Β-adrenergic Agonist Research Articles

Sympathetic Nerves Coordinate Corneal Epithelial Wound Healing by Controlling the Mobilization of Ly6Chi Monocytes From the Spleen to the Injured Cornea.

This study aims to investigate the potential involvement of spleen-derived monocytes in the repair process following corneal epithelial abrasion. A corneal epithelial abrasion model was established in male C57BL/6J mice, and the dynamic changes of monocyte subpopulations in the injured cornea were analyzed using flow cytometry. The effects of Ly6Chi monocyte depletion and local adoptive transfer of purified Ly6Chi monocytes on wound closure and neutrophil recruitment to the injured cornea were observed. The effect of sympathetic nerves on the recruitment of spleen-derived Ly6Chi monocytes to the injured cornea was also investigated using multiple methods. The emigration of fluorescence-labeled monocytes to the injured cornea was validated through intravital microscopy. Finally, differential genes between different groups were identified through high-throughput RNA sequencing and analyzed for functional enrichment, followed by verification by quantitative PCR. Ly6Chi monocytes were present in large numbers in the injured cornea prior to neutrophil recruitment. Predepletion of Ly6Chi monocytes significantly inhibited neutrophil recruitment to the injured cornea. Furthermore, surgical removal of the spleen significantly reduced the number of Ly6Chi monocytes in the injured cornea. Further observations revealed that sympathetic blockade significantly reduced the number of Ly6Chi monocytes recruited to the injured cornea. In contrast, administration of the β2-adrenergic receptor agonist significantly increased the number of Ly6Chi monocytes recruited to the injured cornea in animals treated with sympathectomy and catecholamine synthesis inhibition. Our results suggest that spleen-derived Ly6Chi monocytes, under the control of the sympathetic nervous system, play a critical role in the inflammatory response following corneal injury.

Daily injection of the β2 adrenergic agonist clenbuterol improved poor muscle growth and body composition in lambs following heat stress-induced intrauterine growth restriction.

Background: Intrauterine growth restriction (IUGR) is associated with reduced β2 adrenergic sensitivity, which contributes to poor postnatal muscle growth. The objective of this study was to determine if stimulating β2 adrenergic activity postnatal would rescue deficits in muscle growth, body composition, and indicators of metabolic homeostasis in IUGR offspring. Methods: Time-mated ewes were housed at 40°C from day 40 to 95 of gestation to produce IUGR lambs. From birth, IUGR lambs received daily IM injections of 0.8μg/kg clenbuterol HCl (IUGR+CLEN; n = 11) or saline placebo (IUGR; n = 12). Placebo-injected controls (n = 13) were born to pair-fed thermoneutral ewes. Biometrics were assessed weekly and body composition was estimated by ultrasound and bioelectrical impedance analysis (BIA). Lambs were necropsied at 60days of age. Results: Bodyweights were lighter (p ≤ 0.05) for IUGR and IUGR+CLEN lambs than for controls at birth, day 30, and day 60. Average daily gain was less (p ≤ 0.05) for IUGR lambs than controls and was intermediate for IUGR+CLEN lambs. At day 58, BIA-estimated whole-body fat-free mass and ultrasound-estimated loin eye area were less (p ≤ 0.05) for IUGR but not IUGR+CLEN lambs than for controls. At necropsy, loin eye area and flexor digitorum superficialis muscles were smaller (p ≤ 0.05) for IUGR but not IUGR+CLEN lambs than for controls. Longissimus dorsi protein content was less (p ≤ 0.05) and fat-to-protein ratio was greater (p ≤ 0.05) for IUGR but not IUGR+CLEN lambs than for controls. Semitendinosus from IUGR lambs had less (p ≤ 0.05) β2 adrenoreceptor content, fewer (p ≤ 0.05) proliferating myoblasts, tended to have fewer (p = 0.08) differentiated myoblasts, and had smaller (p ≤ 0.05) muscle fibers than controls. Proliferating myoblasts and fiber size were recovered (p ≤ 0.05) in IUGR+CLEN lambs compared to IUGR lambs, but β2 adrenoreceptor content and differentiated myoblasts were not recovered. Semitendinosus lipid droplets were smaller (p ≤ 0.05) in size for IUGR lambs than for controls and were further reduced (p ≤ 0.05) in size for IUGR+CLEN lambs. Conclusion: These findings show that clenbuterol improved IUGR deficits in muscle growth and some metabolic parameters even without recovering the deficit in β2 adrenoreceptor content. We conclude that IUGR muscle remained responsive to β2 adrenergic stimulation postnatal, which may be a strategic target for improving muscle growth and body composition in IUGR-born offspring.

