Existence of ATP sensitive potassium currents on human periodontal ligament cells.

OBJECTIVE: Potassium channels of the ATP-sensitive family (KATP channel) are inhibited by increase in intracellular ATP. Electrophysiological studies have demonstrated that the kinetics and pharmacological properties of KATP channels vary among different tissues, suggesting structurally and functionally distinct types. There are studies showing human periodontal ligament (PDL) cells respond to mechanical stress by increasing ATP release, which participates in bone resorption or bone homeostasis. So, in this study we investigated the existence of KATP channel subunit and their single channel properties in human periodontal ligaments. MATERIALS & METHOD: The human PDL cells were isolated from healthy erupted third molar. For patch-clamp experiments, human PDL fibroblasts were seeded on 3.5cm plastic dishes. The inside-out patch clamp recordings were performed under voltage clamp mode. Reverse transcriptase polymerase chain reaction (RT-PCR) was conducted to identify the channel subunits. All pair-wise comparisons were performed by Paired t-test. A P value <0.05 was considered significant. RESULTS: We observed mRNA transcripts for Kir6.1, Kir6.2 and Sur2B subuits in the human PDL cells. In inside-out patch mode, the single channel conductance was 163pS at symmetrical K+ concentration of 140mM and inward rectification was seen in ATP-free bath solution. The reversal potential of the currents was found to be 0mV at symmetrical concentration (140mM) of K+ in bath solution. The single channel currents were almost blocked by adding 5mM ATP in the bath solution. However, the currents were not blocked by 100µM glibenclamide, a subunit specific KATP channel blocker. CONCLUSIONS: These results indicate that human PDL cells express KATP channels subunit including Sur2B and Kir6.1 and Kir6.2 which are sensitive to ATP but insensitive to glibenclamide.

Effects of hypotaurine on substantia gelatinosa neurons of the trigeminal subnucleus caudalis in immature mice.

To understand the action and mechanism of hypotaurine, an immediate precursor of taurine, on orofacial nociceptive processing, we examined the direct effects and receptor types involved in hypotaurine-induced responses using the whole-cell patch clamp technique in the substantia gelatinosa (SG) neurons of the trigeminal subnucleus caudalis (Vc) of immature mice. Under the condition of high-chloride pipette solution, hypotaurine elicited inward currents or upward deflections of membrane potential, which increased in a concentration-dependent manner (30-3000 µM) with the EC50 of 663.8 and 337.6 µM, respectively. The responses to 300 µM hypotaurine were reproducible and recovered upon washout. The 300 µM hypotaurine-induced currents were maintained in the presence of TTX, CNQX, and AP5, indicating direct postsynaptic action of hypotaurine on SG neurons. Responses to both low (300 µM) and high (1 or 3 mM) concentrations of hypotaurine were completely and reversibly blocked by the glycine receptor antagonist strychnine (2 µM), but unaffected by the GABAA receptor antagonist gabazine (3 µM) which blocks synaptic GABAA receptors at low concentration. Furthermore, responses to 300 µM hypotaurine and a maximal concentration of glycine (3 mM) were not additive, indicating that hypotaurine and glycine act on the same receptor. Hypotaurine-induced currents were partially antagonized by picrotoxin (50 µM) which blocks homomeric glycine receptors and by bicuculline (10 µM) which is an antagonist of α2 subunit-containing glycine receptors. These results suggest that hypotaurine-induced responses were mediated by glycine receptor activation in the SG neurons and hypotaurine might be used as an effective therapeutics for orofacial pain.

Effects of cisplatin on potassium currents in CT26 cells.

