The T-type calcium channel Ca V 3.2 regulates bladder afferent responses to mechanical stimuli.

ABSTRACT: The bladder wall is innervated by a complex network of afferent nerves that detect bladder stretch during filling. Sensory signals, generated in response to distension, are relayed to the spinal cord and brain to evoke physiological and painful sensations and regulate urine storage and voiding. Hyperexcitability of these sensory pathways is a key component in the development of chronic bladder hypersensitivity disorders including interstitial cystitis/bladder pain syndrome and overactive bladder syndrome. Despite this, the full array of ion channels that regulate bladder afferent responses to mechanical stimuli have yet to be determined. Here, we investigated the role of low-voltage-activated T-type calcium (Ca V 3) channels in regulating bladder afferent responses to distension. Using single-cell reverse-transcription polymerase chain reaction and immunofluorescence, we revealed ubiquitous expression of Ca V 3.2, but not Ca V 3.1 or Ca V 3.3, in individual bladder-innervating dorsal root ganglia neurons. Pharmacological inhibition of Ca V 3.2 with TTA-A2 and ABT-639, selective blockers of T-type calcium channels, dose-dependently attenuated ex-vivo bladder afferent responses to distension in the absence of changes to muscle compliance. Further evaluation revealed that Ca V 3.2 blockers significantly inhibited both low- and high-threshold afferents, decreasing peak responses to distension, and delayed activation thresholds, thereby attenuating bladder afferent responses to both physiological and noxious distension. Nocifensive visceromotor responses to noxious bladder distension in vivo were also significantly reduced by inhibition of Ca V 3 with TTA-A2. Together, these data provide evidence of a major role for Ca V 3.2 in regulating bladder afferent responses to bladder distension and nociceptive signalling to the spinal cord.

Time-of-day dependent changes in guinea pig bladder afferent mechano-sensitivity.

The voiding of urine has a clear circadian rhythm with increased voiding during active phases and decreased voiding during inactive phases. Bladder spinal afferents play a key role in the regulation of bladder storage and voiding, but it is unknown whether they exhibit themselves a potential circadian rhythm. Therefore, this study aimed to determine the mechano- and chemo- sensitivity of three major bladder afferent classes at two opposite day-night time points. Adult female guinea pigs underwent conscious voiding monitoring and bladder ex vivo single unit extracellular afferent recordings at 0300 h and 1500 h to determine day-night modulation of bladder afferent activity. All guinea pigs voided a higher amount of urine at 1500 h compared to 0300 h. This was due to an increased number of voids at 1500 h. The mechano-sensitivity of low- and high-threshold stretch-sensitive muscular-mucosal bladder afferents to mucosal stroking and stretch was significantly higher at 1500 h compared to 0300 h. Low-threshold stretch-insensitive mucosal afferent sensitivity to stroking was significantly higher at 1500 h compared to 0300 h. Further, the chemosensitivity of mucosal afferents to N-Oleoyl Dopamine (endogenous TRPV1 agonist) was also significantly increased at 1500 h compared to 0300 h. This data indicates that bladder afferents exhibit a significant time-of-day dependent variation in mechano-sensitivity which may influence urine voiding patterns. Further studies across a 24 h period are warranted to reveal potential circadian rhythm modulation of bladder afferent activity.

Endocannabinoids in Bladder Sensory Mechanisms in Health and Diseases.

The recent surge in research on cannabinoids may have been fueled by changes in legislation in several jurisdictions, and by approval for the use of cannabinoids for treatment of some chronic diseases. Endocannabinoids act largely, but not exclusively on cannabinoid receptors 1 and 2 (CBR1 and CBR2) which are expressed in the bladder mainly by the urothelium and the axons and endings of motor and sensory neurons. A growing body of evidence suggests that endocannabinoid system constitutively downregulates sensory bladder function during urine storage and micturition, under normal physiological conditions. Similarly, exogenous cannabinoid agonists have potent modulatory effects, as do inhibitors of endocannabinoid inactivation. Results suggest a high potential of cannabinoids to therapeutically ameliorate lower urinary tract symptoms in overactive bladder and painful bladder syndromes. At least part of this may be mediated via effects on sensory nerves, although actions on efferent nerves complicate interpretation. The sensory innervation of bladder is complex with at least eight classes identified. There is a large gap in our knowledge of the effects of endocannabinoids and synthetic agonists on different classes of bladder sensory neurons. Future studies are needed to reveal the action of selective cannabinoid receptor 2 agonists and/or peripherally restricted synthetic cannabinoid receptor 1 agonists on bladder sensory neurons in animal models of bladder diseases. There is significant potential for these novel therapeutics which are devoid of central nervous system psychotropic actions, and which may avoid many of the side effects of current treatments for overactive bladder and painful bladder syndromes.

