Comparison of effects of formoterol and BRL 37344 on isolated term-pregnant rat myometrial strips in vitro

This study was designed to compare the effects of β-adrenoceptor agonists formoterol and BRL 37344 on spontaneous contractions and the levels of cAMP and cGMP of myometrial strips isolated from timed-pregnant rats. Myometrial strips were obtained from term-pregnant Wistar albino rats (n = 12), mounted in organ baths and tested for changes in isometric tension in response to formoterol and BRL 37344. We evaluated the effect of increasing concentrations of formoterol and BRL 37344 on oxytocin-induced myometrial contractions and on contractions of myometrial smooth muscle pretreated with metoprolol, ICI 118.551 and SR 59230A (β₁, β₂, β₃-adrenoceptor antagonist, respectively, 10^− 6 M). Effects of formoterol and BRL 37344 on cAMP and cGMP levels in isolated myometrial strips (n = 6) were evaluated by radioimmunoassay kits. Formoterol (10^− 12–10^− 8 M) and BRL 37344 (10^− 11–10^− 5 M) concentration-dependently decreased the amplitude of oxytocin-induced contractions. E_max value (100%) of formoterol was increased significantly more than E_max value (70.6%) of BRL 37344 (P < 0.05), with no change in pD₂ value (9.54 ± 0.12 and 9.12 ± 0.12, respectively). The inhibition of the amplitude of oxytocin-induced contractions by formoterol was antagonized with ICI 118.551 (10^− 6 M), but they were not changed by metoprolol (10^− 6 M) or SR 59230A (10^− 6 M). The inhibition of the amplitude of oxytocin-induced contractions by BRL 37344 were antagonized with SR 59230A (10^− 6 M), but they were not changed by metoprolol (10^− 6 M) or ICI 118.551 (10^− 6 M). Formoterol and BRL 37344 increased cAMP levels. BRL 37344 increased cGMP levels in BRL 37344 group more than control group, but this increase is less significant than cAMP levels (P > 0.05). Formoterol and BRL 37344 decreased amplitude of myometrial contractions with similar potency, but efficacy of formoterol was better than BRL 37344.     

Paraoxon inhibits GABA uptake in brain synaptosomes

To investigate possible effect of paraoxon (10⁻⁹–10⁻³ M) on GABA uptake, we used rat cerebral cortex synaptosomes. K_m and V_max of GABA uptake were determined in presence of paraoxon (10⁻³ M). Acetylcholine and its antagonists (atropine and mecamylamine) were used for evaluating cholinergic-dependency of uptake. Type of transporter involved was determined by using glial (beta-alanine) and neuronal (DABA) GABA uptake inhibitors. The results of the study showed that paraoxon at low doses (10⁻⁹–10⁻⁶ M) increased and at high doses (10⁻⁵–10⁻³ M) decreased GABA uptake. One millimolar paraoxon significantly decreased V_max (175.2 ± 4.23 vs. 80.4 ± 2.03, P < 0.001) of GABA uptake while had no effect on its K_m. DABA significantly decreased GABA uptake (P < 0.001) while beta-alanine had no effect. In conclusion, present data suggests that paraoxon probably acts as non-competitive antagonist of GABA uptake.     

alpha2B-adrenoceptor agonist ST-91 antagonizes beta2-adrenoceptor-mediated relaxation in rat mesenteric artery rings

