Cytotoxicity studies of 2‐hydroxymethyl‐1‐naphthol diacetate on K+ currents in neoplastic plasma cells

The present study investigated the effects of 2‐hydroxymethyl‐1‐naphthol diacetate (TAC) on cell proliferation and K⁺ currents in RPMI‐8226 human myeloma cells. In cells with intracellular Ca²⁺ concentration ([Ca²⁺]_i) = 10 nM, depolarizing square pulses from a holding potential of –80 mV elicited instantaneous outward current with slow inactivation, corresponding to voltage‐activated K⁺ current. TAC (1–100 μM) inhibited I_K(V) in a concentration‐dependent manner. A23187 (1 μM), a Ca²⁺ ionophore, can potentiate Ca²⁺‐activated K⁺ current (I_K(Ca)). Tetraethylammonium chloride (10 mM) caused a small decrease in the amplitude of I_K(Ca) elicited by A23187, whereas TAC (30 μM) and quinidine (10 μM) decreased I_K(Ca) more effectively. The present results show that TAC directly blocks voltage‐ and Ca²⁺‐activated K⁺ currents in human myeloma cells. TAC inhibited both cell proliferation and voltage‐activated K⁺ current with an effective dose inducing half‐maximum effects at 3.8 ± 0.8 μM and 10 ± 1.5 μM, respectively. The present study suggests that the cytotoxic effect of TAC in cancer cells may be partially explained by blockade of K⁺ channels. The delocalization energy of TAC and other analogs was employed to compare their ability to block the voltage‐activated K⁺ channel in myeloma cells. It was found that naphthol derivatives‐mediated blockade of voltage‐activated K⁺ channel might relate to the level of delocalization energy and molecular volume. Drug Dev. Res. 47:1–8, 1999. © 1999 Wiley‐Liss, Inc.     

Modification of potassium-evoked release of noradrenaline by various ions and agents

1 Release of noradrenaline (NA) from isolated spleen slices of the cat by high K⁺ and tetraethylammonium (TEA) was investigated. Studies were conducted with spleen slices whose tissue stores were prelabelled with [³H]-noradrenaline ([³H]-NA).

2 Release by high K⁺ was related to the K⁺ concentration of the incubation medium. Release of [³H]-NA by 28.5 mM K⁺ was only barely detectable over the background, while 70 mM K⁺ enhanced release to more than 600% of the background output. Tetrodotoxin (TTX) did not block responses to 28.5 or 35 mM K⁺.

3 Background release was not modified by 1 or 3 mM TEA, but 10 and 30 mM TEA enhanced the release of [³H]-NA by about 50% and 150%, respectively, over the background level. Neither TTX nor hexamethonium (C₆) blocked the TEA response. Release by TEA was also not blocked in Ca²⁺-free medium or in Ca²⁺-free medium containing up to 3 mM EGTA. Release by TEA was blocked in Ca²⁺—free medium containing 5 mM EGTA, and by La³⁺ or Mn²⁺.

4 The response to 35 mM K⁺ was not modified by 1 or 3 mM TEA; 10 mM TEA had an additive effect; and 30 mM TEA with 35 mM K⁺ produced a response which was greater than the simple sum of responses to 35 mM K⁺ and 30 mM TEA. At 45 mM K⁺, 3 and 10 mM TEA potentiated the response, and at 30 mM K⁺ only 1 mM TEA showed potentiation. TTX did not alter the response to high K⁺ plus TEA.

5 When TEA (30 mM) was added during prolonged incubation with 140 mM K⁺, the response was only slightly enhanced. This suggests that a large part of the secretory response to TEA is mediated through mobilization of Ca²⁺ activated by depolarization.

6 Phenoxybenzamine (3.3 μM) potentiated responses to 35 and 140 mM K⁺ by about 50%, and TTX did not influence this potentiation. Acetylcholine (ACh) blocked responses to 28.5 and 35 mM K⁺, and 1 mM TEA antagonized this ACh blockade.

7 In the perfused adrenal gland of the cat, the secretory response to TEA was related to its concentration. The response was not diminished by low Na⁺, TTX, or C₆, but was markedly attenuated when TEA was applied 10 min after the start of perfusion with high K⁺.

