Characterization of Intermediate Conductance K+ Channels in Submandibular Gland Acinar Cells

There are some evidences that K+ efflux evoked by muscarinic stimulation is not mainly mediated by large conductance K+ (BK) channels in salivary gland. In this experiment, we therefore characterised non BK channels in rat submandibular gland acinar cells and examined the possibility of agonist effect on this channel using a patch clamp technique. Two types of K+ channels were observed in these cells. BK channels were observed in 3 cells from total 6 cells and its average conductance was 152±7 pS (n=3). The conductance of the another types of K+ channel was estimated as 71±7 pS (n=6). On the basis of the conductance of this channel, we defined this channel as intermediate conductance K+ (IK) channels, which were observed from all 6 cells we studied. When we increased Ca2+ concentration of the bath solution in inside-out mode, the IK channel activity was greatly increased, suggesting this channel is Ca2+ sensitive. We next examined the effect of carbachol (CCh) and isoproterenol on the activity of the IK channels. 10-5 M isoproterenol significantly increased the open probability (Po) from 0.08±0.02 to 0.21±0.03 (n=4, P＜0.05). Application of 10-5 M CCh also increased Po from 0.048±0.03 to 0.55±0.33 (n=5, P＜0.05) at the maximum channel activity. The degree of BK channel activation induced by the same concentration of CCh was lower than that of IK channels; Po value was 0.011±0.003 and 0.027±0.005 in control and during CCh stimulation (n=3), respectively. The result suggests that IK channels exist in salivary acinar cells and its channel activity is regulated by muscaricinic and β- adrenergic agonist. We conclude that IK channels also play a putative role in secretion as well as the BK channels in rat submandibular gland acinar cells.This work was supported by a grant of the Korean Health 21 R&D project, Ministry of Health & Welfare, Republic of Korea (Grant No 00-PJ1-PG1-CH10-0002)

GABAergic amd serotonergic modulation of calcium currents in rat trigeminal motoneurons

We investigated the effects of a GABAB agonist baclofen, and serotonin, on the high voltage-activated Ca channel (HVACC) currents in trigeminal motoneurons. Immunohistochemical and reverse transcription-polymerization chain reaction (RT-PCR) studies demonstrated the expression of α1C, α1B, α1A, and α1E subunits in the trigeminal motoneurons, which form L-, N-, P/Q-, and R-type Ca channels, respectively. By use of specific Ca blockers, it was found that N-type (38%), P/Q-type (27%), L-type (16 %), and R-type Ca currents (19%) contribute to HVACC IBa. Baclofen inhibited HVACC IBa in the majority of trigeminal motoneurons tested (n=15 outof 16), whereas serotonin only did in a small population (n=5 outof 18). The IBa inhibition by baclofen and serotonin was associated with slowing of activation kinetics, relieved by strong prepulse, and prevented by N-ethylmaleimide (NEM), indicative of mediation of Gi/Go. These data provide evidence that GABAergic and serotonergic inputs to trigeminal motoneurons regulate neuronal activities through the inhibition of HVACC currents.This work waas supported by Grant-in-Aid for scientific research from the Ministry of Health and Welfare, Republic of Korea, 00-PJ1-PG1-CH11-0004

Mechanosensitivity of voltage-gated K+ currents in rat trigeminal ganglion neurons.

We investigated the mechanosensitivity of voltage-gated K+ channel (VGPC) currents by using whole-cell patch clamp recording in rat trigeminal ganglion (TG) neurons. On the basis of biophysical and pharmacological properties, two types of VGPC currents were isolated. One was transient (IK,A), the other sustained (IK,V). Hypotonic stimulation (200 mOsm) markedly increased both IK,A and IK,V without affecting their activation and inactivation kinetics. Gadolinium, a well-known blocker of mechanosensitive channels, failed to block the enhancement of IK,A and IK,V induced by hypotonic stimulation. During hypotonic stimulation, cytochalasin D, an actin-based cytoskeletal disruptor, further increased IK,A and IK,V, whereas phalloidin, an actin-based cytoskeletal stabilizer, reduced IK,A and IK,V. Confocal imaging with Texas red-phalloidin showed that actin-based cytoskeleton was disrupted by hypotonic stimulation, which was similar to the effect of cytochalasin D. Our results suggest that both IK,A and IK,V are mechanosensitive and that actin-based cytoskeleton is likely to regulate the mechanosensitivity of VGPC currents in TG neurons. © 2006 Wiley-Liss, Inc

