A novel isolated Langendorff perfused rabbit heart preparation with intact dual autonomic innervation is described. This preparation allows the study of the effects of direct sympathetic and vagus nerve stimulation on the physiology of the whole heart. These hearts (n = 10) had baseline heart rates of 146 ± 2 beats min-1 which could be increased to 240 ±11 beats min-1 by sympathetic stimulation (15 Hz) and decreased to 74 ± 11 beats min-1 by stimulation of the vagus nerve (right vagus, 7 Hz). This model has the advantage of isolated preparations, with the absence of influence from circulating hormones and haemodynamic reflexes, and also that of in vivo preparations where direct nerve stimulation is possible without the need to use pharmacological agents. Data are presented characterising the preparation with respect to the effects of autonomic nerve stimulation on intrinsic heart rate and atrioventricular conduction at different stimulation frequencies. We show that stimulation of the right and left vagus nerve have differential effects on heart rate and atrioventricular conduction. Experimental Physiology (2001) 86.3, 319-329.

The present investigation was designed to examine whether calmodulin is involved in the inhibition of the ATP-sensitive K+ (KATP) channel by glucagon-like peptide 1(7-36) amide (GLP-1) in mouse pancreatic β-cells. Membrane potential, single channel and whole-cell currents through the KATP channels, and intracellular free Ca2+ concentration ([Ca2+]i) were measured in single mouse pancreatic β-cells. Whole-cell patch-clamp experiments with amphotericin-perforated patches revealed that membrane conductance at around the resting potential is predominantly supplied by the KATP channels in mouse pancreatic β-cells. The addition of 20 nM GLP-1 in the presence of 5 mM glucose significantly reduced the membrane KATP conductance, accompanied by membrane depolarization and the generation of electrical activity. A calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7, 20 µM) completely reversed the inhibitory actions of GLP-1 on the membrane KATP conductance and resultant membrane depolarization. Cell-attached patch recordings confirmed the inhibition of the KATP channel activity by 20 nM GLP-1 and its restoration by 20 µM W-7 or 10 µM calmidazolium at the single channel level. Bath application of 20 µM W-7 also consistently abolished the GLP-1-evoked increase in [Ca2+]i in the presence of 5 mM glucose. These results strongly suggest that the mechanisms by which GLP-1 inhibits the KATP channel activity accompanied by the initiation of electrical activity in mouse pancreatic β-cells include a calmodulin-dependent mechanism in addition to the well-documented activation of the cyclic AMP-protein kinase A system. Experimental Physiology (2001) 86.3, 331-339.

The effects of low-intensity, prolonged swimming on functional recovery of the rat heart (Langendorff preparations) from ischaemia-reperfusion (I/R) were investigated. Three groups of rats (120 days old) were used: sedentary rats (S) and rats exercised by a single bout of swimming lasting 5 (E5) or 8 h (E8), respectively. The effect of exercise on the response to I/R was related to an index of oxidative damage such as lipid peroxidation, as well as to the tissue antioxidant capacity and the response of heart tissue to in vitro oxidative stress. The intrinsic performance of E5 Langendorff preparations paced at 220 beats min-1 was also determined. A group of sedentary animals was used for H2O2-treated preparations. The effect of antioxidant treatment on inotropic recovery during reperfusion was studied on preparations from 5 or 8 h swimming vitamin E-treated (EVT5 and EVT8) and 5 or 8 h swimming untreated (EVU5 and EVU8) rats. Hearts from exercised animals displayed a reduced preischaemic inotropism, which in E5 rats was accompanied by an increase in the intrinsic heart rate. The lower intrinsic cardiac inotropism of E5 animals was confirmed in the paced preparations. The reduced contractility found in control hearts after addition of H2O2 to perfusion medium suggested that the low inotropism of E5 and E8 hearts was due to an exercise-induced increase in reactive oxygen species. Inotropic recovery during reperfusion was low in the S hearts, was significantly increased in the E5 hearts, and was again reduced to the S level in the E8 hearts. In the E8 hearts the indexes of cellular damage (LDH release) and oxidative stress increased, and antioxidant capacity decreased, while in E5 hearts there was no evidence of significant changes in such parameters. Performance and reperfusion recovery of hearts from 5 h swimming rats was not affected by vitamin E treatment, while those of hearts from 8 h swimming rats was the highest observed. We suggest that the higher inotropic recovery during reperfusion in the hearts from the E5 rats is related to the negative inotropic effect of exercise. The fall in recovery following the 8 h exercise was instead related to the increased oxidative stress. Experimental Physiology (2001) 86.3, 341-348.