     

Activation of large‐conductance Ca2+‐activated K+ channels by pinacidil in human umbilical vascular endothelial cells

The effect of pinacidil, an opener of ATP‐sensitive K⁺ (K_ATP) channels, on large‐conductance Ca²⁺‐activated K+ (BK_Ca) channels was investigated in cultured endothelial cells of human umbilical veins. In whole cell configuration, pinacidil (30 μM) increased the amplitude of K⁺ outward currents (I_K). Charybdotoxin (100 nM), but not glibenclamide (10 μM), suppressed pinacidil‐induced increase in I_K. Neither carbonyl cyanide m‐chlorophenyl hydrazone (CCCP; 10 μM), an inhibitor of mitochondrial Ca²⁺‐uniporter, nor cyclosporin A (200 nM), an inhibitor of the mitochondrial permeability transition pore, affected pinacidil‐induced increase in I_K. In inside‐out patch configuration, bath application of pinacidil (30 μM) did not change single channel conductance but increased the activity of BK_Ca channels. Pinacidil (30 μM) shifted the activation curve of BK_Ca channels to less positive membrane potential by approximately 15 mV. Pinacidil stimulated the activity of these channels in a concentration‐dependent manner. The EC₅₀ value for pinacidil‐induced channel activity was 20 μM. After BK_Ca channels had been enhanced by Evans blue (100 μM), subsequent application of pinacidil (100 μM) did not further increase the channel activity. These results clearly indicate that in addition to the activation of K_ATP channels, pinacidil can also stimulate BK_Ca channels in endothelial cells. These effects could contribute to the regulation of vascular tone if similar results were found in endothelial cells in vivo. Drug Dev. Res. 48:6–16, 1999. © 1999 Wiley‐Liss, Inc.     

The Influences of G Proteins, Ca2+, and K+ Channels on Electrical Field Stimulation in Cat Esophageal Smooth Muscle

NO released by myenteric neurons controls the off contraction induced by electrical field stimulation (EFS) in distal esophageal smooth muscle, but in the presence of nitric oxide synthase (NOS) inhibitor, L-NAME, contraction by EFS occurs at the same time. The authors investigated the intracellular signaling pathways related with G protein and ionic channel EFS-induced contraction using cat esophageal muscles. EFS-induced contractions were significantly suppressed by tetrodotoxin (1 µM) and atropine (1 µM). Furthermore, nimodipine inhibited both on and off contractions by EFS in a concentration dependent meaner. The characteristics of ''on'' and ''off'' contraction and the effects of G-proteins, phospholipase, and K⁺ channel on EFS-induced contraction in smooth muscle were also investigated. Pertussis toxin (PTX, a G_i inactivator) attenuated both EFS-induced contractions. Cholera toxin (CTX, G_s inactivator) also decreased the amplitudes of EFS-induced off and on contractions. However, phospholipase inhibitors did not affect these contractions. Pinacidil (a K⁺ channel opener) decreased these contractions, and tetraethylammonium (TEA, K⁺_Ca channel blocker) increased them. These results suggest that EFS-induced on and off contractions can be mediated by the activations Gi or Gs proteins, and that L-type Ca²⁺ channel may be activated by G-protein α subunits. Furthermore, K⁺_Ca-channel involve in the depolarization of esophageal smooth muscle. Further studies are required to characterize the physiological regulation of Ca²⁺ channel and to investigate the effects of other K⁺ channels on EFS-induced on and off contractions.     

Inhibitory effects of large Ca2+-activated K+-channel blockers on β-adrenergic- and NO-donor-mediated relaxations of human and guinea-pig airway smooth muscles

In human bronchi, relaxations to salbutamol and sodium nitroprusside were performed in the presence or absence of blockers of the large Ca²⁺-activated K⁺-channels (BK_Ca): charybdotoxin (Chtx), iberiotoxin (Ibtx) or tetraethylammonium (TEA). In bronchi under basal tone in presence of indomethacin (1 μM) or precontracted with acetylcholine (in presence or absence of indomethacin), the relaxations to salbutamol or sodium nitroprusside were unaffected or weakly inhibited by pretreatment with the BK_Ca blockers (Chtx (100 nM), Ibtx (100 and 300 nM) and TEA (1 mM)). Significant inhibitions were mainly observed with TEA (1 mM) and iberiotoxin at high concentration (300 nM). These results contrasts with the potent inhibitory effects exerted by Chtx (100 nM) or Ibtx (100 nM) in guinea-pig trachea precontracted with acetylcholine in absence or presence of indomethacin indicating that human airways are less susceptible to BK_Ca blockade than guinea-pig airways. In addition, the BK_Ca blockers induced slowly developing contractions of human bronchi at basal tone. The contraction induced by TEA (1 mM) was abolished by verapamil (10 μM) suggesting that BK_Ca blockade promotes an increase in membrane Ca²⁺-conductance through activation of voltage-gated Ca²⁺-channels. Verapamil also reversed the effects of TEA on salbutamol-induced relaxations in human bronchi as well as the effects of Ibtx on salbutamol- or sodium nitroprusside-induced relaxations in guinea-pig trachea. These data suggest that BK_Ca blockers induce activation of voltage-gated Ca²⁺-channels and therefore influx of Ca²⁺ which in turn cause a functional antagonism of β₂-adrenoceptor-agonist- and NO-donor-induced relaxations. Moreover, the BK_Ca opener, NS-1619, induced weak relaxations in human bronchi and guinea-pig trachea which were not blocked by TEA or Ibtx suggesting that BK_Ca opening is of minor significance for the relaxation of human airway smooth muscles. In conclusion, although a wealth of studies have demonstrated that β-adrenoceptor agonists or NO-donors activate BK_Ca, the present study provides evidence that in human bronchi, as recently suggested in guinea-pig trachea, opening of BK_Ca does not appear to functionally participate in the relaxation to these relaxant agents. Received: 25 April 1997 / Accepted: 15 September 1997     

