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.

     

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.

     

Stellettamide-A,a novel inhibitor of calmodulin,isolated from a marine sponge

1. Stellettamide A (ST-A), a novel marine toxin isolated from a marine sponge, inhibited high K⁺(72.7 mM)-induced contraction in the smooth muscle of guinea-pig taenia coli with an IC₅₀ of 88 μM.
2. In the taenia permeabilized with Triton X-100, ST-A inhibited Ca²⁺ (3 and 10 μM)-induced contractions with an IC₅₀ of 46 μM for 3 μM Ca²⁺ and 105 μM for 10 μM Ca²⁺. In the permeabilized taenia, calyculin-A (300 nM), a potent inhibitor of type-1 and type-2A phosphatases, induced sustained contraction in the absence of Ca²⁺. ST-A had no effect on this contraction.
3. ST-A inhibited Mg²⁺-ATPase activity in native actomyosin prepared from chicken gizzard with an IC₅₀ of 25 μM.
4. In a reconstituted smooth muscle contractile system containing calmodulin, myosin light chain (MLC) and MLC kinase, ST-A inhibited MLC phosphorylation with an IC₅₀ of 152 μM. The inhibitory effect of ST-A was antagonized by increasing the concentration of calmodulin.
5. ST-A inhibited calmodulin activity, assessed by Ca²⁺/calmodulin-dependent enzymes, (Ca²⁺-Mg²⁺)-ATPase of erythrocyte membrane, with an IC₅₀ of 100 μM and phosphodiesterase prepared from bovine cardiac muscle with an IC₅₀ of 52 μM. The inhibitory effect on phosphodiesterase activity was antagonized by increasing the calmodulin concentration.
6. Interaction between ST-A and calmodulin was demonstrated by instantaneous quenching of the intrinsic tyrosine fluorescence of calmodulin by ST-A (3–300 μM). Similar results were obtained in the presence or absence of Ca²⁺ suggesting that ST-A binds to calmodulin and that Ca²⁺ is not essential for the binding of ST-A to calmodulin.
7. These results suggest that ST-A, isolated from marine metabolites, is a novel inhibitor of calmodulin.

     

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 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.

     

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.

     

The involvement of a novel mechanism distinct from the thrombin receptor in the vasocontraction induced by trypsin

1. The vasocontracting effect of a serine protease trypsin and its mechanisms were investigated by monitoring the isometric tension in endothelium-denuded rings of rabbit thoracic aortae and its effects on intracellular free Ca²⁺ concentrations ([Ca²⁺]_i) in dispersed rabbit vascular smooth muscle cells with a Ca²⁺ indicator fura-2. The actions of trypsin were compared with those of thrombin.
2. Both thrombin and trypsin reversibly contracted aortic rings without endothelium in a concentration-dependent manner. The vasocontraction induced by trypsin was well correlated with the protease activity of trypsin actually added to the tissue baths containing the aortic rings and was completely blocked by soybean trypsin inhibitor and phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor.
3. The trypsin-induced contractions of the aortic rings were not the result of irreversible damage to vascular smooth muscle cells, since the contractile responses induced by noradrenaline or 30 mM KCl were unaffected by pretreatment with trypsin.
4. The contractions induced by either thrombin or trypsin were reduced to about 30% of control responses after removal of extracellular Ca²⁺, indicating that most of the contraction is dependent on extracellular Ca²⁺. By contrast, the contractions induced by either of the proteases were reduced by an antagonist of L-type voltage-operated Ca²⁺ channels, nifedipine, to about 70% of control responses, indicating that both nifedipine-sensitive and -resistant Ca²⁺ channels are involved in these contractions.
5. In the aortic rings precontracted by a maximally effective concentration of thrombin, the second application of thrombin virtually failed to induce contractions but trypsin could still induce contractions amounting to 10% of control values by it''s protease activity.
6. After the first application of a maximal concentration of thrombin, the second application of thrombin could not induce an increase in [Ca²⁺]_i, but an application of trypsin could still induce an increase in [Ca²⁺]_i in dispersed rabbit vascular smooth muscle cells.
7. These data suggest that in addition to activation of a thrombin receptor, trypsin can contract rabbit aortae by a proteinase-activated receptor 2 or a novel mechanism.

     

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.     

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.

