clomethiazole (CMZ; 414 mg/kg/day via subcutaneously implanted mini-pumps), a GABAA receptor agonist with known neuroprotective properties. Mitochondrial FAD-linked (succinate-driven) respiration and complex kinetics were assessed 1 and 3 days post-HI. Results are expressed as the mean ± SEM for n = 6-8 separate observations. Statistical analysis was performed using two-way ANOVA and Newman-Keuls’ multiple pair-wise post-hoc comparisons.
Mitochondrial function was impaired contralaterally at 3 days (2.76 ± 0.23 versus 3.74 ± 0.06; P < 0.01) post-HI compared to controls. CMZ treatment resulted in improved mitochondrial function contralaterally compared to HI + saline treatment at 3 days (3.32 ± 0.22; P < 0.05), and was not different compared to controls at either 1 or 3 days post-HI.
This study demonstrates for the first time that HI induces impaired mitochondrial function in hippocampi contralateral to a carotid artery occlusion, and further extends the range of neuroprotective properties previously described for CMZ.
This work was supported by an Otago Medical Research Foundation Grant and an Otago University Research Grant.
Adult phytoestrogen exposure reduces the fertility of male rats. Amy Glover and S Assinder. Department of Anatomy and Structural Biology, Otago School of Medical Sciences, University of Otago, Dunedin.
Phytoestrogens are plant-derived compounds that are able to activate oestrogen receptors α (ERα) and β (ERβ). While oestrogen is vital for male reproductive function, exposure to endogenous oestrogen has been implicated in declining male fertility. The aim of this study was to determine whether phytoestrogen exposure reduces male fertility, and via what mechanism.
Male Wistar rats were bred and raised on a low phytoestrogen diet containing 112 μg.g-1 isoflavanoid. Six adult males were transferred to a high phytoestrogen diet (465 μg.g-1 isoflavanoid), while 9 adult males remained on the low phytoestrogen diet. After 3, 6 and 12 days all males were mated with low phytoestrogen fed females, and the litter sizes recorded. A second group of male rats was kept on the same dietary regimen and euthanased 3, 6 or 12 days after the change in diet. The mRNA levels of ERα, ERβ and androgen receptor (AR) in the epididymides of the male rats were measured by real time PCR.
The average litter size (mean ± SEM) was significantly reduced from 13.0 ± 1.1 to 8.6 ± 1.7 pups when male rats were fed the high phytoestrogen diet for 3 days (P < 0.02; paired t-test), but were at control levels by day 12. ERα and AR mRNA levels (mean ± SEM) were significantly reduced from 0.572 ± 0.042 to 0.244 ± 0.045 ng.g-1 of total RNA (P < 0.001) and from 7.92 ± 1.8 to 2.67 ± 0.67 ng.g-1 of total RNA (P < 0.02) respectively in the cauda region of the epididymides of rats fed the high phytoestrogen diet for 3 days.
Acute exposure of adult male rats to a high phytoestrogen diet transiently reduces their fertility. We propose that the alteration in steroid hormone receptor mRNA in the epididymis results in decreased steroid hormone receptor protein which increases sperm oxidative stress and leads to reduced fertility.
Supported by a University of Otago Research Grant, the Community Trust of Otago and a University of Otago Postgraduate Scholarship.
Community behaviour of stem cells: Quantitative evidence from cellular patterning during myogenesis in vivo. Antonio S J Lee, N Yoon, M Zhang. Department of Anatomy and Structural Biology, Otago School of Medical Sciences, University of Otago, Dunedin.
The fate of stem cells is controlled by intrinsic and extrinsic factors. Integration of both factors is achieved by single cells and across cell populations, the so-called “community effect”. This study examined the in vivo behaviour of stem cell communities during skeletal myogenesis.
Isolated cells from extensor digitorum longus (EDL) and tibialis anterior (TA) muscles of 229 Wistar rat embryos in 15 litters at embryonic days (E) 15 ~ E19 were prepared for immunocytochemistry. Anti-Pax3, anti-Pax7, anti-myosin heavy chain (MHC) and a cocktail of antibodies against myogenic regulatory factors (MRF), including anti-MyoD, anti-Myf5 and anti-myogenin antibodies, were employed to mark various myogenic stem cell (MSC) communities at various differentiation stages.