The signalling role of action potential depolarization in insulin secretion: Metabolism-dependent dissociation between action potential increase and secretion increase by TEA

The K⁺ channel blocker, TEA is known to increase action potential amplitude and insulin secretion of mouse β-cells when added to a nutrient secretagogue. In the presence of a maximally effective sulfonylurea concentration (2.7 μM glipizide) the nutrient secretagogue α-ketoisocaproic acid (KIC, 10 mM) strongly increased insulin secretion (about elevenfold). Instead of enhancing the effect of KIC, TEA reduced the KIC-induced secretion by more than 50%. Also, the secretion rate produced by 2.7 μM glipizide alone was significantly reduced by TEA. In contrast, TEA enhanced the insulinotropic effect of glipizide when a basal glucose concentration (5 mM) was present. In the presence as well as in the absence of glucose glipizide produced a plateau depolarization with superimposed action potentials. Under both conditions, TEA increased the glipizide-induced action potential amplitude and further elevated the cytosolic free calcium concentration ([Ca²⁺]_i) with an oscillatory characteristic. These effects depended on the activity of L-type Ca²⁺ channels, even though the effect of TEA differed from that of 1 μM of the Ca²⁺ channel opener, Bay K8644, which primarily increased action potential duration. TEA did not negatively affect parameters of β-cell energy metabolism (NAD(P)H fluorescence and ATP/ADP ratio), rather, it slightly increased NAD(P)H fluorescence. Apparently, TEA inhibits insulin secretion in the absence of glucose in spite of a persistent ability to block K⁺ ion conductance. Thus, the signalling role of action potential depolarization in insulin secretion may require reconsideration and ion conductance-independent actions of K⁺ channels may be involved in this paradox effect of TEA.     

Modulation of Ca2+-activated K+ current by isoprenaline,carbachol, and phorbol ester in cultured (and fresh) rat aortic vascular smooth muscle cells

• 1.1. Effects of isoprenaline (ISO), carbachol, and phorbol ester on the outward K⁺ currents in single cultured (or fresh) rat aortic vascular smooth muscle (A7r5 and A-10) cells were examined using a whole-cell voltage-clamp (at room temperature 22°C).
• 2.2. With 10 mM EGTA in the pipette solution, the delayed rectifier K⁺ current (I_K) was activated by Ca²⁺ at pCa 7 more than at pCa 10, and was TEA (10 mM) and apamin (200 nM) sensitive, which represents a Ca²⁺-activated K⁺ current (I_KCa).
• 3.3. In cultured A7r5 cells, isoprenaline (1 and 5 μM) and carbachol (0.1 and 1 μM) inhibited I_KCa. Phorbol ester, 4-β-phorbol-12, 13-dibutyrate (PDB), at 0.1 and 1 μM also inhibited I_KCa and increased the inhibitory effects induced by isoprenaline (1 μM).
• 4.4. In fresh aortic cells, these drugs, at the same concentrations, also produced the similar effects.
• 5.5. In A-10 cells, PDB (1 μM) enhanced the transient outward current (4-AP-sensitive), but ISO (1 μM) inhibited the current.
• 6.6. These results suggest that the I_KCa current would be inhibited by cyclic nucleotides (cAMP and cGMP) and also by PK-C stimulation, and thereby be directly contributed to excitation-contraction coupling of the vascular smooth muscle cells.

