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Dynamics of a transgene expression in acute rat brain slices transfected with adenoviral vectors
- C. E. L. Stokes, D. Murphy, J. F. R. Paton, S. Kasparov
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- Published online by Cambridge University Press:
- 20 June 2003, pp. 459-466
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We present a quantitative account of the expression dynamics of a transgene (enhanced green fluorescent protein, EGFP) in acute brain slices transfected with an adenoviral vector (AVV) under control of the human cytomegalovirus (HCMV) promoter. Micromolar concentrations of EGFP could be detected in brainstem and hippocampal slices as early as 7 h after in vitro transfection with a viral titre of 4.4 × 109 plaque-forming units (pfu) ml-1. Although initially EGFP appeared mainly in glia, it could be detected in neurones with longer incubation times of 10-12 h. However, fluorescence was never detected within some populations of neurones, such as hippocampal pyramidal cells, or within the hypoglossal motor nucleus. The density of cells expressing EGFP peaked at 10 h and then decreased, possibly suggesting that high concentrations of EGFP are toxic. The age of the animal significantly affected the speed of EGFP accumulation: after 10 h of incubation in 30-day-old rats only 4.88 ± 0.51 cells/10 000 µm2 were fluorescent compared to 7.28 ± 0.39 cells/10 000 µm2 in 12-day-old rats (P < 0.05). HCMV promoter-driven transgene expression depended on the activity of protein kinase A, and was depressed with a cAMP/protein kinase A antagonist (20 µM Rp-cAMPS; P < 0.0005). This indicates that expression of HCMV-driven constructs is likely to be skewed towards cellular populations where cAMP-dependent signalling pathways are active. We conclude that acute transfection of brain slices with AVVs within hours causes EGFP expression in micromolar concentrations and that such transfected cells may remain viable for use in physiological experiments. Experimental Physiology (2003) 88.4, 459-466.
Endothelial-independent prevention of high blood pressure in L-NAME-treated rats by angiotensin II type I receptor antisense gene therapy
- Phyllis Y. Reaves, Caren R. Beck, Hong-Wei Wang, Mohan K. Raizada, Michael J. Katovich
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- 20 June 2003, pp. 467-473
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It has previously been established that a single systemic administration of retroviral vector containing angiotensin II type I receptor antisense (AT1R-AS) in the neonatal spontaneously hypertensive rat (SHR) prevents development of hypertension, and in addition cardiac hypertrophy and endothelial dysfunction. However, these studies could not determine whether the effects of AT1R-AS on high blood pressure (BP) and endothelial function were independent. Angiotensin receptor blockers have been shown to reduce BP in the L-NAME (N ω-nitro-L-arginine methyl ester hydrochloride)-induced rat model of hypertension. Our objective in the present study was to use the L-NAME model of hypertension to determine whether AT1R-AS treatment would lower high BP and attenuate cardiac hypertrophy under conditions of permanent endothelial damage. A single bolus of LNSV-AT1R-AS viral particles in neonatal Wistar-Kyoto (WKY) rats was without affect on basal BP. Efficacy of the transgene incorporation was assessed by observing a significant reduction in angiotensin-induced dipsogenic response in the AT1R-AS-treated animals. Introduction of L-NAME in the drinking water for 10 weeks resulted in the establishment of hypertension only in the WKY rats treated with vector alone. These hypertensive (BP, 179 ± 4 mmHg) animals showed a 17 % increase in heart weight/body weight ratio and a 60 % reduction in ACh-induced vasorelaxation in phenylephrine-preconstricted arteries. The L-NAME-induced high BP and cardiac hypertrophy were attenuated in rats expressing AT1R-AS. However, endothelial dysfunction could not be prevented with the antisense therapy. These observations demonstrate that attenuation of endothelial dysfunction is not a prerequisite for the antihypertensive effects of AT1R-AS treatment. Experimental Physiology (2003) 88.4, 467-473.
