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Hypoxia-induced pulmonary vasoconstriction (HPV) is critical for matching of ventilation/perfusion in lungs. Although hypoxic inhibition of K+ channels has been a leading hypothesis for depolarization of pulmonary arterial smooth muscle cells (PASMCs) under hypoxia, pharmacological inhibition of K+ channels does not induce significant contraction in rat pulmonary arteries. Because a partial contraction by thromboxane A2 (TXA2) is required for induction of HPV, we hypothesize that TXA2 receptor (TP) stimulation might activate depolarizing nonselective cation channels (NSCs). Consistently, we found that 5–10 nM U46619, a stable agonist for TP, was indispensible for contraction of rat pulmonary arteries by 4-aminopyridine, a blocker of voltage-gated K+ channel (Kv). Whole cell voltage clamp with rat PASMC revealed that U46619 induced a NSC current ( INSC,TXA2) with weakly outward rectifying current-voltage relation. INSC,TXA2 was blocked by ruthenium red (RR), an antagonist of the transient receptor potential vanilloid-related channel (TRPV) subfamily. 2-Aminoethoxydiphenyl borate, an agonist for TRPV1–3, consistently activated NSC channels in PASMCs. In contrast, agonists for TRPV1 (capsaicin), TRPV3 (camphor), or TRPV4 (α-PDD) rarely induced an increase in the membrane conductance of PASMCs. RT-PCR analysis showed the expression of transcripts for TRPV2 and -4 in rat PASMCs. Finally, it was confirmed that pretreatment with RR largely inhibited HPV in the presence of U46619. The pretreatment with agonists for TRPV1 (capsaicin) and TRPV4 (α-PDD) was ineffective as pretone agents for HPV. Taken together, it is suggested that the concerted effects of INSC,TXA2 activation and Kv inhibition under hypoxia induce membrane depolarization sufficient for HPV. TRPV2 is carefully suggested as the TXA2-activated NSC in rat PASMC.

This study examined the mechanisms of hypertension in diabetes. We investigated the effects of serotonin (5-HT) on voltage-dependent K(+) (Kv) channel activity, vasoconstriction, 5-HT receptor expression levels, and the involvement of protein kinase C (PKC) in mesenteric arteries of Otsuka Long-Evans Tokushima fatty (OLETF) rats compared with Long-Evans Tokushima Otsuka (LETO) rats. Blood pressure, body weight, blood glucose level, and mesenteric arterial wall thickness were greater in OLETF rats. The 5-HT-induced vasoconstriction of mesenteric arteries was greater in OLETF rats than in LETO rats and inhibited by the 5-HT(2A) inhibitor inhibitor, ketanserin. The Kv currents in mesenteric arterial smooth muscle cells (MASMCs), determined using a perforated patch clamp technique, was inhibited by 1 mM 4-AP (42.5 +/- 4.1% vs. 63.5 +/- 2.3% in LETO vs. OLETF rats at +40 mV), but was insensitive to 1 mM TEA and 100 nM iberiotoxin. The inhibition of Kv current by 1 microM 5-HT in MASMCs was greater in OLETF rats than in LETO rats (17.1 +/- 2.2% vs. 33.2 +/- 2.7% in LETO vs. OLETF rats at +40 mV), and the inhibition was prevented by treatment with the PKCalpha- and beta- selective inhibitor, Gö6976. The expression level of 5-HT(2A), but not 5-HT(2B), receptor and the expression levels of total PKC, PKCbeta, and PKCepsilon, but not PKCalpha, were higher in the mesenteric arteries of OLETF rats compared with LETO rats. The enhanced expression of 5-HT(2A) receptor together with PKCbeta may promote mesenteric vasoconstriction and increase vascular resistance in OLETF rats.

Mechanosensitive nonselective cation channels (NSC(ms)), protein kinase C (PKC), and Rho kinase (ROCK) are suggested as underlying mechanisms for the myogenic contractile response (MR) to luminal pressure (P(lum)). Here we compared relative contributions from these mechanisms using pharmacological inhibitors in rabbit middle cerebral (RbCA), rat middle cerebral (RtCA), rat femoral (RtFA), and rat mesenteric (RtMA) small arteries. Inner diameters of pressurized arteries under various P(lum) were video-analyzed. 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 10 microM) was used as a blocker of NSC(ms). In general, RbCA and RtCA showed higher P(lum) sensitivity of MR than RtFA and RtMA. Ten micromolars of DIDS commonly decreased MRs more effectively at low P(lum) (40-60 mmHg) in all tested arteries except RtCA. In RbCA, PKC inhibitors (100 nM of Go6976 or Go6983) decreased the MR at relatively high P(lum) (80-100 mmHg) whereas ROCK inhibitor (Y-27632, 1 microM) showed a P(lum)-independent inhibition. In RtMA and RtCA, PKC inhibitors (Go6976 and Go6983) had no significant effect whereas Y-27632 generally inhibited the MR. In RtFA, neither PKC inhibitor nor Y-27632 alone affected MRs. Interestingly, in the presence of 10 microM DIDS, Go6983 and Y-27632 decreased the MR of RtFA. In RtMA, it was notable that the MR decreased spontaneously on repeated protocol of P(lum) increase, and the 'run-down' could be effective reversed by maxi-K(+) channel blocker (tetraethylammonium or iberiotoxin). In summary, our study shows the variability of MRs according to the arterial types in terms of their pressure sensitivity and underlying mechanisms that are recruited according to P(lum).

Mouse B cells and their cell line (WEHI-231) express large-conductance background K+ channels (LKbg) that are activated by arachidonic acids, characteristics similar to TREK-2. However, there is no evidence to identify the molecular nature of LKbg; some properties of LKbg were partly different from the reported results of TREK type channels. In this study, we compared the properties of cloned TREK-2 and LKbg in terms of their sensitivities to ATP, phosphatidylinositol 4,5-bisphosphate (PIP2), intracellular pH (pHi), and membrane stretch. Similar to the previous findings of LKbg, TREK-2 showed spontaneous activation after membrane excision (i-o patch) and were inhibited by MgATP or by PIP2. The inhibition by MgATP was prevented by wortmannin, suggesting membrane-delimited regulation of TREKs by phosphoinositide (PI) kinase. The same was observed with the property of LKbg; the activation of TREK-2 by membrane stretch was suppressed by U73122 (PLC inhibitor). As with the known properties of TREK-2, LKbg were activated by acidic pHi and inhibited by PKC activator. Finally, we confirmed the expression of TREK-2 in WEHI-231 by using RT-PCR and immunoblot analyses. The amplitude of background K+ current and the TREK-2 expression in WEHI-231 were commonly decreased by genetic knockdown of TREK-2 using small interfering RNA. The downregulation of TREK-2 attenuated Ca2+-influx induced by arachidonic acid in WEHI-231. As a whole, these results strongly indicate that TREK-2 encodes LKbg in mouse B cells. We also newly suggest that the low activity of TREK-2 in intact cells is due to the inhibition by intrinsic PIP2.

The polypeptide hormone atrial natriuretic peptide (ANP) plays vital roles in maintaining blood volume and arterial blood pressure. The recognition of clinical benefits of ANP both in healthy and diseased heart identifies ANP as a potential candidate for therapeutic strategy in the treatment of heart disease. ANP is synthesized and stored in cardiac myocytes and it is released through the exocytosis of ANP granules both constitutively and in response to stimuli. It is well known that mechanical stretch is the predominant stimulus for ANP secretion. However, the mechanistic link between mechanical stimuli and exocytosis of ANP vesicles in single atrial myocyte has not yet been demonstrated. Over the last decade, compelling evidence suggested that stretch-activated ion channels might function as mechanosensors. We showed previously that direct stretch of single atrial myocyte using two micro-electrodes activated a non-selective cation channel (SAC). So far it is not known whether activation of SAC is involved in stretch-induced ANP secretion. The present article aims to give an overview of the mechanism of mechanical stretch-stimulated ANP secretion and describes an innovative technique to detect ANP secretion from isolated rat atrial myocytes with high time-resolution. Combined with capacitance measurement and patch-clamp technique in conjunction with in situ ANP bioassay, we were able to demonstrate that SAC in rat atrial myocytes acts as a mechanosensor to transduce stretch signals into the ANP secretion pathway.