     

Large- and small-conductance Ca2+-activated K+ channels: their role in the nicotinic receptor-mediated catecholamine secretion in bovine adrenal medulla

In cultured bovine adrenal chromaffln cells, charybdotoxin and iberiotoxin (inhibitors of the large-conductance Ca²⁺-activated K⁺ channel) as well as apamin (an inhibitor of the small-conductance Ca²⁺-activated K⁺ channel), at 1–100 nM, suppressed carbachol-induced ⁸⁶Rb⁺ efflux, augmented carbachol-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels and catecholamine secretion and had no effect on carbachol-induced ²²Na⁺ influx via nicotinic receptors, a prerequisite for Ca²⁺ channel activation by carbachol. ⁴⁵Ca²⁺ influx caused by high K⁺ (a direct activation of voltage-dependent Ca²⁺ channels) was also enhanced by these K⁺ channel inhibitors, with the concentration-response curves being similar to those for carbachol-induced ⁴⁵Ca²⁺ influx. Dendrotoxin and mast cell degranulating peptide (inhibitors of voltage-dependent K⁺ channels), on the other hand, did not alter carbachol-induced ⁸⁶Rb⁺ efflux or ⁴⁵Ca²⁺ influx.These results suggest that the stimulation of nicotinic receptors eventually opens large- and small-conductance Ca²⁺-activated K⁺ channels, and that the blockade of these Ca²⁺-activated K⁺ channels results in gating of voltage-dependent Ca²⁺ channels and thereby augments catecholamine secretion from bovine adrenal chromaffln cells.     

Increased inward rectifier K+ current of coronary artery smooth muscle cells in spontaneously hypertensive rats; partial compensation of the attenuated endothelium-dependent relaxation via Ca2+-activated K+ channels

Endothelium-dependent vasorelaxation is partly mediated by small-conductance (SK3) and intermediate-conductance Ca²⁺-activated K⁺ channels (SK4) in the endothelium that results in endothelium-dependent hyperpolarization (EDH). Apart from the electrical propagation through myoendothelial gap junctions, the K⁺ released from the endothelium facilitates EDH by increasing inward rectifier K⁺ channel (Kir) conductance in smooth muscle cells. The EDH-dependent relaxation of coronary artery (CA) and Kir current in smooth muscle cells (CASMCs) of hypertensive animals are poorly understood despite the critical role of coronary flow in the hypertrophic heart. In spontaneously hypertensive (SHR) and control (WKY) rats, we found attenuation of the CA relaxation by activators of SK3 and SK4 (NS309 and 1-EBIO) in SHR. In isolated CASMCs, whole-cell patch-clamp study revealed larger I_Kir in SHR than WKY, whereas the myocytes of skeletal and cerebral arteries showed smaller I_Kir in SHR than WKY. While the treatment with I_Kir inhibitor (0.1 mmol/L Ba²⁺) alone did not affect the WKY-CA, the SHR-CA showed significant contractile response, suggesting relaxing influence of the higher I_Kir in the CASMCs of SHR. Furthermore, the attenuation of NS309-induced relaxation of CA by the combined treatment with 0.1 mmol/L Ba²⁺ was more prominent in SHR than WKY. Our study firstly shows a distinct increase of I_Kir in the CASMCs of SHR, which could partly compensate for the attenuated relaxation via endothelial SK3 and SK4.     

Loss of Ca2+-mediated ion transport during colitis correlates with reduced ion transport responses to a Ca2+-activated K+ channel opener

Background and purpose:

Epithelial surface hydration is critical for proper gut function. However, colonic tissues from individuals with inflammatory bowel disease or animals with colitis are hyporesponsive to Cl⁻ secretagogues. The Cl⁻ secretory responses to the muscarinic receptor agonist bethanechol are virtually absent in colons of mice with dextran sodium sulphate (DSS)-induced colitis. Our aim was to define the mechanism underlying this cholinergic hyporesponsiveness.

Experimental approach:

Colitis was induced by 4% DSS water, given orally. Epithelial ion transport was measured in Ussing chambers. Colonic crypts were isolated and processed for mRNA expression via RT-PCR and protein expression via immunoblotting and immunolocalization.

Key results:

Expression of muscarinic M₃ receptors in colonic epithelium was not decreased during colitis. Short-circuit current (I_SC) responses to other Ca²⁺-dependent secretagogues (histamine, thapsigargin, cyclopiazonic acid and calcium ionophore) were either absent or severely attenuated in colonic tissue from DSS-treated mice. mRNA levels of several ion transport molecules (a Ca²⁺-regulated Cl⁻ channel, the intermediate-conductance Ca²⁺-activated K⁺ channel, the cystic fibrosis transmembrane conductance regulator, the Na⁺/K⁺-ATPase pump or the Na⁺/K⁺/2Cl⁻ co-transporter) were not reduced in colonic crypts from DSS-treated mice. However, protein expression of Na⁺/K⁺-ATPase α1 subunits was decreased twofold during colitis. Activation of Ca²⁺-activated K⁺ channels increased I_SC significantly less in DSS colons compared with control, as did the protein kinase C activator, phorbol 12-myristate 13-acetate.

