Decreased calcium-activated potassium channels by hypoxia causes abnormal firing in the spontaneous firing medial vestibular nuclei neurons.

Vertebrobasilar insufficiency (VBI) presents complex varied clinical symptoms, including vertigo and hearing loss. Little is known, however, about how Ca(2+)-activated K(+) channel attributes to the medial vestibular nucleus (MVN) neural activity in VBI. To address this issue, we performed whole-cell patch clamp and quantitative polymerase chain reaction (qPCR) to examine the effects of hypoxia on neural activity and the changes of the large conductance Ca(2+) activated K(+) channels (BKCa channels) in the MVN neurons in brain slices of male C57BL/6 mice. Brief hypoxic stimuli of the brain slices containing MVN were administrated by switching the normoxic artificial cerebrospinal fluid (ACSF) equilibrated with 21% O2/5% CO2 to hypoxic ACSF equilibrated with 5% O2/5% CO2 (balance N2). 3-min hypoxia caused a depolarization in the resting membrane potential (RM) in 8/11 non-spontaneous firing MVN neurons. 60/72 spontaneous firing MVN neurons showed a dramatic increase in firing frequency and a depolarization in the RM following brief hypoxia. The amplitude of the afterhyperpolarization (AHPA) was significantly decreased in both type A and type B spontaneous firing MVN neurons. Hypoxia-induced firing response was alleviated by pretreatment with NS1619, a selective BKCa activator. Furthermore, brief hypoxia caused a decrease in the amplitude of iberiotoxin-sensitive outward currents and mRNA level of BKCa in MVN neurons. These results suggest that BKCa channels protect against abnormal MVN neuronal activity induced by hypoxia, and might be a key target for treatment of vertigo and hearing loss in VBI.

Inhibitory effects of capsaicin on voltage-gated potassium channels by TRPV1-independent pathway.

Previously we observed that capsaicin, a transient receptor potential vanilloid 1 (TRPV1) receptor activator, inhibited transient potassium current (IA) in capsaicin-sensitive and capsaicin-insensitive trigeminal ganglion (TG) neurons from rats. It suggested that the inhibitory effects of capsaicin on IA have two different mechanisms: TRPV1-dependent and TRPV1-independent pathways. The main purpose of this study is to further investigate the TRPV1-independent effects of capsaicin on voltage-gated potassium channels (VGPCs). Whole cell patch-clamp technique was used to record IA and sustained potassium current (IK) in cultured TG neurons from trpv1 knockout (TRPV1(-/-)) mice. We found that capsaicin reversibly inhibited IA and IK in a dose-dependent manner. Capsaicin (30 µM) did not alter the activation curve of IA and IK but shifted the inactivation-voltage curve to hyperpolarizing direction, thereby increasing the number of inactivated VGPCs at the resting potential. Administrations of high concentrations capsaicin, no use-dependent block, and delay of recovery time course were found on IK and IA. Moreover, forskolin, an adenylate cyclase agonist, selectively decreased the inhibitory effects of IK by capsaicin, whereas none influenced the inhibitions of IA. These results suggest that capsaicin inhibits the VGPCs through TRPV1-independent and PKA-dependent mechanisms, which may contribute to the capsaicin-induced nociception.

Cannabinoid WIN 55,212-2 inhibits TRPV1 in trigeminal ganglion neurons via PKA and PKC pathways.

Although the inhibitory effect of cannabinoids on transient receptor potential vanilloid 1 (TRPV1) channel may explain the efficacy of peripheral cannabinoids in antihyperalgesia and antinociceptive actions, the mechanism for cannabinoid-induced inhibition of TRPV1 in primary sensory neurons is not understood. Therefore, we explored how WIN55,212-2 (WIN, a synthetic cannabinoid) inhibited TRPV1 in rat trigeminal ganglion neurons. A "bell"-shaped concentration-dependent curve was obtained from the effects of WIN on TRPV1 channel. The maximal inhibition on capsaicin-induced current (I (cap)) by WIN was at a concentration of 10(-9) M, and at this concentration I (cap) was reduced by 95 ± 1.6%. When the concentration of WIN was at 10(-6) M, it displayed a stimulatory effect on I (cap). In this study, several intracellular signaling transduction pathways were tested to study whether they were involved in the inhibitory effects of WIN on I (cap). We found that the inhibitory effect of WIN on I (cap) was completely reversed by PKA antagonists H-89 and KT5720 as well as by PKC antagonists BIM and staurosporine. It was also found that the inhibitory effect was partly reversed by PKG antagonist PKGi, while G-protein antagonist GDP-ßs/pertussis toxin (PTX) and PLC antagonist U-73122 had no effect on the inhibitory effect of WIN on I(cap). These results suggest that several intracellular signaling transduction pathways including PKA and PKC systems underlie the inhibitory effects of WIN on I (cap); however, G protein-coupled receptors CB1 or CB2 were not involved.