Evidence is growing of a relationship between atrial dilation and atrial fibrillation (AF), the most prevalent type of arrhythmia. Pulmonary veins, which are important ectopic foci for provoking AF, are of increasing interest in relation to the early development of AF. Here, using single cardiomyocytes isolated from rabbit pulmonary veins, we characterised the stretch-activated currents induced by swelling and axial mechanical stretching. Swelling induced both a stretch-activated nonselective cationic current (NSC) and a Cl(-) current. The swelling-induced Cl(-) current (I Cl,swell) was inhibited by DIDS, whereas the swelling-induced NSC (I NSC,swell) was inhibited by Gd3+. The cationic selectivity of the I NSC,swell was K+ >Cs+ >Na+ >Li+, whilst the PK/PNa, PCs/PNa, and PLi/PNa permeability ratios were 2.84, 1.86, and 0.85, respectively. Activation of the I NSC,swell was faster than that of the I Cl,swell. Given a high K+ concentration in the bath solution, the I NSC,swell showed limited amplitude (

K+ channels play critical roles in the proliferation and activation of lymphocytes. Mouse B cells express large-conductance background K+ channel (LK bg) in addition to the voltage-gated K+ channel (Kv) and Ca2+-activated K+ channel current (IKCa1). Mibefradil, a blocker of T-type Ca2+ channels, has been reported to affect the proliferation of immune cells. In this study, we investigated the effects of mibefradil on the membrane potential and ion channels in murine B cell lines, WEHI-231 and Bal-17. In the whole-cell patch clamp experiments, mibefradil blocked Kv and LK bg current with half inhibitory concentration (IC50), 1.9 and 2.3 microM, respectively. Interestingly, IKCa1 current was increased by mibefradil. In the inside-out patch clamp study with cloned murine IKCa1 (mIKCa1) in HEK-293, mibefradil increased both Ca2+ sensitivity and maximum activity of mIKCa1. At high concentrations (10 microM), mibefradil inhibited mIKCa1 in a voltage-dependent manner. Application of anti-IgM antibody to stimulate B cell receptors (BCR-ligation) induced transient hyperpolarization of Bal-17 and WEHI-231 cells, which became persistent with 1 microM mibefradil. The hyperpolarizing response was abolished by charybdotoxin, a selective blocker for SK4/IKCa1. In summary, our study firstly reports the ion channel-activating effects of mibefradil. The selective potent activation of IKCa1 suggests that mibefradil-derived drugs might be useful in the control of cell responses related with IKCa1.

The mechanosensitive nonselective cation channel (NSC(MS)) and endothelin-1 (ET-1) play critical roles in the regulation of vascular tone. This study was undertaken to investigate the effect of ET-1 on NSC(MS) and on the myogenic response of arteries.Cell-attached patch-clamp techniques were applied to rabbit pulmonary and cerebral arterial smooth muscle cells using a 140 mM CsCl pipette and bath solutions (Ca(2+)-free, 1 mM EGTA). Myogenic responses were determined by video analysis of pressurized arteries.The application of negative pressures through the pipette activated NSC(MS), and this was augmented by bath application of ET-1 (1 pM-30 nM). ET-1 lowered the lowest pressure required for NSC(MS) activation. NSC(MS) facilitation by ET-1 was prevented by BQ-123 (1 microM, an ET(A) antagonist) but not by BQ-788 (1 microM, an ET(B) antagonist). Phorbol 12-myristate 13-acetate (PMA, 100 nM), a protein kinase C activator, also increased the activity of NSC(MS). ET-1- or PMA-induced facilitation of NSC(MS) was abolished by GF109203X (10 microM), a protein kinase C inhibitor. Video analysis of pressurized cerebral artery showed inhibition of the myogenic response by the NSC(MS) channel blockers GsMTx-4 (5 microM) and DIDS (3-100 microM). Treatment with ET-1 (10 pM) augmented the myogenic response and this was inhibited by DIDS (30 microM).Stimulation of ET-1 receptor (ET(A)) facilitates NSC(MS) via a protein kinase C-dependent signaling pathway in rabbit arterial myocytes. Our findings suggest that NSC(MS) play a role in the myogenic response and its augmentation by ET-1.

Ion channels play critical roles in immunological responses. Although the most widely known players are Ca2+-permeable channels (Gallo et al. 2006), K+ channels provide the electrical driving force required for Ca2+ influx, cell volume regulation, and for apoptotic volume decrease (Beeton & Chandy, 2005). B cells are the principal cellular mediators of specific humoral immune response to infection. In a mouse B cell line (WEHI-231), we previously reported novel voltage-independent, large conductance background-type, K+ (LKbg) channels that are inhibited by phosphatidyl inositol 4,5-bisphosphate (PIP2). While LKbg was originally named BKbg (Nam et al. 2004), the acronym was changed to LKbg here to prevent confusion with the calcium-activated K+ channel (BKCa, maxi-K). LKbg channels show low activity in the intact cell membrane while their activity increases spontaneously after excising the membrane (inside-out (i-o) configuration). In i-o patches, LKbg channels are reversibly inhibited by applying MgATP to the cytoplasmic side, and the inhibition was found to be blocked by wortmannin, an inhibitor of phosphoinositide (PI)-kinase. Thus, it has been suggested that the molecular systems that regulate PIP2 levels are tightly associated with LKbg channels (Nam et al. 2004). PIP2 is increasingly being recognized as a key physiological regulator of ion channel/transporters (Hilgemann et al. 2001; Horowitz et al. 2005; Suh & Hille, 2005). While most PIP2-sensitive ion channels are positively regulated, LKbg channels are inhibited by PIP2 along with TRPV1 and cyclic nucleotide-gated cation channels (Hilgemann et al. 2001; Suh & Hille, 2005). In spite of the conspicuous large conductance (∼300 pS), LKbg-like channels with PIP2 sensitivity have not yet been found in other types of cells, and the molecular identity is still unknown. Although LKbg were originally termed ‘background’ K+ channels due to their voltage independence, their activity in the intact resting B cells is very low, which is most likely due to the PIP2 generated from the phosphorylation of phosphoinositides (Nam et al. 2004). Therefore, in pilot studies, we explored the activating conditions for LKbg channels. In these trials we found that mechanical stimulation by negative pressure of patch pipette or osmotic swelling of B cells reversibly activates LKbg channels. It has been recently reported that osmotic swelling and shear stress activate tyrosine kinase (Syk) in DT40 B cells (Miah et al. 2004; Qin et al. 1997), and elevate [Ca2+]i in mouse B cells by activating phospholipase Cγ (PLCγ) and non-selective cation channels (Liu et al. 2005; Zhu et al. 2005). The PLC-mediated hydrolysis of PIP2 by membrane receptor activation is a widely observed regulatory mechanism of PIP2-sensitive ion channels (Suh & Hille, 2005). Considering that membrane stretch is also an activating condition for PLC (Moore et al. 2002; Ruwhof et al. 2001; Zhu et al. 2005; Liu et al. 2005), ion channels regulated by PIP2 might be affected by membrane stretch in a PLC-dependent manner. However, no study has been performed to investigate whether stretch-dependent PLC activation regulates the PIP2-sensitive ion channels. Since cells are exposed to various levels of mechanical stimuli, the elucidation of any such signalling mechanism would provide an intriguing insight into ion channel regulation. In the present study, we propose that the mechanosensitive activation of LKbg channels are mediated by PLC-dependent hydrolysis of PIP2.

We examined the effects of acute hypoxia on vascular tone and coronary blood flow (CBF) in rabbit coronary arteries. In the pressurized arterial preparation of small arteries (100 mum) and the Langendorff-perfused rabbit hearts, hypoxia induced coronary vasodilation and increased CBF in the presence of glibenclamide (K(ATP) channel blocker), Rp-8-Br-PET-cGMPs [cyclic guanosine monophosphate (cGMP)-dependent protein kinase inhibitor, Rp-cGMPs], and methionyl transfer RNA synthetase (MRS) 1334 (adenosine A(3) receptor inhibitor); these increases were inhibited by the inward rectifier K(+) (Kir) channel inhibitor, Ba(2+). These effects were blocked by the adenylyl cyclase inhibitor SQ 22536 and by the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) inhibitors Rp-8-CPT-cAMPs (Rp-cAMPs) and KT 5720. However, cGMP-dependent protein kinase was not involved in the hypoxia-induced increases of the vascular diameter and CBF. In summary, our results suggest that acute hypoxia can induce the opening of Kir channels in coronary artery that has small diameter (100 mum) by activating the cAMP and PKA signalling pathway, which could contribute to vasodilation and, therefore, increased CBF.

The effects of the protein kinase A (PKA) inhibitor H-89 on ATP-sensitive K+ (KATP) and inward rectifier K+ (Kir) currents were examined in rabbit coronary arterial smooth muscle cells using the patch clamp technique. The H-89, in a dose-dependent manner, inhibited KATP and Kir currents with apparent Kd values of 1.19+/-0.18 and 3.78+/-0.37 microM, respectively. H-85, which is considered as an inactive form of H-89, inhibited KATP and Kir currents, similar to the result of H-89. KATP and Kir currents were not affected by either Rp-8-CPT-cAMPs, which is a membrane-permeable selective PKA inhibitor, or KT 5720, which is also known as a PKA inhibitor. Also, these two drugs did not significantly alter the effects of H-89 on the KATP and Kir currents. These results suggest that H-89 directly inhibits the KATP and Kir currents of rabbit coronary arterial smooth muscle cells independently of PKA inhibition.

