Effects of diltiazem and verapamil on ADP-induced rabbit platelet shape change and aggregation

• 1.1. The effects of diltiazem and verapamil (two structurally different calcium channel blockers) were examined on the rabbit platelets shape change and aggregation induced by adenosine-5′-diphosphate (ADP).
• 2.2. ADP was a much more potent stimulator on inducing platelet shape change (ED₅₀ = 1 × 10⁻⁷) than platelet aggregation (ED₅₀ = 1.78 × 10⁻⁶).
• 3.3. Both drugs similarly inhibited ADP-induced platelet shape change and aggregation at concentrations more than 300 μM.
• 4.4. There were no significant differences in inhibitory effects of either diltiazem or verapamil on ADP-induced platelet shape change and aggregation.
• 5.5. The inhibitory effects of diltiazem and verapamil on ADP-induced platelet shape change and aggregation at high concentrations may be due to their non specific properties.

     

Different roles of endothelium-derived substances on inhibiting platelet aggregation in whole blood

• 1.1. The inhibitory effects of adenosine, nitroprusside (a nitric oxide donor) and prostacyclin on collagen induced rabbit platelet aggregation were studied under two different conditions: in whole blood with an impedance method and in platelet-rich plasma (PRP) with a turbidimetric method.
• 2.2. All substances tested were less potent in whole blood than in PRP, and the differences in IC₅₀ value between whole blood and PRP were not of the same order of magnitude; adenosine (669-fold), nitroprusside (54-fold) and prostacyclin (2-fold).
• 3.3. These results imply that (a) some other, as yet unknown, factors in blood modulate the platelet aggregation; (b) adenosine and nitric oxide act close to the endothelium, and (c) prostacyclin acts as a relatively long lasting circulating hormone.

     

Pharmacological properties of MM-706, a new prostacyclin derivative

• 1.1. In human platelet-rich plasma, platelet aggregation induced in vitro by collagen (10 μg/ml) or thrombin (50 mU/ml) was dose-dependently inhibited by increasing concentrations of prostacyclin or of the new derivative (±)(5E)-13,14-didehydro-ω-hexanor(1-hydroxycyclohexyl)-9a-carbaprostacyclin (MM-706) with an IC₅₀ of 20–50 nM and 250–500 nM, respectively. In human platelets loaded with fura-2, the intracellular rise of [Ca²⁺] induced by thrombin was dose-dependently inhibited by MM-706 with an approximate IC₅₀ of 100 μM.
• 2.2. In rabbit isolated femoral artery, MM-706 (10nM–10μM) was completely ineffective in relaxing the vessel, which was different to prostacyclin which was able to relax vessels at the same concentrations.
• 3.3. In in vitro guinea-pig ileum, prostacyclin produced a contractile effect in the concentration range 1 nM-10μM, but the derivative MM-706 was ineffective at the same concentrations. Preventive addition of MM-706 did not inhibit prostacyclin contraction.
• 4.4. On isolated guinea-pig tracheal preparation, prostacyclin induced a concentration-dependent contraction but the new compound MM-706 showed a lower activity, in the concentration range 10nM–10μM. The activity of prostacyclin was not affected by the contemporary presence of MM-706.
• 5.5. It is concluded that MM-706 is a prostacyclin analogue with antiaggregating properties but without evident effects on smooth muscle of different regions.

     

Influence of diethylcarbamazine and mefloquine on PGI2 synthesis by the rat thoracic aorta and myometrial tissues

