Mechanism of prostaglandin E2-, F2α- and latanoprost acid-induced relaxation of submental veins

The mechanism of prostaglandin E₂-, prostaglandin F_2α- and latanoprost acid (13,14-dihydro-17-phenyl-18,19,20-trinor-prostaglandin F_2α)-induced relaxation of the rabbit submental vein was studied. Prostaglandin E₂ caused maximum relaxation of endothelin-1 precontracted vessels (EC₅₀: 1.8×10⁻⁸ M). Much of the relaxation could be abolished by denuding the endothelium with the nitric oxide synthase inhibitor,

Full-size image

-NAME (N^G-Nitro-

Full-size image

-arginine methylester). CGRP-(8–37) (calcitonin gene-related peptide fragment (8–37)), a calcitonin gene-related peptide receptor antagonist, exhibited a partial blocking effect, whereas the tachykinin NK₁ receptor blocker, GR 82334 ([

Full-size image

-Pro⁹[Spiro-γ-Lactam]Leu¹⁰,Trp¹¹]physalaemin (1–11)), markedly attenuated the response. Both prostaglandin F_2α and the relatively selective FP receptor agonist, latanoprost acid, caused relaxation of the veins to about 50% of the precontracted state in the presence of GR 32191B ([1R-[1α(Z),2β,3β,5α]]-(+)-7-[5-([1,1′-biphenyl]-4-ylmethoxy)-3-hydroxy-2-(1-piperidinyl)cyclopentyl]-4-heptenoic acid), a thromboxane receptor antagonist (EC₅₀: for prostaglandin F_2α 7.9×10⁻⁹ M, and for latanoprost acid 4.9×10⁻⁹ M).

Full-size image

-NAME, as well as denuding the endothelium, completely abolished the effect. In addition, most or at least a large part of the relaxation was also blocked by CGRP-(8–37) as well as GR 82334. These results indicate that the FP receptor-mediated relaxation of veins is based on release of nitric oxide in addition to involvement of calcitonin gene-related peptide and substance P, or some other tachykinin, probably released from perivascular sensory nerves. The more pronounced relaxation induced by prostaglandin E₂ could be due to vasodilator EP receptors in the smooth muscle layer of the veins.     

Tyrosine kinase inhibitors suppress prostaglandin F2α-induced phosphoinositide hydrolysis, Ca elevation and contraction in iris sphincter smooth muscle

We investigated the effects of the protein tyrosine kinase inhibitors, genistein, tyrphostin 47, and herbimycin on prostaglandin F_2α- and carbachol-induced inositol-1,4,5-trisphosphate (IP₃) production, [Ca²⁺]_i mobilization and contraction in cat iris sphincter smooth muscle. Prostaglandin F_2α and carbachol induced contraction in a concentration-dependent manner with EC₅₀ values of 0.92×10⁻⁹ and 1.75×10⁻⁸ M, respectively. The protein tyrosine kinase inhibitors blocked the stimulatory effects of prostaglandin F_2α, but not those evoked by carbachol, on IP₃ accumulation, [Ca²⁺]_i mobilization and contraction, suggesting involvement of protein tyrosine kinase activity in the physiological actions of the prostaglandin. Daidzein and tyrphostin A, inactive negative control compounds for genistein and tyrphostin 47, respectively, were without effect. Latanoprost, a prostaglandin F_2α analog used as an antiglaucoma drug, induced contraction and this effect was blocked by genistein. Genistein (10 μM) markedly reduced (by 67%) prostaglandin F_2α-stimulated increase in [Ca²⁺]_i but had little effect on that of carbachol in cat iris sphincter smooth muscle cells. Vanadate, a potent inhibitor of protein tyrosine phosphatase, induced a slow gradual muscle contraction in a concentration-dependent manner with an EC₅₀ of 82 μM and increased IP₃ generation in a concentration-dependent manner with an EC₅₀ of 90 μM. The effects of vanadate were abolished by genistein (10 μM). Wortmannin, a myosin light chain kinase inhibitor, reduced prostaglandin F_2α- and carbachol-induced contraction, suggesting that the involvement of protein tyrosine kinase activity may lie upstream of the increases in [Ca²⁺]_i evoked by prostaglandin F_2α. Further studies aimed at elucidating the role of protein tyrosine kinase activity in the coupling mechanism between prostaglandin F_2α receptor activation and increases in intracellular Ca²⁺ mobilization and identifying the tyrosine-phosphorylated substrates will provide important information about the role of protein tyrosine kinase in the mechanism of smooth muscle contraction, as well as about the mechanism of the intraocular pressure lowering effect of the prostaglandin in glaucoma patients.     

