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1.  Effect of exercise and calorie restriction on biomarkers of aging in mice 
Unlike, calorie restriction, exercise fails to extend maximum life span, but the mechanisms that explain this disparate effect are unknown. We used a 24-wk protocol of treadmill running, weight matching, and pair feeding to compare the effects of exercise and calorie restriction on biomarkers related to aging. This study consisted of young controls, an ad libitum-fed sedentary group, two groups that were weight matched by exercise or 9% calorie restriction, and two groups that were weight matched by 9% calorie restriction + exercise or 18% calorie restriction. After 24 wk, ad libitum-fed sedentary mice were the heaviest and fattest. When weight-matched groups were compared, mice that exercised were leaner than calorie-restricted mice. Ad libitum-fed exercise mice tended to have lower serum IGF-1 than fully-fed controls, but no difference in fasting insulin. Mice that underwent 9% calorie restriction or 9% calorie restriction + exercise, had lower insulin levels; the lowest concentrations of serum insulin and IGF-1 were observed in 18% calorie-restricted mice. Exercise resulted in elevated levels of tissue heat shock proteins, but did not accelerate the accumulation of oxidative damage. Thus, failure of exercise to slow aging in previous studies is not likely the result of increased accrual of oxidative damage and may instead be due to an inability to fully mimic the hormonal and/or metabolic response to calorie restriction.
PMCID: PMC4332519  PMID: 18321952
energetics; obesity; energy balance
2.  Glucocorticoids increase osmotic water permeability (Pf) of neonatal rabbit renal brush border membrane vesicles 
Glucocorticoids increase osmotic water permeability (Pf) of neonatal rabbit renal brush border membrane vesicles. Am J Physiol Regul Integr Comp Physiol 288: R1417–R1421, 2005. First published January 20, 2005; doi:10.1152/ajpregu.00448.2004.— During postnatal maturation, there is an increase in renal brush border membrane vesicle (BBMV) osmotic water permeability and a parallel increase in aquaporin-1 (AQP1) protein abundance. The mechanisms responsible for these changes remain unknown. Because serum glucocorticoid levels rise postnatally and have previously been linked to other maturational changes in renal function, we examined the effects of glucocorticoids on osmotic (Pf) and diffusional (PDW) water permeability and AQP1 protein abundance of renal BBMV. Neonatal rabbits were treated with dexamethasone (10 μg/100 g) for three days and compared with control neonates and adults. Pf and PDW were measured at 20°C with a stopped-flow apparatus using light-scattering and aminonaphthalene trisulfonic acid (ANTS) fluorescence, respectively. Pf was significantly higher in BBMV from dexamethasone-treated neonates compared with vehicle-treated neonates, but remained lower than in BBMV from adults (P < 0.05). PDW in dexamethasone and vehicle-treated neonatal BBMV was lower than in adult BBMV. Pf/PDW ratio increased from neonate (5.1 ± 0.3) to dexamethasone (7.0 ± 0.1) and adult BBMV (6.3 ± 0.1). AQP1 expression was increased by dexamethasone treatment to adult levels. Membrane fluidity, which is inversely related to generalized polarization (GP) of steady-state laurdan fluorescence, was significantly higher in neonatal BBMV than both dexamethasone and adult BBMV (GP: neonate 0.285 ± 0.002, dexamethasone treatment 0.302 ± 0.006, and adult 0.300 ± 0.005; P < 0.05). These combined results show that dexamethasone-treatment during days 4–7 of life increases BBMV water permeability despite a decrease in membrane fluidity. This occurs by increasing channel-mediated water transport, as reflected in an increase in AQP1 protein abundance and a higher Pf/PDW ratio. This mimics the maturational changes and suggests a physiological role for glucocorticoids in maturation of proximal tubule water transport.
PMCID: PMC4131715  PMID: 15661970
stop-flow kinetics; diffusional water permeability; development; aquaporin
3.  Maturation of rat proximal tubule chloride permeability 
We have previously shown that neonate rabbit tubules have a lower chloride permeability but comparable mannitol permeability compared with adult proximal tubules. The surprising finding of lower chloride permeability in neonate proximals compared with adults impacts net chloride transport in this segment, which reabsorbs 60% of the filtered chloride in adults. However, this maturational difference in chloride permeability may not be applicable to other species. The present in vitro microperfusion study directly examined the chloride and mannitol permeability using in vitro perfused rat proximal tubules during postnatal maturation. Whereas there was no maturational change in mannitol permeability, chloride permeability was 6.3 ± 1.3 × 10−5 cm/s in neonate rat proximal convoluted tubule and 16.1 ± 2.3 × 10−5 cm/s in adult rat proximal convoluted tubule (P < 0.01). There was also a maturational increase in chloride permeability in the rat proximal straight tubule (5.1 ± 0.6 × 10−5 cm/s vs. 9.3 ± 0.6 × 10−5 cm/s, P < 0.01). There was no maturational change in bicarbonate-to-chloride permeabilities (PHCO3/PCl) in the rat proximal straight tubules (PST) and proximal convoluted tubules (PCT) or in the sodium-to-chloride permeability (PNa/PCl) in the proximal straight tubule; however, there was a significant maturational decrease in proximal convoluted tubule PNa/PCl with postnatal development (1.31 ± 0.12 in neonates vs. 0.75 ± 0.06 in adults, P < 0.001). There was no difference in the transepithelial resistance measured by current injection and cable analysis in the PCT, but there was a maturational decrease in the PST (7.2 ± 0.8 vs. 4.6 ± 0.1 Ω· cm2, P < 0.05). These studies demonstrate there are maturational changes in the rat paracellular pathway that impact net NaCl transport during development.