     

Stereospecific effects of ketamine enantiomers on canine tracheal smooth muscle

1. Ketamine is a potent bronchodilator which relaxes airway smooth muscle (ASM). Clinically, ketamine is used as a 1:1 racemic mixture of enantiomers that differ in their analgesic and anaesthetic effects. The aim of this study was to determine whether there was a difference between the enantiomers in their ability to relax isolated ASM and to explore mechanisms responsible for any observed differences.
2. Canine tracheal smooth muscle strips were loaded with fura-2 and mounted in a photometric system to measure simultaneously force and [Ca²⁺]_i. Calcium influx was estimated by use of a manganese quenching technique.
3. In strips stimulated with 0.1 μM ACh (EC₅₀) R(−)-ketamine (1–100 μM) caused a significantly greater concentration-dependent decrease in force (P<0.0001) and [Ca²⁺]_i than S(+)-ketamine (1–100 μM) (P<0.0005). In contrast, there was no significant difference between the enantiomers in their ability to inhibit calcium influx (45% decrease in influx rate for R(−)-ketamine and 44% for S(+)-ketamine, P=0.782). In strips contracted with 24 mM isotonic KCl (which activates voltage-operated calcium channels), the enantiomers modestly decreased force and [Ca²⁺]_i; there was no significant difference between the enantiomers in their effects on force (P=0.425) or [Ca²⁺]_i (P=0.604).
4. The R(−)-enantiomer of ketamine is a more potent relaxant of ACh-induced ASM contraction than the S(+)-enantiomer. This difference appears to be caused by differential actions on receptor-operated calcium channels.

     

The actions of the cannabinoid receptor antagonist,SR 141716A,in the rat isolated mesenteric artery

1. The actions of the cannabinoid receptor antagonist, SR 141716A, were examined in rat isolated mesenteric arteries. At concentrations greater than 3 μM, it caused concentration-dependent, but endothelium-independent, relaxations of both methoxamine- and 60 mM KCl-precontracted vessels.
2. SR 141716A (at 10 μM, but not at 1 μM) inhibited contractions to Ca²⁺ in methoxamine-stimulated mesenteric arteries previously depleted of intracellular Ca²⁺ stores. Neither concentration affected the phasic contractions induced by methoxamine in the absence of extracellular Ca²⁺.
3. SR 141716A (10 μM) caused a 130 fold rightward shift in the concentration-response curve to levcromakalim, a K⁺ channel activator, but had no effect at 1 μM.
4. SR 141716A (10 μM) attenuated relaxations to NS 1619 (which activates large conductance, Ca²⁺-activated K⁺ channels; BK_Ca). The inhibitory effect of SR 141716A on NS 1619 was not significantly different from, and was not additive with, that caused by a selective BK_Ca inhibitor, iberiotoxin (100 nM). SR 141716A (1 μM) did not effect NS 1619 relaxation.
5. SR 141716A (10 μM) had no effect on relaxations to the nitric oxide donor S-nitroso-N-acetylpenicillamine, or relaxations to carbachol in the presence of 25 mM KCl.
6. The results show that, at concentrations of 10 μM and above, SR 141716A causes endothelium-independent vasorelaxation by inhibition of Ca²⁺ entry. It also inhibits relaxations mediated by K⁺ channel activation. This suggests that such concentrations of SR 141716A are not appropriate for investigation of cannabinoid receptor-dependent processes.

     

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.

     

Vasorelaxant effects induced by the antiangiogenic drug linomide in aortic and saphenous vein preparations of the rabbit

1. Linomide (N-phenylmethyl-1,2-dihydro-4-hydroxyl-1-methyl-2-oxoquinoline-3-carboxamide) inhibits vascular proliferation and has been proposed as an antiangiogenic drug. We have investigated the vascular effect of linomide in rabbit aortic and saphenous vein ring preparations and in rat cultured vascular smooth muscle cells (VSMCs).
2. Linomide (25–300 μg ml⁻¹) did not alter the basal tone of the preparations. The drug induced a concentration-dependent relaxant effect in aortic rings with endothelium, preconstricted by noradrenaline (NA), 5-hydroxytryptamine (5-HT) and by the thromboxane mimetic U46619.
3. The degree of relaxation induced by linomide was significantly reduced by exposure to the cyclo-oxygenase inhibitors indomethacin (3 μM) and acetylsalicylic acid (500 μM), and was not influenced by pretreatment with the nitric oxide synthase inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) (100 μM) in aortic rings with endothelium, preconstricted with NA.
4. Endothelium removal significantly reduced the relaxant response to linomide in aortic ring preparations.
5. A concentration-dependent relaxant response was observed also in rabbit saphenous vein preparations deprived of endothelium and preconstricted either by NA or U46619. The degree of relaxation obtained in a high potassium solution was consistently smaller than that observed in NA-pretreated venous preparations.
6. The vasorelaxant effect of linomide was consistently blunted by the adenylate cyclase inhibitor SQ 22536 (50 μM), both in intact aortic rings and in those deprived of endothelium.
7. In rat cultured vascular smooth muscle cells, linomide (100–200 μg ml⁻¹) induced a significant increase in cyclic AMP levels, which was blocked by exposure to 50 μM SQ 22536.
8. In endothelium-deprived aortic ring preparations, the linomide-induced relaxant effect was greatly reduced in high potassium medium (KCl=25 mM). Pretreatment with the ATP potassium channel inhibitor glibenclamide (3 μM) significantly reduced the linomide-induced relaxation.
9. The results show that linomide possesses a vasorelaxant effect which is attributable to both endothelium-dependent and -independent properties. While the former component of the drug''s activity is apparently due to the release of a prostanoid from endothelial cells, the endothelium-independent mechanism involved in linomide relaxation is linked to cyclic AMP accumulation and to ATP-sensitive potassium channel activation in VSMCs.