CB2 cannabinoid receptor agonist selectively inhibits the mechanosensitivity of mucosal afferents in the guinea pig bladder.

Bladder afferents play a pivotal role in bladder function such as urine storage and micturition as well as conscious sensations such as urgency and pain. Endocannabinoids are ligands of cannabinoid 1 and 2 (CB1 and CB2) receptors but can influence the activity of a variety of G protein-coupled receptors as well as ligand-gated and voltage-gated channels. It is still not known which classes of bladder afferents are influenced by CB1 and CB2 receptor agonists. This study aimed to determine the role of CB2 receptors in two major classes of afferents in the guinea pig bladder: mucosal and muscular-mucosal. The mechanosensitivity of these two classes was determined by an ex vivo extracellular electrophysiological recording technique. A stable analog of endocannabinoid anandamide, methanandamide (mAEA), potentiated the mechanosensitivity of mucosal bladder afferents in response to stroking. In the presence of a transient receptor potential vanilloid 1 antagonist (capsazepine), the effect of mAEA switched from excitatory to inhibitory. A selective CB2 receptor agonist, 4-quinolone-3-carboxyamide (4Q3C), significantly inhibited the mechanosensitivity of mucosal bladder afferents to stroking. In the presence of a CB2 receptor antagonist, the inhibitory effect of 4Q3C was lost. mAEA and 4Q3C did not affect responses to stretch and/or mucosal stroking of muscular-mucosal afferents. Our findings revealed that agonists of CB2 receptors selectively inhibited the mechanosensitivity of capsaicin-sensitive mucosal bladder afferents but not muscular-mucosal afferents. This may have important implications for understanding of the role of endocannabinoids in modulating bladder function and sensation in health and diseases.NEW & NOTEWORTHY This article describes, for the first time, to our knowledge, the direct inhibitory effect of cannabinoid 2 receptor agonists on guinea pig mucosal bladder afferents. The cannabinoid 2 receptor is involved in pain and inflammation, suggesting that this may be a viable target for treatment of bladder disorders such as cystitis.

Pregnancy-related plasticity of gastric vagal afferent signals in mice.

Gastric vagal afferents (GVAs) sense food-related mechanical stimuli and signal to the central nervous system, to integrate control of meal termination. Pregnancy is characterized by increased maternal food intake, which is essential for normal fetal growth and to maximize progeny survival and health. However, it is unknown whether GVA function is altered during pregnancy to promote food intake. This study aimed to determine the mechanosensitivity of GVAs and food intake during early, mid-, and late stages of pregnancy in mice. Pregnant mice consumed more food compared with nonpregnant mice, notably in the light phase during mid- and late pregnancy. The increased food intake was predominantly due to light-phase increases in meal size across all stages of pregnancy. The sensitivity of GVA tension receptors to gastric distension was significantly attenuated in mid- and late pregnancy, whereas the sensitivity of GVA mucosal receptors to mucosal stroking was unchanged during pregnancy. To determine whether pregnancy-associated hormonal changes drive these adaptations, the effects of estradiol, progesterone, prolactin, and growth hormone on GVA tension receptor mechanosensitivity were determined in nonpregnant female mice. The sensitivity of GVA tension receptors to gastric distension was augmented by estradiol, attenuated by growth hormone, and unaffected by progesterone or prolactin. Together, the data indicate that the sensitivity of GVA tension receptors to tension is reduced during pregnancy, which may attenuate the perception of gastric fullness and explain increased food intake. Further, these adaptations may be driven by increases in maternal circulating growth hormone levels during pregnancy.NEW & NOTEWORTHY This study provides first evidence that gastric vagal afferent signaling is attenuated during pregnancy and inversely associated with meal size. Growth hormone attenuated mechanosensitivity of gastric vagal afferents, adding support that increases in maternal growth hormone may mediate adaptations in gastric vagal afferent signaling during pregnancy. These findings have important implications for the peripheral control of food intake during pregnancy.

