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.

     

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.

     

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.

     

Pharmacological properties of the Ca2+-release mechanism sensitive to NAADP in the sea urchin egg

1. The sea urchin egg homogenate is an ideal model to characterize Ca²⁺-release mechanisms because of its reliability and high signal-to-noise-ratio. Apart from the InsP₃- and ryanodine-sensitive Ca²⁺-release mechanisms, it has been recently demonstrated that this model is responsive to a third independent mechanism, that has the pyridine nucleotide, nicotinic acid adenine dinucleotide phosphate (NAADP), as an endogenous agonist.
2. The sea urchin egg homogenate was used to characterize the pharmacological and biochemical characteristics of the novel Ca²⁺-releasing agent, NAADP, compared to inositol trisphosphate (InsP₃) and cyclic ADP ribose (cyclic ADPR), an endogenous activator of ryanodine receptors.
3. NAADP-induced Ca²⁺-release was blocked by L-type Ca²⁺-channel blockers and by Bay K 8644, while InsP₃- and cyclic ADPR-induced Ca²⁺-release were insensitive to these agents. L-type Ca²⁺-channel blockers did not displace [³²P]-NAADP binding, suggesting that their binding site was different. Moreover, stopped-flow kinetic studies revealed that these agents blocked NAADP in a all-or-none fashion.
4. Similarly, a number of K⁺-channel antagonists blocked NAADP-induced Ca²⁺-release selectively over InsP₃- and cyclic ADPR-induced Ca²⁺-release. Radioligand studies showed that these agents were not competitive antagonists.
5. As has been shown for InsP₃ and ryanodine receptors, NAADP receptors were sensitive to calmodulin antagonists, suggesting that this protein could be a common regulatory feature of intracellular Ca²⁺-release mechanisms.
6. The presence of K⁺ was not essential for NAADP-induced Ca²⁺-release, since substitution of K⁺ with other monovalent cations in the experimental media did not significantly alter Ca²⁺ release by NAADP. On the contrary, cyclic ADPR and InsP₃-sensitive mechanisms were affected profoundly, although to a different extent depending on the monovalent cation which substituted for K⁺. Similarly, modifications of the pH in the experimental media from 7.2 to 6.7 or 8.0 only slightly affected NAADP-induced Ca²⁺-release. While the alkaline condition permitted InsP₃ and cyclic ADPR-induced Ca²⁺-release, the acidic condition completely hampered both Ca²⁺-release mechanisms.
7. The present results characterize pharmacologically and biochemically the novel Ca²⁺-release mechanism sensitive to NAADP. Such characterization will help future research aimed at understanding the role of NAADP in mammalian systems.

     

Ca2+ mobilization in the aortic endothelium in streptozotocin-induced diabetic and cholesterol-fed mice

1. Experiments were performed to compare Ca²⁺ mobilization in the aortic endothelium in streptozotocin (STZ)-induced diabetic and cholesterol-fed mice with that in age-matched controls.
2. The intracellular free Ca²⁺ ([Ca²⁺]_i) in the fura PE-3 loaded endothelium of aortic rings was dose-dependently increased by cumulative administration of acetylcholine (ACh). ACh caused a transient rise in [Ca²⁺]_i in Ca²⁺-free medium. The ACh-induced increase in [Ca²⁺]_i in normal or Ca²⁺-free medium was significantly weaker in both STZ-induced diabetic and cholesterol-fed mice.
3. The weaker [Ca²⁺]_i response in Ca²⁺-containing medium in STZ-induced diabetic and cholesterol-fed mice was normalized by chronic administration of cholestyramine.
4. The increased low density lipoprotein (LDL) levels seen in both STZ-induced diabetic and cholesterol-fed mice were normalized by the same chronic administration of cholestyramine (300 mg kg⁻¹, p.o. daily for 10 weeks). Chronic administration of cholestyramine had no effect on the plasma glucose level.
5. Lysophosphatidylcholine (LPC) decreased the [Ca²⁺]_i responses to ACh in the aortic endothelium from normal mice.
6. These results suggest that ACh increases both Ca²⁺ influx and Ca²⁺ release from storage in the aortic endothelium. The weaker [Ca²⁺]_i influx seen in the endothelium of aortae from both STZ-induced diabetic and cholesterol-fed mice was improved by the chronic administration of cholestyramine, and we suggest that this improvement is due, at least in part, to a lowering of the plasma LDL level. It is further suggested that LPC may have an important influence over Ca²⁺ mobilization in the endothelium.