Microwave-Assisted Reductive Amination under Heterogeneous Catalysis for the Synthesis of β-Adrenergic Agonist and Related Structures

Reductive amination is a powerful tool in sustainable organic synthesis that allows chemists to access a wide range of valuable amine products using renewable feedstocks and mild reaction conditions, with minimal waste generation. Practical applications can be found in various fields, including pharmaceuticals, contributing to greener and more sustainable chemical processes. In this work, we present a heterogeneous (Rh and Pt) catalyzed protocol for the fast and efficient synthesis of ractopamine hydrochloride (β-adrenergic drug) under microwave-assisted reductive amination protocol starting from raspberry ketone and octopamine. Microwave (MW) successfully accelerated the hydrogenation reaction and reduced the reaction time from 13 h to only 3 h under mild conditions (50 °C at 10 bar). The best catalysts were Pt/C and Rh/C, which led to high conversion and selectivity towards ractopamine:HCl. Different solvents and ketone substrates were also experimented. Acetophenone, cyclohexanone, and 2-butanone reacted at lower H2 pressure (5 bar), and highest selectivity was observed with cyclohexanone (99%). These preliminary experiments may be useful for further process improvements in the synthesis of β-adrenergic agonists and related structures and underline the positive synergy between MW and heterogeneous catalysis.

Multiomics Approaches Identify Biomarkers for BAT Thermogenesis.

Brown adipose tissue (BAT) thermogenesis confers beneficial effects on metabolic diseases such as obesity and type-2 diabetes. Nevertheless, the mechanism and lipid driving the process that evokes this response have not been investigated yet. Here, a multiomics approach of integrative transcriptomics and lipidomics is used to explore the mechanism of regulating thermogenesis in BAT and providing promising lipid biomarkers and biomarker genes for thermogenic activators as antiobesity drugs. Lipidomics analysis demonstrated that a high abundance of glycerophospholipids and sphingolipids was more significant in BAT than in WAT. Enrichment analysis of upregulated DEGs between WAT and BAT screened suggested that the differences were mainly involved in lipid metabolism. Besides, β3-adrenergic agonist stimulation reduced the levels of TAG and DAG and increased the content of PC, PE, CL, and LPC and expression of genes involved in thermogenesis, fatty acid elongation, and glycerophospholipid metabolism in BAT. In this study, based on interpreting the inherent characterization of BAT as thermogenic tissue through comparison with WAT as fat storage tissue, adrenergic stimulation-induced BAT thermogenesis further identified specific lipid biomarkers (7 TAG species, 10 PC species, 1 LPC species, and 1 CL species) and Elovl3 and Crat gene biomarkers, which may provide targets for combating obesity by boosting BAT thermogenesis.

A COMPREHENSIVE REVIEW ON ASTHMA: PATHOPHYSIOLOGY, TREATMENT AND ROLE OF GENETICS

Background: To date, several studies conducted to assess the role of genetics in the pathophysiology and treatment of asthma. Objectives: However, the findings were not consistent and remain uncertain. Thus, it is the need of the hour to assess the recent literature, observe limitations and finally propose future research directions in this field. Methodology: The relevant literature published in the English language was retrieved by using different electronic databases including Cochrane Library, Web of Science, PubMed, Ovid, EMBASE, and MEDLINE. Results: Leukotriene modifiers, β-adrenergic receptor agonists, and inhaled corticosteroids are the three most important classes. Data suggested that the heterogeneity in the treatment response among asthma patients is partly due to genetic factors. Conclusion: Large-scale genetic investigations can be improved by using the high throughput technologies that could have promising potential for personalized treatment of asthma, which in turn will minimize side effects, better therapeutic outcomes, and finally lead to more cost-effective care.

