The aim of this study was to evaluate myofibrillar creatine kinase (CK) activity and to quantify the substrate channelling of ATP between CK and myosin ATPase under different pH conditions within the integrity of myofibrils. A pure myofibrillar fraction was prepared using differential centrifugation. The homogeneity of the preparation and the purity of the fraction were confirmed microscopically and by enzymatic assays for contaminant enzyme activities. The specific activity of myofibrillar CK reached 584 ± 33 nmol PCr min-1 mg-1 at pH 6.75. Two methods were used to detect CK activity: (1) measurement of direct ATP production, and (2) measurement of PCr consumption. This method of evaluation has been tested in experiments with isolated creatine kinase. No discrepancy in CK activity between the methods was observed in the pH range tested (6.0-7.5). However, the same procedures resulted in a significant discrepancy between the amounts of reacted PCr and produced ATP within the pure myofibrillar fraction. This discrepancy represents the portion of ATP produced by the CK reaction, which is preferentially channelled to the myosin ATPase before diffusing into the bulk solution. The maximum evaluated difference reached 42.3 % at pH 6.95. The substrate channelling between myofibrillar-bound CK and myosin ATPase was evaluated under various pH levels within the physiological range and it reached a maximum value in a slightly acidic environment. These results suggest that ATP/ADP flux control by the CK system is more important at lower pH, corresponding to the physiological state of muscle fatigue. Experimental Physiology (2003) 88.1, 1-6.

Subjects sat on a 'Balans' chair supported by the shaft of a large torque generator. The lower but not the upper part of the body was free to make to/fro movements maintained by feeding back a modified velocity signal to the generator. The stiffness could be increased by the use of a position signal. Using the equations for a torsion pendulum the good linearity observed in relationship to the applied forces allowed stiffness, inertia, damping, critical damping and the damping factor to be determined in absolute terms. The method enables parameters relevant to back function and problems to be evaluated. Eleven adults acted as subjects. Experimental Physiology (2003) 88.3, 305-308.

It has previously been postulated that rapid red cell capillary transit through the human lung plays a role in the mechanism of diffusion limitation in some endurance athletes. Methodological limitations currently prevent researchers from directly measuring pulmonary capillary transit times in humans during exercise; however, first pass radionuclide cardiography allows direct measurement of red blood cell (RBC) transit times through the whole lung at various exercise intensities. We examined the relationship between mean whole lung red cell pulmonary transit times (cardiopulmonary transit times or CPTT) and different levels of flow in 88 healthy humans (76 males, 12 females) from several studies (mean age 31 years). The pooled data suggest that the relationship between CPTT and cardiac index (CI), beginning at rest and progressing through to maximum exercise demonstrates that CPTT reaches its minimum value when CI is about 8.1 l m2 min-1 (2.5-3 times the CI value at rest), and does not significantly change with further increases in CI. Cardiopulmonary blood volume (CPBV) index also does not change significantly until CI reaches 2.5 to 3 times the CI value at rest and then increases roughly linearly after that point. Consequently, the systematic increase in CPBV index with increasing pulmonary blood flow between 8.1 and 20 l m2 min-1 displays an adaptive response of the cardiopulmonary system by augmenting CPBV (and perhaps pulmonary capillary blood volume through distension and recruitment) to offset the reduction in CPTT, as no significant difference in mean CPTT is observed between these levels of flow (P > 0.05). Therefore, these data demonstrate that CPBV does not reach maximum capacity during strenuous or maximum exercise. This does not support the principle of quarter-power allometric scaling for flow when explaining modifications during exercise. Therefore, we speculate that the observed relationships between CPTT, CBPV index and flow may prevent mean CPTT (and perhaps mean pulmonary capillary transit times) from decreasing below the threshold time required for oxygenation. Experimental Physiology (2003) 88.2, 191-200.

