Exposure of cardiac muscle to metabolic poisons reduces the availability of cellular ATP and cardiac dysfunction ensues. In this study rat ventricular myocytes were exposed to 2-deoxyglucose, iodoacetate and cyanide to induce complete metabolic blockade. Changes in contraction, cytosolic Ca2+ and pH were determined during metabolic blockade and following restoration of mitochondrial ATP production. Metabolic blockade resulted in a rapid failure of contractions and Ca2+ transients, a rise of diastolic Ca2+, a cytosolic acidosis and ultimately a rigor contracture. Washing out cyanide during the development of the rigor contracture led to a rapid relaxation of the contracture, a fall in cytosolic Ca2+ and a rapid, partial reversal of the cytosolic acidosis. The partial reversal of the cytosolic acidosis and fall of cytosolic Ca2+ were abolished in the presence of oligomycin. This suggests that the rapid partial recovery of cytosolic acidosis could result from the rephosphorylation of ADP to ATP by the mitochondrial F1,F0-ATPase (a reaction that consumes protons).

There is a great need for closer integration between physiologists and molecular biologists. These two research fields might soon form a single research discipline under the umbrella of molecular physiology. One of the areas where this interaction may be particularly fruitful is with somatic gene transfer using replication-deficient viral vectors. We applied this approach to study the central control of the cardiovascular system at the level of the nucleus of the solitary tract (NTS). We assess critically this new methodology as applied to experiments in an integrative environment (such as the whole animal). The usefulness of in vivo gene transfer is illustrated by an experiment where viral gene delivery helped to circumvent the problem imposed by an absence of a specific pharmacological blocker of the enzyme, endothelial nitric oxide synthase (eNOS). The pros and cons of using adenoviral vectors as opposed to conventional pharmacological approaches are discussed. We conclude that the use of adenoviruses to manipulate genes offers a new avenue for physiologists studying neuronal mechanisms in integrative models.

Growing lambs were fed the same diet at intakes supporting mean live weight gains of 0·1, 0·2 and 0·3 kg day-1, representing slow, intermediate and fast growth groups, respectively. The effects on bone growth and composition, and on blood and urinary bone marker concentrations or excretion rates were monitored. Compared with the slow-growing lambs, the higher intake group grew twice as fast, had higher rates of bone growth (indicated by external metatarsal length), and larger and heavier bones at slaughter. Bones from fast-growing animals had higher collagen and deoxypyridinoline concentrations, and lower Ca:collagen, Ca :P and pyridinoline : deoxypyridinoline ratios, indicating a less mature bone compared with the slow-growing lambs. Bone growth rate had no effect on plasma osteocalcin, bone-specific alkaline phosphatase or growth hormone concentrations, nor on the urinary excretion of pyridinoline and deoxypyridinoline. The results for plasma markers may be explained by an increase in blood volume linked with increased body weight.

Previous studies have suggested that the optimum length for force generation of the parasternal intercostal (PS) muscles is well above functional residual capacity (FRC). We further explored this issue by examining the pressure-generating capacity of the PS muscles as a function of lung volume in anaesthetized dogs. Upper thoracic spinal cord stimulation (SCS) was used to electrically activate the PS muscles. Changes in airway pressure and parasternal resting length (LR) during airway occlusion were monitored over a wide range of lung volumes during SCS. To assess the effects of parasternal contraction alone, SCS was performed following phrenicotomy and section of the external intercostal, levator costae and triangularis sterni muscles. With increasing lung volume, there were progressive decrements in the capacity of the PS muscles to produce changes in airway pressure. The relationship between PS pressure generation and lung volume was similar to a previous comparable assessment of the external intercostal muscles. The PS muscles shortened during passive inflation and also shortened further (by > 20 % of LR) during SCS. Total shortening (passive plus active) increased progressively with increasing lung volume. Our results indicate that the capacity of the PS muscles to produce changes in airway pressure (a) falls progressively with increasing lung volume and (b) is similar to that of the external intercostal muscles. We speculate that the fall in PS pressure-generating capacity is related, in part, to progressive reductions in end-inspiratory length. Experimental Physiology (2000) 85.3, 331-337.