     

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.

     

Role of Ca2+-activated K+ channels in acetylcholine-induced dilatation of the basilar artery in vivo

1. We tested the hypothesis that activation of large conductance calcium-activated potassium channels is involved in dilator responses of the basilar artery to acetylcholine in vivo. Using a cranial window in anaesthetized rats, we examined responses of the basilar artery to acetylcholine.
2. Topical application of acetylcholine (10⁻⁶ and 10⁻⁵ M) increased diameter of the basilar artery from 238±7 μm to 268±7 and 288±7 μm, respectively (P<0.05 vs. baseline diameter). Iberiotoxin (10⁻⁸ M), an inhibitor of large conductance calcium-activated potassium channels, did not affect baseline diameter of the basilar artery. In the presence of 10⁻⁸ M iberiotoxin, 10⁻⁶ and 10⁻⁵ M acetylcholine increased diameter of the basilar artery from 239±7 μm to 246±7 and 261±7 μm, respectively. Thus, iberiotoxin attenuated acetylcholine-induced dilatation of the basilar artery (P<0.05).
3. Sodium nitroprusside (10⁻⁷ and 10⁻⁶ M) increased diameter of the basilar artery from 242±9 μm to 310±12 and 374±13 μm, respectively (P<0.05 vs. baseline diameter). In the presence of iberiotoxin (10⁻⁸ M), sodium nitroprusside (10⁻⁷ and 10⁻⁶ M) increased diameter of the basilar artery from 243±6 μm to 259±9 and 311±12 μm, respectively. Thus, iberiotoxin attenuated dilator responses of the basilar artery to sodium nitroprusside (P<0.05).
4. Iberiotoxin partly inhibited dilator responses of the basilar artery to forskolin, a direct activator of adenylate cyclase, but did not affect vasodilatation produced by levcromakalim, a potassium channel opener.
5. These results suggest that dilator responses of the basilar artery to acetylcholine and sodium nitroprusside are mediated, in part, by activation of large conductance calcium-activated potassium channels. Because both acetylcholine and sodium nitroprusside have been shown to activate guanylate cyclase via nitric oxide, activation of large conductance calcium-activated potassium channels may be one of the major mechanisms by which cyclic GMP causes dilatation of the basilar artery in vivo.

     

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.     

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.

     

ATP,a partial agonist for the P2Z receptor of human lymphocytes

1. Although extracellular adenosine 5′-triphosphate (ATP) is the natural ligand for the P2Z receptor of human lymphocytes it is less potent than 3′-O-(4-benzoylbenzoyl)-ATP (BzATP) in opening the associated ion channel, which conducts a range of permeants including Ba²⁺ and ethidium⁺. We have quantified the influx of ethidium⁺ into lymphocytes produced by BzATP, ATP, 2-methylthio-ATP (2MeSATP) and ATPγS, studied competition between ATP and BzATP and investigated the effects of KN-62, a new and potent inhibitor of the P2Z receptor.
2. BzATP and ATP stimulated ethidium⁺ influx with EC₅₀ values of 15.4±1.4 μM (n=5) and 85.6±8.8 μM (n=5), respectively. The maximal response to ATP was only 69.8±1.9% of that for BzATP. Hill analysis gave n_H of 3.17±0.24 (n=3) and 2.09±0.45 (n=4) for BzATP and ATP, suggesting greater positive cooperativity for BzATP than for ATP in opening the P2Z receptor-operated ion channel.
3. A rank order of agonist potency of BzATP>ATP=2MeSATP>ATPγS was observed for agonist-stimulated ethidium⁺ influx, while maximal influxes followed a rank order of BzATP>ATP>2MeSATP>ATPγS.
4. Preincubation with 30–50 μM oxidized ATP (ox-ATP), an irreversible P2Z inhibitor, reduced the maximal response but did not change the steepness of the Ba²⁺ influx-response curve produced by BzATP (n_H 3.2 and 2.9 for 30 and 50 μM ox-ATP, respectively (n=2)).
5. ATP (300–1000 μM) added simultaneously with 30 μM BzATP (EC₉₀) inhibited both ethidium⁺ and Ba²⁺ fluxes to a maximum of 30–40% relative to the values observed with BzATP alone. Moreover, ATP (300 μM) shifted the concentration-response curve to the right for BzATP-stimulated Ba²⁺ influx, confirming competition between ATP and BzATP.
6. KN-62, a new and powerful inhibitor of the lymphocyte P2Z receptor, showed less potency in antagonizing BzATP-mediated fluxes than ATP-induced fluxes when maximal concentrations of both agonists (BzATP, 50 μM; ATP, 500 μM) were used.
7. These data suggest that the natural ligand, ATP, is a partial agonist for the P2Z receptor while BzATP is a more efficacious agonist. Moreover the competitive studies show that only a single class of P2-receptor (P2Z class) is expressed on human leukaemic lymphocytes.

