Evans blue dye (EBD) conjugates with albumin in the circulation and is frequently used to measure vascular protein leakage. The fluorescence of the dye from tissue sections can be used to measure its uptake at very specific anatomical locations, but problems arise with dye quantification because tissue components also fluoresce; so-called autofluorescence. We have measured uptake of EBD by blood vessel walls at various points around the aorto-renal branch of rabbits. High resolution, digitised, fluorescence images of histological sections of artery wall allowed detailed microscopic analysis of EBD accumulation; and a ratiometric method was developed to enable autofluorescence to be separated from EBD fluorescence. When EBD-free tissue sections were illuminated with blue light, the ratio of red to green fluorescence was constant throughout the tissue (0.59 ± 0.03, mean ± S.D., n = 32). Therefore, at each individual pixel, the level of red autofluorescence could be determined by multiplying the green intensity at that pixel by the calculated red to green ratio. Since EBD fluorescence was detected only in the red region of the spectrum, intensity values of the dye alone were obtained from EBD-exposed tissue by subtracting the red autofluorescence estimated by this ratiometric method. In such cases the red to green fluorescence ratio was measured from adjacent sites known to be free of EDB (0.59 ± 0.02, mean ± S.D., n = 56). We were therefore able to increase the sensitivity of tracer quantification by complete elimination of background autofluorescence on a pixel-by-pixel basis. Use of EBD standards allowed calibration of corrected fluorescence intensities and calculation of mass transfer coefficients for albumin into the artery wall. Spatial variations in the permeability of the artery wall around the renal ostium were detected with the present high resolution technique, with an average mass transfer coefficient of (6.8 ± 0.9)×10-8 cm s-1 for all sites combined (n = 56). The present ratiometric method could potentially be applied to other quantitative fluorescence-based techniques. Experimental Physiology (2002) 87.2, 163-170.

Arterially perfused, decerebrate preparations of the insectivore, Suncus murinus were made to determine whether the emetic reflex could be activated in such a preparation using a range of stimuli shown to be emetic in conscious or anaesthetised Suncus. Efferent phrenic and vagus nerve activities and electromyograms (EMGs) from the temporalis, abdominal oesophagus and trapezius muscles were recorded, as well as longitudinal shortening of the oesophagus and dorso-ventral movements of the thorax. The preparations swallowed spontaneously every 0.6 to 6.5 min. The duration of a swallow was 3.1 ± 0.3 s (recorded as the time taken for the oesophagus to shorten and recover to its resting position) and the oesophagus shortened by 3.5 ± 0.4 mm during a swallow. The emetic reflex was activated by electrical stimulation (30 Hz, 10-20 V, 0.2 ms pulse width, for 30 s) of abdominal vagal afferents (latency < 30 s) or by arterial perfusion with either 40 nM of the capsaicin analogue resiniferatoxin (latency 1.7 ± 0.6 min), 6 µM nicotine (latency 1.6 ± 0.1 min) or 1 µM of the phosphodiesterase IV inhibitor CP-80,633 (latency 8.9 ± 3.9 min). These emetic stimuli produced somatic and visceral movements in Suncus preparations indicative of activation of the emetic reflex. There were pronounced contractions of the thorax that occurred simultaneously with oesophageal shortening and mouth opening, separated by thorax expansion and a burst of phrenic nerve activity. During emetic-like episodes, oesophageal shortenings were only 0.84 ± 0.1 s in duration, faster than the duration of shortening observed during swallowing (cf. swallowing, 3.1 ± 0.3 s; P < 0.0001). The shortening of the oesophagus during emetic-like episodes was 6.2 ± 0.4 mm, which was greater than the shortening seen during swallowing (cf. swallowing, 3.5 ± 0.4 mm; P < 0.0001). We conclude that the emetic reflex can be activated in our Suncus preparations and that this non-sentient small adult animal model can now be used to study the neurophysiology and pharmacology of swallowing and emesis. Experimental Physiology (2002) 87.5, 563-574.

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.

