Inhibition of intracellular Ca2+ mobilization and potassium channels activation are involved in the vasorelaxation induced by 7-hydroxycoumarin.

Coumarins exhibit a wide variety of biological effects, including activities in the cardiovascular system and the aim of this study was to evaluate the vascular therapeutic potential of 7-Hydroxicoumarin (7-HC). The vascular effects induced by 7-HC (0.001 µM-300 µM), were investigated by in vitro approaches using isometric tension measurements in rat superior mesenteric arteries and by in silico assays using Ligand-based analysis. Our results suggest that the vasorelaxant effect of 7-HC seems to rely on potassium channels, notably through large conductance Ca2+-activated K+ (BKCa) channels activation. In fact, 7-HC (300 µM) significantly reduced CaCl2-induced contraction as well as the reduction of intracellular calcium mobilization. However, the relaxation induced by 7-HC was independent of store-operated calcium entry (SOCE). Moreover, in silico analysis suggests that potassium channels have a common binding pocket, where 7-HC may bind and hint that its binding profile is more similar to quinine's than verapamil's. These results are compatible with the inhibition of Ca2+ release from intracellular stores, which is prompted by phenylephrine and caffeine. Taken together, these results demonstrate a therapeutic potential of 7-HC on the cardiovascular system, making it a promising lead compound for the development of drugs useful in the treatment of cardiovascular diseases.

Aromatic interactions with membrane modulate human BK channel activation.

Large-conductance potassium (BK) channels are transmembrane (TM) proteins that can be synergistically and independently activated by membrane voltage and intracellular Ca2+. The only covalent connection between the cytosolic Ca2+ sensing domain and the TM pore and voltage sensing domains is a 15-residue 'C-linker'. To determine the linker's role in human BK activation, we designed a series of linker sequence scrambling mutants to suppress potential complex interplay of specific interactions with the rest of the protein. The results revealed a surprising sensitivity of BK activation to the linker sequence. Combining atomistic simulations and further mutagenesis experiments, we demonstrated that nonspecific interactions of the linker with membrane alone could directly modulate BK activation. The C-linker thus plays more direct roles in mediating allosteric coupling between BK domains than previously assumed. Our results suggest that covalent linkers could directly modulate TM protein function and should be considered an integral component of the sensing apparatus.

Activation of transient receptor potential vanilloid 4 channels dilates rat retinal arterioles through nitric oxide- and BKCa channel-dependent mechanisms in vivo.

Transient receptor potential vanilloid 4 (TRPV4) channel, a cation channel expressed in nearly all cell types, plays an important role in the regulation of vascular tone. In the present study, we examined the effect of GSK1016790A, an activator of TRPV4 channels, on the diameter of retinal blood vessels in rats and the underlying mechanisms. Ocular fundus images were captured with an original high-resolution digital fundus camera in vivo and diameters of retinal blood vessels were measured. Intravenous infusion of GSK1016790A (0.2-2 µg kg-1 min-1) increased retinal arteriolar diameter in a dose-dependent manner. The higher dose of GSK1016790A (2 µg kg-1 min-1) slightly decreased blood pressure. These responses to GSK1016790A were significantly attenuated by intravenous injection of GSK2193874 (0.3 mg/kg), an antagonist of TRPV4 channels. Intravitreal injection of Nω-nitro-L-arginine methyl ester, an inhibitor of nitric oxide (NO) synthase or iberiotoxin, an inhibitor of large-conductance Ca2+-activated K+ (BKCa) channel, significantly attenuated the GSK1016790A-induced increases in retinal arteriolar diameter. These results suggest that activation of TRPV4 channels dilates rat retinal arterioles through NO- and BKCa channel-dependent mechanisms in vivo.

Dynamic- and Frequency-Specific Regulation of Sleep Oscillations by Cortical Potassium Channels.