Reduced number of α- and β-adrenergic receptors in the myocardium of rats exposed to tobacco smoke

The concentration of α- and β-adrenergic receptors-as measured by specific [³H]WB-4101 and (−)-[³H]-dihydroalprenolol binding-was diminished by 60% below control values in the hearts of hearts of rats exposed to tobacco smoke. These changes in receptor numbers took place almost immediately after tobacco smoke exposure and were rapidly reversible after termination of the exposure. The dissociation constant, K , for [³H]WB-4101 was identical in exposed (K_D = 0.34 ± 0.09 nM) and control (K_D = 0.35 ± 0.07 nM) hearts but was significantly different i in the case of (-)-[³H] dihydroalprenolol binding (exposed, (K_D = 2.83 ± 0.30 nM vs control K_D = 5.22 ± 0.61 nM). For β-receptor binding there was no significant difference between exposed and control animals in the K_i values (−)-epinephrine, (−)-norepinephrine, (−)-alprenolol, (±)-propranolol or timolol. (−)-Isoproterenol, however, was found to bind with lower affinity in exposed compared with control hearts. For α-receptor binding there was no significant difference between control and ‘smoked’ animals in the K_i values for (−)-epinephrine, (−)-norepinephrine or phentolamine. The decrease in α- and β-adrenergic receptor concentration may be related to the phenomenon of receptor desensitization resulting from a release of catecholamines in rats exposed to tobacco smoke.     

Kinetic analysis of glutamate-induced chloride current in Aplysia neurones: a ‘concentration clamp’ study

L-Glutamate (Glu) applied by the ‘concentration clamp’ technique to isolated neurones of Aplysia induced a chloride current (I_Cl) by activating a single population of the channel. The concentration-response curve for the peak I_Cl gave a dissociation constant of 1.3 × 10⁻⁴ M and a Hill coefficient of 1.8. The current-voltage relationship was linear in the voltage range examined (−60 to +10 mV). The activation phase of the I_Cl followed a single-exponential time course and desensitization was complete with a double-exponential time course. The time constant for activation and desensitization decreased with increasing concentrations of Glu but were voltage-independent. The process of recovery from desensitization was also double-exponential. The single-channel conductance estimated by ensemble noise analysis was 50±4.7 pS (n=4). These results suggest that the Glu receptor-Cl channel complex in Aplsia neurones consists of a single population with two binding sites for the agonist.     

Trace iron determination in aminoisophthalic acid using differential-pulse cathodic stripping voltammetry at carbon paste electrodes

Application of differential-pulse cathodic stripping voltammetry using a carbon paste electrode (consisting of carbon powder and liquid paraffin) have been investigated for trace determination of iron in 5-aminoisophthalic acid (AIPA). Samples were dissolved in 1 M HCl, pH was adjusted to 4–5 after addition of EDTA. Voltammetric measurements were performed after filtration. No sample decomposition (mineralization) was necessary. The method showed a good linearity between current and concentration from 3×10⁻⁷ to 5×10⁻⁵ mol dm⁻³ of iron, with a detection limit of 3×10⁻⁷ mol dm⁻³ (resp. 1 ppm in solid AIPA). The results agreed well to those obtained by atomic absorption spectrometry (AAS) using electrothermic atomisation. For AAS measurement, however, microwave digestion of samples was necessary.     

