Dental caries, tobacco usage and associated risk factor of dental caries in patients visiting a government hospital in Western, Nepal.

BACKGROUND: This study was conducted to assess the prevalence of dental caries, tobacco usage, and associated risk factors for dental caries in patients who visited a government hospital in Western, Nepal. METHODS: This analytical cross-sectional study was conducted from January to April 2022. Patients above 18 years visiting the dental OPD of a government hospital, and who had provided informed consent were enrolled in the study using a convenience sampling technique. As the study also involved an illiterate population, in that case, informed consent was obtained from their respective legal guardian as well. A pretested standardized, close-ended questionnaire was administered by researchers to gather information regarding the associated risk factors and oral hygiene practices. Clinical examination was done for dental caries according to the criteria by the World Health Organization (WHO) using the "DMFT" index (WHO modification 1987). Bivariate and multivariable logistic regression was done and the odds ratio and p-value was calculated. For all tests, statistical significance was set at p < 0.05. RESULTS: A total of 219 participants completed the study with a mean age of 31.73 ± 12.46. The prevalence of dental caries and tobacco was found to be 80.36% and 5.02% respectively. Participants without health insurance had 2.35 times higher odds of dental caries (95% CI: 1.03-5.36). Not rinsing the mouth after eating sweets was associated with 3.07 times higher odds of dental caries (95% CI: 1.31-7.18). Those who hadn't visited a dentist in the past 12 months had lower odds (0.42; 95% CI: 0.18-0.94). Eating fresh fruit daily showed statistically higher odds (2.70; 95% CI: 1.04-6.99) of dental caries. Non-tobacco users had higher odds (14.19; 2.55-78.99) of dental caries. CONCLUSION: Dental caries is highly prevalent, while tobacco usage is relatively low. Factors associated with dental caries included lack of health insurance coverage, consumption of fruits once daily, recent dental visits within the past year, not rinsing the mouth with water after consuming sweets, and non-tobacco users.

Oral health status and barriers to utilization of dental services among pregnant women in Sunsari, Nepal: A cross-sectional study.

OBJECTIVES: This study was conducted to assess oral hygiene practices, oral health status and barriers to utilization of oral health care services among pregnant mothers attending two family health care clinics in Sunsari, Nepal. METHODS: A cross-sectional study was conducted among 139 women using a purposive sampling technique. The data collection was done using a pretested standard semi-structured questionnaire. Face-to-face interviews of the participants were done by a single investigator in the local language (Nepali). The examination was done using a mouth mirror and CPI probe for periodontal status, loss of attachment and dentition status, and treatment needs. RESULTS: The majority of pregnant mothers brushed their teeth once a day or less than once a day (n = 106, 76.3%) and self-reported perceived oral health status was poor/fair (n = 93, 66.9%). The prevalence of dental caries was found to be 69.8%. Bleeding on probing was present in all participants. DMFT, presence of bleeding on probing and increased periodontal pocket was significantly high among women who had self-reported their oral health problems in comparison to those who had not reported any problem. The most common barriers reported by the participants were a lack of knowledge of dental checkups and a lack of perceived need for dental care during pregnancy. CONCLUSIONS: There was a high prevalence of dental caries and periodontal disease. Lack of knowledge and perceived need for dental care were the major barriers found in this study. Hence, this directs towards the utmost need for improvement in awareness level as well as oral hygiene practices.

Jejunojejunal intussusception in an adult due to adenoma: a case report.

Intussusception is a rare condition in adults and presents a diagnostic challenge. Clinical presentation tends to be chronic and non-specific. Unlike the pediatric population, most adult intussusceptions have structural lesions as lead points. Here, we present a case of jejunojejunal intussusception in a 27-year female due to adenoma of small bowel.

Effectiveness of oral health education intervention among 12-15-year-old school children in Dharan, Nepal: a randomized controlled trial.

