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To understand the direct impact of bradykinin in autonomic control of circulation through baroreflex afferent pathway.The mean arterial pressure (MAP) was monitored while bradykinin and its agonists were applied via nodose (NG) microinjection, the expression of bradykinin receptors (BRs) in the NG (1Under physiological condition, bradykinin-induced dose- and estrogen-dependent reductions of MAP with lower estimated ECThese datasets have demonstrated for the first time that bradykinin mediates direct activation of baroreflex afferent function to trigger estrogen-dependent depressor response, which is due mainly to the direct activation/neuroexcitation of female-specific myelinated Ah-type baroreceptor neurons leading to a sexual dimorphism in parasympathetic domination of blood pressure regulation via activation of B

It is increasingly recognized that a single protein can have multiple, sometimes paradoxical, roles in cell functions as well as pathological conditions depending on its cellular locations. Here we report that moesins (MSNs) in the intracellular and extracellular domains present opposing roles in pro-tumorigenic signaling in breast cancer cells. Using live cell imaging with fluorescence resonance energy transfer (FRET)- and green fluorescent protein (GFP)-based biosensors, we investigated the molecular mechanism underlying the cellular location-dependent effect of MSN on Src and β-catenin signaling in MDA-MB-231 breast cancer cells. Inhibition of intracellular MSN decreased the activities of Src and FAK, whereas overexpression of intracellular MSN increased them. By contrast, extracellular MSN decreased the activities of Src, FAK, and RhoA, as well as β-catenin translocation to the nucleus. Consistently, Western blotting and MTT-based analysis showed that overexpression of intracellular MSN elevated the expression of oncogenic genes, such as p-Src, β-catenin, Lrp5, MMP9, Runx2, and Snail, as well as cell viability, whereas extracellular MSN suppressed them. Conditioned medium derived from MSN-overexpressing mesenchymal stem cells or osteocytes showed the anti-tumor effects by inhibiting the Src activity and β-catenin translocation to the nucleus as well as the activities of FAK and RhoA and MTT-based cell viability. Conditioned medium derived from MSN-inhibited cells increased the Src activity, but it did not affect the activities of FAK and RhoA. Silencing CD44 and/or FN1 in MDA-MB-231 cells blocked the suppression of Src activity and β-catenin accumulation in the nucleus by extracellular MSN. Collectively, the results suggest that cellular location-specific MSN is a strong regulator of Src and β-catenin signaling in breast cancer cells, and that extracellular MSN exerts tumor-suppressive effects via its interaction with CD44 and FN1.

Female-specific subpopulation of myelinated Ah-type baroreceptor neurons (BRNs) in nodose ganglia is the neuroanatomical base of sexual-dimorphic autonomic control of blood pressure regulation, and KCa1.1 is a key player in modulating the neuroexcitation in nodose ganglia. In this study we investigated the exact mechanisms underlying KCa1.1-mediated neuroexcitation of myelinated Ah-type BRNs in the presence or absence of estrogen. BRNs were isolated from adult ovary intact (OVI) or ovariectomized (OVX) female rats, and identified electrophysiologically and fluorescently. Action potential (AP) and potassium currents were recorded using whole-cell recording. Consistently, myelinated Ah-type BRNs displayed a characteristic discharge pattern and significantly reduced excitability after OVX with narrowed AP duration and faster repolarization largely due to an upregulated iberiotoxin (IbTX)-sensitive component; the changes in AP waveform and repetitive discharge of Ah-types from OVX female rats were reversed by G1 (a selective agonist for estrogen membrane receptor GPR30, 100 nM) and/or IbTX (100 nM). In addition, the effect of G1 on repetitive discharge could be completely blocked by G15 (a selective antagonist for estrogen membrane receptor GPR30, 3 μM). These data suggest that estrogen deficiency by removing ovaries upregulates KCa1.1 channel protein in Ah-type BRNs, and subsequently increases AP repolarization and blunts neuroexcitation through estrogen membrane receptor signaling. Intriguingly, this upregulated KCa1.1 predicted electrophysiologically was confirmed by increased mean fluorescent intensity that was abolished by estrogen treatment. These electrophysiological findings combined with immunostaining and pharmacological manipulations reveal the crucial role of KCa1.1 in modulation of neuroexcitation especially in female-specific subpopulation of myelinated Ah-type BRNs and extend our current understanding of sexual dimorphism of neurocontrol of BP regulation.

Rationale: The progression of cancer cells depends on the soil and building an inhibitory soil might be a therapeutic option. We previously created tumor-suppressive secretomes by activating Wnt signaling in MSCs. Here, we examined whether the anti-tumor secretomes can be produced from tumor cells. Methods: Wnt signaling was activated in tumor cells by overexpressing β-catenin or administering BML284, a Wnt activator. Their conditioned medium (CM) was applied to cancer cells or tissues, and the effects of CM were evaluated. Tumor growth in the mammary fat pad and tibia in C57BL/6 female mice was also evaluated through μCT imaging and histology. Whole-genome proteomics analysis was conducted to determine and characterize novel tumor-suppressing proteins, which were enriched in CM. Results: The overexpression of β-catenin or the administration of BML284 generated tumor-suppressive secretomes from breast, prostate and pancreatic cancer cells. In the mouse model, β-catenin-overexpressing CM reduced tumor growth and tumor-driven bone destruction. This inhibition was also observed with BML284-treated CM. Besides p53 and Trail, proteomics analysis revealed that CM was enriched with enolase 1 (Eno1) and ubiquitin C (Ubc) that presented notable tumor-suppressing actions. Importantly, Eno1 immunoprecipitated CD44, a cell-surface adhesion receptor, and its silencing suppressed Eno1-driven tumor inhibition. A pan-cancer survival analysis revealed that the downregulation of MMP9, Runx2 and Snail by CM had a significant impact on survival outcomes (p < 0.00001). CM presented a selective inhibition of tumor cells compared to non-tumor cells, and it downregulated PD-L1, an immune escape modulator. Conclusions: The tumor-suppressive secretome can be generated from tumor cells, in which β-catenin presented two opposing roles, as an intracellular tumor promoter in tumor cells and a generator of extracellular tumor suppressor in CM. Eno1 was enriched in CM and its interaction with CD44 was involved in Eno1's anti-tumor action. Besides presenting a potential option for treating primary cancers and metastases, the result indicates that aggressive tumors may inhibit the growth of less aggressive tumors via tumor-suppressive secretomes.

Large conductance of Ca2+-activated K+ channel (KCa1.1) plays an inhibitory role in neuroexcitation. However, the expression of KCNMB4/β4-subunit in the nodose ganglia (NG) and nucleus tractus solitarius (NTS), and its effect and regulation on baroreflex afferent function at post-transcriptional level of female rats remains unknown. Here, we demonstrated that the expression of β4-subunit encoded by KCNMB4 was significantly lower in females vs. males and ovariectomized (OVX) rats in the NG. Although all baroreceptor neurons (BRNs) expressed β4-subunit, altered discharge characteristics were only observed in Ah-type neurons after ovariectomy. Notably, the decreased excitability of Ah-types was restored by paxilline and further enhanced by iberiotoxin. The consistent changes were observed in excitatory post-synaptic currents. The level of miR-504 was higher in females, which was predicted to bind to the 3'UTR of KCNMB4. In consistent, an inverse expression pattern between miR-504 and KCNMB4 was observed in baroreflex afferents. The paxilline-sensitive β4-subunits is less in Ah-types and up-regulated by ovariectomy. These data indicated that KCa1.1 β4-subunit is the key regulator in neuroexcitation of Ah-types and sexual-dimorphism in baroreflex afferent function through estrogen-dependent inhibition of KCNMB4 expression via miR-504.