The Effect of High-Fat Diet-Induced Obesity on the Expression of Nutrient Chemosensors in the Mouse Stomach and the Gastric Ghrelin Cell.

The stomach is the primary source of the orexigenic and adiposity-promoting hormone, ghrelin. There is emerging evidence on the nutrient-mediated modulation of gastric ghrelin secretion. However, limited information is available on gastric nutrient-sensing mechanisms in high-fat diet (HFD)-induced obesity. This study investigated the impact of HFD-induced obesity on the expression of nutrient chemosensors in mouse stomach, particularly ghrelin cells. Male C57BL/6 mice were fed either a standard laboratory diet (SLD) or HFD for 12 weeks. The expression of ghrelin, enzymes involved in ghrelin production (PC1/3, GOAT) and nutrient chemosensors (CD36, FFAR2&4, GPR93, CaSR, mGluR4 and T1R3) was determined by quantitative RT-PCR in the mouse corpus and antrum. Immunohistochemistry assessed the protein expression of CaSR and ghrelin in the corpus and antrum. Antral mRNA levels of CaSR and PC1/3 were increased in HFD compared to SLD mice, while mRNA levels of all other nutrient chemosensors examined remained unchanged. CaSR immunolabelling was observed in the gastric antrum only. Nearly 80% of antral ghrelin cells expressed CaSR, with a similar cell density and co-expression in SLD and HFD mice. In conclusion, HFD-induced obesity increased CaSR mRNA expression in mouse antrum. However, the high antral co-expression of CaSR and ghrelin was unaltered in HFD compared to SLD mice.

High fat diet induced obesity alters endocannabinoid and ghrelin mediated regulation of components of the endocannabinoid system in nodose ganglia.

BACKGROUND: Ghrelin and anandamide (AEA) can regulate the sensitivity of gastric vagal afferents to stretch, an effect mediated via the transient receptor potential vanilloid 1 (TPRV1) channel. High fat diet (HFD)-induced obesity alters the modulatory effects of ghrelin and AEA on gastric vagal afferent sensitivity. This may be a result of altered gastric levels of these hormones and subsequent changes in the expression of their receptors. Therefore, the current study aimed to determine the effects of ghrelin and AEA on vagal afferent cell body mRNA content of cannabinoid 1 receptor (CB1), ghrelin receptor (GHSR), TRPV1, and the enzyme responsible for the breakdown of AEA, fatty acid amide hydrolase (FAAH). METHODS: Mice were fed a standard laboratory diet (SLD) or HFD for 12wks. Nodose ganglia were removed and cultured for 14 h in the absence or presence of ghrelin or methAEA (mAEA; stable analogue of AEA). Relative mRNA content of CB1, GHSR, TRPV1, and FAAH were measured. RESULTS: In nodose cells from SLD-mice, mAEA increased TRPV1 and FAAH mRNA content, and decreased CB1 and GHSR mRNA content. Ghrelin decreased TRPV1, CB1, and GHSR mRNA content. In nodose cells from HFD-mice, mAEA had no effect on TRPV1 mRNA content, and increased CB1, GHSR, and FAAH mRNA content. Ghrelin decreased TRPV1 mRNA content and increased CB1 and GHSR mRNA content. CONCLUSIONS: AEA and ghrelin modulate receptors and breakdown enzymes involved in the mAEA-vagal afferent satiety signalling pathways. This was disrupted in HFD-mice, which may contribute to the altered vagal afferent signalling in obesity.

Modulatory effect of methanandamide on gastric vagal afferent satiety signals depends on nutritional status.

