Parotid secretion has been investigated in anaesthetized lambs in the presence and absence of N[omega]- nitro-L-arginine methyl ester (L-NAME) and sodium nitroprusside to block de novo synthesis of nitric oxide (NO). Following administration of L-NAME the basal rate of flow was unaffected and changes in electrolyte secretion failed to achieve statistical significance but there was a significant fall in the basal rate of protein secretion. The flow of parotid saliva which occurred in response to stimulation of the parasympathetic innervation was reduced by 34 % and sodium output was reduced in approximately the same proportion. L-NAME had no significant effect on these parameters during stimulation of the sympathetic innervation. During combined stimulation of the parasympathetic and sympathetic innervations L-NAME caused a reduction in parotid salivary flow and sodium output which was roughly the same as that observed during parasympathetic stimulation alone. However, L-NAME caused a much greater reduction in protein output during each of these experimental protocols: -92 % during parasympathetic stimulation, -63 % during sympathetic stimulation, and -60 % during combined stimulation. Whereas the absolute amount of protein secreted was reduced after L-NAME in each instance, the extent of potentiation of protein output recorded during combined stimulation was increased roughly fivefold. It is concluded that the output of protein in response to autonomic stimulation exhibits a greater NO dependence than either the flow of saliva or secretion of electrolytes in this gland.

Changes in extracellular calcium (CaB) concentration ([Ca2+]o) affect kidney function both under basal and hormone-stimulated conditions. The molecular identification of an extracellular Ca2+-sensing receptor (CaR) has confirmed a direct role of CaB on parathyroid and kidney function (i.e. independent of calciotropic hormones)s as a modulator of Ca2+ homeostasis. In addition, evidence accumulated over the last 10 years has shown that CaR is also expressed in regions outside the calcium homeostatic system where its role is largely undefined but seems to be linked to regulation of local ionic homeostasis. The parathyroid and kidney CaRs are 1081 and 1079 amino acids long, respectively, and belong to the type III family of G protein-coupled receptors (GPCRs), which includes other CaRs, metabotropic glutamate receptors and putative vomeronasal organ receptors. For the CaR, its low (millimolar) affinity for Ca2+, its positive cooperativity and its large ionsensing extracellular domain, indicate that the receptor is more sensitive to changes in net cationic charge rather than to a specific ligand. Mg2+, trivalent cations of the lanthanide series and polyvalent cations such as spermine and aminoglycoside antibiotics can all activate the receptor in vitro with EC50 values in the micromolar range for trivalent and polyvalent cations or in the millimolar range for Ca2+ and Mg2+. In addition to true CaR agonists, CaR sensitivity to CaBis also susceptible to allosteric modulation by ionic strength, L-amino acids and by pharmacological agents. This review will address endogenous and exogenous CaR agonists, the role of the receptor in the calcium homeostatic system and some speculation on possible role(s) of the CaR in regions not involved in mineral ion homeostasis.

We showed recently that post-frusemide (furosemide) natriuresis was associated with a major depression of medullary circulation. In the present study, prior to administration of frusemide the tubular transport of NaCl was modified by loading the animals with 5 % saline to elucidate a possible interrelation between the tubular and vascular effects of the drug. Moreover, a possible involvement of the renin-angiotensin system was examined by pharmacological blockade using captopril, an inhibitor of angiotensin converting enzyme (1 mg kg-1, I.V.), or losartan, a selective inhibitor of angiotensin AT1 receptor (10 mg kg-1, I.V.). The effects of frusemide (0.25 mg kg-1 I.V., then the same dose given over 1 h) on renal medullary and cortical circulation (using laser-Doppler flowmetry) and renal excretion of sodium (UNaV), water and total solutes were measured in anaesthetised rats. With no pre-treatment, frusemide decreased the medullary flow (36.6 ± 6.0 %) significantly more than the cortical flow (10.1 ± 1.0 %; P < 0.001). The difference between the medulla and cortex was not significant in rats which showed high UNaV after hypertonic saline loading (2.0 ± 0.4 vs. 0.4 ± 0.1 µmol min-1 in non-loaded rats): 21.1 ± 3.9 % and 15.8 ± 1.5 %, respectively. At very high UNaV (9.5 ± 1.1 µmol min-1) the post-frusemide decrease in blood flow tended to be smaller in the medulla (7.6 ± 7.7 %) than in the cortex (16.2 ± 2.6 %). The fall in medullary blood flow was attenuated by pre-treatment with captopril (22.0 ± 3.3 %) and abolished by pre-treatment with losartan (2.8 ± 11.8 %). The decrease in cortical blood flow was not changed by hypertonic saline or angiotensin II blockers. The abolition of the post-frusemide depression of medullary blood flow by previous salt loading confirms the proposed link between tubular transport status and vasoconstriction. A similar modification of the response by blockade of the renin-angiotensin system suggests that the system is involved in the mechanism of medullary vasoconstriction. Experimental Physiology (2001) 86.5, 611-616.

