Intense exercise or myocardial ischaemia can significantly increase both the concentration of extracellular potassium ([K+]o) and cardiac sympathetic nerve activity. Since changes in [K+]o modulate membrane currents involved in sino-atrial node pacemaking, in particular the voltage-sensitive hyperpolarization-activated current (If), we investigated whether raised [K+]o (from 4 mM to 8 or 12 mM) could directly affect the heart rate response to cardiac sympathetic nerve stimulation (SNS). In the isolated guinea-pig atrial-right stellate ganglion preparation, raised [K+]o significantly decreased the maximum diastolic potential, amplitude and maximum rate of rise of the upstroke of sino-atrial node pacemaker action potentials in 8 and 12 mM [K+]o (P < 0.05). At 12 mM [K+]o these effects were associated with significant decreases in baseline heart rate (4 mM [K+]o = 187 ± 5 beats min-1 (bpm); 12 mM = 144 ± 11 bpm; P < 0.05) and the heart rate response to SNS (1, 3 and 5 Hz; P < 0.05). A 10 % increase in the baseline heart rate with sympathetic activation (3 Hz) was associated with a significant enhancement of the slope of the pacemaker diastolic depolarization at 4 mM [K+]o (increased by 16 ± 6 %; n = 7; P < 0.05), but not with raised [K+]o. When the If current was blocked with 2 mM caesium (n = 8), 12 mM [K+]o had no effect on baseline heart rate and the heart rate response to 3 Hz SNS. The heart rate response to bath-applied noradrenaline (0.01-100 µM) was significantly attenuated by 12 mM [K+]o (at 4 mM [K+]o, EC50 = -6.31 ± 0.18; at 12 mM [K+]o, EC50 = -5.80 ± 0.10; n = 6, ANOVA, P < 0.05). In conclusion, extreme physiological levels of [K+]o attenuate the positive chronotropic response to cardiac sympathetic activation due to decreased activation of the If current. This is consistent with raised [K+]o protecting the myocardium from potentially adverse effects of excessive noradrenaline. Experimental Physiology (2001) 86.1, 19-25.

The present study was undertaken to explore the role of nitric oxide (NO) in the pathogenesis of experimental non-steroidal anti-inflammatory drug (NSAID)-induced gastropathy. We assessed the role of NO inhibition and donation in indomethacin-induced gastric mucosal dysfunction. The stomach was perfused with vehicle (control) for 20 min, followed by indomethacin (10 mg ml-1 in 1.25 % sodium bicarbonate, pH 8.4) for 120 min. NG-nitro-L-arginine methyl ester (L-NAME, 5 and 10 mg kg-1, I.V. bolus), L-arginine, D-arginine (100 mg kg-1I.V. bolus, 10 mg kg-1 h-1, 2 h infusion) and the NO donor glyceryl trinitrate (GTN) were given at the same time (20, 40 and 80 µg kg-1 min-1, 15 min infusion) as perfusion with indomethacin was started. Epithelial permeability was quantified by measuring blood-to-lumen clearance of 51Cr-labelled EDTA. Indomethacin caused a 20-fold increase in 51Cr-EDTA leakage compared with that of the control group. Treatment with L-NAME or L-arginine did not affect the indomethacin-induced alterations in mucosal permeability. Administration of GTN (20 µg kg-1 min-1) significantly reduced the indomethacin-induced mucosal dysfunction. By contrast, higher doses of GTN (80 µg kg-1 min-1) exacerbated epithelial dysfunction induced by indomethacin. Elevated levels of carbonyls and myeloperoxidase (MPO) observed after indomethacin administration were significantly reduced, to the control values, when GTN (20 µg kg-1 min-1) was administered along with indomethacin. These data suggest that NO from exogenous sources can exert a dual action on the integrity of the gastric mucosa challenged by indomethacin. Low doses of GTN can prevent mucosal dysfunction induced by indomethacin, while higher doses of GTN may exacerbate the increases in epithelial permeability.