AIMS: Cisplatin, a platinum-based drug, is an important weapon against many types of cancer. It is well-known that cisplatin induces apoptosis. Potassium channel plays very important role in several signaling pathways. To investigate the possibility that potassium channels also have a role in the cellular response to cisplatin, we examined the effect of cisplatin on the activity of potassium channels on CT26 cell, the colon carcinoma cell line. MATERIALS AND METHODS: The cells were cultured in DMEM, supplemented with 10< heat-inactivated fetal bovine serum. At mid-log phase, cultures were harvested, washed twice in phosphate-buffered saline, and resuspended in culture medium before use. Cells were voltage-clamped using the whole-cell patch clamp technique. Membrane current data were collected and amplified. STATISTICAL ANALYSIS: Differences between two groups were assessed by paired t-test and one sample t-test to compare the relative values. One-way ANOVA was used for all experiment with more than two groups. RESULTS: Potassium currents were detected in CT26 cells and the currents were reduced by the application of tetraethylammonium (TEA) chloride, iberiotoxin, a big conductance calcium-activated potassium channel blocker and barium. The potassium currents were enhanced to 192< by the application of cisplatin (0.5 mM). Moreover, the increase of potassium currents by cisplatin was further inhibited by the application of TEA confirming the action of cisplatin on potassium channels. In addition, relative current induced by cisplatin in CT26 cells was bit larger than in normal IEC-6 cells. CONCLUSION: Potassium currents were detected in CT26 cells and the currents were reduced by the application of tetraethylammonium (TEA) chloride, iberiotoxin, a big conductance calcium-activated potassium channel blocker and barium. The potassium currents were enhanced to 192< by the application of cisplatin (0.5 mM). Moreover, the increase of potassium currents by cisplatin was further inhibited by the application of TEA confirming the action of cisplatin on potassium channels. In addition, relative current induced by cisplatin in CT26 cells was bit larger than in normal IEC-6 cells.

Baicalin Activates Glycine and γ-Aminobutyric Acid Receptors on Substantia Gelatinosa Neurons of the Trigeminal Subsnucleus Caudalis in Juvenile Mice.

The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) receives nociceptive afferent inputs from thin-myelinated A[Formula: see text] fibers and unmyelinated C fibers and has been shown to be involved in the processing of orofacial nociceptive information. Scutellaria baicalensis Georgi (Huang-Qin, SbG), one of the 50 fundamental herbs of Chinese herbology, has been used historically as anti-inflammatory and antineoplastic medicine. Baicalin, one of the major compounds of SbG, has been reported to have neuroprotective, anti-inflammatory and analgesic effects. However, the receptor type activated by baicalin and its precise action mechanism on the SG neurons of Vc have not yet been studied. The whole-cell patch clamp technique was performed to examine the ion channels activated by baicalin on the SG neurons of Vc. In high Cl[Formula: see text] pipette solution, the baicalin (300[Formula: see text][Formula: see text]M) induced repeatable inward currents ([Formula: see text][Formula: see text]pA, [Formula: see text]) without desensitization on all the SG neurons tested. Further, the inward currents showed a concentration (0.1-3[Formula: see text]mM) dependent pattern. The inward current was sustained in the presence of tetrodotoxin (0.5[Formula: see text][Formula: see text]M), a voltage sensitive Na[Formula: see text] channel blocker. In addition, baicalin-induced inward currents were reduced in the presence of picrotoxin (50[Formula: see text][Formula: see text]M), a GABAA receptor antagonist, flumazenil (100[Formula: see text][Formula: see text]M), a benzodiazepine-sensitive GABAA receptor antagonist, and strychnine (2[Formula: see text][Formula: see text]M), a glycine receptor antagonist, respectively. These results indicate that baicalin has inhibitory effects on the SG neurons of the Vc, which are due to the activation of GABAA and/or the glycine receptor. Our results suggest that baicalin may be a potential target for orofacial pain modulation.

Activation of Strychnine-Sensitive Glycine Receptors by Shilajit on Preoptic Hypothalamic Neurons of Juvenile Mice.