Primary electroencephalographic (EEG) features of sleep arise in part from thalamocortical neural assemblies, and cortical potassium channels have long been thought to play a critical role. We have exploited the regionally dynamic nature of sleep EEG to develop a novel screening strategy and used it to conduct an adeno-associated virus (AAV)-mediated RNAi screen for cellular roles of 31 different voltage-gated potassium channels in modulating cortical EEG features across the circadian sleep-wake cycle. Surprisingly, a majority of channels modified only electroencephalographic frequency bands characteristic of sleep, sometimes diurnally or even in specific vigilance states. Confirming our screen for one channel, we show that depletion of the KCa1.1 (or "BK") channel reduces EEG power in slow-wave sleep by slowing neuronal repolarization. Strikingly, this reduction completely abolishes transcriptomic changes between sleep and wake. Thus, our data establish an unexpected connection between transcription and EEG power controlled by specific potassium channels. We postulate that additive dynamic roles of individual potassium channels could integrate different influences upon sleep and wake within single neurons.

KCa1.1 and Kv1.3 channels regulate the interactions between fibroblast-like synoviocytes and T lymphocytes during rheumatoid arthritis.

BACKGROUND: Fibroblast-like synoviocytes (FLS) and CCR7- effector memory T (TEM) cells are two of the major cell types implicated in the progression of rheumatoid arthritis (RA). In particular, FLS become highly invasive, whereas TEM cells proliferate and secrete proinflammatory cytokines, during RA. FLS and T cells may also interact and influence each other's phenotypes. Inhibition of the pathogenic phenotypes of both FLS and TEM cells can be accomplished by selectively blocking the predominant potassium channels that they upregulate during RA: KCa1.1 (BK, Slo1, MaxiK, KCNMA1) upregulated by FLS and Kv1.3 (KCNA3) upregulated by activated TEM cells. In this study, we investigated the roles of KCa1.1 and Kv1.3 in regulating the interactions between FLS and TEM cells and determined if combination therapies of KCa1.1- and Kv1.3-selective blockers are more efficacious than monotherapies in ameliorating disease in rat models of RA. METHODS: We used in vitro functional assays to assess the effects of selective KCa1.1 and Kv1.3 channel inhibitors on the interactions of FLS isolated from rats with collagen-induced arthritis (CIA) with syngeneic TEM cells. We also used flow cytometric analyses to determine the effects of KCa1.1 blockers on the expression of proteins used for antigen presentation on CIA-FLS. Finally, we used the CIA and pristane-induced arthritis models to determine the efficacy of combinatorial therapies of KCa1.1 and Kv1.3 blockers in reducing disease severity compared with monotherapies. RESULTS: We show that the interactions of FLS from rats with CIA and of rat TEM cells are regulated by KCa1.1 and Kv1.3. Inhibiting KCa1.1 on FLS reduces the ability of FLS to stimulate TEM cell proliferation and migration, and inhibiting Kv1.3 on TEM cells reduces TEM cells' ability to enhance FLS expression of KCa1.1 and major histocompatibility complex class II protein, as well as stimulates their invasion. Furthermore, we show that combination therapies of selective KCa1.1 and Kv1.3 blockers are more efficacious than monotherapies at reducing signs of disease in two rat models of RA. CONCLUSIONS: Our results demonstrate the importance of KCa1.1 and Kv1.3 in regulating FLS and TEM cells during RA, as well as the value of combined therapies targeting both of these cell types to treat RA.

Inhibition of BKCa negatively alters cardiovascular function.

Large conductance calcium and voltage-activated potassium channels (BKCa ) are transmembrane proteins, ubiquitously expressed in the majority of organs, and play an active role in regulating cellular physiology. In the heart, BKCa channels are known to play a role in regulating the heart rate and protect it from ischemia-reperfusion injury. In vascular smooth muscle cells, the opening of BKCa channels results in membrane hyperpolarization which eventually results in vasodilation mediated by a reduction in Ca2+ influx due to the closure of voltage-dependent Ca2+ channels. Ex vivo studies have shown that BKCa channels play an active role in the regulation of the function of the majority of blood vessels. However, in vivo role of BKCa channels in cardiovascular function is not completely deciphered. Here, we have evaluated the rapid in vivo role of BKCa channels in regulating the cardiovascular function by using two well-established, rapid-acting, potent blockers, paxilline and iberiotoxin. Our results show that BKCa channels are actively involved in regulating the heart rate, the function of the left and right heart as well as major vessels. We also found that the effect on BKCa channels by blockers is completely reversible, and hence, BKCa channels can be exploited as potential targets for clinical applications for modulating heart rate and cardiac contractility.