The modulation of the excitability of primary sensory neurons by Ca²âº-CaM-CaMKII pathway.

Ca(2+)-calmodulin (CaM) dependent protein kinase II (CaMKII) is an important intracellular signal transduction pathway. CaMKII is rich in the primary sensory neurons and specifically presents in the small- and medium-sized neurons. It remains unclear about the modulation on the excitability of primary sensory neurons by Ca(2+)-CaM-CaMKII pathway. By current clamp recording, we found that the excitability of capsaicin-sensitive small and medium trigeminal ganglion (TG) neurons was significantly reduced by a CaM specific antagonist (W-7) and a CaMKII antagonist (KN-93). The inhibition is represented as the reduction of numbers of action potential (AP), decrease of the amplitude of AP, increase of threshold, and prolongation of duration of AP. Consistently, by voltage clamp recording, we found that both voltage-gated sodium channels (VGSCs) and voltage-gated potassium channels (VGPCs) were inhibited by W-7 and KN-93 in the order of total sodium (Na(+)) current (INa-T) > sustained potassium (K(+)) current (IK) > A-type K(+) current (IA). In addition, AIP (a selective CaMKII peptide inhibitor) and KN-93 caused a similar inhibition of INa-T and IK. Those evidences show that the excitability of capsaicin sensitive small and medium TG neurons can be regulated by Ca(2+)-CaM-CaMKII pathway through modulating VGSCs and VGPCs. Considering the specific distribution of CaMKII and its susceptibility to many analgesic stimuli, Ca(2+)-CaM-CaMKII pathway may play an important role in the peripheral sensory transduction, especially in nociception.

Osmolality-induced tuning of action potentials in trigeminal ganglion neurons.

The present study explored the effect of anisotonicity on action potential (AP) in cultured trigeminal ganglion (TG) neurons. We demonstrate that the number of evoked APs was increased by both hypo- and hypertonic treatment. Transient Receptor Potential Vanilloid 4 receptor (TRPV4) activator increased the number of APs, but only hypotonic-response was markedly blocked in TRPV4-/- mice. Additionally, inhibition of PKC attenuated hypotonicity-induced increase, whereas antagonism of PKA attenuated hypertonicity-response. We conclude that anisotonicity increases excitability of nociceptors, which might be involved in anisotonicity-induced nociception. The increase of APs by hypo- and hypertonicity is mediated through different receptor and intracellular signaling pathways.

Changes in osmolality modulate voltage-gated calcium channels in trigeminal ganglion neurons.

Voltage-gated calcium channels (VGCCs) participate in many important physiological functions. However whether VGCCs are modulated by changes of osmolarity and involved in anisotonicity-induced nociception is still unknown. For this reason by using whole-cell patch clamp techniques in rat and mouse trigeminal ganglion (TG) neurons we tested the effects of hypo- and hypertonicity on VGCCs. We found that high-voltage-gated calcium current (I(HVA)) was inhibited by both hypo- and hypertonicity. In rat TG neurons, the inhibition by hypotonicity was mimicked by Transient Receptor Potential Vanilloid 4 receptor (TRPV4) activator but hypotonicity did not exhibit inhibition in TRPV4(-/-) mice TG neurons. Concerning the downstream signaling pathways, antagonism of PKG pathway selectively reduced the hypotonicity-induced inhibition, whereas inhibition of PLC- and PI3K-mediated pathways selectively reduced the inhibition produced by hypertonicity. In summary, although the effects of hypo- and hypertonicity show similar phenotype, receptor and intracellular signaling pathways were selective for hypo- versus hypertonicity-induced inhibition of I(HVA).

