The baroreflex afferent pathway plays a critical role in H2S-mediated autonomic control of blood pressure regulation under physiological and hypertensive conditions.

Hydrogen sulfide (H2S), which is closely related to various cardiovascular disorders, lowers blood pressure (BP), but whether this action is mediated via the modification of baroreflex afferent function has not been elucidated. Therefore, the current study aimed to investigate the role of the baroreflex afferent pathway in H2S-mediated autonomic control of BP regulation. The results showed that baroreflex sensitivity (BRS) was increased by acute intravenous NaHS (a H2S donor) administration to renovascular hypertensive (RVH) and control rats. Molecular expression data also showed that the expression levels of critical enzymes related to H2S were aberrantly downregulated in the nodose ganglion (NG) and nucleus tractus solitarius (NTS) in RVH rats. A clear reduction in BP by the microinjection of NaHS or L-cysteine into the NG was confirmed in both RVH and control rats, and a less dramatic effect was observed in model rats. Furthermore, the beneficial effects of NaHS administered by chronic intraperitoneal infusion on dysregulated systolic blood pressure (SBP), cardiac parameters, and BRS were verified in RVH rats. Moreover, the increase in BRS was attributed to activation and upregulation of the ATP-sensitive potassium (KATP) channels Kir6.2 and SUR1, which are functionally expressed in the NG and NTS. In summary, H2S plays a crucial role in the autonomic control of BP regulation by improving baroreflex afferent function due at least in part to increased KATP channel expression in the baroreflex afferent pathway under physiological and hypertensive conditions.

Estrogen-dependent MicroRNA-504 Expression and Related Baroreflex Afferent Neuroexcitation via Negative Regulation on KCNMB4 and KCa1.1 ß4-subunit Expression.

Large conductance of Ca2+-activated K+ channel (KCa1.1) plays an inhibitory role in neuroexcitation. However, the expression of KCNMB4/ß4-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 ß4-subunit encoded by KCNMB4 was significantly lower in females vs. males and ovariectomized (OVX) rats in the NG. Although all baroreceptor neurons (BRNs) expressed ß4-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 ß4-subunits is less in Ah-types and up-regulated by ovariectomy. These data indicated that KCa1.1 ß4-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.

Direct activation of tachykinin receptors within baroreflex afferent pathway and neurocontrol of blood pressure regulation.

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.

Spontaneous activities in baroreflex afferent pathway contribute dominant role in parasympathetic neurocontrol of blood pressure 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.

Neuropeptide Y-mediated sex- and afferent-specific neurotransmissions contribute to sexual dimorphism of baroreflex afferent function.

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 Y1R, not Y2R protein expression, however, both were upregulated upon ovariectomy and canceled by estrogen replacement. Immunostaining confirmed Y1R and Y2R expression in myelinated and unmyelinated afferents. Single-cell PCR demonstrated that Y1R expression/distribution was identical between A- and C-types, whereas, expressed level of Y2R was ~15 and ~7 folds higher in Ah- and C-types than A-types despite similar distribution. Activation of Y1R in nodose elevated BP, while activation of Y2R did the opposite. Activation of Y1R did not alter action potential duration (APD) of A-types, but activation of Y2R- and Y1R/Y2R in Ah- and C-types frequency-dependently prolonged APD. N-type ICa was reduced in A-, Ah- and C-types when either Y1R, Y2R, or both were activated. The sex-difference in Y1R 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.

Unique Expression of Angiotensin Type-2 Receptor in Sex-Specific Distribution of Myelinated Ah-Type Baroreceptor Neuron Contributing to Sex-Dimorphic Neurocontrol of Circulation.

This study aims to understand the special expression patterns of angiotensin-II receptor (AT1R and AT2R) 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 AT1R and AT2R 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 17ß-estradiol on blood pressure and AT2R expression were also tested. The data showed that AT2R, rather than AT1R, expression was higher in female than age-matched male rats. Moreover, AT2R was downregulated in ovariectomized rats, which was restored by the administration of 17ß-estradiol. Single-cell real time-polymerase chain reaction data indicated that AT2R was uniquely expressed in Ah-type BRNs. Functional study showed that long-term administration of 17ß-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 AT2R was identified in Ah-type BRNs, and AT2R-mediated KCa1.1 inhibition in Ah-type BRNs may exert great impacts on baroreflex afferent function and blood pressure regulation in females.

