This study was undertaken to determine the reflex cardiovascular and respiratory responses to discrete stimulation of pulmonary arterial baroreceptors using a preparation in which secondary modulation of responses from other reflexes was prevented. Dogs were anaesthetised with α-chloralose, artificially ventilated, the chests widely opened and a cardiopulmonary bypass established. The main pulmonary arterial trunk, bifurcation and extrapulmonary arteries as far as the first lobar arteries on each side were vascularly isolated and perfused through the left pulmonary artery and drained via the right artery through a Starling resistance which controlled pulmonary arterial pressure. Pressures distending systemic baroreceptors and reflexogenic regions in the heart were controlled. Reflex vascular responses were assessed from changes in perfusion pressures to a vascularly isolated hind limb and to the remainder of the subdiaphragmatic systemic circulation, both of which were perfused at constant flows. Respiratory responses were assessed from recordings of efferent phrenic nerve activity. Increases in pulmonary arterial pressure consistently evoked increases in both perfusion pressures and in phrenic nerve activity. Both vascular and respiratory responses were obtained when pulmonary arterial pressure was increased to above about 30 mmHg. Responses increased at higher levels of pulmonary arterial pressures. In 13 dogs increases in pulmonary arterial pressure to 45 mmHg increased systemic perfusion pressure by 24 ± 7 mmHg (mean ± S.E.M.) from 162 ± 11 mmHg. Setting carotid sinus pressure at different levels did not influence the vascular response to changes in pulmonary arterial pressure. The presence of a negative intrathoracic pressure of -20 mmHg resulted in larger vascular responses being obtained at lower levels of pulmonary arterial pressure. This indicates that the reflex may be more effective in the intact closed-chest animal. These results demonstrate that stimulation of pulmonary arterial baroreceptors evokes a pressor reflex and augments respiratory drive. This reflex is likely to be elicited in circumstances where pulmonary arterial pressure increases and the negative excursions of intrathoracic pressure become greater. They are likely, therefore, to be involved in the cardio-respiratory response to exercise as well as in pathological states such as pulmonary hypertension or restrictive or obstructive lung disease.

To test the hypothesis that angiotensin (ANG) II modulates the arterial baroreflex control of heart rate (HR) in an age-dependent manner, various parameters governing the arterial baroreflex control of HR were assessed before and after removal of endogenously produced ANG II by administration of the angiotensin-converting enzyme (ACE) inhibitor, captopril, to conscious, chronically instrumented lambs aged ~1 week (8 ± 1 days; n = 8) or ~6 weeks (46 ± 5 days; n = 8). After administration of captopril, systolic, diastolic and mean arterial pressures decreased significantly from control levels and HR increased; however, the effects were greater in 1- than in 6-week-old lambs. In 1-week-old lambs, after administration of captopril, there was also a significant increase in the slope coefficient, a decrease in minimum HR and a decrease in the point of maximum gain. In 6-week-old lambs, there were no effects of captopril on any of the parameters governing the arterial baroreflex. Therefore, we accept our hypothesis and conclude that the role of ANG II in modulating cardiovascular homeostasis appears to be more predominant in the newborn than later in life. Experimental Physiology (2003) 88.6, 761-768.