We examined the effects of the protein kinase C (PKC) inhibitor, bisindolylmaleimide (BIM) (I), on voltage-dependent K+ (K(V)) channels in rabbit coronary arterial smooth muscle cells using whole-cell patch clamp technique. BIM (I) reversibly and dose-dependently inhibited the K(V) currents with an apparent Kd value of 0.27 microM. The inhibition of the K(V) current by BIM (I) was highly voltage-dependent between -30 and +10 mV (voltage range of channel activation), and the additive inhibition of the K(V) current by BIM (I) was voltage-dependence in the full activation voltage range. The rate constants of association and dissociation for BIM (I) were 18.4 microM(-1) s(-1) and 4.7 s(-1), respectively. BIM (I) had no effect on the steady-state activation and inactivation of K(V) channels. BIM (I) caused use-dependent inhibition of K(V) current, which was consistent with the slow recovery from inactivation in the presence of BIM (I) (recovery time constants were 856.95 +/- 282.6 ms for control, and 1806.38 +/- 110.0 ms for 300 nM BIM (I)). ATP-sensitive K+ (K(ATP)), inward rectifier K+ (K(IR)), Ca2+-activated K+ (BK(Ca)) channels, which regulate the membrane potential and arterial tone, were not affected by BIM (I). The PKC inhibitor, chelerythrine, and protein kinase A (PKA) inhibitor, PKA-IP, had little effect on the K(V) current and did not significantly alter the inhibitory effects of BIM (I) on the K(V) current. These results suggest that BIM (I) inhibits K(V) channels in a phosphorylation-independent, and voltage-, time- and use-dependent manner.

A whole-cell patch recording was used to determine the effects of staurosporine (ST), a potent protein kinase C (PKC) inhibitor, on L-type Ca(2+) channel (LTCC) activity in rabbit atrial myocytes. Bath application of ST (300 nM) caused a significant reduction in peak I-V relationship of LTCC (from -16.8+/-2.55 to -3.74+/-1.22pApF(-1) at 0 mV). The level of L-type Ca(2+) current (I(Ca,L)) inhibition produced by ST was independent of the voltage at which the effect was measured. ST inhibited the I(Ca,L) in a dose-dependent manner with a K(d) value of 61.98+/-6.802 nM. ST shifted the activation curve to more positive potentials, but did not have any significant effect on the voltage dependence of the inactivation curve. Other PKC inhibitors, GF 109203X (1 microM) and chelerythrine (3 microM), and PKA inhibitor, PKA-IP (5 microM), did not show any inhibitory effect on I(Ca,L). Additional application of ST in the presence of isoproterenol (1 microM), a selective beta-adrenoreceptor agonist, reduced peak I(Ca,L) (78.2%) approximately to the same level with single application of ST (77.8%). In conclusion, our results indicate that ST directly blocks the LTCC in a PKC or PKA-independent manner on LTCC and it should be taken into consideration when ST is used in functional studies of ion channel modulation by protein phosphorylation.

We investigated the effect of bisindolylmaleimide (I), a widely used protein kinase C (PKC) inhibitor, on the voltage-dependent K(+) (Kv) currents of rat mesenteric arterial smooth muscle cells using the whole-cell patch-clamp technique. Bisindolylmaleimide (I) reversibly and dose-dependently inhibited the Kv currents with an apparent K(d) value of 0.23+/-0.001 microM. The blockade was apparently through the acceleration of the decay rate of the Kv currents. The apparent rate constants of association and dissociation for bisindolylmaleimide (I) were 17.9+/-1.6 microM(-1) s(-1) and 4.1+/-1.5 s(-1), respectively. The inhibition of Kv current by bisindolylmaleimide (I) was steeply voltage-dependent between -30 and 0 mV (voltage range of channel activation). Bisindolylmaleimide (I) had no effect on the steady-state activation and inactivation of the Kv currents. Applications of trains of pulses at 1 or 2 Hz lead to a progressive increase in the bisindolylmaleimide (I)-blockade, and the recovery from bisindolylmaleimide (I)-block at -80 mV exhibited a time constant of 577.2+/-52.7 ms. Bisindolylmaleimide (V), an inactive analogue of bisindolylmaleimide (I), similarly inhibited the Kv currents with an apparent K(d) value of 1.48+/-0.004 microM, but other PKC inhibitor chelerythrine little affected the Kv currents. These results suggest that bisindolylmaleimide (I) directly inhibits the Kv currents of rat mesenteric arterial smooth muscle cells independently of PKC inhibition, in a state-, voltage-, time- and use-dependent manner.

Mechanosensitive cation channels may be involved in the development of the myogenic tone of arteries. The molecular identity of these channels is not clear, but transient receptor potential channels (TRPCs) are good candidates. In the present study, we searched for mechanosensitive channels at the single-channel level in arterial smooth muscle cells using the patch-clamp technique and investigated the channel properties in the light of properties of TRPCs. With 140 mmol/L CsCl in the pipette solution, application of negative pressures to the back of the pipette induced the activation of channels the open probability of which increased with the amount of negative pressure. The current-voltage relationship was linear in symmetrical ionic conditions, and the single-channel conductances for Cs + , K + , and Na + were 30, 36, and 27 pS, respectively. When NMDG + was substituted for Cs + in the pipette solution, inward currents were abolished, whereas outward currents remained active, indicating that the channels were nonselective to cations. The channel activity was blocked by intracellular Gd 3+ and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid and increased by diacylglycerol and by cyclopiazonic acid. Phospholipase C inhibitor (U73122) inhibited not only channel activity but also the development of myogenic tone induced by stretching of the basilar arteries. These results suggest that the ion channel responsible for the development of myogenic tone is the 30-pS mechanosensitive cation channel that exhibits properties similar to those of TRPCs.

Using the inside-out patch-clamp technique, large-conductance Ca2+ -activated K+ channel (BK(Ca)) currents were recorded from coronary artery smooth muscle cells. Cytochalasin D, an actin filament disrupter, increased channel activity ( NP(o), where N is the number of channels and P(o) the open probability), and this increase was reversed by phalloidin, an actin filament stabilizer. NP(o) was also increased by colchicine, a microtubule disrupter, and decreased by taxol, a microtubule stabilizer. With the stepwise increase of negative pressure in the patch pipettes, the activity of BK(Ca) gradually increased: the maximum effect (527% increase in NP(o)) was achieved at -40 cmH(2)O and the half-maximum effect at -25 cmH(2)O. The increase in NP(o) in response to negative pressure was abolished by phalloidin but not by taxol. These results imply that both actin filaments and microtubules inhibit the opening of BK(Ca) in coronary artery smooth muscle cells, but that only actin filaments are involved in the stretch sensitivity of BK(Ca).

The present investigation tested the hypothesis that nitric oxide (NO) potentiates ATP-sensitive K+(KATP) channels by protein kinase G (PKG)-dependent phosphorylation in rabbit ventricular myocytes with the use of patch-clamp techniques. Sodium nitroprusside (SNP; 1 mM) potentiated KATPchannel activity in cell-attached patches but failed to enhance the channel activity in either inside-out or outside-out patches. The 8-(4-chlorophenylthio)-cGMP Rp isomer (Rp-CPT-cGMP, 100 μM) suppressed the potentiating effect of SNP. 8-(4-Chlorophenylthio)-cGMP (8-pCPT-cGMP, 100 μM) increased KATPchannel activity in cell-attached patches. PKG (5 U/μl) added together with ATP and cGMP (100 μM each) directly to the intracellular surface increased the channel activity. Activation of KATPchannels was abolished by the replacement of ATP with ATPγS. Rp-pCPT-cGMP (100 μM) inhibited the effect of PKG. The heat-inactivated PKG had little effect on the KATPchannels. Protein phosphatase 2A (PP2A, 1 U/ml) reversed the PKG-mediated KATPchannel activation. With the use of 5 nM okadaic acid (a PP2A inhibitor), PP2A had no effect on the channel activity. These results suggest that the NO-cGMP-PKG pathway contributes to phosphorylation of KATPchannels in rabbit ventricular myocytes.

Two different families of Na(+)/Ca(2+) exchangers, K(+)-independent NCX and K(+)-dependent NCKX, are known. Exploiting the outward K(+) gradient, NCKX is able to extrude Ca(2+) more efficiently than NCX, even when the Na(+) gradient is reduced. The NCKX, which was originally thought to be limited to the retinal photoreceptor, was shown recently to be widely distributed in the brain. We investigated the contribution of Na(+)/Ca(2+)exchange to Ca(2+) clearance mechanisms in neurohypophysial (NHP) axon terminals, using patch-clamp and microfluorometry techniques. In the presence of internal K(+), Ca(2+) decay was significantly slowed by the removal of external Na(+), indicative of the role of Na(+)/Ca(2+) exchange. As internal [K(+)] was decreased, Ca(2+) decay rate and its dependence on Na(+) were greatly attenuated. In the absence of internal K(+), Ca(2+) decay rate was little affected by Na(+) removal. Quantitative analysis using Ca(2+) decay rate constant indicated that >60% of Ca(2+) extrusion is mediated by Na(+)/Ca(2+) exchange when peak [Ca(2+)] level is higher than 500 nm, and ∼90% of Na(+)/Ca(2+) exchange activity is K(+) dependent. In situhybridization confirmed the expression of NCKX2 transcripts in the supraoptic nucleus in which soma of NHP axon terminals are located. To our knowledge, this is the first report to show the significant role of K(+)-dependent Na(+)/Ca(2+) exchange in neuronal cells other than photoreceptors. Considering that axon terminals are subject to an invasion by high-frequency Na(+) spikes, which may lower Na(+) gradients, the presence of NCKX may have a functional significance in intracellular Ca(2+) regulation.