Effect of polyphenolic compounds on the renal Na+,K+-ATPase during the restoration of normotension after experimentally induced hypertension in rats
- Veronika Javorková, Olga Pechánová, Ramaroson Andriantsitohaina, Norbert Vrbjar
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- Published online by Cambridge University Press:
- 20 June 2003, pp. 475-482
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It is commonly known that consumption of foods and beverages rich in polyphenols is associated with a lower incidence of cardiovascular disease. The purpose of this study was to assess whether the application of red wine polyphenols influences the kinetic properties of renal Na+,K+-ATPase in rats in which hypertension has been experimentally induced by the nitric oxide synthase inhibitor L-NAME. Treatment with polyphenols during the recovery from hypertension to normotension resulted in the complete revival of the functional properties of the Na+,K+-ATPase, as indicated by the total restoration of Km, KNa (concentration of Na+ necessary to achieve half-maximal reaction velocity) and Vmax for enzyme activation by ATP and/or Na+ to pre-hypertension values. Two positive effects of polyphenols during the recovery period are indicated: a restoration of the affinity of the ATP and Na+ binding sites to control values and a probable increase in the number of Na+,K+-ATPase molecules to a level comparable to that in control conditions, as suggested by the complete renewal of Vmax. Experimental Physiology (2003) 88.4, 475-482.
Effect of endothelin antagonists on the renal haemodynamic and tubular responses to ischaemia-reperfusion injury in anaesthetised rats
- Adam Ajis, Nigel M. Bagnall, Michael G. Collis, Edward J. Johns
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- 20 June 2003, pp. 483-490
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In this investigation we have evaluated whether blockade of endothelin receptors influenced the renal haemodynamic and excretory responses to a period of ischaemia and reperfusion in the anaesthetised rat. The renal artery was occluded for 30 min and renal haemodynamic and excretory function followed for 90 min of reperfusion while either saline, the non-selective endothelin 1 receptor (ETA/ETB) antagonist SB209670 or the selective ETA receptor antagonist UK-350,926 was infused. In the post-ischaemic period, renal cortical and medullary perfusions were reduced by 40-50 %. When SB209670 was administered (30 µg kg-1 min-1 I.V.) for 30 min before, during and for 90 min after renal artery occlusion, cortical and medullary perfusions returned to baseline levels, responses different from those obtained during saline infusion (both P < 0.05). In the presence of UK-350,926 (30 µg kg-1 min-1 I.V.), perfusion in the medulla returned to baseline on clamp removal whereas that in the cortex remained depressed (P < 0.05). Renal ischaemia for 30 min decreased glomerular filtration rate during reperfusion and increased urine flow and sodium excretion 5- to 15-fold. UK-350,926 (30 µg kg-1 min-1 I.V.) reduced (P < 0.05) fluid excretion prior to ischaemia but during reperfusion, glomerular filtration rate returned to basal levels and there were progressive increases in fluid excretion which were smaller compared to the saline-treated group (all P < 0.05). The ischaemic challenge may cause release of endothelin, which acts on ETB receptors in the cortex and ETA receptors in the medulla to decrease perfusion. The blunted natriuresis and diuresis during blockade of ETA receptors may result from either a vascular or tubular action of endothelin. Experimental Physiology (2003) 88.4, 483-490.
Hindquarters vasoconstriction through central GABAB receptors in conscious rats
- Yumi Takemoto
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- 20 June 2003, pp. 491-496
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The exogenous application of GABA into the cisterna magna of the freely moving rat decreases hindquarters vascular tone as well as arterial pressure. GABA could influence GABA receptor subtypes A, B or C. However, the hindquarters vascular response to the stimulation of each receptor subtype has not yet been investigated. The present study therefore characterized the response to the GABAB receptor agonist baclofen injected into the cisterna magna of the conscious rat. Intracisternally injected baclofen induced long-lasting increases in hindquarters vascular resistance and arterial pressure in a dose-dependent manner. Both actions induced by baclofen were completely blocked by a prior intracisternal injection with the GABAB receptor antagonist CGP 35348 (p-[3-aminopropyl]-p-diethoxymethylphosphinic acid), and systemically by ganglionic blockade. These actions of baclofen were also abolished centrally by sodium pentobarbital anaesthesia. The results suggest that GABAB receptor stimulation via the cisterna magna induced hindquarters vasoconstriction, probably due to central disinhibition of sympathetic activity. Experimental Physiology (2003) 88.4, 491-496.