     

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.

     

Actions of 4-chloro-3-ethyl phenol on internal Ca2+ stores in vascular smooth muscle and endothelial cells

1. Recently, 4-chloro-3-ethyl phenol (CEP) has been shown to cause the release of internally stored Ca²⁺, apparently through ryanodine-sensitive Ca²⁺ channels, in fractionated skeletal muscle terminal cisternae and in a variety of non-excitable cell types. Its action on smooth muscle is unknown. In this study, we characterized the actions of CEP on vascular contraction in endothelium-denuded dog mesenteric artery. We also determined its ability to release Ca²⁺, by use of Ca²⁺ imaging techniques, on dog isolated mesenteric artery smooth muscle cells and on bovine cultured pulmonary artery endothelial cells.
2. After phenylephrine-(PE, 10 μM) sensitive Ca²⁺ stores were depleted by maximal PE stimulation in Ca²⁺-free medium, the action of CEP on refilling of the emptied PE stores was tested, by first pre-incubating the endothelium-denuded artery in CEP for 15 min before Ca²⁺ was restored for a 30 min refilling period. At the end of this period, Ca²⁺ and CEP were removed, and the arterial ring was tested again with PE to assess the degree of refilling of the internal Ca²⁺ store.
3. In a concentration-dependent manner (30, 100 and 300 μM), CEP significantly reduced the size of the post-refilling PE contraction (49.4, 28.9 and 5.7% of control, respectively) in Ca²⁺-free media. This suggests that Ca²⁺ levels are reduced in the internal stores by CEP treatment. CEP alone did not cause any contraction either in Ca²⁺-containing or Ca²⁺-free Krebs solution.
4. Restoring Ca²⁺ in the presence of PE caused a large contraction, which reflects PE-induced influx of extracellular Ca²⁺. The contraction of tissues pretreated with 300 μM CEP was significantly less compared with controls. However, tissues pretreated with 30 and 100 μM CEP were unaffected. Washout of CEP over 30 min produced complete recovery of responses to PE in Ca²⁺-free and Ca²⁺-containing medium suggesting a rapid reversal of CEP effects.
5. Concentration-response curves were constructed for PE, 5-hydroxytryptamine (5-HT) and K⁺ in the absence of and after 30 min pre-incubation with 30, 100 and 300 μM CEP. In all cases, CEP caused a concentration-dependent depression of the maximum response to PE (84.8, 43.4 and 11.6% of control), 5-HT (65.4, 25.7 and 6.9% of control) and K⁺ (77.6, 41.1 and 10.8% of control).
6. Some arterial rings were pre-incubated with ryanodine (30 μM) for 30 min before the construction of PE concentration-response curves. In Ca²⁺-free Krebs solution, ryanodine alone did not cause any contraction. However, 58% (11 out of 19) of the tissues tested with ryanodine developed contraction (6.9±1.2% of 100 mM K⁺ contraction, n=11) in the presence of external Ca²⁺. EC₅₀ values for PE in ryanodine-treated tissues (1.7±0.25 μM, n=16) were not significantly different from controls (2.5±0.41 μM, n=22). Maximum contractions to PE (118.5±4.4% of 100 mM K⁺ contraction, n=16) were also unaffected by ryanodine when compared to controls (129±4.2%, n=23).
7. When fura-2 loaded smooth muscle cells (n=13) and endothelial cells (n=27) were imaged for Ca²⁺ distribution, it was observed that 100 and 300 μM CEP in Ca²⁺-free medium caused Ca²⁺ release in both cell types. Smooth muscle cells showed a small decrease in cell length. Addition of EGTA (5 mM) reversed the effect of CEP on intracellular Ca²⁺ to control values.
8. These data show, for the first time in vascular smooth muscle and endothelial cells, that CEP releases Ca²⁺ more rapidly than ryanodine. Unlike ryanodine, CEP caused no basal contraction but depressed contractions to PE, 5-HT and K⁺. The lack of basal contraction may result from altered responsiveness of the contractile system to intracellular Ca²⁺ elevation.