PMCID: PMC4131720  PMID: 16051720
paracellular pathway; mannitol permeability
4.  Ontogeny of rabbit proximal tubule urea permeability 
Urea transport in the proximal tubule is passive and is dependent on the epithelial permeability. The present study examined the maturation of urea permeability (Purea) in in vitro perfused proximal convoluted tubules (PCT) and basolateral membrane vesicles (BLMV) from rabbit renal cortex. Urea transport was lower in neonatal than adult PCT at both 37 and 25°C. The PCT Purea was also lower in the neonates than the adults (37°C: 45.4 ± 10.8 vs. 88.5 ± 15.2 × 10−6 cm/s, P < 0.05; 25°C: 28.5 ± 6.9 vs. 55.3 ± 10.4 ± 10−6 cm/s; P > 0.05). The activation energy for PCT Purea was not different between the neonatal and adult groups. BLMV Purea was determined by measuring vesicle shrinkage, due to efflux of urea, using a stop-flow instrument. Neonatal BLMV Purea was not different from adult BLMV Purea at 37°C [1.14 ± 0.05 × 10−6 vs. 1.25 ± 0.05 × 10−6 cm/s; P = not significant (NS)] or 25°C (0.94 ± 0.06 vs. 1.05 ± 0.10 × 10−6 cm/s; P = NS). There was no effect of 250 μM phloretin, an inhibitor of the urea transporter, on Purea in either adult or neonatal BLMV. The activation energy for urea diffusion was also identical in the neonatal and adult BLMV. These findings in the BLMV are in contrast to the brush-border membrane vesicles (BBMV) where we have previously demonstrated that urea transport is lower in the neonate than the adult. Urea transport is lower in the neonatal proximal tubule than the adult. This is due to a lower rate of apical membrane urea transport, whereas basolateral urea transport is the same in neonates and adults. The lower Purea in neonatal proximal tubules may play a role in overall urea excretion and in developing and maintaining a high medullary urea concentration and thus in the ability to concentrate the urine during renal maturation.
PMCID: PMC4126161  PMID: 11353675
apical membrane; transport; stop-flow kinetics; glycerol
5.  Exercise training normalizes enhanced glutamate-mediated sympathetic activation from the PVN in heart failure 
Exercise training (ExT) normalizes the increased sympathetic outflow in heart failure (HF), but the mechanisms are not known. We hypothesized ExT would normalize the augmented glutamatergic mechanisms mediated by NMDA receptors within the PVN that occurs with HF. Four groups of rats were used: 1) Sham Sedentary (Sed); 2) Sham ExT; 3) HF Sed; and 4) HF ExT. HF was induced by left coronary artery ligation, and ExT consisted of three weeks of treadmill running. In α-chloralose-urethane-anesthetized rats, the increase in renal sympathetic nerve activity (RSNA) in response to the highest dose of NMDA (200 pmol) injected into the PVN in the HF Sed group was approximately twice that of the Sham Sed group. In the HF ExT group the response was not different from the Sham Sed or Sham ExT groups. Relative NMDA receptor subunit NR1 mRNA expression was 63% higher in the HF Sed group compared to the Sham Sed group, but in the HF ExT group was not different from the Sham Sed or Sham ExT groups. NR1 receptor subunit protein expression was increased 87% in the HF Sed group compared to the Sham Sed group but in the HF ExT group was not significantly different from the Sham Sed or Sham ExT groups. Thus, one mechanism by which ExT alleviates elevated sympathetic outflow in HF may be through normalization of glutamatergic mechanisms within the PVN.
PMCID: PMC4107908  PMID: 18385465
paraventricular nucleus; heart failure; NMDA receptors; exercise training; sympathetic nerve activity
6.  Prenatal programming of rat proximal tubule Na+/H+ exchanger by dexamethasone 
Prenatal administration of dexamethasone causes hypertension in rats when they are studied as adults. Although an increase in tubular sodium reabsorption has been postulated to be a factor programming hypertension, this has never been directly demonstrated. The purpose of this study was to examine whether prenatal programming by dexamethasone affected postnatal proximal tubular transport. Pregnant Sprague-Dawley rats were injected with intraperitoneal dexamethasone (0.2 mg/kg) daily for 4 days between the 15th and 18th days of gestation. Prenatal dexamethasone resulted in an elevation in systolic blood pressure when the rats were studied at 7–8 wk of age compared with vehicle-treated controls: 131 ± 3 vs. 115 ± 3 mmHg (P < 0.001). The rate of proximal convoluted tubule volume absorption, measured using in vitro microperfusion, was 0.61 + 0.07 nl·mm−1·min−1 in control rats and 0.93 + 0.07 nl·mm−1·min−1 in rats that received prenatal dexamethasone (P < 0.05). Na+/H+ exchanger activity measured in perfused tubules in vitro using the pH-sensitive dye BCECF showed a similar 50% increase in activity in proximal convoluted tubules from rats treated with prenatal dexamethasone. Although there was no change in abundance of NHE3 mRNA, the predominant luminal proximal tubule Na+/H+ exchanger, there was an increase in NHE3 protein abundance on brush-border membrane vesicles in 7- to 8-wk-old rats receiving prenatal dexamethasone. In conclusion, prenatal administration of dexamethasone in rats increases proximal tubule transport when rats are studied at 7–8 wk old, in part by stimulating Na+/H+ exchanger activity. The increase in proximal tubule transport may be a factor mediating the hypertension by prenatal programming with dexamethasone.