Small size at birth has been associated with increased blood pressure in adult men and women. In rats, isocaloric protein restriction reduces fetal growth and increases systolic blood pressure in adult offspring. Balanced maternal undernutrition in the rat also increases adult blood pressure, but not consistently. The aim of this study was to determine the effect of moderate balanced maternal undernutrition (85% of ad libitum intake from 4 weeks before, and throughout pregnancy) on blood pressure of adult offspring in the guinea pig, a species that is relatively mature at birth. Blood pressure was measured in chronically catheterised offspring of ad libitum fed or feed-restricted mothers, at 3 months of age (young adult). Maternal feed restriction reduced birth weight (−17%) and increased systolic blood pressure (+9%, P < 0.03) in young adult male offspring. In offspring of ad libitum fed and feed-restricted mothers, combined data showed that systolic blood pressure and mean arterial pressure correlated negatively with head width at birth (P = 0.02 and P = 0.04, respectively, n = 28). Systolic blood pressure also correlated negatively with birth weight and the ratio birth weight/birth length, but only in offspring of ad libitum fed mothers (P = 0.04 and P = 0.03, respectively, n = 22). The effect of maternal feed restriction on systolic blood pressure in male offspring was not significant when adjusted for these measures of size at birth. Thus, moderate balanced undernutrition in the guinea pig increases systolic blood pressure in young adult male offspring; however, these effects may be mediated, at least in part, through effects on fetal growth.

In rings of rat iliac artery, contractions were evoked by noradrenaline (NA), the selective α1 adrenoceptor agonist phenylephrine (PE), and K+, which causes depolarisation-induced contraction. There was no evidence of α2 adrenoceptor-evoked contraction. Hypoxia, induced by reducing PO2 in the bath from 100 mmHg to 70, 55 or 40 mmHg, had similar effects on rings with (E+) and without (E-) endothelium. In E- rings, the NA concentration-response curve was biphasic, whereas that for PE was monophasic. Hypoxia reduced maximum contractions in response to NA and PE (NAmax and PEmax, respectively) without affecting the concentrations that evoked 50 % of maximum contraction (EC50). At PO2 of 70 mmHg, NAmax of the high affinity α1 receptor for NA (NAmaxh) and PEmax were reduced by ~15 %, but at PO2 of 55 and 40 mmHg, NAmaxh was severely attenuated while PEmax fell by 45 and 75 %, respectively. Similarly, the Ca2+ channel blocker nicardipine depressed NAmaxh and PEmax, but PO2 of 55 mmHg further reduced NAmax and PEmax. Hypoxia also reduced contractions evoked by NA, PE or K+ at the concentrations required to produce 80 % of the maximum contraction (EC80), receptor-mediated contractions being more affected. Ca2+-free conditions reduced the contractions evoked by NA and PE, at the EC80, to ~10 % of control. The K+ channel inhibitors glibenclamide and tetraethylammonium did not prevent hypoxia-induced depression of PE-evoked contraction. Thus, contractions evoked in iliac artery by the high affinity subtype of α1 adrenoceptor for NA, which may respond to circulating levels of NA, and by the single α1 adrenoceptor subtype for PE, are especially vulnerable to PO2 levels ≤ 55 mmHg. We propose that this reflects hypoxia-induced inhibition of Ca2+ influx through L-type and receptor-operated Ca2+ channels; K+ channel opening makes little contribution. Experimental Physiology (2002) 87.2, 171-184.