     

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.

     

Ca2+ signalling by P2Y receptors in cultured rat aortic smooth muscle cells

Background and purpose:

P2Y receptors evoke Ca²⁺ signals in vascular smooth muscle cells and regulate contraction and proliferation, but the roles of the different P2Y receptor subtypes are incompletely resolved.

Experimental approach:

Quantitative PCR was used to define expression of mRNA encoding P2Y receptor subtypes in freshly isolated and cultured rat aortic smooth muscle cells (ASMC). Fluorescent indicators in combination with selective ligands were used to measure the changes in cytosolic free [Ca²⁺] in cultured ASMC evoked by each P2Y receptor subtype.

Key results:

The mRNA for all rat P2Y receptor subtypes are expressed at various levels in cultured ASMC. Four P2Y receptor subtypes (P2Y1, P2Y2, P2Y4 and P2Y6) evoke Ca²⁺ signals that require activation of phospholipase C and comprise both release of Ca²⁺ from stores and Ca²⁺ entry across the plasma membrane.

Conclusions and implications:

Combining analysis of P2Y receptor expression with functional analyses using selective agonists and antagonists, we isolated the Ca²⁺ signals evoked in ASMC by activation of P2Y1, P2Y2, P2Y4 and P2Y6 receptors.     

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.

     

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.

     

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.

     

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.

     

Endothelin-1 induced contraction of rat aorta: contributions made by Ca2+ influx and activation of contractile apparatus associated with no change in cytoplasmic Ca2+ level

Summary The modes by which Endothelin-1 (ET) induces Ca²⁺-influx and the relative functional importance of the different sources of Ca²⁺ for ET-induced contraction were studied using fura 2-loaded and unloaded rat aortic strips. ET caused an increase in the cytosolic free Ca²⁺ level ([Ca²⁺]_i) followed by a tonic contraction in Ca²⁺-containing solution, and produced a transient elevation of [Ca²⁺]_i followed by a small sustained contraction in Ca²⁺-free medium. ET also stimulated ⁴⁵Ca influx into La²⁺-inaccessible fraction significantly. With the same change of [Ca²⁺]_i, ET caused a larger tension than that induced by high K. ET-induced contraction and [Ca²⁺]_i elevation were not significantly inhibited by 0.1–0.3 M nicardipine which nearly abolished the contraction and [Ca⁺]_i elevation produced by high K. During treatment of the strips with high K, addition of ET induced further increases in [Ca²⁺]_i and muscle tension, and vice versa. In Ca²⁺-free medium, ET-induced contraction was influenced neither by ryanodine-treatment nor by high K-treatment, although the former attenuated and the latter potentiated the [Ca²⁺]_i transient induced by ET. Further, the ET-induced sustained contraction under Ca²⁺-free conditions began to develop after the [Ca²⁺]_i level returned to the baseline. Thus, it seems that the Ca²⁺ released from the ryanodine-sensitive and -insensitive Ca²⁺ stores by ET may provide only a minor or indirect contribution, if any, to the tension development. ET might cause a contraction mainly by stimulating Ca²⁺-influx through Ca²⁺ channel(s) other than voltage-dependent Ca²⁺ channels in character, and by increasing the sensitivity of the contractile filaments to Ca²⁺ or activating them Ca²⁺-independently.Visiting from Zun Yi Medical College, China Send offprint requests to I. Takayanagi at the above address