The actions of arachidonic acid (AA) were investigated in guinea-pig isolated ventricular myocytes. Exposure of myocytes to 10 µM AA reduced the amplitude of contractions and calcium transients accompanying action potentials at a frequency of 1 Hz. AA (10 µM) also reduced the amplitude of calcium currents recorded under voltage-clamp conditions. The suppression of contraction by AA was not prevented by either 10 µM trihydroindomethicin (to inhibit cyclo-oxygenase) or 10 µM ETYA (5,8,11,14-eicosatetraynoic acid, to inhibit AA metabolising enzymes), showing that the actions of AA appeared not to be mediated by these metabolites. The reduction of contraction by 10 µM AA was also not prevented by the protein kinase C inhibitor, Ro31-8220 (1 µM), showing that this pathway appeared not to be required for the observed effect. Direct effects of AA may be involved. A further action of 10 µM AA was to suppress spontaneous electrical activity induced by either the β-adrenergic agonist isoprenaline or the Na+ pump inhibitor, ouabain. This effect of AA on spontaneous activity might be associated with the observed reduction of calcium entry through L-type calcium channels, although additional effects of AA on calcium release from the sarcoplasmic reticulum might also be involved. Experimental Physiology (2001) 86.4, 437-449.

Effects of prostaglandin E2 (PGE2) on exocytosis of mucin were studied in mucous cells isolated from guinea-pig antrum using video-microscopy. Stimulation with PGE2 elicited a sustained increase in the frequency of exocytotic events in a dose-dependent manner, which was under regulation by both Ca2+ and cAMP. Stimulation with a selective prostanoid EP4 receptor agonist (ONO-AEI-329, 10 µM), which activates cAMP signals, elicited a sustained increase in the frequency of exocytotic events (30 % of that evoked by 1 µM PGE2). Stimulation with an EP1 agonist (17-P-T-PGE2, 1 µM), which activates Ca2+ signals, increased the frequency of exocytotic events to a lesser extent (5 % of that evoked by 1 µM PGE2), while addition of an EP1 antagonist (ONO-8713, 10 µM) decreased the frequency of exocytotic events (approximately 40 % of that evoked by 1 µM PGE2). However, addition of the EP1 agonist potentiated the frequency of exocytotic events evoked by the EP4 agonist or forskolin (which elevates cAMP levels) and increased the sensitivity of the exocytotic events to forskolin. These results suggest that the Ca2+ signal activated via the EP1 receptor potentiates the cAMP-regulated exocytotic events activated via the EP4 receptor during PGE2 stimulation, by increasing the sensitivity of the exocytotic response to cAMP. In conclusion, exocytotic events in PGE2-stimulated antral mucous cells were regulated by interactions between EP1 and EP4 receptors. Experimental Physiology (2001) 86.4, 451-460.

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

In vitro evidence suggests that the 'recycling' of K+ ions through luminal K+ channels in the thick ascending limb of the loop of Henle (TALH) is essential for the normal operation of the luminal Na+-K+-2Cl- co-transporter. In the present study these channels were investigated in vivo by perfusing superficial loops of Henle in anaesthetised rats with and without the K+ channel blocker barium. Using a standard perfusate, intraluminal barium (5 mmol l-1) reduced sodium reabsorption (JNa) from 1887 ± 50 to 1319 ± 53 pmol min-1 (P < 0.001). When the experiment was repeated using a low-Na+ perfusate, designed to inhibit reabsorption in the pars recta (the initial segment of the loop of Henle), a similar reduction in JNa was observed (from 698 ± 47 to 149 ± 23 pmol min-1, P < 0.001), strongly suggesting that the effect of barium is localised to the TALH. The magnitude of the reduction in JNa during blockade of K+ channels confirms the importance of K+ recycling in facilitating Na+ reabsorption in the TALH in vivo. However, the reduction in JNa was not associated with a fall in the K+ concentration of the fluid collected at the early distal tubule. When bumetanide, an inhibitor of the Na+-K+-2Cl- co-transporter, was included in the low-Na+ perfusate, net K+ secretion was observed. Addition of barium to this perfusate reduced, but did not abolish, the secretion, suggesting that bumetanide-induced K+ secretion results partly from paracellular transport. Experimental Physiology (2001) 86.4, 469-474.

