Receptor Stimulation Research Articles

Interventions to Reduce Intra-Operative and Post-Operative Pain Associated with Routine Dental Procedures in Children: A Systematic Review and Meta-Analysis

Objective: implementing appropriate pharmacological and non-pharmacological interventions to alleviate pain related to routine dental procedures in paediatric patients could enhance children’s ability to manage dental care. The aim of this review was to investigate the effectiveness of and provide recommendations for interventions that can be used to reduce intra-operative and post-operative pain associated with routine paediatric dental procedures. Methods: A systematic review of randomised controlled clinical trials (RCT) was conducted. Multiple electronic databases were systematically searched. The Cochrane risk-of-bias tool for RCTs was used to evaluate the quality of the included studies. A meta-analysis was performed to determine the effectiveness of the interventions using the Cohen’s d standardised mean differences (SMD) and 95% confidence intervals (CIs) for continuous outcomes. The GRADE tool was used to assess the certainty of evidence to make recommendations. Results: The review included forty-five RCTs comprising 3093 children. Thirty-seven RCTs were included in the meta-analysis, which showed the effectiveness of behavioural interventions (SMD = −0.50, 95% CI −0.83 to −0.18), mechanoreceptor and thermal receptor stimulation (SMD = −1.38, 95% CI −2.02 to −0.73) for intra-operative pain, and pre-emptive oral analgesics (SMD = −0.77, 95% CI −1.21 to −0.33) for reducing post-operative pain in children receiving routine dental care. Conclusion: The GRADE results for these interventions were strong recommendation (IB) for their use, based on moderate evidence and their benefits far outweighing the harm, and they can be delivered readily with minimal training to reduce the pain experience of paediatric patients.

Transcriptional control of metabolism by interferon regulatory factors.

Interferon regulatory factors (IRFs) comprise a family of nine transcription factors in mammals. IRFs exert broad effects on almost all aspects of immunity but are best known for their role in the antiviral response. Over the past two decades, IRFs have been implicated in metabolic physiology and pathophysiology, partly as a result of their known functions in immune cells, but also because of direct actions in adipocytes, hepatocytes, myocytes and neurons. This Review focuses predominantly on IRF3 and IRF4, which have been the subject of the most intense investigation in this area. IRF3 is located in the cytosol and undergoes activation and nuclear translocation in response to various signals, including stimulation of Toll-like receptors, RIG-I-like receptors and the cGAS-STING pathways. IRF3 promotes weight gain, primarily by inhibiting adipose thermogenesis, and also induces inflammation and insulin resistance using both weight-dependent and weight-independent mechanisms. IRF4, meanwhile, is generally pro-thermogenic and anti-inflammatory and has profound effects on lipogenesis and lipolysis. Finally, new data are emerging on the role of other IRF family members in metabolic homeostasis. Taken together, data indicate that IRFs serve as critical yet underappreciated integrators of metabolic and inflammatory stress.

Bioinformatics-Based Identification of Human B-Cell Receptor (BCR) Stimulation-Associated Genes and Putative Promoters

Genome engineered B-cells are being developed for chronic, systemic in vivo protein replacement therapies and for localized, tumor cell-actuated anticancer therapeutics. For continuous systemic engineered protein production, expression may be driven by constitutively active promoters. For actuated payload delivery, B-cell conditional expression could be based on transgene alternate splicing or heterologous promotors activated after engineered B-cell receptor (BCR) stimulation. This study used a bioinformatics-based approach to identify putative BCR-stimulated gene promoters. Gene expression data at four timepoints (60, 90, 210, and 390 min) following in vitro BCR stimulation using an anti-IgM antibody in B-cells from six healthy donors were analyzed using R (4.2.2). Differentially upregulated genes were stringently defined as those with adjusted p-value < 0.01 and a log2FoldChange > 1.5. The most upregulated and statistically significant genes were further analyzed to find those with the lowest unstimulated B-cell expression. Of the 46 significantly upregulated genes at 390 min post-BCR stimulation, 6 had average unstimulated expression below the median unstimulated expression at 390 min for all 54,675 gene probes. This bioinformatics-based identification of 6 relatively quiescent genes at baseline that are upregulated by BCR-stimulation (“on-switch”) provides a set of promising promotors for inclusion in future transgene designs and engineered B-cell therapeutics development.

Protein carbonylation causes sarcoplasmic reticulum Ca2+ overload by increasing intracellular Na+ level in ventricular myocytes.