Protein corona of airborne nanoscale PM2.5 induces aberrant proliferation of human lung fibroblasts based on a 3D organotypic culture.

Exposure to PM2.5 has become one of the most important factors affecting public health in the world. Both clinical and research studies have suggested that PM2.5 inhalation is associated with impaired lung function. In this study, material characterization identified the existence of nanoscale particulate matter (NPM) in airborne PM2.5 samples. When coming into contact with protein-rich fluids, the NPM becomes covered by a protein layer that forms a "protein corona". Based on a 3D organotypic cell culture, the protein corona was shown to mitigate NPM cytotoxicity and further stimulate the proliferation of human lung fibroblasts (HLFs). ROS-activated alpha-smooth muscle actin (α-SMA) is considered to be one of the proliferation pathways. In this research, 3D cell cultures exhibited more tissue-like properties compared with the growth in 2D models. Animal models have been widely used in toxicological research. However, species differences make it impossible to directly translate discoveries from animals to humans. In this research, the 3D HLF model could partly simulate the biological responses of NPM-protein corona-induced aberrant HLF proliferation in the human lung. Our 3D cellular results provide auxiliary support for an animal model in research on PM2.5-induced impaired lung function, particularly in lung fibrosis.

Author Correction: Protein corona of airborne nanoscale PM2.5 induces aberrant proliferation of human lung fibroblasts based on a 3D organotypic culture.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Correction: Functional human 3D microvascular networks on a chip to study the procoagulant effects of ambient fine particulate matter.

[This corrects the article DOI: 10.1039/C7RA11357A.].

Effects of capsaicin on VGSCs in TRPV1-/- mice.

Two different mechanisms by which capsaicin blocks voltage-gated sodium channels (VGSCs) were found by using knockout mice for the transient receptor potential V1 (TRPV1(-/-)). Similar with cultured rat trigeminal ganglion (TG) neurons, the amplitude of tetrodotoxin-resistant (TTX-R) sodium current was reduced 85% by 1 muM capsaicin in capsaicin sensitive neurons, while only 6% was blocked in capsaicin insensitive neurons of TRPV1(+/+) mice. The selective effect of low concentration capsaicin on VGSCs was reversed in TRPV1(-/-) mice, which suggested that this effect was dependent on TRPV1 receptor. The blockage effect of high concentration capsaicin on VGSCs in TRPV1(-/-) mice was the same as that in capsaicin insensitive neurons of rats and TRPV1(+/+) mice. It is noted that non-selective effect of capsaicin on VGSCs shares many similarities with local anesthetics. That is, firstly, both blockages are concentration-dependent and revisable. Secondly, being accompanied with the reduction of amplitude, voltage-dependent inactivation curve shifts to hyperpolarizing direction without a shift of activation curve. Thirdly, use-dependent blocks are induced at high stimulus frequency.

Cannabinoids inhibit ATP-activated currents in rat trigeminal ganglionic neurons.

The present study aimed to investigate whether cannabinoids could modulate the response mediated by ATP receptor (P2X purinoceptor). Whole-cell patch-clamp recording was performed on cultured rat trigeminal ganglionic (TG) neurons. The majority of TG neurons were sensitive to ATP (67/75, 89.33%). Extracellular pretreatment with WIN55212-2, a cannabinoid receptor 1 (CB1 receptor) agonist, reduced ATP-activated current (I(ATP)) significantly. This inhibitory effect was concentration-dependent and was blocked by AM281, a specific CB1 receptor antagonist. Pretreatment with WIN55212-2 at 1×10(-13), 1×10(-12), 1×10(-11), 1×10(-10), 1×10(-9) and 1×10(-8) mol/L reduced I(ATP) (induced by 1×10(-4) mol/L ATP) by (8.14±3.14)%, (20.11±2.72)%, (46.62±3.51)%, (72.16±5.64)%, (80.21±2.80)% and (80.59±3.55)%, respectively. The concentration-response curves for I(ATP) pretreated with and without WIN55212-2 showed that WIN55212-2 shifted the curve downward, and decreased the maximal amplitude of I(ATP) by (58.02±4.21)%. But the threshold value and EC(50) (1.15×10(-4) mol/L vs 1.27×10(-4) mol/L) remained unchanged. The inhibition of I(ATP) by WIN55212-2 was reversed by AM281, suggesting that the inhibition was mediated via the CB1 receptor. Pretreatment with forskolin [an agonist of adenylyl cyclase (AC)] or 8-Br-cAMP reversed the inhibition of I(ATP) by WIN55212-2. These results suggest that the inhibitory effect of cannabinoids on I(ATP) is mediated via the CB1 receptors, that lead to inhibition of the AC-cAMP-PKA signaling pathway.