In the presence of the muscarinic blocker atropine, the α-adrenoceptor blocker phentolamine and the β-adrenoceptor blocker propranolol (2 mg kg-1 of each, I.P.), the numerical density of parotid acinar secretory granules was reduced by 32 % in response to ascorbic acid (0.5 M) applied on the tongue every 30 s over 30 min in awake rats. This non-adrenergic, non-cholinergic (NANC) response was entirely dependent on an intact auriculo-temporal nerve supply. The NANC mechanisms were found to be potentially responsible for almost all of the exocytotic response that occurs in the absence of the three autonomic receptor blockers. No sympathetic contribution to the exocytotic response was found and furthermore, studies in parasympathetically denervated glands showed that the sympathetic contribution to the salivary flow was small. Experimental Physiology (2001) 86.4, 475-480.

The renal excretory responses to volume expansion (VE), by 10 % body wt, were determined in groups of anaesthetised streptozotocin-induced diabetic rats with one denervated and one innervated kidney in the presence and absence of nitric oxide synthase (NOS) inhibitors. VE in diabetic rats increased (P < 0.001) cumulative urine sodium excretion (CuUNaV) to 104 ± 9 and 69 ± 6 µmol min-1 (g kidney wt)-1 in the denervated and in the innervated kidneys, respectively, which were both less (P < 0.001) than in the non-diabetic rats, at 225 ± 14 and 148 ± 14 µmol min-1 (g kidney wt)-1, respectively, in the denervated and the innervated kidney. The non-selective NOS inhibitor, NG-nitro-L-arginine-methyl-ester (L-NAME) given to the diabetic rats with intact renal innervation enhanced CuUNaV after VE by 43 % (P < 0.001), while the combination of L-NAME and renal denervation restored CuUNaV to a value comparable to that of non-diabetic rats. In diabetic rats treated with either a relatively selective inhibitor for the neuronal isoform of NOS, 7-nitroindazole, or a relatively selective inhibitor for the inducible isoform of NOS, aminoguanidine, CuUNaV after VE was similar to the untreated diabetic rats irrespective of whether or not the renal nerves were present. This investigation demonstrated that NO production contributed, at least partly, to the depressed ability to excrete a saline load in diabetes mellitus. The endothelial isoform of NOS was most probably responsible for generating NO which caused the blunted excretory responses. The ability of NO to attenuate the excretory responses to volume expansion was an action independent of the renal innervation status. Experimental Physiology (2001) 86.4, 481-488.

In a previous report, it has been shown that water deprivation significantly affects the two-bottle taste preferences and one-bottle taste acceptance in rats when no food was available during tests. Since no food was available, the course of drinking was never interrupted by eating. Theoretically, if a rat faces a simultaneous choice between food and fluid, and if the course of drinking is interrupted by eating, these conditions might interfere with taste preferences, total fluid intake and eating in thirsty rats. The aims of the present experiments were: to ascertain whether food intake during both two-bottle preference and one-bottle acceptance tests in thirsty rats might be influenced by the palatability of the solutions; to verify whether the availability of food during tests influences taste preference and acceptance, and total fluid intake; to detect variations induced by dehydration on body weight and some plasma and urinary parameters that might interfere with food and fluid intake, taste preference and acceptance. Using naive rats, five groups of rats showing the same taste preferences for one of four prototypical tastes and water were selected. Then, both two-bottle preference (Expt 1) and one-bottle acceptance tests (Expt 2) were performed in rats deprived of water for either 12, 24, 36 or 48 h. The results showed that in both Expt 1 and Expt 2, inhibition of feeding and decrease of body weight during dehydration was very similar in all rats. The presence of food during the tests did not affect taste preference and acceptance. During Expt 1, after severe water deprivation (36 and 48 h), food intake was related to the palatability of the solution paired with water. When rats drank either NaCl or sucrose, they ate less food than rats drinking HCl, quinine, or water. In Expt 2, rats drinking NaCl solution as the only source of fluid ate significantly less food than all other groups. The intake of sucrose and/or NaCl solutions be may explained by two different post-ingestion effects (energetic and osmotic). Since rats drinking either sucrose or NaCl ate less food but drank more fluid, they had a significantly higher fluid/food intake ratio than that of rats who drank water, quinine, or HCl, who ate more food but drank less fluid. The increase of the fluid/food intake ratio in rats drinking sucrose or NaCl was directly correlated with the length of dehydration. Self-denial of food during dehydration may be responsible for overeating and overdrinking during the recovery period after tests. After dehydration lasting for 24 and 48 h, plasma [Na+], [protein], osmolality and haematocrit values increased but [K+] decreased. Urinary volume decreased but urinary [Na+] increased. These results are related to food and fluid intake, taste preference and acceptance after dehydration periods. Experimental Physiology (2001) 86.4, 489-498.