BACKGROUND: School-aged adolescents are in particular need of preventive program to ensure positive long-term oral health and hygiene. The objective of this study was to assess the effectiveness of an oral health education (OHE) intervention on oral hygiene knowledge, attitude and practices (KAP), plaque control and gingival health among 12-15 years old school children in Dharan sub-metropolitan city, Nepal. METHODS: A randomized controlled trial was conducted with parallel study groups, comprising 12-15-year-old school children, 120 in each group. OHE was given to the experimental group at baseline, third and sixth months and to the control group after completion of the study. Interview of the participants were done using a 23-item questionnaire for assessment of oral hygiene KAP. For each question, correct answer was scored as 1 and wrong answer was scored zero. An overall composite score was then created, by adding the individual scores. Oral examination was done using mouth mirror and WHO probe to record Turesky-Gilmore-Glickman modification of the Quigley-Hein plaque index, Gingival index and Dentition status and treatment needs. Analysis was done using chi-square test for categorical data and independent t test, Mann-Whitney U test, repeated measures ANOVA and post hoc Tukey's test for quantitative data. The level of significance was set at P < 0.05. RESULTS: There was 54.58% improvement in overall oral hygiene KAP in experimental group (P = 0.001) whereas no improvement was seen in control group at the end of the study. The mean plaque score was improved by 57.67% (P = 0.001) in experimental group in comparison to 4.56% in control group. Gingival index was improved by 49.90% (P = 0.001) in experimental group in comparison to 0.7% in control group. Caries experience was increased in both groups but no significant difference was seen. CONCLUSIONS: The study concluded that oral health education was effective in improving oral hygiene KAP, plaque control and gingival health. Trial registration The trial was retrospectively registered with Clinical Trial Registry India (CTRI) with identifier no. CTRI/2018/05/013985, registered on 05/21/2018. ( http://www.ctri.nic.in/Clinicaltrials/pdf_generate.php?trialid=23651&EncHid=&modid=&compid=%27,%2723651det%27 ). Institutional Review Committee, B. P. Koirala Institute of Health Sciences (BPKIHS), Dharan, Nepal provided the ethical approval (Ref. No.: 292/074/075-IRC).

Optimization of Growth Conditions and Biological Activities of Nepalese Ganoderma lucidum Strain Philippine.

Ganoderma lucidum has been extensively studied for its valuable medicinal importance. In this study, the artificial cultivation of G. lucidum strain Philippine in different culture media, including sawdust substrate, was performed and optimized on the Potato Dextrose Agar (PDA) media. Phytochemical, antibacterial, and antioxidant analyses were performed and compared between the ethanol extracts prepared from two different cultures (fruit from synthetic log culture and mycelia from PDA media culture). Both the 200 mg/mL and 100 mg/mL concentrations of extracts inhibited all the tested bacteria, and the results were promising than the corresponding control using antibiotics. The fruit extract showed higher antioxidant potential (150.6 ± 56.92 mg ascorbic acid equivalent/g extract) than mycelial extract (144.28 ± 81.72 mg ascorbic acid equivalent/g extract). The results indicate that fruiting bodies of G. lucidum cultivated in a complex dust medium possess higher antioxidant properties than mycelia culture, which can be further explored for therapeutic applications.

Assessment of nicotine dependence among tobacco users visiting outreach programs in Dharan, Nepal: a cross-sectional study.

BACKGROUND: Nicotine is a highly addictive substance present in tobacco. This study was conducted to assess the level of nicotine dependence among smokers and smokeless tobacco users visiting dental outreach programs of B.P. Koirala Institute of Health Sciences -Dharan, Nepal. METHODS: A cross sectional study was conducted from June 2018 to April 2019. A total of 726 people were selected from participants of dental outreach programs of 6 districts using convenience sampling technique. The data collection was done using semi-structured questionnaire through face-to-face interview by a single researcher. History of tobacco use and level of nicotine dependency was measured using Nepali translated and validated form of Fagerström Test for Nicotine Dependence for smoking and smokeless tobacco. The mean age of the tobacco users was 39.55 ± 15.57. Descriptive statistics including the mean, median, percentage, standard deviations and interquartile range were computed. Chi-square test, Fisher's exact test, univariate and bivariate logistic regression were used where needed. RESULTS: Nicotine dependence (moderate and severe) was found in 80% of smokeless tobacco users and 48% of smokers. Among the smokeless tobacco users, nicotine dependency was found to be more with increase in duration of tobacco use (AOR = 50.25, 95%CI = 3.51-718.62, p = 0.004), low socioeconomic status (AOR = 6.27, 95%CI = 1.30-30.31, p = 0.02), less number of tobacco packets used per day and tried to quit tobacco use in last 1 year. Among smokers nicotine dependency was found to be significantly higher with smoking more than 10 cigarettes per day (AOR = 7.14, 95% CI = 2.00-25.40, p = 0.002). CONCLUSIONS: The study concluded that level of nicotine dependence for both smoking and smokeless tobacco was high among the people visiting dental outreach programs. It is high time to develop a policy to control tobacco use along with creating tobacco cessation centers. Currently, tobacco control program is mostly focusing on smoking. However, it is also important to incorporate smokeless tobacco control at policy level.