PMCID: PMC4096979  PMID: 17095646
NHE3; in vitro microperfusion; volume absorption; acidification
7.  Food-entrained circadian rhythms are sustained in arrhythmic Clk/Clk mutant mice 
Daily scheduled feeding is a potent time cue that elicits anticipatory activity in rodents. This food-anticipatory activity (FAA) is controlled by a food-entrainable oscillator (FEO) that is distinct from light-entrained oscillators of the suprachiasmatic nucleus (SCN). Circadian rhythms within the SCN depend on transcription-translation feedback loops in which CLOCK protein is a key positive regulator. The Clock gene is expressed in rhythmic tissues throughout the brain and periphery, implicating its widespread involvement in the functioning of circadian oscillators. To examine whether CLOCK protein is also necessary for the FEO, the effect of daily food restriction was studied in homozygous Clock mutant (Clk/Clk) mice. The results show that Clk/Clk mutant mice exhibit FAA, even when their circadian wheel-running behavior is arrhythmic. As in wild-type controls, FAA in Clk/Clk mutants persists after temporal feeding cues are removed for several cycles, indicating that the FEO is a circadian timer. This is the first demonstration that the Clock gene is not necessary for the expression of a circadian, food-entrained behavior and suggests that the FEO is mediated by a molecular mechanism distinct from that of the SCN.
PMCID: PMC3837688  PMID: 12649127
food-anticipatory activity; feeding; entrainment; clock genes; suprachiasmatic nucleus
8.  Effect of Chronic Endothelin Receptor Antagonism on Cerebrovascular Function in Type 2 Diabetes 
Diabetes increases the risk of stroke and contributes to poor clinical outcomes in this patient population. Myogenic tone of the cerebral vasculature, including basilar arteries, plays a key role in controlling cerebral blood flow. Increased myogenic tone is ameliorated with ET receptor antagonism in Type 1 diabetes. However, the role of ET-1 and its receptors in cerebrovascular dysfunction in Type-2 diabetes, a common comorbidity in stroke patients, remains poorly elucidated. Therefore, we hypothesized that 1) cerebrovascular dysfunction occurs in the Goto-Kakizaki (GK) model of Type-2 diabetes, and 2) pharmacological antagonism of ETA receptors ameliorates while ETB receptor blockade augments vascular dysfunction. GK or control rats were treated with antagonists to either ETA (Atrasentan, 5mg/kg/d) or ETB (A-192621, 15 or 30 mg/kg/d) receptors for four weeks and vascular function of basilar arteries was assessed using a wire myograph. GK rats exhibited increased sensitivity to ET-1. ETA receptor antagonism caused a rightward shift indicating decreased sensitivity in diabetes while it increased sensitivity to ET-1 in control rats. Endothelium-dependent relaxation was impaired in diabetes. ETA receptor blockade restored relaxation to control values in the GK animals with no significant effect in Wistars and ETB blockade with 30 mg/kg/d A-192621 caused paradoxical constriction in diabetes. These studies demonstrate that cerebrovascular dysfunction occurs and may contribute to altered regulation of myogenic tone and cerebral blood flow in diabetes. While ETA receptors mediate vascular dysfunction, ETB receptors display differential effects. These results underscore the importance of ETA/ETB receptor balance and interactions in cerebrovascular dysfunction in diabetes.
PMCID: PMC3791629  PMID: 18287215
9.  Melatonin phase shifts human circadian rhythms in a placebo-controlled simulated night-work study 
There has been scant evidence for a phase-shifting effect of melatonin in shift-work or jet-lag protocols. This study tested whether melatonin can facilitate phase shifts in a simulated night-work protocol. Subjects (n = 32) slept in the afternoons/evenings before night work (a 7-h advance of the sleep schedule). They took melatonin (0.5 mg or 3.0 mg) or placebo before the first four of eight afternoon/evening sleep episodes at a time when melatonin has been shown to phase advance the circadian clock. Melatonin produced larger phase advances than placebo in the circadian rhythms of melatonin and temperature. Average phase advances (±SD) of the dim light melatonin onset were 1.7 ± 1.2 h (placebo), 3.0 ± 1.1 h (0.5 mg), and 3.9 ± 0.5 h (3.0 mg). A measure of circadian adaptation, shifting the temperature minimum enough to occur within afternoon/evening sleep, showed that only subjects given melatonin achieved this goal (73% with 3.0 mg, 56% with 0.5 mg, and 0% with placebo). Melatonin could be used to promote adaptation to night work and jet travel.
PMCID: PMC3696986  PMID: 11792655
body temperature; jet lag; sleep; sleepiness; shift work; work-schedule tolerance; humans; constant routine
10.  Working under pressure: coronary arteries and the endothelin system 
Endogenous endothelin-1-dependent (ET-1) tone in coronary arteries depends on the balance between ETA and ETB receptor-mediated effects and on parameters such as receptor distribution and endothelial integrity. Numerous studies highlight the striking functional interactions that exist between nitric oxide (NO) and ET-1 in the regulation of vascular tone. Many of the cardiovascular complications associated with cardiovascular risk factors and aging are initially attributable, at least in part, to endothelial dysfunction characterized by a dysregulation between NO and ET-1. The contribution of the imbalance between smooth muscle ETA/B and endothelial ETB receptors to this process is poorly understood. An increased contribution of ET-1 that is associated with a proportional decrease in that of NO accompanies the development of coronary endothelial dysfunction, coronary vasospasm, and atherosclerosis. These data form the basis for the rationale of testing therapeutic approaches counteracting ET-1-induced cardiovascular dysfunction. It remains to be determined whether the beneficial role of endothelial ETB receptors declines with age and risk factors for cardiovascular diseases, revealing smooth muscle ETB receptors with proconstricting and proinflammatory activities.