The MSC communities displayed two distinct behaviours during myogenesis. Pax3+ve and MRF+ve communities maintained their relative size throughout myogenesis (51.4 ± 1.6%, mean ± SEM, n = 15 and 56.4 ± 2.0%, n = 15, respectively), contributing to the main stream of the MSC pool in a developing muscle. The MHC+ve community was also constant, yet extremely low in proportion (0.8% ± 0.1%, n = 15) throughout myogenesis. In contrast, the relative size of the Pax7+ve community increased significantly from E15 (5.3 ± 0.3%, n = 3) to E18 (32.8 ± 1.4%, n = 3, P < 0.001; ANOVA single factor) and was maintained at E19 (31.3 ± 1.5%, n = 3).
In MSC, Pax3 and/or Pax7 act upstream of MRF followed by expression of MHC immediately prior to fusion. The observed features of the MSC community behaviour will be essential for normal skeletal myogenesis. Stable Pax3+ve and MRF+ve communities may provide a consistent source of differentiating cells for muscle growth, while an expanding Pax7+ve community may supply cells to meet specific requirements as development proceeds. A small yet stable MHC+ve community may contribute to the consistent rate of growth.
The neuroprotective effects of melatonin in stroke-induced brain damage, via modulation of L-arginine metabolism. Shiva M Nair, RMA Rahman, I Appleton. Department of Pharmacology and Toxicology, Otago School of Medical Sciences, University of Otago, Dunedin.
Currently, there are no agents that can prevent the neurodegeneration which occurs after a stroke. Melatonin has been implicated in numerous physiological processes. Recently it has also been shown to exhibit neuroprotective effects against acute focal cerebral ischaemic damage possibly by acting as a free radical scavenger. However, the mechanisms of these neuroprotective effects have not been determined. Therefore, this project explored the mechanism(s) by which melatonin acts as an acute neuroprotective agent post-stroke.
Male Sprague-Dawley rats, 285 ± 15 g underwent a 2-hour transient occlusion of the middle cerebral artery by filament insertion. Rats were treated with 5 mg/kg i.p. daily melatonin or vehicle (5% DMSO in 0.9% saline) for 3 days. Brain damage was assessed histologically at 72 hours post stroke with the use of 2,3,5-triphenlyltetrazolium chloride stain. We focused on pivotal inflammatory enzymes, namely nitric oxide synthase (NOS) and its product, nitric oxide which breaks down to nitrite, and the enzyme arginase. Enzyme activity was measured in brain homogenates taken 3 days post-stroke.
A decrease in infarct volume in the melatonin group (46 ± 8 mm3, mean ± SEM, n = 6, P < 0.05; Mann Whitney U test) compared to controls (155 ± 33 mm3, n = 7) was observed. No significant effects on arginase and NOS activity were observed with melatonin. However, a significant decrease in nitrite levels occurred with melatonin treatment (9.5 ± 1.1 µM nitrite/mg protein, n = 7, P < 0.001) compared to non-intervention controls (32 ± 5 µM nitrite/mg protein, n = 6). Elevation of inducible NOS activity was seen in the vehicle treated group (510 ± 140 [3H] L-citrulline/mg protein, P < 0.05) in comparison to control (230 ± 60 [3H] L-citrulline/mg protein) and this was reduced towards normal (non-intervention controls) with melatonin treatment.
This study clearly demonstrated that melatonin is a key mediator in the post-stroke neuronal inflammatory response. In addition, this is in part, due to its inhibition of the enzyme inducible NOS as well as its free radical scavenging properties on its product nitric oxide.
Supported by a grant from the Health Research Council of New Zealand (SN), Bright Future Fellowship NZ (RR) and Lottery Health New Zealand (IA).
The role of melatonin on scarring in an incisional model of dermal wound healing in rats. Kamali Pugazhenthi, M Kapoor, P Young, A Clarkson, I Hall, I Appleton. Department of Pharmacology and Toxicology, Otago School of Medical Sciences, University of Otago, Dunedin.
Melatonin possesses anti-oncotic, anti-inflammatory and immunomodulatory effects. However, its role in wound healing has not been established. In this study we determined the effects of melatonin on scarring using a full thickness incisional wound healing model in rats.