We investigated the role of intracellular Mg(2+) ([Mg(2+)](i)) in the regulation of membrane potential ( V(m)) in rabbit coronary artery smooth muscle cells. V(m), membrane currents and intracellular Ca(2+) ([Ca(2+)](i)) were measured using standard patch-clamp and microfluorometry techniques. When [Ca(2+)](i) was increased by caffeine, V(m) depolarized at low [Mg(2+)](i) (0.1 mM), but hyperpolarized at high [Mg(2+)](i) (or =1.2 mM). Effects of [Mg(2+)](i) on caffeine-induced currents were investigated. [Mg(2+)](i) selectively facilitated the activation of Ca(2+)-activated K(+) currents ( I(K,Ca)), while Ca(2+)-activated Cl(-) currents ( I(Cl,Ca)) were unaffected. Simultaneous recording of [Ca(2+)](i) and I(K,Ca) at different [Mg(2+)](i) showed that [Mg(2+)](i) increased the Ca(2+) sensitivity of I(K,Ca). [Ca(2+)](i) also inhibited voltage-dependent K(+) (K(V)) currents, although this effect was significant only at low [Mg(2+)](i). These results imply that the relative contributions of I(K,Ca), I(Cl,Ca) and K(V) currents to V(m) during an increase in [Ca(2+)](i) are affected by [Mg(2+)](i): at low [Mg(2+)](i), activation of I(Cl,Ca) and inhibition of K(V) currents depolarized V(m); at high [Mg(2+)](i) the activation of I(K,Ca) predominated, resulting in hyperpolarization of V(m). In conclusion, [Mg(2+)](i) hyperpolarizes V(m) by selective facilitation of I(K,Ca) and may thus possibly contributes to the relaxation of the coronary artery.

We have investigated the effect of {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, a specific inhibitor of phospholipase C (PLC), on acetylcholine-activated K+ currents (IKACh) in mouse atrial myocytes. In perforated patch clamp mode, IKACh was activated by 10 μM acetylcholine. When atrial myocytes were pretreated with {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 or {"type":"entrez-nucleotide","attrs":{"text":"U73343","term_id":"1688125","term_text":"U73343"}}U73343, IKACh was inhibited dose-dependently (half-maximal inhibition at 0.12±0.0085 and 0.16±0.0176 μM, respectively). The current-voltage relationships for IKACh in the absence and in the presence of {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 showed that the inhibition occurred uniformly from −120 to +40 mV, indicating a voltage-independent inhibition. When {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 was applied after IKACh reached steady-state, a gradual decrease in IKACh was observed. The time course of the current decrease was well fitted to a single exponential, and the rate constant was proportional to the concentration of {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122. When KACh channels were directly activated by adding 1 mM GTPγS to the bath solution in inside-out patches, {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (1 μM) decreased the open probability significantly without change in mean open time. When KACh channels were activated independently of G-protein activation by 20 mM Na+, open probability was also inhibited by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122. Voltage-activated K+ currents and inward rectifying K+ currents were not affected by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122. These findings show that inhibition by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 and {"type":"entrez-nucleotide","attrs":{"text":"U73343","term_id":"1688125","term_text":"U73343"}}U73343 of KACh channels occurs at a level downstream of the action of Gβγ or Na+ on channel activation. The interference with phosphatidylinositol 4,5-bisphosphate (PIP2)-channel interaction can be suggested as a most plausible mechanism. Keywords: {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, {"type":"entrez-nucleotide","attrs":{"text":"U73343","term_id":"1688125","term_text":"U73343"}}U73343, acetylcholine-activated K+ current, phospholipase C, phospholipase C inhibitor, atrial myocytes, phosphatidylinositol 4,5-bisphosphate, patch-clamp Introduction {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 has been shown to inhibit phospholipase C (PLC) at low micromolar concentrations (Smith et al., 1990; Bleasdale et al., 1990), and it is one of the most widely used PLC inhibitors. Very often the structurally related {"type":"entrez-nucleotide","attrs":{"text":"U73343","term_id":"1688125","term_text":"U73343"}}U73343 is used as negative control, since it does not inhibit PLC (Smith et al., 1990; Bleasdale et al., 1990). Recent studies, however, indicate that the action of {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 and {"type":"entrez-nucleotide","attrs":{"text":"U73343","term_id":"1688125","term_text":"U73343"}}U73343 may not be specific as originally thought, since both agents also inhibit phospholipase D activity (Bosch et al., 1998), and since {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 elicits intracellular calcium release (Muto et al., 1997). Furthermore, the non-selective actions of {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 were also shown for a variety of receptor-mediated signal transductions, such as adenosine A1-receptor (Walker et al., 1998) and histamine H1-receptor (Hughes et al., 2000). ACh-activated K+ currents (IKACh) are responsible for the inhibitory effect of cardiac function by ACh (Jan & Jan, 1997; Yamada et al., 1998). Activation of IKACh by ACh receptors (mAChR) is mediated via the pertussis toxin-sensitive G-protein. G-protein-ion channel coupling mechanisms have been widely investigated for IKACh and its molecular equivalent G-protein-gated inwardly rectifying K+ channels (GIRK), and it is now believed that the direct binding of G protein Gβγ subunits to the channel protein opens GIRK channels (Huang et al., 1995; Krapivinsky et al., 1995; Kunkel & Peralta, 1995; Inanobe et al., 1995). In addition to Gβγ subunits, GIRK channels can also be activated by Na+ ions through G-protein-independent pathway (Sui et al., 1996; 1998). Both Gβγ subunits and Na+ ion cause a stabilization of phosphatidylinositol 4,5-bisphosphate (PIP2)-channel interaction, which is absolutely required for channel opening (Huang et al., 1998). In the present study, we investigated the effects of {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 and {"type":"entrez-nucleotide","attrs":{"text":"U73343","term_id":"1688125","term_text":"U73343"}}U73343 on IKACh in mouse atrial myocytes and found that both inhibit IKACh independently of PLC inhibition. The inhibition was almost complete at concentrations similar to or below those used for PLC inhibition. The target for {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 action was investigated using inside-out patch recording, showing that the inhibition by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 occurs even when the channels are activated by Na+ independently of G-protein activation. These results suggest that {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 inhibits KACh channels possibly by the interference with PIP2-channel interaction.

We have investigated the effect of alpha(1)-adrenergic agonist phenylephrine (PE) on acetylcholine-activated K(+) currents (I(KACh)). I(KACh) was recorded in mouse atrial myocytes using the patch clamp technique. I(KACh) was activated by 10 microm ACh and the current decreased by 44.27 +/- 2.38% (n = 12) during 4 min due to ACh-induced desensitization. When PE was applied with ACh, the extent of desensitization was markedly increased to 69.34 +/- 2.22% (n = 9), indicating the presence of PE-induced desensitization. I(KACh) was fully recovered from desensitization after a 6-min washout. PE-induced desensitization of I(KACh) was not affected by protein kinase C inhibitor, calphostin C, but abolished by phospholipase C (PLC) inhibitor, neomycin. When phophatidylinositol 4,5-bisphosphate (PIP(2)) replenishment was blocked by wortmannin (an inhibitor of phophatidylinositol 3-kinase and phophatidylinositol 4-kinase), desensitization of I(KACh) in the presence of PE was further increased (97.25 +/- 7.63%, n = 6). Furthermore, the recovery from PE-induced desensitization was inhibited, and the amplitude of I(KACh) at the second exposure after washout was reduced to 19.65 +/- 2.61% (n = 6) of the preceding level. These data suggest that the K(ACh) channel is modulated by PE through PLC stimulation and depletion of PIP(2).

1. Amitriptyline has been known to induce QT prolongation and torsades de pointes which causes sudden death. We studied the effects of amitriptyline on the human ether-a-go-go-related gene (HERG) channel expressed in Xenopus oocytes and on the rapidly activating delayed rectifier K(+) current (I(Kr)) in rat atrial myocytes. 2. The amplitudes of steady-state currents and tail currents of HERG were decreased by amitriptyline dose-dependently. The decrease became more pronounced at more positive potential, suggesting that the block of HERG by amitriptyline is voltage dependent. IC(50) for amitriptyline block of HERG current was progressively decreased according to depolarization: IC(50) values at -30, -10, +10 and +30 mV were 23.0, 8.71, 5.96 and 4.66 microM, respectively. 3. Block of HERG by amitriptyline was use dependent: exhibiting a much faster block at higher activation frequency. Subsequent decrease in frequency after high activation frequency resulted in a partial relief of HERG blockade. 4. Steady-state block by amitriptyline was obtained while depolarization to +20 mV for 0.5 s was applied at 0.5 Hz: IC(50) was 3.26 microM in 2 mM [K(+)](o). It was increased to 4. 78 microM in 4 mM [K(+)](o), suggesting that the affinity of amitriptyline on HERG was decreased by external K(+). 5. In rat atrial myocytes bathed in 35 degrees C, 5 microM amitriptyline blocked I(Kr) by 55%. However, transient outward K(+) current (I(to)) was not significantly affected. 6. In summary, the data suggest that the block of HERG currents may contribute to arrhythmogenic side effects of amitriptyline.

