Developmental changes in expression of GABA(A) receptor-channels in rat intrinsic cardiac ganglion neurones

The effects of gamma-aminobutyric acid (GABA) on the electrophysiological properties of intracardiac neurones were investigated in the intracardiac ganglion plexus in situ and in dissociated neurones from neonatal, juvenile and adult rat hearts. Focal application of GABA evoked a depolarizing, excitatory response in both intact and dissociated intracardiac ganglion neurones. Under voltage clamp, both GABA and muscimol elicited inward currents at -60 mV in a concentration-dependent manner. The fast, desensitizing currents were mimicked by the GABA(A) receptor agonists muscimol and taurine, and inhibited by the GABA(A) receptor antagonists, bicuculline and picrotoxin. The GABA(A0) antagonist (1,2,5,6-tetrahydropyridin-4-yl)methyl phosphonic acid (TPMPA), had no effect on GABA-induced currents, suggesting that GABA(A) receptor-channels mediate the response. The GABA-evoked current amplitude recorded from dissociated neurones was age dependent whereby the peak current density measured at -100 mV was similar to 20 times higher for intracardiac neurones obtained from neonatal rats (P2-5) compared with adult rats (P45-49). The decrease in GABA sensitivity occurred during the first two postnatal weeks and coincides with maturation of the sympathetic innervation of the rat heart. Immunohistochemical staining using antibodies against GABA demonstrate the presence of GABA in the intracardiac ganglion plexus of the neonatal rat heart. Taken together, these results suggest that GABA and taurine may act as modulators of neurotransmission and cardiac function in the developing mammalian intrinsic cardiac nervous system.

The effect of exercise on large artery haemodynamics in healthy young men

Background Brachial blood pressure predicts cardiovascular outcome at rest and during exercise. However, because of pulse pressure amplification, there is a marked difference between brachial pressure and central (aortic) pressure. Although central pressure is likely to have greater clinical importance, very little data exist regarding the central haemodynamic response to exercise. The aim of the present study was to determine the central and peripheral haemodynamic response to incremental aerobic exercise. Materials and methods Twelve healthy men aged 31 +/- 1 years (mean +/- SEM) exercised at 50%, 60%, 70% and 80% of their maximal heart rate (HRmax) on a bicycle ergometer. Central blood pressure and estimated aortic pulse wave velocity, assessed by timing of the reflected wave (T-R), were obtained noninvasively using pulse wave analysis. Pulse pressure amplification was defined as the ratio of peripheral to central pulse pressure and, to assess the influence of wave reflection on amplification, the ratio of peripheral pulse pressure to nonaugmented central pulse pressure (PPP : CDBP-P-1) was also calculated. Results During exercise, there was a significant, intensity-related, increase in mean arterial pressure and heart rate (P < 0.001). There was also a significant increase in pulse pressure amplification and in PPP : CDBP-P-1 (P < 0.001), but both were independent of exercise intensity. Estimated aortic pulse wave velocity increased during exercise (P < 0.001), indicating increased aortic stiffness. There was also a positive association between aortic pulse wave velocity and mean arterial pressure (r = 0.54; P < 0.001). Conclusions Exercise significantly increases pulse pressure amplification and estimated aortic stiffness.

Strain and excursion of the sciatic, tibial, and plantar nerves during a modified straight leg raising test

A modified straight leg raising (SLR) in which ankle dorsiflexion is performed before hip flexion has been suggested to diagnose distal neuropathies such as tarsal tunnel syndrome. This study evaluates the clinical hypothesis that strain in the nerves around the ankle and foot caused by ankle dorsiflexion can be further increased with hip flexion. Linear displacement transducers were inserted into the sciatic, tibial, and plantar nerves and plantar fascia of eight embalmed cadavers to measure strain during the modified SLR. Nerve excursion was measured with a digital calliper. Ankle dorsiflexion resulted in a significant strain and distal. excursion of the tibial nerve. With the ankle in dorsiflexion, the proximal excursion and tension increase in the sciatic nerve associated with hip flexion were transmitted distally along the nerve from the hip to beyond the ankle. As hip flexion had an impact on the nerves around the ankle and foot but not on the plantar fascia, the modified SLR may be a useful test to differentially diagnose plantar heel pain. Although the modified SLR caused the greatest increase in nerve strain nearest the moving joint, mechanical forces acting on peripheral nerves are transmitted well beyond the moving joint. (c) 2006 Orthopaedic Research Society.