Breathing frequency (fb), tidal volume (VT), and respiratory timing during slow (SR, 8 W min-1) and fast (FR, 65 W min-1) ramp exercise to exhaustion on a cycle ergometer was examined in seven healthy male subjects. Expiratory ventilation (VE), pulmonary gas exchange (VO2 and VCO2) and end-tidal gas tensions (PET,O2 and PET,CO2) were determined using breath-by-breath techniques. Arterialized venous blood was sampled from a dorsal hand vein at 2 min intervals during SR and 30 s intervals during FR and analysed for arterial plasma PCO2 (Pa,CO2). PET,CO2 increased with increasing work rates (WRs) below the ventilatory threshold (VT); at WRs >= 90 % VO2,max, PET,CO2 was reduced (P < 0Σ05) below 0 W values in SR but not in FR. fb and VT were similar for SR and FR at all submaximal WRs, resulting in a similar VE. At exhaustion VE was similar but fb was higher (P < 0Σ05) and VT was lower (P < 0Σ05) in SR ( fb, 51 ± 10 breaths min-1; VT, 2590 ± 590 ml) than in FR ( fb, 42 ± 8 breaths min-1; VT, 3050 ± 470 ml). The time of expiration (TE) decreased with increasing WR, but there was no difference between SR and FR. The time of inspiration (TI) decreased at exercise intensities >= VT; at exhaustion, TI was shorter (P < 0Σ05) during SR (0Σ512 ± 0Σ097 s) than during FR (0Σ753 ± 0Σ100 s). The TI to total breath duration (TI/TTot) and the inspiratory flow (VT/TI) were similar during SR and FR at all submaximal exercise intensities; at VO2,max, TI/TTot was lower (P < 0Σ05) and VT/TI was higher (P < 0Σ05) during SR (TI/TTot, 0Σ473 ± 0Σ030; VT/TI, 5Σ092 ± 0Σ377 l s-1) than during FR (TI/TTot, 0Σ567 ± 0Σ050; VT/TI, 4Σ117 ± 0Σ635 l s-1). These results suggest that during progressive exercise, breathing pattern and respiratory timing may be determined, at least at submaximal work rates, independently of alveolar and arterial PCO2.

The effects of cyclical, passive manipulation (PM, 30 min day-1, 5 days week-1 for 6 months) mimicking the length excursions observed during stepping on the mechanical and associated biochemical properties of the inactive cat soleus muscle were determined in five cats. Inactivity was produced via spinal cord isolation (SI), i.e. complete spinal cord transections at low thoracic and high sacral levels and bilateral dorsal rhizotomy between the transection sites. Passive manipulation was administered to one leg of each SI cat. Compared with normal controls, SI resulted in [similar]70 % decrease in weight, an 80 % decrease in maximum tetanic tension (Po) and an [similar]100 % increase in maximum rate of shortening (Vmax) and myosin adenosine triphosphatase (mATPase) activity of the soleus. The passive manipulation regime partially ameliorated these effects. When compared with the control SI soleus, the SI-PM soleus weight and maximum tetanic tension were 12 and 21 % higher, respectively, and the Vmax and mATPase activity 21 and 12 % (p > 0·05) lower, respectively. Thus, inactivity resulted in a smaller and faster muscle, whereas passive manipulation for only 30 min a day tended to maintain these properties closer to normal control values. The results suggest a potential therapeutic effect of short bouts of cyclical, passive manipulation on otherwise inactive skeletal muscles.

An increase in the concentration of serotonin in the brain has been shown to cause fatigue during exercise in humans and experimental animals. This type of fatigue is referred to as central fatigue and is likely to be mediated by the concentration of serotonin as well as serotonin receptor sensitivity. Serotonin (5-HT) receptor antagonism in humans and experimental animals has been shown to improve endurance performance. A previous report has shown decreased receptor sensitivity in athletes compared to sedentary controls. It is unclear whether this is due to a training adaptation or if individuals are predisposed to enhanced athletic performance due to their inherent decreased receptor sensitivity. The present study investigated changes in 5-HT receptor sensitivity in response to aerobic exercise. Subjects completed 3 × 30 min of stationary cycling at 70 % of their peak aerobic power (V˙O2,peak) for 9 weeks. Serotonin receptor sensitivity was assessed indirectly by measuring the neuroendocrine response following administration of a serotonin agonist (buspirone hydrochloride). The neuroendocrine response following administration of a placebo was also investigated in a blind crossover design. A group of sedentary control subjects was also recruited to control for seasonal variations in central receptor sensitivity. The training caused a significant increase in V˙O2,peak (3.1 ± 0.16 to 3.6 ± 0.15 l min-1, P < 0.05) and endurance capacity (93 ± 8 to 168 ± 11 min, P < 0.05), but there was no change (P > 0.05) in the neuroendocrine response in the presence of a serotonin agonist. However, one-quarter of the subjects in the training group demonstrated decreases in receptor sensitivity. These results suggest that despite increases in V˙O2,peak and endurance performance, there was no measurable change in 5-HT receptor sensitivity in the presence of a serotonin agonist. In addition, it is possible that changes in receptor sensitivity may take longer to occur, that the training stimulus used in the present investigation was inadequate and/or that changes occurred in receptor subtypes that were not probed by the agonist used in the present investigation. Experimental Physiology (2002) 87.1, 83-89.