The whole-cell and perforated patch configurations of the patch-clamp technique were used to characterise the volume-sensitive anion channel in rat pancreatic β-cells. The channel showed high permeability (P ) relative to Cl- to extracellular monovalent organic anions (PSCN/PCll = 1.73, Pacetate/PCll = 0.39, Plactate/PCll = 0.38, Pacetoacetate/PCll = 0.32, Pglutamate/PCll = 0.28) but was less permeable to the divalent anion malate (Pmalate/PCll = 0.14). Channel activity was inhibited by a number of putative anion channel inhibitors, including extracellular ATP (10 mM), 1,9-dideoxyforskolin (100 µM) and 4-OH tamoxifen (10 µM). Inclusion of the catalytic subunit of protein kinase A in the pipette solution did not activate the volume-sensitive anion channel in non-swollen cells. Furthermore, addition of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) or forskolin failed to activate the channel in intact cells under perforated patch conditions. Addition of phorbol 12,13-dibutyrate (200 nM), either before or after cell swelling, also failed to affect channel activation. Our findings do not support the suggestion that the volume-sensitive anion channel in pancreatic β-cells can be activated by protein kinase A. Furthermore, the β-cell channel does not appear to be subject to regulation via protein kinase C. Experimental Physiology (2001) 86.2, 145-150.

The aim of the present study was to examine the pattern of cardiovascular recovery from exercise in 15 women (age, 20.3 ± 1.4 years; body mass, 61.5 ± 4.3 kg) across two phases of oral contraceptive (OC) use: 21 days of consumption and 7 days of withdrawal. Cardiovascular recovery was measured in the supine position for 60 min following 30 min of exercise at 60 % maximal rate of oxygen consumption (V˙O2,max). Central and peripheral haemodynamics were assessed during consumption and withdrawal of the OC pill using occlusion plethysmography, Doppler flowmetry and echocardiography. Significant hypotension occurred following exercise (P < 0.05), returning to baseline values after 60 min. The peak hypotension occurred 5 min into recovery. Cardiac output and heart rate were elevated for 60 min following exercise (P < 0.05), whilst stroke volume remained at baseline values. Heart rate was greater throughout recovery during consumption compared to withdrawal (P < 0.05); however, although there was a trend for greater responses during consumption, phase of OC use did not affect the other central cardiovascular variables (P > 0.05). Post-exercise blood flow parameters were not significantly affected by exercise or OC phase; however, calf blood flow was greater, and resistance to flow lower during consumption (P > 0.05). The pattern of post-exercise fluctuations in cardiovascular parameters may differ from those seen in men, whilst oestrogen variation may influence research findings. Experimental Physiology (2002) 87.5, 623-632.

The aim of the present work was to investigate the role of the serotoninergic system in the control of sodium appetite of hypothyroid rats (HTR) by administering drugs that affect the serotoninergic activity, and to compare the same homeostatic behaviour in euthyroid rats (ETR) also given these drugs. Fenfluramine (FEN; 5.0 mg kg-1, I.P.), which releases serotonin in the brain, significantly reduced the intake of 1.8 % NaCl in HTR subjected to water and sodium depletion (depleted) or water, sodium and food deprivation (deprived) by 31 and 45 %, respectively, 120 min after FEN injection, compared to HTR that received vehicle alone. Similarly, administration of FEN to ETR reduced 1.8 % NaCl intake in depleted and deprived rats by 64 and 46 %, respectively. The presynaptic serotonin reuptake inhibitor fluoxetine (20.0 mg kg-1, I.P.) led to the inhibition of sodium appetite in HTR during the initial 30 min in depleted rats and for up to 60 min post-injection in deprived rats, while sodium appetite inhibition persisted for longer periods in ETR. The 5HT2C receptor agonist mCPP (5.0 mg kg-1, I.P.) caused a drastic reduction in sodium appetite in HTR and ETR in depleted and deprived rats, respectively, after 120 min. Prior administration of the 5HT2C receptor antagonist LY53857 (5.0 mg kg-1, I.P.) completely blocked the inhibitory action of mCPP on sodium appetite in both HTR and ETR. In summary, our results suggest that the recruitment of serotoninergic neurons involved in the modulation of sodium appetite seems to be decreased in hypothyroidism due to a probable deficiency in the cerebral signalling pathway. Experimental Physiology (2003) 88.2, 251-260.

