Journal of Physiology

Ageing is associated with a decline in muscle mass and strength leading to increased physical dependency in old age. Postmenopausal women experience a greater decline than men of similar age in parallel with the decrease in female sex steroid hormone production. We recruited six monozygous female twin pairs (55–59 years old) where only one twin pair was on hormone replacement therapy (HRT use = 7.8 ± 4.3 years) to investigate the association of HRT with the cytoplasmic volume supported by individual myonuclei (myonuclear domain (MND) size,) together with specific force at the single fibre level. HRT use was associated with a significantly smaller (~27%; P < 0.05) mean MND size in muscle fibres expressing the type I but not the IIa myosin heavy chain (MyHC) isoform. In comparison to non-users, higher specific force was recorded in HRT users both in muscle fibres expressing type I (~27%; P < 0.05) and type IIa (~23%; P < 0.05) MyHC isoforms. These differences were fibre-type dependent, i.e. the higher specific force in fast-twitch muscle fibres was primarily caused by higher force per cross-bridge while slow-twitch fibres relied on both a higher number and force per cross-bridge. HRT use had no effect on fibre cross-sectional area (CSA), velocity of unloaded shortening (V0) and relative proportion of MyHC isoforms. In conclusion, HRT appears to have significant positive effects on both regulation of muscle contraction and myonuclei organization in postmenopausal women.

The impact of 17-oestradiol (E2) exposure on autonomic control of orthostasis in young women is unclear. We tested the hypothesis that autonomic cardiovascular regulation is more sensitive to E2 exposure in women with low orthostatic tolerance. Women underwent an initial maximal lower body negative pressure (LBNP) test to place them into a low (LT, n = 7, 22 ± 1 years old, body mass index 22 ± 1 kg m–2) or a high orthostatic tolerance group (HT, n = 7, 22 ± 1 years old, body mass index 24 ± 1 kg m–2). We then suppressed endogenous reproductive hormone production using a gonadotrophin-releasing hormone antagonist (GnRHant) for 10 days, with E2 administration during the last 7 days of GnRHant. We measured R–R interval and beat-by-beat blood pressure during the modified Oxford protocol, and changes in heart rate, blood pressure and forearm vascular resistance (FVR) during submaximal LBNP. During submaximal LBNP, FVR increased in HT (ANOVA P < 0.05) but not in LT (ANOVA P > 0.05), and stroke volume was lower in LT relative to HT at all levels of LBNP (P < 0.05). Compared with GnRHant, E2 administration shifted FVR lower in LT (ANOVA P < 0.05), with no effect in HT. Administration of E2 increased baroreflex control of heart rate (derived from the modified Oxford protocol) in LT (GnRHant 10.7 ± 2.5 ms mmHg–1 vs. E2 16.1 ± 2.4 ms mmHg–1, P < 0.05) but not in HT (GnRHant 13.4 ± 1.9 ms mmHg–1 vs. E2 15.3 ± 2.4 ms mmHg–1, n.s.). In conclusion, blunted peripheral vasoconstriction and lower stroke volume contribute to compromised orthostatic tolerance in women; this inability to vasoconstrict is further exacerbated by exposure to E2. Furthermore, E2 administration increases baroreflex-mediated heart rate responses to orthostasis in low orthostatic tolerant women, which is likely to be a compensatory mechanism for the blunted peripheral vascular resistance and lower central volume.