Dependent upon the relative speed of pulmonary oxygen consumption (V˙O2) and blood flow (Q˙) kinetics, the exercise off-transient may represent a condition of sub- or supra-optimal perfusion. To date, there are no direct measurements of the dynamics of the V˙O2/Q˙ relationship within the muscle at the onset of the work/recovery transition. To address this issue, microvascular PO2 (PO2,m) dynamics were studied in the spinotrapezius muscles of 11 female Sprague-Dawley rats (weight ~220 g) during and following electrical stimulation (1 Hz) to assess the adequacy of Q˙ relative to V˙O2 post exercise. The exercise blood flow response (radioactive microspheres: muscle Q˙ increased ~240 %), and post-exercise arterial blood pH (7.40 ± 0.02) and blood lactate (1.3 ± 0.4 mM l-1) values were consistent with moderate-intensity exercise. Recovery PO2,m (i.e. off-transient) rose progressively until baseline values were achieved (Δend-recovery exercise PO2,m, 14.0 ± 1.9 Torr) and at no time fell below exercising PO2,m. The off-transient PO2,m was well fitted by a dual exponential model with both fast (τ = 25.4 ± 5.1 s) and slow (τ = 71.2 ± 34.2 s) components. Furthermore, there was a pronounced delay (54.9 ± 10.7 s) before the onset of the slow component. These data, obtained at the muscle microvascular level, support the notion that muscle V˙O2 falls with faster kinetics than muscle Q˙ during the off-transient, such that PO2,m increases systematically, though biphasically, during recovery. Experimental Physiology (2001) 86.3, 349-356.

Blast injuries represent a problem for civilian and military populations. Primary thoracic blast injury causes a triad of bradycardia, hypotension and apnoea. The objective of this study was to investigate the reflex nature of this response and its modulation by vagotomy or administration of atropine. The study was conducted on terminally anaesthetised (alphadolone/alphaxalone, 18-24 mg kg h-1, I.V.) male Wistar rats randomly allocated to the groups indicated below. Blast injuries were produced with compressed air while sham blast involved the sound of a blast only. Primary blast injury to the thorax resulted in a bradycardia (measured as an increase in the interval between beats, or heart period (HP) to 489 ± 37 ms from 133 ± 3 ms with a latency of onset of 4.3 ± 0.3 s, mean ± S.E.M.), hypotension (fall in mean arterial blood pressure (MBP) from 128.1 ± 3.7 mmHg to 34.8 ± 4.1 mmHg, latency of onset 2.0 ± 0.1 s) and apnoea lasting 28.3 ± 2.3 s. Sham blast had no effect. The bradycardia and apnoea following thoracic blast were abolished by cervical vagotomy while the hypotension was attenuated. Atropine (0.3 mg kg-1, I.V.) caused a significant reduction in the bradycardia (HP increasing from 124 ± 3 ms to 142 ± 4 ms) but did not modulate either the hypotension or apnoea. It is concluded that a reflex involving the vagus nerve mediates the bradycardia, apnoea and a component of the hypotension associated with thoracic blast. The pattern of this response is similar to effects that follow stimulation of the pulmonary afferent C-fibres. Experimental Physiology (2001) 86.3, 357-364.

