Presently, many researchers have adopted a reductionistic approach to physiology. Specifically, this involves studies on acutely isolated or cultured cells. While this is essential for our understanding of important cellular and subcellular mechanisms, it should not be forgotten that physiology is about the function of an integrating body. One view point is that definitive interpretations can only be made by crossing boundaries of physiology from the molecular/ cellular to the systems/behavioural level. An aim of this lecture will stress the importance of a multidisciplinary approach to physiology, that is, 'putting the cell back into the system'. The lecture attempts to demonstrate why this is important for enhancing the functional significance and relevance of physiological data as well as for validating predictions.

Bronchoconstriction of small bronchioles plays a major role in the increase in airway resistance following agonist challenge. There is evidence that the airway smooth muscle (ASM) of small bronchioles differs functionally from that in larger airways. Little is known however about the electrophysiology of small bronchioles. Ion currents were therefore studied in airway smooth muscle cells freshly dissociated from human intralobular bronchioles, with a diameter between 0·3 and 1·0 mm. As previously reported for human large airways, the major outward current in these cells was due to activity of large conductance K+ (BK) channels, with a relatively minor component due to a voltage-gated delayed rectifier current (IDR), which was only observed in [sim]30 % of cells. Three distinct types of iberiotoxin- and TEA-sensitive large conductance K+ channel contributed to large conductance K+ current (IBK). These included a highly voltage- and Ca2+-sensitive 200 pS channel previously reported in human large airways, and two smaller channels of 150 and 100 pS previously seen only in human fetal or cultured ASM. In contrast to large airways, ASM cells from bronchioles also demonstrated a voltage-gated inward rectifier current (IIR). IIR was activated by hyperpolarisation below the K+ equilibrium potential and could be blocked by submillimolar concentrations of Cs+ or Ba2+, and partially by physiological concentrations of Na+. Corresponding single channels with a conductance of [sim]>17 pS could also be recorded in the cell-attached configuration. A small voltage-independent current was also observed which was resistant to classic K+ and Cl- channel blockers but which could be abolished by replacement of Na+ with the impermeant cation N-methyl-D-glucamine (NMDG+). Corresponding non-selective single channels of [sim]20 pS could be seen in inside-out mode. These results demonstrate that ASM from small bronchioles differs in terms of ion currents and channels from ASM derived from large airways, with possible implications for function.

Xenopus laevis oocytes were used to analyse the effects of serotonin (5-hydroxytryptamine, 5-HT) and serotonergic agents on ionic currents elicited by the activation of mammalian muscle nicotinic acetylcholine receptors (AChRs). 5-HT as well as other serotonergic agents, such as ketanserin, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), methysergide, spiperone, or fluoxetine alone (up to 1 mM), did not elicit membrane currents in Xenopus oocytes expressing AChRs, but they reversibly reduced the current elicited by acetylcholine (ACh-current). Serotonin was applied before, together with or after ACh application, and its effects were examined on desensitizing and non-desensitizing ACh-currents. 5-HT reduced the amplitude and accelerated the desensitization of the desensitizing currents. In contrast, non-desensitizing currents were reduced in amplitude but their time course was not significantly affected. With the same concentration of 5-HT the inhibition was stronger on desensitizing than on non-desensitizing ACh-currents. For example, 100 µM 5-HT reduced the peak of a desensitizing ACh-current to 0·48 ± 0·06 (peak current ratio) and after 40 s the current was reduced to a ratio of 0·25 ± 0·04, whereas a non-desensitizing ACh-current was reduced to a ratio of 0·66 ± 0·01. All the serotonergic agents tested inhibited the ACh-currents rapidly and reversibly, suggesting that they are acting directly on the AChRs. The half-inhibitory concentration, IC50, of 5-HT acting on non-desensitizing currents elicited by 250 nM ACh was 247 ± 26 µM and the Hill coefficient was [sim]0.88, suggesting a single site for the interaction of 5-HT with the receptor. It appears that 5-HT inhibits AChRs non-competitively because neither the half-effective concentration of ACh, EC50, for ACh-current nor the Hill coefficient were affected by 5-HT. Furthermore, the extent of inhibition of 5-HT on AChRs did not depend on the nicotinic agonist (suberyldicholine, ACh or nicotine). The inhibition of AChRs by serotonergic agents was voltage-dependent. The electrical distance of the binding site for 5-HT was [sim]0.75, whereas for the other serotonergic agents tested it was [sim]0.22, suggesting that ketanserin, 8-OH-DPAT, methysergide, spiperone and fluoxetine act within the ion channel, but at a site more external than that for 5-HT. These substances inhibited the ACh-current more potently than 5-HT. We conclude that 5-HT and serotonergic agents inhibit, in a non-competitive manner, the ACh-current in muscle AChRs by blocking the open receptor-channel complex. Moreover, 5-HT appears to promote the desensitized state of the receptor when the current is elicited by high ACh concentrations.