The variable pressure neck chamber has provided an invaluable research tool for the non-invasive assessment of carotid baroreflex (CBR) function in human investigations. The ability to construct complete stimulus-response curves and define specific parameters of the reflex function curve permits statistical comparisons of baroreflex function between different experimental conditions, such as rest and exercise. Results have convincingly indicated that the CBR stimulus-response curve is reset during exercise in an intensity-dependent manner to functionally operate around the prevailing pressure elicited by the exercise workload. Furthermore, both at rest and during exercise, alterations in stroke volume do not contribute importantly to the maintenance of arterial blood pressure by the carotid baroreceptors, and therefore, any reflex-induced changes in cardiac output (Q) are the result of CBR-mediated changes in heart rate. However, more importantly, the CBR-induced changes in mean arterial pressure (MAP) are primarily mediated by alterations in vascular conductance with only minimal contributions from Q to the initial reflex MAP response. Thus, the capacity of the CBR to regulate blood pressure depends critically on its ability to alter vascular tone both at rest and during exercise. This review will emphasize the utility of the variable pressure neck chamber to assess CBR function in human experimental investigations and the mechanisms by which the CBR responds to alterations in arterial blood pressure both at rest and during exercise. Experimental Physiology (2003) 88.6, 671-680.

The specificity in location of angiogenesis to either glycolytic or oxidative fibre types, or muscle regions, was examined in the tibialis anterior (TA) and extensor digitorum longus (EDL) muscles of rat. Angiogenesis was induced by mechanical means either with (chronic muscle stimulation) or without (muscle stretch by overload) changes in blood flow, treatments which invoked only minor changes in fibre type and fibre size. Proliferation estimated by PCNA labelling of cells co-localised with capillaries was very rare in control muscles, where it occurred mainly in the glycolytic regions, but was increased in both models of angiogenesis. However, when labelled capillaries were scored according to the type of surrounding fibres, only muscle stimulation significantly accentuated proliferation of capillaries surrounded by glycolytic fibres. We conclude that while mechanical stimuli are important for proliferation in glycolytic regions in both models, capillary growth occurs specifically around glycolytic fibres in that region when the angiogenic stimulus includes increased blood flow and/or increased metabolic demand. Experimental Physiology (2003) 88.5, 565-568.

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.

Rodent models of sepsis differ from clinical human disease in that humans make substantially less whole-body nitric oxide and have different cellular responses to endotoxin. Sheep, when exposed to endotoxin, behave in a manner more similar to humans. Many studies of rodent peripheral blood mononuclear cells (PBMCs) exposed to endotoxin demonstrate increased cationic amino acid transporter function (particularly through the y+ transporter) to supply arginine substrate to upregulated nitric oxide synthase. Whether this is true in sheep is not known. We have studied cationic amino acid transport in sheep PBMCs stimulated with endotoxin, using labelled lysine. PBMCs stimulated both in vitro and in vivo show an initial reduction in total and y+ lysine transport (after 1-2 h exposure to endotoxin): a previously undescribed effect of endotoxin. In in vitro activated cells, the reduction in y+ transport was prevented by the lipoxygenase inhibitor, nordihydroguaretic acid (NDGA), and the phospholipase inhibitor 4-bromophenacyl bromide (4-BPAB), but not cyclohexamide or a number of other inhibitors of intracellular second-messenger pathways. In contrast after 14 h incubation, the expected increase in total and y+ lysine transport was seen. The increase in y+ transport could be prevented by cyclohexamide, dexamethasone, ibuprofen, the protein kinase C inhibitor sphingosine, NDGA and 4-BPAB. These results suggest that in response to endotoxin exposure there is an initial decrease in y+ activity mediated by a lipoxygenase product, followed by a substantial increase in y+ activity mediated by the products of either cyclo-oxygenase or lipoxygenase. Cyclo-oxygenase and/or lipoxygenase inhibition might be useful in reducing arginine transport, and hence nitric oxide production, in these cells. Experimental Physiology (2003) 88.2, 201-208.