The length of cooperative units along the thin filament of rabbit psoas single muscle fibres was determined by reducing filament length by treatment with the thin filament severing protein, gelsolin, in the presence of Ca2+ and 2,3-butanedione 2-monoxime (BDM). The average time for 50% reduction in isometric tension was 6.7 min at 22°C. The pCa–tension relationship was measured at 22°C, pH 7.00 and ionic strength 200 mM, and the data were fitted to the Hill equation to determine the half-saturation point (K) and the cooperativity (n). Our results demonstrate that the cooperativity does not change much when the remaining isometric tension was in the range 20–100%. The cooperativity quickly diminished when the remaining tension was reduced to less than 20%. Our results further demonstrate that the change in the pK value was minimal and averaged 0.075 (less Ca2+ sensitive) as the thin filament length was reduced. We infer from the first observation that the thin filament cooperativity extends up to 0.2 μm, which includes the maximum of about four basic cooperative units consisting of seven actin molecules, one tropomyosin dimer and one troponin complex. We infer from the second observation that the Ca2+ sensitivity is slightly reduced by removal of the cooperative interaction between neighbouring cooperative units.

Dilator products of nitric oxide synthase (NOS) and cyclooxygenase (COX) may contribute to the low normal pulmonary artery pressure (Ppa). In isolated perfused lungs of ferrets, rabbits and rats we investigated this hypothesis by blockade of NOS with L-NAME (L-nitro-arginine methyl ester) and COX with meclofenamate. There were species differences. Inhibition of either enzyme caused little rise in Ppa in ferrets and rats but inhibition of both enzymes caused huge increases in Ppa. We suggest this might be due to intracellular connections between the excitatory pathways for NOS and COX dilators, such that inhibition of one enzyme leads to activation of the other. Impairment of these endothelial-based enzymes in pulmonary vascular disease might lead to severe pulmonary hypertension. By contrast, in rabbits, comparable doses of L-NAME lead to large rises in Ppa which were reversed rather than amplified by COX blockade. NO seems to protect against some pressor/oedema forming product of COX in this species.

L-Arginine transport by the fetal side of human placenta was investigated through the characterization of L-[3H]arginine uptake in isolated perfused cotyledon. Competitive inhibition experiments suggest the presence of at least two transport systems: a Na+-independent, pH-insensitive system inhibitable by cationic amino acids, similar to system y+, and a Na+-dependent system which recognizes both cationic and neutral amino acids only in the presence of Na+, i.e. a Bo,+-like system. The kinetic analysis of L-arginine uptake in the presence of Na+ revealed that the process is mediated by saturable components: a high-affinity system (Km = 167 ± 18·0 μM; Vmax = 0·174 ± 0·012 μmol min-1) and a low-affinity carrier (Km = 980 ± 112 μM; Vmax = 1·60 ± 0·12 μmol min-1). In the absence of Na+, L-arginine uptake was fitted by one model with a Michaelis-Menten constant of 200 ± 24·8 μM. These results suggest that the high-affinity component corresponds to the Na+-independent system y+, whilst the low-affinity system may represent the activity of the Na+-dependent Bo,+ transporter. Kinetic studies in placentae taken from aspirin-treated pregnancies showed that L-arginine is transported with a significantly higher affinity (Km = 42·5 ± 5·7 μM), but with a lower capacity (Vmax = 0·064 ± 0·003 μmol min-1) than in the non-treated group. The latter finding suggests that aspirin would facilitate the uptake of the NO precursor only at very low arginine concentrations.