Diet-induced obesity (DIO) has been shown to alter the biophysical properties and pharmacological responsiveness of vagal afferent neurones and fibres, although the effects of DIO on central vagal neurones or vagal efferent functions have never been investigated. The aims of this study were to investigate whether high-fat diet-induced DIO also affects the properties of vagal efferent motoneurones, and to investigate whether these effects were reversed following weight loss induced by Roux-en-Y gastric bypass (RYGB) surgery. Whole-cell patch-clamp recordings were made from rat dorsal motor nucleus of the vagus (DMV) neurones in thin brainstem slices. The DMV neurones from rats exposed to high-fat diet for 12–14 weeks were less excitable, with a decreased membrane input resistance and decreased ability to fire action potentials in response to direct current pulse injection. The DMV neurones were also less responsive to superfusion with the satiety neuropeptides cholecystokinin and glucagon-like peptide 1. Roux-en-Y gastric bypass reversed all of these DIO-induced effects. Diet-induced obesity also affected the morphological properties of DMV neurones, increasing their size and dendritic arborization; RYGB did not reverse these morphological alterations. Remarkably, independent of diet, RYGB also reversed age-related changes of membrane properties and occurrence of charybdotoxin-sensitive (BK) calcium-dependent potassium current. These results demonstrate that DIO also affects the properties of central autonomic neurones by decreasing the membrane excitability and pharmacological responsiveness of central vagal motoneurones and that these changes were reversed following RYGB. In contrast, DIO-induced changes in morphological properties of DMV neurones were not reversed following gastric bypass surgery, suggesting that they may be due to diet, rather than obesity. These findings represent the first direct evidence for the plausible effect of RYGB to improve vagal neuronal health in the brain by reversing some effects of chronic high-fat diet as well as ageing. Vagovagal neurocircuits appear to remain open to modulation and adaptation throughout life, and understanding of these mechanisms may help in development of novel interventions to alleviate environmental (e.g. dietary) ailments and also alter neuronal ageing.

Studies of regulation of the haematopoietic growth factor erythropoietin led to the unexpected discovery of a widespread system of direct oxygen sensing that regulates gene expression in animals. The oxygen-sensitive signal is generated by a series of non-haem Fe(II)- and 2-oxoglutarate-dependent dioxygenases that catalyse the post-translational hydroxylation of specific residues in the transcription factor hypoxia-inducible factor (HIF). These hydroxylations promote both oxygen-dependent degradation and oxygen-dependent inactivation of HIF, but are suppressed in hypoxia, leading to the accumulation of HIF and assembly of an active transcriptional complex in hypoxic cells. Hypoxia-inducible factor activates an extensive transcriptional cascade that interfaces with other cell signalling pathways, microRNA networks and RNA–protein translational control systems. The relationship of these cellular signalling pathways to the integrated physiology of oxygen homeostasis and the implication of dysregulating these massive physiological pathways in diseases such as cancer are discussed.

This review focuses on how smooth muscle sarcoplasmic reticulum (SR), the major releasable Ca2+ store in these cells, performs its many functions by communicating with the plasma membrane (PM) and other organelles across cytoplasmic nanospaces, defined by membrane–membrane junctions less than 50 nm across. In spite of accumulating evidence in favour of the view that cytoplasmic nanospaces are a prerequisite for effective control of diverse cellular functions, our current understanding of how smooth muscle cells accomplish site- and function-specific Ca2+ signalling remains in its infancy. We first present evidence in support of the view that effective Ca2+ signalling depends on the restricted diffusion of Ca2+ within cytoplasmic nanospaces. We then develop an evidence-based model of the smooth muscle SR – the ‘pan-junctional SR' model – that incorporates a network of tubules and quilts that are capable of auto-regulating their Ca2+ content and determining junctional [Ca2+]i through loading and unloading at membrane–membrane nanojunctions. Thereby, we provide a novel working hypothesis in order to inform future investigation into the control of a variety of cellular functions by local Ca2+ signals at junctional nanospaces, from contraction and energy metabolism to nuclear transcription. Based on the current literature, we discuss the molecular mechanisms whereby the SR mediates these multiple functions through the interaction of ion channels and pumps embedded in apposing membranes within inter-organellar junctions. We finally highlight the fact that although most current hypotheses are qualitatively supported by experimental data, solid quantitative simulations are seriously lacking. Considering that at physiological concentrations the number of calcium ions in a typical junctional nanospace between the PM and SR is of the order of 1, ion concentration variability plays a major role as the currency of information transfer and stochastic quantitative modelling will be required to both test and develop working hypotheses.

The genotype–phenotype map (GP map) concept applies to any time point in the ontogeny of a living system. It is the outcome of very complex dynamics that include environmental effects, and bridging the genotype–phenotype gap is synonymous with understanding these dynamics. The context for this understanding is physiology, and the disciplinary goals of physiology do indeed demand the physiological community to seek this understanding. We claim that this task is beyond reach without use of mathematical models that bind together genetic and phenotypic data in a causally cohesive way. We provide illustrations of such causally cohesive genotype–phenotype models where the phenotypes span from gene expression profiles to development of whole organs. Bridging the genotype–phenotype gap also demands that large-scale biological (‘omics') data and associated bioinformatics resources be more effectively integrated with computational physiology than is currently the case. A third major element is the need for developing a phenomics technology way beyond current state of the art, and we advocate the establishment of a Human Phenome Programme solidly grounded on biophysically based mathematical descriptions of human physiology.