Under the conditions of both an increased red cell affinity for O2 at a constant rate of O2 delivery (arterial O2 content × flow) and a decrease in the rate of O2 delivery induced by hypoxic hypoxia at constant blood flow, we have obtained a linear relationship between the partial pressure of O2 in the muscle venous effluent (Pv,O2) and O2 uptake (V˙O2). The relationship is described by the equation V˙O2 = Da × Pv,O2 + V˙O2,conv where Da is the apparent O2 diffusion capacity and V˙O2,conv is O2 delivery-limited V˙O2, and Da × Pv,O2 represents the O2 diffusion-limited V˙O2 (V˙O2,diff). From these observations, we propose the hypothesis that V˙O2 consists of two additive values, V˙O2,conv and V˙O2,diff. The mechanism underlying the reduction in V˙O2 that is induced by reducing O2 delivery to markedly below the V˙O2,conv value has only been investigated using a model based on the single compartment of diffusion-limited V˙O2, and has not been investigated in terms of this additive V˙O2 model. The single compartment analysis appears to overestimate the role of O2 diffusion in limiting the reduction of V˙O2 that occurs in response to a decrease in O2 diffusion capacity, as reflected by the V˙O2/Pv,O2 ratio. To gain better insight into the mechanism involved, we altered the rate of O2 delivery by changing arterial PO2 from normoxia (with inhalation of air) to hypoxia (by inhalation of 10-11 % O2) and blood flow (with high and low flow rates (n = 7 for both groups), and very low and ischaemic flow rates (n = 4 for both groups)) in pump-perfused dog gastrocnemius preparations during tetanic isometric contractions at 1 Hz. As rates of O2 delivery were reduced from 23.2 to 10.9 ml min-1 (100 g)-1, significant decreases in Pv,O2 and V˙O2 were observed (P < 0.05). From the data of Pv,O2 and V˙O2 values within this range of O2 delivery rates, we obtained the regression equation V˙O2 = 0.22 × Pv,O2 + 8.14 (r = 0.58). From the equation, the intercept of the V˙O2-axis was significantly different from zero (P < 0.05), in accordance with the observation that the V˙O2/Pv,O2 ratio (ml min-1 (100 g)-1 Torr-1) increased from 0.54 to 1.35 (P < 0.05). However, at extremely low rates of O2 delivery (5.6 and 7.3 ml min-1 (100 g)-1 the V˙O2/Pv,O2 ratio was 1.51 and 2.80 (P < 0.05), respectively. This indicates a break in the linear V˙O2-Pv,O2 relationship as the rate of O2 delivery was reduced to below the V˙O2,conv value of the V˙O2-axis intercept. These results suggest that the reduction in V˙O2 caused by extreme reductions in the rate of O2 delivery is not attributable to a reduction in O2 diffusion capacity, as expected from the V˙O2/Pv,O2 ratio, but to a reduction in the O2 delivery-limited V˙O2 component, as evaluated by the V˙O2-axis intercept of the linear V˙O2-Pv,O2 relationship. Experimental Physiology (2002) 87.1, 53-61.

We have examined the influence of innervation on the expression of different isoforms of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) in regenerating rat slow twitch muscle. The process of degeneration/ regeneration was induced by injection of bupivacaine into rat soleus muscle under four different conditions: (1) in the presence of intact motor nerves, (2) after surgical denervation, (3) with nerve impulse conduction blocked by the Na+-channel blocker tetrodotoxin (TTX), and (4) with the axoplasmic flow blocked by vinblastine. Expression of SERCA isoforms was visualized by immunohistochemical and Western blot analysis. In regenerating innervated muscle, SERCA1, the isoform normally expressed in fast twitch fibres, was present after 5 days and was then progressively replaced by SERCA2, the isoform typical of slow twitch fibres. The maximum Ca2+ uptake, measured in single skinned fibres regenerating for 10-21 days, was similar to that of slow adult fibres and significantly lower than that of fast adult fibres. Denervation or TTX treatment prevented the expression of the SERCA2 isoform. Conversely, vinblastine did not affect the expression of SERCA isoforms. These data indicate that nerve impulses play a determinant role in the expression of the SERCA2 isoform. Experimental Physiology (2002) 87.5, 575-583.

We have previously shown that extracellular fluid (ECF) is translocated by atrial contraction. Following on from this finding we have proposed a two-step sequential mechanism for the regulation of stretch-activated secretion of atrial natriuretic peptide (ANP): myocytic release of ANP into the surrounding paracellular space followed by the translocation of ECF with the released ANP into the bloodstream. This latter step is induced by atrial contraction, and is therefore controlled by atrial workload. However, the mechanism that regulates the changes in translocation of the ECF has not been defined. To define the relationship between the atrial workload, ECF translocation, size of the extracellular space (ECS) and ANP secretion, experiments have been performed in isolated perfused beating rabbit atria. Atrial workload, transendocardial translocation of the ECF and the secretion of ANP were quantified. Changes in the size of the atrium and the ECS were determined by a newly developed methodology in the beating atria. Atrial workload determined the translocation of the ECF and released ANP with waning of the translocation at higher myocardial workloads. Atrial workload inversely determined the size of the atrium and the ECS. The latter directly determined the translocation of the ECF in terms of atrial workload. From these data we suggest that the size of the ECS is an intimate modulator of the translocation of the ECF and released ANP, and that the phenomenon of waning of the transendocardial translocation that appeared at higher atrial workloads is closely related to the shrinkage of the ECS. Experimental Physiology (2002) 87.2, 185-194.