SIGNIFICANCE STATEMENT: Gastric vagal afferent responses to tension are dampened in high fat diet-induced obesity. Endocannabinoids are known to dose-dependently inhibit and excite gastric vagal afferents but their effect on gastric vagal afferents in diet-induced obesity are unknown. In individual gastric vagal afferent neurons of diet-induced obese mice the co-expression of components of the endocannabinoid system, including CB1, GHSR, TRPV1 and FAAH, was increased compared with lean mice. In high fat diet-induced obese mice, methanandamide only inhibited gastric vagal afferent responses to tension, possibly due to the observed change in the balance of receptors, hormones and breakdown enzymes in this system. Collectively, these data suggest that endocannabinoid signalling, by gastric vagal afferents, is altered in diet-induced obesity which may impact satiety and gastrointestinal function. ABSTRACT: Gastric vagal afferents (GVAs) play a role in appetite regulation. The endocannabinoid anandamide (AEA) dose-dependently inhibits and excites tension-sensitive GVAs. However, it is also known that high fat diet (HFD) feeding alters GVA responses to stretch. The aim of this study was to determine the role of AEA in GVA signalling in lean and HFD-induced obese mice. Male C57BL/6 mice were fed (12 weeks) a standard laboratory diet (SLD) or HFD. Protein and mRNA expression of components of the cannabinoid system was determined in individual GVA cell bodies and the gastric mucosa. An in vitro GVA preparation was used to assess the effect of methanandamide (mAEA) on tension-sensitive GVAs and the second messenger pathways involved. In individual GVA cell bodies, cannabinoid 1 (CB1) and ghrelin (GHSR) receptor mRNA was higher in HFD mice than SLD mice. Conversely, gastric mucosal AEA and ghrelin protein levels were lower in HFD mice than SLD mice. In SLD mice, mAEA exerted dose-dependent inhibitory and excitatory effects on tension-sensitive GVAs. Only an inhibitory effect of mAEA was observed in HFD mice. The excitatory effect of mAEA was dependent on CB1, transient receptor potential vanilloid 1 (TRPV1) and the protein kinase C. Conversely, the inhibitory effect was dependent on CB1, growth hormone secretagogue receptor, TRPV1 and the protein kinase A. Endocannabinoids, acting through CB1 and TRPV1, have a pivotal role in modulating GVA satiety signals depending on the second messenger pathway utilised. In HFD mice only an inhibitory effect was observed. These changes may contribute to the development and/or maintenance of obesity.

Circadian regulation of appetite and time restricted feeding.

The circadian system plays an important role in the temporal regulation of metabolic processes as well as food intake to ensure energy efficiency. The 'master' clock is located within the superchiasmatic nucleus and receives input from the retina so that it can be entrained by the light:dark cycle. In turn, the master clock entrains other clocks in the central nervous system, including areas involved in energy homeostasis such as the arcuate nucleus, and the periphery (e.g. adipose tissue and the gastrointestinal tract). This master clock is reinforced by other zeitgebers such as the timing of food intake and activity. If these zeitgebers desynchronise, such as occurs in high fat diet-induced obesity or shift work conditions, it can lead to a misalignment of circadian clocks, disruption of metabolic processes and the development of metabolic disorders. The timing of food intake is a strong zeitgeber, particularly in the gastrointestinal tract, and therefore time restricted feeding offers potential for the treatment of diet and shift work induced metabolic disorders. This review will focus on the role of the circadian system in food intake regulation and the effect of environment factors, such as high fat diet feeding or shift work, on the temporal regulation of food intake along with the benefits of time restricted feeding.

Biphasic effects of methanandamide on murine gastric vagal afferent mechanosensitivity.

