The effects of recombinant rat μ-opioid receptor activation in CHO cells on phospholipase C, [Ca2+]i and adenylyl cyclase

1. The rat μ-opioid receptor has recently been cloned, yet its second messenger coupling remains unclear. The endogenous μ-opioid receptor in SH-SY5Y cells couples to phospholipase C (PLC), increases [Ca²⁺]_i and inhibits adenylyl cyclase (AC). We have examined the effects of μ-opioid agonists on inositol(1,4,5)trisphosphate (Ins(1,4,5)P₃), [Ca²⁺]_i and adenosine 3′ : 5′-cyclic monophosphate (cyclic AMP) formation in Chinese hamster ovarian (CHO) cells transfected with the cloned μ-opioid receptor.
2. Opioid receptor binding was assessed with [³H]-diprenorphine ([³H]-DPN) as a radiolabel. Ins(1,4,5)P₃ and cyclic AMP were measured by specific radioreceptor assays. [Ca²⁺]_i was measured fluorimetrically with Fura-2.
3. Scatchard analysis of [³H]-DPN binding revealed that the B_max varied between passages. Fentanyl (10 pM–1 μM) dose-dependently displaced [³H]-DPN, yielding a curve which had a Hill slope of less than unity (0.6±0.1), and was best fit to a two site model, with pK_i values (% of sites) of 9.97±0.4 (27±4.8%) and 7.68±0.07 (73±4.8%). In the presence of GppNHp (100 μM) and Na⁺ (100 mM), the curve was shifted to the right and became steeper (Hill slope=0.9±0.1) with a pK_i value of 6.76±0.04.
4. Fentanyl (0.1 nM–1 μM) had no effect on basal, but dose-dependently inhibited forskolin (1 μM)-stimulated, cyclic AMP formation (pIC₅₀=7.42±0.23), in a pertussis toxin (PTX; 100 ng ml⁻¹ for 24 h)-sensitive and naloxone-reversible manner (K_i=1.7 nM). Morphine (1 μM) and [D-Ala², MePhe⁴, gly(ol)⁵]-enkephalin (DAMGO, 1 μM) also inhibited forskolin (1 μM)-stimulated cyclic AMP formation, whilst [D-Pen², D-Pen⁵], enkephalin (DPDPE, 1 μM) did not.
5. Fentanyl (0.1 nM–10 μM) caused a naloxone (1 μM)-reversible, dose-dependent stimulation of Ins(1,4,5)P₃ formation, with a pEC₅₀ of 7.95±0.15 (n=5). PTX (100 ng ml⁻¹ for 24 h) abolished, whilst Ni²⁺ (2.5 mM) inhibited (by 52%), the fentanyl-induced Ins(1,4,5)P₃ response. Morphine (1 μM) and DAMGO (1 μM), but not DPDPE (1 μM), also stimulated Ins(1,4,5)P₃ formation. Fentanyl (1 μM) also caused an increase in [Ca²⁺]_i (80±16.4 nM, n=6), reaching a maximum at 26.8±2.5 s. The increase in [Ca²⁺]_i remained elevated until sampling ended (200 s) and was essentially abolished by the addition of naloxone (1 μM). Pre-incubation with naloxone (1 μM, 3 min) completely abolished fentanyl-induced increases in [Ca²⁺]_i.
6. In conclusion, the cloned μ-opioid receptor when expressed in CHO cells stimulates PLC and inhibits AC, both effects being mediated by a PTX-sensitive G-protein. In addition, the receptor couples to an increase in [Ca²⁺]_i. These findings are consistent with the previously described effector-second messenger coupling of the endogenous μ-opioid receptor.

     

Mg2+ and ATP dependence of KATP channel modulator binding to the recombinant sulphonylurea receptor,SUR2B

1. The binding of modulators of the ATP-sensitive K⁺ channel (K_ATP channel) to the murine sulphonylurea receptor, SUR2B, was investigated. SUR2B, a proposed subunit of the vascular K_ATP channel, was expressed in HEK 293 cells and binding assays were performed in membranes at 37°C using the tritiated K_ATP channel opener, [³H]-P1075.
2. Binding of [³H]-P1075 required the presence of Mg²⁺ and ATP. MgATP activated binding with EC₅₀ values of 10 and 3 μM at free Mg²⁺ concentrations of 3 μM and 1 mM, respectively. At 1 mM Mg²⁺, binding was lower than at 3 μM Mg²⁺.
3. [³H]-P1075 saturation binding experiments, performed at 3 mM ATP and free Mg²⁺ concentrations of 3 μM and 1 mM, gave K_D values of 1.8 and 3.4 nM and B_MAX values of 876 and 698 fmol mg⁻¹, respectively.
4. In competition experiments, openers inhibited [³H]-P1075 binding with potencies similar to those determined in rings of rat aorta.
5. Glibenclamide inhibited [³H]-P1075 binding with K_i values of 0.35 and 2.4 μM at 3 μM and 1 mM free Mg²⁺, respectively. Glibenclamide enhanced the dissociation of the [³H]-P1075-SUR2B complex suggesting a negative allosteric coupling between the binding sites for P1075 and the sulphonylureas.
6. It is concluded that an MgATP site on SUR2B with μM affinity must be occupied to allow opener binding whereas Mg²⁺ concentrations ⩾10 μM decrease the affinities for openers and glibenclamide. The properties of the [³H]-P1075 site strongly suggest that SUR2B represents the drug receptor of the openers in vascular smooth muscle.