Effect of interleukin-1beta on I(A) and I(K) currents in cultured murine trigeminal ganglion neurons.

To investigate the effect of interleukin-1beta (IL-1beta) on I(A) and I(K) currents in cultured murine trigeminal ganglion (TG) neurons, whole-cell patch clamp technique was used to record the I(A) and I(K) currents before and after 20 ng/mL I(L)-1beta perfusion. Our results showed that 20 ng/mL IL-1beta inhibited I(A) currents (18.3 +/- 10.7)% (n=6, P<0.05). I(L)-1beta at 20 ng/mL had no effect on G-V curve of I(A) but moved the H-infinity curve V0.5 from -36.6+/-6.1 mV to -42.4+/-5.2 mV (n=5, P<0.01). However, 20 ng/mL IL-1beta had effect on neither the amplitude nor the G-V curve of I(K). IL-1beta was found to selectively inhibit I(A) current in TG neurons and the effect may contribute to hyperalgesia under various inflammatory conditions.

Effects of phorbol-12,13-dibuterate on sodium currents and potassium currents in rat trigeminal ganglion neurons.

The effects of phorbol-12,13-dibuterate (PDBu) on total sodium current (I(Na)-total), tetrodotoxin-resistant sodium current (I(Na)-TTXr), 4-AP-sensitive potassium current (I(A)) and TEA-sensitive potassium current (I(K)) in trigeminal ganglion (TG) neurons were investigated. Whole-cell patch clamp techniques were used to record ion currents in cultured TG neurons of rats. Results revealed that 0.5 micromol/L PDBu reduced the amplitude of I(Na)-total by (38.3+/-4.5)% (n=6, P<0.05), but neither the G-V curve (control: V (0.5)=-17.1+/-4.3 mV, k=7.4+/-1.3; PDBu: V (0.5)=-15.9+/-5.9 mV, k=5.9+/-1.4; n=6, P>0.05) nor the inactivation rate constant (control: 3.6+/-0.9 ms; PDBu: 3.6+/-0.8 ms; n=6, P>0.05) was altered. 0.5 micromol/L PDBu could significantly increase the amplitude of I(Na)-TTXr by (37.2+/-3.2)% (n=9, P<0.05) without affecting the G-V curve (control: V (0.5)=-14.7+/-6.0 mV, k=6.9+/- 1.4; PDBu: V (0.5)=-11.1+/-5.3 mV, k=8.1+/-1.5; n=5, P>0.05) or the inactivation rate constant (control: 4.6+/-0.6 ms; PDBu: 4.2+/-0.5 ms; n=5, P>0.05). 0.5 mumol/L PDBu inhibited I(K) by (15.6+/-5.0) % (n=16, P<0.05), and V (0.5) was significantly altered from - 4.7+/-1.4 mV to -7.9 +/-1.8 mV (n=16, P<0.05). I(A) was not significantly affected by PDBu, 0.5 mumol/L PDBu decreased I(A) by only (0.3+/-3.2)% (n=5, P>0.05). It was concluded that PDBu inhibited I(Na)-total but enhanced I(Na)-TTXr, and inhibited I(K) without affecting I(A). These data suggested that the activation of PKC pathway could exert the actions.

Different effects of capsaicin on I(A) and I(K) in pain-conduct neurons of rats.

The different effects of capsaicin on I(A) and I(K) currents in pain-conduct neurons of trigeminal ganglia (TG) were investigated. In cultured TG neurons of rats, whole-cell patch clamp techniques were used to record the I(A) and I(K) before and after capsaicin perfused. Results revealed that 1 micromol/L capsaicin could inhibit the amplitude of I(A) by 48.2% (n = 10, P < 0.05), but had no inhibitory effect on I(K) (n = 7, P > 0.05). Ten micromol/L capsaicin could significantly inhibit the amplitude of I(A) by 93.2% (n = 8, P < 0.01), but only slightly inhibit the amplitude of I(K) by 13.2% (n = 7, P < 0.05). Neither 1 micromol/L nor 10 micromol/L capsaicin had effects on the active curve of I(A) and I(K). It was concluded that capsaicin could selectively inhibit the I(A) current, and this effect might involve in the analgesic mechanisms of capsaicin.

