Nystatin-perforated Patch-clamp Technique Research Articles

Inhibition of suplatast tosilate on EPSC in the single paratracheal ganglion neurons attached with presynaptic boutons.

Using single neurons of rat paratracheal ganglia (PTG) attached with presynaptic boutons, the effects of suplatast tosilate on excitatory postsynaptic currents (EPSCs) were investigated with nystatin-perforated patch-clamp recording technique. We found that suplatast concentration dependently inhibited the EPSC amplitude and its frequency in single PTG neurons attached with presynaptic boutons. EPSC frequency was higher sensitive to suplatast than EPSC amplitude. IC50 for EPSC frequency was 1.1 × 10-5 M, being similar to that for the effect on histamine release from mast cells and lower than that for the inhibitory effect on cytokine production. Suplatast also inhibited the EPSCs potentiated by bradykinin (BK), but it did not affect the potentiation itself by BK. Thus suplatast inhibited the EPSC of PTG neurons attached with presynaptic boutons at both the presynaptic and postsynaptic sites.NEW & NOTEWORTHY In this study, using single neurons of rat paratracheal ganglia (PTG) attached with presynaptic boutons, the effects of suplatast tosilate on excitatory postsynaptic currents (EPSCs) were investigated with patch-clamp recording technique. We found that suplatast concentration dependently inhibited the EPSC amplitude and its frequency in single PTG neurons attached with presynaptic boutons. Thus suplatast inhibited the function of PTG neurons at both of presynaptic and postsynaptic sites.

Abstract 18503: Geranylgeranylacetone (gga) And Gga-derivatives Inhibit Acetylcholine-activated K+ Current In Mouse Atrial Cardiomyocyte ; Possible Mechanism For Af Suppression

Atrial fibrillation (AF) is the most common sustained arrhythmia. AF is caused by inflammation, oxidative stress, and various structural heart diseases. An important key factor in AF promotion is that atrial cardiomyocytes have constitutively active acetylcholine-activated K+ (IKACh) current that is enhanced by the tachycardia. Furthermore, it is known that heat shock proteins (HSPs) are involved in the protection against different forms of cellular stress and recent investigators reported that the HSP inducing compound geranylgeranylacetone (GGA, also known as teprenone) prevents atrial remodeling and attenuates the promotion of AF. Whether GGA and GGA-derivatives prevent the acetylcholine-activated K+ current is unknown. Methods: We examined the effect of HSP inducer GGA and 5 derivatives on IKACh current in mouse atrial cardiomyocytes. In the present study, the IKACh current was recorded using a nystatin-perforated whole cell patch-clamp technique following activation by acetylcholine (10 μM for 2 min). After the measurement of the baseline IKACh current, atrial cardiomyocytes were treated with GGA 10μM or a derivative of GGA (NYK9274, NYK9308, NYK9354, NYK9356 or NYK9376) for 10min and the IKACh current was re-measured. Results: IKACh current in mouse atrial cardiomyocyte treated with acetylcholine (10 μM) was significantly increased, which was markedly attenuated by GGA 10μM treatment (n=5, peak; 54±11.2%, quasi-steady-state; 58±11.4%, p<0.001). In addition, the effects of the GGA derivatives on IKACh were evaluated with same experimental protocol. Our results showed that next to GGA, also NYK9308 and NYK9356 significantly inhibit both peak and quasi-steady-state IKACh. In contrast, NYK9274, NYK9354 and NYK9376 had no effect on IKACh. Conclusion: This study provides important evidences that the HSP inducer GGA facilitates its potent antiarrhythmic effect by inhibiting IKAch, which underlies electrical remodeling in mammalian atrial cardiomyocytes. Furthermore, two GGA derivatives, NYK9308 and NYK9356, with improved pharma-chemical properties compared to GGA, reveal similar protective effects compared to GGA, and provide important background information for the use of GGA and GGA-like compounds in patients with AF.

Attenuation of acetylcholine activated potassium current (I KACh) by simvastatin, not pravastatin in mouse atrial cardiomyocyte: possible atrial fibrillation preventing effects of statin.