We sought an isolated vascular preparation and experimental setting where the function of _2B-adrenoceptors could be demonstrated by non-recombinant technique. ST-91 (2-[2,6-diethylphenylamino]-2-imidazoline), an _2B-adrenoceptor agonist with a mixed adrenergic receptor type/subtype selection profile antagonized the relaxant effect of isoproterenol in endothelium-denuded rat mesenteric artery rings precontracted with phenylephrine. At 10^− 7 M of ST-91, the antagonism was characterized by a rightward shift of isoproterenol dose–response curve (A₅₀ = 6.81 ± 1.40 e− 7 (n = 4) vs the control 1.29 ± 0.25 e− 7 M (n = 4)) with no E_max depression. At 10^− 6 M the E_max depression was prevalent (36.1 ± 7.0% (n = 4) vs the control 79.9 ± 5.1% (n = 4)); both actions could be antagonized by the ₂-adrenoceptor antagonist yohimbine. The not subtype-selective ₂-adrenoceptor agonist xylazine (10^− 7 M) did not affect the relaxant action of isoproterenol. Present findings are discussed in the light of previously reported hemodynamic effects attributed to _2B-adrenoceptors in receptor subtype-knockout animals.     

Rho kinase expression and its central role in ovine gallbladder contractions elicited by a variety of excitatory stimuli

Rho kinase has contractile activity, which induces Ca2+ sensitization in various cells. Several receptors are linked to the Rho/Rho-kinase pathway. Therefore, in this study we aimed to demonstrate the central importance of this novel pathway for diverse excitatory stimuli in the smooth muscle of the sheep gallbladder. Accordingly, the effects of a Rho kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, 10(-8)-3 x 10(-5) M), were investigated on cholecystokinin-8 (CCK-8, 10(-8) M), endothelin-1 (10(-8) M), carbachol (10(-6)-10(-5) M), 5-hydroxytryptamine (5-HT, 10(-6)-10(-5) M), histamine (10(-6)-10(-5) M), phenylephrine (10(-5)-10(-4) M), neurokinin A (10(-7)-10(-6) M), electrical field stimulation (40 V, 0.5 ms, 2, 4, 8, 16, 32 Hz, 15 s, 3 min intervals) and potassium chloride (KCl, 25-50 mM)-induced contractions as well as spontaneous contractile activity. Electrical field stimulation evoked tetrodotoxin (3 x 10(-6) M)-sensitive reproducible contractions, which were inhibited by atropine (2 x 10(-6) M) and potentiated by eserine (5 x 10(-7) M). EFS-induced contraction was significantly inhibited by Y-27632 (10(-5) M). In addition, spontaneous contractile activity was suppressed in the presence of the compound (10(-6)-10(-5) M). This Rho kinase inhibitor also dramatically decreased the contractions elicited by 5-HT, neurokinin A and carbachol. KCl-induced contraction, which was not atropine-sensitive, was also conspicuously attenuated by Y-27632. Moreover, Y-27632 (10(-8)-3 x 10(-5) M) relaxed gallbladder strips that were contracted by histamine, endothelin-1, CCK-8 and phenylephrine in a concentration-dependent manner. pEC50 values for Y-27632 were 6.25+/-0.10, 5.79+/-0.12, 5.83+/-0.09 and 5.70+/-0.13 for the contraction elicited by histamine, CCK-8, endothelin-1 and phenylephrine, respectively. Furthermore, we also demonstrated Rho kinase protein expression (ROCK-1 and ROCK-2) by Western blot analysis. In conclusion, ROCK is expressed in the smooth muscle of the ovine gallbladder, and it has a central role in the contractile activity induced by diverse excitatory stimuli.     

KATP channels do not mediate vasodilation by 3-morpholinosydnonimine in goat coronary artery