     

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.

     

Pharmacological characterization of type 1α metabotropic glutamate receptor-stimulated [35S]-GTPγS binding

1. The activation of G proteins by type 1α metabotropic glutamate receptors (mGluRs) in membranes from recombinant baby hamster kidney cells expressing the cloned rat mGluR1α receptor has been studied by use of a [³⁵S]-guanosine 5′-[γ-thio]triphosphate ([³⁵S]-GTPγS) binding assay.
2. L-Glutamate increased the rate of [³⁵S]-GTPγS binding in a concentration-dependent manner (−logEC₅₀ (M) 5.25±0.07), with an optimal (62.4±1.6%) increase over basal binding being observed following 60 min incubation at 30°C with 70 pM [³⁵S]-GTPγS, 1 μM GDP, 10 mM MgCl₂, 100 mM NaCl and 100 μg membrane protein ml⁻¹. The L-glutamate (100 μM)-stimulated increase in [³⁵S]-GTPγS binding was totally prevented in the presence of the group I mGluR antagonist (S)-4-carboxy-3-hydroxyphenylglycine (300 μM).
3. Quantitative analysis of the affinity and number of G proteins activated by a maximally effective concentration of L-glutamate revealed an equilibrium dissociation constant (K_D) for [³⁵S]-GTPγS binding of 0.76±0.20 nM and a maximal number of GTPγS-liganded G proteins (B_max) of 361±30 fmol mg⁻¹ protein.
4. Metabotropic glutamate receptor agonists, quisqualate (−logEC₅₀ (M) 6.74±0.06), 1S,3R-ACPD (4.64±0.08) and (S)-3,5-dihydroxyphenylglycine (5.16±0.23) also increased [³⁵S]-GTPγS binding in a concentration-dependent manner, with the latter two agents behaving as partial agonists.
5. (+)-α-Methylcarboxyphenylglycine (300 μM) caused a parallel rightward shift of the L-glutamate concentration-effect curve for [³⁵S]-GTPγS binding, allowing an antagonist equilibrium dissociation constant (K_D) of 34.0±7.8 μM to be calculated for this mGluR antagonist.
6. Pretreatment of BHK-mGluR1α cells with a concentration of pertussis toxin (PTX) shown to be maximally effective (100 ng ml⁻¹, 24 h) before membrane preparation resulted in a marked decrease in agonist-stimulated [³⁵S]-GTPγS binding (by 66.0±0.9%), and an altered concentration-effect relationship for agonist-stimulated [³⁵S]-GTPγS binding by the residual PTX-insensitive G-protein population.
7. The modulation of [³⁵S]-GTPγS binding by agonists and antagonists in membranes from recombinant cells provides an excellent system in which to study mGluR interactions with PTX-sensitive and -insensitive G proteins.

     

The spasmogenic effects of vanadate in human isolated bronchus

1. Inhalation of vanadium compounds, particularly vanadate, is a cause of occupational bronchial asthma. We have now studied the action of vanadate on human isolated bronchus. Vanadate (0.1 μM–3 mM) produced concentration-dependent, well-sustained contraction. Its −logEC₅₀ was 3.74±0.05 (mean±s.e.mean) and its maximal effect was equivalent to 97.5±4.2% of the response to acetylcholine (ACh, 1 mM).
2. Vanadate (200 μM)-induced contraction of human bronchus was epithelium-independent and was not inhibited by indomethacin (2.8 μM), zileuton (10 μM), a mixture of atropine, mepyramine and phentolamine (each at 1 μM), or by mast cell degranulation with compound 48/80.
3. Vanadate (200 μM)-induced contraction was unaltered by tissue exposure to verapamil or nifedipine (each 1 μM) or to a Ca²⁺-free, EGTA (0.1 mM)-containing physiological salt solution (PSS). However, tissue incubation with ryanodine (10 μM) in Ca²⁺-free, EGTA (0.1 mM)-containing PSS reduced vanadate-induced contraction. A series of vanadate challenges was made in tissues exposed to Ca²⁺-free EGTA (0.1 mM)-containing PSS with the object of depleting intracellular Ca²⁺ stores. In such tissues cyclopiazonic acid (CPA; 10 μM) prevented Ca²⁺-induced recovery of vanadate-induced contraction.
4. Tissue incubation in K⁺-rich (80 mM) PSS, K⁺-free PSS, or PSS containing ouabain (10 μM) did not alter vanadate (200 μM)-induced contraction. Ouabain (10 μM) abolished the K⁺-induced relaxation of human bronchus bathed in K⁺-free PSS. This action was not shared by vanadate (200 μM). The tissue content of Na⁺ was increased and the tissue content of K⁺ was decreased by ouabain (10 μM). In contrast, vanadate (200 μM) did not alter the tissue content of these ions. Tissue incubation in a Na⁺-deficient (25 mM) PSS or in PSS containing amiloride (0.1 mM) markedly inhibited the spasmogenic effect of vanadate (200 μM).
5. Vanadate (200 μM)-induced contractions were markedly reduced by tissue treatment with each of the protein kinase C (PKC) inhibitors H-7 (10 μM), staurosporine (1 μM) and calphostin C (1 μM). Genistein (100 μM), an inhibitor of protein tyrosine kinase, also reduced the response to vanadate.
6. Vanadate (0.1–3 mM) and ACh (1 μM–3 mM) each increased inositol phosphate accumulation in bronchus. Such responses were unaffected by a Ca²⁺-free medium either alone or in combination with ryanodine (10 μM).
7. In human cultured tracheal smooth muscle cells, histamine (100 μM) and vanadate (200 μM) each produced a transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i).
8. Intracellular microelectrode recording showed that the contractile effect of vanadate (200 μM) in human bronchus was associated with cellular depolarization.
9. It is concluded that vanadate acts directly on human bronchial smooth muscle, promoting the release of Ca²⁺ from an intracellular store. The Ca²⁺ release mechanism involves both the production of inositol phosphate second messengers and inhibition of Ca-ATPase. The activation of PKC plays an important role in mediating vanadate-induced contraction at values of [Ca²⁺]_i that are close to basal.