Ca2+-activated K+ currents of pancreatic duct cells in guinea-pig

There are numerous studies on transepithelial transports in duct cells including Cl- and/or HCO3-. However, studies on transepithelial K+ transport of normal duct cells in exocrine glands are scarce. In the present study, we examined the characteristics of K+ currents in single duct cells isolated from guinea pig pancreas, using a whole-cell patch clamp technique. Both Cl- and K+ conductance were found with KCl rich pipette solutions. When the bath solution was changed to low Cl-, reversal potentials shifted to the negative side, -75±4 mV, suggesting that this current is dominantly selective to K+. We then characterized this outward rectifying K+ current and examined its Ca22+ dependency. The K+ currents were activated by intracellular Ca22+. 100 nM or 500 nM Ca22+ in pipette significantly (P<0.05) increased outward currents (currents were normalized, 76.8?7.9 pA, n=4 or 107.9?35.5 pA, n=6) at +100 mV membrane potential, compared to those with 0 nM Ca2+ in pipette (27.8?3.7 pA, n=6). We next examined whether this K+ current, recorded with 100 nM Ca2+ in pipette, was inhibited by various inhibitors, including Ba2+, TEA and iberiotoxin. The currents were inhibited by 40.4±% (n=3), 87.0±% (n=5) and 82.5±% (n=9) by 1 mM Ba2+, 5 mM TEA and 100 nM iberiotoxin, respectively. Particularly, an almost complete inhibition of the current by 100 nM iberiotoxin further confirmed that this current was activated by intracellular Ca2+. The K+ current may play a role in secretory process, since recycling of K+ is critical for the initiation and sustaining of Cl- or HCO32+ secretion in these cells.This work was supported by a grant of the Korea Health 21 R&D project, Ministry of Health & Welfare, Republic of Korea (Grant No 01-PJ5-PG1-01CH12-0002)

Activation of glia and microglial p38 MAPK in medullary dorsal horn contributes to tactile hypersensitivity following trigeminal sensory nerve injury

Glial activation is known to contribute to pain hypersensitivity following spinal sensory nerve injury. In this study, we investigated mechanisms by which glial cell activation in medullary dorsal horn (MDH) would contribute to tactile hypersensitivity following inferior alveolar nerve and mental nerve transection (IAMNT). Activation of microglia and astrocytes was monitored at 2 h, 1, 3, 7, 14, 28, and 60 days using immunohistochemical analysis with OX-42 and GFAP antibodies, respectively. Tactile hypersensitivity was significantly increased at 1 day, and this lasted for 28 days after IAMNT. Microglial activation, primarily observed in the superficial laminae of MDH, was initiated at 1 day, maximal at 3 days, and maintained until 14 days after IAMNT. Astrocytic activation was delayed compared to that of microglia, being more profound at 7 and 14 days than at 3 days after IAMNT. Both tactile hypersensitivity and glial activation appeared to gradually reduce and then return to the basal level by 60 days after IAMNT. There was no significant loss of trigeminal ganglion neurons by 28 days following IAMNT, suggesting that degenerative changes in central terminals of primary afferents might not contribute to glial activation. Minocycline, an inhibitor of microglial activation, reduced microglial activation, inhibited p38 mitogen-activated protein kinase (MAPK) activation in microglia, and significantly attenuated the development of pain hypersensitivity in this model. These results suggest that glial activation in MDH plays an important role in the development of neuropathic pain and activation of p38 MAPK in hyperactive microglia contributes to pain hypersensitivity in IAMNT model.This research was supported by grant (M103KV010009-04K2201-00930) from Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology and grant (RO1-2004-000-103 84-0) from the Basic Research Program of the Korea Science & Engineering Foundation, Republic of Korea

Systemic administration of minocycline inhibits formalin-induced inflammatory pain in rat

It has been demonstrated that spinal microglial activation is involved in formalin-induced pain and that minocycline, an inhibitor of microglial activation, attenuate behavioral hypersensitivity in neuropathic pain models. We investigated whether minocycline could have any anti-nociceptive effect on inflammatory pain, after intraperitonial administration of minocycline, 1 h before formalin (5%, 50 μl) injection into the plantar surface of rat hindpaw. Minocycline (15, 30, and 45 mg/kg) significantly decreased formalin-induced nociceptive behavior during phase II, but not during phase I. The enhancement in the number of c-Fos-positive cells in the L4–5 spinal dorsal horn (DH) and the magnitude of paw edema induced by formalin injection during phase II were significantly reduced by minocycline. Minocycline inhibited synaptic currents of substantia gelatinosa (SG) neurons in the spinal DH, whereas membrane electrical properties of dorsal root ganglion neurons were not affected by minocycline. Analysis with OX-42 antibody revealed the inhibitory effect of minocycline on microglial activation 3 days after formalin injection. These results demonstrate the anti-nociceptive effect of minocycline on formalin-induced inflammatory pain. In addition to the well-known inhibitory action of minocycline on microglial activation, the anti-edematous action in peripheral tissue, as well as the inhibition of synaptic transmission in SG neurons, is likely to be associated with the anti-nociceptive effect of minocycline.This research was supported by grant (M103KV010009- 04K2201-00930) from Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, Republic of Korea