Conclusions and implications:

Decreased Na⁺/K⁺-ATPase expression probably contributes to overall epithelial hyporesponsiveness during colitis, while dysfunctional K⁺ channels may account, at least partially, for lack of epithelial secretory responses to Ca²⁺-mediated secretagogues.     

Relaxant Effect of Spermidine on Acethylcholine and High K+-induced Gastric Contractions of Guinea-Pig

In our previous study, we found that spermine and putrescine inhibited spontaneous and acetylcholine (ACh)-induced contractions of guinea-pig stomach via inhibition of L-type voltage-dependent calcium current (VDCC_L). In this study, we also studied the effect of spermidine on mechanical contractions and calcium channel current (I_Ba), and then compared its effects to those by spermine and putrescine. Spermidine inhibited spontaneous contraction of the gastric smooth muscle in a concentration-dependent manner (IC₅₀=1.1±0.11 mM). Relationship between inhibition of contraction and calcium current by spermidine was studied using 50 mM high K⁺-induced contraction: Spermidine (5 mM) significantly reduced high K⁺ (50 mM)-induced contraction to 37±4.7% of the control (p<0.05), and inhibitory effect of spermidine on I_Ba was also observed at a wide range of test potential in current/voltage (I/V) relationship. Pre- and post-application of spermidine (5 mM) also significantly inhibited carbachol (CCh) and ACh-induced initial and phasic contractions. Finally, caffeine (10 mM)-induced contraction which is activated by Ca²⁺-induced Ca²⁺ release (CICR),'' was also inhibited by pretreatment of spermidine (5 mM). These findings suggest that spermidine inhibits spontaneous and CCh-induced contraction via inhibition of VDCC_L and Ca²⁺ releasing mechanism in guinea-pig stomach.     

Presynaptic muscarinic receptors and the release of acetylcholine from cerebrocortical prisms: roles of Ca2+ and K+ concentrations

Summary The mechanism by which presynaptic muscarinic autoreceptors inhibit the release of acetylcholine (ACh) from cerebrocortical cholinergic fibres has not been clarified. To test the view that muscarinic autoreceptors act by decreasing Ca²⁺ influx, we performed experiments in which rat cerebrocortical prisms were preloaded with (¹⁴C)choline, washed, depolarized with 14–65 mM K⁺ in the absence of Ca²⁺ and then exposed (still under depolarization) to various concentrations of Ca²⁺ to evoke the release of (¹⁴C)ACh. The muscarinic agonist, oxotremorine, used at a 100 M concentration, inhibited the release of (¹⁴C)ACh by 59–86% in experiments with 14 and 26.5 mM K⁺ but had no significant effect at 65.5 mM K⁺. No systematic changes in the inhibitory effects of oxotremorine could be found at any of the K⁺ concentrations used when the concentration of Ca²⁺ was varied in the range of 0.25–4.0 mM. At 2 mM Ca ²⁺ and K⁺ concentrations above 14 mM, the inhibitory effect of oxotremorine was inversely related to the concentration of K⁺. The inhibitory effect of oxotremorine on (¹⁴C)ACh release was not blocked by 100 M 4-aminopyridine. The fact that the inhibitory effect of oxotremorine could not be overcome by an increase in the concentration of Ca²⁺ suggests that, under the conditions used, a restriction of the influx of Ca²⁺ did not play a major role in the muscarinic inhibition of ACh release; rather, oxotremorine appeared to act by decreasing membrane depolarization. Our observations are best explained in terms of the Ca²⁺-voltage hypothesis of neurotransmitter release, supposing that a voltage-activated molecule is involved in the control of the release process at a post-Ca²⁺-entry stage. Correspondence to S. Tuek at the above address     

Action potential bursts in central snail neurons elicited by paeonol: roles of ionic currents

Aim:

To investigate the effects of 2′-hydroxy-4′-methoxyacetophenone (paeonol) on the electrophysiological behavior of a central neuron (right parietal 4; RP4) of the giant African snail (Achatina fulica Ferussac).

Methods:

Intracellular recordings and the two-electrode voltage clamp method were used to study the effects of paeonol on the RP4 neuron.