Studies into Slo1 K+ channels and their ligand docosahexaenoic acid in murine sepsis to delineate off-target effects of immunonutrition.

AIMS: Studies on omega-3 fatty acids, including docosahexaenoic acid (DHA), reveal diverging results: Their intake is recommended in cardiovascular disease and major surgery, while evidence argues against use in septic patients. DHA mediates its blood-pressure-lowering effect through Slo1 channels that are expressed on cardiovascular and immune cells. We hypothesised that conflicting effects of immunonutrition could be explained by the influence of omega-3 fatty acids on systemic blood pressure or immune effector cells through Slo1. MAIN METHODS: The effect of DHA on blood pressure was analysed in septic wild-type (WT) mice. Septic WT and Slo1 knockout (KO) mice were compared regarding survival, clinical presentation, haematology, cytokine release and bacterial burden. Cytokine expression and release of bone marrow derived macrophages (BMDM) from WT and Slo1 KO mice was assessed in response to LPS. KEY FINDINGS: The significant blood-pressure-lowering effect of DHA in healthy animals was blunted in already hypotensive septic mice. Septic Slo1 KO mice displayed moderately lower bacterial burden in blood and lungs compared with WT, which did not translate into improved survival. Slo1 KO BMDM presented lower IL-6 levels in response to LPS, an effect that was abolished in the presence of DHA. More importantly, the strong inhibitory effect of DHA on IL-6 release was also observed in Slo1 KO BMDM. SIGNIFICANCE: The controversial effects of immunonutrition in sepsis are unlikely to be primarily explained by the influence of DHA on blood pressure or effects on immune response mediated through Slo1 channels.

The role of BKCa in endometrial cancer HEC-1-B cell proliferation and migration.

BKCa is a large conductance calcium activated potassium channel ubiquitously expressed in various cell types. Accumulating evidence demonstrates that BKCa is aberrantly expressed in many malignancies, involving in cancerous behaviors such as cell proliferation and migration. In this study, we investigated the functional role of BKCa in endometrial cancer HEC-1-B cells. Overexpression of BKCa by plasmid transfection enhanced endometrial cancer cell proliferation and migration. Conversely, silence of BKCa by lentivirus mediated RNAi system not only inhibited proliferation and migration but also impaired tumor growth in vivo. Patch clamp assay identified the BKCa currents in HEC-1-B cells, which was supported by the observation of channel activation or inhibition in response to the specific opener (NS1619) or blocker (IBTX) of BKCa. Moreover, NS1619 significantly increased cell proliferation and migration while IBTX exhibited the opposite effects. In summary, these data suggested an important role of BKCa in proliferation and migration of endometrial cancer HEC-1-B cells. Thus, BKCa may be established as a potential therapeutic target in endometrial cancer.

Voltage-gated and stretch-activated potassium channels in the human heart : Pathophysiological and clinical significance.

Ion channels are essential for electrical signaling and contractility in cardiomyocytes. Detailed knowledge about the molecular function and regulation of cardiac ion channels is crucial for understanding cardiac physiology and pathophysiology especially in the field of arrhythmias. This review aims at providing a general overview on the identity, functional characteristics, and roles of voltage-gated as well as stretch-activated potassium selective channels in the heart. In particular, we will highlight potential therapeutic targets as well as the emerging fields of future investigations.

The Cardioprotective Effect of Dexmedetomidine in Rats Is Dose-Dependent and Mediated by BKCa Channels.