Statins, 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors, are associated with the prevention of atrial fibrillation (AF) by pleiotropic effects. Recent clinical trial studies have demonstrated conflicting results on anti-arrhythmia between lipophilic and hydrophilic statins. However, the underlying mechanisms responsible for anti-arrhythmogenic effects of statins are largely unexplored. In this study, we evaluated the different roles of lipophilic and hydrophilic statins (simvastatin and pravastatin, respectively) in acetylcholine (100 µM)-activated K+ current (IKACh, recorded by nystatin-perforated whole cell patch clamp technique) which are important for AF initiation and maintenance in mouse atrial cardiomyocytes. Our results showed that simvastatin (1–10 µM) inhibited both peak and quasi-steady-state IKACh in a dose-dependent manner. In contrast, pravastatin (10 µM) had no effect on IKACh. Supplementation of substrates for the synthesis of cholesterol (mevalonate, geranylgeranyl pyrophosphate or farnesyl pyrophosphate) did not reverse the effect of simvastatin on IKACh, suggesting a cholesterol-independent effect on IKACh. Furthermore, supplementation of phosphatidylinositol 4,5-bisphosphate, extracellular perfusion of phospholipase C inhibitor or a protein kinase C (PKC) inhibitor had no effect on the inhibitory activity of simvastatin on I KACh. Simvastatin also inhibits adenosine activated IKACh, however, simvastatin does not inhibit IKACh after activated by intracellular loading of GTP gamma S. Importantly, shortening of the action potential duration by acetylcholine was restored by simvastatin but not by pravastatin. Together, these findings demonstrate that lipophilic statins but not hydrophilic statins attenuate IKACh in atrial cardiomyocytes via a mechanism that is independent of cholesterol synthesis or PKC pathway, but may be via the blockade of acetylcholine binding site. Our results may provide important background information for the use of statins in patients with AF.

Growth Hormone Secretagogues Reduce Transient Outward K+ Current via Phospholipase C/Protein Kinase C Signaling Pathway in Rat Ventricular Myocytes

Endogenous ghrelin and its synthetic counterpart hexarelin are peptide GH secretagogues (GHS) that exert a positive ionotropic effect in the cardiovascular system. The mechanism by which GHS modulate cardiac electrophysiology properties to alter myocyte contraction is poorly understood. In the present study, we examined whether GHS regulates the transient outward potassium current (I(to)) as well as the putative intracellular signaling cascade responsible for such regulation. GHS and experimental agents were applied locally onto freshly isolated adult Sprague-Dawley rat ventricular myocytes and action potential morphology and I(to) was recorded using nystatin-perforated whole-cell patch-clamp recording technique. Under current clamp, ghrelin and hexarelin (10 nm) significantly prolonged action potential duration. Under voltage clamp, hexarelin and ghrelin inhibited I(to) in a concentration-dependent manner. This inhibition was abolished in the presence of the GHS receptor (GHS-R) antagonist [D-Lys(3)]GH-releasing peptide-6 (10 microm) and GHS-R1a-specific antagonist BIM28163 (1 microm). GHS-induced I(to) inhibition was totally reversed by the phospholipase C inhibitor U73122 (5 microm) and protein kinase C inhibitors GO6983 (1 microm) and calphostin C (0.1 microm) but not by the cAMP antagonist Rp-cAMP (100 microm) or the PKA inhibitor H89 (1 microm). We conclude that hexarelin and ghrelin activate phospholipase C and protein kinase C signaling cascade through the stimulation of the GHS-R, resulting in a decrease in the I(to) current and subsequent prolongation of action potential duration.

Acetylsalicylic acid enhances purinergic receptor-mediated outward currents in rat megakaryocytes