Silent myocardial infarction (MI) is critical for clinical practice with increasing risk for women and the cause remains a medical mystery. Upon the discovery of female-specific Ah-type baroreceptor neurons (BRNs), we hypothesize that glutamate mediates depressor response through afferent-specific expression of particular glutamate receptors (mGluRs) leading descending inhibition of cardiac nociception. In vivo, tail-flick reflex and electromyography were assessed to evaluate glutamate-mediated blood pressure regulation, peripheral and cardiac nociception. The results showed that glutamate decreased mean arterial pressure (MAP) and increased peripheral nociception. Interestingly, glutamate-mediated capsaicin-induced cardiac nociception was strongly reduced in female rats compared with males. Furthermore, Nodose (NG) microinjection of mGluR7 agonist significantly increased MAP in males and slightly decreased that in females. Even though mGluR8 direct activation intensified baroreceptor activation, the sensitivity was similar between sexes. In vitro, the expression profiles of mGluRs were investigated using Western blot and identified BRNs using single-cell qRT-PCR under ischemic conditions. Glutamate in serum, NG and nucleus tractus solitary (NTS) was raised significantly in the model rats of both sexes vs. sham-controls. Female-specific expression of mGluR7 in the baroreflex afferent pathway, especially higher expression in Ah-type BRNs, contributes significantly to cardiac analgesia, which may explain that the pathogenesis of silent MI occurs mainly in female patients. Therefore, higher expression of mGluR7 in female-specific subpopulation of Ah-type BRNs plays a critical role in cardiac analgesia and peripheral nociception.

Recent studies suggest that melatonin (Mel) plays an important role in the regulation of blood pressure (BP) via the aortic baroreflex pathway. In this study, we investigated the interaction between the baroreflex afferent pathway and Mel-mediated BP regulation in rats under physiological and hypertensive conditions. Mel (0.1, 0.3, and 1.0 mg/mL) was microinjected into the nodose ganglia (NG) of rats. We showed that Mel-induced reduction of mean arterial pressure in female rats was significantly greater than that in male and in ovariectomized rats under physiological condition. Consistently, the expression of Mel receptors (MTNRs) in the NG of female rats was significantly higher than that of males. In L-NAME-induced hypertensive and spontaneously hypertensive rat models, MTNRs were upregulated in males but downregulated in female models. Interestingly, Mel-induced BP reduction was found in male hypertensive models. In whole-cell recording from identified baroreceptor neurons (BRNs) in female rats, we found that Mel (0.1 μM) significantly increased the excitability of a female-specific subpopulation of Ah-type BRNs by increasing the Na

Background: Bone is a frequent site of metastases from breast cancer, but existing therapeutic options are not satisfactory. Although osteoblasts have active roles in cancer progression by assisting the vicious bone-destructive cycle, we employed a counterintuitive approach of activating pro-tumorigenic Wnt signaling and examined the paradoxical possibility of developing osteoblast-derived tumor-suppressive, bone-protective secretomes. Methods: Wnt signaling was activated by the overexpression of Lrp5 and β-catenin in osteoblasts as well as a pharmacological agent (BML284), and the therapeutic effects of their conditioned medium (CM) were evaluated using in vitro cell cultures, ex vivo breast cancer tissues, and a mouse model of osteolysis. To explore the unconventional regulatory mechanism of the action of Wnt-activated osteoblasts, whole-genome proteomics analysis was conducted, followed by immunoprecipitation and gain- and loss-of-function assays. Results: While osteoblasts did not present any innate tumor-suppressing ability, we observed that the overexpression of Lrp5 and β-catenin in Wnt signaling made their CM tumor-suppressive and bone-protective. The growth of breast cancer cells and tissues was inhibited by Lrp5-overexpressing CM (Lrp5 CM), which suppressed mammary tumors and tumor-driven bone destruction in a mouse model. Lrp5 CM also inhibited the differentiation and maturation of bone-resorbing osteoclasts by downregulating NFATc1 and cathepsin K. The overexpression of Lrp5 upregulated osteopontin that enriched Hsp90ab1 (Hsp90 beta) and moesin (MSN) in Lrp5 CM. Hsp90ab1 and MSN are atypical tumor-suppressing proteins since they are multi-tasking, moonlighting proteins that promote tumorigenesis in tumor cells. Importantly, Hsp90ab1 immuno-precipitated latent TGFβ and inactivated TGFβ, whereas MSN interacted with CD44, a cancer stem-cell marker, as well as fibronectin 1, an ECM protein. Furthermore, Hsp90ab1 and MSN downregulated KDM3A that demethylated histones, together with PDL1 that inhibited immune responses. Conclusion: In contrast to inducing tumor-enhancing secretomes and chemoresistance in general by inhibiting varying oncogenic pathways in chemotherapy, this study presented the unexpected outcome of generation tumor-suppressive secretomes by activating the pro-tumorigenic Wnt pathway. The results shed light on the contrasting role of oncogenic signaling in tumor cells and osteoblast-derived secretomes, suggesting a counterintuitive option for the treatment of breast cancer-associated bone metastasis.

Background: Advanced breast cancer metastasizes to many organs including bone, but few effective treatments are available. Here we report that induced tumor-suppressing (iTS) MSCs protected bone from metastases while un-induced MSCs did not. Methods: iTS MSCs were generated by overexpressing Lrp5, β-catenin, Snail, or Akt. Their tumor-suppressing capability was tested using a mouse model of mammary tumors and bone metastasis, human breast cancer tissues and cancer cell lines. Results: In a mouse model, the induced MSC-derived conditioned medium (MSC CM) reduced mammary tumors and suppressed tumor-induced osteolysis. Tumor-promoting genes such as CXCL2 and LIF, as well as PDL1, a blocker of T-cell-based immune responses were downregulated. Proteomics analysis revealed that heat shock protein 90 (Hsp90ab1), calreticulin (Calr) and peptidylprolyl isomerase B (Ppib), which are highly expressed intracellular proteins in many cancers, were enriched in MSC CM as atypical tumor suppressors. Thus, overexpressing selected genes that were otherwise tumorigenic rendered MSCs the tumor-suppressing capability through the atypical suppressors, as well as p53 and Trail. Notably, the inhibitory effect of Lrp5- and Akt-overexpressing MSC CMs, Hsp90ab1 and Calr presented selective inhibition to tumor cells than non-tumor cells. The development of bone-resorbing osteoclasts was also suppressed by MSC CMs. Conclusion: Collectively, the results showed an anti-tumor effect of iTS MSCs and suggested novel therapeutic approaches to suppress the progression of tumors into the bone.