Folate supplementation fails to affect vascular function and carotid artery intima media thickness in cyclosporin A-treated renal transplant recipients

Background: Cyclosporin A (CsA)-treated renal transplant recipients (RTR) exhibit relative hyperhomocystinemia and vascular dysfunction. Folate supplementation lowers homocysteine and has been shown to improve vascular function in healthy subjects and patients with coronary artery disease. The aim of this study was to assess the effects of 3 months of folate supplementation (5 mg/day) on vascular function and structure in RTR. Methods: A double-blind, placebo-controlled crossover study was conducted in 10 CsA-treated RTR. Vascular structure was measured as carotid artery intima media thickness (IMT) and function was assessed as changes in brachial artery diameter during reactive hyperemia (RE) and in response to glyceryl trinitrate (GTN). Function data were analyzed as absolute and percent change from baseline and area under the diameter/time curve. Blood samples were collected before and after supplementation and analyzed for total plasma homocysteine, folate, vitamin B-12 and asymmetric dimethyl arginine (ADMA) in addition to regular measures of hemoglobin, hematocrit, mean corpuscular volume (MCV) and serum creatinine. Results: Folate supplementation significantly increased plasma folate by 687% (p < 0.005) and decreased homocysteine by 37% (p < 0.05) with no changes (p > 0.05) in vitamin B 12 or ADMA. There were no significant (p > 0.05) changes in vascular structure or function during the placebo or the folate supplementation phases; IMT; placebo pre mean +/- SD, 0.52 +/- 0.12, post 0.50 +/- 0.11; folate pre 0.55 +/- 0.17, post 0.49 +/- 10.20 mm 5% change in brachial artery diameter (RH, placebo pre 10 +/- 8, post 6 +/- 5; folate pre 9 +/- 7, post 7 +/- 5; GTN, placebo pre 18 +/- 10, post 17 +/- 9, folate pre 16 +/- 9, post-supplementation 18 +/- 8). Conclusion: Three months of folate supplementation decreases plasma homocysteine but has no effect on endothelial function or carotid artery IMT in RTR.

Implantation of a scaffold following bulbectomy induces laminar organization of regenerating olfactory axons

Primary olfactory axons expressing different odorant receptors are interspersed within the olfactory nerve. However, upon reaching the outer nerve fiber layer of the olfactory bulb they defasciculate, sort out, and refasciculate prior to targeting glomeruli in fixed topographic positions. While odorant receptors are crucial for the final targeting of axons to glomeruli, it is unclear what directs the formation of the nerve fiber and glomerular layers of the olfactory bulb. While the olfactory bulb itself may provide instructive cues for the development of these layers, it is also possible that the incoming axons may simply require the presence of a physical scaffold to establish the outer laminar cytoarchitecture. In order to begin to understand the underlying role of the olfactory bulb in development of the outer layers of the olfactory bulb, we physically ablated the olfactory bulbs in OMP-IRES-LacZ and P2-IRES-tau-LacZ neonatal mice and replaced them with artificial biological scaffolds molded into the shape of an olfactory bulb. Regenerating axons projected around the edge of the cranial cavity at the periphery of the artificial scaffold and were able to form an olfactory nerve fiber layer and, to some extent, a glomerular layer. Our results reveal that olfactory axons are able to form rudimentary cytoarchitectonic layers if they are provided with an appropriately shaped biological scaffold. Thus, the olfactory bulb does not appear to provide any tropic substance that either attracts regenerating olfactory axons into the cranial cavity or induces these axons to form a plexus around its outer surface. (c) 2006 Elsevier B.V. All rights reserved.