Mitochondria-targeted antioxidant protects against irradiation-induced salivary gland hypofunction.

A severe consequence of radiation therapy in patients with head and neck cancer is persistent salivary gland hypofunction which causes xerostomia and oral infections. We previously showed that irradiation (IR) of salivary glands in mice triggers initial transient increases in mitochondrial reactive oxygen species (ROSmt), mitochondrial [Ca2+] ([Ca2+]mt), and activated caspase-3 in acinar cells. In contrast, loss of salivary secretion is persistent. Herein we assessed the role of ROSmt in radiation-induced irreversible loss of salivary gland function. We report that treatment of mice with the mitochondrial-targeted antioxidant, MitoTEMPO, resulted in almost complete protection of salivary gland secretion following either single (15 Gy) or fractionated (5 × 3 Gy) doses of irradiation. Salivary gland cells isolated from MitoTEMPO-treated, irradiated, mice displayed significant attenuation of the initial increases in ROSmt, ([Ca2+]mt, and activated caspase-3 as compared to cells from irradiated, but untreated, animals. Importantly, MitoTEMPO treatment prevented radiation-induced decrease in STIM1, consequently protecting store-operated Ca2+ entry which is critical for saliva secretion. Together, these findings identify the initial increase in ROSmt, that is induced by irradiation, as a critical driver of persistent salivary gland hypofunction. We suggest that the mitochondrially targeted antioxidant, MitoTEMPO, can be potentially important in preventing IR-induced salivary gland dysfunction.

Unnatural Deaths among Autopsy Cases Brought at Tertiary Care Hospital of Western Nepal during COVID-19 Pandemic Period: A Descriptive Cross-sectional Study.

INTRODUCTION: The outbreak of COVID-19  as changed patterns of mortality in different setups. The rate of suicide has increased in some countries during the pandemic while the overall death rates  have decreased. The study was conducted with objective to find out the prevalence of unnatural deaths among the autopsy cases brought at tertiary care hospital during COVID-19 pandemic period. METHODS: This is a descriptive cross-sectional study using the records of the medico legal autopsies conducted from 24th March 2020 to 23rd August 2020 during the COVID-19 pandemic in Pokhara Academy of Health Sciences. Ethical approval was taken from Institutional Review Committee of Pokhara Academy of Health Sciences (Reference number 28.2077/78). Whole sampling method was used. Records which were available were included in the study whereas those cases whose complete records were not available were excluded. Point estimate at 95% confidence interval was calculated along with frequency and proportion for binary data. RESULTS: Out of 188 deaths studied at the autopsy during the COVID-19 pandemic period, the prevalence of unnatural deaths was 147 (78.19%) (71.04-85.33 at 95% Confidence Interval). Among these deaths, 109 (74.14%) were males and 38 (25.85%) were females. Suicide was the most common manner attributing to 78 (53.06%) of the unnatural deaths. CONCLUSIONS: The prevalence of suicide was more than those demonstrated by earlier observations in similar settings before the pandemic period. Suicidal deaths were more common during the COVID 19 pandemic. This is an indicator of frustration of the people and necessary steps have to be taken to decrease such deaths in similar conditions to come.

STIM2 targets Orai1/STIM1 to the AKAP79 signaling complex and confers coupling of Ca2+ entry with NFAT1 activation.