1. Stretch-activated channels (SACs) were studied in isolated rat atrial myocytes using the whole-cell and single-channel patch clamp techniques. Longitudinal stretch was applied by using two patch electrodes. 2. In current clamp configuration, mechanical stretch of 20 % of resting cell length depolarised the resting membrane potential (RMP) from -63.6 +/- 0.58 mV (n = 19) to -54.6 +/- 2.4 mV (n = 13) and prolonged the action potential duration (APD) by 32.2 +/- 8.8 ms (n = 7). Depolarisation, if strong enough, triggered spontaneous APs. In the voltage clamp configuration, stretch increased membrane conductance in a progressive manner. The current-voltage (I-V ) relationship of the stretch-activated current (ISAC) was linear and reversed at -6.1 +/- 3.7 mV (n = 7). 3. The inward component of ISAC was abolished by the replacement of Na+ with NMDG+, but ISAC was hardly altered by the Cl- channel blocker DIDS or removal of external Cl-. The permeability ratio for various cations (PCs:PNa:PLi = 1.05:1:0.98) indicated that the SAC current was a non-selective cation current (ISAC,NC). The background current was also found to be non-selective to cations (INSC,b); the permeability ratio (PCs:PNa:PLi = 1.49:1:0.70) was different from that of ISAC,NC. 4. Gadolinium (Gd3+) acted on INSC,b and ISAC,NC differently. Gd3+ inhibited INSC,b in a concentration-dependent manner with an IC50 value of 46.2 +/- 0.8 microM (n = 5). Consistent with this effect, Gd3+ hyperpolarised the resting membrane potential (-71.1 +/- 0.26 mV, n = 9). In the presence of Gd3+ (0.1 mM), stretch still induced ISAC,NC and diastolic depolarisation. 5. Single-channel activities were recorded in isotonic Na+ and Cs+ solutions using the inside-out configuration. In NMDG+ solution, outward currents were abolished. Gd3+ (100 microM) strongly inhibited channel opening both from the inside and outside. In the presence of Gd3+ (100 microM) in the pipette solution, an increase in pipette pressure induced an increase in channel opening (21.27 +/- 0.24 pS; n = 7), which was distinct from background activity. 6. We concluded from the above results that longitudinal stret in rat atrial myocytes induces the activation of non-selective cation channels that can be distinguished from background channels by their different electrophysiology and pharmacology.

We investigated the permeability of Cs+ and Na+ through various ion channels in rat atrial myocytes using the whole-cell voltage-clamp technique. With isotonic CsCl (140 mM) on both sides of the membrane and nominally [Ca2+]o-free conditions, depolarising clamp pulses induced an increase of outward currents which showed a biphasic time course. Repolarisation to the holding potential induced inward tail currents. With isotonic NaCl, depolarisation also induced outward currents which showed a monotonic decay, but inward tail currents were not observed. Both in NaCl and CsCl, currents were hardly affected by TEA (10 mM), 4-AP (5 mM) and DIDS (100 µM). Nicardipine (1 M) almost completely blocked time-dependent outward currents in isotonic NaCl solution, leaving only time-independent currents which showed linear I-V relationship. In isotonic CsCl conditions, nicardipine blocked outward current considerably, but there still remained time-dependent outward currents and inward tail currents. Addition of E-4031 (2-20 M) which is known as a specific blocker of the rapidly activating delayed rectifier K+ current (IKr) completely blocked these time-dependent outward and inward currents, leaving only a time-independent current. Time-independent currents recorded in the presence of nicardipine and E-4031 were inhibited by GdCl3, which is known to block non-selective cation (NSC) currents. From these results, it was suggested that NSC current in atrial myocytes can be investigated in isotonic Cs+ or Na+ solution in the presence of Ca2+ channel and IKr blockers.

Stretch-activated cation channels have been suggested to mediate various intracellular physiological events. In this study, inside-out excised-patch recording technique was used to identify stretchactivated cation channels in adult rat atrial myocytes. In symmetrical NaCl or CsCl (140 mM) solution, spontaneous opening of single channel with relatively small conductance (- 9 pS) was observed. When negative pressure was applied to the pipette, single channel opening with larger conductance (21. 3 ± 0.2 pS, n=7) was observed. In symmetrical KCl (140 mM) solution, increase of channel opening with different characteristics was also observed by negative pressure. This channel was selectively permeable to K+. The slope conductance at -80 mV was 50.6 ± 1.7 pS (n=5) . These results demonstrate that the mechanical stretch activates a non-selective and K+ -selective cation channel and that the net results should be determined by the modulation or relative contribution of them.

We investigated the actions of various divalent cations on the delayed rectifier K+ currents (IKr) in rabbit sinoatrial node cells using the whole-cell voltage-clamp technique in isotonic K+ solutions. External divalent cations decreased the amplitude of currents, accelerated the time course of deactivation and shifted the activation to positive potentials in a dose-dependent manner. The concentrations for half-maximum inhibition of the steady-state currents (KM) obtained at 0 mV were 0.63, 1.36, 1.65 and 2.16 mM for Ni2+, Co2+, Mn2+ and Ba2+, respectively. The effect was voltage dependent (KM decreased e-fold for 12.2-16.8 mV hyperpolarization), but the dependence did not vary significantly among different cations. Acceleration of the time course of current deactivation by the increase of divalent cation concentration was well fitted by the voltage-dependent block model, and the binding rate constant (k1) was obtained. The binding rates for the ions took the following order: Ni2+ >Co2+ >Mn2+ >Ba2+. The degree of the shift of activation occurred in the same order: Ni2+ >Co2+ >Mn2+ >Ba2+. From these results, we conclude that IKr channels are non-selectively blocked by most divalent cations from the external side and that the binding site is located deep inside the channel, resulting in a steep voltage dependence of the blockade.

We have investigated the effect of external H+ concentration ([H+]o)on the human-ether-a-go-go-related gene (HERG) current (I HERG), the molecular equivalent of the cardiac delayed rectifier potassium current (I Kr), expressed in Xenopus oocytes, using the two-microelectrode voltage-clamp technique. When [H+]o was increased, the amplitude of the I HERG elicited by depolarization decreased, and the rate of current decay on repolarization was accelerated. The activation curve shifted to a more positive potential at lower external pH (pHo) values (the potential required for half-maximum activation, V 1/2, was: –41.8 mV, –38.0 mV, –33.7 mV, –26.7 mV in pHo 8.0, 7.0, 6.6, 6.2, respectively). The maximum conductance (g max) was also affected by [H+]o: a reduction of 7.9%, 14.6%, and 22.8% was effected by decreasing pHo from 8.0 to 7.0, 6.6, and 6.2, respectively. We then tested whether this pH effect was affected by the external Ca2+ concentration, which is also known to block HERG channels. When the extracellular Ca2+ concentration was increased from 0.5 mM to 5 mM, the shift in V 1/2 caused by increasing [H+]o was attenuated, suggesting that these two ions compete for the same binding site. On the other hand, the decrease in g max caused by increasing [H+]o was not significantly affected by changing external Ca2+ levels. The results indicate that HERG channels are inhibited by [H+]o by two different mechanisms: voltage-dependent blockade (shift of V 1/2) and the decrease in g max. With respect to the voltage-dependent blockade, the interaction between H+ and Ca2+ is competitive, whereas for the decreasing g max, their interaction is non-competitive.

We have investigated actions of various divalent cations (Ba2+, Sr2+, Mn2+, Co2+, Ni2+, Zn2+) on human ether-a-go-go related gene (HERG) channels expressed in Xenopus laevis oocytes using the voltage clamp technique. All divalent cations inhibited HERG current dose-dependently in a voltage-dependent manner. The concentration for half-maximum inhibition (Ki) decreased at more negative potentials, indicating block is facilitated by hyperpolarization. Ki at 0 mV for Zn2+, Ni2+, Co2+, Ba2+, Mn2+, and Sr2+ was 0.19, 0.36, 0. 50, 0.58, 2.36, and 6.47 mM, respectively. The effects were manifested in four ways: 1) right shift of voltage dependence of activation, 2) decrease of maximum conductance, 3) acceleration of current decay, and 4) slowing of activation. However, each parameter was not affected by each cation to the same extent. The potency for the shift of voltage dependence of activation was in the order Zn2+ > Ni2+ >/= Co2+ > Ba2+ > Mn2+ > Sr2+, whereas the potency for the decrease of maximum conductance was Zn2+ > Ba2+ > Sr2+ > Co2+ > Mn2+. The kinetics of activation and deactivation were also affected, but the two parameters are not affected to the same extent. Slowing of activation by Ba2+ was most distinct, causing a marked initial delay of current onset. From these results we concluded that HERG channels are nonselectively blocked by most divalent cations from the external side, and several different mechanism are involved in their actions. There exist at least two distinct binding sites for their action: one for the voltage-dependent effect and the other for reducing maximum conductance.