Resistance arteries isolated from patients with critical limb ischaemia (CLI), a hypotensive/hypoperfusion state of the lower leg, have been shown to undergo morphological changes opposite to those observed in hypertension, that is, decreased wall thickness, reduced cross-sectional area and a decreased wall : lumen ratio. The aim of this present study was to use laser scanning confocal microscopy (LSCM) to study intact resistance arteries isolated from patients with CLI, specifically to identify the cellular aspects of the morphological changes identified in ischaemic subcutaneous and skeletal muscle resistance vessels. Using LSCM, a significant reduction in adventitial and medial thickness, cross-sectional area and wall : lumen ratio was confirmed in resistance arteries from both distal ischaemic subcutaneous and skeletal muscle vascular beds when compared with corresponding arteries from the proximal non-ischaemic sites. The cellular composition of the adventitial, medial and intimal layers of these distal ischaemic arteries was significantly different compared with proximal non-ischaemic arteries. These differences in the distal arteries were characterised by hypoplasia in the adventitial and medial layers of the arterial wall and hypertrophy in the intimal layer. The differences observed in both distal ischaemic vascular beds (subcutaneous and skeletal muscle) were similar. Experimental Physiology (2003) 88.4, 547-554.

For synaptic and cellular analyses of the mammalian respiratory network, intracellular recording and good pharmacological access to respiratory neurones is required. Using an existing arterially perfused in situ preparation of neonatal rat, we report on a method allowing stable intracellular recordings of ventrolateral medullary respiratory neurones. The in situ preparation generates a recognizable eupnoeic respiratory motor pattern similar to that reported in vivo. Using this preparation, we have developed a methodology for the use of patch pipettes to record from neurones within the ventral respiratory group. This technique in the arterially perfused neonatal rat is novel and has the advantage that neurones can be recorded from an intact and well-oxygenated brainstem in which the pontine regions are known to be viable. We describe the methods, present the first whole cell recordings of numerous types of respiratory neurones from neonatal rats in such a preparation, and demonstrate the applicability of the model for neuropharmacological experiments. Experimental Physiology (2003) 88.6, 725-732.

Chewing and acid on the tongue evoked a flow of saliva from the duct-cannulated parotid gland of the conscious rat despite pretreatment with atropine and adrenoceptor antagonists. This non-adrenergic, non-cholinergic (NANC) response depended on an intact parasympathetic innervation and was abolished by a tachykinin antagonist. The present findings are consistent with a physiological role for the secretory NANC mechanisms of the salivary glands.

The purpose of the study was to investigate whether jaw reflexes evoked by selective stimulation of periodontal ligament mechanoreceptors are susceptible to modulation by remote noxious stimulation. Experiments were performed on 10 volunteer subjects. Skin surface recordings were made from the jaw-closing masseter muscle. The subjects activated the muscle to approximately 10% of maximum by biting on a rubber impression of their molar teeth while they received visual feedback of the electromyogram (EMG) of the muscle. Reflexes were produced by the application of gentle mechanical stimuli to an upper central incisor tooth. The stimuli were in the form of ‘ramp and hold’ forces with a 5 ms rise-time and a 1.5 N plateau which lasted 350ms. The resulting reflexes were recorded both under control conditions and while the subjects received a remote noxious stimulus (immersion of a hand in water at 3°C). In all 10 subjects, the stimuli produced a single period of inhibition of masseteric activity (latency, 12.8±0.4 ms; duration, 18.1±1.3 ms; means ± S.E.M.), which was usually followed by a period of increased masseteric activity. The period of inhibition constituted a downward wave in full-wave rectified, averaged signals. The integrals of such waves were significantly smaller (by 17±6.5 %; P = 0.027; Student's t test) when the reflex was evoked during remote noxious stimulation rather than under control conditions. As such reflexes are believed to play a modulatory role during normal oral function, this finding may be relevant to disorders of mastication associated with pain.