The role of endothelins in the renal damage associated with ischaemic-reperfusion (I-R) injury during organ transplantation was determined by selective blockade of the ETA receptors with the receptor antagonist ABT-627. The integrity of kidney function was determined 2 and 8 weeks after transplantation by investigation of the renal response to angiotensin II. Under pentobarbitone anaesthesia (70 mg kg-1, I.P.), rats underwent a right nephrectomy. Transplantation of the left kidney was performed after 2 h cold ischaemia without or with ABT-627 treatment. Control animals underwent left renal denervation. The renal response to angiotensin II was measured 2 weeks later following blockade of endogenous production of angiotensin II with captopril. A further transplant group was allowed to recover for 8 weeks before the terminal study. In the control group, angiotensin II reduced renal blood flow (RBF), glomerular filtration rate (GFR), urine flow rate (UV), and fractional sodium excretion (FENa) by 29 ± 5 %, 19 ± 4 %, 25 ± 4 % and 32 ± 7 %, respectively. Conversely, in the transplant group, angiotensin II left RBF unchanged and increased GFR (59 ± 12 %) and UV (93 ± 8 %). FENa decreased by 24 ± 9 %. In both the transplant group treated with ABT-627 and the long-term recovery group, the renal response to angiotensin II was normalised. In conclusion, renal transplantation following 2 h cold I-R injury resulted in a temporary abnormal renal response to angiotensin II, which was reversed by ETA receptor antagonism at the time of transplantation. Experimental Physiology (2001) 86.3, 365-372.

Prostaglandins (PGs) promote both bone resorption and formation in vitro and in vivo. In a synchronised model of bone remodelling, indomethacin, an inhibitor of PG synthesis, given from the start of the sequence, transiently impaired bone resorption. In this study we further explored the involvement of PGs in this model by treating rats with indomethacin (7.5 mg kg-1 day-1) for 6 days from the peak of resorption (day 4 after activation in this model) or during reversal (day 6 after activation). In rats treated from day 4, the resorption surface (Oc.S/BS) and the number of osteoclasts (N.Oc/BPm) were higher on day 10 (+69 %, P < 0.01, and +60 %, P < 0.02 compared with controls, respectively); no effect on cell resorptive activity was observed. The bone formation surface (OS/BS) was reduced (-50 %, P < 0.01). The inactive surface (In/BS) was not modified. In rats treated from day 6, the Oc.S/BS was also higher than in controls (P < 0.02), as was the N.Oc/BPm (P < 0.05). Osteoclast activity appeared to be increased, as the osteoclast-bone interface was larger (P < 0.02), but the mean lacuna area was reduced (-23 %, P < 0.05). Bone formation was also strongly affected: the OS/BS was decreased (-66 %, P < 0.01), as was the osteoid seam thickness (-24 %, P < 0.05). The In/BS was increased 1.5-fold (P < 0.05). These data indicate that PGs intervene at various stages of this remodelling sequence, as both resorption and formation were affected by indomethacin. Although resorption resumed in the two treatment groups despite treatment continuation, the timing of treatment was clearly important. Only inhibition of PG synthesis at the peak of resorption delayed all phases of the remodelling sequence. In contrast, inhibition during the reversal phase prevented activation of a significant part of the bone surface usually involved at this stage of remodelling; this treatment schedule reduced the resorptive capacity of the system, and depressed osteoblast activity. Experimental Physiology (2001) 86.3, 373-379.

Stimulation of left ventricular mechanoreceptors was believed not only to exert important effects on the circulation, but also to influence the responses to baroreceptor reflexes. However, most previous work is flawed due to inadequate localisation of stimuli to specific reflexogenic areas. In this study, we applied a discrete stimulus to left ventricular mechanoreceptors to examine other reflexes known to effect the circulation. Dogs were anaesthetised, artificially ventilated and a cardiopulmonary bypass established. The pressure distending the left ventricle was controlled through an apical cannula with the aortic valve obstructed by a balloon. Changes in ventricular systolic and end-diastolic pressure had only a small effect on vascular resistance, assessed as perfusion pressure in the systemic circulation (flow constant). Responses to changes in carotid or coronary pressure or to stimulation of chemosensitive afferents by injecting veratridine into the coronary circulation were always much larger. Responses to stimulation of these reflexes were little affected by the level of stimulus to the ventricular receptors. These experiments confirm that responses to stimulation of ventricular mechanoreceptors are very small and show that they remain small at different levels of input to other baroreceptive regions. There was no evidence of interaction between ventricular mechanoreceptor reflexes and carotid or coronary baroreceptors or ventricular chemosensitive reflexes. Experimental Physiology (2001) 86.3, 381-390.