Purinergic receptor activation increases cytosolic Ca(2+) concentration in a fluctuating fashion, triggering oscillatory outward Ca(2+)-activated K(+) currents in rat megakaryocytes (MKs). Whole cell and nystatin-perforated patch-clamp techniques were used to analyze changes in ionic conductance in MK with acetylsalicylic acid (ASA), a cyclooxygenase-1 inhibitor and antithrombotic agent. MKs are a model for platelet reactivity, particularly in ASA treatment failure (ASA resistance). Freshly isolated MKs were incubated 30 min in the absence or presence of 1 mM ASA. Using a K(+)-rich internal solution, we recorded outward currents in response to 10 microM ATP, 10 microM ADP, and 5 microM 2-methyl-thio-ADP (2MeSADP) in the voltage-clamp mode. Agonist-induced currents decreased in amplitude over time, but this decline was attenuated by ASA in both continuous and repeated agonist challenge, indicating increased MK reactivity with ASA treatment. In separate experiments, heterologous desensitization was observed when MKs were stimulated with ADP after exposure to a thromboxane receptor agonist (U46619), indicating cross talk between thromboxane and purinergic pathways. Different cells, treated with ASA or MRS2179 (P2Y1 receptor antagonist), were stimulated with 2MeSADP. The dose-response curve was shifted to the left in both cases, suggesting increased MK reactivity. ASA also caused an increased interval between currents (delay). ASA attenuated desensitization of purinergic receptors and increased delay, again suggesting cross talk between purinergic and thromboxane pathways. These findings may be relevant to ASA resistance, because individual variations in sensitivity to the multiple effects of ASA on signaling pathways could result in insensitivity to its antiplatelet effects in some patients.

Modulation of the aqueous extract of Bupleuri radix on glycine-induced current in the acutely dissociated rat periaqueductal gray neurons

SUMMARY Bupleuri radix (Umbelliferae), the dried root of Bupleurum Chinense DC, has been clinically used tomitigate pain sensation. The descending pain control system consists of three major components,and modulation of pain in the periaqueductal gray is the most extensively studied descendingpain control system. However, the relation of Bupleuri radix on the descending pain controlsystem has not been clarified. In the present study, modulation of the aqueous extract of Bupleuriradix on glycine-induced ion current in the acutely dissociated periaqueductal gray neurons wasinvestigated by using nystatin-perforated patch-clamp technique under voltage-clamp condition.In the present results, the glycine-induced ion current was significantly suppressed by 0.1 mg/mlBupleuri radix, while treatment with 10 -5 M naltrexone, opioid receptor antagonist, alleviatedBupleuri radix-induced inhibition on glycine-induced ion current. The present study showed thatthe aqueous extract of Bupleuri radix may activate descending pain control system throughinhibition on glycine-induced ion current in the periaqueductal gray neurons and this effect ismediated by opioid receptors.Key words: Bupleuri radix; Glycine; Periaqueductal gray; Naltrexone; Patch Clamp

A selective T-type Ca2+ channel blocker R(−) efonidipine

Recently, novel compound R(-) efonidipine was reported to selectively block low-voltage-activated (LVA or T-type) Ca2+ channels in peripheral organs. We examined how R(-) efonidipine acts on T-type and high-voltage-activated (HVA) Ca2+ channels in mammalian central nervous system (CNS) neurons. Furthermore, we compared the effects of R(-) efonidipine with those of flunarizine and mibefradil on both T-type and HVA Ca2+ channels in rat hippocampal CA1 neurons by using the nystatin perforated-patch clamp technique. Flunarizine and mibefradil nonselectively inhibited both T-type and HVA Ca2+ channels, though the dose-dependent blocking potency of flunarizine on T-type Ca2+ channels was slightly stronger than that of mibefradil. In contrast, R(-) efonidipine inhibited only T-type Ca2+ channels and did not show any effect on HVA Ca2+ channels. The inhibitory actions of R(-) efonidipine or flunarizine were similar on both Ba2+ and Ca2+ current components passing through T-type Ca2+ channels. In addition, flunarizine but not R(-) efonidipine inhibited voltage-dependent Na+ channels and Ca2+-activated K+ channels. Thus, it appears that R(-) efonidipine is a selective blocker for T-type Ca2+ channels. It could be used as a pharmacological tool in future studies on T-type Ca2+ channels.