KEY POINTS: The fine control of food intake is important for the maintenance of a healthy metabolic state. Gastric vagal afferents (GVAs) are involved in the peripheral regulation of food intake via signalling the degree of distension of the stomach which ultimately leads to feelings of fullness and satiety. This study provides evidence that endocannabinoids such as anandamide are capable of regulating GVA sensitivity in a concentration-dependent biphasic manner. This biphasic effect is dependent upon interactions between the CB1, TRPV1 and GHSR receptors. These data have important implications for the peripheral control of food intake. ABSTRACT: Gastric vagal afferents (GVAs) signal to the hindbrain resulting in satiety. Endocannabinoids are endogenous ligands of cannabinoid 1 receptor (CB1) and transient receptor potential vanilloid-1 (TRPV1) channels. The endocannabinoid anandamide (AEA) is expressed in the stomach, and its receptor CB1 is expressed in ghrelin-positive gastric mucosal cells. Further, TRPV1, CB1 and growth hormone secretagogue receptor (ghrelin receptor, GHSR) are expressed in subpopulations of GVA neurons. This study aimed to determine the interaction between TRPV1, CB1, GHSR and endocannabinoids in the modulation of GVA signalling. An in vitro electrophysiology preparation was used to assess GVA mechanosensitivity in male C57BL/6 mice. Effects of methanandamide (mAEA; 1-100 nm), on GVA responses to stretch were determined in the absence and presence of antagonists of CB1, TRPV1, GHSR, protein kinase-A (PKA), protein kinase-C (PKC) and G-protein subunits Gαi/o , or Gαq . Low doses (1-10 nm) of mAEA reduced GVA responses to 3 g stretch, whereas high doses (30-100 nm) increased the response. The inhibitory and excitatory effects of mAEA (1-100 nm) were reduced/lost in the presence of a CB1 and TRPV1 antagonist. PKA, Gαi/o or GHSR antagonists prevented the inhibitory effect of mAEA on GVA mechanosensitivity. Conversely, in the presence of a PKC or Gαq antagonist the excitatory effect of mAEA was reduced or lost, respectively. Activation of CB1, by mAEA, can activate or inhibit TRPV1 to increase or decrease GVA responses to stretch, depending on the pathway activated. These interactions could play an important role in the fine control of food intake.

Disruption of the light cycle ablates diurnal rhythms in gastric vagal afferent mechanosensitivity.

BACKGROUND: Gastric vagal afferents (GVAs) respond to mechanical stimulation, initiating satiety. These afferents exhibit diurnal fluctuations in mechanosensitivity, facilitating food intake during the dark phase in rodents. In humans, desynchrony of diurnal rhythms (eg, shift work) is associated with a higher risk of obesity. To test the hypothesis that shift work disrupts satiety signaling, the effect of a rotating light cycles on diurnal rhythms in GVA mechanosensitivity in lean and high-fat diet (HDF)-induced obese mice was determined. METHODS: Male C57BL/6 mice were fed standard laboratory diet (SLD) or HFD for 12 weeks. After 4 weeks, mice were randomly allocated to a normal light (NL; 12 hour light: 12 hour dark; lights on at zeitgeber time [ZT] 0) or rotating light (RL; 3-day NL cycle, 4-day reversed light cycle [lights on: ZT12] repeated) cycle for 8 weeks. At week 12, eight mice from each group were housed in metabolic cages. After 12 weeks, ex vivo GVA recordings were taken at 3 hour intervals starting at ZT0. KEY RESULTS: SLD-RL and HFD-RL gained more weight compared to SLD-NL and HFD-NL mice, respectively. Gonadal fat pad mass was higher in SLD-RL compared to SLD-NL mice. In SLD-NL mice, tension and mucosal receptor mechanosensitivity exhibited diurnal rhythms with a peak at ZT9. These rhythms were lost in SLD-RL, HFD-NL, and HFD-RL mice and associated with dampened diurnal rhythms in food intake. CONCLUSIONS & INFERENCES: GVA diurnal rhythms are susceptible to disturbances in the light cycle and/or the obese state. This may underpin the observed changes in feeding behavior.

Chronic stress induces hypersensitivity of murine gastric vagal afferents.

BACKGROUND: Stress exposure is known to trigger and exacerbate functional dyspepsia (FD) symptoms. Increased gastric sensitivity to food-related stimuli is widely observed in FD patients and is associated with stress and psychological disorders. The mechanisms underlying the hypersensitivity are not clear. Gastric vagal afferents (GVAs) play an important role in sensing meal-related mechanical stimulation to modulate gastrointestinal function and food intake. This study aimed to determine whether GVAs display hypersensitivity after chronic stress, and whether its interaction with leptin was altered by stress. METHODS: Eight-week-old male C57BL/6 mice were exposed to unpredictable chronic mild stress or no stress (control) for 8 weeks. The metabolic rate, gastric emptying rate, and anxiety- and depression-like behaviors were determined. GVA mechanosensitivity, and its modulation by leptin, was determined using an in vitro single fiber recording technique. QRT-PCR was used to establish the levels of leptin and leptin receptor mRNA in the stomach and nodose ganglion, respectively. KEY RESULTS: The stressed mice had lower body weight and food intake, and increased anxiety-like behavior compared to the control mice. The mechanosensitivity of mucosal and tension-sensitive GVAs was higher in the stressed mice. Leptin potentiated mucosal GVA mechanosensitivity in control but not stressed mice. The expression of leptin mRNA in the gastric mucosa was lower in the stressed mice. CONCLUSIONS AND INFERENCES: In conclusion, chronic stress enhances GVA mechanosensitivity, which may contribute to the gastric hypersensitivity in FD. In addition, the modulatory effect of leptin on GVA signaling is lost after chronic stress exposure.