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.

Multiphoton microscopy is based on the simultaneous absorption of two photons emitted by a pulsed infrared laser source. In this technique, the excitation is restricted to a very small focus and thus results in optical sectioning a priori without the need of a confocal aperture. Multiphoton microscopy was introduced in live cell imaging as an alternative to confocal microscopy due to its superior qualities, such as the deep penetration depth, the reduced photodamage and the lack of out-of-focus bleaching. However, during the past years, examinations revealed severe limitations to the initial expectations. In the focal plane, photodamage and photobleaching can be worse than in single photon microscopy. However, studies showed that with low excitation intensity and by special technical adaptations photodamage could be avoided successfully. For functional biological imaging, multiphoton excitation provides an excellent tool such as the release of caged compounds in a diffraction-limited volume combined with multiphoton or confocal imaging.

The development of the reaction to thermal nociceptive stimulation was studied in neonatal rats of two strains (albino Wistar and pigmented Long-Evans). Using the tail-flick method in neonates of both strains the effects of daily repeated thermal stimulation (sensitisation) were compared to those of acute stimulation applied on 1 day only. During the whole postnatal development period studied (3-28 days) the pigmented rat pups were more sensitive to a repeated thermal nociceptive stimulation than the albinos. The latency of the response did not change linearly during the developmental time studied, but had a pronounced peak (decreased sensitivity) in the period of eye-lid opening. In the pigmented animals the peak appeared 1 day earlier than in the albinos. At the time of the weaning period the latencies of the response were again shorter. Experimental Physiology (2002) 87.1, 63-67.

Alleviation of muscle ischaemia by improving capillary supply has proved difficult, possibly reflecting the inability to substantially increase blood flow. We reasoned that muscle overload, which induces angiogenesis in the absence of altered blood flow, may be an alternative to drug therapy. Male Wistar rats underwent unilateral ligation of the common iliac artery, with or without ipsilateral extirpation of the tibialis anterior muscle. Six weeks later ischaemic (I) extensor digitorum longus (EDL) had a 10% (P< 0.05) decrease in relative muscle mass, while overloaded muscles (O) had undergone hypertrophy of 39% and 52% relative to contralateral (CL) and control (C) muscle masses, respectively (P<0.01). Muscle atrophy was prevented by the combination of overload and ischaemia (O/I), with hypertrophy of 24% (vs. CL) and 35% (vs. C), respectively (P<0.01). Changes in muscle fibre cross-sectional area paralleled the changes in muscle mass, with means of 1898 ± 59, 1531 ± 90, 2253 ± 155 and 2292 ± 80 mm2 for C, I, O and O/I, respectively (P<0.01 vs. C and I). Capillary to fibre ratio (C:F) was significantly increased in overloaded (2.58 ± 0.09) compared to contralateral (1.78 ± 0.04), control (1.61 ± 0.05) and ischaemic (1.73 ± 0.06) muscles (P<0.001). A similar increase in C:F was seen in overloaded plus ischaemic muscle (2.59 ± 0.07) compared to contralateral (1.40 ± 0.01) and control or ischaemic values (P<0.01). In both O and O/I muscle groups, C:F and capillary density (CD) increased most in the region of EDL where fibre size was largest, while hypertrophy of fibres was least in the same region for both groups. These data suggest that the microvascular deficit evident in chronic muscle ischaemia may be alleviated by angiogenesis that is induced by mechanical stimuli via chronic muscle overloaded.