The alpha-2 receptor agonist Dexmedetomidine (Dex) protects the heart against ischemia-reperfusion injury. We investigated the signaling cascade underlying Dex-induced acute cardioprotection, with special emphasis on large-conductance Ca2+-sensitive potassium (BKCa) channels. Rats were anesthetized with pentobarbital. Hearts were isolated, mounted on a Langendorff system and perfused with Krebs-Henseleit buffer. Hearts underwent 33 minutes of ischemia followed by 60 minutes of reperfusion. Before the beginning of ischemia, Dex was administered at different doses (0.1-30 nM) for characterization of a dose-effect relationship. In another set of experiments, Dex (3 nM) was administered together with the BKCa channel inhibitor paxilline and the connexin-43 inhibitor peptide Gap27. Also, the BKCa channel opener NS1619 was administered. In control animals, infarct size was 49% ± 5%. Dex at 3-30 nM reduced infarct size to ∼22%, whereas lower (0.1-1 nM) doses reduced infarct size to ∼38%. Paxilline (1 µM) and GAP27 (6 µM) blocked the Dex-induced cardioprotection. NS1619 (10 µM) reduced infarct size to about the same magnitude as did the higher doses of Dex. Functional heart parameters and coronary flow were not different between the study groups. In male rats, the Dex-induced protection against ischemia-reperfusion injury involves connexin-43 and activation of BKCa channels.

Guide to the Pharmacology of Mitochondrial Potassium Channels.

This chapter provides a critical overview of the available literature on the pharmacology of mitochondrial potassium channels. In the first part, the reader is introduced to the topic, and eight known protein contributors to the potassium permeability of the inner mitochondrial membrane are presented. The main part of this chapter describes the basic characteristics of each channel type mentioned in the introduction. However, the most important and valuable information included in this chapter concerns the pharmacology of mitochondrial potassium channels. Several available channel modulators are critically evaluated and rated by suitability for research use. The last figure of this chapter shows the results of this evaluation at a glance. Thus, this chapter can be very useful for beginners in this field. It is intended to be a time- and resource-saving guide for those searching for proper modulators of mitochondrial potassium channels.

Glabridin-induced vasorelaxation: Evidence for a role of BKCa channels and cyclic GMP.

BACKGROUND AND PURPOSE: Glabridin is a major flavonoid in Glycyrrhiza glabra (licorice) root, a traditional Asian medicine. Glabridin is reported to have anti-atherogenic, anti-inflammatory and anti-nephritic properties; however its effects on vascular tone remain unexplored. EXPERIMENTAL APPROACH: We examined the effect of glabridin on rat main mesenteric artery using isometric myography and also ELISA to measure cGMP levels. KEY RESULTS: Glabridin (30µM) relaxed arteries pre-constricted with the thromboxane A2 analog U46619 (0.2µM) by ~60% in an endothelium-independent manner. Relaxation to 30µM glabridin was abolished by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (1µM) and by the BKCa channel blocker tetraethyammonium (1mM) but was unaffected by the estrogen receptor antagonist ICI182780. The concentration-response curve to glabridin (0.1 to 30µM) was downshifted by the KATP channel blocker glibenclamide (10µM), the KV channel blocker 4-aminopyridine (300µM), and the KIR blocker BaCl2 (30µM). In U46619-contracted arteries partially relaxed by 0.1µM sodium nitroprusside, application of 10 and 30nM glabridin caused additional vasorelaxation. Glabridin (30µM) approximately doubled tissue [cyclic GMP]. Application of the phosphodiesterase inhibitor isobutylmethylxanthine caused a much larger rise in [cyclic GMP], and glabridin failed to cause vasorelaxation or a further rise in [cGMP] when co-applied with IBMX. CONCLUSIONS AND IMPLICATIONS: Vasorelaxation to glabridin is dependent on the opening of K+ channels, particularly BKCa, probably caused by a rise in cellular [cyclic GMP] owing to phosphodiesterase inhibition. In the presence of sodium nitroprusside an effect of glabridin is observed at nM concentrations, similar those measured in plasma following human ingestion of licorice flavonoid oil.

Cytoplasmic vacuolization in cell death and survival.

Cytoplasmic vacuolization (also called cytoplasmic vacuolation) is a well-known morphological phenomenon observed in mammalian cells after exposure to bacterial or viral pathogens as well as to various natural and artificial low-molecular-weight compounds. Vacuolization often accompanies cell death; however, its role in cell death processes remains unclear. This can be attributed to studying vacuolization at the level of morphology for many years. At the same time, new data on the molecular mechanisms of the vacuole formation and structure have become available. In addition, numerous examples of the association between vacuolization and previously unknown cell death types have been reported. Here, we review these data to make a deeper insight into the role of cytoplasmic vacuolization in cell death and survival.