Mechanical stimulations can prevent bone loss, but their effects on the tumor-invaded bone or solid tumors are elusive. Here, we evaluated the effect of knee loading, dynamic loads applied to the knee, on metastasized bone and mammary tumors. In a mouse model, tumor cells were inoculated to the mammary fat pad or the proximal tibia. Daily knee loading was then applied and metabolic changes were monitored mainly through urine. Urine samples were also collected from human subjects before and after step aerobics. The result showed that knee loading inhibited tumor progression in the loaded tibia. Notably, it also reduced remotely the growth of mammary tumors. In the urine, an altered level of cholesterol was observed with an increase in calcitriol, which is synthesized from a cholesterol derivative. In urinary proteins, knee loading in mice and step aerobics in humans markedly reduced WNT1-inducible signaling pathway protein 1, WISP1, which leads to poor survival among patients with breast cancer. In the ex vivo breast cancer tissue assay, WISP1 promoted the growth of cancer fragments and upregulated tumor-promoting genes, such as Runx2, MMP9, and Snail. Collectively, the present preclinical and human study demonstrated that mechanical stimulations, such as knee loading and step aerobics, altered urinary metabolism and downregulated WISP1. The study supports the benefit of mechanical stimulations for locally and remotely suppressing tumor progression. It also indicated the role of WISP1 downregulation as a potential mechanism of loading-driven tumor suppression.

While urine has been considered as a useful bio-fluid for health monitoring, its dynamic changes to physical activity are not well understood. We examined urine's possible antitumor capability in response to medium-level, loading-driven physical activity. Urine was collected from mice subjected to 5-minute skeletal loading and human individuals before and after 30-minute step aerobics. Six cancer cell lines (breast, prostate, and pancreas) and a mouse model of the mammary tumor were employed to evaluate the effect of urine. Compared to urine collected prior to loading, urine collected post-activity decreased the cellular viability, proliferation, migration, and invasion of tumor cells, as well as tumor weight in the mammary fat pad. Detection of urinary volatile organic compounds and ELISA assays showed that the loading-conditioned urine reduced cholesterol and elevated dopamine and melatonin. Immunohistochemical fluorescent images presented upregulation of the rate-limiting enzymes for the production of dopamine and melatonin in the brain. Molecular analysis revealed that the antitumor effect was linked to the reduction in molecular vinculin-linked molecular force as well as the downregulation of the Lrp5-CSF1-CD105 regulatory axis. Notably, the survival rate for the high expression levels of Lrp5, CSF1, and CD105 in tumor tissues was significantly lowered in the Cancer Genome Atlas database. Collectively, this study revealed that 5- or 10-minute loading-driven physical activity was sufficient to induce the striking antitumor effect by activating the neuronal signaling and repressing cholesterol synthesis. The result supported the dual role of loading-conditioned urine as a potential tumor suppressor and a source of diagnostic biomarkers.

Aim Substance P (SP) causes vasodilation and blood pressure (BP) reduction. However, the involvement of tachykinin receptors (NKRs) within baroreflex afferent pathway in SP-mediated BP regulation is largely unknown. Methods Under control and hypertensive condition, NKRs' expressions were evaluated in nodose (NG) and nucleus of tractus solitary (NTS) of male, female, and ovariectomized (OVX) rats; BP was recorded after microinjection of SP and NKRs agonists into NG; Baroreceptor sensitivity (BRS) was tested as well. Results Immunostaining and immunoblotting data showed that NK1R and NK2R were estrogen-dependently expressed on myelinated and unmyelinated afferents in NG. A functional study showed that BP was reduced dose-dependently by SP microinjection, which was more dramatic in males and can be mimicked by NK1R and NK2R agonists. Notably, further BP elevation and BRS dysfunction were confirmed in desoxycorticosterone acetate (DOCA)-salt model in OVX compared with DOCA-salt model in intact female rats. Additionally, similar changes in NKRs' expression in NG were also detected using DOCA-salt and SHR. Compared with NG, inversed expression profiles of NKRs were also found in NTS with either gender. Conclusion The estrogen-dependent NKRs' expression in baroreflex afferent pathway participates at least partially in sexual-dimorphic and SP-mediated BP regulation under physiological and hypertensive conditions.

Neuropeptide Y (NPY), a metabolism-related cardiovascular factor, plays a crucial role in blood pressure (BP) regulation via peripheral and central pathways. The expression of NPY receptors (Y(1)R/Y(2)R) specific to baroreflex afferents impacts on the sexually dimorphic neural control of circulation. This study was designed to investigate the expression profiles of NPY receptors in the nodose ganglion (NG) and nucleus tractus solitary (NTS) under hypertensive conditions. To this end, rats with hypertension induced by N(G)-nitro-L-arginine methylester (L-NAME) or high fructose drinking (HFD), and spontaneously hypertensive rats (SHRs) were used to explore the effects/mechanisms of NPY on BP using functional, molecular, and electrophysiological approaches. The data showed that BP was elevated along with baroreceptor sensitivity dysfunction in model rats; Y(1)R was up- or down-regulated in the NG or NTS of male and female HFD/L-NAME groups, while Y(2)R was only down-regulated in the HFD groups as well as in the NG of the male L-NAME group. In SHRs, Y(1)R and Y(2)R were both down-regulated in the NTS, and not in the NG. In addition to NPY-mediated energy homeostasis, leptin-melanocortin activation may be essential for metabolic disturbance-related hypertension. We found that leptin and α-melanocyte stimulating hormone (α-MSH) receptors were aberrantly down-regulated in HFD rats. In addition, α-MSH concentrations were reduced and NPY concentrations were elevated in the serum and NTS at 60 and 90 min after acute leptin infusion. Electrophysiological recordings showed that the decay time-constant and area under the curve of excitatory post-synaptic currents were decreased by Y(1)R activation in A-types, whereas, both were increased by Y(2)R activation in Ah- or C-types. These results demonstrate that sex- and afferent-specific NPY receptor expression in the baroreflex afferent pathway is likely to be a novel target for the clinical management of metabolism-related and essential hypertension. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12264-019-00438-w) contains supplementary material, which is available to authorized users.

Bone is a frequent site of metastasis from breast cancer, and a desirable drug could suppress tumor growth as well as metastasis-linked bone loss. Currently, no drug is able to cure breast cancer–associated bone metastasis. In this study, we focused on statins that are known to inhibit cholesterol production and act as antitumor agents. After an initial potency screening of 7 U.S. Food and Drug Administration–approved statins, we examined pitavastatin as a drug candidate for inhibiting tumor and tumor-induced bone loss. In vitro analysis revealed that pitavastatin acted as an inhibitor of tumor progression by altering stress to the endoplasmic reticulum, down-regulating peroxisome proliferator–activated receptor γ, and reducing Snail and matrix metalloproteinase 9. In bone homeostasis, it blocked osteoclast development by suppressing transcription factors c-Fos and JunB, but stimulated osteoblast mineralization by regulating bone morphogenetic protein 2 and p53. In a mouse model, pitavastatin presented a dual role in tumor inhibition in the mammary fat pad, as well as in bone protection in the osteolytic tibia. In mass spectrometry–based analysis, volatile organic compounds (VOCs) that were linked to lipid metabolism and cholesterol synthesis were elevated in mice from the tumor-grown placebo group. Notably, pitavastatin-treated mice reduced specific VOCs that are linked to lipid metabolites in the mevalonate pathway. Collectively, the results lay a foundation for further investigation of pitavastatin’s therapeutic efficacy in tumor-induced bone loss, as well as VOC-based diagnosis of tumor progression and treatment efficacy.—Wang, L., Wang, Y., Chen, A., Teli, M., Kondo, R., Jalali, A., Fan, Y., Liu, S., Zhao, X., Siegel, A., Minami, K., Agarwal, M., Li, B.-Y., Yokota, H. Pitavastatin slows tumor progression and alters urine-derived volatile organic compounds through the mevalonate pathway.