Carbohydrate ingestion increases both endurance time to exhaustion during prolonged exercise, and the ability to perform resistance exercise. The mechanism(s)underlying the increased performance following glucose ingestion remain(s) unclear. The purpose of the present study was to verify the hypothesis that glucose infusion could attenuate peripheral muscle fatigue in the anaesthetized rat during prolonged indirect electrical stimulation in situ. For this purpose the plantaris muscle was electrically stimulated (50 Hz for 200 ms every 2.7 s; 5 V; pulse width, 0.05 ms) in situ through the sciatic nerve to perform concentric contractions for 60 min while infusing intravenously either saline alone (7.25 ml kg-1 h-1), or saline and glucose (1 g kg-1 h-1: plasma glucose 11 ± 1.1, vs. 4.9 ± 0.2 mM with infusion of saline) (8 rats per group). Glucose infusion attenuated the reduction in submaximal peak dynamic force (55 % decrease vs. 70 % decrease in rats infused with saline alone, P < 0.05). In a third group of rats (n = 8), infusion of glucose 30 min after the start of stimulation partially restored submaximal peak dynamic force (P < 0.05). Maximum dynamic and isometric forces at the end of the period of stimulation were also higher (P < 0.05) in rats infused with glucose (4.0 ± 0.2 and 4.3 ± 0.2 N, respectively) than saline alone (3.0 ± 0.2 and 3.5 ± 0.2 N, respectively). The beneficial effect of glucose infusion on peripheral muscle force during prolonged stimulation was not associated with a reduction in muscle glycogen utilisation, nor with a reduction of fatigue at the neuromuscular junction, as assessed through maximal direct muscle stimulation (200 Hz for 200 ms; 150 V; pulse width, 0.05 ms). However, changes in M-wave peak-to-peak amplitude, duration and total area suggest that glucose infusion, and/or the associated increase in plasma insulin concentration, may prevent the deterioration of electrical properties of the muscle fibre membrane. Experimental Physiology (2002) 87.5, 585-592.

The aim of the present study was to investigate the mechanism of the retrograde transfer of ovarian steroid hormones from the ovarian lymphatic and venous effluent to the arterial blood supplying the ovary. In the first experiment, reproductive organs were collected from gilts in the luteal (n = 10) and follicular (n = 10) phase of the oestrous cycle. The ovary with the mesovarium was isolated and perfused through the ovarian artery with warmed, oxygenated autologous blood. The concentrations of progesterone and oestradiol in ovarian arterial blood increased on passing through the ovarian artery to the ovary, in the luteal phase, from 20.3 ± 2.1 to 31.4 ± 3.9 ng ml-1 (P < 0.001) and from 6.2 ± 0.8 to 11.4 ± 1.4 pg ml-1 (P < 0.001), respectively, and in the follicular phase, from 1.2 ± 0.2 to 2.2 ± 0.4 ng ml-1 (P < 0.001) and from 8.2 ± 1.8 to 13.2 ± 2.3 pg ml-1 (P < 0.001), respectively. Approximately 17.5 ± 3.9 % of the progesterone and 12.6 ± 1.7 % of the oestradiol found in the ovarian venous effluent was retrogradely transferred from the ovarian venous blood to the ovary in the luteal phase. In the follicular phase, these values were 10.1 ± 2.0 % and 8.6 ± 1.4 %, respectively. The efficiency of retrograde transfer of oestradiol and the rate of retrograde transfer of progesterone differed between phases of the oestrous cycle (P < 0.05 and P < 0.0001, respectively). A direct relationship between the concentration of the steroids in the venous effluent and the efficiency and rate of the retrograde transfer to the ovary was not found. In the second experiment (luteal phase, n = 10; follicular phase, n = 5), the concentration of progesterone and oestradiol increased in both ovarian arterial blood (P < 0.0001) and in the venous effluent (P < 0.0001) after administration of the steroids into the lymphatic vessels of the isolated mesovarium with separated ovary. In the third experiment (follicular phase, n = 5), with the mesovarium isolated after the ovary was removed and ovarian venous blood flowing out under the force of gravity (without the blood pressure in the ovarian vein), it was demonstrated that the veno-venous network covering the branches of the ovarian artery was supplied with the blood flowing out from the mesovarian tissue and that the filling of the veno-venous network was dependent on the blood pressure in the ovarian artery. We conclude that the effective retrograde transfer of steroid hormones from ovarian venous and lymphatic effluent to the ovary is accomplished not only by the classical counter-current exchange mechanism, but also as a result of complex processes that may be dependent on a specific part of the circulation of the blood and lymph in the periovarian vascular complex of the mesovarium. Experimental Physiology (2002) 87.3, 361-371.