Physiological changes occurring in the mother during pregnancy can determine the outcome of pregnancy in terms of birthweight and neonatal viability. Maternal adaptations include plasma volume expansion linked to enhanced activity of the renin-angiotensin system (RAS). The present study was designed to determine whether these changes occur very early in gestation, and the extent to which maternal nutrient restriction may compromise the maternal RAS. Using sheep, we have investigated the effects of pregnancy per se, maternal nutrient restriction and later restoration of maternal diet on maternal body weight, plasma volume and plasma renin concentration (PRC), and angiotensinogen (Aogen) and arginine vasopressin (AVP) concentration. During the period of placental growth (i.e. 28-80 days gestation) ewes were fed either a nutrient-restricted (NR) diet or were well fed (WF). NR ewes consumed between 3.2 and 3.8 MJ day-1 of metabolisable energy (ME) which is close to 60 % of requirements taking into account the ME required for both ewe maintenance and growth of the conceptus in order to produce a 4.5 kg lamb at term. WF ewes consumed 150 % of ME requirements. Restoration of maternal diet between 80 and 140 days gestation (i.e. fed to satiety and consuming between 8 and 10.9 MJ day-1, which is close to 150 % of ME requirements) followed previous nutrient restriction. Between pre-conception and 28 days gestation, plasma volume increased in conjunction with a decline in PRC and Aogen concentration. During the period of nutrient restriction ewe body weight did not increase and plasma volume was lower in NR than WF ewes. During this time there was no effect of maternal nutrition on PRC; however, Aogen concentration was lower in the NR group. From 80 days gestation following the rise in food intake for previously NR ewes, greater increases in ewe body weight, plasma volume and PRC occurred up to term compared with ewes that were well fed throughout gestation. Plasma AVP concentration was not significantly affected by either maternal nutrition or gestational age. In conclusion, the stimulus of moderately severe maternal nutrient restriction evoked smaller rises in maternal weight, plasma volume and Aogen concentration than occurred in ewes that were well fed throughout gestation. Following the restoration of maternal diet after 80 days gestation, PRC gradually rose to peak at term. These adaptations in the maternal RAS during the critical period of placental growth may have long-term effects on fetal development. Experimental Physiology (2002) 87.3, 353-359.

A stress protocol - 6 h of daily immobilization - was applied throughout male rat sexual development. Immobilization caused a small reduction in food intake and body weight gain whereas pair-fed animals had a marginal decrease only in body weight gain. Stress, confirmed by increased plasma adrenocorticotrophic hormone (ACTH) and corticosterone, caused a decrease in plasma luteinizing hormone (LH) after 15 and 60 days of immobilization and in plasma testosterone after 60 days, but produced an opposite androgenic response in pubertal animals (15 days of immobilization). A presumed sympathetic over-stimulation is suggested to account for increased testosterone levels in pubertal stressed rats.

One or more episodes of sublethal ischaemia and reperfusion delay infarct development during subsequent, sustained ischaemia in the heart and skeletal muscle. The present study tested whether or not such ischaemic preconditioning (IP) also protects the kidney. Enflurane-anaesthetized pigs underwent 60 min of right renal vessel occlusion (RVO), followed by 8 h of reperfusion without (placebo group, n = 8) or with three preceding cycles of 10 min RVO and 10 min reperfusion (IP group, n = 8). After 8 h of reperfusion, kidneys were oliguric in both groups (placebo group: 23 ± 21 ml h-1, IP group: 24 ± 27 ml h-1). A transient polyuric phase occurred in the IP group at 2 h reperfusion. The reperfused kidneys did not excrete inulin, creatinine or urea in both groups, although renal blood flow during reperfusion was similar to baseline. Morphological damage ranged in both groups from single cell necrosis to disseminated patchy necrosis; the number of pyknotic cells tended to be higher in the IP group than in the placebo group (27.0 ± 7.1 vs. 15.6 ± 5.6 %, n.s.). In anaesthetized pigs, IP did not therefore attenuate renal dysfunction and morphological damage resulting from 60 min of renal normothermic ischaemia followed by 8 h of reperfusion.