Role of DNA methylation during recovery from spinal cord injury with and without β2-adrenergic receptor agonism

Daily treatment with the FDA-approved β2-adrenergic receptor agonist formoterol beginning 8 h after severe spinal cord injury (SCI) induces mitochondrial biogenesis and improves recovery in mice. We observed decreased DNA methyltransferase (DNMT) expression, global DNA methylation and methylation of the mitochondrial genes PGC-1α and NDUFS1 in the injury site of formoterol-treated mice 1 DPI, but this effect was lost by 7 DPI. To investigate the role of DNA methylation on recovery post-SCI, injured mice were treated daily with formoterol or vehicle, plus the DNMT inhibitor decitabine (DAC) on days 7–9. While DAC had no apparent effect on formoterol-induced recovery, mice treated with vehicle plus DAC exhibited increased BMS scores compared to vehicle alone beginning 15 DPI, reaching a degree of functional recovery similar to that of formoterol-treated mice by 21 DPI. Furthermore, DAC treatment increased injury site mitochondrial protein expression in vehicle-treated mice to levels comparable to that of formoterol-treated mice. The effect of DNMT inhibition on pain response with and without formoterol was assessed following moderate SCI. While all injured mice not treated with DAC displayed thermal hyperalgesia by 21 DPI, mice treated with formoterol exhibited decreased thermal hyperalgesia compared to vehicle-treated mice by 35 DPI. Injured mice treated with DAC, regardless of formoterol treatment, did not demonstrate thermal hyperalgesia at any time point assessed. Although these data do not suggest enhanced formoterol-induced recovery with DNMT inhibition, our findings indicate the importance of DNA methylation post-SCI and support both DNMT inhibition and formoterol as potential therapeutic avenues.

Browning of perivascular adipose tissue prevents vascular dysfunction and reduces hypertension in angiotensin II-infused mice.

Hypertension is a world-leading cause of cardiovascular disease and premature deaths. Vascular tone is in part regulated by perivascular adipose tissue (PVAT) that releases pro and anticontractile factors. In hypertension, dysfunctional PVAT is observed and studies have indicated a causal relationship between dysfunctional PVAT and vascular damage in hypertension. The phenotype of PVAT on resistance vessels is primarily white adipose tissue. The present study investigates the impact of a changed phenotype, i.e., browning of PVAT, on vascular function and the development of hypertension. Browning was induced by β3-adrenergic agonist in control and angiotensin II-induced hypertensive mice. Studied parameters included blood pressure by tail-cuff plethysmography and vascular function by wire myography. Browning was confirmed through an immunohistochemical and gene analysis approach. The anticontractile effect of PVAT is lost in untreated hypertensive mice and vascular tone and blood pressure are increased. Browning of PVAT resulted in a maintained anticontractile effect, improved endothelial function, and reduced development of hypertension. Phenotype of PVAT is a major determinant of PVAT health during hypertensive conditions. Our data clearly demonstrates that browning of PVAT, i.e. changing the phenotype of PVAT, protects the vascular function and counteract the development of hypertension. This study provides novel insights into how PVAT can be protected in pathologies and thus limit the development of hypertension.