This study utilized a Förster resonance energy transfer (FRET)-based molecular tension sensor and live cell imaging to evaluate the effect of osteocytes, a mechanosensitive bone cell, on the migratory behavior of tumor cells. Two cell lines derived from MDA-MB-231 breast cancer cells were transfected with the vinculin tension sensor to quantitatively evaluate the force in focal adhesions of the tumor cell. Tumor cells treated with MLO-A5 osteocyte-conditioned media (CM) decreased the tensile forces in their focal adhesions and decreased their migratory potential. Tumor cells treated with media derived from MLO-A5 cells exposed to fluid flow-driven shear stress (FFCM) increased the tensile forces and increased migratory potential. Focal adhesion tension in tumor cells was also affected by distance from MLO-A5 cells when the two cells were co-cultured, where tumor cells close to MLO-A5 cells exhibited lower tension and decreased cell motility. Overall, this study demonstrates that focal adhesion tension is involved in altered migratory potential of tumor cells, and tumor-osteocyte interactions decrease the tension and motility of tumor cells.

// Yang Liu 1, * , Di Wu 2, * , Mei-Yu Qu 2, * , Jian-Li He 2 , Mei Yuan 1 , Miao Zhao 2 , Jian-Xin Wang 2 , Jian He 2 , Lu-Qi Wang 2 , Xin-Jing Guo 1 , Meng Zuo 1 , Shu-Yang Zhao 2 , Mei-Na Ma 2 , Jun-Nan Li 1 , Weinian Shou 3 , Guo-Fen Qiao 1, 2 , Bai-Yan Li 1 1 Department of Pharmacology, Harbin Medical University, Harbin, China 2 Key Laboratory of Cardiovascular Research of Ministry of Education, Harbin Medical University, Harbin, China 3 Riley Heart Research Center, Division of Pediatric Cardiology, Herman B. Wells Center for Pediatric Research, Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN, USA * These authors have contributed equally to this work Correspondence to: Bai-Yan Li, email: liby@ems.hrbmu.edu.cn Guo-Fen Qiao, email: qiaogf88@163.com Keywords: neuropeptide Y, baroreflex, nodose ganglion, nucleus tractus solitarii, whole-cell patch techniques Received: October 27, 2015 Accepted: July 16, 2016 Published: September 07, 2016 ABSTRACT Background: Molecular and cellular mechanisms of neuropeptide-Y (NPY)-mediated gender-difference in blood pressure (BP) regulation are largely unknown. Methods: Baroreceptor sensitivity (BRS) was evaluated by measuring the response of BP to phenylephrine/nitroprusside. Serum NPY concentration was determined using ELISA. The mRNA and protein expression of NPY receptors were assessed in tissue and single-cell by RT-PCR, immunoblot, and immunohistochemistry. NPY was injected into the nodose while arterial pressure was monitored. Electrophysiological recordings were performed on nodose neurons from rats by patch-clamp technique. Results: The BRS was higher in female than male and ovariectomized rats, while serum NPY concentration was similar among groups. The sex-difference was detected in Y 1 R, not Y 2 R protein expression, however, both were upregulated upon ovariectomy and canceled by estrogen replacement. Immunostaining confirmed Y 1 R and Y 2 R expression in myelinated and unmyelinated afferents. Single-cell PCR demonstrated that Y 1 R expression/distribution was identical between A- and C-types, whereas, expressed level of Y 2 R was ~15 and ~7 folds higher in Ah- and C-types than A-types despite similar distribution. Activation of Y 1 R in nodose elevated BP, while activation of Y 2 R did the opposite. Activation of Y 1 R did not alter action potential duration (APD) of A-types, but activation of Y 2 R- and Y 1 R/Y 2 R in Ah- and C-types frequency-dependently prolonged APD. N-type I Ca was reduced in A-, Ah- and C-types when either Y 1 R, Y 2 R, or both were activated. The sex-difference in Y 1 R expression was also observed in NTS. Conclusions: Sex- and afferent-specific expression of Neuropeptide-Y receptors in baroreflex afferent pathway may contribute to sexual-dimorphic neurocontrol of BP regulation.

Aim To study the dominant role of parasympathetic inputs at cellular level of baroreflex afferent pathway and underlying mechanism in neurocontrol of blood pressure regulation. Methods Whole-cell patch-clamp and animal study were conducted. Results For the first time, we demonstrated the spontaneous activities from resting membrane potential in myelinated A- and Ah-type baroreceptor neurons (BRNs, the 1st-order), but not in unmyelinated C-types, using vagus-nodose slice of adult female rats. These data were further supported by the notion that the spontaneous synaptic currents could only be seen in the pharmacologically and electrophysiologically defined myelinated A- and Ah-type baroreceptive neurons (the 2nd-order) of NTS using brainstem slice of adult female rats. The greater frequency and the larger amplitude of the spontaneous excitatory postsynaptic currents (EPSCs) compared with the inhibitory postsynaptic currents (IPSCs) were only observed in Ah-types. The ratio of EPSCs:IPSCs was estimated at 3:1 and higher. These results confirmed that the afferent-specific spontaneous activities were generated from baroreflex afferent pathway in female-specific subpopulation of myelinated Ah-type BRNs in nodose and baroreceptive neurons in NTS, which provided a novel insight into the dominant role of sex-specific baroreflex-evoked parasympathetic drives in retaining a stable and lower blood pressure status in healthy subjects, particularly in females. Conclusion The data from current investigations establish a new concept for the role of Ah-type baroreceptor/baroreceptive neurons in controlling blood pressure stability and provide a new pathway for pharmacological intervention for hypertension and cardiovascular diseases.

While bone is a frequent target of breast cancer–associated metastasis, little is known about the effects of tumor–bone interactions on the efficacy of tumor-suppressing agents. Here we examined the effect of two FDA-approved dopamine modulators, fluphenazine and trifluoperazine, on mammary tumor cells, osteoclasts, osteoblasts, and osteocytes. These agents suppressed proliferation and migration of mammary tumor cells chiefly by antagonizing dopamine receptor D2 and reduced bone resorption by downregulating nuclear factor of activated T cells, cytoplasmic 1 (Nfatc1). Three-dimensional spheroid formation assays revealed that tumor cells have high affinity to osteocytes and type I collagen, and interactions with osteocytes as well as administration of fluphenazine and trifluoperazine downregulated Snail and suppressed migratory behaviors. Unlike the inhibitory action of fluphenazine and trifluoperazine on tumor growth, tumor–osteocyte interactions stimulated tumor proliferation by upregulating NFκB and Akt. In the bone microenvironment, osteocytes downregulated Snail and acted as an attractant as well as a stimulant to mammary tumor cells. These results demonstrate that tumor–osteocyte interactions strengthen dopamine receptor–mediated suppression of tumor migration but weaken its inhibition of tumor proliferation in the osteocyte-rich bone microenvironment. Significance: These findings provide novel insight into the cellular cross-talk in the bone microevironment and the effects of dopamine modulators on mammary tumor cells and osteocytes. Cancer Res; 78(14); 3865–76. ©2018 AACR.