Shilajit, a mineral pitch, has been used in Ayurveda and Siddha system of medicine to treat many human ailments, and is reported to contain at least 85 minerals in ionic form. This study examined the possible mechanism of Shilajit action on preoptic hypothalamic neurons using juvenile mice. The hypothalamic neurons are the key regulator of many hormonal systems. In voltage clamp mode at a holding potential of -60 mV, and under a high chloride pipette solution, Shilajit induced dose-dependent inward current. Shilajit-induced inward currents were reproducible and persisted in the presence of 0.5 µM tetrodotoxin (TTX) suggesting a postsynaptic action of Shilajit on hypothalamic neurons. The currents induced by Shilajit were almost completely blocked by 2 µM strychnine (Stry), a glycine receptor antagonist. In addition, Shilajit-induced inward currents were partially blocked by bicuculline. Under a gramicidin-perforated patch clamp mode, Shilajit induced membrane depolarization on juvenile neurons. These results show that Shilajit affects hypothalamic neuronal activities by activating the Stry-sensitive glycine receptor with α2/α2ß subunit. Taken together, these results suggest that Shilajit contains some ingredients with possible glycine mimetic activities and might influence hypothalamic neurophysiology through activation of Stry-sensitive glycine receptor-mediated responses on hypothalamic neurons postsynaptically.

Activation of synaptic and extrasynaptic glycine receptors by taurine in preoptic hypothalamic neurons.

Taurine is an essential amino-sulfonic acid having a fundamental function in the brain, participating in both cell volume regulation and neurotransmission. Using a whole cell voltage patch clamp technique, the taurine-activated neurotransmitter receptors in the preoptic hypothalamic area (PHA) neurons were investigated. In the first set of experiments, different concentrations of taurine were applied on PHA neurons. Taurine-induced responses were concentration-dependent. Taurine-induced currents were action potential-independent and sensitive to strychnine, suggesting the involvement of glycine receptors. In addition, taurine activated not only α-homomeric, but also αß-heteromeric glycine receptors in PHA neurons. Interestingly, a low concentration of taurine (0.5mM) activated glycine receptors, whereas a higher concentration (3mM) activated both glycine and gamma-aminobutyric acid A (GABAA) receptors in PHA neurons. These results suggest that PHA neurons are influenced by taurine and respond via glycine and GABAA receptors.

Enhanced GABA action on the substantia gelatinosa neurons of the medullary dorsal horn in the offspring of streptozotocin-injected mice.

Peripheral neuropathy is a frequent complication of diabetes mellitus and a common symptom of neuropathic pain, the mechanism of which is complex and involves both peripheral and central components of the sensory system. The lamina II of the medullary dorsal horn, called the substantia gelatinosa (SG), is well known to be a critical site for processing of orofacial nociceptive information. Although there have been a number of studies done on diabetic neuropathy related to the orofacial region, the action of neurotransmitter receptors on SG neurons in the diabetic state is not yet fully understood. Therefore, we used the whole-cell patch clamp technique to investigate this alteration on SG neurons in both streptozotocin (STZ)-induced diabetic mice and offspring from diabetic female mice. STZ (200 mg/kg)-injected mice showed a small decrease in body weight and a significant increase in blood glucose level when compared with their respective control group. However, application of different concentrations of glycine, gamma-aminobutyric acid (GABA) and glutamate on SG neurons from STZ-injected mice did not induce any significant differences in inward currents when compared to their control counterparts. On the other hand, the offspring of diabetic female mice (induced by multiple injections of STZ (40 mg/kg) for 5 consecutive days) led to a significant decrease in both body weight and blood glucose level compared to the control offspring. Glycine and glutamate responses in the SG neurons of the offspring from diabetic female mice were similar to those of control offspring. However, the GABA response in SG neurons of offspring from diabetic female mice was greater than that of control offspring. Furthermore, the GABA-mediated responses in offspring from diabetic and control mice were examined at different concentrations ranging from 3 to 1,000 µM. At each concentration, the GABA-induced mean inward currents in the SG neurons of offspring from diabetic female mice were larger than those of control mice. These results demonstrate that SG neurons in offspring from diabetic mice are more sensitive to GABA compared to control mice, suggesting that GABA sensitivity may alter orofacial pain processing in offspring from diabetic female mice.