Effects of fluid flow on voltage-dependent calcium channels in rat vascular myocytes: Fluid flow as a shear stress and a source of artifacts during patch-clamp studies

We examined the effects of fluid flow on L-type voltage-dependent Ca 2+ channel (VDCC L) currents in rat vascular myocytes using the nystatin perforated patch-clamp technique. The effect of fluid flow on the liquid (bathing solution)–metal (Ag/AgCl ground electrode) junction potential was also studied. With a fluid flow of 0–10 ml/min, changes in the junction potential of up to 5 mV were observed in proportion to the flow rate. Accordingly, fluid flow shifted the current–voltage ( I– V) relationship of the recorded VDCC L currents in a positive direction. In addition to these shifts, fluid flow also increased the peak VDCC L current, suggesting some modulatory role for fluid flow in VDCC L currents. The use of a 3-M KCl agar-bridge between the ground electrode and bathing solution abnegated the potential shifts, and fluid flow increased the VDCC L currents in a voltage-independent manner. These results suggest that the bathing fluid flow can be both a source of erroneous voltage shift between liquid and metal junctions in the patch-clamp configuration and an important shear stress for the cells. The facilitation of VDCC L currents by fluid flow in vascular myocytes may contribute to the myogenic contraction of blood vessels. The mechanism by which fluid flow causes the voltage shift is vigorously discussed.

Enhancement of receptor-operated cation current and TRPC6 expression in arterial smooth muscle cells of deoxycorticosterone acetate-salt hypertensive rats

In deoxycorticosterone acetate (DOCA)-salt hypertensive rats, altered reactivity of blood vessels to vasoactive agonists is frequently associated with an elevation in blood pressure. Canonical transient receptor potential (TRPC) channels are believed to encode receptor-operated cation channels (ROC), the activation of which is involved in smooth muscle depolarization and vasoconstriction. The aims of the present study were to investigate whether the ROC current is increased in DOCA-hypertensive rats and determine whether aldosterone directly enhances the expression of TRPC. The nystatin-perforated patch-clamp technique was used for the recording of receptor-stimulated ion currents in mesenteric arterial smooth muscle cells, which were enzymatically dispersed from sham-operated and DOCA-salt hypertensive rats. Expressions of TRPCs were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) and by Western blot analysis. Receptor-stimulated currents activated by 5-hydroxytryptamine (serotonin) and norepinephrine were increased significantly in the mesenteric arterial smooth muscle cells of DOCA-salt hypertensive rats compared to sham-operated rats. Ion-substitution experiments revealed that the enhanced currents were cation currents (ROC currents). Enhanced expression of TRPC6 in mesenteric arteries from DOCA-salt hypertensive rats was demonstrated by real-time RT-PCR. Up-regulation of TRPC6 by aldosterone treatment in vitro was also observed in A7r5 cells by RT-PCR and in western blots. These results suggest that aldosterone enhances TRPC6 expression and ROC currents in vascular smooth muscle cells, and that this may in turn contribute to altered vascular reactivity and to hypertension.

Potentiation of nicotinic currents by bradykinin in the paratracheal ganglia neurons of rats

The effects of bradykinin on nicotine-induced responses were investigated in neurons dissociated from rat paratracheal ganglia using the nystatin-perforated patch-clamp recording technique. When bradykinin (10 − 9 to 10 − 8 M) was pretreated and then simultaneously applied with 10 − 5 M nicotine, bradykinin potentiated the nicotine-induced currents. The potentiation was mimicked by [Hyp 3]-bradykinin and inhibited by HOE-140, pertussis toxin, neomycin and U-73122, but not U-73433. These results suggest that bradykinin potentiates nicotinic currents via bradykinin B 2 receptor, pertussis toxin-sensitive G-protein and phospholipase C. Since bradykinin inhibits the M-current via bradykinin B 2 receptor and pertussis toxin-insensitive G-protein [Mochidome, T., Ishibashi, H., Takahama, K., 2001. Bradykinin activates airway parasympathetic ganglion neurons by inhibiting M-currents. Neuroscience 105, 785–791.], it seemed that bradykinin B 2 receptor activated two distinct signal transduction pathways in the paratracheal ganglia neurons. This effect of bradykinin might cause enhanced synaptic transmission in paratracheal ganglia neurons and contribute to the aggravation of pathological conditions of the lower airway via enhanced acetylcholine release from the postganglionic nerve terminals.