ß1-subunit-induced structural rearrangements of the Ca2+- and voltage-activated K+ (BK) channel.

Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels are involved in a large variety of physiological processes. Regulatory ß-subunits are one of the mechanisms responsible for creating BK channel diversity fundamental to the adequate function of many tissues. However, little is known about the structure of its voltage sensor domain. Here, we present the external architectural details of BK channels using lanthanide-based resonance energy transfer (LRET). We used a genetically encoded lanthanide-binding tag (LBT) to bind terbium as a LRET donor and a fluorophore-labeled iberiotoxin as the LRET acceptor for measurements of distances within the BK channel structure in a living cell. By introducing LBTs in the extracellular region of the α- or ß1-subunit, we determined (i) a basic extracellular map of the BK channel, (ii) ß1-subunit-induced rearrangements of the voltage sensor in α-subunits, and (iii) the relative position of the ß1-subunit within the α/ß1-subunit complex.

Equol increases cerebral blood flow in rats via activation of large-conductance Ca(2+)-activated K(+) channels in vascular smooth muscle cells.

The present study was designed to investigate the effect of equol on cerebral blood flow and the underlying molecular mechanisms. The regional cerebral blood flow in parietal lobe of rats was measured by using a laser Doppler flowmetry. Isolated cerebral basilar artery and mesenteric artery rings from rats were used for vascular reactivity measurement with a multi wire myography system. Outward K(+) current in smooth muscle cells of cerebral basilar artery, large-conductance Ca(2+)-activated K(+) (BK) channel current in BK-HEK 293 cells stably expressing both human α (hSlo)- and ß1-subunits, and hSlo channel current in hSlo-HEK 293 cells expressing only the α-subunit of BK channels were recorded with whole cell patch-clamp technique. The results showed that equol significantly increased regional cerebral blood flow in rats, and produced a concentration-dependent but endothelium-independent relaxation in rat cerebral basilar arteries. Both paxilline and iberiotoxin, two selective BK channel blockers, significantly inhibited equol-induced vasodilation in cerebral arteries. Outward K(+) currents in smooth muscle cells of cerebral basilar artery were increased by equol and fully reversed by washout or blockade of BK channels with iberiotoxin. Equol remarkably enhanced human BK current in BK-HEK 293 cells, but not hSlo current in hSlo-HEK 293 cells, and the increase was completely abolished by co-application of paxilline. Our findings provide the first information that equol selectively stimulates BK channel current by acting on its ß1 subunit, which may in turn contribute to the equol-mediated vasodilation and cerebral blood flow increase.

[Topology of the mitochondrial potassium ion channels]. / Topologia mitochondrialnych kanalów potasowych.

In the inner mitochondrial membrane several potassium channels have been identified whose activation lead to cytoprotection during ischemic event. It was found that activation of mitochondrial large conductance calcium activated potassium channel (mitoBKCa) and ATP regulated potassium channel (mitoKATP) preserves brain and heart muscle cells against ischemia/reperfusion induced damage. However the detailed cytoprotection mechanism remains unclear. Similarly, the molecular structures and protein interactions of the mitochondrial potassium channels are still unknown. In this article, we summarize the current knowledge of the mitoKATP and mitoBKCa channels topology. Different aspects of this topic are discussed like import and assembly of the channel subunits and biophysical properties of mitochondrial compartments. Additionally, the consequences of different topology models on the cytoprotective function of the mitochondrial potassium channels were analyzed.

Identification and Characterization of GAL-021 as a Novel Breathing Control Modulator.