The properties of capacitative Ca2+ influx were studied using the whole-cell patch-clamp technique in crypts isolated from rat distal colon. Store-operated cation influx was evoked by increasing the intracellular buffering capacity for Ca2+ in the pipette solution; contamination by Cl- currents was reduced by the use of NMDG gluconate as the main electrolyte in the pipette solution. The permeability of the non-selective cation conductance stimulated by store depletion had the following sequence for monovalent cations: Cs+ > Na+ [ges ] Li+. The store-operated conductance is permeable to Na+ and Ca2+, but in contrast to Na+, Ca2+ also exerts a (feedback) inhibition on its own influx. Other divalent cations shared this inhibitory action with the sequence: Ca2+ [ges ] Mg2+ [ges ] Ba2+ [ges ] Sr2+. Fura-2 experiments revealed that replacement of extracellular Na+ by NMDG+ induced an increase in the intracellular Ca2+ concentration, which was suppressed by the Na+-Ca2+ exchange inhibitor, dichlorobenzamil, indicating the presence of a Na+-Ca2+ exchanger within the colonic crypt cells. In Ussing chamber experiments dichlorobenzamil induced an increase in short-circuit current (Isc) in the majority of tissues tested indicating that this exchanger acts as a Ca2+-extruding transporter under physiological conditions. When Ca2+-dependent anion secretion was stimulated by the acetylcholine analogue carbachol, dichlorobenzamil no longer evoked an increase in Isc, indicating that after stimulation of the store-operated cation conductance the Na+-Ca2+ exchanger is turned off. Therefore, it is concluded that the influx of Na+ across the non-selective store-operated cation conductance serves to reduce the driving force for Ca2+ extrusion via the Na+-Ca2+ exchanger and thereby maintains the increase in the intracellular Ca2+ concentration during induction of secretion. Experimental Physiology (2001) 86.4, 461-468.

The purpose of this study was to use an allometric model (maximal oxygen uptake (VO2,max) = FFMb1 × PAb2 × exp(b0 + b3 age + b4 sex) × ) to determine the influence of fat-free mass (FFM), physical activity (PA), sex and age on VO2,max in older men (n = 152) and women (n = 146) aged 55-86 years. VO2,max was measured during a fatigue-limited treadmill test, FFM was determined from skinfold thickness and physical activity by the Minnesota Leisure Time Physical Activity questionnaire. The model was linearised by taking the natural logarithm of VO2,max, FFM and physical activity. Variables were selected using multiple linear regression (P < 0.05). The sex variable was not significant (P = 0.062). The model explained 72.1 % of the variance in VO2,max. Significant individual coefficients were incorporated into the model yielding the following expression: VO2,max = FFM0.971 × PA0.026 × exp(-2.48 - 0.015age). Therefore, FFM and physical activity were significant factors contributing to the changes in VO2,max with age. In addition, controlling for FFM and physical activity abolished sex differences in VO2,max. The rate of decline in VO2,max (after accounting for FFM and physical activity) with age, was approximately 15 % per decade.

In this study, we investigated the ability of acute infusions of isoprenaline to alter renin and angiotensinogen gene expression in the kidney of rats anaesthetised with chloralose-urethane. Groups of rats received I.V. infusions of either saline or the β-adrenoceptor agonist isoprenaline at 400 ng kg-1 min-1 for 4 h. The isoprenaline infusion caused a sustained decrease in mean blood pressure of approximately 20 mmHg (P < 0.01), an increase in heart rate of 50 beats min-1 (P < 0.01) and reductions in urine flow and sodium excretion of 80-90 % (both P < 0.01). Renal blood flow and glomerular filtration rate were transiently reduced by 21 % (P < 0.01) and 61 % (P < 0.001), respectively, in the first hour, recovering to baseline levels after 4 h of infusion. At the end of the study, plasma renin activity was raised approximately 6-fold (P < 0.01) while renal renin and angiotensinogen mRNA levels were 1.8- and 1.5-fold higher (both P < 0.05) compared to the control group (saline infusion). The isoprenaline-induced renin secretion could have been mediated via the activation of β-adrenoceptors resulting in the exocytosis of renin-containing granules, with a smaller contribution being due to reduced renal haemodynamics. The increase in renal renin gene expression in response to isoprenaline was probably due primarily to the intracellular signalling processes acting directly on nuclear mechanisms. Similarly, the increased renal angiotensinogen gene expression most probably reflected a direct action of the isoprenaline. These findings provide evidence that catecholamines are involved in mechanisms that rapidly alter the expression of the genes of the renin-angiotensin system within the kidney.Experimental Physiology (2003) 88.2, 221-227.