PMCID: PMC3693981  PMID: 20237301 CAMSID: cams2990
vascular tone; inflammation; shear stress; coronary blood flow
11.  Conditioned preference for sweet stimuli in OLETF rat: effects of food deprivation 
The Otsuka Long-Evans Tokushima Fatty (OLETF) rat, an outbred strain of Long Evans Tokushima Otsuka rat (LETO) that lacks CCK-1 receptor expression, is hyperphagic and develops obesity and type-2 diabetes. The present study sought to assess how OLETF rats alter intake, preference, and conditioned preference of palatable solutions after acute food deprivation. Our results show that after 24 hr chow restriction LETO rats increase both sucrose intake and two-bottle sucrose preference relative to their free-fed baseline, whereas OLETF rats do not increase sucrose intake (0.3M or 1.0M sucrose) or preference (1.0M vs. 0.3M sucrose) when food-deprived. In contrast, OLETF rats exhibit a higher conditioned flavor preference when sucrose is used as unconditioned stimulus (US) relative to LETO rats, whether overnight food-restricted (81% vs. 71% for OLETF and LETO rats, respectively) or free-fed (82% vs. 54% for OLETF and LETO rats, respectively) on test. When a non-caloric saccharin solution is used as US, OLETF rats show a higher preference for the saccharin-associated flavor relative to LETO rats when non-deprived (76% vs. 58% for OLETF and LETO rats, respectively), however neither strain shows differential conditioned flavor preference for saccharin in the deprivation state on test. These findings suggest that OLETF rats fail to integrate post-absorptive and orosensory effects of sucrose in a conditioning setting to influence intake. Thus, it appears that OLETF rats form preferences for sucrose based largely on orosensory and hedonic properties of the solution, rather than caloric value.
PMCID: PMC3594830  PMID: 17272669
food intake; CCK-1 receptor; hyperphagia; diet-induced obesity; palatability
12.  Organum vasculosum laminae terminalis contributes to increased sympathetic nerve activity induced by central hyperosmolality 
The contribution of the organum vasculosum laminae terminalis (OVLT) in mediating central hyperosmolality-induced increases of sympathetic nerve activity (SNA) and arterial blood pressure (ABP) was assessed in anesthetized rats. Solutions of graded NaCl concentration (150, 375, and 750 mM) were injected (150 μl) into the forebrain vascular supply via an internal carotid artery (ICA). Time-control experiments (n = 6) established that ICA NaCl injections produced short-latency, transient increases of renal SNA (RSNA) and mean ABP (MAP) (P < 0.05– 0.001). Responses were graded, highly reproducible, and unaltered by systemic blockade of vasopressin V1 receptors (n = 4). In subsequent studies, stimulus-triggered averaging of RSNA was used to accurately locate the OVLT. Involvement of OVLT in responses to ICA NaCl was assessed by recording RSNA and MAP responses before and 15 min after electrolytic lesion of the OVLT (n = 6). Before lesion, NaCl injections increased RSNA and MAP (P < 0.05– 0.001), similar to time control experiments. After lesion, RSNA responses were significantly reduced (P < 0.05– 0.001), but MAP responses were unaltered. To exclude a role for fibers of passage, the inhibitory GABA-A receptor agonist muscimol was microinjected into the OVLT (50 pmol in 50 nl) (n = 6). Before muscimol, hypertonic NaCl increased RSNA, lumbar SNA (LSNA), and MAP (P < 0.05– 0.001). After muscimol, both RSNA and LSNA were significantly reduced in response to 375 and 750 mM NaCl (P < 0.05). MAP responses were again unaffected. Injections of vehicle (saline) into OVLT (n = 6) and muscimol lateral to OVLT (n = 5) each failed to alter responses to ICA NaCl. We conclude that OVLT neurons contribute to sympathoexcitation by central hyperosmolality.
PMCID: PMC3575105  PMID: 17898124
sympathetic nerve discharge; body fluid homeostasis; blood pressure; osmolality
13.  Cerebral energetic effects of creatine supplementation in humans 
There has been considerable interest in the use of creatine (Cr) supplementation to treat neurological disorders. However, in contrast to muscle physiology, there are relatively few studies of creatine supplementation in the brain. In this report, we use high-field MR 31P and 1H spectroscopic imaging of human brain with a 7-day protocol of oral Cr supplementation to examine its effects on cerebral energetics (phosphocreatine, PCr; ATP) and mitochondrial metabolism (N-acetyl aspartate, NAA; and Cr). We find an increased ratio of PCr/ATP (day 0, 0.80 ± 0.10; day 7, 0.85 ± 09), with this change largely due to decreased ATP, from 2.7 ± 0.3 mM to 2.5 ± 0.3 mM. The ratio of NAA/Cr also decreased (day 0, 1.32 ± 0.17; day 7 1.18 ± 0.13), primarily from increased Cr (9.6 ± 1.9 to 10.1 ± 2.0 mM). The Cr-induced changes significantly correlated with the basal state, with the fractional increase in PCr/ATP negatively correlating with the basal PCr/ATP value (R = −0.74, P < 0.001). As NAA is a measure of mitochondrial function, there was also a significant negative correlation between basal NAA concentrations with the fractional change in PCr and ATP. Thus healthy human brain energetics is malleable and shifts with 7 days of Cr supplementation, with the regions of initially low PCr showing the largest increments in PCr. Overall, Cr supplementation appears to improve high-energy phosphate turnover in healthy brain and can result in either a decrease or an increase in high-energy phosphate concentrations.