A rotating light cycle promotes weight gain and hepatic lipid storage in mice.

Processes involved in regulation of energy balance and intermediary metabolism are aligned to the light-dark cycle. Shift-work and high-fat diet (HFD)-induced obesity disrupt circadian rhythmicity and are associated with increased risk of nonalcoholic fatty liver disease. This study aimed to determine the effect of simulating shift work on hepatic lipid accumulation in lean and HFD mice. C57BL/6 mice fed a standard laboratory diet (SLD) or HFD for 4 wk were further allocated to a normal light (NL) cycle (lights on: 0600-1800) or rotating light (RL) cycle [3 days NL and 4 days reversed (lights on: 1800-0600) repeated] for 8 wk. Tissue was collected every 3 h beginning at 0600. HFD mice gained more weight than SLD mice, and RL mice gained more weight than NL mice. SLD-NL and HFD-NL mice, but not RL mice, were more active, had higher respiratory quotients, and consumed/expended more energy during the dark phase compared with the light phase. Blood glucose and plasma cholesterol and triglyceride concentrations were elevated in HFD and SLD-RL compared with SLD-NL mice. Hepatic glycogen was elevated in HFD compared with SLD mice. Hepatic triglycerides were elevated in SLD-RL and HFD mice compared with SLD-NL. Circadian rhythmicity of hepatic acetyl-CoA carboxylase (ACACA) mRNA was phase shifted in SLD-RL and HFD-NL and lost in HFD-RL mice. Hepatic ACACA protein was reduced in SLD-RL and HFD mice compared with SLD-NL mice. Hepatic adipose triglyceride lipase was elevated in HFD-NL compared with SLD-NL but lower in RL mice compared with NL mice irrespective of diet. In conclusion, an RL cycle model of shift work promotes weight gain and hepatic lipid storage even in lean conditions. NEW & NOTEWORTHY In this publication we describe the effects of a rotating light cycle model of shift work in lean and high-fat diet-induced obese mice on body mass, diurnal patterns of energy intake and expenditure, and hepatic lipid storage. The data indicate that modeling shift work, via a rotating light cycle, promotes weight gain and hepatic lipid accumulation even in mice on a standard laboratory diet.

Involvement of TRPV1 Channels in Energy Homeostasis.

The ion channel TRPV1 is involved in a wide range of processes including nociception, thermosensation and, more recently discovered, energy homeostasis. Tightly controlling energy homeostasis is important to maintain a healthy body weight, or to aid in weight loss by expending more energy than energy intake. TRPV1 may be involved in energy homeostasis, both in the control of food intake and energy expenditure. In the periphery, it is possible that TRPV1 can impact on appetite through control of appetite hormone levels or via modulation of gastrointestinal vagal afferent signaling. Further, TRPV1 may increase energy expenditure via heat production. Dietary supplementation with TRPV1 agonists, such as capsaicin, has yielded conflicting results with some studies indicating a reduction in food intake and increase in energy expenditure, and other studies indicating the converse. Nonetheless, it is increasingly apparent that TRPV1 may be dysregulated in obesity and contributing to the development of this disease. The mechanisms behind this dysregulation are currently unknown but interactions with other systems, such as the endocannabinoid systems, could be altered and therefore play a role in this dysregulation. Further, TRPV1 channels appear to be involved in pancreatic insulin secretion. Therefore, given its plausible involvement in regulation of energy and glucose homeostasis and its dysregulation in obesity, TRPV1 may be a target for weight loss therapy and diabetes. However, further research is required too fully elucidate TRPV1s role in these processes. The review provides an overview of current knowledge in this field and potential areas for development.