     

Isoform-specific regulation of the Na+-K+ pump by adenosine in guinea pig ventricular myocytes

Aim:

The present study investigated the effect of adenosine on Na⁺-K⁺ pumps in acutely isolated guinea pig (Cavia sp.) ventricular myocytes.

Methods:

The whole-cell, patch-clamp technique was used to record the Na⁺-K⁺ pump current (I_p) in acutely isolated guinea pig ventricular myocytes.

Results:

Adenosine inhibited the high DHO-affinity pump current (I_h) in a concentration-dependent manner, which was blocked by the selective adenosine A₁ receptor antagonist DPCPX and the general protein kinase C (PKC) antagonists staurosporine, GF 109203X or the specific δ isoform antagonist rottlerin. In addition, the inhibitory action of adenosine was mimicked by a selective A₁ receptor agonist CCPA and a specific activator peptide of PKC-δ, PP114. In contrast, the selective A_2A receptor agonist {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 and A₃ receptor agonist Cl-IB-MECA did not affect I_h. Application of the selective A_2A receptor antagonist {"type":"entrez-protein","attrs":{"text":"SCH58261","term_id":"1052882304","term_text":"SCH58261"}}SCH58261 and A₃ receptor antagonist MRS1191 also failed to block the effect of adenosine. Furthermore, H89, a selective protein kinase A (PKA) antagonist, did not exert any effect on adenosine-induced I_h inhibition.

Conclusion:

The present study provides the electrophysiological evidence that adenosine can induce significant inhibition of I_h via adenosine A₁ receptors and the PKC-δ isoform.     

Effect of the triaminopyridine flupirtine on calcium uptake,membrane potential and ATP synthesis in rat heart mitochondria

1. Flupirtine is an analgesic agent which exhibits neuronal cytoprotective activity and may have value in the treatment of conditions involving cell injury and apoptosis. Since flupirtine has no action on known receptor sites we have investigated the effect of this drug on mitochondrial membrane potential, and the changes in intramitochondrial calcium concentration in particular.
2. The findings show that flupirtine increases Ca²⁺ uptake in mitochondria in vitro. At clinically relevant flupirtine concentrations, corresponding to flupirtine levels in vitro of 0.2 to 10 nmol mg⁻¹ mitochondrial protein, there was a 2 to 3 fold increase in mitochondrial calcium levels (P<0.01). At supra-physiological flupirtine concentrations of 20 nmol mg⁻¹ mitochondrial protein and above, the mitochondrial calcium concentrations were indistinguishable from those in untreated mitochondria.
3. Mitochondrial membrane potential closely paralleled the changes in mitochondrial calcium levels showing a 20% (P<0.01) increase when the flupirtine concentration was raised from 0.2 nmol to 10 nmol mg⁻¹ mitochondrial protein and a return to control values at 20 nmol mg⁻¹ protein.
4. The increase in mitochondrial calcium uptake and membrane potential were accompanied by an increase in mitochondrial ATP synthesis (30%; P<0.05) and a similar percentage reduction in mitochondrial volume.
5. Calcium at 80 and 160 nmol mg⁻¹ mitochondrial protein decreased ATP synthesis by 20–25% (P<0.001). This decrease was prevented or diminished if flupirtine at 10 nmol mg⁻¹ protein was added before the addition of calcium.
6. Since intracellular levels of flupirtine in intact cells never exceeded 10 nmol mg⁻¹ mitochondrial protein, these findings are supportive evidence for an in vivo cytoprotective action of flupirtine at the mitochondrial level.