The Orai1 channel is regulated by stromal interaction molecules STIM1 and STIM2 within endoplasmic reticulum (ER)-plasma membrane (PM) contact sites. Ca2+ signals generated by Orai1 activate Ca2+-dependent gene expression. When compared with STIM1, STIM2 is a weak activator of Orai1, but it has been suggested to have a unique role in nuclear factor of activated T cells 1 (NFAT1) activation triggered by Orai1-mediated Ca2+ entry. In this study, we examined the contribution of STIM2 in NFAT1 activation. We report that STIM2 recruitment of Orai1/STIM1 to ER-PM junctions in response to depletion of ER-Ca2+ promotes assembly of the channel with AKAP79 to form a signaling complex that couples Orai1 channel function to the activation of NFAT1. Knockdown of STIM2 expression had relatively little effect on Orai1/STIM1 clustering or local and global [Ca2+]i increases but significantly attenuated NFAT1 activation and assembly of Orai1 with AKAP79. STIM1ΔK, which lacks the PIP2-binding polybasic domain, was recruited to ER-PM junctions following ER-Ca2+ depletion by binding to Orai1 and caused local and global [Ca2+]i increases comparable to those induced by STIM1 activation of Orai1. However, in contrast to STIM1, STIM1ΔK induced less NFAT1 activation and attenuated the association of Orai1 with STIM2 and AKAP79. Orai1-AKAP79 interaction and NFAT1 activation were recovered by coexpressing STIM2 with STIM1ΔK. Replacing the PIP2-binding domain of STIM1 with that of STIM2 eliminated the requirement of STIM2 for NFAT1 activation. Together, these data demonstrate an important role for STIM2 in coupling Orai1-mediated Ca2+ influx to NFAT1 activation.

Cancer Registration in Nepal: Current Status and Way Forward.

Cancer registration is an organization for the systematic collection, storage, analysis, interpretation and reporting of data on subjects with cancer. Cancer Registry was initiated in 1995 and expanded as National Cancer Registry Program since 2003 by B.P. Koirala Memorial Cancer Hospital with the support of World Health Organization. National cancer registry program currently includes 12 hospital-based registries. First time in Nepal, B.P. Koirala Memorial Cancer Hospital piloted population-based cancer registry in 2013, which included 15 districts covering 25.8% of total population of Nepal. National cancer registry program is important to assure the quality of data from all the registries to ensure the availability of reliable and valid data of cancer cases. This will further help policymakers to develop preventive and control strategies against cancer. This paper reviews the current status of cancer registries in Nepal and discusses challenges and future perspectives related to national cancer registry program. National cancer registry should further include major hospitals in Nepal to give scientific information on cancer trends by community, provinces and regions and to analyze on survival of cancer cases. Keywords: cancer; national cancer registry program; Nepal.

Cross-talk between N-terminal and C-terminal domains in stromal interaction molecule 2 (STIM2) determines enhanced STIM2 sensitivity.

Store-operated Ca2+ entry (SOCE) is a ubiquitous pathway for Ca2+ influx across the plasma membrane (PM). SOCE is mediated by the endoplasmic reticulum (ER)-associated Ca2+-sensing proteins stromal interaction molecule 1 (STIM1) and STIM2, which transition into an active conformation in response to ER Ca2+ store depletion, thereby interacting with and gating PM-associated ORAI1 channels. Although structurally homologous, STIM1 and STIM2 generate distinct Ca2+ signatures in response to varying strengths of agonist stimulation. The physiological functions of these Ca2+ signatures, particularly under native conditions, remain unclear. To investigate the structural properties distinguishing STIM1 and STIM2 activation of ORAI1 channels under native conditions, here we used CRISPR/Cas9 to generate STIM1-/-, STIM2-/-, and STIM1/2-/- knockouts in HEK293 and colorectal HCT116 cells. We show that depending on cell type, STIM2 can significantly sustain SOCE in response to maximal store depletion. Utilizing the SOCE modifier 2-aminoethoxydiphenyl borate (2-APB), we demonstrate that 2-APB-activated store-independent Ca2+ entry is mediated exclusively by endogenous STIM2. Using variants that either stabilize or disrupt intramolecular interactions of STIM C termini, we show that the increased flexibility of the STIM2 C terminus contributes to its selective store-independent activation by 2-APB. However, STIM1 variants with enhanced flexibility in the C terminus failed to support its store-independent activation. STIM1/STIM2 chimeric constructs indicated that coordination between N-terminal sensitivity and C-terminal flexibility is required for specific store-independent STIM2 activation. Our results clarify the structural determinants underlying activation of specific STIM isoforms, insights that are potentially useful for isoform-selective drug targeting.

Tuning store-operated calcium entry to modulate Ca2+-dependent physiological processes.