Radical oxygen species are a byproduct of normal energy metabolism in mitochondria. The short-lived radicals cause damage to their immediate surrounding, i.e. the mitochondria. While most of this damage will be removed by normal mitochondrial turnover, damage to mitochondrial DNA (mtDNA) can persist and may accumulate with age. Recent evidence indicates that mutant mtDNA molecules can accumulate within individual cells, potentially hampering mitochondrial function. Experimental Physiology (2003) 88.1, 167-174.

Bladder, or detrusor, instability is a symptomatic condition which is characterized by uncontrollable rises in pressure within the bladder lumen leading to a feeling of urgency of micturition and, if severe enough, urinary incontinence. The rises in pressure are brought about by contraction of the detrusor smooth muscle which forms the major tissue component of the urinary bladder wall. This abnormality can be associated with a number of clinical conditions and when there is evidence of associated neurological deficits it is termed detrusor hyper-reflexia. An increase in bladder outflow resistance is also associated with detrusor instability and this is especially prevalent in men due to the age-related hypertrophy of the prostate gland which surrounds the urethra. However, in the majority of cases the cause is idiopathic and probably multifactorial. No matter what the cause, detrusor instability is one the largest causes of morbidity in the community; in the UK its prevalence increases from about 2 % in men and 9 % in women aged 15-64 years to 7 % and 12 %, respectively, in the over 65 years age group (Thomas et al. 1980; Brocklehurst, 1993).

The two neurohypophysial hormones arginine vasopressin (AVP) and oxytocin have actions in the inner medullary collecting duct (IMCD) where both peptides induce an increase in cAMP accumulation. The present study has employed a novel IMCD cell line to determine whether these two hormones induce cAMP accumulation via common or separate receptors, and to characterize the potential receptors responsible. Equal volumes of vehicle (150 mM NaCl) or hormone/antagonist solutions were added to aliquots of 104 IMCD cells in the presence of 10-3 M 3-isobutylmethylxanthine (IBMX) and incubated at 37°C for 4 min. cAMP levels were determined by radioimmunoassay and protein concentration by Bradford assay. Both AVP and oxytocin elicited dose-dependent increases in cAMP generation, though oxytocin was less potent than AVP (EC50 = 1Σ6 × 10-8 M vs. 7Σ4 × 10-10 M). AVP at 10-8 M and oxytocin at 10-6 M, concentrations sufficient to elicit near-maximal cAMP accumulation, resulted in cAMP levels of 73Σ4 ± 1Σ7 and 69Σ0 ± 3Σ3 pmol (mg protein)-1 (4 min)-1, respectively (n = 10), compared with the vehicle-treated basal value of 37Σ7 ± 2Σ2 pmol (mg protein)-1 (4 min)-1 (P < 0Σ001, n = 10). Combined AVP (10-8 M) and oxytocin (10-6 M) resulted in cAMP accumulation of 63Σ8 ± 3Σ1 pmol (mg protein)-1 (4 min)-1 (n = 10), which was not significantly different from the effect of oxytocin alone, but slightly less than that for AVP alone (P < 0Σ05). A submaximal concentration of AVP (10-10 M) induced cAMP accumulation of 48Σ6 ± 2Σ5 pmol (mg protein)-1 (4 min)-1 (P < 0Σ01 compared with basal level of 34Σ9 ± 2Σ4 pmol (mg protein)-1 (4 min)-1, n = 10), which was blocked in the presence of a vasopressin V2 receptor antagonist (10-7 M OPC-31260) but not by the oxytocin receptor antagonist (10-6 M [Pen1,pMePhe2,Thr4,Orn8]oxytocin) (36Σ3 ± 6Σ1 and 45Σ1 ± 1Σ3 pmol (mg protein)-1 (4 min)-1 respectively, P < 0Σ05, n = 10). A submaximal concentration of oxytocin (10-7 M) induced a cAMP accumulation of 45Σ8 ± 1Σ8 pmol (mg protein)-1 (4 min)-1 (n = 10), which was reduced by addition of 10-6 M oxytocin antagonist (36Σ3 ± 2Σ1 pmol (mg protein)-1 (4 min)-1, P < 0Σ05, n = 10), whereas co-incubation with 10-6 M of the V2 receptor antagonist had no effect (43Σ2 ± 1Σ3 pmol (mg protein)-1 (4 min)-1, n = 10). These results indicate that AVP and oxytocin induce cAMP accumulation from a common ATP pool in IMCD cells, and that separate vasopressin V2 and oxytocin receptor systems are involved, perhaps coupled to a common adenylate cyclase system.