Effects of chronic systemic hypoxia on contraction evoked by noradrenaline in the rat iliac artery
- I. S. Bartlett, Janice M. Marshall
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- Published online by Cambridge University Press:
- 20 June 2003, pp. 497-507
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Comparisons were made between responses evoked by noradrenaline (NA) in iliac artery rings from normoxic (N) rats and chronically hypoxic (CH) rats kept in 12 % O2 for 3-4 weeks. At PO2 of 100 mmHg, cumulative concentration-response curves (CCRC) to NA were greatly depressed in endothelium-intact (E+) rings, but not endothelium-denuded (E-) rings, of CH rats relative to N rats. However, CCRCs evoked by NA in E+ and E- rings during nitric oxide (NO) synthase inhibition were similar in N and CH rats. Reducing PO2 to 55 mmHg depressed CCRCs to NA in E+ and E- rings of CH and N rats in the absence and presence of NO synthase inhibition. At PO2 of 100 mmHg, CCRCs evoked by phenylephrine were comparable in E+ and E- rings of N and CH rats as were CCRCs for the relaxation evoked by isoprenaline, which were similarly rightward shifted by NO synthase inhibition. However, CCRCs evoked by the NO donor sodium nitroprusside were leftward shifted in E- rings of CH rats relative to N rats. Further, in the presence of the α2 adrenoceptor inhibitor rauwolscine, CCRCs to NA were comparable in E+ rings of CH and N rats. Thus, the depressive effects of chronic hypoxia on NA-evoked contractions of iliac artery are additional to those of acute hypoxia. We propose that they reflect a facilitation of the contribution of NO to α2 adrenoceptor-evoked relaxation that includes an increased sensitivity of the vascular smooth muscle of arteries from CH rats to NO. Experimental Physiology (2003) 88.4, 497-507.
Characterization of hypoxia-induced ventilatory depression in newborn piglets
- Rene St-Jacques, James J. Filiano, Robert A. Darnall, Walter M. St-John
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- Published online by Cambridge University Press:
- 20 June 2003, pp. 509-515
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We evaluated the hypothesis that a 'central oxygen detector' in the brainstem is necessary for depressions of ventilatory activity to be manifested in the newborn. Decerebrate piglets, ventilated with 100 % O2, were studied following neuromuscular blockade. The vagi and carotid sinus nerves were sectioned bilaterally in order to remove the influence of the peripheral chemoreceptors. Activity of the phrenic nerve was recorded as the index of the central respiratory rhythm. This activity declined and, in some preparations, ceased upon ventilation with air or a hypoxic gas, at either normocapnia or hypercapnia. The degree of depression in hypercapnic hypoxia was greatest in the youngest piglets. Following a medial section of the brainstem, extending to the caudal pons, the depression was reduced. In some preparations, a similar reduction followed the placement of radiofrequency lesions in the caudal ventromedial pons. We conclude that a region of the caudal mesencephalon or pons is necessary for the manifestation of depressions of ventilatory activity in the newborn pig. Experimental Physiology (2003) 88.4, 509-515.
Dynamic exercise attenuates spontaneous baroreceptor reflex sensitivity in conscious rats
- Hidefumi Waki, Sergey Kasparov, Kiyoaki Katahira, Tsuyoshi Shimizu, David Murphy, Julian F. R. Paton
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- Published online by Cambridge University Press:
- 20 June 2003, pp. 517-526
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In humans, it has been reported that dynamic exercise attenuates spontaneous baroreceptor reflex sensitivity (sBRS), which is an index of the gain of the baroreceptor-cardiac reflex. We demonstrated previously that endogenously produced NO from endothelial nitric oxide synthase (eNOS) within the nucleus tractus solitarii (NTS), the central terminal site of baroreceptor afferents, depressed sBRS. In this study, we investigated whether eNOS activity within the NTS plays any role in down-modulating the sBRS during dynamic exercise. In conscious Wistar rats arterial pressure and heart rate (HR) were monitored continuously and chronically using radiotelemetry before and during wheel cage running at 6 m min-1 for 10 min. sBRS was determined by a time-series method. During dynamic exercise systolic blood pressure (SBP) and HR were significantly increased (SBP: 138 ± 2 vs. 125 ± 2 mmHg, P < 0.001; HR: 447 ± 6 vs. 362 ± 8 beats min-1, P < 0.001) while sBRS was significantly decreased (0.53 ± 0.03 vs. 1.08 ± 0.08 ms mmHg-1, P < 0.001). In sino-aortic denervated rats the change in SBP in response to dynamic exercise was significantly larger than that in baroreceptor-intact rats (denervated: 21.6 ± 2.5 mmHg; intact: 12.0 ± 2.8 mmHg, P < 0.05). In contrast, denervation made no difference to the change in HR. Although disabling eNOS activity in the NTS by adenoviral-directed expression of a dominant negative mutant form of eNOS increased resting sBRS (1.48 ± 0.20 vs. 1.09 ± 0.15 ms mmHg-1, P < 0.05), the absolute level reached during dynamic exercise was identical to control. These results demonstrate that during dynamic exercise (i) the sBRS decreases around the operating point of the baroreceptor-cardiac reflex function curve in normotensive rats, (ii) the baroreceptor reflex operates to limit the rise in arterial pressure, and (iii) the attenuation of sBRS is not mediated by changes in eNOS activity within the NTS. Experimental Physiology (2003) 88.4, 517-526.