The present study investigated the role of ATP-sensitive potassium (K_ATP) channels in mediating relaxation to the nitric oxide (NO) donor, 3-morpholinosydnonimine (SIN-1) in goat coronary arteries. SIN-1 (10⁻⁸–10⁻⁵ M) caused concentration-dependent relaxations of the coronary artery ring segments contracted with K⁺ (30 mM) with an EC₅₀ of 6.61×10⁻⁷ M. Methylene blue (3×10⁻⁶ M) caused a rightward shift in the concentration–response curve of SIN-1 (10⁻⁸–3×10⁻⁵ M) with a corresponding increase in the EC₅₀ (3.62×10⁻⁶ M) of the nitrovasodilator. While the K_ATP channel blocker, glibenclamide (1 and 3×10⁻⁶ M) caused dose-dependent inhibition of vasorelaxations produced by pinacidil (10⁻⁸–10⁻⁴ M), it had no effect on the vasodilations elicited by SIN-1 (10⁻⁸–10⁻⁵ M) in the coronary arterial smooth muscle. Increasing the extracellular K⁺ concentration from 30 mM to 80 mM to reduce the K⁺ gradient across the cell membrane, inhibited the relaxations elicited by pinacidil (10⁻⁸–10⁻⁴ M). On the other hand, SIN-1 (10⁻⁸–10⁻⁵ M)-induced relaxations were potentiated in high K⁺ (80 mM) compared to those observed at K⁺ (30 mM). These results suggest that goat coronary artery vasodilations caused by the NO donor, SIN-1, do not involve K_ATP channels.     

In vitro and in vivo effects of endothelin-1 and YM598, a selective endothelin ET A receptor antagonist, on the lower urinary tract

We investigated the contractile response of the lower urinary tract to endothelin-1 in vitro (rabbits) and in vivo (dogs). We also assessed the effects of a selective endothelin ET_A receptor antagonist, (E)-N-[6-methoxy-5-(2-methoxyphenoxy)[2, 2′-bipyrimidin]-4-yl]-2-phenylethenesulfonamide monopotassium salt (YM598), on endothelin-1-induced contractile responses. In the in vitro study, endothelin-1 induced contractile responses in isolated rabbit bladder base, urethra, and prostate tissues. YM598 (10^− 7–10^− 5 M) antagonized these endothelin-1-induced contractile responses without affecting the maximal responses. In the in vivo study, endothelin-1 induced the elevation of non-prostatic urethral pressure as well as prostatic urethral pressure even in the presence of tamsulosin (10 μg/kg, i.v.) in anesthetized male dogs. YM598 (0.1–3 mg/kg, i.v.) inhibited these endothelin-1-induced contractile responses in a dose-dependent fashion. These results suggest that endothelin ET_A receptors play an important role in the lower urinary tract contraction, and that the selective endothelin ET_A receptor antagonist YM598 has ameliorating effects on various urinary dysfunctions, including benign prostatic hyperplasia.     

Role of Rho-kinase in guinea-pig gallbladder smooth muscle contraction

Guinea-pig gallbladder smooth muscle contractions can be elicited pharmacologically by a range of mechanisms. The involvement of Rho-kinase in contractions mediated by receptor-dependent and receptor-independent mechanisms was investigated using the Rho-kinase inhibitor (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide (Y-27632). In a separate series of experiments, the role of Rho-kinase in the contractile response to Ca2+ entry through store-operated Ca2+ channels and to electrical field stimulation was also examined. Y-27632 (10 microM), which caused a significant decrease (P<0.0005) in basal resting tone, significantly inhibited gallbladder contractions evoked by cumulative additions of the G-protein-coupled agonists, carbachol (1 nM-100 microM; P<0.05) and cholecystokinin (10 nM-1 microM; P<0.005). Y-27632 also inhibited the contractions evoked by a single addition of the sarcoplasmic reticulum ATPase inhibitor, thapsigargin (1 microM; P<0.0005) and cumulative additions of KCl (10-85 mM; P<0.0005). The contractile response to Ca2+ entry through store-operated Ca2+ channels was significantly inhibited by Y-27632 (P<0.05) as were the contractile responses evoked by electrical field stimulation (2-25 Hz; P<0.0005). In contrast, Y-27632 had no significant effect on contractions evoked by phorbol 12,13-dibutyrate (0.1 nM-1 microM; a protein kinase C activator) or by the phosphatase inhibitor, cantharidin (100 microM). In conclusion, Rho-kinase contributes to the contractile response in guinea-pig gallbladder smooth muscle evoked by both G-protein-coupled and non-G-protein-coupled mechanisms in addition to contributing to the maintenance of basal tone. It also contributes to the contractile responses resulting from electrical field stimulation and store-operated Ca2+ channel entry.     