Diabetes is commonly associated with an elevated level of reactive carbonyl species due to alteration of glucose and fatty acid metabolism. These metabolic changes cause an abnormality in cardiac Ca2+ regulation that can lead to cardiomyopathies. In this study, we explored how the reactive α-dicarbonyl methylglyoxal (MGO) affects Ca2+ regulation in mouse ventricular myocytes. Analysis of intracellular Ca2+ dynamics revealed that MGO (200 μM) increases action potential (AP)-induced Ca2+ transients and sarcoplasmic reticulum (SR) Ca2+ load, with a limited effect on L-type Ca2+ channel-mediated Ca2+ transients and SERCA-mediated Ca2+ uptake. At the same time, MGO significantly slowed down cytosolic Ca2+ extrusion by Na+/Ca2+ exchanger (NCX). MGO also increased the frequency of Ca2+ waves during rest and these Ca2+ release events were abolished by an external solution with zero [Na+] and [Ca2+]. Adrenergic receptor activation with isoproterenol (10nM) increased Ca2+ transients and SR Ca2+ load, but it also triggered spontaneous Ca2+ waves in 27% of studied cells. Pretreatment of myocytes with MGO increased the fraction of cells with Ca2+ waves during adrenergic receptor stimulation by 163%. Measurements of intracellular [Na+] revealed that MGO increases cytosolic [Na+] by 57% from the maximal effect produced by the Na+-K+ ATPase inhibitor ouabain (20 μM). This increase in cytosolic [Na+] was a result of activation of a tetrodotoxin-sensitive Na+ influx, but not an inhibition of Na+-K+ ATPase. An increase in cytosolic [Na+] after treating cells with ouabain produced similar effects on Ca2+ regulation as MGO. These results suggest that protein carbonylation can affect cardiac Ca2+ regulation by increasing cytosolic [Na+] via a tetrodotoxin-sensitive pathway. This, in turn, reduces Ca2+ extrusion by NCX, causing SR Ca2+ overload and spontaneous Ca2+ waves.

Chemogenetic inhibition of the lateral hypothalamus effectively reduces food intake in rats in a translational proof-of-concept study

Despite the therapeutic potential of chemogenetics, the method lacks comprehensive preclinical validation, hindering its progression to human clinical trials. We aimed to validate a robust but simple in vivo efficacy assay in rats which could support chemogenetic drug discovery by providing a quick, simple and reliable animal model. Key methodological parameters such as adeno-associated virus (AAV) serotype, actuator drug, dose, and application routes were investigated by measuring the food-intake-reducing effect of chemogenetic inhibition of the lateral hypothalamus (LH) by hM4D(Gi) designer receptor stimulation. Subcutaneous deschloroclozapine in rats transfected with AAV9 resulted in a substantial reduction of food-intake, comparable to the efficacy of exenatide. We estimated that the effect of deschloroclozapine lasts 1–3 h post-administration. AAV5, oral administration of deschloroclozapine, and clozapine-N-oxide were also effective but with slightly less potency. The strongest effect on food-intake occurred within the first 30 min after re-feeding, suggesting this as the optimal experimental endpoint. This study demonstrates that general chemogenetic silencing of the LH can be utilized as an optimal, fast and reliable in vivo experimental model for conducting preclinical proof-of-concept studies in order to validate the in vivo effectiveness of novel chemogenetic treatments. We also hypothesize based on our results that universal LH silencing with existing and human translatable genetic neuroengineering techniques might be a viable strategy to affect food intake and influence obesity.

Susceptibility to innate immune activation in genetically-mediated myocarditis.

Myocarditis is clinically characterized by chest pain, arrhythmias, and heart failure, and treatment for myocarditis is often supportive. Mutations in DSP, a gene encoding the desmosomal protein desmoplakin, have been increasingly implicated in myocarditis with biomarkers and pathological features indistinguishable from other forms of myocarditis. DSP-associated myocarditis can progress to dilated cardiomyopathy with heightened arrhythmia risk. To model the cardiomyocyte aspects of DSP-associated myocarditis and assess the role of innate immunity, we generated engineered heart tissues (EHTs) from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients and gene-edited healthy control hiPSC lines. Homozygous and heterozygous DSP disrupted EHTs were generated to contain 90% hiPSC-CMs and 10% healthy control human cardiac fibroblasts. We measured innate immune activation and function at baseline and in response to Toll-like receptor (TLR) stimulation in EHTs. At baseline, DSP-/- EHTs displayed a transcriptomic signature of immune activation which was mirrored by EHT cytokine release. Importantly, DSP-/- EHTs were hypersensitive to TLR stimulation demonstrating greater contractile function impairment compared to isogenic controls. Compared to homozygous DSP-/- EHTs, heterozygous DSP patient-derived EHTs had less functionally impairment but also displayed heightened sensitivity to TLR stimulation. When subjected to strain, heterozygous DSP EHTs developed greater functional deficit indicating reduced contractile reserve compared to healthy control. Colchicine or NFΚB inhibitors improved baseline force production and strain-induced force deficits in DSP EHTs. Genomic correction of DSP p.R1951X using adenine base editing reduced inflammatory biomarker release from EHTs. Genetic reduction of DSP renders cardiomyocytes susceptible to innate immune activation and strain-dependent contractile deficits. EHTs replicate electrical and contractile phenotypes seen in human myocarditis implicating cytokine release as a key part of the myogenic susceptibility to inflammation. This heightened innate immune activation and sensitivity is a target for clinical intervention.

The role of regulatory T-cells in the development of endometriosis.

