Activation by high potassium of a novel voltage-operated Ca2+ channel in rat spleen

1. High potassium produced a concentration-dependent contraction in rat isolated spleen.
2. The high potassium-induced contraction of rat spleen was abolished in Ca²⁺-free Krebs solution containing 1 mM EGTA, and the subsequent addition of 3 mM Ca²⁺ restored the high potassium-induced contraction to the control level.
3. Nifedipine, verapamil, diltiazem, Cd²⁺, Ni²⁺, Co²⁺, R-(+)-Bay K 8644 and pimozide inhibited and relaxed high potassium-induced contraction of rat spleen with IC₅₀ and EC₅₀ values much higher than those values in rat aorta.
4. In addition, high potassium-stimulated contraction of rat spleen was insensitive to ω-conotoxin GVIA, ω-conotoxin MVIIC and ω-agatoxin IVA.
5. The high potassium-induced contraction of rat spleen was also unaffected by tetrodotoxin (TTX), prazosin, chloroethylclonidine (CEC), yohimbine, propranolol, atropine, diphenhydramine, cimetidine, ketanserin, 3-tropanyl-indole-3-carboxylate, saralasin, indomethacin, nordihydroguaiaretic acid, GR32191B, domperidone, naloxone, chlorpromazine, suramin, (±)-2-amino-5-phosphonopentanoic acid, 6,7-dinitroquinoxaline-2,3-dione (DNQX), L-659,877, L-703,606, lorglumide, PD 135,158 N-methyl-D-glucamine, benextramine, amiloride, dantrolene, TMB-8, econazole, staurosporine and neomycin.
6. Forskolin and sodium nitroprusside relaxed high potassium-induced contraction of rat spleen with EC₅₀ values of 0.55±0.04 and 20.0±2.7 μM, respectively.
7. It is concluded that high potassium may activate a novel, pharmacologically uncharacterized voltage-operated Ca²⁺ channel in rat spleen.

     

Effects of docosahexaenoic acid on large-conductance Ca2+-activated K+ channels and voltage-dependent K+ channels in rat coronary artery smooth muscle cells

Aim:

To investigate the effects of docosahexaenoic acid (DHA) on large-conductance Ca²⁺-activated K⁺(BK_Ca) channels and voltage-dependent K⁺ (K_V) channels in rat coronary artery smooth muscle cells (CASMCs).

Methods:

Rat CASMCs were isolated by an enzyme digestion method. BK_Ca and K_V currents in individual CASMCs were recorded by the patch-clamp technique in a whole-cell configuration at room temperature. Effects of DHA on BK_Ca and K_V channels were observed when it was applied at 10, 20, 30, 40, 50, 60, 70, and 80 μmol/L.

Results:

When DHA concentrations were greater than 10 μmol/L, BK_Ca currents increased in a dose-dependent manner. At a testing potential of +80 mV, 6.1%±0.3%, 76.5%±3.8%, 120.6%±5.5%, 248.0%±12.3%, 348.7%±17.3%, 374.2%±18.7%, 432.2%±21.6%, and 443.1%±22.1% of BK_Ca currents were increased at the above concentrations, respectively. The half-effective concentration (EC₅₀) of DHA on BK_Ca currents was 37.53±1.65 μmol/L. When DHA concentrations were greater than 20 μmol/L, K_V currents were gradually blocked by increasing concentrations of DHA. At a testing potential of +50 mV, 0.40%±0.02%, 1.37%±0.06%, 11.80%±0.59%, 26.50%±1.75%, 56.50%±2.89%, 73.30%±3.66%, 79.70%±3.94%, and 78.1%±3.91% of K_V currents were blocked at the different concentrations listed above, respectively. The EC₅₀ of DHA on K_V currents was 44.20±0.63 μmol/L.

Conclusion:

DHA can activate BK_Ca channels and block K_V channels in rat CASMCs, and the EC₅₀ of DHA for BK_Ca channels is lower than that for K_V channels; these findings indicate that the vasorelaxation effects of DHA on vascular smooth muscle cells are mainly due to its activation of BK_Ca channels.     

Multiple effects of nordihydroguaiaretic acid on ionic currents in rat isolated type I carotid body cells

1. The effects of the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) on the ionic currents of rat carotid body type I cells were investigated by use of whole-cell and outside-out patch clamp techniques.
2. NDGA (5–50 μM) produced a concentration-dependent inhibition of whole-cell K⁺ currents at all activating test potentials (holding potential −70 mV). The time-course of the inhibition was also concentration-dependent and the effects of NDGA were only reversible following brief periods of exposure (<2 min). Another lipoxygenase inhibitor, phenidone (5 μM), was without effect on whole-cell K⁺ currents in carotid body type I cells.
3. NDGA (5–50 μM) also inhibited whole-cell Ca²⁺ channel currents (recorded with Ba²⁺ as charge carrier) in a concentration-dependent manner.
4. Isolation of voltage-gated K⁺ channels by use of high [Mg²⁺] (6 mM), low [Ca²⁺] (0.1 mM) solutions revealed a direct inhibition of the voltage-sensitive component of the whole-cell K⁺ current by NDGA (50 μM).
5. In excised, outside-out patches NDGA (20–50 μM) increased large conductance, Ca²⁺ activated K⁺ channel activity approximately 10 fold, an effect which could be reversed by either tetraethylammonium (10 mM) or charybdotoxin (30 nM).
6. It is concluded that NDGA activates maxi-K⁺ channels in carotid body type I cells and over the same concentration range inhibits voltage-sensitive K⁺ and Ca²⁺ channels. The inhibition of whole cell K⁺ currents seen is most likely due to a combination of direct inhibition of the voltage-sensitive K⁺ current and indirect inhibition of maxi-K⁺ channel activity through blockade of Ca²⁺ channels.