Fluorimetric determination of some antibiotics in raw material and dosage forms through ternary complex formation with terbium (Tb(3+))

A highly sensitive and specific fluorimetric method was developed for the determination of cefazolin sodium I, cefoperazone sodium II, ceftriaxone sodium III, and cefixime IV. The proposed method involves the formation of ternary complex with Tb³⁺ in the presence of Tris buffer. The quenching of the terbium fluorescence due to the complex formation was quantitative for the four studied drugs. The effect of pH, concentration of Tris buffer and terbium were studied. The formation of the complex was highly dependent on the pH. The optimum pH was found to be pH 8 for cefazolin sodium I, ceftriaxone sodium III, cefixime IV and pH 10 for cefoperazone sodium II. The optimum concentration for Tb³⁺ was found 1 ml of 10⁻⁴ M solution and for Tris buffer 1 ml of the prepared solution. Under the described conditions, the proposed method was applicable over the concentration range 8.79×10⁻⁶–7.91×10⁻⁵, 9.7×10⁻⁶–4.49×10⁻5, 6.10×10⁻⁶–2.50×10⁻⁵, and 4.92×10⁻⁶–2.95×10⁻⁵ mol with mean percentage accuracy of 99.79±0.24, 98.97±1.25, 100.05±0.79, and 100.15±0.54 for I, II, III, and IV, respectively. The proposed method was applied successfully for the determination of studied drugs in bulk powder and in pharmaceutical formulations. The results obtained by applying the described method were statistically analyzed and compared with those obtained by applying the official method. The proposed method was used as stability indicating method for the determination of the studied drugs in the presence of their degradation products.     

Voltammetric investigation of diethylstilbestrol

In this work electrooxidation of diethylstilbestrol (DES) was investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) using a glassy carbon (GC) electrode. It was statistically shown that both methods could be used for the determination of DES in the concentration range of 2×10⁻⁵–6×10⁻⁴ M by CV and 1×10⁻⁵–1×10⁻³ M in methanol (MeOH) and 4×10⁻⁵–6×10⁻⁴ M in acetonitrile (ACN) by DPV and both of the methods could be applied to human serum. A mechanism was proposed about the electrooxidation of this substance.     

8-isoprostaglandin E(2) activates Ca(2+)-dependent K(+) current via cyclic AMP signaling pathway in murine renal artery

The inhibitory pathway of 8-isoprostaglandin E₂ was investigated in murine renal arterial smooth muscle. K⁺ current was augmented in a concentration-dependent fashion, with an average increase of 123 ± 28% (n = 6) following application of 10^− 5 M 8-isoPGE₂. This augmentation was observed in the presence of 4-aminopyridine (4-AP, 10^− 3 M) but not that of charybdotoxin (ChTx, 10^− 7 M). Fluorimetric recordings showed marked concentration-dependent increase of cytosolic Ca²⁺ levels by 8-isoPGE₂, while an enzyme-linked immunosorbent assay (ELISA)-based cyclic AMP assay showed increased cAMP levels by 10^− 7 M 8-isoPGE₂ challenge. The isoprostane-induced augmentation was prevented by the ryanodine receptor blocker ruthenium red (10^− 5 M) or the adenylate cyclase blocker SQ 22536 (10^− 4 M). The protein kinase A (PKA) inhibitor H89 (10^− 5 M) inhibited resting K⁺ currents (78 ± 5%, n = 5) but did not prevent 8-isoPGE₂ from augmenting the remaining K⁺ current. We conclude that 8-isoPGE₂ enhances Ca²⁺-dependent K⁺ currents in murine renal artery through a cAMP-dependent pathway which may involve internally sequestered Ca²⁺.     