Endometriosis is a benign disease of the female reproductive tract, characterized by the process of chronic inflammation and alterations in immune response. It is estimated to affect 2-19% of women in the general population and is commonly associated with symptoms of chronic pelvic pain and infertility. Regulatory T cells (Treg) are a subpopulation of T lymphocytes that are potent suppressors of inflammatory immune response, essential in preventing destructive immunity in all tissues. In endometriosis, several studies have investigated the possible role of Treg cells in the development of the disease. Most studies to date are heterogeneous in methodology and are based on a small number of cases, which means that it is impossible to define their exact role at present. Based on current knowledge, it seems that disturbed Treg homeostasis, leading to increased systemic and local inflammation within ectopic and eutopic endometrium, is present in women who eventually develop endometriosis. It is also evident that different subsets of human Treg cells have different roles in suppressing the immune response. Recent studies in patients with endometriosis have investigated naive/resting FOXP3lowCD45RA+ Treg cells, which upon T cell receptor stimulation, differentiate into activated/effector FOXP3highCD45RA- Treg cells, characterized by a strong immunosuppressive activity. In addition, critical factors controlling expression of Treg/effector genes, including reactive oxygen species and heme-responsive master transcription factor BACH2, were found to be upregulated in endometriotic lesions. As shown recently for cancer microenvironments, microbial inflammation may also contribute to the local composition of FOXP3+ subpopulations in endometriotic lesions. Furthermore, cytokines, such as IL-7, which control the homeostasis of Treg subsets through the tyrosine phosphorylation STAT5 signalling pathway, have also been shown to be dysregulated. To better understand the role of Treg in the development of endometriosis, future studies should use clear definitions of Tregs along with specific characterization of the non-Treg (FOXP3lowCD45RA-) fraction, which itself is a mixture of follicular Tregs and cells producing inflammatory cytokines.

Comment on 'Accumbens cholinergic interneurons dynamically promote dopamine release and enable motivation'.

Acetylcholine is widely believed to modulate the release of dopamine in the striatum of mammals. Experiments in brain slices clearly show that synchronous activation of striatal cholinergic interneurons is sufficient to drive dopamine release via axo-axonal stimulation of nicotinic acetylcholine receptors. However, evidence for this mechanism in vivo has been less forthcoming. Mohebi, Collins and Berke recently reported that, in awake behaving rats, optogenetic activation of striatal cholinergic interneurons with blue light readily evokes dopamine release measured with the red fluorescent sensor RdLight1 (Mohebi et al., 2023). Here, we show that blue light alone alters the fluorescent properties of RdLight1 in a manner that may be misconstrued as phasic dopamine release, and that this artefactual photoactivation can account for the effects attributed to cholinergic interneurons. Our findings indicate that measurements of dopamine using the red-shifted fluorescent sensor RdLight1 should be interpreted with caution when combined with optogenetics. In light of this and other publications that did not observe large acetylcholine-evoked dopamine transients in vivo, the conditions under which such release occurs in behaving animals remain unknown.

Mast cell degranulation and bradykinin-induced angioedema - searching for the missing link

Initiation of the bradykinin generation cascade is responsible for the occurrence of attacks in some types of angioedema without wheals. Hereditary angioedema due to C1 inhibitor deficiency (HAE-C1-INH) is one such clinical entity. In this paper, we explore the existing evidence that mast cells (MCs) degranulation may contribute to the activation of the kallikrein-kinin system cascade, followed by bradykinin formation and angioedema. We present the multidirectional effects of MC-derived heparin and other polyanions on the major components of the kinin-kallikrein system, particularly on the factor XII activation. Although, bradykinin- and histamine-mediated symptoms are distinct clinical phenomena, they share some common features, such as some similar triggers and a predilection to occur at sites where mast cells reside, namely the skin and mucous membranes. In addition, recent observations indicate a high incidence of hypersensitivity reactions associated with MC degranulation in the HAE-C1-INH patient population. However, not all of these can be explained by IgE-dependent mechanisms. Mast cell-related G protein-coupled receptor-X2 (MRGPRX2), which has recently attracted scientific interest, may be involved in the activation of MCs through a different pathway. Therefore, we reviewed MRGPRX2 ligands that HAE-C1-INH patients may be exposed to in their daily lives and that may affect MCs degranulation. We also discussed the known inter- and intra-individual variability in the course of HAE-C1-INH in relation to factors responsible for possible variability in the strength of the response to MRGPRX2 receptor stimulation. The above issues raise several questions for future research. It is not known to what extent a prophylactic or therapeutic intervention targeting the pathways of one mechanism (mast cell degranulation) may affect the other (bradykinin production), or whether the number of mast cells at a specific body site and their reactivity to triggers such as pressure, allergens or MRGPRX2 agonists may influence the occurrence of HAE-C1-INH attacks at that site.

Therapeutic Inducers of Natural Killer cell Killing (ThINKK): preclinical assessment of safety and efficacy in allogeneic hematopoietic stem cell transplant settings

BackgroundAllogeneic hematopoietic stem cell transplantation (HSCT) remains the standard of care for chemotherapy-refractory leukemia patients, but cure rates are still dismal. To prevent leukemia relapse following HSCT, we aim to...