     

Characterization of action potential-triggered [Ca2+]i transients in single smooth muscle cells of guinea-pig ileum

1. To characterize increases in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) associated with discharge of action potentials, membrane potential and [Ca²⁺]_i were simultaneously recorded from single smooth muscle cells of guinea-pig ileum by use of a combination of nystatin-perforated patch clamp and fura-2 fluorimetry techniques.
2. A single action potential in response to a depolarizing current pulse elicited a transient rise in [Ca²⁺]_i. When the duration of the current pulse was prolonged, action potentials were repeatedly discharged during the early period of the pulse duration with a progressive decrease in overshoot potential, upstroke rate and repolarization rate. However, such action potentials could each trigger [Ca²⁺]_i transients with an almost constant amplitude.
3. Nicardipine (1 μM) and La³⁺ (10 μM), blockers of voltage-dependent Ca²⁺ channels (VDCCs), abolished both the action potential discharge and the [Ca²⁺]_i transient.
4. Charybdotoxin (ChTX, 300 nM) and tetraethylammonium (TEA, 2 mM), blockers of large conductance Ca²⁺-activated K⁺ channels, decreased the rate of repolarization of action potentials but increased the amplitude of [Ca²⁺]_i transients.
5. Thapsigargin (1 μM), an inhibitor of SR Ca²⁺-ATPase, slowed the falling phase and somewhat increased the amplitude, of action potential-triggered [Ca²⁺]_i transients without affecting action potentials. In addition, in voltage-clamped cells, the drug had little effect on the voltage step-evoked Ca²⁺ current but exerted a similar effect on its concomitant rise in [Ca²⁺]_i to that on the action potential-triggered [Ca²⁺]_i transient.
6. Similar action potential-triggered [Ca²⁺]_i transients were induced by brief exposures to high-K⁺ solution. They were not decreased, but rather increased, after depletion of intracellular Ca²⁺ stores by a combination of ryanodine (30 μM) and caffeine (10 mM) through an open-lock of Ca²⁺-induced Ca²⁺ release (CICR)-related channels.
7. The results show that action potentials, discharged repeatedly during the early period of a long membrane depolarization, undergo a progressive change in configuration but can each trigger a constant rise in [Ca²⁺]_i. Intracellular Ca²⁺ stores have a role, especially in accelerating the falling phase of the action potential-triggered [Ca²⁺]_i transients by replenishing cytosolic Ca²⁺. No evidence was provided for the involvement of CICR in the action potential-triggered [Ca²⁺]_i transient.

     

The effect of a secreted form of β-amyloid-precursor protein on intracellular Ca2+ increase in rat cultured hippocampal neurones

1. The effects of secreted forms of β-amyloid-precursor proteins (APP^Ss) on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) were investigated in rat cultured hippocampal neurones. APP695^S, a secretory form of APP695, attenuated the increase in [Ca²⁺]_i evoked by glutamate. In addition, APP695^S itself evoked an increase in [Ca²⁺]_i in 1 or 2 day-cultured hippocampal cells, but not in 7 to 13 day-cultured cells.
2. Eighty-one percent of neurones which were immunocytochemically positive for microtubule-associated protein 2 responded to APP695^S with an increase in [Ca²⁺]_i.
3. APP695^S induced a transient rise in [Ca²⁺]_i even in the absence of extracellular Ca²⁺ and produced an elevation in inositol-1,4,5-trisphosphate (IP₃) in a concentration-dependent manner from 100 to 500 ng ml⁻¹. In the presence of extracellular Ca²⁺, APP695^S caused a transient rise in [Ca²⁺]_i followed by a sustained phase at high [Ca²⁺]_i, suggesting Ca²⁺ entry from the extracellular space.
4. The [Ca²⁺]_i elevation was mimicked by amino terminal peptides of APP^S, but not by carboxy terminal peptides.
5. These results taken together suggest that APP695^S induces an increase in [Ca²⁺]_i in hippocampal neurones through an IP₃-dependent mechanism that changes according to the stage of development.