Acute hypoxia regulates the activity of specific ion channels in a rapid and reversible manner. Such effects underlie appropriate cellular responses to hypoxia which are designed to initiate cardiorespiratory reflexes and contribute importantly to other tissue responses, all of which are designed to improve tissue O2 supply. These responses include excitation of chemoreceptors as well as pulmonary vasoconstriction and systemic vasodilatation. However, such responses may also contribute to the adverse responses to hypoxia, such as excitotoxicity in the central nervous system. Whilst numerous ion channel types are known to be modulated by acute hypoxia, the nature of the O2 sensor in most tissues remains to be identified. Prolonged (chronic) hypoxia regulates functional expression of ion channels, and so remodels excitability of various cell types. Whilst this may contribute to adaptive responses such as high-altitude acclimatization, such altered channel expression may also contribute to the onset of pathological disorders, including Alzheimer's disease. Indeed, evidence is emerging that production of pathological peptides associated with Alzheimer's disease is increased during prolonged hypoxia. Such effects may account for the known increased incidence of this disease in patients who have previously endured hypoxic episodes, such as congestive heart failure and stroke. Identification of the mechanisms coupling hypoxia to the increased production of these peptides is likely to be of therapeutic benefit.

The interstitial fluid pressure (Pisf) has been measured in the exposed superfused mesenteries of anaesthetised rats using the micropipette servo-null technique. When mesenteries were superfused with Ringer-Locke solutions, Pisf was close to atmospheric pressure with mean ± S.E.M. values of -0.46 ± 0.14 cmH2O (n = 22). Superfusing with paraffin oil did not alter Pisf significantly, but Pisf could be lowered considerably by removing fluid from the upper surface of the mesentery. Measurements of Pisf were also made in the tissues immediately outside mesenteric venules as the pressure inside these vessels and the filtration of fluid through their walls was varied. No significant changes in perivascular Pisf could be detected even though the intravascular pressure varied from 20 to 70 cmH2O. Addition of histamine or the mast cell degranulating agent compound 48/80 to the superfusate had no significant effect on Pisf. The findings are relevant to experiments on the permeability of single perfused mesenteric microvessels. They strengthen the assumption, which is made in these studies, that Pisf is close to atmospheric pressure and does not change significantly with changes in the filtration and reabsorption of fluid through the vessel walls. Experimental Physiology (2001) 86.1, 33-38.

In addition to its known effect on renal tubular transport, frusemide (furosemide) has been shown to affect renal circulation. This study in the anaesthetised rat examined the influence of frusemide (bolus 0.25 or 0.5 mg kg-1 I.V., then infusion delivering the same dose over 1 h) on renal cortical and medullary circulation measured as laser-Doppler blood (cell) flux. The responses were compared with simultaneously measured changes in renal excretion and in the tissue admittance, an index of medullary ionic hypertonicity of the interstitium. Renal vascular responses to frusemide were significant but not dose dependent. During low-dose frusemide infusion cortical flux decreased 11.5 ± 0.9 % and medullary flux decreased 32.3 ± 3.5 % (difference significant at P < 0.001). During high-dose infusion the decreases were by 13.5 ± 1.4 and 29.3 ± 3.8 %, respectively (difference significant at P < 0.001). Sodium excretion increased 15-fold (by 3.7 ± 0.4 µmol min-1) and 30-fold (by 5.9 ± 1.1 µmol min-1) during low- and high-rate infusion of frusemide, respectively. By contrast, medullary tissue admittance decreased similarly with the two doses: maximally by 13.4 ± 1.4 and 10.9 ± 0.9 %, respectively. The observations that an exaggerated post-frusemide decrease in blood flow within the medulla coincided with decreasing tissue admittance in this zone and that neither medullary blood flow nor admittance changes were related to the dose suggest a causal relationship between interstitial ionic hypertonicity and vascular resistance. We propose that the post-frusemide decrease in medullary tissue NaCl depressed medullary circulation by inhibiting local generation of vasodilator prostaglandins.