This study was performed to determine whether the absorption of liquid from the lungs of postnatal sheep is dependent on pulmonary perfusion pressure, blood gases or blood flow. Relationships between perfusion pressure, rate of lung liquid absorption and perfusate PO2, PCO2 and pH were examined by linear regression analysis from in situ perfused lungs from sheep aged 6 weeks to 6 months. The airspaces of the lungs were filled with liquid containing an impermeant tracer, to allow measurement of the rate of liquid absorption. There was no significant relationship between the rate of lung liquid absorption and pulmonary blood flow (n = 36, r = -0.01, P > 0.1), pulmonary perfusion pressure (n = 36, r = 0.28, P > 0.05) or perfusate PO2, PCO2 or pH. No significant relationships were found between pulmonary blood flow and perfusate PO2, PCO2 or pH. There was no evidence to suggest that the absorption of liquid from the lungs of postnatal sheep is dependent on pulmonary blood flow, blood gases or perfusion pressure, within the limits studied, indicating that lung liquid absorption is dependent on the pulmonary epithelium and not on the pulmonary vasculature. The findings that lung liquid absorption continues in hypoxic environments and despite severe reductions in blood flow may be relevant to the field of transplant surgery. Experimental Physiology (2001) 86.3, 391-398.

The aim of this study was to investigate the role of the parasympathetic (cholinergic and peptidergic) nervous system in the regulation of exocrine pancreas function in piglets during their early postnatal development. The cholinergic and peptidergic regulatory pathways of exocrine pancreatic function were tested by the specific muscarinic receptor blocker 4-diphenylacetoxy-N-methylpiperidine-methiodide (4-DAMP) and bombesin, respectively. At the age of 2 weeks, piglets were surgically fitted with a chronic pancreatic duct catheter, a duodenal re-entrant cannula and a jugular vein catheter. The experiments comprised a pre-weaning period, and a post-weaning period that commenced at the beginning of the 5th week of age. Intravenous infusion of 4-DAMP (100 pmol kg-1 h-1) reduced the outflow of pancreatic juice, the output of total protein and the activity of trypsin, chymotrypsin, carboxyl ester hydrolase and amylase during preprandial and postprandial pancreatic secretion, in both the pre- and post-weaning periods. However, the inhibitory effect of 4-DAMP during postprandial secretion was significantly greater (P < 0.05) in suckling piglets. The infusion of bombesin (10, 100 and 1000 pmol kg-1 h-1) stimulated exocrine pancreatic secretion in a dose-dependent manner during both the pre- and post-weaning periods. However, the stimulatory effect of 1000 pmol kg-1 h-1 bombesin on total protein output and the activities of trypsin, chymotrypsin and amylase were significantly higher (P < 0.05) in suckling piglets. In summary, our study showed that cholinergic and peptidergic mechanisms are involved in the regulation of exocrine pancreas function in piglets in both the pre- and post-weaning stages. 4-DAMP had a greater inhibitory effect on exocrine pancreatic secretion in piglets during the pre-weaning period. Thus, these observations suggest that the parasympathetic nervous system plays a dominant role in the functioning of the exocrine pancreas at this time. The action of bombesin suggests that it is a potent secretagogue for the exocrine pancreas in pigs during their postnatal development. Experimental Physiology (2001) 86.3, 399-409.