     

ATP stimulation of Ca2+-dependent plasminogen release from cultured microglia

1. ATP (10–100 μM), but not glutamate (100  μM), stimulated the release of plasminogen from microglia in a concentration-dependent manner during a 10 min stimulation. However, neither ATP (100 μM) nor glutamate (100 μM) stimulated the release of NO. A one hour pretreatment with BAPTA-AM (200 μM), which is metabolized in the cytosol to BAPTA (an intracellular Ca²⁺ chelator), completely inhibited the plasminogen release evoked by ATP (100 μM). The Ca²⁺ ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 induced plasminogen release in a concentration-dependent manner (0.3 μM to 10 μM).
2. ATP induced a transient increase in the intracellular calcium concentration ([Ca²⁺]_i) in a concentration-dependent manner which was very similar to the ATP-evoked plasminogen release, whereas glutamate (100 μM) had no effect on [Ca²⁺]_i (70 out of 70 cells) in microglial cells. A second application of ATP (100 μM) stimulated an increase in [Ca²⁺]_i similar to that of the first application (21 out of 21 cells).
3. The ATP-evoked increase in [Ca²⁺]_i was totally dependent on extracellular Ca²⁺, 2-Methylthio ATP was active (7 out of 7 cells), but α,β-methylene ATP was inactive (7 out of 7 cells) at inducing an increase in [Ca²⁺]_i. Suramin (100 μM) was shown not to inhibit the ATP-evoked increase in [Ca²⁺]_i (20 out of 20 cells). 2′- and 3′-O-(4-Benzoylbenzoyl)-adenosine 5′-triphosphate (BzATP), a selective agonist of P2X₇ receptors, evoked a long-lasting increase in [Ca²⁺]_i even at 1 μM, a concentration at which ATP did not evoke the increase. One hour pretreatment with adenosine 5′-triphosphate-2′, 3′-dialdehyde (oxidized ATP, 100 μM), a selective antagonist of P2X₇ receptors, blocked the increase in [Ca²⁺]_i induced by ATP (10 and 100 μM).
4. These data suggest that ATP may transit information from neurones to microglia, resulting in an increase in [Ca²⁺]_i via the ionotropic P2X₇ receptor which stimulates the release of plasminogen from the microglia.

     

Inhibition by ATP of calcium oscillations in rat cultured hippocampal neurones

1. The effect of adenosine 5′-triphosphate (ATP) on glutamatergic synaptic transmission in hippocampus was examined by an indicator of intracellular Ca²⁺ oscillations. These oscillations were postsynaptic responses by glutamate released from presynaptic sites. ATP completely inhibited the oscillations in a concentration-dependent manner.
2. The ATP-induced inhibition was mediated via P2-purinoceptors since ATP exhibited the inhibitory action even in the presence of P1-purinoceptor antagonists. Also non-hydrolysable ATP analogues and uridine 5′-triphosphate (UTP) inhibited the oscillation.
3. The rank order of agonist potency of ATP analogues for inhibition of the Ca²⁺ oscillation was as follows: 2-methyl-thio-adenosine 5′-triphosphate⩾ATP>adenosine 5′-O-(3-thiotriphosphate)>UTP>α,β-methylene-adenosine 5′-triphosphate. These inhibitory effects were insensitive to suramin. Judging from this rank order of potency, the inhibitory P2-purinoceptor could be assigned to a subclass of GTP-binding protein coupled-type receptors.
4. The site of action of ATP was thought to be presynaptic since ATP did not affect the postsynaptic Ca²⁺ responses by glutamate. These results suggest the existence of a presynaptic inhibitory P2-receptor that inhibits glutamate release in the hippocampus.

     

Regulation of apical and basolateral K+ conductances in the rat colon

1. Apical administration of an ionophore, nystatin, and basolateral depolarization by K⁺ were used to investigate the regulation of apical and basolateral electrogenic transport pathways for K⁺ in the rat proximal and distal colon.
2. Administration of nystatin (100 μg ml⁻¹ at the mucosal side), in the presence of Na⁺ and in the presence of a serosally directed K⁺ gradient, stimulate a large increase in short-circuit current (I_SC) and tissue conductance in both colonic segments. This response was composed of a pump current generated by the Na⁺-K⁺-ATPase and of a current across a quinine-sensitive basolateral K⁺ conductance.
3. The pump current, measured as Na⁺-dependent or scilliroside-sensitive current in the absence of a K⁺ gradient, was significantly greater in the distal than in the proximal colon. The pump current was unaltered by pretreatment of the tissue with forskolin (5×10⁻⁶ mol l⁻¹).
4. The current across the basolateral K⁺ conductance, measured as current in the presence of a serosally directed K⁺ gradient either in the absence of Na⁺ or in the presence of scilliroside, was increased by the cholinoreceptor agonist, carbachol (5×10⁻⁵ mol l⁻¹), but inhibited by forskolin (5×10⁻⁶ mol l⁻¹).
5. Basolateral K⁺ depolarization induced a negative I_SC in both colonic segments, which was inhibited by the K⁺ channel blocker quinine (10⁻³ mol l⁻¹ at the mucosal side), but was resistant to tetraethylammonium (5×10⁻³ mol l⁻¹ at the mucosal side). This K⁺ current across an apical K⁺ conductance was stimulated in both colonic segments by carbachol, whereas forskolin had no effect, although control experiments revealed that forskolin was still able to open an apical Cl⁻ conductance under these conditions.
6. These results demonstrate that an increase in intracellular Ca²⁺ concentration induced by carbachol causes an increase in the basolateral and the apical K⁺ conductance, thereby inducing K⁺ secretion in parallel with an indirect support for Cl⁻ secretion due to the hyperpolarization of the cell membrane. In contrast, the dominating effect of an increase in the intracellular cyclic AMP concentration is inhibition of a basolateral K⁺ conductance; a mechanism which might contribute to the inhibition of K⁺ absorption.