Isolated cardiac ventricular myocytes from guinea-pig were used to investigate the effect of voltage clamp protocols on background Na+ current (ib.Na) and inward rectifier current (iK1). Using long (4 s) clamp pulses and very long step clamps, the i-V relations showed that removal of Na+ reduces the amplitude and shifts the voltage dependence of iK1 (Spindler et al. 1998). Ramp clamps, however, gave more complicated results, with slower ramps more often giving the same results as steps and pulses. Both iK1 itself and, during faster ramps, other currents show hysteresis, so masking the steady-state changes. Using pulses, TTX had no effect on steady-state current. Small differences occurred in the ramps, but even at fast ramp speeds the effects are very much smaller than in Purkinje tissue. Only part of ib,Na is TTX sensitive and the effect does not occur in all cells.

The patch-clamp recording technique was used to examine the properties of the K+ channels in cultured equine sweat gland epithelial cells. With symmetric K+ solutions (140 mM), a single population of K+ channels was identified with a slope conductance of 187 pS and a reversal potential of around 0 mV. The channel was selective for K+ over Na+. Channel activity was increased by membrane depolarization. A 10-fold increase in [Ca2+]i produced an approximate 60 mV negative shift in the open state probability (Popen)-voltage curve. Externally applied tetraethylammonium ions (TEA+) caused a rapid and flickery block of the channel and reduced the unitary current amplitude. TEA+ bound to the blocking site with stoichiometry of 1:1 and with a dissociation constant (Kd) of 186 ± 27 µM at +40 mV. A weak voltage dependence of Kd was observed. Iberiotoxin (100 nM) reduced Popen but had no effect on single-channel conductance. Neither glibenclamide (10 µM) nor intracellular adenosine 5'-triphosphate (ATP, 1 mM) altered channel activity. In addition, ATP, when applied extracellularly, transiently activated the channel by increasing Popen. Channel activity was low around the resting membrane potential in the intact epithelia, indicating that these channels might not contribute to the resting K+ conductance. However, the channel could be activated in a regulated manner. The K+ channels may play a role in transepithelial fluid secretion in sweat gland.

We reported recently that hypoxia inhibits baroreflex vagal bradycardia (BVB) in rats and that this inhibition persists following chemoreceptor denervation. However, since it is possible that hypoxia also affects the central processing of chemoreceptive input, the existence of chemoreceptor-mediated inhibition of BVB cannot be ruled out. Therefore, we have studied whether selective chemoreceptor activation affects BVB in normoxic conditions. In pentobarbital-urethane-anaesthetized, succinylcholine-immobilized, artificially ventilated rats, BVB was provoked by electrical stimulation of the aortic depressor nerve. Arterial chemoreceptors were selectively activated by intracarotid injection of a minute amount of sodium cyanide. Cyanide injection consistently increased blood pressure while changing heart rate variably. BVB was inhibited in a dose-dependent manner. This inhibition, as well as changes in blood pressure and heart rate, was abolished following transection of the carotid sinus nerve (CSN) ipsilateral to the injection. Spinal cord transection at the C2 level did not affect the inhibition. On the other hand, intracarotid cyanide had no effect on bradycardia elicited by electrical stimulation of a peripheral cut end of the cervical vagus nerve. We conclude that chemoreceptor activation definitely inhibits BVB and that this inhibition is mediated by the CSN, and predominantly occurs in the central nervous system. The possibility is suggested that severe hypoxia suppresses not only BVB but also the chemoreceptor-mediated inhibition of BVB, both via the direct, central action.