Changes in high-energy phosphate levels in single human skeletal muscle fibres after 10 s of maximal (all-out) dynamic exercise were investigated. Muscle biopsies from vastus lateralis of two volunteers were collected at rest and immediately post exercise. Single muscle fibres were dissected from dry muscle and were assigned into one of four groups according to their myosin heavy chain (MyHC) isoform content: that is type I, IIA, IIAx and IIXa (the latter two groups containing either less or more than 50 % IIX MyHC). Fragments of characterised fibres were analysed by HPLC for ATP, inosine-monophosphate (IMP), phosphocreatine (PCr) and creatine levels. After 10 s of exercise, PCr content ([PCr]) declined by approximately 46, 53, 62 and 59 % in type I, IIA, IIAx and IIXa fibres, respectively (P < 0.01 from rest). [ATP] declined only in type II fibres, especially in IIAx and IIXa fibres in which [IMP] reached mean values of 16 ± 1 and 18 ± 4 mmol (kg dry mass)-1, respectively. While [PCr] was reduced in all fibre types during the brief maximal dynamic exercise, it was apparent that type II fibres expressing the IIX myosin heavy chain isoform were under a greatest metabolic stress as indicated by the reductions in [ATP]. Experimental Physiology (2001) 86.3, 411-415.

Prior heavy exercise (above the lactate threshold, LT) reduces the amplitude of the pulmonary oxygen uptake (V˙O2) slow component during heavy exercise, yet the precise effect of prior heavy exercise on the phase II V˙O2 response remains to be established. This study was designed to test the hypotheses that (1) prior heavy exercise increases the amplitude of the phase II V˙O2 response independently of changes in the baseline V˙O2 value and (2) the effect of prior exercise depends on the amount of external work done during prior exercise, irrespective of the intensity of the prior exercise. Nine subjects performed two 6 min bouts of heavy cycling exercise separated by 6 min baseline pedalling recovery (A), two 6 min heavy exercise bouts separated by 12 min recovery (6 min rest and 6 min baseline pedalling, B), and a bout of moderate exercise (below the LT) in which the same amount of external work was performed as during the prior heavy exercise, followed by 6 min heavy exercise (C). In both tests A and B, prior heavy exercise significantly increased the absolute V˙O2 amplitude at the end of phase II (by ~150 ml min-1), and reduced the amplitude of the V˙O2 slow component by a similar amount. Following 12 min of recovery (B), baseline V˙O2, but not blood [lactate], had returned to pre-exercise levels, indicating that these effects occurred independently of changes in baseline V˙O2. Prior moderate exercise (C) had no effect on either the V˙O2 or blood [lactate] responses to subsequent heavy exercise. The V˙O2 response to heavy exercise was therefore dependent on the intensity of prior exercise, and the effects on the amplitudes of the phase II and slow V˙O2 components persisted for at least 12 min following prior heavy exercise. Experimental Physiology (2001) 86.3, 417-425.

Lower body negative pressure (LBNP) reduces central venous pressure (CVP) and cardiac output. The elderly are reported to have a limited capacity to increase cardiac output by increasing heart rate (HR), are especially dependent on end diastolic volume to maintain stroke volume and therefore should be especially vulnerable to LBNP. The present study compared the effects of LBNP in the young and old. Stroke volume was assessed non-invasively as stroke distance (SD) by aortovelography. Two groups of healthy male volunteers were studied: eight young (29.7 ± 2.0 years, mean ± S.E.M.) and nine old (70.1 ± 0.9 years). LBNP was applied progressively at 17.5, 35 and 50 mmHg in 20 min steps, with measurements taken during each steady state. There were similar, significant, falls in CVP in both groups. SD fell significantly in both groups from respective control values of 24.8 ± 1.6 and 16.6 ± 0.9 cm to 12.5 ± 1.3 and 8.9 ± 0.4 cm at a LBNP of 50 mmHg. Although SD in the elderly was significantly lower than in the young, the LBNP-induced changes were not different between groups. Both groups produced similar significant increases in vascular resistance, HR, plasma vasopressin (AVP) and noradrenaline. Mean arterial blood pressure (MBP) and plasma adrenaline did not change significantly. Therefore healthy old men respond to LBNP in a similar manner to the young, although MBP and SD are regulated around different baselines in the two groups. Experimental Physiology (2001) 86.3, 427-435.