PMCID: PMC3570026  PMID: 17185404
high-energy phosphates; brain; N-acetyl aspartate; metabolism
14.  Chronic angiotensin II infusion attenuates the renal sympathoinhibitory response to acute volume expansion 
In this study the hypothesis was tested that chronic infusion of ANG II attenuates acute volume expansion (VE)-induced inhibition of renal sympathetic nerve activity (SNA). Rats received intravenous infusion of either vehicle or ANG II (12 ng·kg−1 ·min−1) for 7 days. ANG II-infused animals displayed an increased contribution of SNA to the maintenance of mean arterial pressure (MAP) as indicated by ganglionic blockade, which produced a significantly (P < 0.01) greater decrease in MAP (75 ± 3 mmHg) than was observed in vehicle-infused (47 ± 8 mmHg) controls. Rats were then anesthetized, and changes in MAP, mean right atrial pressure (MRAP), heart rate (HR), and renal SNA were recorded in response to right atrial infusion of isotonic saline (20% estimated blood volume in 5 min). Baseline MAP, HR, and hematocrit were not different between groups. Likewise, MAP was unchanged by acute VE in vehicle-infused animals, whereas VE induced a significant bradycardia (P < 0.05) and increase in MRAP (P < 0.05). MAP, MRAP, and HR responses to VE were not statistically different between animals infused with vehicle vs. ANG II. In contrast, VE significantly (P < 0.001) reduced renal SNA by 33.5 ± 8% in vehicle-infused animals but was without effect on renal SNA in those infused chronically with ANG II. Acutely administered losartan (3 mg/kg iv) restored VEinduced inhibition of renal SNA (P < 0.001) in rats chronically infused with ANG II. In contrast, this treatment had no effect in the vehicle-infused group. Therefore, it appears that chronic infusion of ANG II can attenuate VE-induced renal sympathoinhibition through a mechanism requiring AT1 receptor activation. The attenuated sympathoinhibitory response to VE in ANG II-infused animals remained after arterial barodenervation and systemic vasopressin V1 receptor antagonism and appeared to depend on ANG II being chronically increased because ANG II given acutely had no effect on VE-induced renal sympathoinhibition.
PMCID: PMC3564518  PMID: 12626370
sympathetic nerve activity; cardiopulmonary reflex; body fluid balance; arterial baroreceptor reflex
15.  Effects of hindbrain melanin-concentrating hormone and neuropeptide Y administration on licking for water, saccharin, and sucrose solutions 
Melanin-concentrating hormone (MCH) and neuropeptide Y (NPY) are orexigenic peptides found in hypothalamic neurons that project throughout the forebrain and hindbrain. The effects of fourth ventricle (4V) infusions of NPY (5 μg) and MCH (5 μg) on licking for water, 4 mM saccharin, and sucrose (0.1 and 1.0 M) solutions were compared to identify the contributions of each peptide to hindbrain-stimulated feeding. NPY increased mean meal size only for the sucrose solutions, suggesting that caloric feedback or taste quality is pertinent to the orexigenic effect; MCH infusions under identical testing conditions failed to produce increases for any tastant. A second experiment also observed no intake or licking effects after MCH doses up to 15 μg, supporting the conclusion that MCH-induced orexigenic responses require forebrain stimulation. A third experiment compared the 4V NPY results with those obtained after NPY infusions (5 μg) into the third ventricle (3V). In contrast to the effects observed after the 3V NPY injections and previously reported forebrain intracerebroventricular (ICV) NPY infusion studies, 4V NPY failed to increase meal frequency for any taste solution or ingestion rate in the early phases of the sucrose meals. Overall, 4V NPY responses were limited to intrameal behavioral processes, whereas forebrain ICV NPY stimulation elicited both consummatory and appetitive responses. The dissociation between MCH and NPY effects observed for 4V injections is consistent with reports that forebrain ICV injections of MCH and NPY produced nearly dichotomous effects on the pattern of licking microstructure, and, collectively, the results indicate that the two peptides have separate sites of feeding action in the brain.
PMCID: PMC3522464  PMID: 17989139
taste; feeding; microstructure; intake
16.  Interaction between muscarinic receptor subtype signal transduction pathways mediating bladder contraction 
M3 muscarinic receptors mediate cholinergic-induced contraction in most smooth muscles. However, in the denervated rat bladder, M2 receptors participate in contraction because M3-selective antagonists [para-fluoro-hexahydro-sila-diphenidol (p-F-HHSiD) and 4-DAMP] have low affinities. However, the affinity of the M2-selective antagonist methoctramine in the denervated bladder is consistent with M3 receptor mediating contraction. It is possible that two pathways interact to mediate contraction: one mediated by the M2 receptor and one by the M3 receptor. To determine whether an interaction exists, the inhibitory potencies of combinations of methoctramine and p-F-HHSiD for reversing cholinergic contractions were measured. In normal bladders, all combinations gave additive effects. In denervated bladders, synergistic effects were seen with the 10:1 and 1:1 (methoctramine:p-F-HHSiD wt/wt) combinations. After application of the sarcoplasmic reticulum ATPase inhibitor thapsigargin to normal tissue, the 10:1 and 1:1 ratios became synergistic, mimicking denervated tissue. Thus in normal bladders both M2 and M3 receptors can induce contraction. In the denervated bladder, the M2 and the M3 receptors interact in a facilitatory manner to mediate contraction.