The aim of this study was to test the hypothesis that Huntington's disease is associated with impaired phospholipid-related signal transduction using the niacin skin flush test. This is the first reported use of this test in this patient group. The response to topical aqueous methyl nicotinate solution was recorded at 5 min intervals over 20 min in six in-patients with advanced (stage III) Huntington's disease and in 14 age- and sex-matched normal individuals with no history of this or any other major neurological disorder. The volumetric niacin response (VNR) (mean ± S.E.M.) in the patients with Huntington's disease, 16.3 ± 2.6 mol s l-1, was significantly lower than the mean VNR of 28.3 ± 2.1 mol s l-1 in the control group (P = 0.004). These results are consistent with the conclusion that Huntington's disease may be associated with an abnormality of neuronal membrane fatty acid metabolism, possibly as a consequence of an as yet unidentified action of huntingtin. Experimental Physiology (2001) 86.5, 683-685.

The rapid increase in body mass that often occurs following creatine (Cr) supplementation is believed to be due to intracellular water retention. The purpose of this study was to determine whether Cr consumption alters the magnetic resonance (MR) transverse relaxation (T2) distribution of skeletal muscle. Transverse relaxation can be used to model water compartments within a cell or tissue. In this double-blind study, subjects were asked to supplement their normal diet with creatine monohydrate (20 g day-1 for 5 days) mixed with a grape drink (Creatine group, n = 7), or the grape drink alone (Placebo group, n = 8). Phosphorous MR spectroscopy was used to determine the effectiveness of the supplementation protocol. Subjects that responded to the Cr supplementation (i.e. showed a > 5 % increase in the ratio of the levels of phosphocreatine (PCr) and ATP) were placed in the Creatine group. Both proton MR imaging and spectroscopy were used to acquire T2 data, at 1.89 T, from the flexor digitorum profundus muscle of each subject before and after supplementation. Following the supplementation period, the Creatine group showed a gain in body mass (1.2 ± 0.8 kg, P < 0.05, mean ± S.D.), and an increase in PCr/ATP ratio (23.8 ± 16.4 %, P < 0.001). Neither group showed any changes in intracellular pH or T2 calculated from MR images. However, the spectroscopy data revealed at least three components (> 5 ms) at approximately 20, 40 and 125 ms in both groups. Only in the Creatine group was there an increase in the apparent proton concentration of the two shorter components combined (+5.0 ± 4.7 %, P < 0.05). According to the cellular water compartment model, the changes observed in the shorter T2 components are consistent with an increase in intracellular water. Experimental Physiology (2002) 87.3, 383-389.