     

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.

     

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.

     

Effects of Ca2+ channel blocker neurotoxins on transmitter release and presynaptic currents at the mouse neuromuscular junction

1. The effects of the voltage-dependent calcium channel (VDCC) blockers ω-agatoxin IVA (ω-AgaIVA), ω-conotoxin GVIA (ω-CgTx), ω-conotoxin MVIIC (ω-MVIIC) and ω-conotoxin MVIID (ω-MVIID) were evaluated on transmitter release in the mouse diaphragm preparation. The effects of ω-AgaIVA and ω-MVIIC were also evaluated on the perineurial calcium and calcium-dependent potassium currents, I_Ca and I_K(Ca), respectively, in the mouse levator auris preparation.
2. The P- and Q-type VDCC blocker ω-AgaIVA (100 nM) and P- Q- and N-type channel blockers ω-MVIIC (1 μM) and ω-MVIID (3 μM) strongly reduced transmitter release (>80–90% blockade) whereas the selective N-type channel blocker ω-CgTx (5 μM) was ineffective.
3. The process of release was much more sensitive to ω-MVIIC (IC₅₀=39 nM) than to ω-MVIID (IC₅₀=1.4 μM). After almost completely blocking transmitter release (quantal content ∼0.3% of its control value) with 3 μM ω-MVIIC, elevating the external [Ca²⁺] from 2 to 10 mM induced an increase of ∼20 fold on the quantal content of the endplate potential (e.p.p.) (from 0.2±0.04 to 4.8±1.4).
4. Nerve-evoked transmitter release in a low Ca²⁺-high Mg²⁺ medium (low release probability, quantal content = 2±0.1) had the same sensitivity to ω-AgaIVA (IC₅₀=16.8 nM) as that in normal saline solutions. In addition, K⁺-evoked transmitter release was also highly sensitive to the action of this toxin (IC₅₀=11.5 nM; 100 nM >95% blockade). The action of ω-AgaIVA on transmitter release could be reversed by toxin washout if the experiments were carried out at 31–33°C. Conversely, the effect of ω-AgaIVA persisted even after two hours of toxin washout at room temperature.
5. Both the calcium and calcium-dependent potassium presynaptic currents, I_Ca and I_K(Ca), respectively, were highly sensitive to low concentrations (10–30 nM) of ω-AgaIVA. The I_Ca and the I_K(Ca) were also strongly reduced by 1 μM ω-MVIIC. The most marked difference between the action of these two toxins was the long incubation times required to achieve maximal effects with ω-MVIIC.
6. In summary these results provide more evidence that synaptic transmission at the mammalian neuromuscular junction is mediated by Ca²⁺ entry through P- and/or Q-type calcium channels.

     

Modulation of IL-1-induced cartilage injury by NO synthase inhibitors: a comparative study with rat chondrocytes and cartilage entities