The guinea-pig has been used extensively to investigate adrenal steroidogenesis. However, very little is known about adrenocortical responses to corticotrophin-releasing hormone (CRH) and adrenocorticotrophin (ACTH) in this species, in vivo. In the present study, we have developed a stress-free sampling system, in the chronically catheterized adult guinea-pig, that has allowed us to investigate basal and activated adrenocortical activity. Indwelling carotid artery and jugular vein catheters were surgically implanted into female guinea-pigs (n = 5). Each animal was treated with vehicle, human CRH (0·2 or 2 µg kg-1) and ACTH1-24 (0·2 or 2 µg kg-1), and serial plasma samples removed for analysis of ACTH and cortisol concentrations by radioimmunoassay. There was no effect of serial sampling on pituitary-adrenocortical activity, indicating that the animals remain in an unstressed state. Basal plasma ACTH and cortisol concentrations were 703·9 ± 24·5 pg ml-1 and 117·9 ± 5·2 ng ml-1, respectively. Both CRH and ACTH significantly increased adrenocortical activity in a dose-dependent manner. ACTH (2 µg kg-1) was the most potent activator leading to plasma cortisol concentrations of 647 ± 116 ng ml-1. In conclusion, we have shown that basal plasma cortisol concentrations in the guinea-pig are low compared to those obtained in previous studies by cardiac puncture or following decapitation. However, plasma ACTH concentrations are high compared to other species. We have also shown that human CRH and ACTH1-24 act as potent activators of the guinea-pig pituitary-adrenocortical axis, leading to response profiles consistent with mild cortisol resistance.

Maintaining the extracellular K+ concentration ([K+]o) between 15 and 60 mM induced oscillations in the intracellular Ca2+ concentration ([Ca2+]i) in rat submandibular acinar cells during stimulation with acetylcholine (ACh, 1 µM). These [Ca2+]i oscillations were also induced by 1 µM thapsigargin and were inhibited by 50 µM La3+, 1 µM Gd3+, or the removal of extracellular Ca2+, indicating that the [Ca2+]i oscillations were generated by store-operated Ca2+ entry (SOC). The frequency of the ACh-evoked [Ca2+]i oscillations increased from 0.8 to 2.3 mHz as [K+]o was increased from 15 to 50 mM. TEA (an inhibitor of K+ channels) also induced [Ca2+]i oscillations at [K+]o of 4.5 or 7.5 mM in ACh-stimulated cells. These data suggest that depolarization causes [Ca2+]i to oscillate in ACh-stimulated submandibular acinar cells. Pertussis toxin (PTX, an inhibitor of G proteins) caused [Ca2+]i to be sustained at a high level in ACh-stimulated cells at 25 mM or 60 mM [K+]o. This suggests that the [Ca2+]i oscillations are generated by a periodic inactivation of the SOC channels via PTX-sensitive G proteins, which are stimulated by depolarization. Moreover, in the presence of DBcAMP or forskolin which accumulated cAMP the frequency of the [Ca2+]i oscillations remained constant (approximately 1.2 mHz) when [K+]o was maintained in the range 25-60 mM. Based on these observations in ACh-stimulated submandibular acinar cells, we conclude that depolarization stimulates the PTX-sensitive G proteins, which inactivate the SOC channels periodically ([Ca2+]i oscillation), while hyperpolarization or PTX inhibits the G proteins, maintaining the activation of the SOC channels. Accumulation of cAMP is likely to modulate the PTX-sensitive G proteins. Experimental Physiology (2003) 88.3, 369-379.

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.