Results:

The RP4 neuron generated spontaneous action potentials. Bath application of paeonol at a concentration of ≥500 μmol/L reversibly elicited action potential bursts in a concentration-dependent manner. Immersing the neurons in Co²⁺-substituted Ca²⁺-free solution did not block paeonol-elicited bursting. Pretreatment with the protein kinase A (PKA) inhibitor KT-5720 or the protein kinase C (PKC) inhibitor Ro 31-8220 did not affect the action potential bursts. Voltage-clamp studies revealed that paeonol at a concentration of 500 μmol/L had no remarkable effects on the total inward currents, whereas paeonol decreased the delayed rectifying K⁺ current (I_KD) and the fast-inactivating K⁺ current (I_A). Application of 4-aminopyridine (4-AP 5 mmol/L), an inhibitor of I_A, or charybdotoxin 250 nmol/L, an inhibitor of the Ca²⁺-activated K⁺ current (I_K(Ca)), failed to elicit action potential bursts, whereas tetraethylammonium chloride (TEA 50 mmol/L), an I_KD blocker, successfully elicited action potential bursts. At a lower concentration of 5 mmol/L, TEA facilitated the induction of action potential bursts elicited by paeonol.

Conclusion:

Paeonol elicited a bursting firing pattern of action potentials in the RP4 neuron and this activity relates closely to the inhibitory effects of paeonol on the I_KD.     

Forskolin Changes the Relationship between Cytosolic Ca2+ and Contraction in Guinea Pig Ileum

This study was designed to clarify the mechanism of the inhibitory effect of forskolin on contraction, cytosolic Ca²⁺ level ([Ca²⁺]_i), and Ca²⁺ sensitivity in guinea pig ileum. Forskolin (0.1 nM~10 µM) inhibited high K⁺ (25 mM and 40 mM)- or histamine (3 µM)-evoked contractions in a concentration-dependent manner. Histamine-evoked contractions were more sensitive to forskolin than high K⁺-evoked contractions. Spontaneous changes in [Ca²⁺]_i and contractions were inhibited by forskolin (1 µM) without changing the resting [Ca²⁺]_i. Forskoln (10 µM) inhibited muscle tension more strongly than [Ca²⁺]_i stimulated by high K⁺, and thus shifted the [Ca²⁺]_i-tension relationship to the lower-right. In histamine-stimulated contractions, forskolin (1 µM) inhibited both [Ca²⁺]_i and muscle tension without changing the [Ca²⁺]_i-tension relationship. In α-toxin-permeabilized tissues, forskolin (10 µM) inhibited the 0.3 µM Ca²⁺-evoked contractions in the presence of 0.1 mM GTP, but showed no effect on the Ca²⁺-tension relationship. We conclude that forskolin inhibits smooth muscle contractions by the following two mechanisms: a decrease in Ca²⁺ sensitivity of contractile elements in high K⁺-stimulated muscle and a decrease in [Ca²⁺]_i in histamine-stimulated muscle.     

Neomycin blocks dihydropyridine-insensitive Ca2+ influx in bovine adrenal chromaffin cells

There is evidence that bovine adrenal chromaffin cells are provided with both dihydropyridine-sensitive and -resistant voltage-sensitive Ca²⁺ influx pathways. Although recent electrophysiological work indicates that the dihydropyridine-resistant pathway is partially mediated by w-conotoxin-sensitive and -insensitive Ca²⁺ channels, the pharmacological sensitivity of the latter channels remains elusive. We have now found that combined incubations with nitrendipine (1 μM) and neomycin (0.5 mM) reduced high K⁺ (50 mM)-evoked intracellular Ca²⁺ concentration ([Ca²⁺]_i) transients to a larger extent than each drug separately. [Ca²⁺]_i was measured using the fluorescent intracellular Ca²⁺ indicator fura-2. Neomycin (0.05−2 mM) reduced high K⁺-evoked ⁴⁵Ca²⁺ uptake in a dose-dependent manner (IC₅₀ = 0.09 mM). In the presence of nitrendipine (1 μM), the minimal neomycin concentration necessary for total blockade of ⁴⁵Ca²⁺ uptake was reduced to 0.3 mM. Moreover, in the absence of nitrendipine the ⁴⁵Ca²⁺ uptake remaining in 0.3 mM neomycin (26% of maximum) was similar to the fractional inhibition by nitrendipine alone (29%). Neomycin (0.05−2 mM) inhibited the [Ca²⁺]_i transient induced by the L-type Ca²⁺ channel agonist Bay K 8644 (1 μM) much more extensively at 2 mM than at 0.3 mM (percent inhibition = 59% and 15%, respectively). Neomycin (0.05−2 mM) blocked high K⁺-evoked noradrenaline and adrenaline release in a dose-dependent fashion (IC₅₀ = 0.8−1.1 mM), the blockade efficiency being enhanced in the presence of 1 μM nitrendipine (IC₅₀ = 0.17−0.19 mM). It is concluded that neomycin (≤ 0.3 mM) blocks preferentially the dihydropyridine-insensitive Ca²⁺ influx pathway of the chromaffin cell. Moreover, both the dihydropyridine-sensitive and the dihydropyridine-resistant, neomycin-sensitive Ca²⁺ influx pathways contribute strongly to depolarization-evoked catecholamine secretion.     