Connexins (Cx) form intercellular junctional channels which are responsible for metabolic and electrical coupling. We report here on the biochemical and immunohistochemical characterization of zebrafish connexin zfCx43.4, an orthologue of mammalian and avian Cx45, and the electrophysiological properties of junctional channels formed by this protein. The investigations were performed on transfected COS-7 cells or HeLa cells. Using site-directed antibodies, zfCx43.4 cDNA (GenBank accession no. X96712) was demonstrated to code for a protein with a Mr of 45 000. In transfected cells, zfCx43.4 was localized in cell-cell contact areas as expected for a gap junction protein. zfCx43.4 channels were shown to transfer Lucifer Yellow. The multichannel currents were sensitive to the transjunctional voltage (Vj). Their properties were consistent with a two-state model and yielded the following Boltzmann parameters for negative/positive Vj: Vj,0 = -38.4/41.9 mV; gj,min = 0.19/0.18; z = 2.6/2.3. These parameters deviate somewhat from those of zfCx43.4 channels expressed in Xenopus oocytes and from those of Cx45, an orthologue of zfCx43.4, expressed in mammalian cells or Xenopus oocytes. Conceivably, the subtle differences may reflect differences in experimental methods and/or in the expression system. The single channel currents yielded two prominent levels attributable to a main conductance state (γj,main = 33.2 ± 1.5 pS) and a residual conductance state (γj,residual = 11.9 ± 0.6 pS). Experimental Physiology (2003) 88.6, 681-690.

Salivary fluid secretion is dependent upon reflex stimuli mediated by autonomic nerves. In order to determine if immunoglobulin A (IgA) and salivary proteins are secreted in the absence of nerve stimulation, small volumes (< 2 µl) of saliva were consecutively collected from the submandibular duct of anaesthetised rats following rest pauses in order to sample the protein contents of the ductal system. Within the first 5 µl of such saliva collected by parasympathetic nerve stimulation, IgA and other salivary proteins reached peak concentrations that were over 20-fold greater than levels in parasympathetically stimulated saliva subsequently collected during a 5 min period of stimulation. Confocal microscopy of TRITC-labelled IgA added to live, acutely isolated submandibular acini indicated that it did not enter the lumina by paracellular leakage. IgG is thought to enter saliva by paracellular leakage but did not accumulate in luminal saliva in the present study. Electrophoresis suggested that the major proteins secreted in the absence of stimulation were the same as those present in subsequently stimulated saliva. It can be concluded that IgA and other major submandibular proteins are secreted into glandular lumina in the absence of nerve stimulation. The functional significance of such unstimulated protein secretion is at present unclear. Experimental Physiology (2003) 88.1, 7-12.

Modulation at the level of the nucleus tractus solitarii (NTS) appears to be an effective way of controlling cardiovascular reflexes. Angiotensin II acting on angiotensin AT1 receptors at the central nervous system appears to have an important role in these modulatory processes. The hypothalamic defence area (HDA) is a potential source of descending fibres containing angiotensin II that innervate the NTS. We investigated the effect of AT1 receptor blockade in the NTS on the response to stimulation of HDA in anaesthetised rats treated with the neuromuscular blocking agent pancuronium bromide. The characteristic increase in heart rate, blood pressure and phrenic nerve activity evoked by electrical stimulation of HDA is decreased by the microinjection of the AT1 receptor antagonist losartan into the NTS and the cardiovascular response to carotid body chemical stimulation is also reduced. These results support the hypothesis that AT1 receptors in the NTS play a role in the modulation of cardiovascular reflexes, and modify the influence exerted on the processing of these reflexes by other areas of the central nervous system. Experimental Physiology (2003) 88.3, 309-314.