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.

This study examined the effect of erythropoietin (EPO) on resting tension and on the responses of rat mesenteric and renal arcuate arteries in vitro to a number of agonists as a possible cause of its blood pressure elevating properties when used therapeutically. Noradrenaline and potassium chloride induced concentration-dependent vasoconstrictions in both vessel types but the basal tension, maximum tension, and the -log concentration producing half-maximal response (pEC50) were altered in the presence of 0·1 or 20 U ml-1 EPO. The thromboxane A2 receptor agonist U46619 induced a constriction of the renal arcuate arteries which was enhanced by EPO at 20 U ml-1, from 1·68 ± 0·34 to 2·64 ± 0·39 mN mm-1 (P < 0·01), but which was unchanged by NG-nitro-L-arginine methyl ester (10-4 m). Serotonin (10-9-10-5 M) caused a concentration-related vasoconstriction in renal arcuate arteries which was shifted to the right in the time control study (P < 0·001) but this was abolished by both 0·1 and 20 U ml-1 of EPO. Acetylcholine induced a relaxation of precontracted mesenteric arteries, by 95·4 ± 1·64 % with an EC50 of 7·08 ± 0·08 M which was reduced (P < 0·001) by 20 U ml-1 EPO to 81·7 ± 3·56 % and 6·10 ± 0·11 M, respectively. The sodium nitroprusside-induced relaxations were unaffected by EPO. The acetylcholine-mediated relaxations in renal arcuate arteries were unchanged by EPO. Bradykinin-induced relaxations in mesenteric and renal arcuate arteries were unaffected by both EPO concentrations. Together these data showed that EPO over a large concentration range had only minor effects on basal tension and the vascular responsiveness of both mesenteric and renal arcuate arteries. The mechanism whereby EPO causes a chronic elevation in blood pressure is unlikely to be due to acute interactions with agonist-mediated responses.

Mechanical properties of the vascular smooth muscle from normal and dystrophin-deficient (mdx) mice were examined. Changes in resting and developed tensions in response to stretch were recorded in isolated portal vein. The vascular segments were elongated in 5 % increments of the 'in situ' length (Lr) up to 1á30Lr. The resting length-tension curves in male mdxmice were similar to normal mice, while a marked decrease in the slope of the curve was noted in female mdx mice. These findings were not affected by atropine, phentolamine, tetrodotoxin or [Ca2+] in the surrounding media. At Lr, the tension of isolated portal vein was characterized by spontaneous synchronized uniform force waves in normal mouse. In contrast, in mdxmouse portal veins an irregular motor pattern characterized by desynchronized force waves with a decrease of amplitude and an increase in frequency was recorded. Extension of the length of the portal vein segment did not increase the spontaneous phasic activity developed in female mdx mice although this was noted with male mdx mice and normal mice. Experiments with chemical depolarizing agents indicated that spontaneous myogenic excitation activated the great majority of vascular smooth muscle cells in normal mouse portal vein, whereas in mdx mice only a reduced number of these cells were excited suggesting that in the mdx mouse the intercellular electronic coupling is altered. In conclusion this study provides the first description of the mechanical activities of portal vein longitudinal muscle and shows that in mdx mice the motor activity is severely disrupted.

The effect of stimulation frequency on intact muscle blood flow was measured by radiolabelled microsphere entrapment. Maximum flow occurred at 4 Hz in both extensor digitorum longus and tibialis anterior (156 ± 13 and 205 ± 24 ml min-1 (100 g)-1, respectively). Regional differences in fibre composition were reflected in the relative flow rates, with functional hyperaemia in the oxidative tibialis anterior core being twice that of the glycolytic cortex at 10 Hz. A consistent difference in flow between legs was noted that may originate from an unequal division of the common iliac artery in rats.