During prolonged exercise in a cold environment, fatigue is generally associated with a depletion of endogenous glycogen stores. This has lead many authors to hypothesize that the carbohydrate (CHO) content of fluids ingested in cool environments should be high, yet this hypothesis has not been specifically examined. In the present study, six healthy males cycled to exhaustion at approximately 80 % of their maximum oxygen consumption ([Vdot]O2,max) with either no drink (ND), a 15 % CHO-electrolyte drink (15 % CHO) or a 2 % CHO-electrolyte drink (2 % CHO). Dietary intake and exercise were replicated 2 days prior to each trial. Mean (S.D.) ambient temperature was 10·0 (0·3)°C with a relative humidity of 72 (2) % and an air velocity of approximately 0·7 m s-1 on all trials. Weighted mean skin temperature was calculated, and rectal temperature and heart rate were recorded at rest, during exercise and at exhaustion. Venous samples were drawn before and during exercise and at exhaustion for determination of haemoglobin, haematocrit, blood metabolites and serum electrolytes and osmolality. Expired air was collected for calculation of [Vdot]O2 and respiratory exchange ratio which were used to estimate rates of fuel oxidation. Ratings of perceived exertion (RPE) were also obtained. Exercise capacity was not different (P = 0·49) between trials, with median (range) times to exhaustion of 90·6 (66·8-106·0), 97·7 (60·9-112·0) and 102·0 (77·1-170·7) min for the ND, 15 % CHO and 2 % CHO trials, respectively. The 15 % CHO drink significantly (P < 0·05) elevated blood glucose concentration and total CHO oxidation compared with the ND trial. The 2 % CHO drink significantly (P < 0·05) reduced the estimated change in plasma volume and resulted in a lower serum osmolality than the other two trials. No differences were observed in any thermoregulatory or cardiorespiratory responses between trials. These data suggest that maintenance of blood-borne substrate and/or plasma volume does not influence exercise capacity in the cold, as is commonly reported during this type and intensity of exercise in warmer environments.

The effects of osmotic stress on the cytosolic Ca2+ concentration ([Ca2+]i) in the endothelium of excised intact rat aorta were investigated using the indicator fura-2 and the patch clamp technique. Hyperosmotic stress evoked a reversible rise in endothelial [Ca2+]i in the presence but not absence of extracellular Ca2+, indicating that it evoked Ca2+ entry without release from intracellular stores. Hyposmotic stress was without significant effect. Cytochalasins B and D reduced the effect of hyperosmotic stress but not acetylcholine on endothelial [Ca2+]i. In endothelium isolated from underlying smooth muscle, hyperosmotic stress produced a rise in the [Ca2+]i and depolarisation of the endothelial membrane potential whereas hyposmotic stress was without effect. Mechanosensitive cationic channels recorded in cell-attached patches were activated by hyperosmotic solutions applied to the endothelium and inhibited by hyposmotic solutions. These data suggest that shrinkage of endothelial cells evokes an increase in [Ca2+]i by opening a pathway for Ca2+ entry from the extracellular space. The mechanosensitive ionic channels which we have previously described may be responsible for this response.

Activation of purinergic P2X receptors, putatively P2X1, may be important in the initiation of contraction in human detrusor. Purinergic transmission may be more important in muscle taken from patients with bladder instability. In this study the presence of the P2X1 receptor subtype was confirmed using RT-PCR. In addition, the results indicate, at the mRNA level, the presence of a splice variant of P2X1 that is lacking part of the second transmembrane domain. It is therefore possible that human bladder expresses multiple isoforms of the P2X1 receptor which may be potential sites for modifying or regulating putative purinergic activation of the human bladder.

This study was designed to investigate the effect of both hypertension and ageing on the efficiency of glucose metabolism. A 12-sample, 120 min intravenous glucose tolerance test (IVGTT) was applied to 36 rats: two groups of nine young (12 weeks) spontaneously hypertensive and Wistar Kyoto rats (Y-SHR and Y-WKY group, respectively) and two groups of nine old (40 weeks) SHR and WKY rats (O-SHR and O-WKY group, respectively). Insulinaemia and glycaemia data were interpreted in terms of estimates of glucose effectiveness, SG, and insulin sensitivity, SI, provided by the minimal model of glucose kinetics. The possible link between insulin resistance and hypertension was investigated by comparing Y-SHR vs. Y-WKY and O-SHR vs. O-WKY groups. Comparison of O-SHR vs. Y-SHR and O-WKY vs. Y-WKY groups enabled us to investigate the role of age in the development of abnormalities in glucose metabolism. No significant differences (P > 0.05) were observed in the mean SG and SI estimates between SHR and age-matched WKY groups. This finding indicates that exposure of SHR to high blood pressure levels does not necessarily lead to the development of insulin resistance and impaired glucose effectiveness. Similarly, no significant differences (P > 0.05) were observed in SG and SI estimates between old and young SHR and WKY groups. This finding indicates that, in this animal model of hypertension, insulin sensitivity and glucose effectiveness do not even deteriorate with ageing. Experimental Physiology (2003) 88.3, 399-404.