Modulation of ATP-Induced Inward Currents by Docosahexaenoic Acid and Other Fatty Acids in Rat Nodose Ganglion Neurons

The effects of docosahexaenoic acid (DHA) and other fatty acids on P2X-receptor-mediated inward currents in rat nodose ganglion neurons were studied using the nystatin perforated patch-clamp technique. DHA accelerated the desensitization rate of the ATP-induced current. DHA showed use-dependent inhibition of the peak ATP-induced current. Other polyunsaturated fatty acids, such as arachidonic acid and eicosapentaenoic acid, displayed a similar use-dependent inhibition. The inhibitory effects of saturated fatty acids including palmitic acid and arachidic acid were weaker than those of polyunsaturated fatty acids. The results suggest that fatty acids may modulate the P2X receptor-mediated response when the channel is in the open-state.

ClC-2 channels get new partners. Focus on “Association between Hsp90 and the ClC-2 chloride channel upregulates channel function”

clc-2 is a chloride channel that belongs to the ClC family of anion channels and transporters. ClC-2 is activated by hyperpolarization, leading to strict inward rectification, and is further activated by intracellular chloride and extracellular acidification. Work with recombinant ClC-2 in different

Endothelin-1 increases intracellular Ca2+in rabbit pulmonary artery smooth muscle cells through phospholipase C

In freshly isolated rabbit pulmonary artery smooth muscle cells, endothelin (ET)-1 induced a transient increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) followed by a return to the initial [Ca(2+)](i). This response was not abolished by the voltage-dependent Ca(2+) channel blocker nicardipine or removal of Ca(2+) from the bath solution but was inhibited by ryanodine and thapsigargin. This finding suggested that the increase in [Ca(2+)](i) induced by ET-1 was attributable to release of Ca(2+) from ryanodine- and inositol 1,4,5-trisphosphate-sensitive intracellular Ca(2+) stores. The transient increase in [Ca(2+)](i) induced by ET-1 was also inhibited by pretreatment with antagonists of ET type A and B (ET(A) and ET(B)) receptors (BQ-123 and BQ-788, respectively). Furthermore, the ET(B) receptor agonist IRL-1620 induced an increase in [Ca(2+)](i) that was followed by a sustained increase in [Ca(2+)](i); the sustained increase in [Ca(2+)](i) was blocked by nicardipine. Using the nystatin-perforated patch-clamp technique, we found that IRL-1620 caused an increase in Ca(2+) current that was inhibited by addition of ET-1. ET-1 did not inhibit Ca(2+) current when cells were pretreated with BQ-123. These results suggested that when both receptor types are activated, the opposing responses lead to abolition of the sustained [Ca(2+)](i) increases induced by ET(B) receptor activation. Western blot analysis confirmed expression of ET(A) and ET(B) receptors. Finally, U-73122 inhibited the ET-1-induced [Ca(2+)](i) increase, indicating that phospholipase C was involved in modulation of the ET-1-induced [Ca(2+)](i) increase in rabbit pulmonary artery smooth muscle cells.

Oxytocin Inhibits T-Type Calcium Current of Human Decidual Stromal Cells

Little is known about the crosstalk between the decidua and myometrium in relation to human labor. The hormone oxytocin (OT) is considered to be a key mediator of uterine contractility during parturition, exerting some of its effects through calcium channels. The objective was to characterize the effect of OT on the T-type calcium channel in human decidual stromal cells before and after the onset of labor. The nystatin-perforated patch-clamp technique was used to record inward T-type calcium current (I(Ca(T))) from acutely dispersed decidual stromal cells obtained from women at either elective cesarean section [CS (nonlabor)] or after normal spontaneous vaginal delivery [SVD (labor)]. These studies took place at the University of Nottingham Medical School. I(Ca(T)) of both SVD and CS cells were blocked by nickel (IC(50) of 5.6 microm) and cobalt chloride (1 mm) but unaffected by nifedipine (10 microm). OT (1 nm to 3.5 microm) inhibited I(Ca(T)) of SVD cells in a concentration-dependent manner, with a maximal inhibition of 79.0% compared with 26.2% in decidual cells of the CS group. OT-evoked reduction of I(Ca(T)) was prevented by preincubation with the OT antagonist L371,257 in the SVD but not CS group. OT, in a concentration-dependent manner, displaced the steady-state inactivation curve for I(Ca(T)) to the left in the SVD group with no significant effect on curves of the CS group. Inhibition of I(Ca(T)) by OT in decidual cells obtained during labor may signify important functional remodeling of uterine signaling during this period.