Social isolation increases metabolic rate in the transition of light- to dark- phase and advances Rev-erb-α expression in brown adipose tissue to regulate daily rhythm of core body temperature in mice

Mammals use social thermoregulation to maintain the core body temperature (Tc) at a lower energy cost. Brown adipose tissue (BAT) plays a crucial role in thermoregulation. This study tested the hypothesis that social isolation induces alterations in thermogenesis and metabolism to maintain the rhythm of Tc throughout the day. Adult male mice (C57BLJ/6) were maintained in groups of 4–5 (group-housed) or isolated (single-housed) for 28 days. Telemetric probes recorded spontaneous locomotor activity (SLA) and Tc to analyze SLA and Tc daily rhythms. Body weight was measured weekly. VO2 consumption was analyzed at zeitgeber time (ZT) 1–3 (beginning of light phase) or ZT10–14 (beginning of dark phase). The expression of thermogenic-related and clock genes in the BAT occurred at ZT2, ZT8, ZT14, and ZT20. The β3 adrenergic agonist CL-316,243 was i.p. injected in the group- or single-housed mice. Social isolation increased the amplitude of Tc rhythm but decreased the amplitude of SLA oscillation. Nevertheless, in single-housed mice, the circadian peak of Tc and SLA was unaltered, with reduced body weight gain, increased VO2 consumption, and BAT Ucp1 expression at ZT14. Isolation advanced the BAT Rev-erb-ɑ peak of expression and phased-shift the peak of expression in BAT Bmal1, while it abolished the daily BAT Per2 oscillation. Isolation also abolished the BAT Ucp1 and hormone-sensitive lipase (Hsl) expression induced by stimulation of the β3ADR, but it increased Rev-erb-ɑ and Pgc1α. Thus, alterations in Ucp1, Reverb-α, Bmal1, and Per2 daily expression may favor an increased metabolic rate at the beginning of the dark, which, in turn, contributes to maintaining the daily Tc rhythm at the expense of reduced body weight gain in isolated mice. In addition, at least at the transcription level, the response of BAT from isolated mice to adrenergic agonist seems to be via a non-canonical mechanism involving Rev-erb-α and Pgc1α.

The β2-adrenergic receptor agonist terbutaline upregulates T helper-17 cells in a protein kinase A-dependent manner

BackgroundT helper 17 (Th17) cells produce IL-17A cytokine and can exacerbate autoimmune diseases and asthma. The β2 adrenergic receptor is a g protein-coupled receptor that induces cAMP second messenger pathways. We tested the hypothesis that terbutaline, a β2-adrenergic receptor-specific agonist, promotes IL-17 secretion by memory Th17 cells in a cAMP and PKA-dependent manner. MethodsVenous peripheral blood mononuclear cells (PBMC) from healthy human participants were activated with anti-CD3 and anti-CD28 antibodies. Secreted IL-17A was measured by enzyme linked immunosorbent assay, intracellular IL-17A, and RORγ were measured using flow cytometry, and RORC by qPCR. Memory CD3+CD4+CD45RA-CD45RO+ T cells were obtained by immunomagnetic negative selection and activated with tri-antibody complex CD3/CD28/CD2. Secreted IL-17A, intracellular IL-17A, RORC were measured, and phosphorylated-serine133-CREB was measured by western blotting memory Th cells. ResultsTerbutaline increased IL-17A (p < 0.001), IL-17A+ cells (p < 0.05), and RORC in activated PBMC and memory Th cells. The PKA inhibitors H89 (p < 0.001) and Rp-cAMP (p < 0.01) abrogated the effects of terbutaline on IL-17A secretion in PBMC and memory T cells. Rolipram increased IL-17A (p < 0.01) to a similar extent as terbutaline. P-Ser133-CREB was increased by terbutaline (p < 0.05) in memory T cells. ConclusionTerbutaline augments memory Th17 cells in lymphocytes from healthy participants. This could exacerbate autoimmune diseases or asthma, in cases where Th17 cells are considered to be pro-inflammatory.

Selective activation of PPARα maintains thermogenic capacity of beige adipocytes

Beige adipocytes are inducible thermogenic adipocytes used for anti-obesity treatment. Beige adipocytes rapidly lose their thermogenic capacity once external cues are removed. However, long-term administration of stimulants, such as PPARγ and β-adrenergic receptor agonists, is unsuitable due to various side effects. Here, we reported that PPARα pharmacological activation was the preferred target for maintaining induced beige adipocytes. Pemafibrate used in clinical practice for dyslipidemia was developed as a selective PPARα modulator (SPPARMα). Pemafibrate administration regulated the thermogenic capacity of induced beige adipocytes, repressed body weight gain, and ameliorated impaired glucose tolerance in diet-induced obese mouse models. The transcriptome analysis revealed that the E-twenty-six transcription factor ELK1 acted as a cofactor of PPARα. ELK1 was mobilized to the Ucp1 transcription regulatory region with PPARα and modulated its expression by pemafibrate. These results suggest that selective activation of PPARα by pemafibrate is advantageous to maintain the function of beige adipocytes.