Phasic and tonic type A γ-Aminobutryic acid receptor mediated effect of Withania somnifera on mice hippocampal CA1 pyramidal Neurons.

BACKGROUND: In Nepali and Indian system of traditional medicine, Withania somnifera (WS) is considered as a rejuvenative medicine to maintain physical and mental health and has also been shown to improve memory consolidation. OBJECTIVE: In this study, a methanolic extract of WS (mWS) was applied on mice hippocampal CA1 neurons to identify the receptors activated by the WS. MATERIALS AND METHODS: The whole cell patch clamp recordings were performed on CA1 pyramidal neurons from immature mice (7-20 postnatal days). The cells were voltage clamped at -60 mV. Extract of WS root were applied to identify the effect of mWS. RESULTS: The application of mWS (400 ng/µl) induced remarkable inward currents (-158.1 ± 28.08 pA, n = 26) on the CA1 pyramidal neurons. These inward currents were not only reproducible but also concentration dependent. mWS-induced inward currents remained persistent in the presence of amino acid receptor blocking cocktail (AARBC) containing blockers for the ionotropic glutamate receptors, glycine receptors and voltage-gated Na(+) channel ( CONTROL: -200.3 ± 55.42 pA, AARBC: -151.5 ± 40.58 pA, P > 0.05) suggesting that most of the responses by mWS are postsynaptic events. Interestingly, these inward currents were almost completely blocked by broad GABAA receptor antagonist, bicuculline- 20 µM (BIC) (BIC: -1.46 ± 1.4 pA, P < 0.001), but only partially by synaptic GABAA receptor blocker gabazine (1 µM) (GBZ: -18.26 ± 4.70 pA, P < 0.01). CONCLUSION: These results suggest that WS acts on synaptic/extrasynaptic GABAA receptors and may play an important role in the process of memory and neuroprotection via activation of synaptic and extrasynaptic GABAA receptors.

Potentiation of NMDA receptors by Withania somnifera on hippocampal CA1 pyramidal neurons.

In Ayurveda,Withania somnifera (WS) is used as a medicine to maintain mental and physical health as well as to enhance memory. In this study, the methanolic extract of WS(mWS) was tested for its electrical influence on hippocampal CA1 pyramidal neurons using a patch clamp technique. In current clamp mode under a high chloride pipette solution, mWS (400 ng/µl) induced remarkable membrane depolarization (9.75 ± 2.54 mV, n = 6) of CA1 neurons. The mWS-induced depolarization was dose-dependent, reproducible, and persistent in the presence of 0.5 µM tetrodotoxin (TTX, 10.17 ± 0.04 mV, n = 6). In voltage clamp mode (holding potential = -60 mV), mWS induced a dose-dependent non-desensitizing inward current that persisted in the presence of TTX (0.5 µM), suggesting that the response induced by mWS was purely a postsynaptic event. Interestingly, these inward currents were partially blocked by strychnine, a glycine receptor blocker. Further, mWS potentiated the NMDA response in hippocampal CA1 neurons at low concentrations. Overall, these results suggest that there are compounds in WS with possible glycine mimetic activities, which may be potential targets for inducing memory consolidation in hippocampal CA1 neurons.

Nitric oxide suppresses L-type calcium currents in basilar artery smooth muscle cells in rabbits.