Molecular Mechanisms and Therapeutic Potential of Gabapentin with a Focus on Topical Formulations to Treat Ocular Surface Diseases.

Gabapentin (GBP) was originally developed as a potential agonist for Gamma-Amino-Butyric-Acid (GABA) receptors, aiming to inhibit the activation of pain-signaling neurons. Contrary to initial expectations, it does not bind to GABA receptors. Instead, it exhibits several distinct pharmacological activities, including: (1) binding to the alpha-2-delta protein subunit of voltage-gated calcium channels in the central nervous system, thereby blocking the excitatory influx of calcium; (2) reducing the expression and phosphorylation of CaMKII via modulation of ERK1/2 phosphorylation; (3) inhibiting glutamate release and interfering with the activation of NMDA receptors; (4) enhancing GABA synthesis; (5) increasing cell-surface expression of δGABA_A receptors, contributing to its antinociceptive, anticonvulsant, and anxiolytic-like effects. Additionally, GBP displays (6) inhibition of NF-kB activation and subsequent production of inflammatory cytokines, and (7) stimulation of the purinergic adenosine A1 receptor, which supports its anti-inflammatory and wound-healing properties. Initially approved for treating seizures and postherpetic neuralgia, GBP is now broadly used for various conditions, including psychiatric disorders, acute and chronic neuropathic pain, and sleep disturbances. Recently, as an eye drop formulation, it has also been explored as a therapeutic option for ocular surface discomfort in conditions such as dry eye, neurotrophic keratitis, corneal ulcers, and neuropathic ocular pain. This review aims to summarize the evidence supporting the molecular effects of GBP, with a special emphasis on its applications in ocular surface diseases.

The Effect of Thyrotropin Suppression on Survival Outcomes in Patients with Differentiated Thyroid Cancer: A Systematic Review and Meta-Analysis.

Background: Long-term management of intermediate- and high-risk differentiated thyroid cancer (DTC) involves thyrotropin (TSH) suppression with thyroid hormone to prevent potential stimulation of TSH receptors on DTC cells, leading to tumor growth. However, the current guidelines recommending TSH suppression are based on low- to moderate-quality evidence. Methods: We performed a systematic review and meta-analysis of studies evaluating the role of TSH suppression in intermediate- and high-risk DTC patients (≥18 years) treated as per regional guideline-based therapy with a follow-up duration of 5 years (PROSPERO #252396). TSH suppression was defined as "below normal reference range" or, when known, <0.5 mIU/L. Primary outcome measures included (i) composite of progression-free survival (PFS), disease-free survival (DFS), and relapse-free survival (RLFS), and (ii) composite of disease-specific survival (DSS), and overall survival (OS). Secondary outcome included a composite of cardiac or skeletal adverse events. All outcomes and comparisons were represented as TSH suppression versus TSH nonsuppression. Randomized controlled trials, cohort studies, and case-control studies were included for analysis. Pooled hazard ratio (HR) and 95% confidence interval (CI) were calculated using random-effects model. Results: Abstract screening was performed on 6,369 studies. After the exclusion of irrelevant studies and full-text screening, nine studies were selected for the final meta-analysis. Based on seven studies (3,591 patients), the composite outcome of PFS, DFS, and RLFS was not significantly different between TSH suppression and nonsuppression groups (HR: 0.75; 95% CI: 0.48-1.17; I2 = 76%). Similarly, a DSS and OS composite outcome assessment based on four studies (3,616 patients) did not favor TSH suppression (HR: 0.69; 95% CI: 0.31-1.52; I2 = 88%). Even after excluding studies of lower quality, the primary outcomes were not significantly different between the TSH suppression and nonsuppression cohorts. The secondary outcome, obtained from two studies (1,294 patients), was significantly higher in the TSH-suppressed groups (HR: 1.82; 95% CI: 1.30-2.55; I2 = 0%). Significant study heterogeneity was noted for primary outcomes. Conclusion: TSH suppression in intermediate- and high-risk DTC may not improve survival outcomes but may increase the risk of secondary complications. However, the limited evidence and study heterogeneity warrant cautious interpretation of our findings. Registration: PROSPERO #252396.