When immune thrombocytopenia gets on your nerves: β2-adrenergic receptor agonists as a potential treatment?

When immune thrombocytopenia gets on your nerves: β2-adrenergic receptor agonists as a potential treatment?

Calcium Handling Remodeling Underlies Impaired Sympathetic Stress Response in Ventricular Myocardium from Cacna1c Haploinsufficient Rats.

CACNA1C encodes the pore-forming α1C subunit of the L-type Ca2+ channel, Cav1.2. Mutations and polymorphisms of the gene are associated with neuropsychiatric and cardiac disease. Haploinsufficient Cacna1c+/- rats represent a recently developed model with a behavioral phenotype, but its cardiac phenotype is unknown. Here, we unraveled the cardiac phenotype of Cacna1c+/- rats with a main focus on cellular Ca2+ handling mechanisms. Under basal conditions, isolated ventricular Cacna1c+/- myocytes exhibited unaltered L-type Ca2+ current, Ca2+ transients (CaTs), sarcoplasmic reticulum (SR) Ca2+ load, fractional release, and sarcomere shortenings. However, immunoblotting of left ventricular (LV) tissue revealed reduced expression of Cav1.2, increased expression of SERCA2a and NCX, and augmented phosphorylation of RyR2 (at S2808) in Cacna1c+/- rats. The β-adrenergic agonist isoprenaline increased amplitude and accelerated decay of CaTs and sarcomere shortenings in both Cacna1c+/- and WT myocytes. However, the isoprenaline effect on CaT amplitude and fractional shortening (but not CaT decay) was impaired in Cacna1c+/- myocytes exhibiting both reduced potency and efficacy. Moreover, sarcolemmal Ca2+ influx and fractional SR Ca2+ release after treatment with isoprenaline were smaller in Cacna1c+/- than in WT myocytes. In Langendorff-perfused hearts, the isoprenaline-induced increase in RyR2 phosphorylation at S2808 and S2814 was attenuated in Cacna1c+/- compared to WT hearts. Despite unaltered CaTs and sarcomere shortenings, Cacna1c+/- myocytes display remodeling of Ca2+ handling proteins under basal conditions. Mimicking sympathetic stress with isoprenaline unmasks an impaired ability to stimulate Ca2+ influx, SR Ca2+ release, and CaTs caused, in part, by reduced phosphorylation reserve of RyR2 in Cacna1c+/- cardiomyocytes.

Pristimerin protects against pathological cardiac hypertrophy through improvement of PPARα pathway

Pristimerin (PM), serving as a biological component mainly obtained from Celastraceae and Hippocrateaceae families, has been extensively explored for its numerous pharmacological activities, especially anti-cancer activity. However, the function of PM on pathological cardiac hypertrophy is poorly understood. This work was intended to investigate the effects of PM on pressure-overload induced myocardial hypertrophy and its potential pathways. Mouse model of pathological cardiac hypertrophy was generated by transverse aortic constriction (TAC) or minipump administration of the β-adrenergic agonist ISO for 4 weeks, and PM (0.5 mg/Kg/d, i.p.) was treated for 2 weeks. PPARα−/− mice received TAC surgery were used for mechanism exploration. Moreover, neonatal rat cardiomyocytes (NRCMs) were utilized to explore the effect of PM following Angiotensin II (Ang II, 1.0 μM) administration. We found that PM attenuated pressure-overload induced cardiac dysfunction, myocardial hypertrophy and fibrosis in mice. Likewise, PM incubation dramatically reversed Ang II-mediated cardiomyocytes hypertrophy in NRCMs. RNA-Sequence showed that PM selectively contributed to improvement of PPARα/PGC1 signaling, while silencing PPARα abrogated the beneficial effects of PM on Ang II-treated NRCMs. Importantly, PM ameliorated Ang II-induced mitochondrial dysfunction and decrease in metabolic genes, whereas knockdown of PPARα eliminated these alterations in NRCMs. Similarly, PM presented limited protective effects on pressure-overload induced systolic dysfunction and myocardial hypertrophy in PPARα deficient mice. Overall, this study revealed that PM exerted protective activity against pathological cardiac hypertrophy through improvement of PPARα/PGC1 pathway.