BACKGROUND: The authors describe the preclinical pharmacological properties of GAL-021, a novel peripheral chemoreceptor modulator. METHODS: The ventilatory effects of GAL-021 were characterized using tracheal pneumotachometry (n = 4 to 6), plethysmography (n = 5 to 6), arterial blood gas analyses (n = 6 to 11), and nasal capnography (n = 3 to 4) in naive animals and those subjected to morphine-induced respiratory depression. Morphine analgesia in rats was evaluated by tail-flick test (n = 6). Carotid body involvement in GAL-021 ventilatory effects was assessed by comparing responses in intact and carotid sinus nerve-transected rats. Hemodynamic effects of GAL-021 were evaluated in urethane-anesthetized rats (n = 7). The pharmacological profile of GAL-021 in vitro was investigated using radioligand binding, enzyme inhibition, and cellular electrophysiology assays. RESULTS: GAL-021 given intravenously stimulated ventilation and/or attenuated opiate-induced respiratory depression in rats, mice, and nonhuman primates, without decreasing morphine analgesia in rats. GAL-021 did not alter mean arterial pressure but produced a modest increase in heart rate. Ventilatory stimulation in rats was attenuated by carotid sinus nerve transection. GAL-021 inhibited KCa1.1 in GH3 cells, and the evoked ventilatory stimulation was attenuated in Slo1 mice lacking the pore-forming α-subunit of the KCa1.1 channel. CONCLUSIONS: GAL-021 behaved as a breathing control modulator in rodents and nonhuman primates and diminished opioid-induced respiratory depression without compromising opioid analgesia. It acted predominantly at the carotid body, in part by inhibiting KCa1.1 channels. Its preclinical profile qualified the compound to enter clinical trials to assess effects on breathing control disorders such as drug (opioid)-induced respiratory depression and sleep apnea.

Unoprostone activation of BK (KCa1.1) channel splice variants.

This investigation was conducted to study the relationship between intracellular Ca(2+) and activation of large conductance Ca(2+)-activated K(+) (BK) currents by unoprostone, the first synthetic docosanoid. We used HEK293 cells stably transfected with two BK channel splice variants, one sensitive to unoprostone and the other insensitive. We examined the effects of unoprostone on channel activity in excised inside-out patches and cell-attached patches. The half-maximal stimulation of the sensitive BK channels by Ca(2+) was shifted from 3.4±0.017 nM to 0.81±.0058 nM in the presence of 10 nM unoprostone. There was no effect on insensitive channels even at unoprostone concentrations as high as 1000 nM. There was no effect of unoprostone on the voltage dependence of the BK channels. Changes in open probability and effects of Ca(2+) and unoprostone were best described by a synergistic binding model. These data would suggest that Ca(2+) and unoprostone were binding to sites close to one another on the channel protein and that unoprostone binding causes the affinity of the calcium binding site to increase. This idea is consistent with three dimensional models of the Ca(2+) binding site and a putative unoprostone binding domain. Our results have important implications for the clinical use of unoprostone to activate BK channels. Channel activation will be limited if intracellular Ca(2+) is not elevated.

Reduction in renal blood flow following administration of norepinephrine and phenylephrine in septic rats treated with Kir6.1 ATP-sensitive and KCa1.1 calcium-activated K+ channel blockers.

We evaluated the effects of K+ channel blockers in the vascular reactivity of in vitro perfused kidneys, as well as on the influence of vasoactive agents in the renal blood flow of rats subjected to the cecal ligation and puncture (CLP) model of sepsis. Both norepinephrine and phenylephrine had the ability to increase the vascular perfusion pressure reduced in kidneys of rats subjected to CLP at 18 h and 36 h before the experiments. The non-selective K+ channel blocker tetraethylammonium, but not the Kir6.1 blocker glibenclamide, normalized the effects of phenylephrine in kidneys from the CLP 18 h group. Systemic administration of tetraethylammonium, glibenclamide, or the KCa1.1 blocker iberiotoxin, did not change the renal blood flow in control or septic rats. Norepinephrine or phenylephrine also had no influence on the renal blood flow of septic animals, but its injection in rats from the CLP 18 h group previously treated with either glibenclamide or iberiotoxin resulted in an exacerbated reduction in the renal blood flow. These results suggest an abnormal functionality of K+ channels in the renal vascular bed in sepsis, and that the blockage of different subtypes of K+ channels may be deleterious for blood perfusion in kidneys, mainly when associated with vasoactive drugs.

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.