The sorting behaviour of olfactory and vomeronasal axons during regeneration

In order to begin to understand how primary olfactory and vomeronasal organ (VNO) axons target specific regions of the olfactory bulb, we examined the sorting behaviour of these axons following neonatal unilateral olfactory bulbectomy. Bulbectomy induced widespread ipsilateral death of the primary olfactory and VNO neurons. After 4 weeks, many new sensory axons had re-grown into the cranial cavity and established a prominent plexus with evidence of dense tufts that were similar in gross appearance to glomeruli. Axons expressing the cell adhesion molecule OCAM, which normally innervate the ventrolateral and rostral halves of the main and accessory olfactory bulbs, respectively, sorted out and segregated from those axons not expressing this molecule within the plexus. In addition, VNO axons formed large discrete bundles that segregated from main olfactory axons within the plexus. Thus, VNO and primary olfactory axons as well as discrete subpopulations of both are able to sort out and remain segregated in the absence of the olfactory bulb. Sorting and convergence of axons therefore occur independently of the olfactory bulb and are probably attributable either to inherent properties of the axons themselves or to interactions between the axons and accompanying glial ensheathing cells.

Inflammation, complement activation and endothelial function in stable and unstable coronary artery disease

Background: Endothelial dysfunction plays an important role in the pathogenesis of coronary artery disease (CAD). Apart from traditional risk factors complement activation and inflammation may trigger and sustain endothelial dysfunction. We sought to assess the association between endothelial function, high sensitivity C-reactive protein (hs-CRP) and markers of complement activation in patients with either stable or unstable coronary artery disease. Methods: We prospectively recruited 78 patients, 35 patients with stable angina pectoris (SAP) and 43 patients with unstable angina pectoris (UAP). Endothelial function was assessed as brachial artery reactivity (BAR). Hs-CRP, C3a, C5a, and C1-Inhibitor (C1 inh.) were measured enzymatically. Results: Patients with IJAP showed higher median levels of hs-CRP and C3a compared to patients with SAP, while BAR was not significantly different between patient groups. In UAP patients, hs-CRP was significantly correlated with cholesterol (r = 0.27, p < 0.02), C3a (r = 0.32, p < 0.001) and C1 INH.(r = 0.41, p < 0.003), but not with flow mediated dilatation (r = 0.09, P = 0.41). Hs-CRP and C1 INH.were found to be independant predictors of IJAP in a backward stepwise logistic regression model. Conclusions: We conclude that both hs-CRP, a marker of inflammation and C3a, a marker of complement activation are elevated in patients with UAP, but not in patients with SAP. (c) 2005 Elsevier B.V. All rights reserved.

Validation of a generalized transfer function to noninvasively derive central blood pressure during exercise

Exercise brachial blood pressure ( BP) predicts mortality, but because of wave reflection, central ( ascending aortic) pressure differs from brachial pressure. Exercise central BP may be clinically important, and a noninvasive means to derive it would be useful. The purpose of this study was to test the validity of a noninvasive technique to derive exercise central BP. Ascending aortic pressure waveforms were recorded using a micromanometer-tipped 6F Millar catheter in 30 patients (56 +/- 9 years; 21 men) undergoing diagnostic coronary angiography. Simultaneous recordings of the derived central pressure waveform were acquired using servocontrolled radial tonometry at rest and during supine cycling. Pulse wave analysis of the direct and derived pressure signals was performed offline (SphygmoCor 7.01). From rest to exercise, mean arterial pressure and heart rate were increased by 20 +/- 10 mm Hg and 15 +/- 7 bpm, respectively, and central systolic BP ranged from 77 to 229 mm Hg. There was good agreement and high correlation between invasive and noninvasive techniques with a mean difference (+/- SD) for central systolic BP of -1.3 +/- 3.2 mm Hg at rest and -4.7 +/- 3.3 mm Hg at peak exercise ( for both r=0.995; P < 0.001). Conversely, systolic BP was significantly higher peripherally than centrally at rest (155 +/- 33 versus 138 +/- 32mm Hg; mean difference, -16.3 +/- 9.4mm Hg) and during exercise (180 +/- 34 versus 164 +/- 33 mm Hg; mean difference, -15.5 +/- 10.4 mm Hg; for both P < 0.001). True myocardial afterload is not reliably estimated by peripheral systolic BP. Radial tonometry and pulse wave analysis is an accurate technique for the noninvasive determination of central BP at rest and during exercise.