Molecular, morphological and behavioral alterations of zebrafish (Danio rerio) embryos/larvae after clorprenaline hydrochloride exposure.

Chlorprenaline hydrochloride (CLOR) is a typical representative of β-adrenergic agonists that may be used illegally as a livestock feed additive and may have adverse impacts on the environment. In the present study, zebrafish embryos were exposed to CLOR to investigate its developmental toxicity and neurotoxicity. The results demonstrated that CLOR exposure led to adverse effects on developing zebrafish, such as morphological changes, a high heart rate, and increased body length, resulting in developmental toxicity. Moreover, the up-regulation of activities of superoxide dismutase (SOD) and catalase (CAT) and the enhancement of malondialdehyde (MDA) content illustrated that CLOR exposure activated oxidative stress in exposed zebrafish embryos. Meanwhile, CLOR exposure also caused alterations in locomotive behavior in zebrafish embryos, including an increase in acetylcholinesterase (AChE) activity. Quantitative polymerase chain reaction (QPCR) results showed that the transcription of genes related to the central nervous system (CNS) development, namely, mbp, syn2a, α1-tubulin, gap43, shha, and elavl3, indicated that CLOR exposure could lead to neurotoxicity in zebrafish embryos. These results showed that CLOR exposure could cause developmental neurotoxicity in the early stages of zebrafish development and that CLOR might induce neurotoxicity by altering the expression of neuro-developmental genes, elevating AChE activity, and activating oxidative stress.

Remodeled connexin 43 hemichannels alter cardiac excitability and promote arrhythmias.

Connexin-43 (Cx43) is the most abundant protein forming gap junction channels (GJCs) in cardiac ventricles. In multiple cardiac pathologies, including hypertrophy and heart failure, Cx43 is found remodeled at the lateral side of the intercalated discs of ventricular cardiomyocytes. Remodeling of Cx43 has been long linked to spontaneous ventricular arrhythmia, yet the mechanisms by which arrhythmias develop are still debated. Using a model of dystrophic cardiomyopathy, we previously showed that remodeled Cx43 function as aberrant hemichannels (non-forming GJCs) that alter cardiomyocyte excitability and, consequently, promote arrhythmias. Here, we aim to evaluate if opening of remodeled Cx43 can serve as a general mechanism to alter cardiac excitability independent of cellular dysfunction associated with a particular cardiomyopathy. To address this issue, we used a genetically modified Cx43 knock-in mouse (S3A) that promotes cardiac remodeling of Cx43 protein without apparent cardiac dysfunction. Importantly, when S3A mice were subjected to cardiac stress using the β-adrenergic agonist isoproterenol (Iso), they displayed acute and severe arrhythmias, which were not observed in WT mice. Pretreatment of S3A mice with the Cx43 hemichannel blocker, Gap19, prevented Iso-induced abnormal electrocardiographic behavior. At the cellular level, when compared with WT, Iso-treated S3A cardiomyocytes showed increased membrane permeability, greater plasma membrane depolarization, and Ca2+ overload, which likely caused prolonged action potentials, delayed after depolarizations, and triggered activity. All these cellular dysfunctions were also prevented by Cx43 hemichannel blockers. Our results support the notion that opening of remodeled Cx43 hemichannels, regardless of the type of cardiomyopathy, is sufficient to mediate cardiac-stress-induced arrhythmogenicity.