Relaxation and cyclic GMP levels in response to sildenafil in human pulmonary arteries from donors

We measured cyclic GMP formation and relaxation response to sildenafil given either alone or in combination with sodium nitroprusside (SNP) in pulmonary arteries obtained from 13 multi-organ donors. Sildenafil (10^− 9–10^− 4 M) caused concentration-dependent relaxations and amplified the relaxation induced by SNP. Relaxation was unaffected by endothelium removal or by pre-treatment with the inhibitor of nitric oxide synthase L-NMMA (10^− 4 M). SNP (10^− 7 M) caused elevation of cyclic GMP levels that was potentiated by sildenafil (10^− 6 M). Thus, the enhancement of SNP-induced relaxation by sildenafil is mainly due to an increase in cyclic GMP accumulation.     

Effects of CD-832, a dihydropyridine derivative with a nitrate ester moiety, on rabbit femoral artery and vein

We investigated the effects of CD-832 ((4R)-(−)-2-(nicotinoyl-amino)ethyl 3-nitroxypropyl 1,4-dihydro-2,6-dimethyl-4,3-nitrophenyl, 3,5-pyridine dicarboxylate), a dihydropyridine derivative with a nitrate ester moiety, on contractile responses in rabbit femoral arteries and veins. CD-832 (10⁻⁸ to 10⁻⁶ M) and nifedipine inhibited the 64 mM KCl-induced and 10⁻⁶ M norepinephrine-induced contractions of rabbit femoral arteries, while nitro compounds had no effect on the contractions. CD-832 (10⁻⁸ to 10⁻⁶ M) and nitro compounds inhibited the 10⁻⁶ M norepinephrine-induced contractions in rabbit femoral veins, while other Ca²⁺ channel antagonists had little effect. The inhibitory effects of CD-832 (10⁻⁷ M) on norepinephrine-induced contractions were antagonized by treatment with methylene blue (10⁻⁵ M). These results indicate that CD-832 potently relaxes venous smooth muscle, and that it may be a useful agent for the treatment of angina pectoris.     

Interaction of ethanol with retinol and retinoic acid in RAR β and GAP-43 expression

Fetal ethanol exposure has many detrimental effects on neural development, which possibly occurs through ethanol-induced disruption of the function of vitamin A. In LAN-5 neuroblastoma cells, retinol (10⁻⁶ M) and retinoic acid (RA; 10⁻⁵–10⁻⁶ M) increased RAR β mRNA expression. Ethanol downregulated RAR β levels, even in the presence of retinol. RAR β mRNA expression was decreased by ethanol in the presence of 10⁻⁶ M RA, but not 10⁻⁵ M RA. With cycloheximide (CX), RA still stimulated RAR β mRNA, but the effect of ethanol was abolished. The mRNA expression of GAP-43, an important factor in neural development, increased with 10⁻⁶ M retinol and 10⁻⁵–10⁻⁹ M RA. Ethanol decreased GAP-43 mRNA expression in the presence or absence of retinol. Ethanol was without effect on GAP-43 mRNA at 10⁻⁵ M RA, but did lower the levels at 10⁻⁶ and 10⁻⁷ M RA. CX prevented the effects of both RA and ethanol on GAP-43 mRNA. These studies provide support for the hypothesis that retinoid function is altered by ethanol.     

Upregulation of Rho-kinase (ROCK-2) expression and enhanced contraction to endothelin-1 in the mesenteric artery from lipopolysaccharide-treated rats