1. Nitric oxide (NO) is produced in diseased joints and may be a key mediator of IL-1 effects on cartilage. Therefore, we compared the potency of new [aminoguanidine (AG), S-methylisothiourea (SMT), S-aminoethylisothiourea (AETU)] and classical [N^ω-monomethyl-L-arginine (L-NMMA), N^ω-nitro-L-arginine methyl ester (L-NAME)] NO synthase (NOS) inhibitors on the inhibitory effect of recombinant human interleukin-1β (rhIL-1β) on rat cartilage anabolism. Three different culture systems were used: (1) isolated chondrocytes encapsulated in alginate beads; (2) patellae and (3) femoral head caps.
2. Chondrocyte beads and cartilage entities were incubated in vitro for 48 h in the presence of rhIL-1β with a daily change of incubation medium to obtain optimal responses on proteoglycan synthesis and NO production. Proteoglycan synthesis was assessed by incorporation of radiolabelled sodium sulphate [Na₂³⁵SO₄] and NO production by cumulated nitrite release during the period of study.
3. Chondrocytes and patellae, as well as femoral head caps, responded concentration-dependently to IL-1β challenge (0 to 250 U ml⁻¹ and 0 to 15 U ml⁻¹ respectively) by a large increase in nitrite level and a marked suppression of proteoglycan synthesis. Above these concentrations of IL-1β (2500 U ml⁻¹ and 30 U ml⁻¹ respectively), proteoglycan synthesis plateaued whereas nitrite release still increased thus suggesting different concentration-response curves.
4. When studying the effect of NOS inhibitors (1 to 1000 μM) on NO production by cartilage cells stimulated with IL-1β (25 U ml⁻¹ or 5 U ml⁻¹), we observed that: (i) their ability to reduce nitrite level decreased from chondrocytes to cartilage samples, except for L-NMMA and AETU; (ii) they could be roughly classified in the following rank order of potency: AETU>L-NMMA⩾SMT>AG⩾L-NAME and (iii) AETU was cytotoxic when used in the millimolar range.
5. When studying the effect of NOS inhibitors on proteoglycan synthesis by cartilage cells treated with IL-1β, we observed that: (i) they had more marked effects on proteoglycan synthesis in chondrocytes than in cartilage samples; (ii) they could be roughly classified in the following rank order of potency: L-NAME⩾L-NMMA>>AG>SMT>>AETU and (iii) potentiation of the IL-1 effect by AETU was consistent with cytotoxicity in the millimolar range.
6. D-isomers of L-arginine analog inhibitors (1000 μM) were unable to correct nitrite levels or proteoglycan synthesis in IL-1β treated cells. L-arginine (5000 μM) tended to reverse the correcting effect of L-NMMA (1000 μM) on proteoglycan synthesis, thus suggesting a NO-related chondroprotective effect. However, data with L-NAME and SMT argued against a general inverse relationship between nitrite level and proteoglycan synthesis.
7. Dexamethasone (0.1 to 100 μM) (i) failed to inhibit NO production in femoral head caps and chondrocytes beads whilst reducing it in patellae (50%) and (ii) did not affect or worsened the inhibitory effect of IL-1β on proteoglycan synthesis. Such results suggested a corticosteroid-resistance of rat chondrocyte iNOS. Data from patellae supported a possible contribution of subchondral bone in NO production.
8. In conclusion, our results suggest that (i) NO may account only partially for the suppressive effects of IL-1β on proteoglycan synthesis, particularly in cartilage samples; (ii) the chondroprotective potency of NOS inhibitors can not be extrapolated from their effects on NO production by joint-derived cells and (iii) L-arginine analog inhibitors are more promising than S-substituted isothioureas for putative therapeutical uses.

     

Involvement of bradykinin B1 and B2 receptors in relaxation of mouse isolated trachea

1. The aim of the present study was to investigate the effects of bradykinin and [des-Arg⁹]-bradykinin and their relaxant mechanisms in the mouse isolated trachea.
2. In the resting tracheal preparations with intact epithelium, bradykinin and [des-Arg⁹]-bradykinin (each drug, 0.01–10 μM) induced neither contraction nor relaxation. In contrast, bradykinin (0.01–10 μM) induced concentration-dependent relaxation when the tracheal preparations were precontracted with methacholine (1 μM). The relaxation induced by bradykinin was inhibited by the B₂ receptor antagonist, D-Arg⁰-[Hyp³,Thi⁵,D-Tic⁷,Oic⁸]-bradykinin (Hoe 140, 0.01–1 μM) in a concentration-dependent manner whereas the B₁ receptor antagonist, [des-Arg⁹,Leu⁸]-bradykinin (0.01–1 μM), had no inhibitory effect on bradykinin-induced relaxation. [des-Arg⁹]-bradykinin (0.01–10 μM) also caused concentration-dependent relaxation after precontraction with methacholine. The relaxation induced by [des-Arg⁹]-bradykinin was concentration-dependently inhibited by the B₁ receptor antagonist, [des-Arg⁹,Leu⁸]-bradykinin (0.01–1 μM), whereas the B₂ receptor antagonist, Hoe 140 (0.01–1 μM) was without effect.
3. In the presence of the cyclo-oxygenase inhibitor, indomethacin (0.01–1 μM), the relaxations induced by bradykinin and [des-Arg⁹]-bradykinin were inhibited concentration-dependently.
4. Two nitric oxide (NO) biosynthesis inhibitors N^G-nitro-L-arginine methyl ester (L-NAME, 100 μM) and N^G-nitro-L-arginine (L-NOARG, 100 μM) had no inhibitory effects on the relaxations induced by bradykinin and [des-Arg⁹]-bradykinin. Neither did the selective inhibitor of the soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 μM) inhibit the relaxations induced by bradykinin and [des-Arg⁹]-bradykinin.
5. Prostaglandin E₂ (PGE₂, 0.01–33 μM) caused concentration-dependent relaxation of the tracheal preparations precontracted with methacholine. Indomethacin (1 μM) and ODQ (10 μM) exerted no inhibitory effects on the relaxation induced by PGE₂.
6. The NO-donor, sodium nitroprusside (SNP; 0.01–100 μM) also caused concentration-dependent relaxation of the tracheal preparations precontracted with methacholine. ODQ (0.1–1 μM) concentration-dependently inhibited the relaxation induced by SNP.
7. These data demonstrate that bradykinin and [des-Arg⁹]-bradykinin relax the mouse trachea precontracted with methacholine by the activation of bradykinin B₂-receptors and B₁-receptors, respectively. The stimulation of bradykinin receptors induces activation of the cyclo-oxygenase pathway, leading to the production of relaxing prostaglandins. The NO pathway is not involved in the bradykinin-induced relaxation. The relaxation caused by NO-donors in the mouse trachea is likely to be mediated via activation of soluble guanylate cyclase.