Docking guided phase display to develop fusion protein with novel scFv and alkaline phosphatase for one-step ELISA salbutamol detection.

Salbutamol (SAL) is a β2 adrenergic receptor agonist which has potential hazardous effects for human health. It is very important to establish a sensitive and convenient method to monitor SAL. Here we introduce a method to combine the information from docking and site specific phage display, with the aim to obtain scFv with high affinity to SAL. First, single chain variable fragment (scFv) antibodies against SAL were generated through phage display. By using molecular docking approach, the complex structure of SAL with antibody was predicted and indicated that H3 and L3 contribute to the binding. Then new libraries were created by randomization specific residues located on H3 and L3 according to the docking results. Anti-SAL scFv antibodies with high efficiency were finally identified. In addition, the selected scFv was fused with alkaline phosphatase and expressed in E coli to develop a rapid and low-cost one step ELISA to detect SAL.

The secretory ability of newly formed secretory granules is regulated by pro-cathepsin B and amylase in parotid glands

Parotid glands are exocrine glands that release saliva into the oral cavity. Acinar cells of parotid glands produce many secretory granules (SGs) that contain the digestion enzyme amylase. After the generation of SGs in the Golgi apparatus, they mature by enlarging and membrane remodeling. VAMP2, which is involved in exocytosis, accumulates in the membrane of mature SGs. The remodeling of SG membranes is regarded as a preparation process for exocytosis but its detailed mechanism remains unknown. To address that subject, we investigated the secretory ability of newly formed SGs. Although amylase is a useful indicator of secretion, the cell leakage of amylase might affect the measurement of secretion. Thus, in this study, we focused on cathepsin B (CTSB), a lysosomal protease, as an indicator of secretion. It has been reported that some procathepsin B (pro-CTSB), which is a precursor of CTSB, is initially sorted to SGs after which it is transported to lysosomes by clathrin-coated vesicles. Because pro-CTSB is processed to mature CTSB after its arrival in lysosomes, we can distinguish between the secretion of SGs and cell leakage by measuring the secretion of pro-CTSB and mature CTSB, respectively. When acinar cells isolated from parotid glands were stimulated with isoproterenol (Iso), a β-adrenergic agonist, the secretion of pro-CTSB was increased. In contrast, mature CTSB was not detected in the medium although it was abundant in the cell lysates. To prepare parotid glands rich in newly formed SGs, the depletion of per-existing SGs was induced by an intraperitoneal injection of Iso into rats. At 5 h after that injection, newly formed SGs were observed in parotid acinar cells and the secretion of pro-CTSB was also detected. We confirmed that the purified newly formed SGs contained pro-CTSB, but not mature CTSB. At 2 h after Iso injection, few SGs were observed in the parotid glands and the secretion of pro-CTSB was not detected, which proved that the Iso injection depleted pre-existing SGs and the SGs observed at 5 h were newly formed after the Iso injection. These results suggest that newly formed SGs have a secretory ability prior to membrane remodeling.

Sexual dimorphism in bidirectional SR-mitochondria crosstalk in ventricular cardiomyocytes