Effects of bacterial lipopolysaccharide (Escherichia coli serotype, 055:B5, 20 mg kg(-1), i.p., for 6 h) and a Rho-kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate, Y-27632 (10(-9)-10(-5) M) were investigated on the contractile responses of the rat mesenteric artery to phenylephrine (10(-9)-3 x 10(-5) M), angiotensin-2 (10(-10)-10(-6) M) and endothelin-1 (10(-10)-10(-7) M). Moreover, alteration in the level of Rho-kinase (ROCK-2) expression was examined in the superior mesenteric artery obtained from saline- and lipopolysaccharide-treated rats by Western blotting. Endotoxemic rat mesenteric rings exhibited no different contractions to phenylephrine and angiotensin-2 but augmented contractile activity to endothelin-1. In the mesenteric artery obtained from the endotoxemic rats, acetylcholine-induced vasorelaxation did not differ; pD2 value for acetylcholine was 7.85+/-0.12 in the endotoxemic rings; however, it was 7.81+/-0.15 in the control rings (P>0.05). Y-27632 induced relaxation, which was the same in the control arteries as in endotoxemic ones when contracting agent was phenylephrine. However, when endothelin-1 was used to precontract the rings, Y-27632 produced enhanced relaxation in endotoxemic vessels. pD2 values for Y-27632 were, respectively, 7.69+/-0.12 and 8.20+/-0.10 in control and endotoxemic rings precontracted by endothelin-1 (10(-8) M) (P<0.01). Moreover, Y-27632 (10(-5) M) suppressed the contraction induced by angiotensin-2 (10(-10)-10(-6) M). Western blot analysis revealed that Rho-kinase was upregulated significantly in the mesenteric artery obtained from the rats treated with LPS for 6 h. In addition, serum NO2-/NO3- level, which was detected by Griess method, was 10.0+/-1.4 microM in endotoxemic rats; however, it was 6.6+/-0.5 microM in control (P<0.05). Taken together, these results show that the expression of the contractile protein Rho-kinase could be upregulated in endotoxemic mesenteric artery and this upregulation may be coincided with an enhanced contraction to endothelin-1 but not phenylephrine and angiotensin-2.     

Preparation of a cimetidine ion-selective electrode and its application to pharmaceutical analysis

A novel cimetidine ion-selective electrode is prepared, characterized and used in pharmaceutical analysis. The electrode incorporates PVC-membrane with cimetidine–phospohotungstate ion pair complex. The electrode exhibits a Nernstian response for cimetidine in the concentration range 1.0×10⁻⁵–1.0×10⁻² M with a slope of 58±1 mV per decade. The limit of detection is 5.0×10⁻⁶ M. The electrode displays a good selectivity for cimetidine with respect to a number of common foreign inorganic and organic species. It can be used in the pH range 3.0–5.5. The membrane sensor was successfully applied to the determination of cimetidine in its tablets as well as its recovery from a urine sample.     

Expression and functional role of Rho-kinase in rat urinary bladder smooth muscle

(1) The involvement of Rho-kinase (ROCK) in the contractile mechanisms mediating smooth muscle contraction of the rat urinary bladder was investigated using expression studies and the ROCK inhibitor Y-27632. (2) Both isoforms of ROCK (ROCK I and ROCK II) were detected in high levels in rat urinary bladder. (3) Y-27632 (10 micro M) significantly attenuated contractions of rat urinary bladder strips evoked by the G-protein coupled receptor agonists carbachol (58.1+/-10.5% at 0.3 micro M) and neurokinin A (68.6+/-12.7% at 1 micro M) without affecting contractions to potassium chloride (10-100 mM). In addition, basal tone was reduced by 47.8+/-2.0% by 10 micro M Y-27632 in the absence of stimulation. (4) Contractions of urinary bladder strips evoked by the P2X receptor agonist alpha,beta-methylene ATP (alpha,beta-mATP; 10 micro M) were also attenuated by Y-27632 (30.0+/-7.2% at 10 micro M). (5) Y-27632 (10 micro M) significantly attenuated contractions evoked by electrical field stimulation (2-16 Hz). The effect of Y-27632 on the tonic portion of the neurogenic response (4-16 Hz) was not significantly different from the effect of atropine (1 micro M) alone. (6) While the mechanism underlying the ability of Y-27632 to inhibit alpha,beta-mATP-evoked contractions remains undetermined, the results of the present study clearly demonstrate a role for ROCK in the regulation of rat urinary bladder smooth muscle contraction and tone.     