We determined the interaction of diet and training on metabolic adaptations in skeletal muscle and liver, and the consequences of these adaptations for endurance. Eighty rats performed a baseline treadmill run to exhaustion at 16 m min-1 (RUN1) and were then divided into two groups and given one of two diets: high carbohydrate (CHO) or high fat (FAT). Each dietary group was then divided into one of four subgroups: sedentary control that performed no training (NT); low-intensity running (8 m min-1; LOW) and two groups who trained at their maximal voluntary running speed without electrical stimulation (28 m min-1; VMAX). Training volume was identical for LOW and VMAX (1000 m session-1) and animals ran 4 days week-1 for 8 weeks. To assess the interaction of the higher intensity exercise with diet, a second endurance test (RUN2) was undertaken after 6 weeks at either 16 m min-1 or 28 m min-1. The NT group ran for a longer duration (increase of 77 %) after FAT than CHO (239 ± 28 vs. 135 ± 30 min, P < 0.05) at 16 m min-1. There were no differences in RUN2 for the LOW group when rats ran at 16 m min-1 (454 ± 86 vs. 427 ± 75 min for CHO and FAT groups, respectively), but rats in the VMAX group fed FAT ran longer than rats fed CHO at 28 m min-1 (100 ± 28 vs. 58 ± 11 min, respectively, P < 0.05). FAT increased the activities of the enzymes citrate synthase, β-hydroxyacyl-CoA dehydrogenase and carnitine palmitoyl-transferase compared to CHO (P < 0.01), but there was no systematic effect of training. We conclude: (1) there was no additive effect of a high-fat diet on endurance performance when rats performed low-intensity training; (2) running performance at 28 m min-1 was only enhanced by a high-fat diet after more intense training; (3) diet-induced and training-induced adaptations that increase exercise capacity may be under independent control. Experimental Physiology (2001) 86.4, 499-508.

In this study we tested the hypothesis that, compared to young adult rats, senescent rats have a reduced ability to respond to muscle unloading. Unloading of the muscles was induced by hindlimb suspension (HS) of young adult and senescent rats for 21 days. Plantaris muscles from young adult rats had significantly higher levels of myogenin mRNA and protein (890 % and 314 %, respectively, P < 0.05) than plantaris muscles from senescent rats and also a higher MyoD mRNA level (280 %, P < 0.05), but ageing did not increase MyoD protein levels. Although HS did not increase plantaris mRNA or protein levels of myogenin or MyoD in senescent rats (P = 0.22), myogenin mRNA and protein levels increased by 850 % and 580 % respectively, and MyoD mRNA and protein levels by 235 % and 1600 %, respectively in young adult rats (P < 0.05). Soleus muscles from senescent rats had 150 % and 85 % greater myogenin and MyoD mRNA levels, respectively (P < 0.05), than soleus muscles from young adult rats, whereas protein levels of myogenin were similar (P > 0.05) and MyoD protein levels were 60 % lower in the muscle of senescent rats (P < 0.05). In young rats, soleus muscle mRNA levels of myogenin and MyoD were not altered by HS but myogenin protein levels decreased by 57 % (P < 0.05) whereas MyoD protein levels increased by 187 % (P < 0.05). In senescent rats, HS decreased soleus muscle myogenin mRNA and protein levels by 42 % and 26 % respectively (P < 0.05), but MyoD protein and mRNA levels were not changed. MRF4 levels were not affected by ageing in either muscle. These data suggest that ageing reduces the ability of fast muscles to increase myogenin protein levels, and prevents both fast and slow muscles from increasing MyoD protein levels during muscle unloading. Experimental Physiology (2001) 86.4, 509-517.