1. Using the perforated patch-clamp or whole-cell clamp technique, we investigated the contribution of Ca2+-activated K+ current (IK(Ca)) and non-selective cation currents (INSC) to the membrane potential in small pulmonary arterial smooth muscle cells of the rabbit. 2. The resting membrane potential (Vm) was -39.2 +/- 0.9 mV (n = 72). It did not stay at a constant level, but hyperpolarized irregularly, showing spontaneous transient hyperpolarizations (STHPs). The mean frequency and amplitude of the STHPs was 5.6 +/- 1. 1 Hz and -7.7 +/- 0.7 mV (n = 12), respectively. In the voltage-clamp mode, spontaneous transient outward currents (STOCs) were recorded with similar frequency and irregularity. 3. Intracellular application of BAPTA or extracellular application of TEA or charybdotoxin suppressed both the STHPs and STOCs. The depletion of intracellular Ca2+ stores by caffeine or ryanodine, and the removal of extracellular Ca2+ also abolished STHPs and STOCs. 4. Replacement of extracellular Na+ with NMDG+ caused hyperpolarization Vm of without affecting STHPs. Removal of extracellular Ca2+ induced a marked depolarization of Vm along with the disappearance of STHPs. 5. The ionic nature of the background inward current was identified. The permeability ratio of K+ : Cs+ : Na+ : Li+ was 1.7 : 1.3 : 1 : 0. 9, indicating that it is a non-selective cation current (INSC). The reversal potential of this current in control conditions was calculated to be -13.9 mV. The current was blocked by millimolar concentrations of extracellular Ca2+ and Mg2+. 6. From these results, it was concluded that (i) hyperpolarizing currents are mainly contributed by Ca2+-activated K+ (KCa) channels, and thus STOCs result in transient membrane hyperpolarization, and (ii) depolarizing currents are carried through NSC channels.

Using the whole-cell voltage-clamp technique, we have investigated the effect of nitric oxide (NO) donor (sodium nitroprusside, SNP) on hyperpolarization-activated inward current, I f , in isolated rabbit sinoatrial node (SAN) cells. I f in the basal state increased when NO was applied but decreased when I f was pre-stimulated by isoproterenol (ISO) or by adding cAMP to the pipette solution. Both the stimulatory and the inhibitory effects of NO were abolished by guanylyl cyclase inhibitor, methylene blue (MB), suggesting that the effect of NO is mediated by cGMP. The inhibitory effect of NO was abolished when I f was pre-stimulated by 3-isobutyl-1-methylxanthine (IBMX), which is a phosphodiesterase (PDE) inhibitor, or by adding 8Br-cAMP (which is resistant to PDE) to the pipette solution. An analogue of cGMP, 8Br-cGMP, which is a potent stimulator of cGMP-dependent protein kinase (PKG) but has little effect on PDE, did not inhibit I f when I f was pre-stimulated by ISO. In its basal state, I f was still increased by 8Br-cGMP, and this effect was not prevented by the pretreatment with H-7, PKG inhibitor. The effect of acetylcholine (ACh) was not identical to that of NO I f decreased when pre-stimulated not only by ISO, but also by IBMX. The above results suggest that via cGMP, NO exerts a dual effect on I f : the inhibitory effect is mediated by cGMP-stimulated PDE, and the stimulatory effect may be attributable to direct binding of cGMP to I f channels.

Brain-derived neurotrophic factor (BDNF) rapidly enhances synaptic transmission among the hippocampal neurons. In order to examine which component of glutamate receptors participates in synaptic potentiation by BDNF, we have studied the effect of glutamate antagonists on excitatory postsynaptic currents (EPSCs) enhanced by BDNF, using cultured embryonic hippocampal neurons. In the presence of AP5, a N-methyl-D-aspartate (NMDA) antagonist, BDNF depressed the EPSCs. In contrast, BDNF enhanced the EPSCs in the presence of a non-NMDA antagonist CNQX. Our results suggest that BDNF acutely activates synaptic transmission via NMDA, but suppresses it via non-NMDA receptors in the hippocampus.

The effect of guanosine 3',5'-cyclic monophosphate (cGMP) on L-type Ca current (ICa) was investigated in a study of rabbit ventricular myocytes using the whole-cell patch-clamp technique. Intracellular application of cGMP (100 MUm) increased ICa in the absence of isoprenaline or forskolin. 8-Bromo-cGMP (100 muM) and 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP, 400 muM), relatively specific stimulators of cGMP-dependent protein kinase (cGMP-PK), also increased ICa. The stimulatory effect of 8-pCPT-cGMP was suppressed by Rp-8-chlorophenylthio-cGMP (400 muM), a phosphodiesterase-resistant cGMP-PK inhibitor. When ICa was increased by bath application of the non-specific phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 100 muM), 8-pCPT-cGMP (400 muM) resulted in additional stimulation of ICa. In the presence of 8-pCPT-cGMP, additional applications of isoprenaline (1 muM) or forskolin (1 muM) induced a further increase in ICa. From these results, it could be concluded that the activation of cGMP-dependent protein kinase is involved in the facilitation of ICa by cGMP in rabbit ventricular myocytes.

Ca(2+)-activated K+ currents (IK(Ca)) and voltage-dependent Ca(2+)-insensitive K+ currents (IK(V)) were recorded using the patch clamp technique to study the pulmonary (PASMC) and ear arterial smooth muscle cells (EASMC) of the rabbit and the possible regulatory mechanisms related to hypoxia. When a hypoxic solution (1 mM Na2S2O4, gassed with 100% N2) was superfused, the activity of Ca(2+)-activated K+ channels (KCa channels) recorded at a pipette potential of -70 mV in cell-attached mode was decreased to 49 +/- 7% in PASMC, whereas EASMC KCa channels did not respond to hypoxia. In inside-out patches (bathed symmetrically in 150 mM KCl), reducing agents such as dithiothreitol (DTT; 5 mM), reduced glutathione (GSH; 5 mM) and NADH (2 mM) decreased KCa channel activity in PASMC, but they did not affect the EASMC KCa channel. However, oxidizing agents such as 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB; 1 mM), oxidized GSH (GSSG; 5 mM) and NAD (2 mM) increased KCa channel activity in both PASMC and EASMC. In the whole-cell configuration, using a pipette solution containing a high concentration of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA; 10 mM), PASMC IK(V) were activated by depolarizing step pulses to voltages more positive than -30 mV (holding potential, -80 mV). IK(V) was increased by application of a membrane-permeable oxidizing agent, 2,2'-dithio-bis(5-nitropyridine) (DTBNP; 200 microM), whereas it was decreased by application of DTT (5 mM). From these results, it could be suggested that hypoxic pulmonary vasoconstriction is attributable, at least in part, to a change of cellular redox state, which decreases outward K+ currents. This hypothesis is further supported by the observation that the basal redox state of EASMC KCa channels is more reduced than that of PASMC KCa channels. The distinct responses to hypoxia of pulmonary and systemic arterial smooth muscle could be explained by this difference.

The periodic oscillations of outward currents were studied in smooth muscle cells of the rabbit pulmonary artery. The combined stimuli of superfusion with 1 mM caffeine and depolarization of the membrane potential to 0 mV evoked periodic oscillations of outward currents with fairly uniform amplitudes and intervals. The oscillating outward currents induced by caffeine were dependent on intracellular Ca2+ concentration ([Ca2+]i) and had a reversal potential near to the equilibrium potential for K+. So the oscillating outward currents are carried by K+ through Ca(2+)-dependent K+ channels (IK(Ca)), and may reflect the oscillations of [Ca2+]i. The oscillating outward currents were abolished, or their frequency reduced, by lowering external [Ca2+], Ca2+ channel blockers, or by 1 microM ryanodine, indicating that: (1) there is a continuous influx of Ca2+ through the plasma membrane at a holding potential of 0 mV; (2) the periodic transient increases of [Ca2+]i are ascribed to the rhythmic release of Ca2+ from ryanodine-sensitive intracellular store by the mechanism of Ca(2+)-induced Ca2+ release (CICR). On the basis of the above results, we simulated the oscillation of [Ca2+]i induced by caffeine, which is known to lower the threshold of CICR. The patterns of peak amplitude histograms of spontaneous transient outward currents (STOC) in the oscillating cells were different from those in non-oscillating cells. The amplitudes of STOC in the latter were more variable than those in the former. The oscillating outward currents were modulated by 1 microM forskolin and 1 microM sodium nitroprusside, but STOC were little affected. The above differences between STOC and oscillating outward currents suggest that the two currents are activated by the Ca2+ originating from different intracellular Ca2+ stores which are functionally heterogeneous.