Cardiac Na+–Ca2+ exchange (NCX) activity is regulated by [Ca2+]i. The physiological role and dynamics of this process in intact cardiomyocytes are largely unknown. We examined NCX Ca2+ activation in intact rabbit and mouse cardiomyocytes at 37°C. Sarcoplasmic reticulum (SR) function was blocked, and cells were bathed in 2 mm Ca2+. We probed Ca2+ activation without voltage clamp by applying Na+-free (0 Na+) solution for 5 s bouts, repeated each 10 s, which should evoke [Ca2+]i transients due to Ca2+ influx via NCX. In rested rabbit myocytes, Ca2+ influx was undetectable even after 0 Na+ applications were repeated for 2–5 min or more, suggesting that NCX was inactive. After external electric field stimulation pulses were applied, to admit Ca2+ via L-type Ca2+ channels, 0 Na+ bouts activated Ca2+ influx efficaciously, indicating that NCX had become active. Calcium activation increased with more field pulses, reaching a maximum typically after 15–20 pulses (1 Hz). At rest, NCX deactivated with a time constant typically of 20–40 s. An increase in [Na+]i, either in rabbit cardiomyocytes as a result of inhibition of Na+–K+ pumping, or in mouse cardiomyocytes where normal [Na+]i is higher vs. rabbit, sensitized NCX to self-activation by 0 Na+ bouts. In experiments with the SR functional but initially empty, the activation time course was slowed. It is possible that the SR initially accumulated Ca2+ that would otherwise cause activation. We modelled Ca2+ activation as a fourth-order highly co-operative process ([Ca]i required for half-activation K0.5act = 375 nm), with dynamics severalfold slower than the cardiac cycle. We incorporated this NCX model into an established ventricular myocyte model, which allowed us to predict responses to twitch stimulation in physiological conditions with the SR intact. Model NCX fractional activation increased from 0.1 to 1.0 as the frequency was increased from 0.2 to 2 Hz. By adjusting Ca2+ activation on a multibeat time scale, NCX might better maintain a stable long-term Ca2+ balance while contributing to the ability of myocytes to produce Ca2+ transients over a wide range of intensity.

We examined junctional conductance (gj) and its dependence on transjunctional voltage in gap junction (GJ) channels formed of wild-type connexin36 (Cx36) or its fusion form with green fluorescent protein (Cx36-EGFP) transfected in HeLa cells or endogenously expressed in primary culture of pancreatic -cells. Only a very small fraction (~0.8%) of Cx36-EGFP channels assembled into junctional plaques of GJs were open under control conditions. We found that short carbon chain n-alkanols (SCCAs) increased gj, while long carbon chain n-alkanols resulted in full uncoupling; cutoff is between heptanol and octanol. The fraction of functional channels and gj increased several fold under an exposure to SCCAs, or during reduction of endogenous levels of arachidonic acid (AA) by exposure to fatty acid-free BSA or cytosolic phospholipase A2 inhibitors. Moreover, uncoupling caused by exogenously applied AA can be rescued by BSA, which binds AA and other polyunsaturated fatty acids (PUFAs), but not by BSA modified with 1,2-cyclohexanedione, which does not bind AA and other PUFAs. We propose that under control conditions, Cx36 GJ channels in HeLa transfectants and -cells are inhibited by endogenous AA, which stabilizes a closed conformational state of the channel that leads to extremely low fraction of functional channels. In addition, SCCAs increase gj by interfering with endogenous AA-dependent inhibition, increasing open probability and the fraction of functional channels.