Viscoelastic properties determine the dynamic behaviour of the arterial wall under pulsatile pressure and flow, suggesting time- or frequency-dependent responses to changes in wall stress and strain. The objectives of the present study were: (i) to develop a simplified model to derive simultaneously the elastic, viscous and inertial wall moduli; (ii) to assess Young's modulus as a function of frequency, in conscious, chronically instrumented dogs. Parametric discrete time models were used to characterise the dynamics of the arterial system based on thoracic aortic pressure (microtransducer) and diameter (sonomicrometry) measurements in control steady state and during activation of smooth muscle with the α-adrenoceptor agonist phenylephrine (5 µg kg-1 min-1, I.V.), in eight conscious dogs. The linear autoregressive model and a physically motivated non-linear model were fitted to the input-output (stress-strain) relationship. The aortic buffering function (complex Young's modulus) was obtained in vivo from the identified linear model. Elastic, viscous and inertial moduli were significantly increased from control state ((44.5 ± 7.7) × 104 Pa; (12.3 ± 4.7) × 104 Pa s; (0.048 ± 0.028) × 104 Pa s2 ) to active state ((85.3 ± 29.5) × 104 Pa, P < 0.001; (22.4 ± 8.3) × 104 Pa s, P < 0.05; (0.148 ± 0.060) × 104 Pa s2, P < 0.05). These moduli, obtained using the linear model, did not present significant differences compared with those derived using the non-linear model. In control conditions, the magnitude of the normalised complex Young's modulus was found to be similar to that reported in previous animal studies ranging from 1 to 10 Hz. During vascular smooth muscle activation, this modulus was found to be increased with regard to control conditions (P < 0.01) in the frequency range used in this study. The frequency-dependent Young's modulus of the aortic wall was obtained for the first time in conscious, unsedated dogs. The parametric modelling approach allows us to verify that vascular smooth muscle activation increases the elastic, viscous and inertial moduli with the advantage of being able to track their time evolution. Furthermore, under activation, the aortic wall remains stiff in the physiological frequency range, suggesting the impairment of the arterial buffering function. Experimental Physiology (2001) 86.4, 519-528.

This study examined the role of muscarinic parasympathetic mechanisms in generating the progressive increases in ventilation (V˙E) and heart rate previously reported with 8 h exposures to hypoxia. The sensitivities of V˙E (Gp) and heart rate (GHR) to acute variations in hypoxia, and V˙E and heart rate during acute hyperoxia were assessed in 10 subjects before and after two 8 h exposures to isocapnic hypoxia (end-tidal PO2 = 50 mmHg). The responses were measured during muscarinic blockade with glycopyrrolate (0.015 mg kg-1) and without glycopyrrolate, as a control. There were significant increases in Gp (P < 0.01) and V˙E during hyperoxia (P < 0.01) following hypoxic exposure, but these were unaffected by glycopyrrolate. GHR increased significantly by 0.29 ± 0.08 beats min-1 %-1 (mean ± S.E.M.) following exposure to hypoxia under control conditions, but only non-significantly by 0.10 ± 0.08 beats min-1 %-1 with glycopyrrolate. This difference was significant. Changes in heart rate during hyperoxia were slight and inconclusive. We conclude that muscarinic mechanisms play little role in the progressive ventilatory changes that occur over 8 h of hypoxia, but that they do mediate much of the progressive increase in heart rate. Experimental Physiology (2001) 86.4, 529-538.