Vagal nerve and sympathetic nerve activity in response to glucagon-like peptide-1 (GLP-1) receptor stimulation in conscious rats

GLP-1 (Glucagon-Like Peptide-1) is a type of gastrointestinal hormone that plays an integral role in regulating glucose homeostasis and appetite control. GLP-1 receptors are expressed throughout the body, and it has been suggested that GLP-1 can be detected by vagal afferent nerve activity, leading to changes in sympathetic nerve activity. However, the details of the effects of GLP-1 receptor stimulation on autonomic nerve activity have not been reported. Therefore, this study investigated the effects of different sites of GLP-1 receptor stimulation on autonomic nerve activity by administering GLP-1 receptor agonists via three routes: intravenous (IV), intraportal vein (iport), and intraperitoneal (iperi). Male Wistar rats were chronically implanted with electrodes, catheters for measuring cervical vagal activity, renal and lumbar sympathetic nerve activity, electroencephalogram, electromyogram, electrocardiogram, arterial pressure, sensors for tissue fluid glucose concentration, and catheters for drug administration in the vein, portal vein, and abdominal cavity. Exendin-4, a GLP-1 receptor agonist, was administered to conscious rats via one of the following routes: intravenous, intra-portal vein, or intra-abdominal. Cervical vagal activity was increased in all routes of Exendin-4 administration. Renal sympathetic nerve activity was rapidly decreased by Exendin-4 administration and gradually recovered to pre-administration levels. There were no significant differences in renal sympathetic nerve activity among the different routes of Exendin-4 administration. On the other hand, lumbar sympathetic nerve activity increased slowly after Exendin-4 administration through the iperi and iport routes, while it decreased initially, then gradually increased following administration through the IV route. These results demonstrate that stimulation of GLP-1 receptors by Exendin-4 increases vagal nerve activity, decreases renal sympathetic nerve activity, and produces regionally different responses in lumbar sympathetic nerve activity depending on the site of stimulation. JST (JPMJMS2023). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Antigen-Independent Activation of CD8 T Cells Promotes Salt Sensitive Hypertension

While CD8+ T cells (CD8Ts) have recently emerged as key players in hypertension, the precise mechanisms underlying their involvement remain incompletely understood. Our previous research uncovered the critical role of CD8T activation during the initial phase of blood pressure elevation, facilitating their direct interaction with the distal convoluted tubule (DCT) via the interferon-gamma (IFNγ)-PDL1 pathway. Importantly, we have now discovered that CD8T activation during this early phase occurs in an antigen-independent manner. In this study, we delve into the contribution of the P2X7 receptor to CD8T activation and its subsequent promotion of sodium retention in the kidney. We hypothesize that blocking this receptor on CD8Ts will disrupt CD8T-tubular interaction and lead to a reduction in blood pressure. To investigate whether CD8T activation in hypertension necessitates T cell receptor (TCR) stimulation, we employed Rag2−/−/OT1mice, which exclusively harbor transgenic OT1 CD8Ts, recognizing only ovalbumin peptides but not other antigens. These mice, without any exposure to ovalbumin, were induced to develop hypertension through the DOCA + salt (DOCA) model. Blood pressure was monitored using radio-biotelemetry, and CD8T activation was confirmed using flow cytometry. In our in-vitro studies, CD8Ts were obtained from both WT and P2X7 KO mice and stimulated with BZATP or ATP. The endpoints include calcium influx (measured via the intracellular calcium indicator Fluo-4), IFNγ transcription, and glycolytic activity (assessed by Seahorse metabolic flux analyzer) as indicators of activation. To validate our findings in an in-vivo context, we measured the activation profiles of CD8Ts obtained from WT and P2X7 KO mice subjected to sham or DOCA treatment, employing RT-PCR and flow cytometry. Additionally, we conducted an adoptive transfer experiment, transferring naïve WT CD8Ts to P2X7 KO mice undergoing DOCA treatment. Blood pressure was consistently monitored via radio-biotelemetry. Our observations revealed that CD8Ts from Rag2−/−/OT1 mice become activated in response to the DOCA pressor, leading to increased IFNγ production and contributing to salt-sensitive hypertension, even in the absence of TCR-mediated antigen stimulation. Agonists of the P2X7 receptor, such as BZATP and ATP, elicited calcium influx in CD8Ts, upregulated IFNγ transcription, and enhanced glycolytic activity, all indicative of T cell activation. Furthermore, CD8Ts pre-stimulated with the P2X7 agonist BZATP displayed increased tubular PDL1 expression and sodium retention in our co-culture model when compared to naïve CD8Ts. Notably, DOCA-treated P2X7 KO CD8Ts were unable to induce an increase in blood pressure in our adoptive transfer model. However, transferring sham WT CD8Ts to DOCA-treated P2X7 KO mice successfully restored hypertension to WT levels. Our findings suggest a novel mechanism in which P2X7 receptor stimulation contributes to CD8T activation in the pathogenesis of hypertension. The deficiency of this receptor on CD8Ts prevents their activation by ATP, resulting in attenuated blood pressure elevation. As such, the CD8T P2X7 receptor presents itself as a promising target for the improvement of blood pressure management. Funding: NIH R01-HL146713, AHA 23TPA1076467. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effect of Cold Adaptation on the State of Cardiovascular System and Cardiac Tolerance to Ischemia/Reperfusion Injury.