One way to identify central cardiovascular neurons is to consider their barosensitivity, for example by plotting changes in their single unit activity as a function of evoked changes in blood pressure. To simplify the genesis of such pressure-activity relationships, a coronary angioplasty catheter was threaded into the aorta of anaesthetized rats and the balloon inflated to raise the blood pressure mechanically. Here, such a procedure is exemplified for cardiac vagal motoneurons in the medulla oblongata in eight rats. This simplification bypasses potentially problematic surgery and minimizes deterioration of the animal. Experimental Physiology (2002) 87.5, 535-538.

We investigated the effects of sepsis, through the lipopolysaccharide (LPS)-induced inflammatory response, on plasma corticosterone and prolactin (PRL) levels during acute immobilization stress in normal and thyroidectomized rats. Thyroidectomized (TX) or sham-operated (N) rats were subjected to 120 min of immobilization stress. Rats were treated with an intraperitoneal injection of either LPS (250 µg (100 g body wt)-1) or the same volume of vehicle (saline solution), 90 min before the induction of stress. Blood samples for hormone assays were collected before sepsis and stress induction for baseline measures (-90 min), and during sepsis and immobilization stress for the measurement of prolactin and corticosterone levels by radioimmunoassay. Our results show that the thyroid hormones are necessary for a proper response of PRL and corticosterone release during immobilization stress. Although sepsis enhanced PRL secretion, this was not true of corticosterone release in either group of rats. Low levels of thyroid hormones partially block the release of PRL, but do not block corticosterone secretion during sepsis. Experimental Physiology (2003) 88.6, 755-760.

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.

We investigated the permeability of Cs+ and Na+ through various ion channels in rat atrial myocytes using the whole-cell voltage-clamp technique. With isotonic CsCl (140 mM) on both sides of the membrane and nominally [Ca2+]o-free conditions, depolarising clamp pulses induced an increase of outward currents which showed a biphasic time course. Repolarisation to the holding potential induced inward tail currents. With isotonic NaCl, depolarisation also induced outward currents which showed a monotonic decay, but inward tail currents were not observed. Both in NaCl and CsCl, currents were hardly affected by TEA (10 mM), 4-AP (5 mM) and DIDS (100 µM). Nicardipine (1 M) almost completely blocked time-dependent outward currents in isotonic NaCl solution, leaving only time-independent currents which showed linear I-V relationship. In isotonic CsCl conditions, nicardipine blocked outward current considerably, but there still remained time-dependent outward currents and inward tail currents. Addition of E-4031 (2-20 M) which is known as a specific blocker of the rapidly activating delayed rectifier K+ current (IKr) completely blocked these time-dependent outward and inward currents, leaving only a time-independent current. Time-independent currents recorded in the presence of nicardipine and E-4031 were inhibited by GdCl3, which is known to block non-selective cation (NSC) currents. From these results, it was suggested that NSC current in atrial myocytes can be investigated in isotonic Cs+ or Na+ solution in the presence of Ca2+ channel and IKr blockers.

We investigated the effects of glucose 6-phosphate (G6P) on skeletal muscle contractile apparatus and sarcoplasmic reticulum (SR) function. Using rat extensor digitorum longus fibres, the presence of 5 mM G6P decreased the Ca2+ sensitivity of both force production and actomyosin ATPase (AM-ATPase) activity. Conversely, maximal Ca2+-activated force was unaffected while maximal AM-ATPase activity was increased by 37 %. In SR vesicles isolated from rat gastrocnemius, G6P markedly altered Ca2+ handling. It increased Ca2+-stimulated Ca2+-ATPase activity but depressed the net rate of Ca2+ uptake. This latter effect appears to be due to G6P-stimulated Ca2+ release. When G6P was added to Ca2+-loaded vesicles, a small, transient release of Ca2+ was elicited. In addition, G6P lowered the threshold for Ca2+-induced Ca2+ release but depressed the net rates of both AgNO3- and caffeine-induced releases. It is possible that the accumulation of G6P during muscular activity may adversely affect muscle force production and contribute to the fatigue process via its action on the contractile apparatus and SR.