Effects of WIN 55,212-2 on I(K) current in cultured trigeminal ganglion neurons of rat.

To investigate the effects of WIN 55,212-2 on I(K) in cultured rat trigeminal ganglion (TG) neurons, whole-cell patch clamp techniques were used to record the I(K) before and after WIN 55,212-2 perfusion at different concentrations. 30 micromol/L WIN 55,212-2 markedly (35.7% +/- 7.3%, P < 0.01, n = 8) inhibited I(K) currents, and the currents were partially recovered after washing. 30 micromol/L WIN 55,212-2 also induced a significant depolarizing shift in conductance-voltage parameters (control: V0.5 = 10.43 +/- 4.25 mV, k = 16.27 +/- 3.86; WIN 55,212-2: V0.5 = 24.71 +/- 3.91 mV, k =16.69 +/- 2.75; n = 8, P < 0.01 for V0.5). 0.01 micromol/L WIN 55,212-2 slightly (27.0% +/- 7. 9%, P < 0.05, n = 7) increased I(K) currents, but had no significant change in conductance voltage parameters (control: V0.5 =10.74 +/- 5.27 mV, k = 17.33 +/- 2.96; WIN 55,212-2: V0.5 = 11.06 +/- 2.05 mV, k = 19.69 +/- 6.60; n = 7, P > 0.05 for V0.5 and k). These results suggested that WIN 55,212-2 has dual action, which might be through different receptors.

Mechanisms of depressor effect of norepinephrine injected into subnucleus commissuriu of nucleus solitarius tractus in rabbits.

This experiment aimed to investigate the effect of adrenergic system in the subnucleus commissuriu of nucleus solitrius tractus (CNTS) on renal nerve discharges. Norepinephrine (NE) was microinjected into the CNTS of rabbits and mean arterial blood pressure (MAP) and renal nerve discharges (FRND) were synchronously recorded. The results indicated that (1) microinjection of norepinephine into the CNTS of rabbit could significantly attenuate the frequency of renal nerve discharge, and at the same time decrease markedly the mean arterial pressure. (2) Microinjection of 0.3 nmol yohimbin into CNTS had no significant influence on FRND and MAP, but could attenuate and even reverse the effects of NE on FRND and MAP. These results suggest that microinjection of NE into CNTS may activate the alpha-adrenorecptor located in CNTS and secondarily produce a depressor effect by attenuating the activity of periphenal sympathetic nervous system.

The protein tyrosine kinase inhibitor, genistein, decreases excitability of nociceptive neurons.

One mechanism by which neurons regulate their excitability is through ion channel phosphorylation. Compounds that increase nociceptive neuron excitability can cause hyperalgesia or allodynia whereas compounds that decrease nociceptive neuron excitability can be used as analgesics to relieve pain arising from inflammation or trauma. To identify targets that may cause a decrease in nociceptive neuron excitability, we have investigated the effects of genistein, a specific inhibitor of protein tyrosine kinases (PTKs), on capsaicin-sensitive neurons from cultured rat trigeminal ganglion neurons. It was found that genistein decreased the number of evoked action potentials, and hence their excitability. To determine whether genistein's effects occur through the inhibition of PTKs, we also tested the effects of two of its inactive analogues, daidzein and genistin. Whereas daidzein decreased excitability, albeit to a lower extent than genistein, excitability was unaffected by genistin. To determine which currents are involved in genistein's reduction in nociceptive neuron excitability, whole-cell voltage-clamp measurements were performed on voltage-gated sodium and potassium currents. One hundred micromolar genistein, daidzein and genistin inhibited tetrodotoxin-resistant voltage-gated sodium currents 74, 42, and 3%, respectively. Genistein markedly inhibited delayed rectifier (IK) and IA potassium currents, whereas daidzein and genistin were comparatively ineffective. In summary, we found that genistein's ability to inhibit nociceptive neuron excitability arises primarily from its non-specific inhibition of voltage-dependent sodium channels.