The role of extracellular Ca2+ (Ca2+o) in the modulation of cardiac Cl- currents (ICl) such as those activated by cAMP or swelling is uncertain. The effects of Ca2+o and extracellular cadmium (Cd2+o) on Cl- currents in cultured chick cardiac myocytes were investigated in Na+- and K+-free internal and external solutions using the whole-cell patch-clamp technique. In the absence of Na+ and K+ internally and externally, the whole-cell current was predominantly ICl. In the absence of cAMP, removal of Ca2+o (+ 1 mM EGTA) resulted in an increase in the current that was suppressed by reduction of Clo- with a rightward shift of the zero-current potential towards the Cl- reversal potential. We designated this current as a Ca2+-inhibitable ICl. Addition of 0·5 mM Cd2+o with or without removal of Ca2+o also resulted in a 1·5- to 2·0-fold increase in ICl that was attenuated by 1 mM DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid). Under similar conditions, ICl activated by Cd2+o (in 1 mM Ca2+o solution) was not further increased by subsequent removal of Ca2+o, suggesting that addition of Cd2+o and removal of Ca2+o activated the same ICl. In contrast, exposure to 1 µM forskolin further enhanced ICl in the presence of Cd2+o. With 10 µM cAMP in the pipette solution, Ca2+-inhibitable ICl could be activated in myocytes that do not possess cAMP-activated Cl- channels, indicating that activation of Ca2+-inhibitable ICl does not require cAMP. In the presence of cAMP, in cells that display the cAMP-activated ICl, removal of Ca2+o resulted in a further increase in ICl, comparable to the Ca2+-inhibitable ICl. The Ca2+-inhibitable ICl was minimized when pipette solutions contained 1·5 µM Ca2+. These results suggest that removal of Ca2+o or application of Cd2+o activates a Ca2+-inhibitable ICl that is distinct from the cAMP-activated ICl.

We used the short-circuit current (Isc) and patch-clamp techniques to investigate the effects of methoxsalen (MTX) on the electrogenic Cl− secretion of the mouse jejunum. MTX stimulated a sustained increase in Isc that was dose dependent. Bumetanide inhibited MTX-stimulated Isc in a dose-dependent manner consistent with activation of Cl− secretion. MTX failed to stimulate Isc following maximal activation of the cAMP pathway by forskolin, but did increase Isc after a submaximal dose of forskolin. Glibenclamide, a blocker of the cystic fibrosis transmembrane conductance regulator (CFTR), reduced the MTX-stimulated increase of Isc by 59 ± 6%. The cAMP-dependent K+ channel blocker 293B did not alter the MTX-activated Isc; however, clotrimazole, an intermediate Ca2+-activated K+ channel (IKCa) blocker, reduced the MTX-stimulated Isc. MTX did not alter Na+–glucose cotransport across the mouse jejunum.In cell-attached membrane patches, MTX increased the open probability of the basolateral IKCa channel of isolated crypts. These data suggest that the CFTR and IKCa channels participate in the MTX-activated, sustained Cl− secretory response of the mouse jejunum.

Experiments were performed to investigate the nature of α- and β-adrenoceptors in blood vessels supplying the posterior capsule of the acutely inflamed rabbit knee joint, and results were compared to findings from previous experiments on the normal joint, to assess any alteration which may occur in the adrenoceptor profile due to the inflammation process. Electrical stimulation of the posterior articular nerve resulted in vasoconstriction which was reversed to vasodilatation by phentolamine and yohimbine. The dose-response curves to close intra-arterial injection of α-adrenoceptor agonists showed a rank-order potency of: adrenaline = phenylephrine = clonidine. The adrenaline dose-response curve was shifted to the right by administration of antagonists with a rank-order potency of: phentolamine = yohimbine = prazosin. At this stage of the experiments there was an equal response of α1- and α2-adrenoceptors in blood vessels of the acutely inflamed rabbit knee joint. In another group of animals the neurally mediated vasodilatation, which appeared after administration of phentolamine, was completely blocked by propranolol, and was reduced by about 50 % by atenolol. The dose-response curves to close intra-arterial injection of β-adrenoceptor agonists showed a rank-order potency of: isoprenaline > salbutamol = dobutamine. The isoprenaline dose-response curve was shifted to the right by administration of antagonists with a rank-order potency of: propranolol > atenolol. These experiments also showed an almost equal response of β1- and β2-adrenoceptors in blood vessels of the acutely inflamed rabbit knee joint. Overall, compared to previous experiments on the normal joint in which α2- and β1-adrenoceptor responses predominated, acute inflammation resulted in a shift from α2- towards α1- and from β1- towards β2-adrenoceptor responses. Experimental Physiology (2000) 85.3, 267-273.