Augmented inositol 1,4,5-trisphosphate receptor (InsP3R) function has been linked to a variety of cardiac pathologies, including cardiac arrhythmia. The contribution of inositol 1,4,5-trisphosphate-induced Ca2+ release (IP3ICR) in excitation-contraction coupling (ECC) under physiological conditions, as well as under cellular remodelling, remains controversial. Here we test the hypothesis that local IP3ICR directly affects ryanodine receptor (RyR) function and subsequent Ca2+-induced Ca2+ release in atrial myocytes. IP3ICR was evoked by UV-flash photolysis of caged InsP3 under whole-cell configuration of the voltage-clamp technique in atrial myocytes isolated from C57/BL6 mice. Photolytic release of InsP3 was accompanied by a significant increase in the Ca2+ release event frequency (4.14 ± 0.72 vs. 6.20 ± 0.76 events (100 m)–1 s–1). These individual photolytically triggered Ca2+ release events were identified as Ca2+ sparks, which originated from RyR openings. This was verified by Ca2+ spark analysis and pharmacological separation between RyR and InsP3R-dependent sarcoplasmic reticulum (SR)-Ca2+ release (2-aminoethoxydiphenyl borate, xestospongin C, tetracaine). Significant SR-Ca2+ flux but eventless SR-Ca2+ release through InsP3R were characterized using SR-Ca2+ leak/SR-Ca2+ load measurements. These results strongly support the idea that IP3ICR can effectively modulate RyR openings and Ca2+ spark probability. We conclude that eventless and highly efficient InsP3-dependent SR-Ca2+ flux is the main mechanism of functional cross-talk between InsP3Rs and RyRs, which may be an important factor in the modulation of ECC sensitivity.

Delayed cord clamping improves circulatory stability in preterm infants at birth, but the underlying physiology is unclear. We investigated the effects of umbilical cord clamping, before and after ventilation onset, on cardiovascular function at birth. Prenatal surgery was performed on lambs (123 days) to implant catheters into the pulmonary and carotid arteries and probes to measure pulmonary (PBF), carotid (CaBF) and ductus arteriosus blood flows. Lambs were delivered at 126 ± 1 days and: (1) the umbilical cord was clamped at delivery and ventilation was delayed for about 2 min (Clamp 1st; n = 6), and (2) umbilical cord clamping was delayed for 3–4 min, until after ventilation was established (Vent 1st; n = 6). All lambs were subsequently ventilated for 30 min. In Clamp 1st lambs, cord clamping rapidly (within four heartbeats), but transiently, increased pulmonary and carotid arterial pressures (by ~30%) and CaBF (from 30.2 ± 5.6 to 40.1 ± 4.6 ml min–1 kg–1), which then decreased again within 30–60 s. Following ventilation onset, these parameters rapidly increased again. In Clamp 1st lambs, cord clamping reduced heart rate (by ~40%) and right ventricular output (RVO; from 114.6 ± 14.4 to 38.8 ± 9.7 ml min–1 kg–1), which were restored by ventilation. In Vent 1st lambs, cord clamping reduced RVO from 153.5 ± 3.8 to 119.2 ± 10.6 ml min–1 kg–1, did not affect heart rates and resulted in stable blood flows and pressures during transition. Delaying cord clamping for 3–4 min until after ventilation is established improves cardiovascular function by increasing pulmonary blood flow before the cord is clamped. As a result, cardiac output remains stable, leading to a smoother cardiovascular transition throughout the early newborn period.

Intrauterine inflammation is associated with preterm birth and poor long-term cardiopulmonary outcomes. We aimed to determine the effect of intrauterine inflammation on the cardiopulmonary and cerebral haemodynamic transition at birth, and the response to subsequent haemodynamic challenge. Fetal instrumentation was performed at ~112 days gestation (term is 147 days) for measurement of cardiopulmonary and cerebral haemodynamics. At 118 days, inflammation was induced by intra-amniotic administration of lipopolysaccharide (LPS; n = 7); controls (n = 5) received intra-amniotic saline. At 125 days lambs were delivered and mechanically ventilated. Arterial blood gases, pulmonary and systemic arterial blood pressures and flows were measured during the perinatal period. At 10 min a haemodynamic challenge was administered by increasing positive end-expiratory pressure. During the first 10 min after birth, LPS-exposed lambs had higher pulmonary vascular resistance and lower pulmonary blood flow and left ventricular output than controls. Carotid arterial blood flow was higher in LPS-exposed lambs than controls between 3 and 7 min after delivery, and cerebral oxygen delivery was higher at 5 min. During the haemodynamic challenge, pulmonary blood flow and left ventricular output were reduced in controls but not in LPS-exposed lambs; a transient reduction in brachiocephalic arterial pressure occurred in LPS-exposed lambs but not in controls. Intrauterine inflammation altered the cardiopulmonary and cerebral haemodynamic transition at birth and reduced the cardiopulmonary response to a haemodynamic challenge after birth. The transient reduction in brachiocephalic arterial pressure suggests intrauterine inflammation may alter cerebrovascular control following an increase in positive end-expiratory pressure.