Potassium transport was measured in isolated chicken colonocytes using 85Rb+ as a tracer for K+. Rb+ was determined by atomic absorption spectrometry. The results revealed that net K+ uptake occurred via at least four mechanisms: (i) Na+,K+-ATPase, (ii) K+-ATPase, (iii) Na+-K+-2Cl- cotransport system and (iv) a mechanism(s) which is resistant to both ouabain and bumetanide. The rate of K+(Rb+) efflux is stimulated by the calcium ionophore A23187, inhibited by either quinine, verapamil or Ba2+, and unaffected by either apamin, 3,4-diaminopyridine (3,4-DAP), H2-DIDS or bumetanide. The A23187-induced increase in K+(Rb+) efflux was abolished by apamin. These findings suggest that K+(Rb+) efflux from chicken colonocytes occurs at least in part through Ca2+-activated K+ channels. The present results also show that all these K+ transport systems are involved in cell volume regulation. Thus, external hyposmolarity decreased net K+(Rb+) uptake mediated by Na+,K+-ATPase, K+-ATPase and the Na+-K+-2Cl- cotransporter and increased K+(Rb+) efflux rate. The opposite was observed under hyperosmotic conditions.

The effects of carbonic anhydrase inhibition on the responsiveness to CO2 of pressure-sensitive laryngeal receptors were examined in anaesthetised, paralysed cats. Laryngeal CO2-sensitive receptors from the superior laryngeal nerve were selected by their responsiveness to intralaryngeal pressure and to perfusion of solution equilibrated with 9 % CO2. The carbonic anhydrase inhibitor, methazolamide, when given intralaryngeally at 10-4 M, diminished or abolished the responses to the CO2-equilibrated solution in four of six pressure-sensitive receptors. Histochemical staining for carbonic anhydrase activity showed that the larynges perfused with methazolamide had diminished carbonic anhydrase activity, especially on the superficial layers of surface epithelium. Compared to untreated controls, when given intravenously (50 mg kg-1) methazolamide diminished or abolished the responses to CO2 of five of the six fibres studied. Histochemical staining of these larynges showed no carbonic anhydrase reactivity at the sites of laryngeal receptors. These results suggest that the responses to CO2 of laryngeal pressure-sensitive receptors are dependent on the presence of carbonic anhydrase. Inhibition of laryngeal receptor carbonic anhydrase activity by methazolamide is more reliably achieved by systemic rather than by luminal administration. Experimental Physiology (2001) 86.5, 641-649.

The mechanisms which underlie the inotropic actions of phorbol dibutyrate (PDBu), a synthetic compound which can directly activate protein kinase C (PKC), were investigated in guinea-pig isolated ventricular myocytes. Exposure of cells to PDBu (10-7 M) reduced myocyte contraction amplitude to 46 ± 3 % of control (n = 8; P < 0.05) with an associated shortening in action potential duration (action potential duration at 90 % repolarisation (APD90) was reduced to 83 ± 1 % of control; P < 0.05). The negative inotropic actions of PDBu and the shortening in action potential duration were abolished in the presence of a selective PKC inhibitor, Ro31-8220. Calcium transients (constructed from calcium-activated tail currents following interruption of action potentials by voltage clamp to -70 mV) were reduced following exposure to 10-7 M PDBu by 38 ± 2 % (n = 9, P < 0.05). L-type calcium currents were not significantly altered following exposure to 10-7 M PDBu (98 ± 2 % of control; P > 0.05). In contrast, delayed rectifier potassium currents (IK) were enhanced to 154 ± 8 % of control (n = 7; P < 0.05) by 10-7 M PDBu. This enhancement of IK may contribute to the observed shortening in action potential duration observed following exposure to PDBu under the conditions of our experiments. When the action potential configuration was maintained throughout the experiment by applying a voltage-clamp waveform, 10-7 M PDBu still reduced contraction amplitude to 57 ± 3 % of control (P < 0.05). Exposure to 10-7 M PDBu also suppressed spontaneous activity (both spontaneous potential fluctuations induced by the β-adrenergic agonist isoprenaline (40 nM), and transient inward currents induced by the cardiac glycoside ouabain (1 µM) under voltage clamp). It therefore appears that the reduction in myocyte contraction amplitude induced by exposure to PDBu may result in part through mechanisms independent of action potential shortening, which may include direct actions of protein kinase C on the function of the sarcoplasmic reticulum (SR) calcium store and/or on contractile proteins (though action potential shortening would be expected to cause a further decrease as a consequence of reduced calcium loading of the SR). The reduction of spontaneous activity caused by PDBu may also result from changes in the function of the SR store mediated by protein kinase C. Experimental Physiology (2001) 86.5, 561-570.