     

Effects of the 5-lipoxygenase activating protein inhibitor MK886 on voltage-gated and Ca2+-activated K+ currents in rat arterial myocytes

1. The effects on the voltage-gated (I_K) and Ca²⁺ activated (I_K,Ca) K⁺ currents in rat arterial myocytes of the 5-lipoxygenase activating protein (FLAP) inhibitor MK886, and its inactive analogue L583,916 were evaluated.
2. In rat pulmonary arterial myocytes (RPAMs), MK886 caused a concentration-dependent reduction of the I_K, with little obvious change in the kinetics of the current. Half maximal current block was observed at 75 nM MK886.
3. MK886 application led to a concentration-dependent increase in the amplitude of the TEA-sensitive I_K,Ca current and single channel activity in RPAMs in whole cell and inside-out configurations, respectively. The threshold concentration for this effect was approximately 300 nM and a maximal 4–5 fold increase was observed at 10 μM MK886. MK886 also increased I_K,Ca in rat mesenteric arterial myocytes (RMAMs).
4. L538,916, an analogue of MK886 which does not block FLAP, had no effect on either I_K or I_K,Ca at a concentration of 10 μM.
5. Leukotriene C₄ (100 nM) had no effect on either I_K or I_K,Ca in RPAMs. MK886 produced its usual increase in I_K,Ca and also blocked I_K, in the presence of leukotriene C₄. Similarly, leukotriene E₄ (100 nM) did not alter the amplitude of I_K. Also, the nonselective leukotriene receptor antagonist ICI 198,615 (3 μM) did not affect I_K in RPAMs, and did not affect the response to MK886.
6. Arachidonic acid (10 μM) enhanced I_K,Ca in both RPAMs and RMAMs.
7. The results show that MK886 markedly affects both I_K and I_K,Ca in a manner similar to that of arachidonic acid and independent of the endogenous production of leukotrienes. It is therefore possible that MK886, which is thought to compete with arachidonic acid for its binding to FLAP, may similarly occupy arachidonic acid binding sites on these K⁺ channels, and mimic its effects. Alternatively, MK886 might act via non-selective effects on other arachidonic acid metabolites which could modify K⁺ channel function.

     

Characterization of the P2 receptors on the human umbilical vein endothelial cell line ECV304

1. To characterize the P2 receptors present on the human umbilical vein endothelial-derived cell line, ECV304, cytosolic Ca²⁺, ([Ca²⁺]_c), responses were recorded in single cells and in cell suspensions to a series of nucleotides and nucleotide agonists.
2. Concentration response curves were obtained in fura-2-loaded ECV304 cell suspensions, with EC₅₀ values of 4.2 μM for ATP, 2.5 μM for UTP and 14 μM for adenosine-5′-O-(3-thio)triphosphate (ATPγS). EC₅₀ values for 2-methylthioATP, ADP, adenosine-5′-O-(2-thio)diphosphate (ADPβS) and AMP were 0.5 μM, 3.5 μM, 15 μM and 4.7 μM respectively, but maximal [Ca²⁺]_c responses were less than those produced by a maximal addition of ATP/UTP. ECV304 cells were unresponsive to UDP and β,γ,methyleneATP.
3. Cross-desensitization studies on ECV304 cells suggested that ATP and UTP recognized the same receptor. However, ADP recognized a receptor distinct from the UTP-sensitive receptor and AMP recognized a third distinct receptor.
4. ECV304 [Ca²⁺]_c responses to 2-methylthioATP were inhibited in the presence of 30 μM pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), whereas [Ca²⁺]_c responses to UTP were unaffected by this treatment.
5. ECV304 cells responded to the diadenosine polyphosphate Ap₃A with rises in [Ca²⁺]_c. Apparent responses to Ap₄A, Ap₅A and Ap₆A, were shown to be due to a minor nucleotide contaminant that could be removed by pre-treatment of the diadenosine samples with either alkaline phosphatase or apyrase.
6. ECV304 cells display a pharmacology consistent with the presence of at least two P2 receptors; a P2Y₂ receptor insensitive to the diadenosine polyphosphates and a P2Y₁ receptor sensitive to Ap₃A. In addition, ECV304 cells respond to AMP with increases in [Ca²⁺]_c via an as yet uncharacterized receptor.