     

The effects of nifedipine and other calcium antagonists on the glibenclamide-sensitive K+ currents in smooth muscle cells from pig urethra

1. The effects of nifedipine on both levcromakalim-induced membrane currents and unitary currents in pig proximal urethra were investigated by use of patch-clamp techniques (conventional whole-cell configuration and cell-attached patches).
2. Nifedipine had a voltage-dependent inhibitory effect on voltage-dependent Ba²⁺ currents at −50 mV (K_i=30.6 nM).
3. In current-clamp mode, subsequent application of higher concentrations of nifedipine (⩾30 μM) caused a significant depolarization even after the membrane potential had been hyperpolarized to approximately −82 mV by application of 100 μM levcromakalim.
4. The 100 μM levcromakalim-induced inward current (symmetrical 140 mM K⁺ conditions, −50 mV) was inhibited by additional application of three different types of Ca antagonists (nifedipine, verapamil and diltiazem, all at 100 μM). In contrast, Bay K 8644 (1 μM) possessed no activating effect on the amplitude of this glibenclamide-sensitive current.
5. When 100 μM nifedipine was included in the pipette solution during conventional whole-cell recording at −50 mV, application of levcromakalim (100 μM) caused a significant inward membrane current which was suppressed by 5 μM glibenclamide. On the other hand, inclusion of 5 μM glibenclamide in the pipette solution prevented levcromakalim from inducing an inward membrane current.
6. The levcromakalim-induced K⁺ channel openings in cell-attached configuration were suppressed by subsequent application of 5 μM glibenclamide but not of 100 μM nifedipine.
7. These results suggest that in pig proximal urethra, nifedipine inhibits the glibenclamide-sensitive 43 pS K⁺ channel activity mainly through extracellular blocking actions on the K⁺ channel itself.

     

Nitric oxide-related cyclic GMP-independent relaxing effect of N-acetylcysteine in lipopolysaccharide-treated rat aorta

1. We have recently demonstrated the formation of protein-bound dinitrosyl-iron complexes (DNIC) in rat aortic rings exposed to lipopolysaccharide (LPS) and shown that N-acetylcysteine (NAC) can promote vasorelaxation in these arteries, possibly via the release of nitric oxide (NO) as low molecular weight DNIC from these storage sites. The aim of the present study was to investigate further the mechanism of the relaxation induced by NAC in LPS-treated vessels.
2. In rings incubated with LPS (10 μg ml⁻¹ for 18 h) and precontracted with noradrenaline (NA, 3 μM) plus N^ω-nitro-L-arginine methylester (L-NAME, 3 mM), the relaxation evoked by NAC (0.1 to 10 mM) was abolished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 μM, a selective inhibitor of soluble guanylyl cyclase) but not affected by Rp-8-bromoguanosine 3′5′-cyclic monophosphorothioate (Rp-8BrcGMPS, 60 μM a selective inhibitor of cyclic GMP-dependent protein kinase). Tetrabutylammonium (TBA, 3 mM, as a non selective K⁺ channels blocker) or elevated concentration of external KCl (25 or 50 mM) significantly attenuated the NAC-induced relaxation. Selective K⁺ channels blockers (10 μM glibenclamide, 0.1 μM charybdotoxin, 0.5 μM apamin or 3 mM 4-aminopyridine) did not affect the NAC-induced relaxation. The relaxing effect of NAC (10 mM) was not associated with an elevation of guanosine 3′ : 5′ cyclic monophosphate (cyclic GMP) in LPS-treated rings.
3. In aortic rings precontracted with NA (0.1 μM), low molecular weight DNIC (with thiosulphate as ligand, 1 nM to 10 μM) evoked a concentration-dependent relaxation which was antagonized by ODQ (1 μM) and Rp-8BrcGMPS (150 μM) but not significantly affected by TBA (3 mM) or by the use of KCl (50 mM) as preconstricting agent. The relaxation produced by DNIC (0.1 μM) was associated with an 11 fold increase in aortic cyclic GMP content, which was completely abolished by ODQ (1 μM).
4. Taken together with our previous data, the main finding of the present study is that the vascular relaxation induced by NAC in LPS-treated aorta, although probably related to NO through an interaction via preformed NO stores, was not mediated by activation of the cyclic GMP pathway. It may involve the activation of TBA-sensitive K⁺ channels. The differences in the mechanism of relaxation induced by NAC and by exogenous DNIC suggest that the generation of low molecular weight DNIC from protein-bound species does not play a major role in the NAC-induced relaxation observed in LPS-treated rat aorta. In addition, it is suggested that ODQ may display other properties than the inhibition of soluble guanylyl cyclase.