Investigations of intestinal secretion are often performed under anaesthesia. This study evaluates the influence of anaesthetic agents on the intestinal secretion induced by cholera toxin (CT) in the pig. CT was instilled for 4 h in ligated jejunal loops under anaesthesia with halothane, saffan, [alpha]-chloralose, or propofol. Cardiovascular parameters, blood gas data, plasma cortisol levels, net fluid accumulation, intraluminal mediators (serotonin (5-HT), prostaglandin E2 (PGE2)) and electrolyte concentrations in the accumulated fluid were determined. The systolic blood pressure and heart rate was highest for saffan-anaesthetized pigs (blood pressure: saffan > [alpha]-chloralose > propofol = halothane; heart rate: saffan > [alpha]-chloralose = propofol = halothane), while blood gases and cortisol levels were within the same range. CT induced a dose-dependent fluid accumulation under all four anaesthetics. The fluid accumulation was significantly higher in pigs treated with saffan, [alpha]-chloralose and propofol than in halothane-treated pigs (saffan = [alpha]-chloralose > propofol > halothane). There was no significant difference in electrolyte concentrations in the accumulated fluid or in the luminal content of 5-HT and PGE2 between anaesthetics. The results demonstrate that anaesthetic agents profoundly influence the secretory response in the small intestine and indicate the importance of the choice of anaesthetic in this type of experiment.

To elucidate the role of corticotrophin-releasing factor (CRF) in the spontaneous rhythmic contractions of pregnant myometrium, isometric contractions of longitudinal myometrial preparations taken from Sprague-Dawley rats (18-21 days gestation) were measured. CRF (0·03-30 nM) significantly increased the amplitude (117 ± 4 % of control at 30 nM, mean ± S.E.M.) and significantly decreased the frequency (74 ± 5 %) in a concentration-dependent manner. The area under the tension curve did not increase (89 ± 8 %). Such effects of CRF were not observed in preparations pretreated with the RP diastereomer of adenosine cyclic 3',5'-phosphorothioate (Rp-cAMPS, 10 µM), an inhibitor of protein kinase A. In preparations pretreated with indomethacin (1 µM), CRF caused little change in the amplitude but significantly decreased the frequency of the contractions. Intracellular cAMP levels in the preparations, measured by enzyme immunoassay, rose when CRF was applied at 30 nM. These results suggest that the slight positive inotropic action of CRF is due to activation of both cAMP and prostaglandin pathways, whereas the negative chronotropic action is due solely to the cAMP signalling pathway.