Wounding was initiated in male Sprague Dawley rats 250 ± 25g (n = 6/group/time point). Four 1cm incisions were made on the dorsum of each rat. The treated and control animals received 1.2 mg/kg melatonin or saline intradermally, at 11 pm (compliant with the rats daily melatonin rhythm), 24 hours prior and daily for a further 7 days post-wounding. Rats were sacrificed on days 0, 1, 3, 7, 14, and 21, at 2 pm (the time of maximal melatonin levels), and biopsies extracted. Homogenates of biopsies were assessed for arginase and nitric oxide synthase (NOS) activity and nitrite levels. Immunohistochemical studies on melatonin receptor expression were also determined. A collagen stain (van Gieson’s) was used to assess the quality of scarring.
Melatonin receptors (MT1 and MT2) were expressed in the epidermis and macrophages on day 1 in both groups. Thereafter the expression of MT1 decreased post day 3. The blind study showed that melatonin treatment significantly reduced scarring on day 21. Melatonin treatment also significantly (P < 0.01) increased arginase activity at 1 and 3 days and iNOS activity at day 7 (P > 0.01). Statistical analysis at each time point for the controls and melatonin group was performed using Mann Whitney-U test.
Arginase generates proline, the building block for collagen synthesis. Melatonin treatment increased arginase activity and thus collagen synthesis from day 1. It has been reported that increased nitric oxide (NO) production prolongs the inflammatory phase of wound healing. We conclude that melatonin improves scarring and that this effect is, in part, mediated by modulation of iNOS and arginase.
Supported by an Otago Special Health Scholarship, Hope Foundation, New Zealand (KP), Otago University International Scholarship (MK) and Lottery Health New Zealand (IA).
Spatial distribution of COX-1, COX-2 and iNOS enzymes in the rat brain following hypoxia-ischaemia. Odette M Shaw, AN Clarkson, BA Sutherland, I Appleton. Department of Error! Bookmark not defined., School of Medical Sciences, University of Otago, Dunedin.
Cyclooxygenase-2 (COX-2) is induced following various stimuli in the brain. As well as an increase in COX-2 mRNA there is a concomitant increase in the inducible isoform of nitric oxide synthase (iNOS) expression in animal models of stroke. Both COX-2 and iNOS are reported to cause cellular injury by mediating inflammation and free radical damage. The aim of this study was to determine the spatial and cellular distribution of COX-1 (the constitutive isoform of COX), COX-2 and iNOS proteins in the hypoxia-ischaemia (HI) model of neurodegeneration.
HI was induced in male Wistar rats (n=4 for both controls and HI) by permanently ligating the left common carotid, followed 2 hours later by 1 hour of hypoxia. Animals were sacrificed 3 days post HI. The distribution of COX-1 and COX-2 in the brain were determined by immunohistochemistry. The colocalisation of COX-2 and iNOS were determined by double immunofluorescence labelling.
COX-1, in control and HI brains, was uniformly expressed throughout all brain cells and regions. In the HI sections, COX-2 positive astrocytes, neuroglia, activated microglia and hippocampal neurons were localised throughout the ispilateral hemisphere. In addition, infiltrating macrophages within the ischaemic penumbra were immunolabelled for COX-2. The double immunolabelling studies demonstrated high levels of COX-2 expression in the nucleus of cells which were colocalised with iNOS. This demonstrates that COX-2 expression is up-regulated in immune cells that are distributed widely throughout the ipsilateral hemisphere. COX-2 and iNOS were colocalised within the ipsilateral hemisphere illustrating the potential neurodegenerative relationship between these two enzymes.
Previous studies have shown that COX-2 can mediate the resolution of inflammation. It is therefore possible that during the time course of HI, COX-2 up regulation may not be detrimental by itself. However, the coexpression of iNOS and COX-2 is more indicative of the greater neurodegenerative potential of the iNOS enzyme.
Funding provided by the Lottery Board and the Neurological Foundation of New Zealand.
Use of the oestrogen receptor antagonist ICI-182,780 to alter central steroid actions. Frederik J Steyn, GM Anderson, DR Grattan. Centre for Neuroendocrinology and Department of Anatomy and Structural Biology, Otago School of Medical Sciences, University of Otago, Dunedin.