Expression of Rho-kinase and its functional role in the contractile activity of the mouse vas deferens

The effects of two Rho-kinase inhibitors, Y-27632 and fasudil, were investigated on the contractions produced by electrical field stimulation (EFS, 40 V, 1 mS, 2, 4, 8 and 16 Hz, for 20 s), KCl (30 - 60 mm), phenylephrine (Phe) (10-5 - 10-4 m), adenosine-3', 5'-triphosphate (ATP) (10-4 - 10-3 m) and alpha,beta-methylene ATP (10-5 m). EFS produced frequency-dependent reproducible contractile activity, which was almost abolished by guanethidine (10-5 m, for 1 h). This contraction consisted of two components (a phasic initial contraction followed by a tonic one), and it was inhibited by Y-27632 and fasudil (both at 10-5 m). However, these inhibitors had no effect on resting tension of the tissue. Contractions elicited by KCl (30 - 60 mm) were insensitive to guanethidine (10-5 m, for 1 h), but suppressed by Y-27632 (10-5 m) and fasudil (10-5 m). In addition, the contractions induced by Phe (an alpha1-adrenoceptor agonist) and ATP (a purinergic agent) were inhibited significantly by Y-27632 (10-5 m). Phasic contractions evoked by the selective P2X purinoceptor agonist alpha,beta-methylene ATP were also suppressed by Y-27632 (10-5 m). Western blot analysis revealed that the mouse vas deferens expresses Rho-kinase (ROKalpha, ROCK-2 isoform) protein with a molecular weight of approximately 160 kDa. As a positive control, the presence of this protein was also shown in homogenates of smooth muscle from the rat mesenteric artery. In conclusion, Rho-kinase protein is expressed in the mouse vas deferens, and it mediates neurogenic contractile activity as well as the contractions induced by KCl, Phe, ATP and alpha,beta-methylene ATP. Owing to the suppressive effects of Rho-kinase inhibitors on the contractile activity of the vas deferens, the possibility that these compounds might impair ejaculation must be taken into account when considering them as potential agents in the treatment of erectile dysfunction.     

Expression of Rho-kinase (ROCK-1 and ROCK-2) and its substantial role in the contractile activity of the sheep ureter

Expression of two isoforms of Rho-kinase (ROCK) and its functional role in the physiological control of smooth muscle contraction in the sheep ureter were investigated. Helical strips of the ureteric smooth muscle were stimulated by electrical field stimulation (EFS, 60 V, 1 mS, 2, 4, 8, 16 and 32 Hz, for 20 S), KCl (80 mm), carbachol (CCh, 10(-8)-10(-4) m) or phenylephrine (Phe, 10(-8)-10(-4) m). EFS produced a reproducible contractile activity, which was abolished by tetrodotoxin (3 x 10(-6) m), a Na(+) channel blocker. A muscarinic receptor antagonist, atropine (2 x 10(-6) m), and an adrenergic neuron blocker, guanethidine (10(-5) m), significantly suppressed the contraction induced by EFS. However, this contraction was augmented in the presence of N(G)-nitro-l-arginine (l-NA, 10(-4) m), a nitric oxide synthase inhibitor. Two Rho-kinase inhibitors, Y-27632 (5 x 10(-5) m) and fasudil (5 x 10(-5) m), markedly attenuated the EFS-elicited contraction. CCh and Phe produced concentration-dependent contraction in the sheep ureter. pD(2) values for Phe and CCh were 5.04+/-0.11 and 5.00+/-0.22, respectively. Y-27632 (5 x 10(-5) m) and fasudil (5 x 10(-5) m) also significantly inhibited CCh- and Phe-induced contractions. Moreover, these ROCK inhibitors produced relaxations in the KCl-elicited contraction in a concentration-dependent manner. pD(2) values for Y-27632 and fasudil were, respectively, 5.17+/-0.07 and 4.58+/-0.08 (P<0.001). Furthermore, the influences of these agents were also tested on spontaneous phasic contractions of the tissue. Among Y-27632, fasudil, TTX, l-NA, guanethidine and atropine, only the ROCK inhibitors (10(-6)-10(-5) m) were able to suppress the spontaneous contractile activity. Western blot analysis has revealed that both isoforms of Rho-kinase (ROCK-1 and ROCK-2) are expressed in the sheep ureter. Densitometric analysis has indicated that these enzymes are less expressed in the sheep ureter than are in the sheep aorta in a significant manner. These results show that a contractile enzyme, Rho-kinase, is expressed, and it mediates agonist- and EFS-induced contractions as well as spontaneous contractile activity of the isolated sheep ureter. Since Y-27632 and fasudil depressed the contractions, it seems plausible to postulate that Rho-kinase inhibitors may be beneficial in the treatment of renal colic.     