Increasing blood bicarbonate content has long been cited as a potential mechanism to improve contractile function. We investigated whether sodium bicarbonate-induced metabolic alkalosis could positively affect force development during the rest-to-work transition in ischaemic skeletal muscle. Secondly, assuming it could, we investigated whether bicarbonate could augment acetyl group availability through the same equilibrium reaction as sodium acetate pre-treatment and whether this underpins, at least in part, its ergogenic effect. Multiple biopsy samples were obtained from the canine gracilis muscle during 5 min of electrically evoked ischaemic contraction, which enabled the determination of the time course of acetyl group accumulation, substrate utilisation, pyruvate dehydrogenase complex activation and tension development in animals treated with saline (control; n = 6) or sodium bicarbonate (n = 5). Treatment with bicarbonate elevated acetylcarnitine content above the control level at rest (P<0.05), but at no time point during subsequent contraction. The pyruvate dehydrogenase complex was activated following 40 s of contraction in both groups, with no differences existing between treatments at any time point. The requirement for ATP resynthesis from non-oxygen-dependent routes was no different between groups at any time point during contraction. No difference in peak twitch force production existed between groups. However, at 3 min of stimulation, tension development was better maintained in the bicarbonate group (P<0.05), being ∼20% greater than control following 5 min of contraction (P<0.05). The results demonstrate, for the first time, that bicarbonate can augment acetyl group availability prior to contraction, independent of pyruvate dehydrogenase complex activation, but cannot influence the requirement for non-oxidative ATP re-synthesis during subsequent contraction. It would appear, therefore, that the bicarbonate-induced improvement in muscle tension development was probably mediated through the metabolic alkalosis and not via the increased availability of acetyl groups within the cell.

This study examined post-exercise changes in corticospinal excitability in five 'elite' rowers and six non-rowers. Transcranial magnetic stimulation (TMS) was delivered to the motor cortex and bilateral electromyographic (EMG) recordings were made from erector spinae (ES) muscles at L3/L4 spinal level and from the first dorsal interosseous (FDI) muscle of the dominant hand. Each subject completed two exercise protocols on a rowing ergometer: a light exercise protocol at a sub-maximal output for 10 min and an intense exercise protocol at maximum output for 1 min. A trial of ten magnetic stimuli was delivered before each of the protocols and, on finishing exercise, further trials of ten stimuli were delivered every 2 min for a 16 min period. Amplitudes of motor-evoked potentials (MEPs) in each of the three test muscles were measured before exercise and during the recovery period after exercise. The non-rowers showed a brief facilitation of MEPs in ES 2 min after light and intense exercise that was only present in the elite rowers after intense exercise. In the period 4-16 min after light exercise, the mean (± S.E.M.) MEP amplitude (relative to pre-exercise levels) was less depressed in the elite rowers (79.4 ± 2.1 %) than in the non-rowers (60.9 ± 2.5 %) in the left ES but not significantly so in the right ES. MEP amplitudes in FDI were significantly larger in the elite rowers, averaging 119.0 ± 3.1 % pre-exercise levels, compared with 101.2 ± 5.8 % in the non-rowers. Pre-exercise MEP latencies were no different in the two groups. After light exercise MEP latencies became longer in the elite rowers (left ES, 16.1 ± 0.5 ms; right ES, 16.1 ± 0.4 ms; dominant FDI, 23.4 ± 0.2 ms) than in the non-rowers (left ES, 15.0 ± 0.3 ms; right ES, 15.2 ± 0.3 ms; dominant FDI, 21.5 ± 0.2 ms). There were no differences in MEP depression or latency between elite rowers and non-rowers after intense exercise. We conclude that the smaller degree of MEP depression in the elite rowers after light exercise reflects less central fatigue within corticospinal control pathways than that seen in the non-rowers. The longer latency of MEPs seen in the elite rowers may reflect recruitment of more slower-conducting fatigue-resistant motor units compared with the non-rowers. These differences may be because the energy requirements for the non-rowers during light exercise are closer to their maximum capacity, leading to more fatigue. This notion is supported by the lack of any difference between groups following intense exercise when both groups were working at their own maximum. Experimental Physiology (2002) 87.5, 593-600.