• 1.1. The influence of the antifilarial drug diethylcarbamazine citrate (D) and dl-erythro mefloquine hydrochloride (Mf) on PGI₂ synthesis by the male rat thoracic aorta and day-20 pregnant rat myometrium was investigated in vitro using a rat platelet antiaggregatory bioassay method.
• 2.2. Pretreatment of the tissues with D (25.5–204 μM) or Mf (24–192 μM) for 30 min at 37°C significantly inhibited PGI₂ synthesis in a concentration-dependent manner.
• 3.3. D exhibited its inhibitory effect even in presence of exogenous arachidonic acid (AA) (16.6 μM) whereas Mf lost its inhibitory effect in presence of AA.
• 4.4. Pretreatment of urethane-anaesthetized rats with D (32 μmol kg⁻¹) but not Mf (7.5 μmol kg⁻¹) for 30 min significantly antagonized AA (4 nmol kg⁻¹)-induced hypotension
• 5.5. Furthermore, D (0.25–0.5 μM) antagonized AA-induced aggregation in rabbit platelet-rich plasma without affecting that of ADP.
• 6.6. D seemed to interfere with the action of the PG endoperoxide synthase (PG cyclooxygenase) whereas Mf seemed to interfere with the action of phospholipase A₂ (PLA₂) enzyme.
• 7.7. D may have exerted its effect via release of toxic O₂ radicals whereas Mf effect may have been due to an interaction with PLA₂ substrate phospholipids.
• 8.8. The demonstrated inherent property of these two drugs to inhibit the synthesis of the potent vasodilator, platelet antiaggregatory, anticonvulsant and antiinflammatory mediator PGI₂ may partly contribute towards better understanding of the biochemical mechanisms that underly some of the previously known but poorly understood actions of these drugs.

     

Antiplatelet and calcium inhibitory properties of eugenol and sodium eugenol acetate

• 1.1. Eugenol (3-methoxy-4-hydroxy-propenylbenzene) or sodium eugenol acetate (4-0-acetic acid sodium-3-methoxy-l-propenylbenzene) (0.25, 0.5, 1 mM) concentration-dependently inhibited arachidonic acid (AA)., collagen-, epinephrine- and ADP-induced platelet aggregation.
• 2.2. Eugenol or sodium eugenol acetate inhibited collagen-induced aggregation of washed rabbit platelets synergistically with creatine phosphatelcreatine phosphokinase (CP/CPK, 5 mM/10 U/ml) or p-bromophenacyl bromide (p-BPB, 10 μM), and they also potentiated the inhibitory action of imidazole (0.5 mM) on AA-induced aggregation.
• 3.3. Eugenol or sodium eugenol acetate (0.25, 0.5, 1 mM) concentration-dependently inhibited AA-induced thromboxane B₂ and prostaglandin E₂ formation.
• 4.4. The rise of intracellular Ca²⁺ caused by collagen, epinephrine, ADP, and AA were inhibited by eugenol or sodium eugenol acetate (1 mM).

     

Blockade of cardiac nicotinic responses by anticholinesterases

• 1.1. Tacrine (10 μM) and physostigmine (10 μM) completely inhibited the positive chronotropic and inotropic actions of acetylcholine (ACh) or nicotine in the atropinized guinea pig right atria.
• 2.2. Edrophonium (6 μM) and soman (0.1 μM) completely inhibited these nicotinic responses, as well as the associated increase in pyridine nucleotide fluorescence and vasodilation induced by ACh in the atropinized guinea pig perfused heart.
• 3.3. The 200-fold increase in noradrenaline release induced by ACh in the perfused heart was blocked by 10 μM tacrine and 6 μM edrophonium.
• 4.4. Tacrine (10 μM) significantly (16–32%) reduced the basal heart rate of both preparations.
• 5.5. Edrophonium (6 μM) induced a five- to sixfold increase in basal 3,4-dihydroxyphenyl-(ethylene) glycol (DOPEG) release.
• 6.6. The inhibition of nicotinic receptor activation in the atria by the anticholinesterases appears mainly non-competitive. IC₅₀ values range from 0.1 to 10 μM in the perfused heart to 1 to 100 μM in atria (in either case tacrine about 2 μM).
• 7.7. The possibility that these compounds have a direct action at nicotinic receptors is discussed.