Despite marked differences in both the extent of physical activity and in muscle metabolism and structure between tetraplegic and control subjects, the glycogen content in the skeletal muscle of both groups is similar. We determined whether this similarity could be explained by the activities of key enzymes of glycogen metabolism. Muscle biopsies were analysed for glycogen synthase (GS) and glycogen phosphorylase (GP) activities, as well as for metabolites. Glycogen content did not differ significantly between the two groups. Total glycogen synthase activity was reduced by almost 60 % in tetraplegics (P < 0.01), whereas total phosphorylase activity did not differ between groups. GS fractional activity did not differ between groups, whereas phosphorylase fractional activity (-/+ AMP) was significantly higher in the tetraplegics (0.08 ± 0.01, control; 0.25 ± 0.02, tetraplegics; P < 0.001). Neither uridine diphosphate (UDP)-glucose nor glucose 6-phosphate (G-6-P) content in muscle differed significantly between groups. These data demonstrate that, in tetraplegics, muscle glycogen content is preserved despite decreases in GS activity and increases in phosphorylase fractional activity. Muscle paralysis has differential effects on the activities of GS and GP. Experimental Physiology (2001) 86.2, 205-209.

Humans, after extensive ileal resection, frequently suffer from diarrhoea, which may be due to an increased delivery of deoxycholate (DOC) to the large intestine. In the frog skin the addition of DOC (0·5 mM) to the apical side induced the activation of amiloride-sensitive Na+ channels and an increase in the unidirectional Cl- fluxes. Here we used two established cell lines (A6 and Caco2) to study the effect of DOC on ion channels at cell and membrane level using the patch-clamp technique. In A6 cells subcultured directly on Petri dishes and studied in the whole-cell configuration, DOC induced an increase in cell conductance of 110·3 ± 4 pS pF-1 (N = 8) which was reduced to 89 ± 14 pS pF-1 (N = 8) by the addition of DIDS (0·5 mM). The absolute values of these two effects were not statistically different (P < 0·2). In Caco2 cells, the addition of DOC (0·5 mM) induced, after 1 min, an increase in cell conductance of 583 ± 16 pS pF-1 (N = 8) which was reduced to 560·4 ± 16 pS pF-1 (N = 8) by DIDS (0·5 mM) and N-phenylanthranilic acid (DPC; 0·5 mM). The two values were not statistically different (P < 0·4). In Caco2 cells subcultured under the same conditions, DOC induced an increase in cell conductance of 1710 ± 64 pS pF-1 (N = 6). Subsequent addition of amiloride (0·1 mM) reduced the cell conductance to 1558 ± 33 pS pF-1 (N = 6). These two mean values were statistically different allowing for an error of the second kind < 0·05. In cells in which DOC produced a conductance increase of 1010 ± 10 pS pF-1, gadolinium (0·5 mM) induced a fall in cell conductance of 1800 ± 10 pS pF-1. In Caco2 cells, addition of DOC (0·5 mM) to the bath reversibly induced the appearance of or an increase in channel activity in patches studied in cell-attached and excised inside-out configuration. In inside-out experiments (N = 13) DOC (0·5 mM) induced the appearance of channel activity with conductances and reversal potentials (Er) of 27·7 ± 1·9 pS and 0·8 ± 5·7 mV, respectively. In cell-attached patches (N = 13) these values were 24·9 ± 4·4 pS and -18·1 ± 6·4 mV. In excised inside-out patches from Caco2 cells, subjected to electrochemical gradients for Na+, K+ and Cl-, (+85, -85 and 0 mV, respectively), addition of DOC also induced an increase in the baseline conductance and a shift in the reversal potential from values around +25 mV to values around 0 mV. Bile salts activated both anionic and cationic channels and did not require the presence of intracellular factors for these effects. We suggest that they act at the membrane level.