The intracellular calcium signaling processes are tightly regulated to ensure the generation of calcium signals with the specific spatiotemporal characteristics required for regulating various cell functions. Compartmentalization of the molecular components involved in the generation of these signals at discrete intracellular sites ensures the signaling specificity and transduction fidelity of the signal for regulating downstream effector processes. Store-operated calcium entry (SOCE) is ubiquitously present in cells and is critical for essential cell functions in a variety of tissues. SOCE is mediated via plasma membrane Ca2+ channels that are activated when luminal [Ca2+] of the endoplasmic reticulum ([Ca2+]ER) is decreased. The ER-resident stromal interaction molecules, STIM1 and STIM2, respond to decreases in [Ca2+]ER by undergoing conformational changes that cause them to aggregate at the cell periphery in ER-plasma membrane (ER-PM) junctions. At these sites, STIM proteins recruit Orai1 channels and trigger their activation. Importantly, the two STIM proteins concertedly modulate Orai1 function as well as the sensitivity of SOCE to ER-Ca2+ store depletion. Another family of plasma membrane Ca2+ channels, known as the Transient Receptor Potential Canonical (TRPC) channels (TRPC1-7) also contribute to sustained [Ca2+]i elevation. Although Ca2+ signals generated by these channels overlap with those of Orai1, they regulate distinct functions in the cells. Importantly, STIM1 is also required for plasma membrane localization and activation of some TRPCs. In this review, we will discuss various molecular components and factors that govern the activation, regulation and modulation of the Ca2+ signal generated by Ca2+ entry pathways in response to depletion of ER-Ca2+ stores. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Nurse's Awareness on Ethico-legal Aspects of Nursing Profession.

BACKGROUND: Nursing practice amicably includes practical efficacy and ethics. Now a days legal and ethical problems associated with client care are arising day by day. Therefore, nurses should have adequate understanding of basic legal concepts and issues relevant to nursing profession in order to protect the rights of the clients and the nurses. METHODS: A cross sectional descriptive design was adopted for the study. 142 nurses were included by using purposive sampling technique. Data was collected with self-administered structured questionnaire. Descriptive statistics was used to reveal demographic information. Kruskal Wallis and Mann Whitney test were used to find out association of selected demographic variables and ethico legal aspects of nursing. RESULTS: Majority of participants were belonging to 20-29 years of age. More than half nurses had complete bachelor's degree and had less than 10 year's experiences. Majority of participants reported that they did not encounter any legal issues in their professional life till date. Similarly, majority of participants had average level knowledge and equate level of practice. Years of experiences and education level did not affect in knowledge level and existing practice related to ethico legal aspect of nursing. There was no significant relationship between level of knowledge and existing practice. CONCLUSIONS: Nurses have average knowledge and practice on ethico legal aspects. There is positive relationship between knowledge and practice though it is not statistically significant.

STIM2 Induces Activated Conformation of STIM1 to Control Orai1 Function in ER-PM Junctions.

Ca2+ entry mediated by the calcium channel, Orai1, provides critical Ca2+ signals that regulate cell function. The ER-Ca2+ sensor protein, STIM1, recruits and strongly activates Orai1 within ER-PM junctions. STIM2 is a poor activator of Orai1, and its physiological role is not well understood. Herein, we report a crucial function for STIM2 in inducing the activated conformation of STIM1. By using conformational sensors of STIM2 and STIM1, together with protein interaction and functional studies, we show that STIM2 is constitutively localized within ER-PM junctions in ER-Ca2+ store replete cells. Importantly, STIM2 traps STIM1 and triggers remodeling of STIM1 C terminus, causing STIM1/Orai1 coupling and enhancement of Orai1 function in cells with relatively high ER-[Ca2+]. The increase in Ca2+ entry controls Ca2+-dependent transcription factor, NFAT, activation at low [agonist]. Our findings reveal that STIM2 modulates STIM1/Orai1 function to tune the fidelity of receptor-evoked Ca2+ signaling and the physiological response of cells.

Radiation inhibits salivary gland function by promoting STIM1 cleavage by caspase-3 and loss of SOCE through a TRPM2-dependent pathway.

Store-operated Ca2+ entry (SOCE) is critical for salivary gland fluid secretion. We report that radiation treatment caused persistent salivary gland dysfunction by activating a TRPM2-dependent mitochondrial pathway, leading to caspase-3-mediated cleavage of stromal interaction molecule 1 (STIM1) and loss of SOCE. After irradiation, acinar cells from the submandibular glands of TRPM2+/+ , but not those from TRPM2-/- mice, displayed an increase in the concentrations of mitochondrial Ca2+ and reactive oxygen species, a decrease in mitochondrial membrane potential, and activation of caspase-3, which was associated with a sustained decrease in STIM1 abundance and attenuation of SOCE. In a salivary gland cell line, silencing the mitochondrial Ca2+ uniporter or caspase-3 or treatment with inhibitors of TRPM2 or caspase-3 prevented irradiation-induced loss of STIM1 and SOCE. Expression of exogenous STIM1 in the salivary glands of irradiated mice increased SOCE and fluid secretion. We suggest that targeting the mechanisms underlying the loss of STIM1 would be a potentially useful approach for preserving salivary gland function after radiation therapy.