Involvement of different calcium channels in K+- and veratridine-induced increases of cytosolic calcium concentration in rat cerebral cortical synaptosomes

Intracellular calcium ion concentrations ([Ca²⁺]_i) in rat cerebral cortical synaptosomes were measured, using the calcium chelating fluorescence dye fura-2. The synaptosomes were depolarized by elevation of the extracellular K⁺ concentration or by addition of veratridine, which opens voltage-dependent Na⁺-channels and prevents their inactivation. Both enhancement of the concentration of extracellular K⁺ (up to 60 mM) and veratridine (1–100 μM) increased the [Ca²⁺]_i in a concentration-dependent manner. In the absence of extracellular Ca²⁺, the K⁺- and veratridine-induced increases in [Ca²⁺]_i were abolished, indicating that the increase in [Ca²⁺]_i was due to an influx of extracellular Ca²⁺. Tetrodotoxin (TTX), a blocker of the voltage-dependent Na⁺ channel, inhibited the veratridine-induced (10 μM) Ca²⁺ influx by more than 80%, while the K⁺-evoked (30 mM) increase of [Ca²⁺]_i was TTX-resistant. Both the K⁺- and the veratridine-induced Ca²⁺ influx were not reduced by nifedipine (1 μM), a blocker of L-type Ca²⁺ channels. Blockade of the voltage dependent N-type Ca²⁺ channels with ω-conotoxin GVIA (ω-CTx GVIA; 0.1 μM) and of the voltage-dependent P/Q-type channels with ω-agatoxin IVA (ω-AgaTx IVA; 0.2 μM) inhibited the K⁺-induced increase in [Ca²⁺]_i by about 30 and 55%, respectively; these effects were additive. ω-Conotoxin MVIIC (ω-CTx MVIIC) at a concentration of 0.2 μM, which may be assumed to block predominantly the Q-type Ca²⁺ channel, inhibited the K⁺-induced increase in [Ca²⁺]_i by 50%. The veratridine-induced increase in [Ca²⁺]_i was reduced by about 25% by ω-CTx GVIA (0.1 μM), but was resistant to ω-AgaTx IVA (0.2 μM) and ω-CTx MVIIC (0.2 μM). Mibefradil (former designation Ro 40-5967), a Ca²⁺ antagonist which blocks all types of voltage-dependent Ca²⁺ channels including the T and R channels, led to a concentration-dependent inhibition of the K⁺- and veratridine-induced increase in [Ca²⁺]_i (abolition at 10 μM mibefradil). Ifenprodil, another non-specific blocker of voltage-dependent Ca²⁺ channels, also inhibited the K⁺- and veratridine-induced increase in [Ca²⁺]_i in concentration-dependent manner and abolished it at 320 μM ifenprodil. In contrast, KB-R 7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate; 1 and 3 μM), a highly potent and selective inhibitor of the Na⁺/Ca²⁺ exchanger (NCX1), failed to inhibit the K⁺- and veratridine-induced increase in [Ca²⁺]_i. It is concluded that the K⁺-induced increase in free cytosolic Ca²⁺ results from Ca²⁺ influx through voltage-dependent N- and, above all, Q-type Ca²⁺ channels. N-type Ca²⁺ channels also play a minor role in the veratridine-induced increase in [Ca²⁺]_i, but P/Q-type channels do not appear to be involved at all. The inhibition of the veratridine-induced, ω-CTx GVIA- and ω-AgaTx IVA-resistant increase in [Ca²⁺]_i by mibefradil and the failure of KB-R 7943 to inhibit this response are compatible with the suggestion that in rat cerebral cortical synaptosomes, Ca²⁺ influx via the R-type Ca²⁺ channel and/or another so far uncharacterized Ca²⁺ channel may substantially contribute to the veratridine-induced increase in [Ca²⁺]_i. Received: 7 March 1997 / Accepted: 9 September 1997     

(-)-Epigallocatechin Gallate Inhibits the Pacemaker Activity of Interstitial Cells of Cajal of Mouse Small Intestine