Despite the unconditional success achieved in the treatment and prevention of AMI over the past 40 years, mortality in this disease remains high. Hence, it is necessary to develop novel drugs with mechanism of action different from those currently used in clinical practices. Studying the molecular mechanisms involved in the cardioprotective effect of adapting to cold could contribute to the development of drugs that increase cardiac tolerance to the impact of ischemia/reperfusion. An analysis of the published data shows that the long-term human stay in the Far North contributes to the occurrence of cardiovascular diseases. At the same time, chronic and continuous exposure to cold increases tolerance of the rat heart to ischemia/ reperfusion. It has been demonstrated that the cardioprotective effect of cold adaptation depends on the activation of ROS production, stimulation of the β2-adrenergic receptor and protein kinase C, MPT pore closing, and KATP channel.

Does menthol independently modify thermoregulatory control during passive heat stress in humans?

Introduction: Physical cooling within the gastrointestinal (GI) tract attenuates sudomotor activity likely by activation of the cold-sensitive transient receptor potential melastatin 8 (TRPM8). In the absence of physical cooling, it remains unknown whether stimulation of TRPM8 receptors within the GI tract during heat stress alters sudomotor control. Objective and Hypothesis: The purpose of this study is to assess whether a TRPM8 agonist (e.g., menthol) independently modulates the sudomotor response during heat stress. Relative to 37°C water, It was hypothesized that menthol would i) delay the sweating onset, and ii) attenuate the sweating response during heat stress. Methods: To date, a total of 9 participants (5 females; 22±5 y; 72.7±10.5 kg; 1.8±0.1 m) have completed three passive heating protocols on separate days. The participant’s rectal temperature was increased by 1.0°C above baseline using a water perfused suit circulating 49°C water. Participants consumed 3.2 ml per kg of body mass of either: i) 37°C water (37C), ii) 1.5°C water (1.5C), or iii) 37°C menthol solution (0.05%, 37M), 5 minutes before passive heating, and following a 0.5°C and 1.0°C increase in rectal temperature. Mean skin temperature (4 sites) and local sweat rate of the forearm and forehead were recorded during the baseline period and passive heating protocol. Mean body temperature was calculated as the weighted average between skin (20%) and rectal (80%) temperature, and whole-body sweat rate expressed as the net difference in body mass relative to heating time (in g/h). Results: Mean body temperature at baseline was not different between the three conditions (37C: 36.21±0.41˚C, 1.5C: 36.24±0.38˚C, and 37M: 36.30±0.43˚C, P=0.32). Forearm sweating onset was attenuated with 37M (36.97±0.42˚C, P=0.04), and trending to be attenuated with 1.5C (36.96±0.26˚C, P=0.07) relative to 37C (36.69±0.40˚C). Similarly, forehead sweating onset was attenuated with 37M (36.95±0.44˚C, P=0.02), and trending to be attenuated with 1.5C (36.84±0.35˚C, P=0.09), relative to 37C (36.70±0.43˚C). Thermosensitivity was not different between all three fluid conditions for the arm (37C: 1.4±0.7 mg/cm2/min/˚C; 1.5C: 1.7±1.0 mg/cm2/min/˚C; 37M: 1.5±1.0 mg/cm2/min/˚C, P=0.59) and head (37C: 1.7±0.9 mg/cm2/min/˚C; 1.5C: 1.6±1.2 mg/cm2/min/˚C; 37M: 1.7±1.1 mg/cm2/min/˚C, P=0.88). Lastly, whole-body sweat rate was not significantly different between all three conditions (37C: 631±288 g/h; 1.5C: 580±270 g/h; 37M: 635±440 g/h, P=0.63). Conclusion: Preliminary results suggest that consumption of a TRPM8 agonist in advance of passive heat stress may attenuate the sudomotor onset similarly to 1.5˚C water relative to 37˚C. However, the sweating response remains largely unaffected during passive heat stress between all three fluids assessed. Funding: This research was supported by Dr. Nicholas Ravanelli’s Natural Sciences and Engineering Research Council of Canada Discovery Grant (PIN#2022-05096). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Urinary Bladder Dysfunction in a Novel Model of Neuroendocrine Stress