The Na+-K+-2Cl- cotransporter (NKCC1) is responsible for ion transport across the secretory and absorptive epithelia, the regulation of cell volume, and possibly the modulation of cell growth and development. It has been reported that a variety of cells, including osteoblasts, contain this cotransporter. In this study, the physiological role of NKCC1 in osteoclastogenesis was exploited in a co-culture system. Bumetanide, a specific inhibitor of NKCC1, reduced the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. In order to investigate the mechanism by which bumetanide inhibits osteoclastogenesis, the mRNA expressions of the receptor activator of nuclear factor (NF)-κB ligand (RANKL) and osteoprotegerin (OPG) were analysed by RT-PCR. Exposure of osteoblastic cells to a medium containing 1 µM bumetanide reduced RANKL mRNA expression induced by 10 nM 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3, in a dose-dependent manner. In addition, RANKL expression was also analysed with enzyme-linked immunosorbant assay (ELISA) using anti-RANKL antibody. The expression of RANKL was decreased with the increase of bumetanide concentration. In contrast, the expression of OPG mRNA, a novel tumour necrosis factor (TNF) receptor family member was increased in the presence of bumetanide. These results imply that bumetanide inhibits osteoclast differentiation by reducing the RANKL/OPG ratio in osteoblastic cells. However, no significant difference in M-CSF mRNA expression was observed when bumetanide was added. Also, we found that the phosphorylation of c-Jun NH2-terminal kinase (JNK), which regulates the activity of various transcriptional factors, was reduced by bumetanide treatment. Conclusively, these findings suggest that NKCC1 in osteoblasts has a pivotal role in 1α,25(OH)2D3-induced osteoclastogenesis partly via the phosphorylation of JNK. Experimental Physiology (2003) 88.5, 569-574.

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.

Interstitial fluid fluxes are much greater in the fetus than in the adult, and filtration rates are increased over control in most tissues of the anaemic fetus. Increased capillary filtration may lead to cardiac oedema which, in turn, severely impacts cardiac function. Mechanisms that underlie these differences in flux are incompletely understood. One possible mechanism is an increase in capillary water permeability. Therefore, the goal of our study was to determine the level of expression of the water channel aquaporin 1 (AQP1) during cardiac development and in the anaemic fetal sheep heart. Hearts from chronically instrumented anaemic sheep fetuses and hearts from normal early fetal, late fetal, neonatal and adult sheep were used for Northern and Western analyses and immunohistochemistry. We found that AQP1 mRNA levels were lower in the young fetal left ventricle than in the adult left ventricle (P < 0.05). We also found that cardiac AQP1 expression was increased in anaemic fetuses compared to age-matched controls (P < 0.05). Expression of AQP1 in all groups was greatest in the microvascular endothelium. These data suggest that AQP1 plays an important role in the physiological accommodation to fetal anaemia. Experimental Physiology (2003) 88.6, 691-698.

In the present study, we have investigated the viability of using tetrodotoxin (TTX) to induce selective blockade of myelinated fibre conduction in rabbit sural nerve, and explored some aspects of reflexes evoked by non-myelinated sural nerve afferents before and after application of TTX. In rabbits decerebrated under halothane-nitrous oxide anaesthesia, application of 30 nM TTX to the desheathed sural nerve completely blocked Aβ and Aδ waves of the compound action potential evoked by electrical stimulation of the nerve at 95 times threshold. The amplitude of C-fibre volleys evoked by these stimuli was reduced to a mean of 60 % of pre-treatment values. Reflexes evoked in medial gastrocnemius motoneurones by sural nerve stimulation showed corresponding changes after TTX treatment, with activation latency increasing from 5-7 ms in the control state to > 100 ms after TTX application. Temporal summation (wind up) in long latency reflexes (> 100 ms) was significantly enhanced after application of TTX. These data show that low concentrations of TTX can selectively block conduction in rabbit sural nerve A-fibres, providing a method for studying the central actions of non-myelinated C-fibres in isolation. Experimental Physiology (2003) 88.1, 13-18.

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.