Sexual-dimorphic neurocontrol of circulation has been described in baroreflex due largely to the function of myelinated Ah-type baroreceptor neurons (BRNs, 1st-order) in nodose. However, it remains unclear if sex- and afferent-specific neurotransmission could also be observed in the central synapses within nucleus of solitary track (NTS, 2nd-order). According to the principle of no mixed neurotransmission among afferents and differentiation of Ah- and A-types to iberiotoxin (IbTX) observed in nodose, the 2nd-order Ah-type BRNs are highly expected. To test this hypothesis, the excitatory post-synaptic currents (EPSCs) were recorded in identified 2nd-order BRNs before and after IbTX using brain slice and whole-cell patch. These results showed that, in male rats, the dynamics of EPSCs in capsaicin-sensitive C-types were dramatically altered by IbTX, but not in capsaicin-insensitive A-types. Interestingly, near 50% capsaicin-insensitive neurons in females showed similar effects to C-types, suggesting the existence of Ah-types in NTS, which may be the likely reason why the females had lower blood pressure and higher sensitivity to aortic depressor nerve stimulation via KCa1.1-mediated presynaptic glutamate release from Ah-type afferent terminals.

a Department of Pharmacology, Harbin Medical University, Harbin, China b Key Laboratory of Cardiovascular Research, Ministry of Education, Harbin Medical University, Harbin, China c Department of Immunology, Capital Medical University School of Basic Medical Science, 10 Xitoutiao, Youanmen, Fengtai District, Beijing, China d Department of Gynecology and Obstetrics, 211 Hospital of People's Liberation Army, Harbin, China

Sex differences in baroreflex (BRx) function are well documented. Hormones likely contribute to this dimorphism, but many functional aspects remain unresolved. Our lab has been investigating a subset of vagal sensory neurons that constitute nearly 50% of the total population of myelinated aortic baroreceptors (BR) in female rats but less than 2% in male rats. Termed “Ah,” this unique phenotype has many of the nonoverlapping electrophysiological properties and chemical sensitivities of both myelinated A-type and unmyelinated C-type BR afferents. In this study, we utilize three distinct experimental protocols to determine if Ah-type barosensory afferents underlie, at least in part, the sex-related differences in BRx function. Electron microscopy of the aortic depressor nerve (ADN) revealed that female rats have less myelin ( P < 0.03) and a smaller fiber cross-sectional area ( P < 0.05) per BR fiber than male rats. Electrical stimulation of the ADN evoked compound action potentials and nerve conduction profiles that were markedly different ( P < 0.01, n = 7 females and n = 9 males). Selective activation of ADN myelinated fibers evoked a BRx-mediated depressor response that was 3–7 times greater in female ( n = 16) than in male ( n = 17) rats. Interestingly, the most striking hemodynamic difference was functionally dependent upon the rate of myelinated barosensory fiber activation. Only 5–10 Hz of stimulation evoked a rapid, 20- to 30-mmHg reduction in arterial pressure of female rats, whereas rates of 50 Hz or higher were required to elicit a comparable depressor response from male rats. Collectively, our experimental results are suggestive of an alternative myelinated baroreceptor afferent pathway in females that may account for, at least in part, the noted sex-related differences in autonomic control of cardiovascular function.

Trabeculation and compaction of the embryonic myocardium are morphogenetic events crucial for the formation and function of the ventricular walls. Fkbp1a (FKBP12) is a ubiquitously expressed cis-trans peptidyl-prolyl isomerase. Fkbp1a-deficient mice develop ventricular hypertrabeculation and noncompaction. To determine the physiological function of Fkbp1a in regulating the intercellular and intracellular signaling pathways involved in ventricular trabeculation and compaction, we generated a series of Fkbp1a conditional knockouts. Surprisingly, cardiomyocyte-restricted ablation of Fkbp1a did not give rise to the ventricular developmental defect, whereas endothelial cell-restricted ablation of Fkbp1a recapitulated the ventricular hypertrabeculation and noncompaction observed in Fkbp1a systemically deficient mice, suggesting an important contribution of Fkbp1a within the developing endocardia in regulating the morphogenesis of ventricular trabeculation and compaction. Further analysis demonstrated that Fkbp1a is a novel negative modulator of activated Notch1. Activated Notch1 (N1ICD) was significantly upregulated in Fkbp1a-ablated endothelial cells in vivo and in vitro. Overexpression of Fkbp1a significantly reduced the stability of N1ICD and direct inhibition of Notch signaling significantly reduced hypertrabeculation in Fkbp1a-deficient mice. Our findings suggest that Fkbp1a-mediated regulation of Notch1 plays an important role in intercellular communication between endocardium and myocardium, which is crucial in controlling the formation of the ventricular walls.

Nodose ganglia are composed of A-, Ah- and C-type neurons. Despite their important roles in regulating visceral afferent function, including cardiovascular, pulmonary, and gastrointestinal homeostasis, information about subtype-specific expression, molecular identity, and function of individual ion transporting proteins is scarce. Although experiments utilizing the sliced ganglion preparation have provided valuable insights into the electrophysiological properties of nodose ganglion neuron subtypes, detailed characterization of their electrical phenotypes will require measurements in isolated cells. One major unresolved problem, however, is the difficulty to unambiguously identify the subtype of isolated nodose ganglion neurons without current-clamp recording, because the magnitude of conduction velocity in the corresponding afferent fiber, a reliable marker to discriminate subtypes in situ, can no longer be determined. Here, we present data supporting the notion that application of an algorithm regarding to microscopic structural characteristics, such as neuron shape evaluated by the ratio between shortest and longest axis, neuron surface characteristics, like membrane roughness, and axon attachment, enables specific and sensitive subtype identification of acutely dissociated rat nodose ganglion neurons, by which the accuracy of identification is further validated by electrophysiological markers and overall positive predictive rates is 89.26% (90.04%, 76.47%, and 98.21% for A-, Ah, and C-type, respectively). This approach should aid in gaining insight into the molecular correlates underlying phenotypic heterogeneity of nodose ganglia. Additionally, several critical points that help for neuron identification and afferent conduction calibration are also discussed.

(1) BACKGROUND: Transient receptor potential vanilloid 3 (TRPV3) is a member of the TRP channels family of Ca(2+)-permeant channels. The proteins of some TRP channels are highly expressed in cancer cells. This study aimed to assess the clinical significance and biological functions of TRPV3 in non-small cell lung cancer (NSCLC); (2) METHODS: Immunohistochemistry was used to detect the expression of TRPV3 in NSCLC tissues and adjacent noncancerous lung tissues. Western blot was used to detect the protein expressions of TRPV3, CaMKII, p-CaMKII, CyclinA, CyclinD, CyclinE1, CDK2, CDK4, and P27. Small interfering RNA was used to deplete TRPV3 expression. A laser scanning confocal microscope was used to measure intracellular calcium concentration ([Ca(2+)]i). Flow cytometry was used to analyze cell cycle; (3) RESULTS: TRPV3 was overexpressed in 65 of 96 (67.7%) human lung cancer cases and correlated with differentiation (p = 0.001) and TNM stage (p = 0.004). Importantly, TRPV3 expression was associated with short overall survival. In addition, blocking or knockdown of TRPV3 could inhibit lung cancer cell proliferation. Moreover, TRPV3 inhibition could decrease [Ca(2+)]i of lung cancer cells and arrest cell cycle at the G1/S boundary. Further results revealed that TRPV3 inhibition decreased expressions of p-CaMKII, CyclinA, CyclinD1, CyclinE, and increased P27 level; (4) CONCLUSIONS: Our findings demonstrate that TRPV3 was overexpressed in NSCLC and correlated with lung cancer progression. TRPV3 activation could promote proliferation of lung cancer cells. TRPV3 might serve as a potential companion drug target in NSCLC.