Calcium transfer into the mitochondrial matrix during sarcoplasmic reticulum (SR) Ca2+ release is essential to boost energy production in ventricular cardiomyocytes (VCMs) and match increased metabolic demand. Mitochondria from female hearts exhibit lower mito-[Ca2+] and produce less reactive oxygen species (ROS) compared to males, without change in respiration capacity. We hypothesized that in female VCMs, more efficient electron transport chain (ETC) organization into supercomplexes offsets the deficit in mito-Ca2+ accumulation, thereby reducing ROS production and stress-induced intracellular Ca2+ mishandling. Experiments using mitochondria-targeted biosensors confirmed lower mito-ROS and mito-[Ca2+] in female rat VCMs challenged with β-adrenergic agonist isoproterenol compared to males. Biochemical studies revealed decreased mitochondria Ca2+ uniporter expression and increased supercomplex assembly in rat and human female ventricular tissues vs male. Importantly, western blot analysis showed higher expression levels of COX7RP, an estrogen-dependent supercomplex assembly factor in female heart tissues vs males. Furthermore, COX7RP was decreased in hearts from aged and ovariectomized female rats. COX7RP overexpression in male VCMs increased mitochondrial supercomplexes, reduced mito-ROS and spontaneous SR Ca2+ release in response to ISO. Conversely, shRNA-mediated knockdown of COX7RP in female VCMs reduced supercomplexes and increased mito-ROS, promoting intracellular Ca2+ mishandling. Compared to males, mitochondria in female VCMs exhibit higher ETC subunit incorporation into supercomplexes, supporting more efficient electron transport. Such organization coupled to lower levels of mito-[Ca2+] limits mito-ROS under stress conditions and lowers propensity to pro-arrhythmic spontaneous SR Ca2+ release. We conclude that sexual dimorphism in mito-Ca2+ handling and ETC organization may contribute to cardioprotection in healthy premenopausal females.

β-adrenergic receptor agonist promotes ductular expansion during 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced chronic liver injury

Intrahepatic nerves are involved in the regulation of metabolic reactions and hepatocyte-based regeneration after surgical resection, although their contribution to chronic liver injury remains unknown. Given that intrahepatic nerves are abundant in the periportal tissue, they may be correlated also with cholangiocyte-based regeneration. Here we demonstrate that isoproterenol (ISO), a β-adrenergic receptor agonist, promoted ductular expansion induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) in vivo. Immunofluorescence analysis shows that nerve fibers positive for tyrosine hydroxylase form synaptophysin-positive nerve endings on epithelial cell adhesion molecule-positive (EpCAM+) cholangiocytes as well as on Thy1+ periportal mesenchymal cells (PMCs) that surround bile ducts, suggesting that the intrahepatic biliary tissue are targeted by sympathetic nerves. In vitro analyses indicate that ISO directly increases cAMP levels in cholangiocytes and PMCs. Mechanistically, ISO expands the lumen of cholangiocyte organoids, resulting in promotion of cholangiocyte proliferation, whereas it increases expression of fibroblast growth factor 7, a growth factor for cholangiocytes, in PMCs. Taken together, the results indicate that intrahepatic sympathetic nerves regulate remodeling of bile ducts during DDC-injury by the activation of β-adrenergic receptors on cholangiocytes and PMCs.

Kleinhovia Hospita Leaf Extract Protects the Heart Against Infarction by Isoproterenol

Background: Isoproterenol, a β-adrenergic agonist, may induce myocardial infarction when used in high dosage in rats. This study was conducted to evaluate the effect of the ethanol extract of Kleinhovia hospita leaves on cardiac biomarkers and myocardial structures of rats induced by isoproterenol. Methods: Male rats (n=30) were assigned as a normal controls or treatment groups. The treatment groups received pretreatments, either placebo or the extract at doses of 250, 500, or 750 mg/kg for 14 days, followed by two isoproterenol injections at 100 mg/kg. After 24 hours, blood samples were taken and the hearts dissected. The tested cardiac biomarkers were creatinine kinase myocardial band (CKMB) and lactate dehydrogenase (LDH). Heart histopathology analysis was performed followed by staining of samples with hematoxylin and eosin. Results: The isoproterenol injections significantly increased CKMB and LDH levels in the placebo group compared to those in normal controls. Pretreatment with the extract at doses of 500 and 750 mg/kg significantly reduced the serum CKMB and LDH levels compared to those of the placebo. The histopathological examinations showed the presence of diffused necrosis and severe inflammation in the placebo group. Pretreatment with the extract at 500 or 750 mg/kg significantly reduced the myocardial tissue damages in rats. Conclusion: The K. hospita extract at doses of 500 or 750 mg/kg significantly reduced the infarctions in the rats’ heart tissue, shown by significantly low levels of CKMB and LDH, and reduced necrotic lesions and inflammation in the rat heart tissue samples induced by isoproterenol pretreatment.