Sumoylation of the Tumor Suppressor Promyelocytic Leukemia Protein Regulates Arsenic Trioxide-Induced Collagen Synthesis in Osteoblasts.

BACKGROUND/AIMS: Promyelocytic leukemia (PML) protein is a tumor suppressor that fuses with retinoic acid receptor-α (PML-RARα) to contribute to the initiation of acute promyelocytic leukemia (APL). Arsenic trioxide (ATO) upregulates expression of TGF-ß1, promoting collagen synthesis in osteoblasts, and ATO binds directly to PML to induce oligomerization, sumoylation, and ubiquitination. However, how ATO upregulates TGF-ß1 expression is uncertain. Thus, we suggested that PML sumoylation is responsible for regulation of TGF-ß1 protein expression. METHODS: Kunming mice were treated with ATO, and osteoblasts were counted under scanning electron microscopy. Masson's staining was used to quantify collagen content. hFOB1.19 cells were transfected with siRNA against UBC9 or RNF4, and then treated with ATO or FBS. TGF-ß1, PML expression, and sumoylation were quantified with Western blot, and collagen quantified via immunocytochemistry. RESULTS: ATO enhanced osteoblast accumulation, collagen synthesis, and PML-NB formation in vivo. Knocking down UBC9 in hFOB1.19 cells inhibited ATO- and FBS-induced PML sumoylation, TGF-ß1 expression, and collagen synthesis. Conversely, knocking down RNF4 enhanced ATO- and FBS-induced PML sumoylation, TGF-ß1 expression, and collagen synthesis. CONCLUSION: These data suggest that PML sumoylation is required for ATO-induced collagen synthesis in osteoblasts.

KCa1.1 is potential marker for distinguishing Ah-type baroreceptor neurons in NTS and contributes to sex-specific presynaptic neurotransmission in baroreflex afferent pathway.

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.

Arsenic trioxide exerts a double effect on osteoblast growth in vitro.

Arsenic trioxide (ATO) is a promising antitumor agent used to treat acute promyelocytic leukemia (APL) and, recently solid tumor. The present study was designed to evaluate the effect of ATO proliferation of osteoblast that plays very important roles in maintaining the structure integrity and function of bone. Cell survives, apoptosis, collagen, and molecular targets were identified by multiple detecting techniques, including MTT assay, electron microscopy, collagen detecting kit, TUNEL kit, and western blot in hFOB1.19 human osteoblasts cell line. The results showed that low dose of ATO (0.25, 0.5, and 1µM) remarkably enhanced the viability of cultured osteoblasts in a concentration- and time-dependent manner. Intriguingly, a dual effect of high dose of ATO (5, 10, and 20µM) was also observed showing significant reduction in viability of culture osteoblasts at concentration- and time-dependent fashion. Moreover, low dose of ATO promoted secretion and synthesis of collagen, whereas high dose of ATO induced typical morphological characteristics of apoptosis in osteoblasts. Mechanically, western blot results demonstrated that low dose of ATO dramatically up-regulated TGF-ß1 protein and activated p-AKT proliferative signaling. And, high dose of ATO increased Bax/Bcl-2 ratio in a time-dependent fashion and activated caspase-3 apoptotic signaling. These results demonstrate at the first time that ATO exerts a double effect on osteoblast function depending upon the concentration and provide a clue to rationally use ATO for clinicians to pay more attention to protect bone from the adverse effects of therapeutic dose of ATO during tumor therapy.

Increase in neuroexcitability of unmyelinated C-type vagal ganglion neurons during initial postnatal development of visceral afferent reflex functions.

BACKGROUND: Baroreflex gain increase up closely to adult level during initial postnatal weeks, and any interruption within this period will increase the risk of cardiovascular problems in later of life span. We hypothesize that this short period after birth might be critical for postnatal development of vagal ganglion neurons (VGNs). METHODS: To evaluate neuroexcitability evidenced by discharge profiles and coordinate changes, ion currents were collected from identified A- and C-type VGNs at different developmental stages using whole-cell patch clamping. RESULTS: C-type VGNs underwent significant age-dependent transition from single action potential (AP) to repetitive discharge. The coordinate changes between TTX-S and TTX-R Na(+) currents were also confirmed and well simulated by computer modeling. Although 4-AP or iberiotoxin age dependently increased firing frequency, AP duration was prolonged in an opposite fashion, which paralleled well with postnatal changes in 4-AP- and iberiotoxin-sensitive K(+) current activity, whereas less developmental changes were verified in A-types. CONCLUSION: These data demonstrate for the first time that the neuroexcitability of C-type VGNs increases significantly compared with A-types within initial postnatal weeks evidenced by AP discharge profiles and coordinate ion channel changes, which explain, at least in part, that initial postnatal weeks may be crucial for ontogenesis in visceral afferent reflex function.