Supervised stationary cycling versus supervised treadmill walking for patients with intermittent claudication: Preliminary results

Introduction: Walking programmes are recommended as part of the initial treatment for intermittent claudication (IC). However, for many patients factors such as frailty, the severe leg discomfort associated with walking and safety concerns about exercising in public areas reduce compliance to such prescription. Thus, there is a need to identify a mode of exercise that provides the same benefits as regular walking while also offering convenience and comfort for these patients. The present study aims to provide evidence for the first time of the efficacy of a supervised cycle training programme compared with a conventional walking programme for the treatment of IC. Methods: Thus far 33 patients have been randomized to: a treadmill-training group (n = 12); a cycle-training group (n = 11); or a control group (n = 10). Training groups participated in three sessions of supervised training per week for a period of 6 weeks. Control patients received no experimental intervention. Maximal incremental treadmill testing was performed at baseline and after the 6 weeks of training. Measures included pain-free (PFWT) and maximal walking time (MWT), continuous heart rate and gas-analysis recording, and ankle-brachial index assessment. Results: In the treadmill trained group MWT increased significantly from 1016.7 523.7 to 1255.2 432.2 s (P < 0.05). MWT tended to increase with cycle training (848.72 333.18 to 939.54 350.35 s, P = 0.14), and remained unchanged in the control group (1555.1 683.23 to 1534.7 689.87 s). For PFWT, there was a non-significant increase in the treadmill-training group from 414.4 262.3 to 592.9 381.9 s, while both the cycle training and control groups displayed no significant change in this time (226.7 147.1 s to 192.3 56.8 and 499.4 503.7 s to 466.0 526.1 s, respectively). Conclusions: These preliminary results might suggest that, unlike treadmill walking, cycling has no clear effect on walking performance in patients with IC. Thus the current recommendations promoting walking based programmes appear appropriate. The present study was funded by the National Heart Foundation of Australia.

Diastolic dysfunction is a manifestation of ventricular-vascular interaction in patients with type 2 diabetes mellitus

Vascular disease is accelerated in patients with Type 2 diabetes mellitus (T2DM). Since the systemic vasculature plays a pivotal role in myocardial loading, this study aimed to determine the effect of arterial characteristics on left ventricular (LV) morphology and function in patients with T2DM. Conventional echocardiography and tissue Doppler imaging were performed in 172 T2DM patients (95 men; aged 55±11y) with preserved ejection fraction (62±5%). Patients were stratified into groups based on LV geometric pattern (normal [n = 79], concentric remodeling [n = 33], concentric hypertrophy [n = 29], eccentric hypertrophy [n = 31]). Total arterial compliance (TAC) was recorded by simultaneous radial tonometry and aortic outflow pulsed wave Doppler. Arterial (brachial and carotid) structure and function were determined by standard ultrasound methods. There were no significant differences between the LV geometric groups in demographic or clinical parameters. The concentric hypertrophy group had significantly increased carotid artery diameter (6.0±0.7mm versus 6.5±0.7mm; p < 0.05) and stiffness (1912±1203 dynes/cm2mm versus 2976±2695 dynes/cm2mm×10−6; p < 0.05) compared to those with normal geometry. However, TAC did not differ between groups. LV diastolic function, as determined by the ratio of diastolic mitral inflow velocity to mitral annulus tissue velocity (E/E_), was significantly associated with carotid artery relative wall thickness and intima media thickness (p < 0.05). Moreover, E/E_ was independently predicted by carotid artery relative wall thickness (β = 22.9; p = 0.007). We conclude that structural characteristics of the carotid artery are associated with abnormal LV structure and function in patients with T2DM. The LV functional irregularities may be a downstream consequence of amplified pressure wave reflections effecting sub-optimal ventricular-vascular interaction.