OBJECTIVES: Nitric oxide (NO) is well known to be a vasodilator, and NO donor compounds are currently used for treating vasospasm following subarachnoid hemorrhage. However, the action mechanism of cerebral vascular relaxation is not yet clear. L-type calcium channels have been determined to play an essential role in smooth muscle contraction. To investigate the role of L-type calcium channels in NO-induced relaxation of basilar smooth muscle cells, we examined the effect of the NO donor, sodium nitroprusside (SNP) on calcium (Ca2+) currents using smooth muscle cells isolated from a rabbit basilar artery. METHOD: The smooth muscle cells were isolated from rabbit basilar artery by enzyme treatment. To identify L-type Ca2+ currents, we used cesium chloride, a potassium channel blocker and Bay K8644, an activator of L-type Ca2+ channel. RESULTS: The L-type calcium currents (91±13.0 pA; n = 11) were significantly reduced by SNP (32±5 pA; n = 11; P<0.05). 1H-[1,2,4] Oxadiazolo [4,3-a] quinoxalin-1-one, a 3',5'-cyclic guanosine monophosphate inhibitor, blocked the effect of SNP on L-type Ca2+ currents, and similar results were obtained after the application of 7-nitroindazole, a specific NO synthase inhibitor. Furthermore, inward currents were enhanced by Bay K8644 (170±22 pA; n = 5) and were suppressed by SNP (54±13 pA; n = 5; P<0.05). DISCUSSION: These results demonstrate that NO suppresses the L-type Ca2+ currents in rabbit basilar smooth muscle cells, and suggest that L-type Ca2+ channels may play a pivotal role in NO-induced vascular relaxation.

Activation of glycine and extrasynaptic GABA(A) receptors by taurine on the substantia gelatinosa neurons of the trigeminal subnucleus caudalis.

The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) has been known for the processing and transmission of orofacial nociceptive information. Taurine, one of the most plentiful free amino-acids in humans, has proved to be involved in pain modulation. In this study, using whole-cell patch clamp technique, we investigated the direct membrane effects of taurine and the action mechanism behind taurine-mediated responses on the SG neurons of the Vc. Taurine showed non-desensitizing and repeatable membrane depolarizations and inward currents which remained in the presence of amino-acid receptors blocking cocktail (AARBC) with tetrodotoxin, indicating that taurine acts directly on the postsynaptic SG neurons. Further, application of taurine at different doses (10 µM to 3 mM) showed a concentration dependent depolarizations and inward currents with the EC50 of 84.3 µM and 723 µM, respectively. Taurine-mediated responses were partially blocked by picrotoxin (50 µM) and almost completely blocked by strychnine (2 µM), suggesting that taurine-mediated responses are via glycine receptor (GlyR) activation. In addition, taurine (1 mM) activated extrasynaptic GABA(A) receptor (GABA(A)R)-mediated currents. Taken together, our results indicate that taurine can be a target molecule for orofacial pain modulation through the activation of GlyRs and/or extrasynaptic GABA(A)Rs on the SG neurons.

Magnesium sulfate suppresses L-type calcium currents on the basilar artery smooth muscle cells in rabbits.

OBJECTIVES: Although magnesium is a well-known treatment for vasospasm following subarachnoid hemorrhage, its mechanism of action for cerebral vascular relaxation is not clear. In addition, it is known that L-type calcium (Ca(2+)) channels play a pivotal role in smooth muscle contraction. To investigate the role of L-type Ca(2+) channels in the magnesium-induced relaxation of basilar smooth muscle cells, we examined the effect of magnesium sulfate on L-type Ca(2+) currents using freshly isolated smooth muscle cells from rabbit basilar arteries. METHOD: Rabbits were anesthetized with ketamine (50 mg/kg) with xylazine (25 mg/kg) and exsanguinated. Smooth muscle cells were isolated from rabbit basilar arteries by enzyme treatment. Cells were stored at 4°C before use. Whole-cell patch clamp technique was used to identify L-type Ca(2+) currents, using the potassium channel blocker, cesium chloride, and nimodipine and Bay K8644 as a blocker and activator of L-type Ca(2+) channels, respectively. RESULTS: Inward currents induced by step pulses were significantly reduced by nimodipine (n = 5, P<0·05) and increased by Bay K8644 (n = 5, P<0·05). The L-type Ca(2+) currents (122±14·0 pA, n = 12) were significantly reduced by the application of 5 mM magnesium sulfate (28±4 pA, n = 12, P<0·05). The inward currents enhanced by Bay K8644 were further suppressed by the application of magnesium sulfate. DISCUSSION: These results demonstrate that L-type Ca(2+) channels are functionally expressed in rabbit basilar smooth muscle cells and suggest that L-type Ca(2+) channels may play a pivotal role for magnesium-induced relaxation.