Signaling Pathway for Endothelin-1- and Phenylephrine-Induced cAMP Response Element Binding Protein Activation in Rat Ventricular Myocytes: Role of Inositol 1,4,5-Trisphosphate Receptors and CaMKII.

BACKGROUND/AIMS: Endothelin-1 (ET-1) and the α₁-adrenoceptor agonist phenylephrine (PE) activate cAMP response element binding protein (CREB), a transcription factor implicated in cardiac hypertrophy. The signaling pathway involved in CREB activation by these hypertrophic stimuli is poorly understood. We examined signaling pathways for ET-1- or PE-induced cardiac CREB activation. METHODS: Western blotting was performed with pharmacological and genetic interventions in rat ventricular myocytes. RESULTS: ET-1 and PE increased CREB phosphorylation, which was inhibited by blockade of phospholipase C, the extracellular-signal-regulated kinase 1/2 (ERK1/2) pathway, protein kinase C (PKC) or Ca2+-calmodulin-dependent protein kinase II (CaMKII). Intracellular Ca2+ buffering decreased ET-1- and PE-induced CREB phosphorylation by ≥80%. Sarcoplasmic reticulum Ca2+ pump inhibitor, inositol 1,4,5-trisphosphate receptor (IP₃R) blockers, or type 2 IP₃R (IP₃R2) knock-out abolished ET-1- or PE-induced CREB phosphorylation. ET-1 and PE increased phosphorylation of CaMKII and ERK1/2, which was eliminated by IP₃R blockade/knock-out or PKC inhibition. Activation of CaMKII, but not ERK1/2, by these agonists was sensitive to Ca2+ buffering or to Gö6976, the inhibitor of Ca2+-dependent PKC and protein kinase D (PKD). CONCLUSION: CREB phosphorylation by ET-1 and PE may be mainly mediated by IP₃R2/Ca2+-PKC-PKD-CaMKII signaling with a minor contribution by ERK1/2, linked to IP₃R2 and Ca2+-independent PKC, in ventricular myocytes.

Assembly of ER-PM Junctions: A Critical Determinant in the Regulation of SOCE and TRPC1.

Store-operated calcium entry (SOCE), a unique plasma membrane Ca2+ entry mechanism, is activated when ER-[Ca2+] is decreased. SOCE is mediated via the primary channel, Orai1, as well as others such as TRPC1. STIM1 and STIM2 are ER-Ca2+ sensor proteins that regulate Orai1 and TRPC1. SOCE requires assembly of STIM proteins with the plasma membrane channels which occurs within distinct regions in the cell that have been termed as endoplasmic reticulum (ER)-plasma membrane (PM) junctions. The PM and ER are in close proximity to each other within this region, which allows STIM1 in the ER to interact with and activate either Orai1 or TRPC1 in the plasma membrane. Activation and regulation of SOCE involves dynamic assembly of various components that are involved in mediating Ca2+ entry as well as those that determine the formation and stabilization of the junctions. These components include proteins in the cytosol, ER and PM, as well as lipids in the PM. Recent studies have also suggested that SOCE and its components are compartmentalized within ER-PM junctions and that this process might require remodeling of the plasma membrane lipids and reorganization of structural and scaffolding proteins. Such compartmentalization leads to the generation of spatially- and temporally-controlled Ca2+signals that are critical for regulating many downstream cellular functions.

Shear stress activates monovalent cation channel transient receptor potential melastatin subfamily 4 in rat atrial myocytes via type 2 inositol 1,4,5-trisphosphate receptors and Ca(2+) release.