The role of nitric oxide (NO) in regulating lymphatic contractile function and, consequently, lymph flow has been the subject of intense study. Despite this, the precise effects of NO on lymphatic contractile activity remain unclear. Recent hypotheses posit that basal levels of endogenous NO increase lymphatic contraction strength as a consequence of lowering frequency (i.e. positive lusitropy), whereas higher agonist-evoked concentrations of NO exert purely inhibitory effects on contractile function. We tested both hypotheses directly by isolating and cannulating collecting lymphatic vessels from genetically modified mice for ex vivo study. The effects of basal NO and agonist-evoked NO were evaluated, respectively, by exposing wild-type (WT), endothelial NO synthase (eNOS)–/– and inducible NO synthase (iNOS)–/– lymphatic vessels to controlled pressure steps followed by ACh doses. To compare with pharmacological inhibition of eNOS, we repeated both tests in the presence of l-NAME. Surprisingly, genetic removal of basal NO enhanced contraction amplitude significantly without increasing contraction frequency. Higher levels of NO production stimulated by ACh evoked dilation, decreased tone, slowed contraction frequency and reduced fractional pump flow. We conclude that basal NO specifically depresses contraction amplitude, and that greater NO production then inhibits all other aspects of contractile function. Further, this work demonstrates definitively that mouse collecting lymphatic vessels exhibit autonomous, large-amplitude contractions that respond to pressure similarly to collecting lymphatics of other mammalian species. At least in the peripheral lymphatic vasculature, NO production depresses contractile function, which influences lymph flow needed for fluid regulation, humoral immunity and cancer metastasis.

Regulation of blood flow in microcirculatory networks depends on spread of local vasodilatation to encompass upstream arteries; a process mediated by endothelial conduction of hyperpolarization. Given that endothelial coupling is reduced in hypertension, we used hypertensive Cx40ko mice, in which endothelial coupling is attenuated, to investigate the contribution of the renin–angiotensin system and reduced endothelial cell coupling to conducted vasodilatation of cremaster arterioles in vivo. When the endothelium was disrupted by light dye treatment, conducted vasodilatation, following ionophoresis of acetylcholine, was abolished beyond the site of endothelial damage. In the absence of Cx40, sparse immunohistochemical staining was found for Cx37 in the endothelium, and endothelial, myoendothelial and smooth muscle gap junctions were identified by electron microscopy. Hyperpolarization decayed more rapidly in arterioles from Cx40ko than wild-type mice. This was accompanied by a shift in the threshold potential defining the linear relationship between voltage and diameter, increased T-type calcium channel expression and increased contribution of T-type (3 mol l–1 NNC 55-0396), relative to L-type (1 mol l–1 nifedipine), channels to vascular tone. The change in electromechanical coupling was reversed by inhibition of the renin–angiotensin system (candesartan, 1.0 mg kg–1 day–1 for 2 weeks) or by acute treatment with the superoxide scavenger tempol (1 mmol l–1). Candesartan and tempol treatments also significantly improved conducted vasodilatation. We conclude that conducted vasodilatation in Cx40ko mice requires the endothelium, and attenuation results from both a reduction in endothelial coupling and an angiotensin II-induced increase in oxidative stress. We suggest that during cardiovascular disease, the ability of microvascular networks to maintain tissue integrity may be compromised due to oxidative stress-induced changes in electromechanical coupling.

Efferent signals from the vagus nerve are thought to mediate both basal and meal-induced gastric acid secretion, and provide trophic support of the mucosa. However, the underlying mechanisms are incompletely understood. Neurturin, signalling via glial cell line-derived neurotrophic factor (GDNF)-family receptor 2 (GFR2), is essential for parasympathetic innervation of many target tissues but its role in gastric innervation is unknown. Here we show that most nerve fibres in wild-type mouse gastric mucosa, including all positive for gastrin-releasing peptide, are cholinergic. GFR2-deficient (KO) mice lacked virtually all cholinergic nerve fibres and associated glial cells in the gastric (oxyntic and pyloric) mucosa but not in the smooth muscle, consistent with the selective expression of neurturin mRNA in the gastric mucosa. 2-Deoxyglucose and hexamethonium failed to affect acid secretion in the GFR2-KO mice indicating the lack of functional innervation in gastric mucosa. Interestingly, basal and maximal histamine-induced acid secretion did not differ between wild-type and GFR2-KO mice. Moreover, circulating gastrin levels in both fasted and fed animals, thickness of gastric mucosa, and density of parietal and different endocrine cells were similar. Carbachol-stimulated acid secretion was higher in GFR2-KO mice, while atropine reduced basal secretion similarly in both genotypes. We conclude that cholinergic innervation of gastric mucosa depends on neurturin-GFR2 signalling but is dispensable for gastrin secretion and for basal and maximal acid output. Basal acid secretion in the KO mice appears to be, at least partly, facilitated by constitutive activity of muscarinic receptors.