Remodeling of hyperpolarization-activated current, Ih, in Ah-type visceral ganglion neurons following ovariectomy in adult rats.

Hyperpolarization-activated currents (Ih) mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels modulate excitability of myelinated A- and Ah-type visceral ganglion neurons (VGN). Whether alterations in Ih underlie the previously reported reduction of excitability of myelinated Ah-type VGNs following ovariectomy (OVX) has remained unclear. Here we used the intact nodose ganglion preparation in conjunction with electrophysiological approaches to examine the role of Ih remodeling in altering Ah-type neuron excitability following ovariectomy in adult rats. Ah-type neurons were identified based on their afferent conduction velocity. Ah-type neurons in nodose ganglia from non-OVX rats exhibited a voltage 'sag' as well as 'rebound' action potentials immediately following hyperpolarizing current injections, which both were suppressed by the Ih blocker ZD7288. Repetitive spike activity induced afterhyperpolarizations lasting several hundreds of milliseconds (termed post-excitatory membrane hyperpolarizations, PEMHs), which were significantly reduced by ZD7288, suggesting that they resulted from transient deactivation of Ih during the preceding spike trains. Ovariectomy reduced whole-cell Ih density, caused a hyperpolarizing shift of the voltage-dependence of Ih activation, and slowed Ih activation. OVX-induced Ih remodeling was accompanied by a flattening of the stimulus frequency/response curve and loss of PEMHs. Also, HCN1 mRNA levels were reduced by ∼30% in nodose ganglia from OVX rats compared with their non-OVX counterparts. Acute exposure of nodose ganglia to 17beta-estradiol partly restored Ih density and accelerated Ih activation in Ah-type cells. In conclusion, Ih plays a significant role in modulating the excitability of myelinated Ah-type VGNs in adult female rats.

Localization of human mesenchymal stem cells from umbilical cord blood and their role in repair of diabetic foot ulcers in rats.

The aim of this study is to explore the localization of human mesenchymal stem cells from umbilical cord matrix (hMSCs-UC) and the role of these cells in the repair of foot ulcerate tissue in diabetic foot ulcers in rats. A diabetic rat model was established by administering Streptozotocin. Diabetic foot ulceration was defined as non-healing or delayed-healing of empyrosis on the dorsal hind foot after 14 weeks. hMSCs-UC were delivered through the left femoral artery. We evaluated the localization of hMSCs-UC and their role in tissue repair in diabetic foot ulcers by histological analysis, PCR, and immunohistochemical staining. A model for diabetes was established in 54 out of 60 rats (90% success rate) and 27 of these rats were treated with hMSCs-UC. The area of ulceration was significantly and progressively reduced at 7 and 14 days following treatment with hMSCs-UC. This gross observation was strongly supported by the histological changes, including newly developed blood vessels and proliferation of inflammatory cells at 3 days post-treatment, significant increase in granulation tissue at 7 days post-treatment and squamous epithelium or stratified squamous epithelium at 14 days post-treatment. Importantly, human leukocyte antigen type-I (HLA-1) was confirmed in ulcerated tissue by RT-PCR. The expression of cytokeratin 19 was significantly increased in diabetic model rats, with no detectable change in cytokeratin 10. Additionally, both collagens I and III increased in model rats treated with hMSCs-UC, but the ratio of collagen I/III was less significant in treated rats compared with control rats. These results suggest that hMSCs-UC specifically localize to the target ulcerated tissue and may promote the epithelialization of ulcerated tissue by stimulating the release of cytokeratin 19 from keratinocytes and extracellular matrix formation.

17Beta-estradiol restores excitability of a sexually dimorphic subset of myelinated vagal afferents in ovariectomized rats.

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.

Electrophysiological and neuroanatomical evidence of sexual dimorphism in aortic baroreceptor and vagal afferents in rat.

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.