Chronic stress can lead to urinary bladder dysfunction and pain either through actions within the central nervous system or by mechanisms that are locally induced in the bladder. Prolonged upregulation of brain-derived neurotrophic factor (BDNF) in the paraventricular nucleus of the hypothalamus (PVN) has been shown to induce hypertension and chronically elevate activities of the sympathetic nervous system and hypothalamus-pituitary-adrenal axis. Taking advantage of this mechanism, we created a novel model of chronic neuroendocrine stress by subjecting male Sprague Dawley rats to bilateral PVN injections of AAV2 viral vectors expressing either BDNF or GFP (for control). We tested the hypothesis that model-induced chronic neuroendocrine stress would impair bladder function. Bladder activity was assessed 10 weeks post-injections by monitoring the number of urinary voids and average urinary void volume along with water intake over a 48-hour period. To further characterize changes in the urinary bladder, bladder weights were recorded, and in vitro bladder strip experiments were conducted 14 weeks after viral vector injections. BDNF overexpression in the hypothalamus led to significantly lower daily urine output even though water intake remained unaffected, suggesting that BDNF-treated animals showed a significantly higher non-urine-related fluid compared to GFP controls (daily urine output = GFP: 33.85±4.94, BDNF: 14.82±2.18, p &lt; 0.01. Interestingly, despite a lower total daily urine volume, BDNF-treated animals had a significantly higher number of urinary voids (GFP: 7.50±1.42, BDNF: 15.38±2.76; GFP vs. BDNF: p &lt; 0.001) and lower average void volume (GFP: 1.19±0.15, BDNF: 0.27±0.07; GFP vs. BDNF: p &lt; 0.01), thus emulating the overactive bladder phenotype, a common complication in humans with chronic stress. These findings were complemented by in vitro experiments where bladder strips from BDNF rats showed significantly higher contractility as tested with high K+ concentration and electric field stimulation. Moreover, Carbachol-induced stimulation of muscarinic receptors in BDNF bladder strips also trended towards higher contractility. However, urinary bladder weights were unaffected by BDNF overexpression. In summary, we demonstrated that prolonged hypothalamic BDNF overexpression, a model of chronic neuroendocrine stress, leads to significant alterations in bladder function such that BDNF-treated rats exhibited a higher number of urinary voids and lower average void volume compared to GFP controls, and bladder strip experiments suggest that enhanced smooth muscle contractility may contribute to this overactive bladder phenotype. These findings from our novel experimental model will help elucidate pathways through which neuroendocrine stress mechanisms contribute to the development of the overactive bladder syndrome. Funded by R01HL166464, R03AG072016 and R01DK125543. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Pkd1RC/RC mice demonstrate remodeling of purinergic receptors in ADPKD cysts

Polycystic kidney diseases (PKD) are characterized by development of multiple cysts, dilations of nephron segments, which replace normal tissues and lead to kidney insuffciency. To identify new gene pathways affected by cyst development in collecting ducts, we used a bulk RNAseq approach comparing gene expression of normal microdissected cortical collecting ducts (n=3 mice) vs cysts microdissected from Pkd1 RC/RC mice (n=4). Bulk-RNA analysis identified 18,000 genes and allowed statistical comparison of over 15,000 genes. Our data reveals that although cysts originate from normal collecting ducts, cystic epithelium show 2692 down-regulated and 2278 up-regulated genes (p&lt;0.05 pAdj. FDR). Ingenuity Pathways Analysis identifies the following intracellular mechanisms mostly affected by transition: Rac and Rho signaling, fibrosis signaling, epithelial-to-mesenchymal transition, cytoskeleton rearrangement and ERK/MAPK signaling. Several of the differentially expressed genes were identified in GWAS of PKD, chronic kidney diseases and hypertension. Our previous publication reported that development of cysts in an autosomal recessive model of PKD is associated with a shift of P2Y to P2X receptor abundance. In the current study we found that in the autosomal dominant Pkd1 RC/RC mice purinergic signaling undergoes similar remodeling. The most abundant ionotropic receptors with reduced expression were P2ry2 -2.25; P2ry4 -1.37, log2 fold change, whereas ionotropic receptors P2rx5 and P2rx7 increased expression (2.88 and 1.53, log2 fold change, respectively). Additionally, analysis detected elevated abundance of P2ry6 and P2ry12 RNA level. We used a model of cystogenesis to test how P2X signaling affects growth of cysts formed by mpkCCDcl4 cells in Matrigel. Stimulation of P2X receptors with α,βMe-ATP significantly increases cyst growth. We hypothesize that the physiological significance of the predominant P2X signaling in the cysts include their role in regulation of ATP release via pannexin-1 channels. Abnormal ATP accumulation in the cyst space was shown earlier to contribute in cystogenesis and we previously showed that pannexin-1 mediates ATP release to the cyst lumen. Co-immunoprecipitatation experiments in mpkCCDcl4 cells revealed that P2X5 and P2X7 receptors bind pannexin-1 whereas P2Y6 and P2Y12 do not interact with pannexin-1. Interestingly, stimulation of cystogenesis with α,βMe-ATP was accompanied by increased abundance of pannexin-1. Lack of functional P2X5 in humans prioritizes P2X7 receptors as a therapeutic target in ADPKD. We conclude that development of ADPKD cysts involves massive transcriptome remodeling of collecting ducts which include a shift in purinergic signaling that facilitates pathogenic pannexin-1 hyperactivity. DK123266 and DK131114. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Secretin Inhibits Small Intestinal Motility via Interstitial Cells of Cajal and cAMP Mechanisms