The acute and long-term effects of blockade of nitric oxide (NO) production were studied in six chronically catheterised fetal sheep aged from 116 and 118 days; six untreated fetal sheep received injections of saline. Injection of 10 mg (kg maternal body wt)-1 of the nitric oxide synthase (NOS) inhibitor Nω-nitro-L-arginine (NOLA) to the fetus, caused an immediate rise in fetal mean arterial pressure (MAP, P < 0.005) and a reflex fall in fetal heart rate (FHR, P < 0.001). Plasma renin concentration (PRC) fell from 8.4 ± 3.3 to 1.5 ± 0.3 ng ml-1 h-1 (P < 0.001) and was dependent on MAP (P = 0.001). Glomerular filtration rate (GFR) tended to increase, but renal blood flow (RBF) velocity decreased (P < 0.001). Thus filtration fraction (FF) increased (P < 0.025). Urine flow and sodium excretion increased (P < 0.001 for both). Fractional sodium reabsorption decreased (P < 0.05). In fetuses treated with NOLA, arterial pressure was found to affect glomerular haemodynamics and renal tubular handling of sodium. No such relationships were observed in untreated fetuses. The vascular responses to acetylcholine tended to be less (P = 0.07) and the responses to noradrenaline were enhanced in NOLA-treated fetuses. There were no changes in untreated fetuses. Fetuses were then injected twice daily with either 5 mg kg-1 NOLA or saline for the next 2 days. On the 4th day, injection of 10 mg kg-1 NOLA did not have any effects on MAP, FHR or renal function. However, the pressor responses to angiotensin II (Ang II) were enhanced (P < 0.005), as was the response to noradrenaline but to a lesser extent. It is concluded that endothelial production of NO maintains normal fetal blood pressure, renal vascular resistance and fetal renal function. When NO production was blocked by repeated injections of NOLA, other vasodilator pathways took over the maintenance of cardiovascular and renal vascular tone. However, alterations in both cardiovascular and renal function were still present. That is, there was increased pressor sensitivity to exogenous Ang II and unmasking of effects of arterial pressure on glomerular and tubular function. Experimental Physiology (2002) 87.3, 343-351.

Extracellular ATP in micromolar concentrations evokes a transient elevation in intracellular free Ca2+ concentration ([Ca2+]i), which arises primarily from a release of Ca2+ from intracellular stores in rat brown adipocytes. We investigated the mechanisms underlying this transient nature of [Ca2+]i elevation during exposure to ATP by using fura-2 fluorescence measurements together with the P2 receptor antagonists pyridoxal-phosphate-6-azophenyl-2’,4’-disulfonic acid (PPADS) and suramin. Extracellular ATP (10 µM) almost completely depressed the thapsigargin (100 nM)-evoked [Ca2+]i elevation mediated through storeoperated Ca2+ entry. The inhibitory effect of ATP was antagonized by PPADS with IC50 of 0.7 µM. In the presence of PPADS at concentrations of more than 5 µM, the ATP-induced [Ca2+]i elevation became sustained during the entire duration of the agonist application, although the magnitude of the sustained [Ca2+]i elevation was reduced in a concentration-dependent manner by PPADS with an IC50 of 200 µM. In contrast, the ATPinduced [Ca2+]i elevation was blocked by suramin in a concentration range similar to that required to antagonize the inhibitory effect of ATP on the store-operated pathway. These results suggest that the [Ca2+]i responses to extracellular ATP in rat brown adipocytes are mediated through the activation of at least two distinct P2 receptors exhibiting different sensitivities to PPADS but similar sensitivities to suramin. Extracellular ATP stimulates the PPADS-resistant P2 receptor to mobilize intracellular Ca2+ stores, which is probably followed by the activation of store-operated Ca2+ entry. Extracellular ATP, however, would inhibit this Ca2+ entry process through the stimulation of the PPADS-sensitive P2-receptor, which may underlie the transient nature of [Ca2+]i elevation in response to extracellular ATP.

We report the first simultaneous measurements of pHi, [Ca2+]i and tension, upon alteration of pHi, in isolated rat mesenteric arteries loaded with both carboxy SNARF and indo-1. In these vessels (pre-contracted by 30 mM KCl) alterations of pHi, by addition and subsequent washout of weak bases, produced complicated effects on tone. Although the changes in contractility did not mirror the changes in pHi they were, at all times, accompanied by parallel changes in [Ca2+]i. Experimental Physiology (2000) 85.3, 349-351.