     

Voltage and pH dependent block of cloned N-type Ca2+ channels by amlodipine

1. Two types of Ca²⁺ channel α₁-subunits were co-expressed in Xenopus oocytes with the Ca²⁺ channel α₂- and β₁-subunits. The Ba²⁺ current through the α_1Cα₂β and the α_1Bα₂β channels had electrophysiological and pharmacological properties of L- and N-type Ca²⁺ channels, respectively.
2. Amlodipine had a strong blocking action on both the L-type and N-type Ca²⁺ channels expressed in the oocyte. The potency of the amlodipine block on the N-type Ca²⁺ channel was comparable to that on the L-type Ca²⁺ channel. At −100 mV holding potential, the IC₅₀ values for amlodipine block on the L-type and N-type Ca²⁺ channel were 2.4 and 5.8 μM, respectively.
3. The blocking action of amlodipine on the N-type Ca²⁺ channel was dependent on holding potential and extracellular pH, as has been observed with amlodipine block on the L-type Ca²⁺ channel. A depolarized holding potential and high pH enhanced the blocking action of amlodipine.
4. The time course of block development by amlodipine was similar for L-type and N-type Ca²⁺ channels. However, it was slower than the time course of block development by nifedipine for the L-type Ca²⁺ channel.

     

Regulation of brain capillary endothelial cells by P2Y receptors coupled to Ca2+, phospholipase C and mitogen-activated protein kinase

1. The blood-brain barrier is formed by capillary endothelial cells and is regulated by cell-surface receptors, such as the G protein-coupled P2Y receptors for nucleotides. Here we investigated some of the characteristics of control of brain endothelial cells by these receptors, characterizing the phospholipase C and Ca²⁺ response and investigating the possible involvement of mitogen-activated protein kinases (MAPK).
2. Using an unpassaged primary culture of rat brain capillary endothelial cells we showed that ATP, UTP and 2-methylthio ATP (2MeSATP) give similar and substantial increases in cytosolic Ca²⁺, with a rapid rise to peak followed by a slower decline towards basal or to a sustained plateau. Removal of extracellular Ca²⁺ had little effect on the peak Ca²⁺-response, but resulted in a more rapid decline to basal. There was no response to α,β-MethylATP (α,βMeATP) in these unpassaged cells, but a response to this P2X agonist was seen after a single passage.
3. ATP (log EC₅₀ −5.1±0.2) also caused an increase in the total [³H]-inositol (poly)phosphates ([³H]-InsP_x) in the presence of lithium with a rank order of agonist potency of ATP=UTP=UDP>ADP, with 2MeSATP and α,βMeATP giving no detectable response.
4. Stimulating the cells with ATP or UTP gave a rapid rise in the level of inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃), with a peak at 10 s followed by a decline to a sustained plateau phase. 2MeSATP gave no detectable increase in the level of Ins(1,4,5)P₃.
5. None of the nucleotides tested affected basal cyclic AMP, while ATP and ATPγS, but not 2MeSATP, stimulated cyclic AMP levels in the presence of 5 μM forskolin.
6. Both UTP and ATP stimulated tyrosine phosphorylation of p42 and p44 mitogen-activated protein kinase (MAPK), while 2MeSATP gave a smaller increase in this index of MAPK activation. By use of a peptide kinase assay, UTP gave a substantial increase in MAPK activity with a concentration-dependency consistent with activation at P2Y₂ receptors. 2MeSATP gave a much smaller response with a lower potency than UTP.
7. These results are consistent with brain endothelial regulation by P2Y₂ receptors coupled to phospholipase C, Ca²⁺ and MAPK; and by P2Y₁-like (2MeSATP-sensitive) receptors which are linked to Ca²⁺ mobilization by a mechanism apparently independent of agonist stimulated Ins (1,4,5)P₃ levels. A further response to ATP, acting at an undefined receptor, caused an increase in cyclic AMP levels in the presence of forskolin. The differential MAPK coupling of these receptors suggests that they exert fundamentally distinct influences over brain endothelial function.

     

Possible mechanisms underlying the midazolam-induced relaxation of the noradrenaline-contraction in rabbit mesenteric resistance artery

1. The mechanisms underlying the midazolam-induced relaxation of the noradrenaline (NA)-contraction were studied by measuring membrane potential, isometric force and intracellular concentration of Ca²⁺([Ca²⁺]_i) in endothelium-denuded muscle strips from the rabbit mesenteric resistance artery. The actions of midazolam were compared with those of nicardipine, an L-type Ca²⁺-channel blocker.
2. Midazolam (30 and 100 μM) did not modify either the resting membrane potential or the membrane depolarization induced by 10 μM NA.
3. NA (10 μM) produced a phasic, followed by a tonic increase in both [Ca²⁺]_i and force. Midazolam (10–100 μM) did not modify the resting [Ca²⁺]_i, but attenuated the NA-induced phasic and tonic increases in [Ca²⁺]_i and force, in a concentration-dependent manner. In contrast, nicardipine (0.3 μM) attenuated the NA-induced tonic, but not phasic, increases in [Ca²⁺]_i and force.
4. In Ca²⁺-free solution containing 2 mM EGTA, NA (10 μM) transiently increased [Ca²⁺]_i and force. Midazolam (10–100 μM), but not nicardipine (0.3 μM), attenuated this NA-induced increase in [Ca²⁺]_i and force, in a concentration-dependent manner. However, midazolam (10 and 30 μM), had no effect on the increases in [Ca²⁺]_i and force induced by 10 mM caffeine.
5. In ryanodine-treated strips, which have functionally lost the NA-sensitive Ca²⁺- storage sites, NA slowly increased [Ca²⁺]_i and force. Nicardipine (0.3 μM) did not modify the resting [Ca²⁺]_i but partly attenuated the NA-induced increases in [Ca²⁺]_i and force. In the presence of nicardipine, midazolam (100 μM) lowered the resting [Ca²⁺]_i and further attenuated the remaining NA-induced increases in [Ca²⁺]_i and force.
6. The [Ca²⁺]_i-force relationship was obtained in ryanodine-treated strips by the application of ascending concentrations of Ca²⁺ (0.16–2.6 mM) in Ca²⁺-free solution containing 100 mM K⁺. NA (10 μM) shifted the [Ca²⁺]_i-force relationship to the left and enhanced the maximum Ca²⁺-induced force. Under these conditions, whether in the presence or absence of 10 μM NA, midazolam (10 and 30 μM) attenuated the increases in [Ca²⁺]_i and force induced by Ca²⁺ without changing the [Ca²⁺]_i-force relationship.
7. It was concluded that, in smooth muscle of the rabbit mesenteric resistance artery, midazolam inhibits the NA-induced contraction through its inhibitory action on NA-induced Ca²⁺ mobilization. Midazolam attenuates NA-induced Ca²⁺ influx via its inhibition of both nicardipine-sensitive and -insensitive pathways. Furthermore, midazolam attenuates the NA-induced release of Ca²⁺ from the storage sites. This effect contributes to the midazolam-induced inhibition of the NA-induced phasic contraction.