FK506 binding protein12.6 (FKBP12.6) binds to the Ca(2+) release channel ryanodine receptor (RyR2) in cardiomyocytes and stabilizes RyR2 to prevent premature sarcoplasmic reticulum Ca(2+) release. Previously, two different mouse strains deficient in FKBP12.6 were reported to have different abnormal cardiac phenotypes. The first mutant strain displayed sex-dependent cardiac hypertrophy, while the second displayed exercise-induced cardiac arrhythmia and sudden death. In this study, we tested whether FKBP12.6-deficient mice that display hypertrophic hearts can develop exercise-induced cardiac sudden death and whether the hypertrophic heart is a direct consequence of abnormal calcium handling in mutant cardiomyocytes. Our data show that FKBP12.6-deficient mice with cardiac hypertrophy do not display exercise-induced arrhythmia and/or sudden cardiac death. To investigate the role of FKBP12.6 overexpression for cardiac function and cardiomyocyte calcium release, we generated a transgenic mouse line with cardiac specific overexpression of FKBP12.6 using α-myosin heavy chain (αMHC) promoter. MHC-FKBP12.6 mice displayed normal cardiac development and function. We demonstrated that MHC-FKBP12.6 mice are able to rescue abnormal cardiac hypertrophy and abnormal calcium release in FKBP12.6-deficient mice.

Persistent tetrodotoxin-resistant (TTX-R) Na(+) (Na(v)1.9/SCN11A) currents are not normally recorded in vagal afferent neurons (VANs) with 50 mM of extracellular Na(+) although the functional expression of this current was observed in the presence of PGE(2) or forskolin. However, it is uncertain whether this current can be seen under physiological condition (150 mM Na(+)). Using the whole-cell patch-clamp technique, we showed that persistent TTX-R Na(+) currents were expressed in 9 out of 38 VANs bathed in 150 mM Na(+). The current density, but not the whole-cell capacitance, was significantly enhanced in the VANs expressing Nav1.9. Persistent TTX-R Na(+) channels were activated at a more hyperpolarized membrane potential near -60 mV, compared with TTX-sensitive (TTX-S at -40 mV) and TTX-R Na(+) channels (at -20 mV). This indicates that persistent TTX-R Na(+) channels provide a wider activation window than TTX-S and TTX-R Na channels to up-regulate neuronal excitability. These results suggest that the persistent TTX-R Na(+) currents may be involved in the neuronal excitability by setting a lower pressure-discharge threshold and higher discharge frequency of VANs, especially the unique subset and gender-specific distribution of myelinated Ah-type VANs, including Ah-type aortic baroreceptor neurons, identified in our previous study.

This study aims to understand the special expression patterns of angiotensin-II receptor (AT 1 R and AT 2 R) in nodose ganglia and nucleus of tractus solitary of baroreflex afferent pathway and their contribution in sex difference of neurocontrol of blood pressure regulation. In this regard, action potentials were recorded in baroreceptor neurons (BRNs) using whole-cell patch techniques; mRNA and protein expression of AT 1 R and AT 2 R in nodose ganglia and nucleus of tractus solitary were evaluated using real time–polymerase chain reaction, Western blot, and immunohistochemistry at both tissue and single-cell levels. The in vivo effects of 17β-estradiol on blood pressure and AT 2 R expression were also tested. The data showed that AT 2 R, rather than AT 1 R, expression was higher in female than age-matched male rats. Moreover, AT 2 R was downregulated in ovariectomized rats, which was restored by the administration of 17β-estradiol. Single-cell real time–polymerase chain reaction data indicated that AT 2 R was uniquely expressed in Ah-type BRNs. Functional study showed that long-term administration of 17β-estradiol significantly alleviated the blood pressure increase in ovariectomized rats. Electrophysiological recordings showed that angiotensin-II treatment increased the neuroexcitability more in Ah- than C-type BRNs, whereas no such effect was observed in A-types. In addition, angiotensin-II treatment prolonged action potential duration, which was not further changed by iberiotoxin. The density of angiotensin-II–sensitive K + currents recorded in Ah-types was equivalent with iberiotoxin-sensitive component. In summary, the unique, sex- and afferent-specific expression of AT 2 R was identified in Ah-type BRNs, and AT 2 R-mediated KCa1.1 inhibition in Ah-type BRNs may exert great impacts on baroreflex afferent function and blood pressure regulation in females.

// Lu-Qi Wang 1,2,* , Sheng-Zhi Liu 1,* , Xin Wen 1,* , Di Wu 2 , Lei Yin 2 , Yao Fan 1 , Ye Wang 3 , Wei-Ran Chen 1 , Pei Chen 2 , Yang Liu 1 , Xiao-Long Lu 1 , Hong-Li Sun 3 , Weinian Shou 4 , Guo-Fen Qiao 1,2 and Bai-Yan Li 1 1 Department of Pharmacology, Harbin Medical University, Harbin, China 2 Key Laboratory of Cardiovascular Medicine Research of Ministry of Education, Harbin Medical University, Harbin, China 3 Department of Pharmacology, Daqing Campus of Harbin Medical University, Daqing, China 4 Riley Heart Research Center, Division of Pediatric Cardiology, Herman B. Wells Center for Pediatric Research, Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN, USA * These authors have contributed equally to this work Correspondence to: Bai-Yan Li, email: // Guo-Fen Qiao, email: // Keywords : nodose ganglia (NG), nucleus of the solitary tract (NTS), presynaptic neurotransmission, baroreflex afferent pathway, ketamine (Ket), Pathology Section Received : July 10, 2015 Accepted : November 29, 2015 Published : December 12, 2015 Abstract Background: Ketamine enhances autonomic activity, and unmyelinated C-type baroreceptor afferents are more susceptible to be blocked by ketamine than myelinated A-types. However, the presynaptic transmission block in low-threshold and sex-specific myelinated Ah-type baroreceptor neurons (BRNs) is not elucidated. Methods: Action potentials (APs) and excitatory post-synaptic currents (EPSCs) were investigated in BRNs/barosensitive neurons identified by conduction velocity (CV), capsaicin-conjugated with Iberiotoxin-sensitivity and fluorescent dye using intact nodose slice and brainstem slice in adult female rats. The expression of mRNA and targeted protein for NMDAR1 was also evaluated. Results: Ketamine time-dependently blocked afferent CV in Ah-types in nodose slice with significant changes in AP discharge. The concentration-dependent inhibition of ketamine on AP discharge profiles were also assessed and observed using isolated Ah-type BRNs with dramatic reduction in neuroexcitability. In brainstem slice, the 2 nd -order capsaicin-resistant EPSCs were identified and ~50% of them were blocked by ketamine concentration-dependently with IC 50 estimated at 84.4 µM compared with the rest (708.2 µM). Interestingly, the peak, decay time constant, and area under curve of EPSCs were significantly enhanced by 100 nM iberiotoxin in ketamine-more sensitive myelinated NTS neurons (most likely Ah-types), rather than ketamine-less sensitive ones (A-types). Conclusions: These data have demonstrated, for the first time, that low-threshold and sex-specific myelinated Ah-type BRNs in nodose and Ah-type barosensitive neurons in NTS are more susceptible to ketamine and may play crucial roles in not only mean blood pressure regulation but also buffering dynamic changes in pressure, as well as the ketamine-mediated cardiovascular dysfunction through sexual-dimorphic baroreflex afferent pathway.