The pathogenesis of chronic fatigue syndrome (CFS) remains unknown. Thresholds and latencies of motor evoked potentials (MEPs) in response to transcranial magnetic stimulation (TMS) are normal but intracortical inhibition has not been investigated. Eleven patients with CFS were compared with 11 control subjects. Each patient completed a questionnaire using visual analogue indices of pain, fatigue, anxiety and depression. Subjects released a button to initiate simple (SRTs) and choice reaction time (CRTs) tasks; for each task, movement times were measured between release of the initiation button and depression of a second button 15 cm away. Subjects held a 10 % maximum voluntary contraction in the thenar muscles of their dominant hand while TMS was applied to the motor cortex; the duration and extent of inhibition of surface electromyographic (EMG) activity were assessed at stimulus strengths above and below the threshold for MEPs. Patients had significantly (P < 0.05) higher mean indices of fatigue than of pain, anxiety or depression. Mean (± S.E.M.) SRTs (but not CRTs) were longer in patients (309 ± 45 ms) than in controls (218 ± 9 ms). Movement times were longer in patients for both SRTs and CRTs. TMS thresholds, expressed as a percentage of the maximum stimulator output, were not significantly (P > 0.05) different in both groups for both MEPs (patients, 34 ± 3 %; controls, 36 ± 3 %) and inhibition of voluntary contraction (patients, 29 ± 2 %; controls, 34 ± 4 %). The duration and extent of inhibition did not differ significantly between groups at any stimulus strength. The pattern of change in duration and extent of inhibition with increasing stimulus intensity was no different in the two groups. The duration and extent of corticospinal inhibition in patients with CFS did not differ from controls, adding further evidence to the notion that the feeling of fatigue and the slowness of movement seen in CFS is not manifest in corticospinal output pathways. Experimental Physiology (2001) 86.5, 547-550.

Muscle cells in the nematode Ascaris suum undergo bouts of oscillation in the behaving worm, these being correlated with progressing waves of contraction along the body of the worm. The bouts have three time scales: a rough period of 7-20 s, 10-20 short bursts of spikes within a bout, and three to eight spikes per burst. This paper has two aims: showing that there is consistency between measurements of individual currents and measurements on whole muscle cells, and creating a building block to eventually explain the locomotion in this 'simple' system. A realistic model for a single Ascaris suum muscle cell is developed using existing data from experiments which have established the types of ionic currents in the muscle cell and many of their kinetic properties. Numerical simulations are carried out. The model cell reproduces the two shorter time scales present. It has some robustness with regard to parameter changes, but also allows for the different numbers of spikes per burst seen in recordings from muscle cells. The third time scale, the length of a bout, may originate from some system effect, a combination of neural and stretch effects and possibly a secondary effect of the calcium-activated chloride channel. Experimental Physiology (2001) 86.5, 551-559.

The involvement of angiotensin AT1 receptors in sodium appetite was studied in hypothyroid rats treated with the angiotensin II antagonist losartan. Losartan was administered chronically by the oral route or acutely by the subcutaneous route after water and sodium depletion or water, sodium and food deprivation. Three days after addition of losartan to the food at the dose of 1.0 mg g-1, the rats significantly reduced (P < 0.02) their spontaneous intake of 1.8 % NaCl. Increasing the dose of losartan to 2.0 and 4.0 mg g-1 did not reduce NaCl intake; in contrast, the intensity of the sodium appetite gradually returned to previous levels. The simultaneous administration of captopril, an angiotensin converting enzyme inhibitor, and losartan significantly increased (P < 0.05) NaCl intake and after captopril removal NaCl intake returned to the levels observed with losartan treatment alone. The administration of losartan 4 days after the beginning of captopril treatment significantly reduced (P < 0.0001) NaCl intake. Following acute administration of losartan, water- and sodium-depleted rats significantly reduced their NaCl and water intake (P < 0.001). The administration of losartan also induced a significant reduction in NaCl and water intake in water, NaCl and food-deprived rats (P < 0.0001 and P < 0.001, respectively). The present results show that chronic treatment with oral losartan inhibited spontaneous sodium appetite in hypothyroid rats. Continuation of treatment rendered rats resistant to the blockade of AT1 receptors. Water and sodium depletion and water, NaCl and food deprivation induced sodium appetite, which in the short term depends on cerebral angiotensinergic activity mediated by the activation of AT1 receptors. Experimental Physiology (2001) 86.5, 621-628.