Electrophysiological characterization of the tetrodotoxin-resistant Na+ channel, Na(v)1.9, in mouse dorsal root ganglion neurons.

Small dorsal root ganglion neurons express preferentially the Na+ channel isoform Na(v)1.9 that mediates a tetrodotoxin-resistant (TTX-R) Na+ current. We investigated properties of the Na+ current mediated by Na(v)1.9 (I(NaN)) using the whole-cell, patch-clamp recording technique. To isolate I(NaN) from heterogeneous TTX-R Na+ currents that also contain another type of TTX-R Na+ current mediated by Na(v)1.8, we used Na(v)1.8-null mutant mice. When F- was used as an internal anion in the patch pipette solution, both the activation and inactivation kinetics for I(NaN) shifted in the hyperpolarizing direction with time. Such a time-dependent shift of the kinetics was not observed when Cl- was used as an internal anion. Functional expression of I(NaN) declined with time after cell dissociation and recovered during culture, implying that Na(v)1.9 may be regulated dynamically by trophic factors or depend on subtle environmental factors for its survival. During whole-cell recordings, the peak amplitude of I(NaN) increased dramatically after a variable delay, as if inactive or silent channels had been "kindled". Such an unusual increase of the amplitude could be prevented by adding ATP to the pipette solution or by recording with the nystatin-perforated patch-clamp technique, suggesting that the rupture of patch membrane affected the behaviour of Na(v)1.9. These peculiar properties of I(NaN) may provide an insight into the plasticity of Na+ channels that are related to pathological functions of Na+ channels accompanying abnormal pain states.

Modulation of Corydalis tuber on glycine-induced ion current in acutely dissociated rat periaqueductal gray neurons.

Traditionally, Corydalis tuber has been used for the control of pain including headache, stomach ache, and neuralgia. In the present study, modulation of the Corydalis tuber on glycine-activated ion current in the acutely dissociated periaqueductal gray (PAG) neurons was studied by a nystatin-perforated patch-clamp technique. High concentrations of Corydalis tuber elicited ion current, which was suppressed by strychnine application, while low concentrations of Corydalis tuber reduced glycine-induced ion current in the PAG neurons. Inhibitory action of Corydalis tuber on glycine-activated ion current was partially abolished by treatment with naltrexone, a non-selective opioid antagonist. Application of N-methylmalemide (NEM), a sulfhydryl alkylating agent, also partially abolished the inhibitory action of Corydalis tuber on glycine-activated ion current in the PAG neurons. These results suggest that the inhibitory effect of Corydalis tuber on glycine-activated ion current in the PAG neurons is one of the analgesic mechanisms of the Corydalis tuber.

The voltage-dependent nonselective cation current in human red blood cells studied by means of whole-cell and nystatin-perforated patch-clamp techniques

Human red blood cells (RBC) can be studied by means of whole-cell and nystatin-perforated patch-clamp techniques. In 85% of the whole-cell experiments (n=86) and 69% of the perforated-patch recordings (n=13), steps to positive potentials, from a holding potential of 0 mV, induced a slow-activating non-inactivating persistent outward current which reverted at about 0 mV. The current activation phase fitted well with a two-component exponential curve. Half-maximal conductance was reached at about 42 mV. Na+ and K+ carried this current, which was not affected by 20 nM charybdotoxin or 20 mM TEA, but was reduced following a partial substitution of extracellular Cl− by tartrate. This current has characteristics similar to the single-channel currents already described in RBC and may be involved in the rapid adaptations of these cells in the circulation.

Modulation of Chelidonii herba on glycine-activated and glutamate-activated ion currents in rat periaqueductal gray neurons

Background: Chelidonii herba is classified as Papaver somniferum L. Aqueous extract from C. herba is traditionally used for disorders with symptoms like pain, bloating, abdominal cramp after meals. Methods: Modulation of C. herba on glycine-activated and glutamate-activated ion currents in the acutely dissociated periaqueductal gray (PAG) neurons was investigated by the nystatin-perforated patch-clamp technique. Results: C. herba inhibited glycine-activated ion current and increased glutamate-activated ion current. C. herba-induced inhibition on glycine-activated ion current is implicated in opioid receptors and GTP-binding proteins (G-proteins). Increased glutamate-activated ion current induced by C. herba is linked neither by opioid receptors nor GTP-binding proteins. Conclusions: Suppressed glycine-induced response and elevated glutamate-induced response by C. herba may increase neuronal excitability in PAG, results in activation of descending pain control system, and this mechanism can be suggested as one of the analgesic actions of C. herba.