Spermine is a low molecular weight polyamine involved in the postnatal maturation of the gut. When it is administered orally to suckling rats, it induces maturation of the intestinal tract (liver, pancreas and small intestine). Here we show that this polyamine is able to induce precocious intestinal and splenic development in suckling mice. In fact, in 15-day-old mice which had received spermine orally twice daily for 3 days we observed an increase in the ratio of white pulp surface to total spleen surface in comparison with untreated mice. The two macrophage subsets of the marginal zone and the B-cell population were more developed and reached the development level of 5- or 10-week-old mice. The proliferation rate of B-cells was increased by spermine administration to pups. These observations suggest that spermine might play a role in immune system development; further investigation of its effects are intended, namely the evaluation of its capacity to enhance defence during the neonatal period. Experimental Physiology (2002) 87.1, 69-75.

The aim of this study was to characterise the arrhythmogenic mechanisms involved in hypokalaemia-induced sustained ventricular fibrillation (SVF), in hypertensive rats. The hearts from rats with hypertension induced by the nitric oxide synthase inhibitor L-NAME, and age-matched normotensive controls, were perfused in Langendorff mode with oxygenated Krebs-Henseleit solution followed by a K+-deficient solution. In additional experiments, free intracellular Ca2+ concentration ([Ca2+]i) was measured using fura-2 in conjunction with an epicardial optical probe. The epicardial electrocardiogram was continuously monitored during all experiments. The gap junction protein connexin-43 and the ultrastructure of the cardiomyocytes were examined, and selected enzyme activities were measured in situ. There was a higher incidence of low-K+-induced SVF in the hearts of hypertensive compared to normotensive rats (83 % vs. 33 %, P < 0.05). Perfusion with a low-K+-containing solution lead to elevation of diastolic [Ca2+]i that was accompanied by premature beats, bigeminy, ventricular tachycardia and transient ventricular fibrillation. These events occurred earlier with increased incidence and duration in the hearts of hypertensive rats (arrhythmia scores: hypertensive, 4.9 ± 0.7; normotensive, 3.1 ± 0.1; P < 0.05), which exhibited apparent remodelling accompanied by a significant decrease in the density of connexin-43-positive gap junctions. Moreover, low-K+-related myocardial changes, including local impairment of intermyocyte junctions, ultrastructural alterations due to Ca2+ overload and intercellular uncoupling, and decreased enzyme activities were more pronounced and more dispersed in hypertensive than normotensive rats. In conclusion, nitric oxide-deficient hypertension is associated with decreased myocardial coupling at gap junctions. The further localised deterioration of junctional coupling, due to low-K+-induced Ca2+ disturbances, as well as spatial heterogeneity of myocardial alterations including interstitial fibrosis, probably provide the mechanisms for re-entry and sustaining ventricular fibrillation. Experimental Physiology (2002) 87.2, 195-205.

Confocal imaging in living brain slices allows the resolution of submicrometre structures of nerve cells, glia and brain vessels. Imaging living brain slices is in many respects different from conventional fixed histological preparations for which confocal microscopes were designed originally. Several problems (i.e. mechanical and thermal drift, and autofluorescence) resulting from the optical and structural properties of brain slices are discussed. Fluorescent indicators may be used to monitor numerous intracellular parameters such as pH and Ca2+ concentration, but not all of them are equally suitable for this type of work. Genetically engineered fluorescent proteins can be used to visualise the fine dendritic structure of neurones or track particular intracellular structures and proteins. They have also been used to generate indicators for Ca2+, cAMP and other molecules. While conventional chemical indicators can be either loaded into neurones via patch pipettes or as membrane-permeable esters, protein indicators can be expressed in various types of cells using adenoviral vectors. Adenoviral transgenesis can be performed in vitro in both acute slices and organotypic slice cultures. Organotypic slice cultures give excellent optical access to neurones loaded with either conventional fluorescent indicators or transfected with adenovirus to express fluorescent proteins. They are most suitable for experiments where both conventional and genetically engineered indicators are combined. Single photon imaging in brain slices is limited to the superficial layers (∼≤50 mm), while multiphoton excitation has a much greater depth of penetration. However, the overall optical resolution achievable in single photon mode is at least as good as when using multiphoton excitation.