The effects of exercise training were investigated on the vascular responses in the isolated guinea-pig saphenous artery. Exercising animals swam 5 days week-1 for 6 weeks (60 min day-1 for weeks 1 and 2; 75 min day-1 for weeks 3 and 4; 90 min day-1 for weeks 5 and 6), while control animals were placed into shallow water for the same duration. Trained animals had significantly higher ventricular:body weight ratios, increased citrate synthase activity in the latissimus dorsi, and enhanced Na+ pump concentrations in the latissimus dorsi and gastrocnemius muscles (P < 0.05). In vitro isometric techniques were used to measure constriction and relaxation responses of saphenous artery rings from trained and control animals. There were no significant differences in the constriction responses to KCl (50 mm) and phenylephrine (0.3-100 µM) in arterial rings from control versus trained animals. Relaxation responses to acetylcholine (10 µM; ACh-relaxation), following preconstriction with phenylephrine (10 µM), were significantly enhanced in rings from trained animals (P < 0.05). Acetylcholine relaxed the vessels to 47 ± 6 % (control) and 18 ± 3 % (trained) of the preconstriction responses to phenylephrine. The nitric oxide synthase inhibitor N G-nitro-L-arginine (L-NA; 50 µM) significantly attenuated the ACh-relaxation in control and trained animals (P < 0.05). The effect of L-NA on the ACh-relaxation was significantly larger in trained (change in ACh-relaxation with L-NA = 29 ± 9 %) than control (14 ± 3 %) animals (P < 0.05). In conclusion, exercise training enhanced the ACh-relaxation of the isolated guinea-pig saphenous artery. Inhibition of nitric oxide synthase attenuated the ACh-relaxation of rings from control and trained animals, but this effect was significantly larger in the vessels from trained animals. These results are consistent with the idea that nitric oxide could contribute to the enhanced ACh-relaxation of the saphenous artery with exercise training.

The shortcomings of diffusion as a mechanism of glucose transfer across cell membranes date back to the last century. The early work, mostly qualitative, was greatly extended by LeFevre in America and by Wilbrandt in Switzerland, who reported their quantitative studies at the Bangor Symposium of The Society of Experimental Biology in 1953 (LeFevre, 1954; Wilbrandt, 1954). Widdas (1952) published simple carrier kinetics for glucose transfer which predicted the phenomenon of uphill transfer by counterflow. However, in that 1952 paper it was carefully pointed out that a good fit to the kinetics did not define the details of the mechanism. Nevertheless, the reverse is true: namely, any viable scheme of working must be consistent with the established kinetic properties. For instance the kinetics of uphill transfer by counterflow (confirmed four years later by Park, Post, Kalman, Wright, Johnson & Morgan, 1956; Rosenherg & Wilbrandt, 1957) clearly exclude a simple water channel through the membrane.The concept of a mobile membrane carrier on which the early kinetics were based was variously modified to meet newer developments, which included asymmetric affinities on the outside and inside of the red cell membrane. The mobile carrier has now been superseded by the concept of membrane protein transporters. Nevertheless, some of the nomenculture, such as carrier transport and carrier catalysis (Krämer, 1994), has persisted although it no longer conveys all the subtleties of the transfer mechanism.

This study seeks to confirm the progressive changes in cardiac output and heart rate previously reported with 8 h exposures to constant hypoxia, and to examine the role of sympathetic mechanisms in generating these changes. Responses of ten subjects to four 8 h protocols were compared: (1) air breathing with placebo; (2) isocapnic hypoxia (end-tidal PO2 = 50 mmHg) with placebo; (3) isocapnic hypoxia with beta-blockade; and (4) air breathing with beta -blockade. Regular measurements of heart rate and cardiac output (using ultrasonography and N2O rebreathing techniques) were made with subjects seated in the upright position. The sensitivity of heart rate to rapid variations in hypoxia (GHR) and heart rate in the absence of hypoxia were measured at times 0, 4 and 8 h. No significant progressive effect of hypoxia on cardiac output was detected. There was a gradual rise in heart rate with hypoxia of 11 ± 2 beats min-1 in the placebo protocol and of 10 ± 2 beats min-1 in the beta-blockade protocol over 8 h, compared to the air breathing protocols. The rise in heart rate was progressive (P < 0.001) and accompanied by progressive increases in both GHR (P < 0.001) and heart rate measured in the absence of hypoxia (P < 0.05). No significant effect of beta-blockade was detected on any of these progressive changes. We conclude that sympathetic mechanisms that act via beta -receptors play little role in the progressive changes in heart rate observed over 8 h of moderate hypoxia. Experimental Physiology (2000) 85.5, 557-565.