Background The roles of arachidonic acid (AA) metabolites in hypoxia-induced pulmonary vasoconstriction (HPV), a critical physiological mechanism that prevents ventilation/perfusion mismatch, are still incompletely understood. Methods Pulmonary arterial pressure was measured in ventilated/perfused rat lungs. Isometric tones of rat intralobar pulmonary arteries were also measured, using a myograph. Results Hypoxia ( P o 2 , 3%)-induced pulmonary arterial pressure increases (ΔPAP hypox ) were stable with blood-mixed perfusate, but decayed spontaneously. ΔPAP hypox was inhibited by 29%, 16%, and 28% by the thromboxane A 2 (TXA 2 ) antagonist SQ-29548, the 5-lipoxygenase inhibitor, MK886, and the leukotriene D 4 antagonist, LY-171883, respectively. The prostacyclin synthase inhibitor tranylcypromine augmented ΔPAP hypox by 5%, whereas inhibition of cytochrome P450 did not affect ΔPAP hypox . Consistently, the TXA 2 analogue U46619 increased ΔPAP hypox whereas prostacyclin abolished ΔPAP hypox . However, leukotriene D 4 had no direct effect on ΔPAP hypox . In the isolated pulmonary arteries, pretreatment with U46619 was essential to demonstrate hypoxia-induced contraction. Conclusions The above results suggest that TXA 2 and cysteinyl leukotrienes, other than leukotriene D 4 , are endogenous factors that facilitate HPV in rats. The indispensable role of TXA 2 -induced pretone in the HPV of isolated pulmonary arteries indicates that the signal from thromboxane receptors might be a critical component of oxygen sensation mechanisms.

A new kinetic model of the Na(+)/H(+) exchanger (NHE) was developed by fitting a variety of major experimental findings, such as ion-dependencies, forward/reverse mode, and the turnover rate. The role of NHE in ion homeostasis was examined by implementing the NHE model in a minimum cell model including intracellular pH buffer, Na(+)/K(+) pump, background H(+), and Na(+) fluxes. This minimum cell model was validated by reconstructing recovery of pH(i) from acidification, accompanying transient increase in [Na(+)](i) due to NHE activity. Based on this cell model, steady-state relationships among pH(i), [Na(+)](I), and [Ca(2+)](i) were quantitatively determined, and thereby the critical level of acidosis for cell survival was predicted. The acidification reported during partial blockade of the Na(+)/K(+) pump was not attributed to a dissipation of the Na(+) gradient across the membrane, but to an increase in indirect H(+) production. This NHE model, though not adapted to the dimeric behavioral aspects of NHE, can provide a strong clue to quantitative prediction of degree of acidification and accompanying disturbance of ion homeostasis under various pathophysiological conditions.

To investigate the kinetics of the inward Na-Ca exchange tail current activated by internal calcium in rabbit atrial cells, the whole-cell patch-clamp technique with intracellular perfusion was used. We recorded the inward phase of this current during repolarizations following a brief 2-5 ms depolarizing pulse to +40 mV from a holding potential of -70 mV. Peak activation of the current occurs about 10 ms from the beginning of the depolarizing pulse, and it decays spontaneously with a slow timecourse. The voltage dependence of the process that activates the inward current from -40 mV to +40 mV has a very steep slope between -40 and -20 mV and then virtually saturates between -10 mV and +40 mV. The voltage dependence of the process that activates the inward current is steeper than that which activates the sarcolemmal calcium current, i$_{\text{Ca.L}}$, and the timecourse of the current relaxation is much slower at low-frequency stimulation and when using low concentrations of Ca-buffer. The magnitude and timecourse of the calcium transients estimated by the inward tail current are smaller and faster, and the slow component of decay was abolished by the presence of high intracellular concentrations of Ca-buffer or by high frequency stimulation. These observations suggest that calcium release from the sarcoplasmic reticulum may be triggered by only a small fraction of the sarcolemmal calcium current.

To investigate the underlying ionic mechanism of the late plateau phase of the action potential in rabbit atrium the whole-cell patch-clamp technique with intracellular perfusion was used. We recorded the inward current during repolarizations following a brief 2 ms depolarizing pulse to +40 mV from a holding potential of between —70 and —80 mV. The development of this current coincides with the onset of the late plateau phase of the action potential. Peak activation of the current occurs about 10 ms from the beginning of the depolarizing pulse, and it decays spon­taneously with a slow timecourse. Its voltage dependency from —40 mV to +40 mV shows very steep activation ( — 40 to —20 mV) and shows almost the same maximum magnitude between —10 mV and +40 mV. This behaviour is quite different from that of the calcium current. The inward current and the late plateau phase of the action potential were both abolished by the application of 5 mM EGTA, 1 μM ryanodine and by reducing the Na + gradient. The fully activated current-voltage relation of the inward current was plotted as the difference current before and after treatment with Ryanodine, Diltiazem, 20 mM Na + inside or 30 % Na + outside and shows an exponential voltage dependence with the largest magnitude of the current occurring at negative potentials. The current-voltage (I-V) curve was well fitted by the Na-Ca exchange equation, i = A exp (— (1 — r ) EF / RT ). The results suggest that the inward current contributes to the generation of the late plateau phase of the rabbit atrial action potential, and is activated by intracellular calcium released from the sarcoplasmic reticulum. Sarcoplasmic reticulum calcium release appears to be triggered both by the membrane voltage and by the calcium current. It is concluded that the inward current is generated by Na-Ca exchange.

Patch-clamp studies of anion channels show a bewildering variety in their properties and regulation mechanisms.2 Amongst them, the anion channels activated by hypo-osmotic cell swelling ( I Cl,swell) have drawn attention because of their wide expression in virtually every vertebrate cell(s), including cardiac myocytes, and their critical roles in cell volume regulation.2–4 Cells need to regulate their volume in the face of several external and internal challenges. For this purpose, cells are endowed with various ion channels and transporters that become activated upon cell swelling or shrinkage. I Cl,swell are also called by different names like volume-regulated anion channels and volume-stimulated osmolyte and anion channels, implicating their permeability to a wide variety of anionic molecules including organic substances and ATP4−. In spite of their importance and ubiquitous expression, their molecular nature is still the subject of controversy and remains unclear.2,3 One of the key issues in I Cl,swell is the activation mechanism, i.e. how is the hypo-osmotic stress transduced into anion channel activation? A direct mechanical gating is unlikely with regard to the considerable time lag between hypotonic challenge and steady-state activation of I Cl,swell. The list of suggested mechanisms or critical factors supported by experimental evidence include … *Corresponding author. Tel: +82 2 740 8224; fax: +82 2 763 9667. E-mail address : earmye{at}snu.ac.kr

Atrial fibrillation is the most prevalent arrhythmia, but the mechanisms by which it develops are not clear. Recently, over 90% of paroxysmal atrial fibrillation was found to be located inside the main pulmonary veins (PVs). We found that single cardiac myocytes isolated from the main PVs of rabbits generate spontaneous action potentials (SAP). We therefore assayed the electrical characteristics of these cardiomyocytes. Among the diverse ionic currents identified were INa, ICa,L, IK1, IKr, IKs, Ito, IKsus, Incx, Ipump, IKH and ICl,Ca. In contrast, IK1 was minimal, IKs could be detected only in the presence of 10 microM forskolin, and we were unable to detect If and ICa,T, the most important currents for pacemaking activity in sinoatrial node cells. To identify the main cause of SAP, we developed a model that can explain the electrical properties of these cardiomyocytes. After reconstructing the ionic currents based on experimental observations, we were able to use our model to successfully reconstruct the characteristics of the SAP of PV cardiomyocytes. The simulation showed that the major currents contributing to pacemaking depolarization were ICaL, IKr, a background current and Na+-K+ pump current. Deactivation kinetics of IKr was one of the major determinants of the rate of pacemaking depolarization. The steady state inactivation of Ito was shifted to the negative voltage and the activity of Ito was minimal in the range of the SAP. The major currents for the repolarization were IKr and Ipump. The amplitude of most currents in these cardiac myocytes was small and no currents did not exceed 30 pA during the SAP, indicating that slight activation of other inward or outward currents will have profound effects on the SAP. To our knowledge, this report is the first to show the simulation of SAP of PV cardiomyocytes. This model may help to study on the electrophysiological basis of paroxysmal atrial fibrillation originating from PVs.

In recent studies, we recorded transiently activated outward currents by the application of three-step voltage pulses to induce a reverse mode of Na + –Ca 2+ exchange (NCX). We found that these currents were mediated by a Ca 2+ -activated Cl − current. Based on the recent reports describing the atrial Ca 2+ transients, the Ca 2+ transient at the subsarcolemmal space was initiated and then diffused into the cytosolic space. Because the myocardium in the pulmonary vein is an extension of the atrium, the Ca 2+ -activated Cl − current may reflect the subsarcolemmal Ca 2+ dynamics. We tried to predict the subsarcolemmal Ca 2+ dynamics by simulating these current traces. According to recent reports on the geometry of atrial myocytes, we assumed that there were three compartments of sarcoplasmic reticulum (SR): a network SR, a junctional SR and a central SR. Based on these structures, we also divided the cytosolic space into three compartments: the junctional, subsarcolemmal and cytosolic spaces. Geometry information and cellular capacitance suggested that there were essentially no T-tubules in these cells. The basic physical data, such as the compartmental volumes, the diffusion coefficients and the stability coefficients of the Ca 2+ buffers, were obtained from the literature. In the simulation, we incorporated the NCX, the L-type Ca 2+ channel, the rapid activating outward rectifier K + channel, the Na + –K + pump, the SR Ca 2+ -pump, the ryanodine receptor, the Ca 2+ -activated Cl − channel and the dynamics of Na + , K + , Ca 2+ and Cl − . In these conditions, we could successfully reconstruct the Ca 2+ -activated Cl − currents. The simulation allowed estimation of the Ca 2+ dynamics of each compartment and the distribution of the Ca 2+ -activated Cl − channel and the NCX in the sarcolemma on the junctional or subsarcolemmal space.