Ocular and systemic markers for vascular function in those at risk of type 2 diabetes mellitus and cardiovascular disease

The devastating impact of Type 2 Diabetes Mellitus (T2DM) -related morbidity and mortality on global healthcare is escalating with higher prevalences of obesity, poor diet, and sedentary lifestyles. Therefore, the clinical need for early diagnosis and prevention in groups of high-risk individuals is necessary. The purpose of this thesis was to investigate the use of surrogate markers, namely retinal vascular function, to determine future vascular endothelial dysfunction, atherosclerosis, large vessel disease and cardiovascular risk in certain groups. This namely covered normoglycaemic and normotensive South Asians (SAs), those with Impaired-Glucose Tolerance (IGT) and individuals with a familial history (FH) of T2DM. Additionally the effect of overweight and obesity was studied. The techniques and modified protocols adopted for this thesis involved the investigation of endothelial function by means of vascular reactivity at the ocular and systemic level. Furthermore, the relationships between retinal and systemic function with circulating markers for endothelial cell function and cardiovascular risk markers were explored. The principal studies and findings of the research were: Vascular Function in Normoglycaemic Individuals with and without a FH of T2DM WE FH individuals exhibited higher levels of total cholesterol levels that correlated well with the retinal arterial dilation amplitude to flicker light stimulus. However this did not extend to noticeable differences in markers for endothelial cell damage and impaired retinal and systemic function. Vascular Function in Normoglycaemic South-Asians vs. White-Europeans without a FH and Vascular Disturbances Compared to healthy WEs (normo -glycaemic and -tensive), SA participants exhibited levels of dyslipidaemia and a state of oxidative stress that extended to impaired vascular function as detected by reduced brachial artery flow-mediated dilation, slower retinal arterial vessel dilation reaction times (Appendix 3) and steeper constriction profiles. Furthermore, gender sub-group analysis presented in a sub-chapter shows that SA males demonstrated 24-hour systemic blood pressure (BP) and heart rate variability (HRV) abnormalities and heightened cardiovascular disease (CVD) risk. Vascular Function in Individuals Newly Diagnosed with IGT as compared to Normoglycaemic Healthy Controls Newly-diagnosed WE and SA IGT patients showed a greater risk for CVD and T2DM progression by means of 24-hour BP abnormalities, dyslipidaemia, increased carotid artery intimal-media thickness (c-IMT), Framingham scores and cholesterol ratios. Additionally, pre-clinical markers for oxidative stress and endothelial dysfunction, as evident by significantly lower levels of plasma glutathione and increased levels of von-Willebrand factor in IGT individuals, extended to impaired vascular systemic and retinal function compared to normal controls. This originally shows retinal, systemic and biochemical disturbances in newly-diagnosed IGT not previously reported before. Vascular Function in Normal, Overweight and Obese Individuals of SA and WE Ethnicity In addition to the intended study chapters, the thesis also investigated the influence of obesity and overweight on vascular function. Most importantly, it was found for the first time that compared to lean individuals it was overweight and not obese individuals that exhibited signs of vascular systemic and ocular dysfunction that was evident alongside markers of atherosclerosis, CVD risk and endothelial damage.