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.

Genetic methodologies have provided powerful means for investigating the cellular and molecular mechanisms of biological functions. Cell-cell and cell-extracellular matrix interactions in particular have been studied in different functional systems with genetically modified animals. In the peripheral and central nervous system, many aspects of specific processes based on such interactions, including myelination, synaptic transmission and plasticity, have been elucidated at the cellular and molecular level. Importantly, genetic approaches have greatly advanced the understanding of pathologies resulting from impaired cellular interactions in the brain and the periphery. In this review, some of the most relevant genetic mouse models in cell biology and the methodologies employed for their production will be described. In addition their usefulness for studies of the mechanisms of hereditary neuropathies, learning and memory, and tumorigenesis will be illustrated.

The inhibition constant of L-sorbose efflux (Ki(sorbose)) from human erythrocytes for inhibition by D-glucose increases from 5·15 ± 0·89 to 12·24 ± 1·9 mM on cooling from 50¡C to 30¡C; the Ki(sorbose) of D-mannose increases similarly on cooling. The activation energy Ea(sorbose) of net L-sorbose exit from human erythrocytes is 62·9 ± 3·1 kJ mol-1; but in the co-presence of 5 mM D-glucose Ea(sorbose) is reduced to 41·7 ± 1·6 kJ mol-1 (P < 0·005). These data are consistent with the view that when D-glucose binds to the hexose transporter it leads to an activated transporter state which remains transiently activated after glucose dissociates; if L-sorbose binds to this excited state it is more mobile than otherwise and consequently the apparent Ki(sorbose) of D-glucose is raised. Cooling prolongs the decay time of the activated state; hence the Ki(sorbose) of D-glucose rises as temperature is reduced.

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.

The present study was carried out to determine whether the increased salt intake induced by increased specific sodium appetite in pregnant rats modifies water-salt homeostasis throughout pregnancy. Two groups of pregnant rats were used, one fed ad libitum with a normal sodium (NS) diet consisting of standard rat chow and distilled water, and the other fed with a high-sodium (HS) diet with free access to chow, distilled water plus saline solution (1·5 % NaCl). Virgin rats in dioestrus were also studied as non-pregnant controls. Pregnant animals were studied on days 4, 9, 14, 20 and 21 of gestation at which time body weight, water and saline intake, sodium excretion, plasma atrial natriuretic peptide (ANP) and arginine vasopressin (AVP) concentrations, as well as plasma osmolality were determined. Data showed that water intake was higher in the NS group, but total fluid intake (water plus saline) was higher in the HS group throughout pregnancy. Dietary sodium intake was the same for both groups but total sodium intake (chow plus saline) was 60-98 % higher in the HS rats. Pregnant HS rats excreted more fluid (35-50 %) and sodium (up to 100 %) compared with NS rats, indicating that the animals could change their renal excretion in response to a 2·5-fold higher dietary sodium intake compared with the control level. Salt satiety during pregnancy did not modify plasma ANP concentration. In both groups of pregnant rats ANP levels increased 3-fold on day 14 without significant alteration in sodium excretion, suggesting that the natriuretic action of ANP is attenuated at least after the second week of pregnancy. High sodium intake did not change plasma AVP concentration or osmolality and both groups showed the same gradual decrease in plasma osmolality ([similar]8 mosmol kg-1) at the end of pregnancy that was not accompanied by decreased plasma AVP concentration. The present data show that rats maintain the special homeostatic equilibrium that occurs in normal pregnancy even when they are allowed to increase sodium intake to satisfy their salt appetite during this period of the reproductive cycle.