Differential effects of age and sex on the postnatal responsiveness of brown adipose tissue to prolactin administration in rats
- Sarah Pearce, Carlos Dieguez, Oreste Gualillo, Michael E. Symonds, Terence Stephenson
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- Published online by Cambridge University Press:
- 20 June 2003, pp. 527-531
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Previous studies have shown that prolactin administration to pregnant rats results in offspring with enhanced abundance of the brown adipose tissue-specific uncoupling protein (UCP) 1. The present study therefore aimed to determine whether a similar effect was observed after birth and if the sex of the animal further influenced the responsiveness of brown adipose tissue mitochondria to prolactin administration. Daily prolactin injections were therefore commenced at 15, 35 or 60 days of age and continued for 4 days. The abundance of UCP1 was unchanged with age in females but decreased between 15 and 35 days in males and was lower in males than females by 60 days of age. Cytochrome c abundance remained unchanged with postnatal age in both males and females and was consistently higher in males at each sampling age. Prolactin decreased the abundance of UCP1 and cytochrome c when administered to female rats at 35 and 60 days of age, but had no effect at 15 days. In contrast, prolactin had no effect on UCP1 in male rats at any age, but did stimulate the abundance of cytochrome c at 15 days of age. In conclusion, the administration of prolactin to postnatal rats over the period in which maturation of the hypothalamic-pituitary axis and brown adipose tissue function is occurring did not enhance UCP1 abundance. In females, prolactin actually caused a reduction in UCP1 suggesting that in rats it is only prior to birth that prolactin has a stimulatory role on brown adipose tissue development. Experimental Physiology (2003) 88.4, 527-531.
Intravenous magnesium reduces the rate of glucose disposal in lactating sheep
- I. F. Gow, E. Mitchell, M. Wait
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- 20 June 2003, pp. 533-540
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Magnesium deficiency is generally associated with an impaired ability to dispose of glucose. In order to test whether or not increasing the peripheral free Mg concentration ([Mg2+]) would enhance glucose disposal, we have carried out glucose infusions in sheep with and without simultaneous infusion of Mg. Basal plasma glucose levels were higher in lactating sheep ('lactators') than in non-lactating sheep (controls) (P < 0.05). The glucose disposal rate (KG) with no added Mg was greater in lactators than controls (P < 0.05). When Mg was added to the infusate, KG in lactators was reduced (P < 0.005). Infusion of Mg depressed basal insulin levels in controls and lactators (P < 0.0001 for both). The insulin response to the intravenous glucose tolerance test (IVGTT) was lower in lactators compared with controls (P < 0.0001); however, after correcting for the reduced basal insulin level when Mg was included during the IVGTT, there was no difference between the two groups. We conclude that intravenous Mg at the doses used in this study leads to a decrease in basal insulin secretion, and that increasing serum [Mg2+] reduces glucose disposal in lactating sheep. Experimental Physiology (2003) 88.4, 533-540.