PMCID: PMC3277793  PMID: 12185001
urinary bladder; synergy; denervation; second messengers
17.  Hypertrophy changes the muscarinic receptor subtype mediating bladder contraction from M3 toward M2 
Major pelvic ganglion electrocautery (MPGE) and spinal cord injury in the rat induce bladder hypertrophy and a change in muscarinic receptor subtypes mediating bladder contraction from predominantly M3 to a combination of M2 and M3. To determine whether this is a result of bladder hypertrophy or denervation, we studied the following groups: sham-operated controls, urinary diversion (DIV), MPGE together with urinary diversion (DIV-DEN), bilateral MPGE (DEN), bladder outlet obstruction (BOO), and MPG decentralization (MPG-DEC). The degree of bladder denervation was determined by the maximal carbachol response normalized to the response to electric field stimulation. Receptor subtype density was determined by immunoprecipitation. The affinity of subtype-selective muscarinic antagonists for inhibition of carbachol-induced contractions was used to determine the subtype-mediating contraction. DEN, MPG-DEC, and BOO bladders were hypertrophic whereas DIV bladders were atrophic compared with sham operated. Bladder contraction in sham-operated, DIV, and DIV-DEN was mediated by the M3 receptor subtype, whereas the M2 subtype participated in contraction in the DEN, MPG-DEC, and BOO groups. The hypertrophied bladders had an increase in total and M2 receptor density while all experimental groups showed a reduction in M3 receptor density. Thus bladder hypertrophy, independent from bladder denervation, causes a shift in the muscarinic receptor subtype mediating bladder contraction from M3 toward M2.
PMCID: PMC3277086  PMID: 12763741
denervation; outlet obstruction; urinary diversion
18.  The M2 muscarinic receptor mediates in vitro bladder contractions from patients with neurogenic bladder dysfunction 
Bladder muscle specimens from seven patients with neurogenic bladder dysfunction were analyzed to determine whether the muscarinic receptor subtype mediating contraction shifts from M3 to the M2 subtype as found in the denervated, hypertrophied rat bladder. Seven bladder specimens were analyzed from six female and one male patients. Six of the patients had traumatic cervical spinal cord injuries (C4–C7), and the other patient had an L1 congenital myelomeningocele. This was compared with results from bladder specimens obtained from eight organ transplant donors. The affinities of three subtype-selective muscarinic receptor antagonists for inhibition of carbachol-induced contractions were determined. The affinity of the M3 selective antagonists darifenacin or p-fluoro-hexahydrosiladifenadol (p-F-HHSiD) was determined in six of the seven spinal injury patient specimens. The affinity was consistent with M2-mediated contractions in four of these six specimens, intermediate between M2 and M3 in one specimen, and within the M3 range in one specimen. The other specimen, tested only with the M2 selective antagonist methoctramine, showed an M3 affinity. In the organ donors, the affinity of p-F-HHSiD was within the M2 range for six of seven specimens, whereas the affinity of darifenacin was within the M3 range for five of six and intermediate between M2 and M3 for the other specimen tested. The affinity of methoctramine in both organ donor specimens tested was within the M3 range. Whereas normal detrusor contractions are mediated by the M3 receptor subtype, in patients with neurogenic bladder dysfunction as well as certain organ transplant donors, contractions can be mediated by the M2 muscarinic receptor subtype.
PMCID: PMC3274770  PMID: 14751843
spinal cord injury
19.  Altered basal and stimulated accumbens dopamine release in obese OLETF rats as a function of age and diabetic status 
The Otsuka Long Evans Tokushima Fatty (OLETF) rat lacking the CCK-1 receptor is hyperphagic, prefers palatable and high caloric meals, and gradually develops obesity and type-2 diabetes. To determine dopamine levels in this strain, we used in-vivo quantitative (no-net flux) microdialyis at three different ages representing non-diabetic (8 weeks), pre-diabetic (18 weeks), and diabetic (56 weeks) stages in OLETF and age-matched lean LETO controls. Results showed significantly elevated basal dopamine levels in the caudomedial nucleus accumbens of OLETF rats compared to LETO at younger ages (8 weeks: 20.10 ± 5.61 nM vs. 15.85 ± 5.63 nM; 18 weeks: 7.37 ± 3.71 nM vs. 4.75 ± 1.25 nM, Mean ± SD). In contrast, at 56 weeks of age, a profound decline in extracellular dopamine concentrations was seen in both strains with a tendency for a greater effect in OLETF rats (1.78 ± 0.40 nM vs. 2.39 ± 0.42 nM). Further, extracellular fraction, an index for reuptake, was higher in 56-week old OLETF compared to LETO (0.648 ± 0.049 vs. 0.526 ± 0.057). Potassium-stimulated dopamine efflux revealed an increased capacity of vesicular pool in OLETF rats compared to LETO across all age groups with an accentuated strain difference at 56 weeks. These findings demonstrate altered striatal dopamine functions (i.e. increased stimulated release and uptake) in obese OLETF rat. This could be due to the lack of functional CCK-1 receptors, or metabolic and hormonal factors associated with the development of obesity and insulin resistance, or both.