KEY POINTS: During each contraction and haemodynamic disturbance, cardiac myocytes are subjected to fluid shear stress as a result of blood flow and the relative movement of sheets of myocytes. The present study aimed to characterize the shear stress-sensitive membrane current in atrial myocytes using the whole-cell patch clamp technique, combined with pressurized fluid flow, as well as pharmacological and genetic interventions of specific proteins. The data obtained suggest that shear stress indirectly activates the monovalent cation current carried by transient receptor potential melastatin subfamily 4 channels via type 2 inositol 1,4,5-trisphosphate receptor-mediated Ca(2+) release in subsarcolemmal domains of atrial myocytes. Ca(2+) -mediated interactions between these two proteins under shear stress may be an important mechanism by which atrial cells measure mechanical stress and translate it to alter their excitability. ABSTRACT: Atrial myocytes are subjected to shear stress during the cardiac cycle under physiological or pathological conditions. The ionic currents regulated by shear stress remain poorly understood. We report the characteristics, molecular identity and activation mechanism of the shear stress-sensitive current (Ishear ) in rat atrial myocytes. A shear stress of ∼16 dyn cm(-2) was applied to single myocytes using a pressurized microflow system, and the current was measured by whole-cell patch clamp. In symmetrical CsCl solutions with minimal concentrations of internal EGTA, Ishear showed an outwardly rectifying current-voltage relationship (reversal at -2 mV). The current was conducted primarily (∼80%) by monovalent cations but not Ca(2+) . It was suppressed by intracellular Ca(2+) buffering at a fixed physiological level, inhibitors of transient receptor potential melastatin subfamily 4 (TRPM4), intracellular introduction of TRPM4 antibodies or knockdown of TRPM4 expression, suggesting that TRPM4 carries most of this current. A notable reduction in Ishear occurred upon inhibition of Ca(2+) release through the ryanodine receptors or inositol 1,4,5-trisphosphate receptors (IP3 R) and upon depletion of sarcoplasmic reticulum Ca(2+) . In type 2 IP3 R (IP3 R2) knockout atrial myocytes, Ishear was 10-20% of that in wild-type myocytes. Immunocytochemistry and proximity ligation assays revealed that TRPM4 and IP3 R2 were expressed at peripheral sites with co-localization, although they are not localized within 40 nm. Peripheral localization of TRPM4 was intact in IP3 R2 knockout cells. The data obtained in the present study suggest that shear stress activates TRPM4 current by triggering Ca(2+) release from the IP3 R2 in the peripheral domains of atrial myocytes.

Extracellular Adenosine Diphosphate Ribose Mobilizes Intracellular Ca2+ via Purinergic-Dependent Ca2+ Pathways in Rat Pulmonary Artery Smooth Muscle Cells.

BACKGROUND/AIMS: Adenosine diphosphate ribose (ADPR), a product of ß-NAD+ metabolism generated by the multifunctional enzyme CD38, is recognized as a novel signaling molecule. The catalytic site of CD38 orients extracellularly or intracellularly, capable of generating ADPR outside and inside the cells. CD38-dependent pathways have been characterized in pulmonary artery smooth muscle cells (PASMCs); however the physiological function of extracellular ADPR is unclear. METHODS: Ca2+ mobilizing and proliferative effects of extracellular ADPR were characterized and compared with the ATP-induced responses in rat PASMCs; and the expression of purinergic receptor (P2X and P2Y) subtypes were examined in pulmonary arteries. RESULTS: ADPR elicited concentration-dependent increase in [Ca2+]i with a fast transient and a sustained phase in PASMCs. The sustained phase was abolished by Ca2+ removal and inhibited by the non-selective cation channel blocker SKF-96365, but was unaffected by TRPM2 antagonists or nifedipine. The purinergic receptor (P2X) antagonist pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonate inhibited partially the transient and the sustained Ca2+ response, while the P2(XY) inhibitor suramin and the phospholipase C inhibitor U73122 abolished the sustained Ca2+ influx. The P2Y1 antagonist MRS2179 had no effect on the response. By contrast, ATP and ADP activated Ca2+ response exhibited a high and a low affinity component, and the pharmacological profile of ATP-induced Ca2+ response was distinctive from that of ADPR. BrdU incorporation assay showed that ADPR caused significant inhibition whereas ATP caused slight stimulation of PASMC proliferation. RT-PCR analysis found that almost all P2X and P2Y subtypes are expressed in PAs. CONCLUSION: ADPR and ATP activate Ca2+ responses through different combinations of multiple purinergic receptor subtypes; and extracellular ADPR may exert an autocrine/paracrine action via purinergic receptors on PASMCs.