An elevated extracellular concentration of D-glucose (i.e. hyperglycaemia) inhibits cell proliferation and incorporation of the endogenous nucleoside thymidine into DNA in human umbilical vein endothelial cells (HUVECs). Cells in their log-phase of growth (3.7 ± 0.3 days, n = 27) incubated for 30 min with 25 mM D-glucose, but not with equimolar concentrations of L-glucose or D-mannitol, exhibited reduced [3H]thymidine incorporation and cell growth rate, with no change in cell viability (> 98 %), total DNA, protein content or cell volume. Incubation with D-glucose activated protein kinase C (PKC), endothelial NO synthase (eNOS), p42 and p44 mitogen-activated protein kinases (p42/44mapk), but inhibited superoxide dismutase (SOD). Incubation with D-glucose also increased cGMP and cAMP levels. The effect of D-glucose was blocked by the PKC inhibitor calphostin C, the MAP kinase kinase 1/2 (MEK1/2) inhibitor PD-98059, the eNOS inhibitor L-NAME, the protein kinase G (PKG) inhibitor KT-5823 and the protein kinase A (PKA) inhibitor KT-5720. In the presence of 5 mM D-glucose, [3H]thymidine incorporation and cell growth were reduced by the PKC activator phorbol 12-myristate 13-acetate (PMA), the NO donor S-nitroso-N-acetyl-L,D-penicillamine (SNAP), dibutyryl cGMP, dibutyryl cAMP and the Ca2+ ionophore A-23187. The effect of A-23187 was blocked by calphostin C and PD-98059. D-Glucose-dependent inhibition of thymidine incorporation and cell proliferation is associated with increased PKC, eNOS, and MEK1/2, but decreased SOD activity, and higher intracellular levels of cGMP, cAMP and Ca2+ in HUVECs. These are cellular mechanisms which may reduce endothelial cell growth in pathological conditions such as in diabetes mellitus or hyperglycaemia. Experimental Physiology (2003) 88.2, 209-219.

Vagal cardioinhibition is exerted through a reduction not only in the heart rate but also in the rate of propagation of the cardiac action potential and in myocardial contractility. In several species, such effects can be produced independently by selective activation of ganglia in identified 'fat pads'. In this study we investigate differential control of heart rate and atrioventricular conduction by two ganglionic clusters in the rat, a species increasingly important in studies of cardiovascular control. Epicardial sites producing low-threshold changes in P-P and P-R interval of the ECG in an arterially perfused preparation were explored with concentric bipolar stimulating electrodes. Stimulation sites centred on two principal ganglia, the sinoatrial (SA) ganglion at the junction of the right superior vena cava and right atrium, and the atrioventricular (AV) ganglion at the junction of the inferior pulmonary veins and left atrium. Stimulation of the SA ganglion decreased heart rate in all preparations, with little or no effect on AV conduction in one-third. Stimulation of the AV ganglion consistently slowed conduction without eliciting a comparable bradycardia. Responses survived blockade of ganglionic transmission by trimetaphan, with an enhanced chronotropic selectivity to SA ganglion stimulation, suggesting that co-excitation of preganglionic elements en passant may have contributed to the earlier mixed responses. Effective stimulation sites were precisely circumscribed and corresponded to principal ganglionic clusters confirmed histologically. We conclude that cardiac vagal ganglia in the rat show a topographical functional organisation and are amenable to investigation using the arterially perfused preparation. Experimental Physiology (2003) 88.3, 315-327.

The effect of glucagon on renal haemodynamics in sheep is controversial. In this study we have examined the effects of perfused glucagon on renal blood flow (RBF) in six conscious sheep bilaterally implanted with transit-time ultrasonic flow metering probes around the renal arteries. Glucagon was perfused intravenously over 90 min at doses of 3.12, 6.25, 12.5, 25, 50 and 100 ng kg-1 min-1. Mean RBF was calculated over 10 min periods. Blood samples were taken to monitor the time course of the changes in glycaemia and glucagonaemia. The perfusions of glucagon induced rapid and progressive dose-dependent increases in RBF (9-19.2 %, P < 0.05) and glycaemia (29-155 %, P < 0.05) for doses of 25-100 ng kg-1 min-1. High positive correlations were found between the increases in RBF and glucagonaemia (R2 = 0.95) and between the increases in RBF and glycaemia (R2 = 0.96). At the lowest doses of glucagon (3.12-12.5 ng kg-1 min-1), the increase in RBF was highly significant; however, the rise in glucose level was not. At the highest doses (25-100 ng kg-1 min-1) the time course of the changes in RBF was parallel to that of glucagonaemia throughout the perfusion time. However, between minutes 45 and 90 of the glucagon perfusion, the increase in RBF was the inverse of the change in glycaemia, which decreased. One hour after the end of the 50 and 100 ng kg-1 min-1 perfusions, the glucose levels were still significantly higher than the baseline, while the RBF values were not. These results are consistent with the idea that the enhanced RBF cannot be attributed to a rise in blood glucose level. They also show that the haemodynamic response to glucagon perfusion was more sensitive than the metabolic response. It is concluded that the intravenous perfusion of physiological doses of glucagon induced a highly sensitive dose-dependent increase in RBF in sheep. Experimental Physiology (2003) 88.5, 575-580.