+channel Gender difference in cardiovascular diseases is one of the most investigated and still unsolved issues. Finding an explanation to this topic might have important implications for the understanding of the differences between men and women in diseases and possibly lead to the development of gender-specific strategies for its management. The autonomic nervous system influences blood pressure and heart rate through adjustments in parasympathetic and sympathetic activity tightly regulated through baroreflex mechanisms, which plays a pivotal role in cardiovascular regulation and buffer-excessive blood pressure swings [1]. Women had a greater baroreceptor reflex sensitivity controlling efferent sympathetic nerve activity [2] and lower tonic autonomic nerve system support of blood pressure contributes to the lower chronic blood pressure levels of premenopausal women [3], suggesting that females may have a broader spectrum of baroreflex functions and that this may partially explain the observed lower incidence of cardiovascular events in premenopausal women compared with men [4]. Our previous study demonstrated low-threshold myelinated Ah-type baroreceptor neurons (BRNs) in adult female rats [5–7], which expresses a large voltage-gated Na + channel including Nav1.9

Potentiation of 17β-estradiol on neuroexcitability by HCN-mediated neuromodulation of fast-afterhyperpolarization and late-afterdepolarization in low-threshold and sex-specific myelinated Ah-type baroreceptor neurons via GPR30 in female rats Jian-Li He , Jun-Nan Li , Chun-Mei Zuo , Lu-Qi Wang , Xin Wen , Meng Zuo , Jian Guan , Di Wu , Dong-Xue Song , Xue Yu , Mei-Yu Qu , Yang Liu , Guo-Fen Qiao ⁎, Bai-Yan Li a,⁎

Evidence has shown gender differences regarding the critical roles of histamine in the prevalence of asthma, anaphylaxis, and angina pectoris. Histamine depolarizes unmyelinated C-type neurons without any effects on myelinated A-type vagal ganglion neurons (VGNs) in male rats. However, little is known if VGNs from females react to histamine in a similar manner. Membrane depolarization and inward currents were tested in VGNs isolated from adult rats using a whole-cell patch technique. Results from males were consistent with the literature. Surprisingly, histamine-induced depolarization and inward currents were observed in both unmyelinated C-type and myelinated A- and Ah-type VGNs from female rats. In Ah-type neurons, responses to 1.0 μM histamine were stronger in intact females than in males and significantly reduced in ovariectomized (OVX) females. In C-type neurons, histamine-induced events were significantly smaller (pA/pF) in intact females compared with males and this histamine-induced activity was dramatically increased by OVX. Female A-types responded to histamine, which was further increased following ovariectomy. Histamine at 300 nM depolarized Ah-types in females, but not Ah-types in OVX females. In contrast, the sensitivity of A- and C-types to histamine was upregulated by OVX. These data demonstrate gender differences in VGN chemosensitivity to histamine for the first time. Myelinated Ah-types showed the highest sensitivity to histamine across female populations, which was changed by OVX. These novel findings improve the understanding of gender differences in the prevalence of asthma, anaphylaxis, and pain. Changes in sensitivity to histamine by OVX may explain alterations in the prevalence of certain pathophysiological conditions when women reach a postmenopausal age.

High conductance calcium-activated potassium (BKCa) channels can modulate cell excitability and neurotransmitter release at synaptic and afferent terminals. BKCa channels are present in primary afferents of most, if not, all internal organs and are an intriguing target for pharmacological manipulation of visceral sensation. Our laboratory has a long-standing interest in the neurophysiological differences between myelinated and unmyelinated visceral afferent function. Here, we seek to determine whether there is a differential distribution of BKCa channels in myelinated and unmyelinated vagal afferents. Immunocytochemistry studies with double staining for the BK-type KCa1.1 channel protein and isolectin B4 (IB4), a reliable marker of unmyelinated peripheral afferents, reveal a pattern of IB4 labeling that strongly correlates with the expression of the KCa1.1 channel protein. Measures of cell size and immunostaining intensity for KCa1.1 and IB4 cluster into two statistically distinct ( P < 0.05) populations of cells. Smaller diameter neurons most often presented with strong IB4 labeling and are presumed to be unmyelinated ( n = 1,390) vagal afferents. Larger diameter neurons most often lacked or exhibited a very weak IB4 labeling and are presumed to be myelinated ( n = 58) vagal afferents. Complimentary electrophysiological studies reveal that the BKCa channel blockers charybdotoxin (ChTX) and iberiotoxin (IbTX) bring about a comparable elevation in excitability and action potential widening in unmyelinated neurons but had no effect on the excitability of myelinated vagal afferents. This study is the first to demonstrate using combined immunohistochemical and electrophysiological techniques that KCa1.1 channels are uniquely expressed in unmyelinated C-type vagal afferents and do not contribute to the dynamic discharge characteristics of myelinated A-type vagal afferents. This unique functional distribution of BK-type KCa channels may provide an opportunity for afferent selective pharmacological intervention across a wide range of visceral pathophysiologies, particularly those with a reflexogenic etiology and pain.

We recently identified a myelinated vagal afferent subpopulation (Ah type) far more prevalent in female than male rats and showed that this difference extends to functionally specific visceral sensory afferents, baroreceptors of the aortic arch. Excitability of myelinated Ah-type afferents is markedly reduced after ovariectomy (OVX). Here we tested the hypothesis that 17beta-estradiol can selectively restore excitability of these sex-specific vagal afferents. Acutely isolated vagal afferent neurons (VGN) from intact and OVX adult female rats were used with patch-clamp technique and current-clamp protocols to assess the effect of acute application of 17beta-estradiol on neuronal excitability. At over physiologically relevant 17beta-estradiol concentrations for rat (1-10 nM) excitability of myelinated Ah-type vagal afferents is restored to discharge frequencies comparable to those in intact females, albeit with some interesting differences related to burst and sustained patterns of neuronal discharge. Restoration of excitability occurs within 3 min of hormone application and is stereo specific, because 1,000 nM 17alpha-estradiol fails to alter excitability. Furthermore, activation of G protein-coupled estrogen receptor GPR30 with highly selective agonist G-1 similarly restores excitability of Ah-type afferents. The effectiveness of 17beta-estradiol and G-1 is completely eliminated by application of high-affinity estrogen receptor ligand ICI-182,780. 17beta-Estradiol conjugated with BSA is approximately 70% as effective as 17beta-estradiol alone in restoring Ah-type VGN excitability. These data support our conclusions that the cellular mechanisms leading to rapid restoration of neuronal excitability of myelinated Ah-type VGN after OVX occur, at least in part, via membrane-bound estrogen receptors. We contend that recovery of high-frequency discharge at physiologically relevant 17beta-estradiol concentrations implies that this unique subtype of low-threshold myelinated vagal afferent may account for some of the sex-related differences in visceral organ system function. Sex differences in cardiovascular and gastrointestinal function and the potential role of GPR30 in modulation of sex-specific myelinated Ah-type vagal afferents are discussed.