The importance of angiotensin type-1 (AT1) receptor stimulation during renal development has recently been established in both pharmacological and knockout models. We have previously reported irreversible and progressive papillary atrophy and a reduced baseline renal interstitial hydrostatic pressure (RIHP) after neonatal angiotensin-converting enzyme (ACE) inhibition. The aim of the present study was to investigate the consequences of these abnormalities on urinary sodium excretion during acute extracellular sodium loading. Rats were treated neonatally with enalapril (10 mg kg-1 day-1) or saline control from days 3 to 23 after birth. Urinary sodium excretion was assessed in relation to mean arterial pressure (MAP) and RIHP responses in adult anaesthetised rats during moderate (1·5 and 3 % body weight) and severe (9 % body weight) saline-induced volume expansion. Control rats responded to the moderate volume expansion by increasing MAP by 16 ± 6 % and RIHP by 40 ± 23 %, respectively. In neonatally enalapril-treated rats, however, MAP and RIHP remained unchanged and were associated with a smaller increase in sodium excretion (44 ± 11 % of the total amount infused versus 71 ± 16 % for controls, P < 0·05). In contrast, severe volume expansion resulted in marked pressure rises in both the enalapril-treated group (36 ± 12 and 112 ± 48 % of baseline for MAP and RIHP, respectively) and the control group (34 ± 21 and 130 ± 34 % of baseline for MAP and RIHP, respectively). Moreover, the increases in MAP and RIHP were associated with complete excretion of the severe sodium challenge within 60 min in both treatment groups. We conclude that a RIHP response appears to be a prerequisite for adequate urinary sodium excretion in this model of papillary atrophy. Hence, an intact renal medulla is not mandatory in the renal handling of sodium during extracellular loading.

This study compared the effects of high frequency oscillatory ventilation (HFOV) and intermittent mandatory ventilation (IMV) on the homeostasis of nitric oxide (NO) in the lower respiratory tract of healthy rabbits. The mechanisms underlying a putative stretch response of NO formation in the airways were further elucidated. Male New Zealand White rabbits were anaesthetized, tracheotomized and ventilated with IMV or HFOV in random order. Total NO excretion increased from 9σ6 ± 0σ8 nl min-1 (mean ± S.E.M.) during IMV to 22σ6 ± 2σ7 nl min-1 during HFOV (P < 0σ001). This increase was not explained by changes of functional residual capacity ([Delta]>FRC). A similar increase in NO excretion during HFOV was seen in isolated buffer-perfused lungs under constant circulatory conditions (P < 0σ05, n = 4). Intratracheal mean CO2 and NO concentrations, measured at 2σ5, 5, 7σ5 and 10 cm below tracheostomy, increased significantly with increasing distance into the lung during both IMV and HFOV (P < 0σ001 for each comparison). At every intratracheal location of the sampling catheter, particularly low in the airways, both CO2 and NO concentrations were significantly higher during HFOV than during IMV (P < 0σ01 for each comparison). We conclude that HFOV increases pulmonary NO production in healthy rabbits. Increased stretch activation of the respiratory system during HFOV is suggested as a possible underlying mechanism. The increase in mean airway NO concentrations may have biological effects in the respiratory tract. Whether it can account for some of the benefits of HFOV treatment needs to be considered.

The guinea-pig has been used extensively to investigate adrenal steroidogenesis. However, very little is known about adrenocortical responses to corticotrophin-releasing hormone (CRH) and adrenocorticotrophin (ACTH) in this species, in vivo. In the present study, we have developed a stress-free sampling system, in the chronically catheterized adult guinea-pig, that has allowed us to investigate basal and activated adrenocortical activity. Indwelling carotid artery and jugular vein catheters were surgically implanted into female guinea-pigs (n = 5). Each animal was treated with vehicle, human CRH (0·2 or 2 µg kg-1) and ACTH1-24 (0·2 or 2 µg kg-1), and serial plasma samples removed for analysis of ACTH and cortisol concentrations by radioimmunoassay. There was no effect of serial sampling on pituitary-adrenocortical activity, indicating that the animals remain in an unstressed state. Basal plasma ACTH and cortisol concentrations were 703·9 ± 24·5 pg ml-1 and 117·9 ± 5·2 ng ml-1, respectively. Both CRH and ACTH significantly increased adrenocortical activity in a dose-dependent manner. ACTH (2 µg kg-1) was the most potent activator leading to plasma cortisol concentrations of 647 ± 116 ng ml-1. In conclusion, we have shown that basal plasma cortisol concentrations in the guinea-pig are low compared to those obtained in previous studies by cardiac puncture or following decapitation. However, plasma ACTH concentrations are high compared to other species. We have also shown that human CRH and ACTH1-24 act as potent activators of the guinea-pig pituitary-adrenocortical axis, leading to response profiles consistent with mild cortisol resistance.