PMCID: PMC3114425  PMID: 17553848
obesity; CCK-1 receptor; type-2 diabetes; overeating; no-net flux microdialysis
20.  Muscarinic regulation of neonatal rat bladder spontaneous contractions 
In vitro preparations of whole urinary bladders of neonatal rats exhibit prominent myogenic spontaneous contractions, the amplitude and frequency of which can be increased by muscarinic agonists. The muscarinic receptor subtype responsible for this facilitation was examined in the present experiments. Basal spontaneous contractions in bladders from 1- to 2-wk-old Sprague-Dawley rats were not affected by M2 or M3 receptor antagonists. However, administration of 0.5 μM physostigmine, an anticholinesterase agent that increases the levels of endogenous acetylcholine, or 50–100 nM carbachol, a cholinergic agonist at low concentrations, which did not cause tonic contractions, significantly augmented the frequency and amplitude of spontaneous contractions. Blockade of M2 receptors with 0.1 μM AF-DX 116 or 1 μM methoctramine or blockade of M3 receptors with 50 nM 4-diphenylacetoxy-N-methylpiperidine methiodide or 0.1 μM 4-diphenylacetoxy-N-(2-chloroethyl)piperidine hydrochloride (4-DAMP mustard) reversed the physostigmine and carbachol responses. M2 and M3 receptor blockade did not alter the facilitation of spontaneous contractions induced by 10 nM BAY K 8644, an L-type Ca2+ channel opener, or 0.1 μM iberiotoxin, a large-conductance Ca2+-activated K+ channel blocker. NS-1619 (30 μM), a large-conductance Ca2+-activated K+ channel opener, decreased carbachol-augmented spontaneous contractions. These results suggest that spontaneous contractions in the neonatal rat bladder are enhanced by activation of M2 and M3 receptors by endogenous acetylcholine released in the presence of an anticholinesterase agent or a cholinergic receptor agonist.
PMCID: PMC3111964  PMID: 16709645
M2 receptors; M3 receptors; overactive bladder; detrusor overactivity
21.  Roles of glutamatergic and serotonergic mechanisms in reflex control of the external urethral sphincter in urethane-anesthetized female rats 
This study was conducted to examine reflex mechanisms that mediate urinary bladder and external urethral sphincter (EUS) coordination in urethane-anesthetized female Sprague-Dawley rats. We investigated the properties of EUS reflexes elicited by electrical stimulation of pelvic nerve afferent axons (pelvic-EUS reflex). The changes in the reflexes induced by bladder distension and administration of agonists or antagonists for glutamatergic or serotonergic receptors were examined. The reflexes consisted of an early response (ER, 18- to 22-ms latency) and a late, long-duration (>100-ms latency) response (LR), which consisted of bursts of activity at 20- to 160-ms interburst intervals. In a few experiments, a reflex with an intermediate (40- to 70-ms) latency was also identified. With the bladder empty, the ER, but not the LR, was detected in the majority of experiments. The LR was markedly enhanced when the bladder was distended. The ER remained, but the LR was abolished, after spinal cord transection at T8–T9. The ER and LR were significantly decreased 75 and 35%, respectively, by the N-methyl-d-aspartate receptor antagonist MK-801 (0.3 mg/kg iv), but only decreased 18 and 14%, respectively, by the α-amino-5-methylisoxazole-4-propionate receptor antagonist LY-215490 (3 mg/kg iv). The serotonin (5-HT1A) receptor agonist 8-hydroxy-2-(din-propylamino)-tetralin (1 mg/kg iv) enhanced spontaneous EUS activity and the pelvic-EUS reflex. WAY-100635 (0.1–1 mg/kg iv), a 5-HT1A antagonist, reversed the effect of 8-hydroxy-2-(di-n-propylamino)-tetralin and suppressed EUS activity and the pelvic-EUS reflex. These results indicate that glutamatergic and serotonergic mechanisms are important in the reflex pathways underlying bladder-sphincter coordination in rats.
PMCID: PMC3111965  PMID: 16469836
pelvic nerve; bladder distension; bursting activity; spinal cord injury
22.  Smooth muscle and neural mechanisms contributing to the downregulation of neonatal rat spontaneous bladder contractions during postnatal development 
Spontaneous bladder contractions (SBCs) in the neonatal rat urinary bladder change from a high-amplitude, low-frequency pattern to a low-amplitude, high-frequency pattern during the first 6 wk of life. Understanding the mechanism of this developmental change may provide insights into the causes of bladder overactivity in adults. In vitro whole bladder preparations from Sprague-Dawley rats were used to study the modulation of SBCs by calcium-activated potassium channels (KCa) and electrical field stimulation from 3 days to 6 wk of life. SBCs in 3-day-old bladders were unmasked by treatment with iberiotoxin (100 nM), an inhibitor of large conductance KCa (BK) channels, or apamin (100 nM), an inhibitor of small conductance KCa (SK) channels. Iberiotoxin significantly increased the magnitude of SBCs at 2–3 wk, whereas apamin was only effective at 6 wk. In 1–2 wk bladders, exposure to room temperature Krebs solution decreased SBCs. This decrease was reversed by activating intramural nerves with electrical field stimulation. The effect of electrical field stimulation was inhibited by atropine (1 µM), suramin (10 µM), or pretreatment with tetrodotoxin (1 µM) but was not reversed by tetrodotoxin applied after electrical field stimulation. BK-α mRNA increased threefold, and BK-α protein increased fivefold from 3 days to 6 wk. These data suggest that BK channels play an important role in the regulation of SBCs in the neonatal bladder and that both increased BK channel activity, as well as changes in smooth muscle sensitivity to locally released neurotransmitters contribute to the downregulation of SBCs during early postnatal development.