This experiment evaluated the effectiveness of intracerebroventricular administration of the oestradiol receptor antagonist ICI-182,780 (ICI) in blocking central oestradiol action without compromising peripheral steroid effects. This was done as part of our studies on the central actions of steroids during late pregnancy. Two centrally regulated endpoints were measured: luteinising hormone (LH) pulse frequency and hypothalamic progesterone receptor (PR) expression.
Crystalline ICI-containing cannulae were implanted into a lateral ventricle of ovariectomised, oestrogen-treated rats (n = 7). Two ovariectomised control groups (no ICI) were treated with oestrogen (n = 9) or placebo (n = 6). At 70 h post-surgery blood samples were collected every 10 min for 3 h and analysed for LH concentration. Animals were paraformaldehyde-perfused, their uterine weight was recorded and their brains were sectioned (40 μm) for PR immunocytochemistry.
In controls oestrogen suppressed LH pulse frequency (2.2 ± 0.4 versus 5.2 ± 0.18 pulses/3 h in placebo rats, mean ± SEM, P < 0.05, Student’s t-test). This effect was blocked by ICI (4.4 ± 0.2 pulses/3 h, P < 0.05 versus oestrogen-treated controls). Oestrogen increased the number of PR positive stained cells in the arcuate nucleus of controls (92.8 ± 3.9 versus 0.1 ± 0.07, P < 0.001). In contrast to the LH results, ICI did not affect oestrogen induced PR expression (82.8 ± 2.1). Oestrogen stimulated uterine growth (P < 0.001) irrespective of ICI treatment (placebo controls: 0.18 ± 0.01 g, oestrogen-treated controls: 0.51 ± 0.05 g, ICI-treated: 0.60 ± 0.07 g).
These data show that ICI can block the central effects of oestrogen’s negative feedback on LH pulses. However the PR results suggest that this central effect was incomplete. The absence of an effect on uterine growth confirmed that central delivery of ICI does not disrupt peripheral steroid actions.
Supported by a University of Otago Postgraduate Scholarship
Domoic acid directly inhibits cardiac mitochondrial respiratory function. Alexandra V Tramoundanas, JC Harrison, DS Kerr and IA Sammut. Department of Pharmacology and Toxicology, Otago School of Medical Sciences, University of Otago, Dunedin.
Domoic acid (DOM) is a potent neurotoxin produced by marine phytoplankton, and has been implicated in numerous cases of animal and human poisoning. Haemodynamic disturbances and cardiac lesions have been documented and questions have been raised as to whether these effects are directly mediated by DOM.
In the present study, embryonic rat H9c2 cardiomyocytes exposed to DOM (0.05 - 10µM; 40 min) were shown (Enzyme linked immunosorbent assay) to contain DOM within the cytosol of cells, with little evidence of binding to the cellular membrane (n = 5, P < 0.001, One way ANOVA, Bonferroni Post hoc). Lactate dehydrogenase (LDH) assays indicated that DOM did not induce cellular damage acutely (20 min, 40 min, 24 h; 0.05 - 10µM) which may indicate active transport of DOM across cellular membrane rather than a physical perturbation of the membrane.
Acute DOM exposure (20 min; 1 - 5µM) did not alter haemodynamic parameters in an ex vivo whole heart preparation. Mitochondria isolated from our ex vivo model however exhibited increased mitochondrial FAD-linked state 4 and decreased state 3 respiratory function following exposure to DOM (n = 7; P < 0.001). The DOM analogue kainic acid (KA) (50μM) exhibited similar effects.
Isolated mitochondrial FAD (n = 5) and NAD+-linked (n = 6) respiratory function was markedly reduced (P < 0.001) by in vitro treatment with DOM (50 - 250nM; 10min). Individual mitochondrial enzyme complex activities were assessed to examine the extent of mitochondrial damage. Complexes I, II/III, IV, V and matrix marker enzyme citrate synthase activities were all decreased by DOM and KA (0.5 - 2µM) in a concentration-dependant manner (P < 0.01). Results obtained using fluorescent probe analysis of superoxide and hydrogen peroxide levels in mitochondria and H9c2 cells indicated reactive oxygen species were not significantly elevated in DOM induced mitochondrial damage. These results confirm the potential for DOM to access and affect mitochondria directly within cells.
This work was supported by the NZ Health Lottery Board and the NZ Heart Foundation.
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