Ca2+ signaling mediated by IP3-dependent Ca2+ releasing and store-operated Ca2+ channels in rat odontoblasts.

In the phospholipase-C (PLC) signaling system, Ca2+ is mobilized from intracellular Ca2+ stores by an action of inositol 1,4,5-trisphosphate (IP3). The depletion of IP3-sensitive Ca2+ stores activates a store-operated Ca2+ entry (SOCE). However, no direct evidence has been obtained about these signaling pathways in odontoblasts. In this study, we investigate the characteristics of the SOCE and IP3-mediated Ca2+ mobilizations in rat odontoblasts using fura-2 microfluorometry and a nystatin-perforated patch-clamp technique. In the absence of extracellular Ca2+ ([Ca2+]o), thapsigargin (TG) evoked a transient rise in intracellular Ca2+ concentration ([Ca2+]i). After TG treatment to deplete the store, the subsequent application of Ca2+ resulted in a rapid rise in [Ca2+]i caused by SOCE. In the absence of TG treatment, no SOCE was evoked. The Ca2+ influx was dependent on [Ca2+]o (KD = 1.29 mM) and was blocked by an IP3 receptor inhibitor, 2-aminoethoxydiphenyl borate (2-APB), as well as La3+ in a concentration-dependent manner (IC50 = 26 microM). In TG-treated cells, an elevation of [Ca2+]o from 0 to 2.5 mM elicited an inwardly rectifying current at hyperpolarizing potentials with a positive reversal potential. The currents were selective for Ca2+ over the other divalent cations (Ca2+ > Ba2+ > Sr2+ >> Mn2+). In the absence of [Ca2+]o, carbachol, bradykinin, and 2-methylthioadenosine 5'triphosphate activated Ca2+ release from the store; these were inhibited by 2-APB. These results indicate that odontoblasts possessed Ca2+ signaling pathways through the activation of store-operated Ca2+ channels by the depletion of intracellular Ca2+ stores and through the IP3-induced Ca2+ release activated by PLC-coupled receptors.

Growth hormone-releasing peptide-2 reduces inward rectifying K+ currents via a PKA-cAMP-mediated signalling pathway in ovine somatotropes.

Inward-rectifying potassium (Kir) channels are essential for maintaining the resting membrane potential near the K(+) equilibrium and they are responsible for hyperpolarisation-induced K(+) influx. We characterised the Kir current in primary cultured ovine somatotropes and examined the effect of growth hormone-releasing peptide-2 (GHRP-2) on this current and its related intracellular signalling pathways. The Kir current was, in most cases, isolated using nystatin-perforated patch-clamp techniques. In bath solution containing 5 mM K(+), the Kir current was composed of both transient (fast activated) and delayed (slowly activated) components. An increase in the external K(+) concentration from 5 to 25 mM induced an augmentation of approximately 4-fold in the delayed part of the Kir current and both BaCl(2) and CsCl dose-dependently inhibited this current, confirming the presence of the Kir current in ovine somatotropes. Moreover, this specific effect of high K(+) on the Kir current was only observed in the cells that showed positive staining with anti-growth hormone (GH) antibodies, or in GC cells that belong to a rat somatotrope cell line. Application of GHRP-2 (100 nM) reversibly and significantly reduced the Kir current in bath solutions with 5 or 25 mM K(+) in ovine somatotropes. In addition, we found that the reduction in the Kir current mediated by GHRP-2 was totally abolished by the pretreatments with H89 (1 microM) or Rp-cAMP (100 microM) or by intracellular dialysis of a specific protein kinase A (PKA) inhibitory peptide PKI (10 microM). The specific PKC blocker chelerythrine (1 microM) or inhibitory peptide PKC(19-36) (10 microM) did not show any effects on the GHRP-2-induced decrease in the Kir current. These results suggest that the inhibition of Kir current through PKA-cAMP pathways may play an integral role in GHRP-2-induced depolarisation and GH release in ovine somatotropes.