     

Contribution of phosphodiesterase isozymes to the regulation of the L-type calcium current in human cardiac myocytes

1. To determine the contribution of the various phosphodiesterase (PDE) isozymes to the regulation of the L-type calcium current (I_Ca(L)) in the human myocardium, we investigated the effect of selective and non-selective PDE inhibitors on I_Ca(L) in single human atrial cells by use of the whole-cell patch-clamp method. We repeated some experiments in rabbit atrial myocytes, to make a species comparison.
2. In human atrial cells, 100 μM pimobendan increased I_Ca(L) (evoked by depolarization to +10 mV from a holding potential of −40 mV) by 250.4±45.0% (n=15), with the concentration for half-maximal stimulation (EC₅₀) being 1.13 μM. I_Ca(L) was increased by 100 μM UD-CG 212 by 174.5±30.2% (n=10) with an EC₅₀ value of 1.78 μM in human atrial cells. These two agents inhibit PDE III selectively.
3. A selective PDE IV inhibitor, rolipram (1–100 μM), did not itself affect I_Ca(L) in human atrial cells. However, 100 μM rolipram significantly enhanced the effect of 100 μM UD-CG 212 on I_Ca(L) (increase with UD-CG 212 alone, 167.9±33.9, n=5; increase with the two agents together, 270.0±52.2%; n=5, P<0.05). Rolipram also enhanced isoprenaline (5 nM)-stimulated I_Ca(L) by 52.9±9.3% (n=5) in human atrial cells.
4. In rabbit atrial cells, I_Ca(L) at +10 mV was increased by 22.1±9.0% by UD-CG 212 (n=10) and by 67.4±12.0% (n=10) by pimobendan (each at 100 μM). These values were significantly lower than those obtained in human atrial cells (P<0.0001). Rolipram (1–100 μM) did not itself affect I_Ca(L) in rabbit atrial cells. However, I_Ca(L) was increased by 215.7±65.2% (n=10) by the combination of 100 μM UD-CG 212 and 100 μM rolipram. This value was almost 10 times larger than that obtained for the effect of 100 μM UD-CG 212 alone.
5. These results imply a species difference: in the human atrium, the PDE III isoform seems dominant, whereas PDE IV may be more important in the rabbit atrium for regulating I_Ca(L). However, PDE IV might contribute significantly to the regulation of intracellular cyclic AMP in human myocardium when PDE III is already inhibited or when the myocardium is under β-adrenoceptor-mediated stimulation.

     

The actions of azelnidipine, a dihydropyridine-derivative Ca antagonist, on voltage-dependent Ba2+ currents in guinea-pig vascular smooth muscle

BACKGROUND AND PURPOSE: Although azelnidipine is used clinically to treat hypertension its effects on its target cells, Ca2+ channels, in smooth muscle have not been elucidated. Therefore, its effects on spontaneous contractions and voltage-dependent L-type Ca2+ channels were investigated in guinea-pig portal vein. EXPERIMENTAL APPROACH: The inhibitory potency of azelnidipine on spontaneous contractions in guinea-pig portal vein was compared with those of other dihydropyridine (DHP)-derived Ca antagonists (amlodipine and nifedipine) by recording tension. Also its effects on voltage-dependent nifedipine-sensitive inward Ba2+ currents (IBa) in smooth muscle cells dispersed from guinea-pig portal vein were investigated by use of a conventional whole-cell patch-clamp technique. KEY RESULTS: Spontaneous contractions in guinea-pig portal vein were reduced by all of the Ca antagonists (azelnidipine, Ki = 153 nM; amlodipine, Ki = 16 nM; nifedipine, Ki = 7 nM). In the whole-cell experiments, azelnidipine inhibited the peak amplitude of IBa in a concentration- and voltage-dependent manner (-60 mV, Ki = 282 nM; -90 mV, Ki = 2 microM) and shifted the steady-state inactivation curve of IBa to the left at -90 mV by 16 mV. The inhibitory effects of azelnidipine on IBa persisted after 7 min washout at -60 mV. In contrast, IBa gradually recovered after being inhibited by amlodipine, but did not return to control levels. Both azelnidipine and amlodipine caused a resting block of IBa at -90 mV. Only nifedipine appeared to interact competitively with S(-)-Bay K 8644. CONCLUSIONS AND IMPLICATIONS: These results suggest that azelnidipine induces long-lasting vascular relaxation by inhibiting voltage-dependent L-type Ca2+ channels in vascular smooth muscle.