Lung tissue binding of iodobenzyl-propanediamine: Involvement of beta-adrenergic receptors

The basic compound N-N-N′-trimethyl-N′-(2-hydroxy-3-methyl-5-iodobenzyl)-1,3-propanediamine (HIPDM) accumulates in human and rabbit lungs, where it forms a slowly effluxable pool. In isolated perfused rat lung, HIPDM is taken up by a saturable, energy-independent mechanism, which is competitively inhibited by imipramine, chlorpromazine and propranolol. To ascertain whether beta-adrenergic receptors are involved in the binding process of HIPDM to lung tissue, the ability of unlabelled HIPDM to displace the beta-adrenergic receptor ligand [¹²⁵I]iodocyano-pindolol (ICYP) from rabbit lung beta-receptors was examined. Lung microsomal membrane fractions (75 μg ml⁻¹) were incubated at 37°C for 3 h with 68 pM ICYP (with or without 1 μM of (±)-propranolol) in the presence of HIPDM (10⁻¹⁰−10⁻³ M). Bound and free radioactivity were separated through glass-fibre filters and the retained radioactivity was counted in a gamma-spectrometer. HIPDM competed with ICYP for beta-adrenoceptors (13% displacement at 10⁻⁵ M, 50% at 5 × 10⁻⁵ M, and 90% at 2 × 10⁻⁴ M). The inhibition curve of ICYP binding by HIPDM was similar to that observed for (−)-noradrenaline. Although the results of the in vitro studies cannot be extrapolated to in vivo conditions, they suggest that beta-adrenergic receptors may be involved in the observed lung uptake of the basic amine HIPDM.     

Determination of dipyridamole in pharmaceutical preparations using square wave voltammetry

An analytical methodology using square wave voltammetry (SWV) at a hanging mercury drop electrode (HMDE) was developed for the quantitative determination of dipyridamole (DIP), a drug used for the treatment of several cardiovascular diseases, in pharmaceutical tablets and injections of Persantin^® in phosphate buffer (pH 3.0; 0.1 M). After optimization of the parameters for SWV, analytical curves were obtained for application in the range of 1.28 × 10⁻⁶ M to 7.02 × 10⁻⁶ M. It was found a detection limit (DL) of 1.88 × 10⁻⁸ M (9.50 ng/ml). The repeatability and the reproducibility of the method were determinated by successive measurements of DIP solutions on the range of the analytical curve with a coefficient variation of 0.97% (n = 5) and 1.15%, respectively. The apparent recoveries were obtained by the IUPAC recommended procedure using the second reduction peak. Recoveries obtained by SWV were compared with the UV–vis spectrophotometric method. It was found that the determination of DIP in Persantin^® tablets gave a mean value of 75.6 ± 0.4 mg (100.8%) and 68.9 ± 0.3 mg (91.8%) for SWV and UV–vis spectrophotometry, respectively. In the case of injections, it was found 10.4 ± 0.1 mg (103.4%) and 9.9 ± 0.2 mg (99.9%) for SWV and UV–vis spectrophotometry. Both apparent recoveries for the two types of formulations are in good accordance with the declared value of 75 mg (tablets) and 10 mg (injections).