GABAA Receptor- and Non-NMDA Glutamate Receptor-Mediated Actions of Korean Red Ginseng Extract on the Gonadotropin Releasing Hormone Neurons.

Korean red ginseng (KRG) has been used worldwide as a traditional medicine for the treatment of various reproductive diseases. Gonadotropin releasing hormone (GnRH) neurons are the fundamental regulators of pulsatile release of gonadotropin required for fertility. In this study, an extract of KRG (KRGE) was applied to GnRH neurons to identify the receptors activated by KRGE. The brain slice patch clamp technique in whole cell and perforated patch was used to clarify the effect of KRGE on the membrane currents and membrane potentials of GnRH neurons. Application of KRGE (3 µg/µL) under whole cell patch induced remarkable inward currents (56.17±7.45 pA, n=25) and depolarization (12.91±3.80 mV, n=4) in GnRH neurons under high Cl(-) pipette solution condition. These inward currents were not only reproducible, but also concentration dependent. In addition, inward currents and depolarization induced by KRGE persisted in the presence of the voltage gated Na(+) channel blocker tetrodotoxin (TTX), suggesting that the responses by KRGE were postsynaptic events. Application of KRGE under the gramicidin perforated patch induced depolarization in the presence of TTX suggesting its physiological significance on GnRH response. Further, the KRGE-induced inward currents were partially blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; non-NMDA glutamate receptor antagonist, 10 µM) or picrotoxin (PIC; GABAA receptor antagonist, 50 µM), and almost blocked by PIC and CNQX mixture. Taken together, these results suggest that KRGE contains ingredients with possible GABA and non-NMDA glutamate receptor mimetic activity, and may play an important role in the endocrine function of reproductive physiology, via activation of GABAA and non-NMDA glutamate receptors in GnRH neurons.

Magnesium increases iberiotoxin-sensitive large conductance calcium activated potassium currents on the basilar artery smooth muscle cells in rabbits.

OBJECTIVES: Magnesium has been known for treating vasospasm following subarachnoid hemorrhage. However, its action mechanism in cerebral vascular relaxation is not clear. Potassium channels play a pivotal role in the relaxation of smooth muscle cells. To investigate their role in magnesium-induced relaxation of basilar smooth muscle cells, we examined the effect of magnesium on potassium channels using the patch clamp technique on acutely isolated smooth muscle cells from rabbit basilar artery. METHOD: Fresh smooth muscle cells were isolated from the basilar artery by enzyme treatment. To identify which potassium channels are involved in the magnesium-induced currents, we used the potassium channel blockers tetraethylammonium (TEA), glibenclamide, apamin and iberiotoxin (IBX). RESULTS: Magnesium (5 mM) increased the step pulse-induced outward K+ currents by 46% over control level (P < 0.01). The outward K+ current was decreased to 22% (P < 0.01) by TEA (10 mM), a non-specific K+ channel blocker, and to 60% of control level (P < 0.01) by IBX (0.1 µM,), a large-conductance Ca2+-activated K+ (BK) channel blocker, but was not inhibited by apamin (1 nM), a small-conductance Ca2+ -activated potassium (SK) channel blocker, or glibenclamide (3 mM), an adenosine triphosphate (ATP)-sensitive K+) channel blocker. Caffeine (3 mM) enhanced outward K+ currents. Magnesium-induced increase of outward K+ currents persisted in the presence of apamin. However, magnesium failed to increase the outward K+ currents in the presence of IBX. DISCUSSION: These results demonstrate that BK channels are functionally expressed in rabbit basilar smooth muscle cells and suggest that BK channels may play a pivotal role in magnesium-induced relaxation.