PMCID: PMC3111975  PMID: 17234952
large-conductance KCa channel; small-conductance KCa channel; cholinergic; purinergic
23.  Carboxyl-terminal and intracellular loop sites for CRF1 receptor phosphorylation and β-arrestin-2 recruitment: a mechanism regulating stress and anxiety responses 
The primary goal was to test the hypothesis that agonist-induced corticotropin-releasing factor type 1 (CRF1) receptor phosphorylation is required for β-arrestins to translocate from cytosol to the cell membrane. We also sought to determine the relative importance to β-arrestin recruitment of motifs in the CRF1 receptor carboxyl terminus and third intracellular loop. β-Arrestin-2 translocated significantly more rapidly than β-arrestin-1 to agonist-activated membrane CRF1 receptors in multiple cell lines. Although CRF1 receptors internalized with agonist treatment, neither arrestin isoform trafficked with the receptor inside the cell, indicating that CRF1 receptor-arrestin complexes dissociate at or near the cell membrane. Both arrestin and clathrin-dependent mechanisms were involved in CRF1 receptor internalization. To investigate molecular determinants mediating the robust β-arrestin-2-CRF1 receptor interaction, mutagenesis was performed to remove potential G protein-coupled receptor kinase phosphorylation sites. Truncating the CRF1 receptor carboxyl terminus at serine-386 greatly reduced agonist-dependent phosphorylation but only partially impaired β-arrestin-2 recruitment. Removal of a serine/threonine cluster in the third intracellular loop also significantly reduced CRF1 receptor phosphorylation but did not alter β-arrestin-2 recruitment. Phosphorylation was abolished in a CRF1 receptor possessing both mutations. Surprisingly, this mutant still recruited β-arrestin-2. These mutations did not alter membrane expression or cAMP signaling of CRF1 receptors. Our data reveal the involvement of at least the following two distinct receptor regions in β-arrestin-2 recruitment: 1) a carboxyl-terminal motif in which serine/threonine residues must be phosphorylated and 2) an intracellular loop motif configured by agonist-induced changes in CRF1 receptor conformation. Deficient β-arrestin-2-CRF1 receptor interactions could contribute to the pathophysiology of affective disorders by inducing excessive CRF1 receptor signaling.
PMCID: PMC3102763  PMID: 17363685
corticotropin-releasing factor; G protein-coupled receptor kinase; receptor phosphorylation; internalization; stress adaptation
24.  Detectable serum flagellin and lipopolysaccharide and upregulated anti-flagellin and lipopolysaccharide immunoglobulins in human short bowel syndrome 
Gut barrier dysfunction may occur in short bowel syndrome (SBS). We hypothesized that systemic exposure to flagellin and lipopolysaccharide (LPS) in SBS might regulate specific immune responses. We analyzed serial serum samples obtained from parenteral nutrition (PN)-dependent patients with SBS versus non-SBS control serum. Serum from 23 adult SBS patients was obtained at baseline and 4, 8, 12, 16, 20, and 24 wk in a trial of modified diet with or without growth hormone. Control serum was obtained from 48 healthy adults and 37 adults requiring PN during critical illness. Serum flagellin was detected by an ELISA recognizing an array of gram-negative flagellins, and LPS was detected by limulus assay. Serum flagellin- and LPS-specific immunoglobulin levels (IgM, IgA, and IgG) were determined by ELISA. Serum flagellin and LPS were undetectable in control subjects. In contrast, serum flagellin, LPS, or both were detected in 14 SBS patients (61%) during one or more time points [flagellin alone, 5/23 (22%); LPS alone, 6/23 (26%); or flagellin + LPS, 3/23 (13%)]. Flagellin-specific serum IgM, IgA, and IgG levels were markedly increased in SBS patients compared with both control populations and remained elevated during the 6-mo study period. LPS-specific IgA was significantly higher in SBS patients compared with healthy controls; LPS-specific IgM, IgA, and IgG levels each decreased over time in association with PN weaning. We conclude that adults with PN-dependent SBS are systemically exposed to flagellin and LPS, presumably from the gut lumen. This likely regulates innate and adaptive immune responses to these specific bacterial products.
PMCID: PMC3064750  PMID: 18003793
parenteral nutrition; intestinal barrier function
25.  Noradrenergic neurons in the rat solitary nucleus participate in the esophageal-gastric relaxation reflex 
Activation of esophageal mechanosensors excites neurons in and near the central nucleus of the solitary tract (NSTc). In turn, NSTc neurons coordinate the relaxation of the stomach [i.e., the receptive relaxation reflex (RRR)] by modulating the output of vagal efferent neurons of the dorsal motor nucleus of the vagus (DMN). The NSTc area contains neurons with diverse neurochemical phenotypes, including a large population of catecholaminergic and nitrergic neurons. The aim of the present study was to determine whether either one of these prominent neuronal phenotypes was involved in the RRR. Immunohistochemical techniques revealed that repetitive esophageal distension caused 53% of tyrosine hydroxylase-immunoreactive (TH-ir) neurons to colocalize c-Fos in the NSTc. No nitric oxide synthase (NOS)-ir neurons in the NSTc colocalized c-Fos in either distension or control conditions. Local brain stem application (2 ng) of α-adrenoreceptor antagonists (i.e., α1-prazosin or α2-yohimbine) significantly reduced the magnitude of the esophageal distension-induced gastric relaxation to ~55% of control conditions. The combination of yohimbine and prazosin reduced the magnitude of the reflex to ~27% of control. In contrast, pretreatment with either the NOS-inhibitor NG-nitro-L-arginine methyl ester or the β-adrenoceptor antagonist propranolol did not interfere with esophageal distension-induced gastric relaxation. Unilateral microinjections of the agonist norepinephrine (0.3 ng) directed at the DMN were sufficient to mimic the transient esophageal-gastric reflex. Our data suggest that noradrenergic, but not nitrergic, neurons of the NSTc play a prominent role in the modulation of the RRR through action on α1- and α2-adrenoreceptors. The finding that esophageal afferent stimulation alone is not sufficient to activate NOS-positive neurons in the NSTc suggests that these neurons may be strongly gated by other central nervous system inputs, perhaps related to the coordination of swallowing or emesis with respiration.
PMCID: PMC3062485  PMID: 12714355
vagus; brain stem; c-Fos

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