Comparison and characterisation of ribozyme delivery in cultured glial and neuronal cells in vivo and in vitro

Ribozymes are short strands of RNA that possess a huge potential as biological tools for studying gene expression and as therapeutic agents to down-regulate undesirable gene expression. Successful application of ribozymes requires delivery to the target site in sufficient amounts for an adequate duration. However, due to their large size and polyanionic character ribozymes are not amenable to transport across biological membranes. In this study a chemically modified ribozyme with enhanced biological stability, targeted against the EGFR mRNA has been evaluated for cellular delivery to cultured glial and neuronal cells with a view to developing treatments for brain tumours. Cellular delivery of free ribozyme was characterised in cultured glial and neuronal cells from the human and rat. Delivery was very limited and time dependent with no consistent difference observed between glial and neuronal cells in both species. Cellular association was largely temperature and energy-dependent with a small component of non-energy dependent association. Further studies showed that ribozyme cellular association was inhibited with self and cross competition with nucleic and non-nucleic acid polyanions indicating the presence of cell surface ribozyme-binding molecules. Trypsin washing experiments further implied that the ribozyme binding surface molecules were protein by nature. Dependence of cellular association on pH indicated that interaction of ribozyme with cell surface molecules was based on ionic interactions. Fluoresence studies indicated that, post cell association, ribozymes were sequestered in sub-cellular vesicles. South-Western blots identified several cell surface proteins which bind to ribozymes and could facilitate cellular association. The limited cellular association observed with free ribozyme required the development and evaluation of polylactide-co-glycolide microspheres incorporating ribozyme for enhanced cellular delivery. Characterisation of microsphere mediated delivery of ribozyme in cultured glial and neuronal cells showed that association increased by 18 to 27-fold in all cell types with no differences observed between cell lines and species. Microsphere mediated delivery was temperature and energy dependent and independent of pH. In order to assess the potential of PLGA micro spheres for the CNS delivery of ribozyme the distribution of ribozyme entrapping microspheres was investigated in rat CNS after intracerebroventricular injection. Distribution studies demonstrated that after 24 hours there was no free ribozyme present in the brain parenchyma, however microsphere entrapped ribozyme was found in the CNS. Microspheres remained in the ventricular system after deposition and passed from the lateral ventricles to the third and fourth ventricle and in the subarachnoid space. Investigation of the influence of microsphere size on the distribution in CNS demonstrated that particles up to 2.5 and O.5f.lm remained in the ventricles around the choroid plexus and ependymal lining.

Abnormal retinal vascular reactivity in individuals with impaired glucose tolerance:a preliminary study

To investigate the relationship between vascular function parameters measured at the retinal and systemic level and known markers for cardiovascular risk in patients with impaired glucose tolerance (IGT). Sixty age- and gender- matched White-European adults (30 IGT and 30 normal glucose tolerance -NGT) were recruited for the study. Fasting plasma glucose, lipids and 24-hour blood pressure (BP) was measured in all subjects. Systemic vascular and endothelial function was assessed using carotid-artery intimal media thickness (cIMT) and flow mediated dilation (FMD). Retinal vascular reactivity was assessed by the Dynamic Retinal Vessel Analyser (DVA). Additionally, blood glutathione (GSH, GSSG and tGSH) and plasma von-Willebrand (vWF) factor levels were also measured. Individuals with IGT demonstrated higher BP values (p<0.001), fasting TG and TG:HDL ratios (p<0.001) than NGT subjects. Furthermore, Total:HDL-C ratios and Framingham scores were raised (p=0.010 and p<0.001 respectively). Blood glutathione levels (GSH, GSSG and tGSH) were lower (p<0.001, p=0.039 and p<0.001 respectively) while plasma vWF was increased (p=0.014) in IGT subjects compared to controls. IGT individuals also demonstrated higher IMT in right and left carotid arteries (p=0.017 and p=0.005, respectively) alongside larger brachial artery diameter (p=0.015), lower FMD% (p=0.026) and GTN induced dilation (GID) (p=0.012) than healthy controls. At the retinal arterial level, the IGT subjects showed higher baseline fluctuations (BDF) (p=0.026), longer reaction time (RT) (p=0.032) and reduced baseline-corrected flicker response (bFR) (p=0.045). In IGT subjects retinal BDF correlated with and Total:HDL (p= 0.003) and HDL-C (p= 0.004). Arterial RT also correlated with FMD (p=0.017) in IGT but not NGT subjects. In IGT individuals there is a relationship between macro- and microvascular function, as well as a direct correlation between the observed retinal microcirculatory changes and established plasma markers for CVD. Multifactorial preventive interventions to decrease vascular risk in these individuals should be considered.