[Effects of capsaicin on IA and IK in cultured trigeminal ganglion neurons of rat].

AIM: To investigate the effect of capsaicin on IA and IK in cultured rat trigeminal ganglion (TG) neurons. METHODS: Whole-cell patch clamp technique was used to record the IA and IK before and after capsaicin perfusion at different concentrations. RESULTS: In capsaicin-sensitive (CS) neurons, capsaicin was shown to selectively inhibit IA in dose-dependent manner, the IC50 was 0.99 micromol x L(-1). Yet capsaicin showed no inhibitory effect on IK, capsaicin (10 micromol x L(-1)) only slightly inhibited IK by 13.2%. In capsaicin-insensitive (CIS) neurons, capsaicin (1 micromol x L(-1)) showed no significant inhibitory effect on IA and IK, capsaicin (10 micromol x L(-1)) only slightly inhibited IA and IK by 16.8% and 15.3%, respectively. Neither 1 micromol x L(-1) nor 10 micromol x L(-1) capsaicin showed effect on the G-V curve of IA and IK. CONCLUSION: Capsaicin was found to selectively inhibit the IA current in CS neurons, and this effect may contribute to hyperalgesia when capsaicin was first used.

Regulatory effects of anandamide on intracellular Ca(2+) concentration increase in trigeminal ganglion neurons.

Activation of cannabinoid receptor type 1 on presynaptic neurons is postulated to suppress neurotransmission by decreasing Ca(2+) influx through high voltage-gated Ca(2+) channels. However, recent studies suggest that cannabinoids which activate cannabinoid receptor type 1 can increase neurotransmitter release by enhancing Ca(2+) influx in vitro. The aim of the present study was to investigate the modulation of intracellular Ca(2+) concentration by the cannabinoid receptor type 1 agonist anandamide, and its underlying mechanisms. Using whole cell voltage-clamp and calcium imaging in cultured trigeminal ganglion neurons, we found that anandamide directly caused Ca(2+) influx in a dose-dependent manner, which then triggered an increase of intracellular Ca(2+) concentration. The cyclic adenosine and guanosine monophosphate-dependent protein kinase systems, but not the protein kinase C system, were involved in the increased intracellular Ca(2+) concentration by anandamide. This result showed that anandamide increased intracellular Ca(2+) concentration and inhibited high voltage-gated Ca(2+) channels through different signal transduction pathways.

Upregulation of T-type Ca2+ channels in primary sensory neurons in spinal nerve injury.

STUDY DESIGN: Painful behavior testing, whole-cell patch clamp recordings, and PCR analysis were served to test the influence of T-type Ca channels in spinal nerve-injured rats. OBJECTIVE: To determine the changes of T-type Ca channels in dorsal root ganglion (DRG) neurons of different sizes and the contribution to neuronal firing and painful behavior in neuropathic pain induced by nerve injury. SUMMARY OF BACKGROUND DATA: T-type and high-voltage-activated Ca channels play an important role in the transmission of nociceptive signals, especially in neuronal hyperexcitability in neuropathic pain. However, little is known about how nerve injury affects T-type Ca channels in DRG neurons of different sizes. METHODS: The effect of intrathecal administration of mibefradil in nerve-ligated rats was examined by painful behavior testing and current clamp. The changes of T-type Ca channels in DRG neurons caused by spinal nerve ligation were determined by RT-PCR analysis and voltage clamp. RESULTS: Spinal nerve injury significantly increased current density of T-type Ca channels in small DRG neurons. In addition, nerve injury significantly increased the percentage of T-type Ca channels in medium and large DRG neurons. Nerve injury significantly increased the mRNA levels of Cav3.2 and Cav3.3 in DRGs. Block of T-type Ca channels on mibefradil administration significantly normalized painful behavior and hyperexcitability in neuronal firing in spinal nerve-injured rats. CONCLUSION: Our study first indicated the upregulation of functional T-type Ca channels in DRG neurons of different sizes and the changes in different subtypes of T-type Ca channels by spinal nerve injury. Considering the effect of blocking T-type Ca channels in painful behavior and abnormal neuronal firing in rats with nerve injury, our results suggest that T-type Ca channels are potential therapeutic targets for the treatment of spinal nerve ligation-induced neuropathic pain.