Leukocyte coping capacity: a novel technique for measuring the stress response in vertebrates
- G. W. McLaren, D. W. Macdonald, C. Georgiou, F. Mathews, C. Newman, R. Mian
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- 20 June 2003, pp. 541-546
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Methods used to quantify the stress response in animals are vital tools in many areas of biology. Here we describe a new method of measuring the stress response, which provides rapid results and can be used in the field or laboratory. After a stressful event, we measure the capacity of circulating leukocytes to produce a respiratory burst in vitro in response to challenge by phorbol myristate acetate (PMA). During the respiratory burst leukocytes produce oxygen free radicals, and the level of production can be measured directly as chemiluminescence. When in vitro PMA-stimulated whole blood chemiluminescence is measured directly after a stressful event, we define the response as the leukocyte coping capacity (LCC). In an experiment badgers (Meles meles), which were caught as part of an on-going population study, were either transported to a central site prior to blood sampling or blood was collected at their site of capture. Transported animals had a significantly lower LCC and showed changes in leukocyte composition that were indicative of stress. We conclude that the stress of transport reduced LCC in badgers and that LCC serves as a quantitative measure of stress. Potential applications of this method are discussed. Experimental Physiology (2003) 88.4, 541-546.
P. Coats *, Y. P. R. Jarajapu , C. Hillier , J. C. McGrath and C. Daly
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- 20 June 2003, pp. 547-554
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Resistance arteries isolated from patients with critical limb ischaemia (CLI), a hypotensive/hypoperfusion state of the lower leg, have been shown to undergo morphological changes opposite to those observed in hypertension, that is, decreased wall thickness, reduced cross-sectional area and a decreased wall : lumen ratio. The aim of this present study was to use laser scanning confocal microscopy (LSCM) to study intact resistance arteries isolated from patients with CLI, specifically to identify the cellular aspects of the morphological changes identified in ischaemic subcutaneous and skeletal muscle resistance vessels. Using LSCM, a significant reduction in adventitial and medial thickness, cross-sectional area and wall : lumen ratio was confirmed in resistance arteries from both distal ischaemic subcutaneous and skeletal muscle vascular beds when compared with corresponding arteries from the proximal non-ischaemic sites. The cellular composition of the adventitial, medial and intimal layers of these distal ischaemic arteries was significantly different compared with proximal non-ischaemic arteries. These differences in the distal arteries were characterised by hypoplasia in the adventitial and medial layers of the arterial wall and hypertrophy in the intimal layer. The differences observed in both distal ischaemic vascular beds (subcutaneous and skeletal muscle) were similar. Experimental Physiology (2003) 88.4, 547-554.
Acute hypervolaemia improves arterial oxygen pressure in athletes with exercise-induced hypoxaemia
- Gerald S. Zavorsky, Keith R. Walley, Garth S. Hunte, Donald C. McKenzie, George P. Sexsmith, James A. Russell
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- Published online by Cambridge University Press:
- 20 June 2003, pp. 555-564
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The aim of this study was to determine the effect of acute plasma volume expansion on arterial blood-gas status during 6.5 min strenuous cycling exercise comparing six athletes with and six athletes without exercise-induced arterial hypoxaemia (EIAH). We hypothesized that plasma volume expansion could improve arterial oxygen pressure in a homogeneous sample of athletes - those with EIAH. In this paper we have extended the analysis and results of our recently published surprising findings that lengthening cardiopulmonary transit time did not improve arterial blood-gas status in a heterogeneous sample of endurance cyclists. One 500 ml bag of 10 % Pentastarch (infusion condition) or 60 ml 0.9 % saline (placebo) was infused prior to exercise in a randomized, double-blind fashion on two different days. Power output, cardiac output, oxygen consumption and arterial blood gases were measured during strenuous exercise. Cardiac output and oxygen consumption were not affected by acute hypervolaemia. There were group × condition interaction effects for arterial oxygen pressure and alveolar-arterial oxygen pressure difference, suggesting that those with hypoxaemia experienced improved arterial oxygen pressure (+4 mmHg) and lower alveolar-arterial oxygen pressure difference (-2 mmHg) with infusion. In conclusion, acute hypervolaemia improves blood-gas status in athletes with EIAH. The impairment of gas exchange occurs within the first minute of exercise, and is not impaired further throughout the remaining duration of exercise. This suggests that arterial oxygen pressure is only minimally mediated by cardiac output. Experimental Physiology (2003) 88.4, 555-564.