Ocular and systemic endothelial function in the off spring of diabetics:a pilot study

Purpose To investigate ocular and systemic correlates of endothelial function in the normoglycaemic offspring of Type 2 Diabetics (T2DM). Methods Healthy participants aged between 25-65 with (n=30) and without (n=39) a family history were recruited. Retinal vessel reactivity was assessed by using the Retinal Vessel Analyser (RVA, Imedos GmBH). In addition, systemic endothelial function was assessed by using the flow mediated dilation (FMD) technique. Results Parametric testing showed no significant differences in anthropometric, blood assay or ocular and systemic function between both groups (p>0.05). The average maximum dilation in the measured retinal artery correlated significantly with the maximum dilation of the measured brachial artery (p=0.002 R=0.55) in healthy controls; however, this was not true for subjects with family history of T2DM. Conclusion Subjects with family history of T2DM show possibly early signs of endothelial dysfunction that, in certain conditions, could contribute to the higher risk of this group of developing similar pathology to their parents.

Phytoestrogens modulate breast cancer resistance protein expression and function at the blood-cerebrospinal fluid barrier

PURPOSE: Breast cancer resistance protein (BCRP/ABCG2) is a drug efflux transporter expressed at the blood cerebrospinal fluid barrier (BCSFB), and influences distribution of drugs into the central nervous systems (CNS). Current inhibitors have failed clinically due to neurotoxicity. Novel approaches are needed to identify new modulators to enhance CNS delivery. This study examines 18 compounds (mainly phytoestrogens) as modulators of the expression/function of BCRP in an in vitro rat choroid plexus BCSFB model. METHODS: Modulators were initially subject to cytotoxicity (MTT) assessment to determine optimal non-toxic concentrations. Reverse-transcriptase PCR and confocal microscopy were used to identify the presence of BCRP in Z310 cells. Thereafter modulation of the intracellular accumulation of the fluorescent BCRP probe substrate Hoechst 33342 (H33342), changes in protein expression of BCRP (western blotting) and the functional activity of BCRP (membrane insert model) were assessed under modulator exposure. RESULTS: A 24 hour cytotoxicity assay (0.001 µM-1000 µM) demonstrated the majority of modulators possessed a cellular viability IC50 > 148 µM. Intracellular accumulation of H33342 was significantly increased in the presence of the known BCRP inhibitor Ko143 and, following a 24 hour pre-incubation, all modulators demonstrated statistically significant increases in H33342 accumulation (P < 0.001), when compared to control and Ko143. After a 24 hour pre-incubation with modulators alone, a 0.16-2.5-fold change in BCRP expression was observed for test compounds. The functional consequences of this were confirmed in a permeable insert model of the BCSFB which demonstrated that 17-β-estradiol, naringin and silymarin (down-regulators) and baicalin (up-regulator) can modulate BCRP-mediated transport function at the BCSFB. CONCLUSION: We have successfully confirmed the gene and protein expression of BCRP in Z310 cells and demonstrated the potential for phytoestrogen modulators to influence the functionality of BCRP at the BCSFB and thereby potentially allowing manipulation of CNS drug disposition.