Cross-correlation of the discharges of muscle spindle afferents in ankle extensor and flexor muscles has been used to reveal the activity of static gamma (γS) motoneurones innervating chain intrafusal muscle fibres during locomotion. In the anaesthetised cat, the cross-correlation of spindle afferents, jointly innervated by a γS-efferent with chain fibre contacts, showed short duration synchrony (2-8 ms) when the efferent was stimulated repetitively. In pre-mammillary decerebrated cats, the cross-correlograms of discharges of some pairs of spindle afferents showed similar short duration peaks of synchronisation and these were interpreted as being due to a common γS drive to chain intrafusal muscle fibres. The incidence of synchrony was low, and was similar at rest (5 % of pairs) and during treadmill locomotion (7 % of pairs). Phase dependence of synchrony was evident during locomotion in the flexor muscle. The synchrony of muscle spindle afferent discharge is discussed in relation to estimates of the numbers of spindles contacted by individual γS-efferents. Experimental Physiology (2003) 88.2, 285-296.

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.

Our laboratory has identified plasma membrane oestrogen receptors on a GH3/B6 rat pituitary tumour cell line and several sublines which produce rapid (within minutes), non-genomic responses to oestrogens. Oestrogen receptors have been identified by their binding to nine different antibodies (Abs) which together recognize at least seven epitopes on the oestrogen receptor-α. GH3/B6/F10 cells, a membrane oestrogen receptor-enriched subline, elevate intracellular calcium levels in response to 10 nM oestradiol. Prolactin release in these cells is triggered by both 1 pM and 1 nM oestradiol and diethylstilbestrol (DES). A membrane oestrogen receptor-α immunocyto-chemical signal rapidly disappears (at 3 min) and reappears (at 12-15 min) when 1 nM oestradiol, 10 nM diethylstilbestrol, or 10 nM nonylphenol is applied to the cells. This suggests that both oestrogens and xenoestrogens can utilize this alternative pathway for oestrogenic action. Xenoestrogens, which have so far shown weak effects in genomic assay systems, should now be retested for activity in eliciting membrane-initiated oestrogenic responses.

The process by which free energy from ATP or an ion gradient is coupled to work - either osmotic work, by a membrane pump, or mechanical work, by a molecular motor - as well as the development of force by a molecular motor, can be explained by mechanisms dependent on substrate binding energy. Coupling involves a reaction sequence that combines the driving and driven reactions and that is controlled at switch points where the mobility of the coupling protein and its specificity in binding and catalysis can be abruptly altered; the altered state is an intermediate (or transition state) in the coupled reaction, the unaltered state an intermediate in the uncoupled reaction (slippage). The balance between these states, which is determined by the increase in substrate binding energy in the transformation, decides the relative importance of the coupled and uncoupled paths. On this basis a general expression for switching may be derived: the tightness of coupling is limited by a ratio of substrate dissociation constants before and after a controlling change in state: Ratecoupled/Rateuncoupled [less than or equal to] Kinitial state/Kfinal state.When binding energy is used in this way to distort the conformation of a protein, mechanical work is done; the work is internal but becomes external if the protein is connected to an external load. External work, force F multiplied by distance d, is then limited by a ratio of substrate dissociation constants before and after the conformational change: W = Fd < RT ln(Kinitial state/Kfinal state).The ratios of dissociation constants, estimated from published values of the force exerted by molecular motors, are > 1 × 104 for myosin and > 2 × 104 for kinesin. From the tightness of coupling of the anion exchange carrier of red cells the ratio of constants for coupling is [greater than or equal to] 4 × 104. These increments can be accounted for by conversion of a surface complex to an enclosed complex, as in chelate formation. The work done by an ATP-driven motor is limited by the free energy of ATP hydrolysis, but the force developed is independent of the ATP reaction, and comes from binding energy with either the driving or driven substrate.