     

Cyclothiazide and AMPA receptor desensitization: analyses from studies of AMPA-induced release of [3H]-noradrenaline from hippocampal slices

1. Responses in brain produced by the activation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype of ionotropic receptor for L-glutamate are often rapidly desensitizing. AMPA-induced desensitization and its characteristics, and the potentiating effect of cyclothiazide were investigated in vitro by analysing AMPA-induced release of [³H]-noradrenaline from prisms of rat hippocampus.
2. AMPA (1–1000 μM) stimulated the release of [³H]-noradrenaline in a concentration-dependent manner that was both calcium-dependent and tetrodotoxin-sensitive, and attenuated by the AMPA-selective antagonists, NBQX (1 and 10 μM), LY 293558 (1 and 10 μM) and GYKI 52466 (10 and 30 μM).
3. By use of an experimental procedure with consecutive applications of AMPA (100 μM, 28 min apart), the second response was reduced, indicative of receptor desensitization, and was reversed by cyclothiazide in a concentration-dependent manner (1–300 μM). The concentration-response curve for AMPA-induced release of [³H]-noradrenaline was shifted leftwards, but the reversal by cyclothiazide of the desensitized response was partial and failed to reach the maximal response of the first stimulus.
4. Observations made with various schedules of cyclothiazide application indicated that the initial AMPA-evoked response was already partially desensitized (150% potentiation by 100 μM cyclothiazide) and that the desensitization was not likely to be due to a time-dependent diminution and was long-lasting (second application of cyclothiazide was ineffective).
5. Co-application of a number of drugs with actions on second messenger systems, in association with the second AMPA stimulus, revealed significant potentiation of the AMPA-induced release of [³H]-noradrenaline: forskolin (10 μM, +78%), Rp-cAMPS (100 μM, +65%), Ro 31-8220 (10 μM, + 163%) and thapsigargin (100 μM, +161%).
6. The AMPA receptor-mediated response regulating the release of [³H]-noradrenaline from rat hippocampal slices was desensitized and cyclothiazide acted to reverse partially the desensitization in a concentration-dependent manner. Since the time-course of desensitization was longer lasting than that noted in previous electrophysiological studies, multiple events may be involved in the down-regulation of AMPA receptor activity including receptor phosphorylation and depletion of intracellular Ca²⁺ stores.

     

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.

     

The ability of a new hypoglycaemic agent,A-4166, compared to sulphonylureas,to increase cytosolic Ca2+ in pancreatic β-cells under metabolic inhibition

1. N-(trans-4-isopropylcyclohexanecarbonyl)-D-phenylalanine (A-4166) is a new non-sulphonylurea oral hypoglycaemic agent which stimulates insulin release by increasing cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in β-cells.
2. We studied comparative effects of A-4166 and sulphonylureas on [Ca²⁺]_i, measured by dual-wavelength fura-2 microfluorometry, in single rat pancreatic β-cells under normal conditions and conditions where glucose metabolism was inhibited.
3. A glucokinase inhibitor, mannoheptulose (10 mM), a mitochondrial respiratory inhibitor, KCN (100 μM), and uncouplers, dinitrophenol (DNP, 50 μM) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, 0.3 μM), were used to abolish glucose-induced increases in [Ca²⁺]_i in a reversible manner.
4. Under control conditions, A-4166 was one order more potent than tolbutamide in increasing [Ca²⁺]_i, and maximal responses were evoked by 30 μM A-4166 and 300 μM tolbutamide. These equipotent concentrations were employed for the comparative study where glucose metabolism was inhibited.
5. In the presence of mannoheptulose, [Ca²⁺]_i responses to tolbutamide, but not those to A-4166, were attenuated in a reversible manner.
6. KCN, DNP and FCCP inhibited [Ca²⁺]_i responses to tolbutamide to a much greater extent than those to A-4166. Responses to tolbutamide even at 3.3 times the equipotent concentration (1000 μM) were also markedly attenuated by these inhibitors. Responses evoked by another sulphonylurea, gliclazide, were inhibited by DNP to a larger extent than A-4166-induced responses.
7. The results indicate that A-4166 acts more effectively than sulphonylureas to increase [Ca²⁺]_i in β-cells during metabolic inhibition.

     

Effects of L-NG-nitro-arginine on noradrenaline induced contraction in the rat anococcygeus muscle

1. The influence of L-N^G-nitro-arginine (L-NOARG, 30 μM) on contractile responses to exogenous noradrenaline was studied in the rat anococcygeus muscle.
2. Noradrenaline (0.1–100 μM) contracted the muscle in a concentration-dependent manner. L-NOARG (30 μM) had no effect on noradrenaline responses.
3. Phenoxybenzamine (Pbz 0.1 μM) depressed by 46% (P<0.001) the maximum response and shifted to the right (P<0.001) the E/[A] curve to noradrenaline (pEC₅₀ control: 6.92±0.09; pEC₅₀ Pbz: 5.30±0.10; n=20).
4. The nested hyperbolic null method of analysing noradrenaline responses after phenoxybenzamine showed that only 0.61% of the receptors need to be occupied to elicit 50% of the maximum response, indicating a very high functional receptor reserve.
5. Contractile responses to noradrenaline after partial α₁-adrenoceptor alkylation with phenoxybenzamine (0.1 μM) were clearly enhanced by L-NOARG.
6. The potentiating effect of L-NOARG on noradrenaline responses after phenoxybenzamine was reversed by (100 μM) L-arginine but not by (100 μM) D-arginine.
7. These results indicate that spontaneous release of NO by nitrergic nerves can influence the α₁-adrenoceptor-mediated response to exogenous noradrenaline.