The pacemaker activity of interstitial cells of Cajal (ICCs) has been known to initiate the propagation of slow waves along the whole gastrointestinal tract through spontaneous and repetitive generation of action potentials. We studied the mechanism of the pacemaker activity of ICCs in the mouse small intestine and tested it using a mathematical model. The model includes ion channels, exchanger, pumps and intracellular machinery for Ca 2+ regulation. The model also incorporates inositol 1,4,5-triphosphate (IP 3 ) production and IP 3 -mediated Ca 2+ release activities. Most of the parameters were obtained from the literature and were modified to fit the experimental results of ICCs from mouse small intestine. We were then able to compose a mathematical model that simulates the pacemaker activity of ICCs. The model generates pacemaker potentials regularly and repetitively as long as the simulation continues. The frequency was set at 20 min −1 and the duration at 50% repolarization was 639 ms. The resting and overshoot potentials were −78 and +1.2 mV, respectively. The reconstructed pacemaker potentials closely matched those obtained from animal experiments. The model supports the idea that cyclic changes in [Ca 2+ ] i and [IP 3 ] play key roles in the generation of ICC pacemaker activity in the mouse small intestine.

We examined the effects of the protein kinase A (PKA) inhibitor H-89 on voltage-dependent K(+) (K(V)) currents in freshly isolated rabbit coronary arterial smooth muscle cells, using a whole-cell patch clamp technique. H-89 inhibited the K(V) current in a concentration-dependent manner, with a K(d) value of 1.02 microM. However, the PKA inhibitors KT 5720 and Rp-8-CPT-cAMPS did not significantly alter the K(V) current or the inhibitory effects of H-89 on the K(V) current. Moreover, H-85, a structurally similar but inactive analog of H-89, showed similar inhibitory effects on the K(V) channel. H-89 had no effect on the voltage-dependency of activation or inactivation, or on recovery kinetics. These results suggest that in rabbit coronary arterial smooth muscle cells, H-89 inhibits the K(V) current directly by blocking the pore cavity, an effect independent of PKA inhibition.

The effects of H-89, a potent and selective inhibitor of protein kinase A (PKA) on Ca(2+)-activated K(+) (BK(Ca)) channels in coronary arterial smooth muscle cells were examined using a patch-clamp technique. In inside-out configuration, H-89 increased the NP(o) of the BK(Ca) channel, but it reduced the dwell time of BK(Ca) currents. In whole-cell configuration, H-89 markedly increased BK(Ca) currents in a concentration-dependent manner. The EC(50) was 0.470+/-0.0741 microM based on dwell time, 0.582+/-0.0691 microM based on the NP(o), and 0.519+/-0.0295 microM based on the whole-cell current, respectively. H-85, which is an inactive form of H-89, increased BK(Ca) currents, similar to the result of H-89. The other PKA inhibitors (Rp-8-CPT-cAMPs and KT 5720) and protein phosphatase inhibitor (okadaic acid, 1 microM) had little effect on BK(Ca) currents and did not significantly alter the stimulatory effects of 1 microM H-89. These findings suggest that H-89 increases the BK(Ca) current independently of PKA.

GsMTx-4, a polypeptide from the spider Grammostola spatulata, is an inhibitor of mechanosensitive channels. It is known to interact with lipid membranes, suggesting it partitions into the membrane to alter the channel gating, but the effect of the membrane charge on GsMTx-4 activity remains unknown. In this study, we found that GsMTx-4 more effectively interacts with anionic lipids than zwitterionic ones. The effect of GsMTx-4 on negatively charged membranes was similar to that of the antimicrobial peptide melittin, which led us to assess GsMTx-4's antimicrobial activity. Interestingly, we found that, in contrast to other neurotoxins, GsMTx-4 exhibited antimicrobial properties and was more active against Gram-positive than Gram-negative bacteria. These results suggest that GsMTx-4 exerts its antimicrobial effect by altering the packing of the membrane and/or inhibiting mechanosensitive channels. These findings could point the way towards a new class of antimicrobial peptides.

We studied inward rectifier K+ (Kir) channels in smooth muscle cells isolated from rabbit coronary arteries. In cells from small- (100 microm, SCASMC) and medium-diameter (100 approximately 200 microm, MCASMC) coronary arteries, Kir currents were clearly identified (11.2 +/- 0.6 and 4.2 +/- 0.6 pA pF at -140 mV in SCASMC and MCASMC, respectively) that were inhibited by Ba(2+) (50 microm). By contrast, a very low Kir current density (1.6 +/- 0.4 pA pF) was detected in cells from large-diameter coronary arteries (200 microm, LCASMC). The presence of Kir2.1 protein was confirmed in SCASMC in a Western blot assay. Endothelin-1 (ET-1) inhibited Kir currents in a dose-dependent manner. The inhibition of Kir currents by ET-1 was abolished by pretreatment with the protein kinase C (PKC) inhibitor staurosporine (100 nM) or GF 109203X (1 microm). The PKC activators phorbol 12,13-dibutyrate (PDBu) and 1-oleoyl-2-acetyl-sn-glycerol (OAG) reduced Kir currents. The ETA-receptor inhibitor BQ-123 prevented the ET-1-induced inhibition of Kir currents. The amplitudes of the ATP-dependent K+ (KATP), Ca(2+)-activated K+ (BKCa), and voltage-dependent K+ (KV) currents, and effects of ET-1 on these channels did not differ between SCASMC and LCASMC. From these results, we conclude that Kir channels are expressed at a higher density in SCASMC than in larger arteries and that the Kir channel activity is negatively regulated by the stimulation of ETA-receptors via the PKC pathway.

We studied the effect of adenosine on the Ba(2+)-sensitive K(IR) channels in the smooth muscle cells isolated from the small-diameter (100microm) coronary arteries of rabbit. Adenosine increased K(IR) currents in concentration-dependent manner (EC(50)=9.4+/-1.4microM, maximum increase of 153%). The adenosine-induced stimulation of K(IR) current was blocked by adenylyl cyclase inhibitor, SQ22536 and was mimicked by adenylyl cyclase activator, forskolin. The adenosine-induced increase of current was blocked by cyclic AMP-dependent protein kinase (PKA) inhibitors, KT 5720 and Rp-8-CPT-cAMPs. The adenosine-induced increase of K(IR) currents was blocked by an A(3)-selective antagonist MRS1334, while the antagonists of other subtypes (DPCPX for A(1), ZM241385 for A(2A), and alloxazine for A(2B)) were all ineffective. Furthermore, an A(3)-selective agonist, 2-Cl-IB-MECA induced increase of K(IR) currents. We also examined the effect of adenosine on coronary blood flow (CBF) rate by using the Langendorff-perfused heart. In the presence of glibenclamide to exclude the effects of ATP-sensitive K(+) (K(ATP)) channels, CBF was increased by adenosine (10microM), which was blocked by the addition of Ba(2+) (50microM). Above results suggest that adenosine increases K(IR) current via A(3) subtype through the activation of PKA in rabbit small-diameter coronary arterial smooth muscle cells.

The role of stretch-activated channels (SACs) on the stretch-induced changes of rat atrial myocytes was studied using a computer model that incorporated various ion channels and transporters including SACs. A relationship between the extent of the stretch and the activation of SACs was formulated in the model based on experimental findings to reproduce changes in electrical activity and Ca(2+) transients by stretch. Action potentials (APs) were significantly changed by the activation of SACs in the model simulation. The duration of the APs decreased at the initial fast phase and increased at the late slow phase of repolarisation. The resting membrane potential was depolarised from -82 to -70 mV. The Ca(2+) transients were also affected. A prolonged activation of SACs in the model gradually increased the amplitude of the Ca(2+) transients. The removal of Ca((2+)) permeability through SACs, however, had little effect on the stretch-induced changes in electrical activity and Ca(2+) transients in the control condition. In contrast, the removal of the Na(+) permeability nearly abolished these stretch-induced changes. Plotting the peaks of the Ca((2+)) transients during the activation of the SACs along a time axis revealed that they follow the time course of the Na(i)(+) concentration. The Ca((2+)) transients were not changed when the Na(i)(+) concentration was fixed to a control value (5.4mM). These results predicted by the model suggest that the influx of Na(+) rather than Ca(2+) through SACs is more crucial to the generation of stretch-induced changes in the electrical activity and associated Ca(2+) transients of rat atrial myocytes.