Duodenal epithelial cells need efficient defence strategies during gastric acidification of the lumen, while colonic mucosa counteracts damage by pathogens by building up a bacteria-free adherent mucus layer. Transport of HCO3– is considered crucial for duodenal defence against acid as well as for mucus release and expansion, but the transport pathways involved are incompletely understood. This study investigated the significance of the electroneutral Na+–HCO3– cotransporter NBCn1 for duodenal defence against acid and colonic mucus release. NBCn1 was localized to the basolateral membrane of duodenal villous enterocytes and of colonic crypt cells, with predominant expression in goblet cells. Duodenal villous enterocyte intracellular pH was studied before and during a luminal acid load by two-photon microscopy in exteriorized, vascularly perfused, indicator (SNARF-1 AM)-loaded duodenum of isoflurane-anaesthetized, systemic acid–base-controlled mice. Acid-induced HCO3– secretion was measured in vivo by single-pass perfusion and pH-stat titration. After a luminal acid load, NBCn1-deficient duodenocytes were unable to recover rapidly from intracellular acidification and could not respond adequately with protective HCO3– secretion. In the colon, build-up of the mucus layer was delayed, and a decreased thickness of the adherent mucus layer was observed, suggesting that basolateral HCO3– uptake is essential for optimal release of mucus. The electroneutral Na+–HCO3– cotransporter NBCn1 displays a differential cellular distribution in the murine intestine and is essential for HCO3–-dependent mucosal protective functions, such as recovery of intracellular pH and HCO3– secretion in the duodenum and secretion of mucus in the colon.

Body water balance is regulated via the water channel aquaporin-2 (AQP2), which is expressed in the renal connecting tubule (CNT) and collecting duct (CD). The relative roles of AQP2 in the CNT and CD are not fully understood. To study the role of AQP2 in the CNT we generated a mouse model with CNT-specific AQP2 deletion (AQP2-CNT-knockout (KO)). Confocal laser scanning microscopy and immunogold electron microscopy demonstrated an absence of AQP2 in the CNT of AQP2-CNT-KO mice. Twenty-four hour urine output was significantly increased (KO: 3.0 ± 0.3 ml (20 g body weight (BW))–1; wild-type (WT): 1.9 ± 0.3 ml (20 g BW)–1) and urine osmolality decreased (KO: 1179 ± 107 mosmol kg–1; WT: 1790 ± 146 mosmol kg–1) in AQP2-CNT-KO mice compared with controls. After 24 h water restriction, urine osmolality was still significantly lower in AQP2-CNT-KO mice (KO: 2087 ± 169 mosmol kg–1; WT: 2678 ± 144 mosmol kg–1). A significant difference in urine osmolality between groups before desmopressin (dDAVP) (KO: 873 ± 129 mosmol kg–1; WT: 1387 ± 163 mosmol kg–1) was not apparent 2 h after injection, with urine osmolality increased significantly in both groups (KO: 2944 ± 41 mosmol kg–1; WT: 3133 ± 66 mosmol kg–1). Cortical kidney fractions from AQP2-CNT-KO mice had significantly reduced AQP2, with no compensatory changes in sodium potassium chloride cotransporter (NKCC2), AQP3 or AQP4. Lithium chloride treatment increased urine volume and decreased osmolality in both WT and AQP2-CNT-KO mice. After 8 days of treatment, the AQP2-CNT-KO mice still had a significantly higher urine volume and lower urine osmolality, suggesting that the CNT does not play a significant role in the pathology of lithium-induced nephrogenic diabetes insipidus. Our studies indicate that the CNT plays a role in regulating body water balance under basal conditions, but not for maximal concentration of the urine during antidiuresis.