     

Interplay between milrinone and adenosine in the inhibition of human platelet response

• 1.1. In this study, we investigated the influence of the isotropic agent and coronary vasodilator milrinone on platelet aggregation and intracellular levels of 3′,5′ cyclic adenosine monophosphate (cAMP) in human platelet-rich plasma (PRP) and whole blood (WB). Furthermore, we evaluated the influence of milrinone on the effects of adenosine, which reduces the platelet aggregation through an elevation of intraplatelet cAMP levels.
• 2.2. Milrinone decreased the platelet aggregation in response to agonists in both PRP and WB. A dose-dependent increase of intraplatelet cAMP levels was demonstrated: this result is in accordance with an effect on platelet phosphodiesterases.
• 3.3. Milrinone at low concentration and adenosine exerted additive effects on platelet aggregation and intraplatelet cAMP levels.
• 4.4. An interplay between milrinone and adenosine was shown in WB. Furthermore, dipyridamole, which prevents the uptake of endogenous adenosine, markedly enhanced the milrinone antiaggregating effect, whereas the adenosine receptor blocker, theophylline, decreased it.
• 5.5. The present data provide evidence that milrinone modulates the platelet function through an influence on intraplatelet levels of cAMP and it is able to interplay with substances stimulating adenylyl cyclase.
• 6.6. The interplay between milrinone and adenosine in the inhibition of the human platelet function could be effective during milrinone administration in the treatment of heart failure, when blood adenosine levels are significantly increased. These milrinone effects could be advantageous from a therapeutic point of view, since patients with heart failure are at risk of thrombosis and ischemic heart disease.

     

The mechanism of action of KBT-3022, a new antiplatelet agent

• 1.1. The mechanism of action of a new antiplatelet agent, KBT-3022 (ethyl 2-[4,5-bis(4-methoxyphenyl)thiazol-2-yl]pyrrol-1-ylacetate) and its active main metabolite, desethyl KBT-3022, was investigated.
• 2.2. KBT-3022 and desethyl KBT-3022 inhibited cyclooxygenase from ovine seminal gland with IC₅₀ values of 0.69 and 0.43 μM, respectively.
• 3.3. At concentrations higher than those required for cyclooxygenase inhibition, desethyl KBT-3022 inhibited cAMP-phosphodiesterase, specific binding of U46619, and release of phosphatidic acid from thrombin-stimulated platelets.
• 4.4. Oral administration of KBT-3022 inhibited the production of thromboxane B₂ during blood coagulation more potently than the production of 6-keto-prostaglandin F_1α from aortic strips in guinea pigs.
• 5.5. These findings suggest that KBT-3022 may inhibit platelet activation principally via the inhibition of cyclooxygenase by desethyl KBT-3022.

     

A comparison of the relaxant effects of pinacidil in rabbit renal and mesenteric artery

• 1.1. The relaxant effects of pinacidil were compared in isolated rabbit renal and mesenteric artery.
• 2.2. Pinacidil (10 nm–300 μM) relaxed renal and mesenteric arterial rings precontracted with phenylephrine with pD₂ values of 5.11 ± 0.03 and 6.27 ± 0.04, respectively.
• 3.3. The inhibitory effect of pinacidil on the rabbit mesenteric artery was competitively antagonized by glibenclamide (1–10 μM). The calculated pK_B value was 6.37 ± 0.04. On the renal artery, glibenclamide (2–20 μM) did not significantly affect pinacidil-induced relaxation (P > 0.05).
• 4.4. Tetraethylammonium (TEA, 1–10 mM) competitively antagonized the pinacaidil induced relaxation of the rabbit renal artery. The pK_B value was 3.22 ± 0.08. On the mesenteric artery TEA antagonized the effect of pinacidil in a noncompetitive manner.
• 5.5. The concentration-response curves for pinacidil on the rabbit renal and mesenteric artery were not affected by apamin (0.1 μM).
• 6.6. It is concluded that ATP-sensitivie K⁺ channels (K_ATP) are not involved in pinacidil action on the rabbit renal artery. On the contrary, K_ATP are probably major sites of pinacidil action on the rabbit mesenteric artery.