Evidence for sexual dimorphism in autonomic control of cardiovascular function is both compelling and confounding. Across healthy and disease populations sex-associated differences in neurocirculatory hemodynamics are far too complex to be entirely related to sex hormones. As an initial step toward identifying additional physiological mechanisms, we investigated whether there is a sex bias in the relative expression of low-threshold-myelinated and high-threshold-unmyelinated aortic baroreceptor afferents in rats. These two types of afferent fibers have markedly different reflexogenic effects upon heart rate and blood pressure and thus the potential impact upon baroreflex dynamics could be substantial. Our results, using a combination of a patch-clamp study of fluorescently identified aortic baroreceptor neurons (ABN) and morphometric analysis of aortic baroreceptor nerve fibers, demonstrate that females exhibit a greater percentage of myelinated baroreceptor fibers (24.8% vs. 18.7% of total baroreceptor fiber population, P < 0.01) and express a functional subtype of myelinated ABN rarely found in age-matched males (11% vs. 2.3%, n = 107, P < 0.01). Interestingly, this neuronal phenotype is more prevalent in the general population of female vagal afferent neurons (17.7% vs. 3.8%, n = 169, P < 0.01), and ovariectomy does not alter its expression but does lessen neuronal excitability. These data suggest there are fundamental neuroanatomical and electrophysiological differences between aortic baroreceptor afferents of female and male rats. Possible explanations are presented as to how such a greater prevalence of low-threshold myelinated afferents could be a contributing factor to the altered baroreflex sensitivity and vagal tone of females compared with males.

An unavoidable consequence of enzymatic dispersion of sensory neurons from intact ganglia is loss of the axon and thus the ability to classify afferent fiber type based upon conduction velocity (CV). An intact rat nodose ganglion preparation was used to randomly sample neurons (n = 76) using the patch clamp technique. Reliable electrophysiological and chemophysiological correlates of afferent fiber type were established for use with isolated neuron preparations. Myelinated afferents (~25%) formed two groups exhibiting strikingly different functional profiles. One group (n = 10) exhibited CVs in excess of 10 m/s and narrow (< 1 ms) action potentials (APs) while the other (n = 9) had CVs as low as 4 m/s and broad (> 2 ms) APs that closely approximated those identified as unmyelinated afferents (n = 57) with CVs less than 1 m/s. A cluster analysis of select measures from the AP waveforms strongly correlated with CV, producing three statistically unique populations (p < 0.05). These groupings aligned with our earlier hypothesis (Jin et al., 2004) that a differential sensitivity to the selective purinergic and vanilloid receptor agonists can be used as reliable pharmacological indicators of vagal afferent fiber type. These metrics were further validated using an even larger population of isolated (n = 240) nodose neurons. Collectively, these indicators of afferent fiber type can be used to provide valuable insight concerning the relavence of isolated cellular observations to integrated afferent function of visceral organ systems.

To investigate the effects of artemisinin (Art) on the action potentials (AP) recorded from identified C-type nodose neurons and study its anti-arrhythmic and anesthetic mechanisms.Neonatal and adult rats were selected for the preparation of isolated nodose ganglia neurons (NGN) and nodose ganglion-vagus slice preparation. Somatic AP were recorded from both isolated and slice NGN using whole-cell patch technique. Conduction velocity (CV) was measured using slice preparation. The effects of Art on AP were evaluated with the reference to ketamine.Effects of Art on AP were that: (1) AP depolarizing profiles were inhibited without changing resting membrane potential (RMP). The peak of AP (AP(peak)) and upstroke velocity (UV(APD50) and UV(max)) decreased markedly (P0.01). (2) The duration of AP at the point of half repolarization (APD(50)) was obviously prolonged (P0.01). (3) Art also slowed down AP repolarization profiles (downstroke velocity, DV(APD50), and DV(max)) and the peak of after-hyperpolarization (AHP(peak)) was less negative. (4) Total inward and outward currents over the course of AP were significantly reduced in the presence of Art. (5) CV did not changed by Art. (6) The effects of Art on AP were concentration-dependent and resembled with those of ketamine except for CV.Art inhibited both depolarization and repolarization of AP, suggesting that the effects of Art were probably, due to the blockade of Na+ and K+ ion channels.

To differentiate the electrophysiological characteristics of somatic action potentials (AP) from isolated Neo and Juv nodose sensory neurons (NSN) and those from slices of intact Juv and adult rat nodose ganglia.For isolated cell recordings nodose ganglia from 3-8 d old Neo and 4 weeks old Juv rats were dissociated using trypsin and collagenase, respectively. Nodose ganglia slices with attached vagus were prepared using a sequential treatment of collagenase and trypsin for both Juv and adult rats. Conduction velocity (CV) was collected by vagal stimulation. Whole-cell patch was applied for somatic AP recordings.(1) 281 NSN from both isolated cells and nodose slices were studied. Across all age groups, there was no difference observed among either C- or A-types. The difference between C- and A-type was significant. (2) Neurons exhibiting AP with prominent repolarization hump, broader APD50 (2.0 ms), upstroke velocity at the point of APD50 (UV(APD50)) and downstroke velocity at the point of APD50 (DV(APD50)) below 100 V . s-1 and 50 V . s-1 were classified as C-type (n = 222). Those without a hump, brief APD50 (1.0 ms), and UV(APD50) and DV(APD50) greater than 200 V . s-1 and 100 V . s-1 were classified as A-type (n = 59). (3) With slices, except for hump, APD, UV, and DV, significant differences were also observed (C- vs A-type) in CV from both Juv (0.56 +/- 0.15 vs 15.6 m.s-1) and adult (0.9 +/- 0.4 vs 14.5 +/- 1.0 m . s-1) nodose slices, discharge rate (single or burst vs repetitive), and frequency follow (20 Hz vs100 Hz). (4) Phase analysis showed that C-type had higher AP firing threshold, and lower total in- and outward peak currents than A-type.C- and A-type AP from isolated NSN of Neo or Juv rats exhibited electrophysiological characteristics that closely followed those of AP recorded in nodose slices. Therefore, isolated NSN can effectively serve as an experimental surrogate for electrophysiological studies of NSN requiring prior identification of afferent fiber type. Features of the AP wave-form such as the repolarization hump, APD50), and UV are strongly correlated with CV and are therefore reliable measures for classifying isolated NSN as either C- or A-type. It is most important that the nodose slice enables us to study the AP and ion channel characteristics on identified NSN by afferent fiber CV with adult animals.

Salubrinal is an agent that reduces the stress to the endoplasmic reticulum by inhibiting de-phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α). We and others have previously shown that the elevated phosphorylation of eIF2α stimulates bone formation and attenuates bone resorption. In this study, we applied salubrinal to a mouse model of osteogenesis imperfecta (Oim), and examined whether it would improve Oim's mechanical property. We conducted in vitro experiments using RAW264.7 pre-osteoclasts and bone marrow derived cells (BMDCs), and performed in vivo administration of salubrinal to Oim (+/−) mice. The animal study included two control groups (wildtype and Oim placebo). The result revealed that salubrinal decreased expression of nuclear factor of activated T cells cytoplasmic 1 (NFATc1) and suppressed osteoclast maturation, and it stimulated mineralization of mesenchymal stem cells from BMDCs. Furthermore, daily injection of salubrinal at 2 mg/kg for 2 months made stiffness (N/mm) and elastic module (GPa) of the femur undistinguishable to those of the wildtype control. Collectively, this study supported salubrinal's beneficial role to Oim's femora. Unlike bisphosphonates, salubrinal stimulates bone formation. For juvenile OI patients who may favor strengthening bone without inactivating bone remodeling, salubrinal may present a novel therapeutic option.