     

Coexistence of purino- and pyrimidinoceptors on activated rat microglial cells

1. Nucleotide-induced currents in untreated (proliferating) and lipopolysaccharide (LPS; 100 ng ml⁻¹) treated (non-proliferating) rat microglial cells were recorded by the whole-cell patch-clamp technique. Most experiments were carried out on non-proliferating microglial cells. ATP (100 nM–1 mM), ADP (10 nM–10 mM) and UTP (1 μM–100 mM), but not uridine (100 μM–10 mM) produced a slow outward current at a holding potential of 0 mV. The effect of UTP (1 mM) did not depend on the presence of extracellular Mg²⁺ (1 mM). The outward current response to UTP (1 mM) was similar in non-proliferating and proliferating microglia.
2. In non-proliferating microglial cells, the ATP (10 μM)-induced outward current was antagonized by suramin (300 μM) or reactive blue 2 (50 μM), whereas 8-(p-sulphophenyl)-theophylline (8-SPT; 100 μM) was inactive. By contrast, the current induced by UTP (1 mM) was increased by suramin (300 μM) and was not altered by reactive blue 2 (50 μM) or 8-SPT (100 μM).
3. The current response to UTP (1 mM) disappeared when K⁺ was replaced in the pipette solution by an equimolar concentration of Cs⁺ (150 mM). However, the effect of UTP (1 mM) did not change when most Cl⁻ was replaced with an equimolar concentration of gluconate⁻ (145 mM). The application of 4-aminopyridine (1 mM) or Cs⁺ (1 mM) to the bath solution failed to alter the UTP (1 mM)-induced current. UTP (1 mM) had almost no effect in a nominally Ca²⁺-free bath medium, or in the presence of charybdotoxin (0.1 μM); the inclusion of U-73122 (5 μM) or heparin (5 mg ml⁻¹) into the pipette solution also blocked the responses to UTP (1 mM). By contrast, the effect of ATP (10 μM) persisted under these conditions.
4. I-V relations were determined by delivering fast voltage ramps before and during the application of UTP (1 mM). In the presence of extracellular Cs⁺ (1 mM) and 4-aminopyridine (1 mM) the UTP-evoked current crossed the zero current level near−75 mV. Omission of Ca²⁺ from the Cs⁺ (1 mM)- and 4-aminopyridine (1 mM)-containing bath medium or replacement of K⁺ by Cs⁺ (150 mM) in the pipette solution abolished the UTP current.
5. Replacement of GTP (200 μM) by GDP-β-S (200 μM) in the pipette solution abolished the current evoked by UTP (1 mM).
6. When the pipette solution contained Cs⁺ (150 mM) instead of K⁺ and in addition inositol 1,4,5,-trisphosphate (InsP₃; 10 μM), an inward current absolutely dependent on extracellular Ca²⁺ was activated after the establishment of whole-cell recording conditions. This current had a typical delay, a rather slow time course and did not reverse its amplitude up to 100 mV, as measured by fast voltage ramps.
7. A rise of the internal free Ca²⁺ concentration from 0.01 to 0.5 μM on excised inside-out membrane patches produced single channel activity with a reversal potential of 0 mV in a symmetrical K⁺ solution. The reversal potential was shifted to negative values, when the extracellular K⁺ concentration was decreased from 144 to 32 mM. By contrast, a decrease of the extracellular Cl⁻ concentration from 164 to 38 mM did not change the reversal potential.
8. Purine and pyrimidine nucleotides act at separate receptors in rat microglial cells. Pyrimidinoceptors activate via a G protein the enzyme phospholipase C with the subsequent release of InsP₃. The depletion of the intracellular Ca²⁺ pool appears to initiate a capacitative entry of Ca⁺ from the extracellular space. This Ca²⁺ then activates a Ca²⁺-dependent K⁺ current.

     

Pharmacological comparison of UTP- and thapsigargin-induced arachidonic acid release in mouse RAW 264.7 macrophages