     

Sodium nonivamide acetate: A non-pungently antinociceptive capsaicin derivative with unusual anti-inflammatory properties

• 1.1. Bradykinin-induced vascular pain in conscious rats, hyperalgesia in the rat hind paw, rat hind paw edema induced by compound 48/80 and carrageenin and dye exudation induced by intraperitoneal injection of 0.7% acetic acid in mice were all inhibited by sodium nonivamide acetate (SNA).
• 2.2. Collagen and arachidonic acid-induced rabbit platelet aggregations were inhibited by SNA and capsaicin. In human platelet microsomes, prostaglandin E₂ formation in arachidonic acid metabolite was not inhibited by SNA but was inhibited by capsaicin and indomethacin; thromboxane B₂ formation and its synthetase activity were inhibited by SNA and capsaicin.
• 3.3. In the extracellular recording, SNA could not decrease the action potential amplitude of the vagus nerve.
• 4.4. The motor activity of mice induced by caffeine (1.0 mg/kg) was inhibited by SNA and capsaicin.

     

Possible Stimulation of Na+-K+-ATPase by NO Produced from Sodium Nitrite by Ultraviolet Light in Mouse Gastric Fundal Strip

• 1.In the present study, we investigated the roles of Na⁺-K⁺-ATPase and extracellular Na⁺ or Ca²⁺ ions in ultraviolet (UV) light-induced photorelaxation of methacholine-contracted mouse isolated gastric fundus in the presence of NaNO₂ (50 μM).
• 2.Ouabain (1–500 μM), sodium vanadate (10 μM to 3 mM) and amiloride (1–100 μM) completely inhibited the photorelaxation in a concentration-dependent manner.
• 3.Metabolic inhibitors, sodium azide (10–100 μM), 2,4-dinitrophenol (100 μM to 1 mM) and sodium fluoride (100 μM to 1 mM) significantly reduced photorelaxation.
• 4.Substitution of sucrose, lithium or KCl with extracellular Na⁺ completely abolished the photorelaxant responses.
• 5.Replacement of all extracellular CaCl₂ with BaCl₂ also completely inhibited UV-induced relaxation.
• 6.Verapamil (1–10 μM) decreased UV-induced relaxation significantly.
• 7.These results suggest that nitric oxide produced from NaNO₂ by UV-light in mouse gastric fundus probably stimulates Na⁺-K⁺-ATPase activity, and photorelaxation of gastric smooth muscle is dependent on extracellular Na⁺ and Ca²⁺ ions.

     

N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) impairs neurotransmission in the vas deferens of the rat

• 1.1. The in vitro effects of N-(2-chloroethyl)-N-ethyl-bromobenzylamine (DSP4) were studied in the rat vas deferens.
• 2.2. DSP4 inhibited the biphasic motor response induced by field stimulation in a concentration-dependent manner. The concentration of DSP4 that elicited 50% of the maximal inhibition of the twitch response induced by 3 Hz was 10 μM.
• 3.3. DSP4 10 μM abolished the motor response induced by exogenously applied noradrenaline and 0.1 mM ATP. Phentolamine (an α-adrenoceptor blocker) prevented DSP4 inhibitory effect.
• 4.4. DSP4 inhibitory effect was no due to the activation of α₂-presynaptic adrenoceptor mechanisms.
• 5.5. DSP4 impairs neurotransmission in the rat vas deferens by a postsynaptic α₁-adrenoceptor blockade and by an inhibition of the purinergic response.