Background: Secretin is a member of the secretin-glucagon-vasoactive intestinal peptide hormone superfamily and is a multifunctional gastrointestinal- (GI) and neuro- peptide hormone. Secretin is primarily secreted postprandially from the crypts of Lieberkühn of duodenal enteroendocrine S cells into circulation where it reaches secretin receptor targets in the central nervous system and periphery. This dynamic hormone has been shown to act as a key signaling molecule in the regulation of digestion, metabolism and energy expenditure, water retention, reproduction, thermogenesis in adipose tissue, and in gastric and intestinal motility. Secretin’s canonical role in the GI tract is to stimulate the secretion of bicarbonate and bile from pancreatic ducts and bile ducts to neutralize acidic chyme exiting the stomach. Secretin has also been shown to slow intestinal motility, but its targets and mechanism of action is poorly understood. Aims: Several studies have proposed that secretin acts to slow intestinal motility in the intestines primarily through the many secretin receptors present on vagal afferents in the GI tract, here we discuss new data that suggests an alternate and complementary signaling pathway via interstitial cells of Cajal (ICC). ICC act as a liaison to facilitate a reduction in force of GI smooth muscle contraction through the activation of the secretin receptor (SCTR) and subsequent stimulation of the second messenger, cyclic adenosine monophosphate (cAMP). Here we provide evidence to show how ICC mediates changes in myogenic activity and motility in the small intestine. Methods: Spinning-disk confocal microscopy was used to monitor Ca2+ signaling in ICC from small intestinal muscles of GCaMP6f x KitiCre mice. Additionally, cAMP levels were evaluated using CAMPER mice. Intestinal muscle contractility was assessed using muscle strip myography experiments. Results: Secretin reduced small intestinal force of contraction in the presence of tetrodotoxin (TTX) and dampened the effect of cholinergic transmission. The secretin receptor (SCTR) is expressed primarily on ICC, specifically ICC within the deep muscular plexus (ICC-DMP) in the small intestine and Ca2+ imaging confirmed the effects are primarily localized within ICC-DMP. Secretin reduced carbachol-induced contractions and Ca2+ transients in ICC-DMP in response to electrical field stimulation (EFS) in the presence of LNNA (NO synthase inhibitor) and MRS2500 (P2Y1 antagonist). Secretin caused an increase in cAMP levels in ICC-DMP in muscles from Kit-iCre-CAMPER mice. PKA inhibitors rescued some of the effects of secretin on ICC-DMP Ca2+ signaling. Measurements of diameter change in large, intact, intestinal segments (4-5 cm) showed a significant decrease in response to Secretin. Conclusions: Secretin can inhibit small intestinal motility through the activation SCTRs on ICC-DMP via cAMP-mediated mechanisms. These results show how novel secretin targets on ICC influence GI muscles and reduce propulsive and segmental motility to facilitate nutrient absorption and digestion after a meal. Funding: This project was supported by R01 DK-120759 from the National Institute of Diabetes and digestive and Kidney (NIDDK). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Loss of Htr1B Leads to a Failure in the Neonate Autoresuscitation Reflex

Sudden Infant Death Syndrome (SIDS) is the leading cause of death in infants under the age of 1, with an unknown etiology, that takes the lives of 3,400 infants per year in the US alone. Interestingly, SIDS cases show males predominance (60%), however this sexual disparity in is not fully understood. SIDS is believed to follow a triple risk hypothesis model where the infant has an innate vulnerability, is at a critical developmental period, and is challenged with an exogenous stressor. A leading hypothesis indicates many SIDS cases are mechanistically driven by a failure in the autoresuscitation reflex. The autoresuscitation reflex is a series of gasps that occur to revitalize normal cardiorespiratory function when an infant falls into a hypoxic coma, characterized by an apnea and bradycardia, due to severe hypoxic conditions (i.e., rebreathing while sleeping in face down or with the face blocked). Although the cause of failure in the autoresuscitation reflex for SIDS remains unknown, post-mortem studies have found abnormalities related to the central serotonin (5-HT) neurotransmitter system, including several 5-HT receptors (5-HTr’s). Follow up studies in animal models show that 5-HT system perturbations result in a significant survival decrease in neonatal animals during anoxic (3% CO2/balance N2) gas challenges that test the autoresuscitation function, including pharmacological receptor manipulations. Furthermore, adult animal studies report that 5-HT receptor stimulation alters various respiratory parameters (i.e., tidal volume, respiratory rate, gasping). What remains to be understood is the cellular and molecular mechanisms through which 5-HT plays a role in the autoresuscitation reflex and how those mechanism may be disrupted in SIDS. We hypothesize that Htr1B is required for successful autoresuscitation. To test this hypothesis, we utilized an automated closed loop robotic platform for neonate cardio-respiratory measurements ( Looper) to assay P7 Htr1B loss of function mice for their abilities to survive repeated anoxic gas challenges testing the autoresuscitation reflex. Using Looper, we find that the loss of Htr1B decreases survival to repeated anoxic challenges compared to wild type (WT) controls in both male and female mice. Additionally, a subset of male heterozygous Htr1B mice survive fewer challenges compared to WT mice, whereas others survive a similar number of challenges as WT mice; a phenotype not observed in female mice. Our data supports the hypothesis that modulations to the serotonin system via Htr1B is important in eliciting the autoresuscitation response. Future studies aim to investigate additional respiratory parameters from respiratory trace data, in addition to recovery parameters, and functionally mapping out where in the brainstem and what neuronal populations require the expression of this receptor for the autoresuscitation reflex. R01 HL161142. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.