     

Potent vasoconstrictor actions of cyclopiazonic acid and thapsigargin on femoral arteries from spontaneously hypertensive rats

1. The Ca²⁺ buffering function of sarcoplasmic reticulum (SR) in the resting state of arteries from spontaneously hypertensive rats (SHR) was examined. Differences in the effects of cyclopiazonic acid (CPA) and thapsigargin, agents which inhibit the Ca²⁺-ATPase of SR, on tension and cellular Ca²⁺ level were assessed in endothelium-denuded strips of femoral arteries from 13-week-old SHR and normotensive Wistar-Kyoto rats (WKY).
2. In resting strips preloaded with fura-PE3, the addition of CPA (10 μM) or thapsigargin (100 nM) caused an elevation of cytosolic Ca²⁺ level ([Ca²⁺]_i) and a contraction. These responses were significantly greater in SHR than in WKY.
3. The addition of verapamil (3 μM) to the resting strips caused a decrease in resting [Ca²⁺]_i, which was significantly greater in SHR than in WKY. In SHR, but not in WKY, this decrease was accompanied by a relaxation from the resting tone, suggesting the maintenance of myogenic tone in the SHR artery.
4. Verapamil (3 μM) abolished differences between SHR and WKY. The effects of verapamil were much greater on the contraction than on the [Ca²⁺]_i.
5. The resting Ca²⁺ influx in arteries measured after a 5 min incubation of the artery with ⁴⁵Ca was not increased by CPA or thapsigargin in either SHR or WKY. The net Ca²⁺ entry measured after a 30 min incubation of the artery with ⁴⁵Ca was decreased by CPA or thapsigargin in both SHR and WKY. The resting Ca²⁺ influx was significantly higher in SHR than in WKY, and was decreased by nifedipine (100 nM) in the SHR artery, but was unchanged in the WKY artery.
6. The resting ⁴⁵Ca efflux from the artery was increased during the addition of CPA (10 μM). This increase was less in SHR than in WKY. The resting ⁴⁵Ca efflux was the same in SHR and WKY.
7. These results suggest that (1) the Ca²⁺ influx via L-type voltage-dependent Ca²⁺ channels (VDCCs) was increased in the resting state of the SHR femoral artery, (2) the greater part of the increased Ca²⁺ influx was buffered by Ca²⁺ uptake into the SR and some Ca²⁺ reached the myofilaments resulting in the maintenance of the myogenic tone, and (3) therefore the functional elimination of SR by CPA or thapsigargin caused a large elevation of [Ca²⁺]_i and a potent contraction in this artery. During this process, the contraction was mainly due to the basal Ca²⁺ influx via L-type VDCCs. The present study also showed the existence of a relatively large compartment of [Ca²⁺]_i which does not contribute to the contraction during the addition of CPA or thapsigargin.

     

Molecular cloning and characterization of rat P2Y4 nucleotide receptor

An intronless open reading frame encoding a protein (361aa in length) was isolated from a rat genomic library probed with a DNA fragment from rat heart. This protein showed 83% sequence identity with the human P2Y₄ (hP2Y₄) receptor and represents a homologue of the human pyrimidinoceptor. However, the rP2Y₄ receptor is not selective for uridine nucleotides and, instead, shows an agonist potency order of ITP=ATP=ADP(pure)=UTP=ATPγS=2-MeSATP=Ap₄A>UDP(pure). ADP, ATPγS, 2-MeSATP and UDP are partial agonists. Thus, in terms of agonist profile, rP2Y₄ is more like the P2U receptor subtype. The rP2Y₄ receptor was reversibly antagonized by Reactive blue 2 but not by suramin which, otherwise, inhibits the hP2Y₂ receptor (a known P2U receptor). Thus, rP2Y₄ and the P2Y₂ subtype appear to be structurally distinct forms of the P2U receptor (where ATP and UTP are equi-active) but can be distinguished as suramin-insensitive and suramin-sensitive P2U receptors, respectively.     