     

Multiple sensory and functional effects of non-phenolic aminodimethylene nonivamide: An approach to capsaicin antagonist

• 1.1. Hexylaminodimethylene nonivamide (CAPCNC6, 0.1–10 μM) inhibited the contractility of isolated guinea pig right atria, toxically revealed positive inotropic, chronotropic and then a cardiac arrest effect at 100 μM and inhibited capsaicin (1.0 μM)-induced cardiotonic effects.
• 2.2. CAPCNC6 (0.1–10 μM)-induced aorta contractions were inhibited in the presence of flunarizine, atropine, phentolamine, Ca²⁺-free solution and pre-treatment of the animal with capsaicin.
• 3.3. CAPCNC6 (1.0–300 μM)-induced trachea contractions were inhibited in the presence of capsaze. pine, ruthenium red, hCGRP_8–37 and pre-treatment of the animal with capsaicin.

     

Calcium and depolarization-dependent effect of pregnenolone derivatives on uterine smooth muscle

• 1.1. The effects of several gestagens (pregnenolone [1 to 30 μm], 20α-hydroxypregnenolone [1 to 30 μM]), and 20β-hydroxypregnenolone [1 to 30 μm]) on rat uterine contraction induced by KC1 (60 mM) and CaCl₂ (30 μM to 6 mM) have been assayed.
• 2.2. The three drugs relaxed the tonic contraction induced by KCI in a concentration-dependent way. The respective EC₅₀ values were: 27.6±1.58 μM (pregnenolone), 4.1±0.12 μM (20α-hydroxypregnenolone), and 11.2±1.04 μM (20β-hydroxypregnenolone). CaCl₂ (1 to 10 mM) totally counteracted the relaxing effect of pregnenolone but only partially compared to that of 20α- or 20β-hydroxy-pregnenolone.
• 3.3. CaC1₂ (30 μM to 6 mM) produced concentration-dependent contraction of rat uterus in medium lacking calcium plus 30, 60, or 90 mM of KC1. The EC₅₀ values of CaCl₂ were: 0.38±0.072, 0.183±0.015, and 0.183±0.015 μM in a medium with 30, 60, or 90 mM of KCI, respectively.
• 4.4. Pregnenolone (10 μM) did not significantly modify the EC₅₀ of CaC1₂ in a medium with 30, 60, or 90 mM of KCI. However, 20β-hydroxypregnenolone (10 μM) antagonized, in a noncompetitive manner, the concentration-response curve to CaC1₂.
• 5.5. 20α-Hydroxypregnenolone (4 μM) antagonized the concentration-response curve to CaCl₂ in a competitive manner. This antagonism was directly related to the concentration of KCI in the medium.
• 6.6. Our results suggest a different calcium antagonist effect of the three gestagens assayed.

     

Adenosine selectively depresses muscarinic compared with non-muscarinic receptor mediated depolarisation of the rat superior cervical ganglion

• 1.1. A grease gap d.c. recording technique was used to measure electrophysiological responses of the isolated rat superior cervical ganglion.
• 2.2. Adenosine at 100 μM depressed depolarisations to the muscarinic agonists carbachol, muscarine and methylfurmethide. In contrast adenosine (100μM) did not alter depolarisations to 1,1-dimethyl-4-phenylpiperazinium, 2-methyl-5-hydroxytryptamine and potassium and enhanced depolarisations to 5-hydroxytryptamine and gamma-aminobutyric acid.
• 3.3. Adenosine-induced depressions of the depolarisations to carbachol, muscarine, and methylfurmethide tended to be increased in the presence of 0.3 μM methoctramine (a muscarinic receptor antagonist with slight selectivity for M₂ receptors). The increase was statistically significant (P < 0.01) for carbachol.
• 4.4. Medium containing 0.1 mM Ca²⁺ and 0.3 μM pirenzepine augmented the hyperpolarising phase of the response to carbachol. Adenosine (10–300μM) hyperpolarised ganglia and did not significantly alter the hyperpolarisation to 0.3 or 1 μM carbachol but selectively reduced the depolarisation response to 3 μM carbachol.
• 5.5. Adenosine-induced hyperpolarisations (100 μM) were enhanced when applied during depolarisations to muscarinic agonists (muscarine, pilocarpine, N-methyl-N-(1-methyl-4-pyrrolidine-2-butynyl)acetamide (BM-5)), and other M-current inhibitors, barium and eledoisin-related-peptide. Adenosine induced hyperpolarisations were not affected by d-Ala⁶-luteinizing-hormone-releasing-hormone or uridine 5′-triphosphate which produced small depolarisations.
• 6.6. It is concluded that adenosine acts selectively in opposing mechanisms of depolarisation of the rat SCG that are due to the action of muscarinic agonists (acting via M₁-receptors) and by other M-current inhibitors.