The cranial venae cavae of the rat heart are composed of cardiac muscle. We tested whether the vagus nerve has an inotropic action on these blood vessels. Stimulation of right or left vagal fibres (n = 7 animals) produced a negative chronotropic and inotropic effect. Before stimulation the basal cardiac interval was 319 ± 25 ms and the vena caval diastolic force was 1.82 ± 0.29 mN and the systolic force was 3.28 ± 0.39 mN. Ten second stimulation increased the cardiac interval to a maximum of 484 ± 77 ms and reduced the systolic force significantly to 2.83 ± 0.39 mN (two-tailed paired t test). The diastolic or baseline force was unaffected by vagal stimulation (1.85 ± 0.29 mN). The vagal negative inotropic action took significantly longer to reach peak effect (9.5 ± 1.0 s versus 3.2 ± 0.9 s) and lasted longer than the chronotropic effect (20.4 ± 2.1 s versus 10.25 ± 1.2 s). The negative inotropic action was still observed in paced preparations and preparations with transient constant-rate tachyarrhythmias. Both the chronotropic and inotropic effects were abolished by atropine (10-6 M) and mimicked by acetylcholine chloride (10 nM). In order to minimize an atrial contribution to the force production a more reduced preparation was used and ganglion clusters at the cavo-atrial junction were stimulated electrically (n = 4 animals). Similar negative inotropic and chronotropic effects sensitive to hexamethonium were seen. After hexamethonium administration, positive inotopic and chronotropic effects were uncovered and these were abolished by atenolol (0.1 mg %). Methylene Blue staining of the preparation at the end of the experiment showed the presence of ganglion cells at the sites of stimulation. Ganglion clusters were never seen on the venae cavae per se. The results of this investigation show that the vagus has a powerful action on the venae cavae resembling that on the atria and mediated by acetylcholine. Experimental Physiology (2003) 88.3, 329-334.

The aim of our study was to develop and validate a simple surgical model in the sheep which allows control of the gas composition of the blood supplying the carotid and central chemosensitive area independently of the rest of the body. This approach was made possible due to the specific features of the cranial circulation in the sheep. An extracorporeal circuit, consisting of a pump and a gas exchanger, was placed at the level of the two common carotid arteries to create a pressure gradient between the carotid and the systemic systems and to reverse blood flow in the vertebral vessels via the occipital arteries. When a pressure gradient of about 40 Torr was created between the systemic and carotid circulation, we found that no blood could reach the carotid bodies and the medulla without passing though the extracorporeal circulation. This was established (1) by measuring vertebral blood flow; and (2) by injecting either a coloured suspension or particles labelled with 99m*Tc into the systemic or the carotid circulation. The slope of the relationship between minute ventilation (V˙E) and systemic arterial PCO2 (Pa,CO2) during high CO2 inhalation in seven hyperoxic vagotomised and anaesthetised sheep was dramatically reduced, but remained above zero, when Pa,CO2 was maintained constant in the cephalic circuit (0.11 ± 0.15 vs. 0.70 ± 0.35 l min-1 Torr-1 for the control tests). This residual V˙E response to CO2 inhalation remains to be explained since it could not be accounted for by any of the chemical or circulatory changes occurring in the cephalic circulation. Nevertheless, this preparation provides an easy method of maintaining chemical and circulatory homeostasis at the chemoreceptor level. Experimental Physiology (2003) 88.5, 581-594.