The action of calcium channel blockers on recombinant L-type calcium channel α1-subunits

1. CHO cells expressing the α_1C-a subunit (cardiac isoform) and the α_1C-b subunit (vascular isoform) of the voltage-dependent L-type Ca²⁺ channel were used to investigate whether tissue selectivity of Ca²⁺ channel blockers could be related to different affinities for α_1C isoforms.
2. Inward current evoked by the transfected α₁ subunit was recorded by the patch-clamp technique in the whole-cell configuration.
3. Neutral dihydropyridines (nifedipine, nisoldipine, (+)-PN200-110) were more potent inhibitors of α_1C-b-subunit than of α_1C-a-subunit. This difference was more marked at a holding potential of −100 mV than at −50 mV. SDZ 207-180 (an ionized dihydropyridine) exhibited the same potency on the two isoforms.
4. Pinaverium (ionized non-dihydropyridine derivative) was 2 and 4 fold more potent on α_1C-a than on α_1C-b subunit at Vh of −100 mV and −50 mV, respectively. Effects of verapamil were identical on the two isoforms at both voltages.
5. [³H]-(+)-PN 200-110 binding experiments showed that neutral dihydropyridines had a higher affinity for the α_1C-b than for the α_1C-a subunit. SDZ 207-180 had the same affinity for the two isoforms and pinaverium had a higher affinity for the α_1C-a subunit than for the α_1C-b subunit.
6. These results indicate marked differences among Ca²⁺ channel blockers in their selectivity for the α_1C-a and α_1C-b subunits of the Ca²⁺ channel.

     

Properties of the pore-forming P2X7 purinoceptor in mouse NTW8 microglial cells

1. We have used whole-cell patch clamping methods to study and characterize the cytolytic P2X₇ (P2_Z) receptor in the NTW8 mouse microglial cell line.
2. At room temperature, in an extracellular solution containing 2 mM Ca²⁺ and 1 mM Mg²⁺, 2′- and 3′-O-(4-benzoylbenzoyl)-adenosine-5′-triphosphate (Bz-ATP; 300 μM), or ATP (3 mM), evoked peak whole cell inward currents, at a holding potential of −90 mV, of 549±191 and 644±198 pA, respectively. Current-voltage relationships generated with 3 mM ATP reversed at 4.6 mV and did not display strong rectification.
3. In an extracellular solution containing zero Mg²⁺ and 500 μM Ca²⁺ (low divalent solution), brief (0.5 s) application of these agonists elicited larger maximal currents (909±138 and 1818±218 pA, Bz-ATP and ATP, respectively). Longer application of ATP (1 mM for 30 s) produced larger, slowly developing, currents which reached a plateau after approximately 15–20 s and were reversible on washing. Under these conditions, in the presence of ATP, ethidium bromide uptake could be demonstrated. Further applictions of 1 mM ATP produced rapid currents of the same magnitude as those observed during the 30 s application. Subsequent determination of concentration-effect curves to Bz-ATP, ATP and 2-methylthio-ATP yielded EC₅₀ values of 58.3, 298 and 505 μM, respectively. These affects of ATP were antagonized by pyridoxal-phosphate-6-azophenyl- 2′, 4′-disulphonic acid (PPADS; 30 μM) but not suramin (100 μM).
4. In low divalent solution, repeated application of 1 mM ATP for 1 s produced successively larger currents which reached a plateau, after 8 applications, of 466% of the first application current. PPADS (30 μM) prevented this augmentation, while 5-(N,N-hexamethylene)-amiloride (HMA) (100 μM) accelerated it such that maximal augmentation was observed after only one application of ATP in the presence of HMA. At a bath temperature of 32°C, current augmentation also occurred in normal divalent cation containing solution.
5. These data demonstrate that mouse microglial NTW8 cells possess a purinoceptor with pharmacological characteristics resembling the P2X₇ receptor. We suggest that the current augmentation phenomenon observed reflects formation of the large cytolytic pore characteristic of this receptor. We have demonstrated that pore formation can occur under normal physiological conditions and can be modulated pharmacologically, both positively and negatively.

     

Mechanism of nitric oxide-induced contraction in the rat isolated small intestine