     

Carbachol-induced desensitization of rat basophilic leukemia (RBL-2H3) cells transfected with human m3 muscarinic acetylcholine receptors

• 1.1. Carbachol-induced homologous desensitization of the secretory response was investigated by transfecting RBL-2H3 cells with cDNA encoding the human m3 muscarinic acetylcholine receptor (RBL-m3).
• 2.2. Exposure of RBL-m3 cells to 100 μM carbachol for 30 min in Ca²⁺-free medium inhibited the secretion induced by the subsequent addition of 10 μM carbachol plus Ca²⁺.
• 3.3. Desensitized cells bound [³H]quinuclidinyl benzilate with a similar B_max and K_d to those of control cells.
• 4.4. The carbachol-induced transient increase in levels of inositol 1,4,5-trisphosphate was not changed by desensitization.
• 5.5. Homologous desensitization persisted when desensitized cells were permeabilized with Staphylococcal α-toxin.

     

Post-junctional excitatory adenosine A1 receptors in the rat vas deferens

• 1.1. At concentrations between 1 nM and 1 μM, the A₁-selective agonists N⁶-cyclopentyladenosine (CPA) and (R)-N⁶-phenylisopropyladenosine (R-PIA) each enhanced contractions of the rat vas deferens induced by ATP (10 μM), and this enhancement was blocked by an A₁-selective concentration (1 nM) of the antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX).
• 2.2. No such enhancement was observed with the non-selective agonists adenosine and 5′-N-ethylcarboxamidoadenosine (NECA) at concentrations between 1 nM and 100 μM, which instead inhibited the contractions.
• 3.3. These results show that in addition to the previously demonstrated inhibitory A₁ and A₂ adenosine receptors, the rat vas deferens also possesses post-junctional excitatory A₁ adenosine receptors.

     

Neuromuscular facilitation induced by muscarinic antagonists in the rat isolated diaphragm

• 1.1. Atropine (EC₅₀ = 87 μM), pirenzepine (447 μM), and AF-DX 116 (95.5 μM), but not 4-DAMP (at concentrations of up to 110 μM), produced neuromuscular facilitation and antagonized the oxotremorine-induced neuromuscular blockade in the rat isolated diaphragm.
• 2.2. Atropine, pirenzepine, and AF-DX 116 did not change the responses of curarized diaphragms to direct stimulation, or the twitch tension produced by retrograde injection of acetylcholine.
• 3.3. These results indicate that neuromuscular facilitation induced by muscarinic antagonists may depend on drug interaction with the M2 subtype of muscarinic autoreceptors to increase acetylcholine output in the neuromuscular junction.

     

Comparative effects of the Dihydropyridine-type calcium-agonists (−)-S-Bay K 8644, (±)-Bay-W 5035 and (±)-Bay-T 5006 on human platelet aggregability

• 1.1. Human platelet aggregation induced by collagen is concentration-dependently inhibited by dihydropyridine (DHP)-type calcium(Ca)-agonists.
• 2.2. There was no significant difference between the maximal anti-aggregatory effects or the anti-aggregatory potencies of (−)-S-Bay-K 8644 (EC₅₀: 5.3 ± 1.5 × 10^-5 M), (±)-Bay-W 5035 (EC₅₀: 14.9 ± 8.8 × 10^-5 M) or (±)-Bay-T 5006 (EC₅₀: 2.7 ± ± 10^-5 M) (P > 0.05).
• 3.3. Antiaggregatory effects of DHP-type Ca-agonists seem to be independent of Ca-channel activation.