1. Although stimulation of mouse RAW 264.7 macrophages by UTP elicits a rapid increase in intracellular free Ca²⁺ ([Ca²⁺]_i), phosphoinositide (PI) turnover, and arachidonic acid (AA) release, the causal relationship between these signalling pathways is still unclear. In the present study, we investigated the involvement of phosphoinositide-dependent phospholipase C (PI-PLC) activation, Ca²⁺ increase and protein kinase activation in UTP-induced AA release. The effects of stimulating RAW 264.7 cells with thapsigargin, which cannot activate the inositol phosphate (IP) cascade, but results in the release of sequestered Ca²⁺ and an influx of extracellular Ca²⁺, was compared with the effects of UTP stimulation to elucidate the multiple regulatory pathways for cPLA₂ activation.
2. In RAW 264.7 cells UTP (100 μM) and thapsigargin (1  μM) caused 2 and 1.2 fold increases, respectively, in [³H]-AA release. The release of [³H]-AA following treatment with UTP and thapsigargin were non-additive, totally abolished in the Ca²⁺-free buffer, BAPTA (30 μM)-containing buffer or in the presence of the cPLA₂ inhibitor MAFP (50 μM), and inhibited by pretreatment of cells with pertussis toxin (100 ng ml⁻¹) or 4-bromophenacyl bromide (100 μM). By contrast, aristolochic acid (an inhibitor of sPLA₂) had no effect on UTP and thapsigargin responses.
3. {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (10 μM) and neomycin (3 mM), inhibitors of PI-PLC, inhibited UTP-induced IP formation (88% and 83% inhibition, respectively) and AA release (76% and 58%, respectively), accompanied by a decrease in the [Ca²⁺]_i rise.
4. Wortmannin attenuated the IP response of UTP in a concentration-dependent manner (over the range 10 nM–3 μM), and reduced the UTP-induced AA release in parallel. RHC 80267 (30 μM), a specific diacylglycerol lipase inhibitor, had no effect on UTP-induced AA release.
5. Short-term treatment with PMA (1 μM) inhibited the UTP-stimulated accumulation of IP and increase in [Ca²⁺]_i, but had no effect on the release of AA. In contrast, the AA release caused by thapsigargin was increased by PMA.
6. The role of PKC in UTP- and thapsigargin-mediated AA release was shown by the blockade of these effects by staurosporine (1 μM), Ro 31-8220 (10 μM), Go 6976 (1 μM) and the down-regulation of PKC.
7. Following treatment of cells with SK&F 96365 (30 μM), thapsigargin-, but not UTP-, induced Ca²⁺ influx, and the accompanying AA release, were down-regulated.
8. Neither PD 98059 (100 μM), MEK a inhibitor, nor genistein (100 μM), a tyrosine kinase inhibitor, had any effect on the AA responses induced by UTP and thapsigargin.
9. We conclude that UTP-induced cPLA₂ activity depends on the activation of PI-PLC and the sustained elevation of intracellular Ca²⁺, which is essential for the activation of cPLA₂ by UTP and thapsigargin. The [Ca²⁺]_i-dependent AA release that follows treatment with both stimuli was potentiated by the activity of protein kinase C (PKC). A pertussis toxin-sensitive pathway downstream of the increase in [Ca²⁺]_i was also shown to be involved in AA release.

     

Release of the antioxidants ascorbate and urate from a nitrergically-innervated smooth muscle

1. The main object of the present study was to determine whether ascorbate, an antioxidant which has been shown to protect nitric oxide (NO) from attack by scavenger molecules, might be released from nitrergically-innervated smooth muscle; ascorbate release from the rat anococcygeus was measured by use of h.p.l.c. with electrochemical detection.
2. Incubation of rat anococcygeus muscles in normal physiological salt solution (PSS; 30 min) resulted in release of ascorbate into the bathing medium (7.7±0.9 nmol g⁻¹ tissue). This release was increased by 96% when muscles were incubated in high K⁺ (70 mM) PSS. The resting release of ascorbate was unaffected by tetrodotoxin (TTX; 1 μM), ω-conotoxin GVIA (10 nM) or omission of calcium ions from the PSS (with addition of 0.2 mM EGTA), but all three procedures attenuated the increased release observed under depolarizing conditions. Resting release of ascorbate was unaffected by glutamate (100 μM), aspartate (100 μM), γ-aminobutyric acid (100 μM) or carbachol (50 μM).
3. A second h.p.l.c. peak, which always preceded the ascorbate peak, was identified as urate. Urate release from the anococcygeus, following 30 min incubation in normal PSS, was 64.6±12.7 nmol g⁻¹ tissue but, unlike ascorbate, urate release was unchanged in high K⁺ PSS. In functional experiments, urate (100–400 μM) partially protected NO (15 μM)-induced relaxations of the rat anococcygeus from inhibition by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO; 50 μM), but not from inhibition by hydroquinone or duroquinone (both 100 μM).
4. Muscles chemically sympathectomized with 6-hydroxydopamine (6-OHDA, 500 μM; 2 h) still exhibited release of ascorbate (2.5±0.4 nmol g⁻¹ tissue) and urate (22.2±2.9 nmol g⁻¹ tissue); in both cases the release was similar to that observed in time-matched control tissues not exposed to 6-OHDA. High K⁺ PSS produced a TTX-sensitive increase in release of ascorbate, but not urate, from 6-OHDA-treated muscles.
5. The results demonstrate that significant amounts of ascorbate and urate are released from the rat anococcygeus muscle. Ascorbate, but not urate, release appears to be enhanced by activation of nerves which are resistant to 6-OHDA pretreatment. Since both antioxidants can protect NO from attack by scavenger molecules, their release in nitrergically-innervated tissues may be important for the provision of the correct redox environment to allow NO to fulfill its proposed neurotransmitter role.

     

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.