1. The contractile response to nitric oxide (NO) in ral ileal myenteric plexus-longitudinal muscle strips was pharmacologically analysed.
2. NO (10⁻⁷ M) induced only contraction while 10⁻⁶ M NO induced contraction followed by relaxation. Methylene blue (up to 10⁻⁴ M) did not affect the NO-induced contractions but significantly reduced the relaxation evoked by 10⁻⁶ M NO. Administration of 8-bromo-cyclic GMP (10⁻⁶–10⁻⁴ M) only induced relaxation.
3. Sodium nitroprusside (SNP; 10⁻⁷–10⁻⁵ M) induced concentration-dependent contractions per se; the contractile response to NO, administered within 10 min after SNP, was concentration-dependently reduced. The guanosine 3′:5′-cyclic monophosphate (cyclic GMP) content of the tissues was not increased during contractions with 10⁻⁸ M NO and 10⁻⁶ M SNP; it was increased by a factor of 2 during contraction with 10⁻⁷ M NO, and by a factor of 12 during relaxation with 3×10⁻⁶ M NO.
4. The NO-induced contractions were not affected by ryanodine (3×10⁻⁵ M) but were concentration-dependently reduced by nifedipine (10⁻⁸–10⁻⁷ M) and apamin (3×10⁻⁹–3×10⁻⁸ M).
5. These results suggest that cyclic GMP is not involved in the NO-induced contraction in the rat small intestine. The NO-induced contraction is related to extracellular Ca²⁺ influx through L-type Ca²⁺ channels, that might be activated in response to the closure of Ca²⁺-dependent K⁺ channels.

     

Blockade of N-type Ca2+ current by cilnidipine (FRC-8653) in acutely dissociated rat sympathetic neurones

1. The inhibitory effects of cilnidipine (FRC-8653) and various organic Ca²⁺ channel blockers on high voltage-activated Ba²⁺ currents (HVA I_Ba) in rat sympathetic neurones were examined by means of the conventional whole-cell patch-clamp recording mode under voltage-clamped conditions.
2. HVA I_Ba was classified into three different current components with subtype selective peptide Ca²⁺ channel blockers. No ω-Agatoxin IVA-sensitive (P-type) or ω-conotoxin MVIIC-sensitive (Q-type) current components were observed. Most (>85%) I_Ba was found to consist of ω-conotoxin GVIA-sensitive N-type components.
3. The application of cilnidipine inhibited HVA I_Ba in a concentration-dependent manner. The K_d value for cilnidipine was 0.8 μM. Cilnidipine did not shift the current-voltage (I-V) relationship for HVA I_Ba, as regards the threshold potential and peak potential where the amplitude reached a maximum.
4. High concentrations of three hypotensive Ca²⁺ channel blockers, nifedipine, diltiazem and verapamil, all inhibited HVA I_Ba in a concentration-dependent manner. The K_d values for nifedipine, diltiazem and verapamil were 131, 151 and 47 μM, respectively. A piperazine-type Ca²⁺ channel blocker, flunarizine, showed a relatively potent blocking action on I_Ba. The K_d value was about 3 μM.
5. These results thus show that cilnidipine potently inhibits the sympathetic Ca²⁺ channels which predominantly consist of an ω-Cg-GVIA-sensitive component. This blockade of the N-type Ca²⁺ channel, as well as the L-type Ca²⁺ channel by cilnidipine suggests that it could be used therapeutically for treatment of hypersensitive sympathetic disorders associated with hypertension.

     

The signal transduction mechanism involved in kazinol B-stimulated superoxide anion generation in rat neutrophils

1. In this study, the underlying mechanism of stimulation of respiratory burst by kazinol B, a natural isoprenylated flavan, in rat neutrophils in vitro was investigated.
2. Kazinol B concentration-dependently stimulated the superoxide anion (O₂[dot over 2]⁻) generation, with a lag but transient activation profile, in neutrophils but not in a cell-free system. The maximum response (13.2±1.4 nmol O₂[dot over 2]⁻ 10 min⁻¹ per 10⁶ cells) was observed at 10 μM kazinol B.
3. Pretreatment of neutrophils with phorbol 12-myristate 13-acetate (PMA) or formylmethionyl-leucyl-phenylalanine (fMLP) significantly enhanced the O₂[dot over 2]⁻ generation following the subsequent stimulation of cells with kazinol B.
4. Cells pretreated with EGTA or a protein kinase inhibitor staurosporine effectively attenuated the kazinol B-induced O₂[dot over 2]⁻ generation. However, a p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and a phosphoinositide 3-kinase (PI3K) inhibitor wortmannin had no effect on the kazinol B-induced response.
5. Kazinol B significantly stimulated [Ca²⁺]_i elevation in neutrophils, with a lag and slow rate of rise activation profile, and this response was attenuated by a phospholipase C (PLC) inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122. Kazinol B also stimulated the inositol bis- and trisphosphate (IP₂ and IP₃) formation with a 1 min lag time.
6. The membrane-associated PKC-α and PKC-θ but not PKC-ι were increased following the stimulation of neutrophils with kazinol B. It was more rapid and sensitive in the activation of PKC-θ than PKC-α by kazinol B. Kazinol B partially inhibited the [³H]phorbol 12,13-dibutyrate ([³H]PDB) binding to the neutrophil cytosolic PKC.
7. Neither the cellular mass of phosphatidic acid (PA) and phosphatidylethanol (PEt), in the presence of ethanol, nor the protein tyrosine phosphorylation were stimulated by kazinol B. In addition, the kazinol B-induced O₂[dot over 2]⁻ generation remained relatively unchanged in cells pretreated with ethanol or a tyrosine kinase inhibitor genistein.
8. Collectively, these results indicate that the stimulation of the respiratory burst by kazinol B is probably mediated by the synergism of PKC activation and [Ca²⁺]_i elevation in rat neutrophils.