We investigated the hypothesis that exogenous stimulation with thyrotrophin-releasing hormone (TRH) immediately prior to umbilical cord clamping can improve thermoregulatory adaptation after birth in near-term lambs delivered by Caesarean section. Lambs received an umbilical vein injection of saline ± TRH (8 µg) prior to cord clamping. The rate of change in colonic temperature and oxygen consumption after birth were not influenced by TRH, but TRH-treated lambs exhibited a greater incidence of shivering compared with controls over the first hour of neonatal life. Two and a half hours after birth, TRH-treated lambs possessed brown adipose tissue (BAT) with a higher thermogenic activity (i.e. GDP binding to mitochondrial protein), but their BAT had a reduced DNA content and they had less hepatic glycogen than control lambs. TRH administration had no effect on iodothyronine 5' deiodinase activity in BAT and liver, or on plasma concentrations of total triiodothyronine, thyroxine, cortisol or free fatty acids. Three TRH-treated but no control lambs, failed to establish continuous breathing, so tissues from these treated lambs together with time-matched controls were sampled 25 min after birth. These 'non-surviving' TRH-treated lambs had very high plasma catecholamine concentrations, but their lung weights were similar to controls. 'Surviving' TRH-treated lambs possessed lungs with less DNA than non-surviving TRH-treated lambs. It is concluded that umbilical vein injection of TRH prior to umbilical cord clamping increases the recruitment of both shivering and non-shivering thermogenesis after birth.

The kinetics and the isozyme composition of lactate dehydrogenase (LDH) were measured in rat plantaris muscle during a 26 week endurance-training program. Alterations in the LDH isozyme pattern were detectable after 6 weeks as the percentage of the M4 isozyme was reduced from 89 to 76 % and the total percentage of M subunits compared with H subunits declined by 8 %. At 16 weeks, M4 accounted for only 62 % of the total. The replacement of M with H subunits continued when training was prolonged as M4 represented only 52 % of the total isozymes at 26 weeks. Conversely, training for 6 and 16 weeks produced no changes in either Vmax or Km. At 26 weeks, these values declined for both the forward (pyruvate to lactate) and backward reactions. The rate constants for both reactions were also reduced. These data suggest that changes in LDH isozyme pattern do not contribute significantly to the enhancement of lactate oxidation that may occur after training. They also suggest that the functional significance of alterations in LDH structure and/or function are best determined from analysis of the overall reaction kinetics as opposed to individual characteristics such as the isozyme pattern, Km or Vmax.

The lactate threshold is a widely used and, at times, controversial construct in exercise physiology and pathophysiology. Its non-invasive estimation during incremental exercise depends upon CO2 output increasing as a function of O2 uptake, i.e. 'V-slope', as a result of bicarbonate buffering during the lactic acidosis. However, we hypothesised that the V-slope deflection could also occur as a consequence of metabolic CO2 being diverted proportionally more into the CO2 stores in the early phase of exercise. Eight healthy males performed two incremental exercise tests on a cycle ergometer, with and without controlled prior hyperventilation; the hyperventilation caused end-tidal PCO2 to decline by [sim]10 mmHg, with the clearance of a CO2 volume averaging 2547 ml. This corresponded to an 'effective' CO2 capacitance of some 3·12 ml mmHg-1 kg-1. Gas exchange was determined breath-by-breath, and blood was sampled from the dorsum of the heated hand. Our results demonstrate that the early dynamics of CO2 wash-in to the previously depleted body stores can result in a 'pseudo-threshold', i.e. significantly before the onset of the actual lactic acidosis. Precautions should therefore be taken to avoid hyperventilation prior to non-invasive estimation of the lactate threshold.