The effects of (-)-epigallocatechin gallate (EGCG) on pacemaker activities of cultured interstitial cells of Cajal (ICC) from murine small intestine were investigated using whole-cell patch-clamp technique at 30℃ and Ca²⁺ image analysis. ICC generated spontaneous pacemaker currents at a holding potential of -70 mV. The treatment of ICC with EGCG resulted in a dose-dependent decrease in the frequency and amplitude of pacemaker currents. SQ-22536, an adenylate cyclase inhibitor, and ODQ, a guanylate cyclase inhibitor, did not inhibit the effects of EGCG. EGCG-induced effects on pacemaker currents were not inhibited by glibenclamide, an ATP-sensitive K⁺ channel blocker and TEA, a Ca²⁺-activated K⁺ channel blocker. Also, we found that EGCG inhibited the spontaneous [Ca²⁺]_i oscillations in cultured ICC. In conclusion, EGCG inhibited the pacemaker activity of ICC and reduced [Ca²⁺]_i oscillations by cAMP-, cGMP-, ATP-sensitive K+ channel-independent manner.     

MECHANISMS FOR REGULATION OF ARTERIAL TONE BY Ca2+- DEPENDENT K+ CHANNELS IN HYPERTENSION

1. The membrane potential and reactivity of arterial smooth muscle cells is regulated by a variety of K⁺ channels, which are highly expressed in vascular smooth muscle meuscle membrances. 2. Of these K⁺ channel types, the high-conductance, Ca²⁺-ependent K⁺ channel appears to be up-regulated in arterial smooth muscle membrances from hypertensive animals. 3. Patch-clamp studies show that whole-cell membrances and membrane patches of arterial smooth muscle obtained from rats with genetic or renal hypertension show an increased macroscopic and single-channel Ca²⁺-activated K⁺ current. Pharmacological block of this K⁺ current profoundly constricts aortic, renal, mesenteric and femoral arteries obtained from the same hypertensive animals, suggesting that Ca²⁺-dependent K⁺ current is a critical determinant of resting membrane potential in arterial muscle exposed to elevated blood pressure. 4. Thus, K⁺ efflux through Ca²⁺-dependent K⁺ channels appears to constitute an important homeostatic mechanism for buffering increases in arterial reactivity in hypertension.     

四肽FMRFa对大鼠心室肌Na+/Ca2+交换的抑制

目的　研究四肽FMRFa对大鼠单个心室肌细胞Na⁺/Ca²⁺交换的作用。方法　用膜片钳全细胞记录法测定成年大鼠心室肌细胞Na⁺/Ca²⁺交换电流(I_Na⁺/Ca²⁺)和其他离子通道电流。结果　FMRFa对大鼠心室肌细胞I_Na⁺/Ca²⁺呈浓度依赖性抑制,100μmol·L^-1浓度时抑制内向和外向I_Na⁺/Ca²⁺密度分别达60.1%和56.5%,对内向电流及外向电流的IC₅₀分别为20μmol·L^-1和34μmol·L^-1。FMRFa5μmol·L^-1抑制I_Na⁺/Ca²⁺内向和外向电流密度分别为38.7%和34.9%,但FMRFa5μmol·L^-1及20μmol·L^-1对L型钙电流、钠电流、瞬时外向电流和内向整流钾电流均无显著抑制作用。结论　FMRFa对大鼠心室肌细胞是一个特异性Na⁺/Ca²⁺交换抑制剂。     

Rhynchophylline-induced vasodilation in human mesenteric artery is mainly due to blockage of L-type calcium channels in vascular smooth muscle cells

Rhynchophylline (Rhy) is a pharmacologically active substance isolated from Uncaria rhynchophylla which has been used to treat cardiovascular diseases and has drawn considerable attention in recent years for its antihypertensive activities. We investigated the actions of Rhy on endothelium-denuded human mesenteric artery by tension measurement and its actions on high conductance Ca²⁺-activated K⁺ channels (BK_Ca) currents and calcium currents (I_Ca) in freshly isolated smooth muscle cells using perforated patch clamp technique. Intracellular Ca²⁺ level was measured in Fura-2-loaded cells. Rhy inhibited both the KCl and BayK-evoked mesenteric artery constrictions in a dose-dependent manner. K⁺ channel blockers (TEA, glibenclamide, IbTX, and 4-AP) did not affect the vasorelaxing effect of Rhy. Rhy inhibited L-type voltage-gated Ca²⁺ current (I_Ca,L) but had no significant effect on macroscopic BK_Ca current. Rhy preincubation markedly reduced the elevation of [Ca²⁺]_i level induced by KCl depolarization. Caffeine-stimulated [Ca²⁺]_i elevation was also decreased to some extent by pretreatment with Rhy for 20 min. Our results show that Rhy relaxes smooth muscles of human mesenteric resistance arteries, mainly due to inhibition of Ca²⁺ influx by blockage of L-type Ca²⁺ channels and thereby the decrease in [Ca²⁺]_i.