Anodic voltammetry of cefotaxime

In this study electrooxidation of cefotaxime was investigated using specially activated glassy carbon (GC), platinum and carbon paste (CP) electrodes in different supporting electrolyte solutions and at different pHs. The data revealed that the shapes of the voltammograms and the numbers of the oxidation steps changed depending on the nature of the electrode. The nature of the supporting electrolyte was also important for the response of the electrode. From an analytical point of view, the activated GC electrode was the most favourable one. In 0.2 M H₃PO₄ with an activated GC electrode the calibration graph gave two lines with different slopes in the concentration ranges of 2×10⁻⁵–1×10⁻⁴ and 2×10⁻⁴–6×10⁻⁴. The results of the recovery test and statistical analysis showed that the voltammetric method could be used for the determination of cefotaxime.     

Photostability of pitavastatin—A novel HMG-CoA reductase inhibitor

The photostability of pitavastatin, an HMG-CoA reductase inhibitor used in the treatment of hypercholesterolemia, was investigated. The sample solution was exposed to UV-A radiation and the photodegradation process was monitored by means of spectrophotometric method and HPLC–DAD. Pitavastatin was shown to be photolabile and its photodegradation reaction followed the first-order kinetics with the rate constant k = 3.54 × 10⁻⁴ ± 9.43 × 10⁻⁶ s⁻¹.The chromatograms revealed the presence of four major photoproducts (PP-1–PP-4). The separated and isolated photolytic products were identified using a mass spectrometer coupled with a time of flight (TOF) analyzer. The main reaction observed during exposure to radiation of pitavastatin was photocyclisation leading to formation of four-ring photoproducts.     

Novel and selective potentiometric membrane sensor for amiloride determination in pharmaceutical compounds and urine

A new PVC membrane sensor is described as a potentiometric sensor for amiloride. The sensor having amiloride–sodium tetraphenyl phthalate (ion-pair) as an electroactive material and dibutyl phthalate (DBP) as an anion excluder in PVC matrix in the percentage ratio of 4:66:30 (ion-pair: DBP:PVC) (w/w). The membrane sensor exhibits suitable response to amiloride in a concentration range of 1.0 × 10⁻² to 1.0 × 10⁻⁶ mol L⁻¹ with a limit of detection of 9.9 × 10⁻⁷ mol L⁻¹. The slope of the system was −54.3 ± 1.0 mV decade⁻¹ over pH range of 2.0–7.0. Selectivity coefficients for amiloride relative to a numbers of potential interfering substances were investigated. The sensor was highly selective for amiloride over a large number of similar compounds. The sensor showing a fast response time of 6 s and was used over a period of 2 months with a good reproducibility. The sensor was successfully applied to determination of amiloride in pharmaceutical samples with satisfactory results.     

Role of angiotensin II in the response to endothelin-1 of goat cerebral arteries after ischemia–reperfusion

As angiotensin II may underlie the deleterious effects of some vascular diseases, we have examined the role of this peptide on the cerbrovascular endothelin-1 action after ischemia–reperfusion. In anesthetized goats, 1 hour-occlusion followed by 1 hour-reperfusion of the left middle cerebral artery (MCA) was induced, and then segments 3-mm in length from branches of the right MCA (control) and the left MCA (ischemic) were obtained for isometric tension recording. Endothelin-1 (10^− 11–10^− 7 M) produced a contraction that was higher in ischemic than in control arteries, and in control but not in ischemic arteries this contraction was potentiated by angiotensin II (10^− 7 M). Losartan (3 × 10^− 6 M), antagonist of AT₁ receptors, did not affect the response to endothelin-1 in control arteries, but reduced it both in ischemic arteries and angiotensin II-treated control arteries. PD123,319 (3 × 10^− 6 M), antagonist of AT₂ receptors, or the inhibitor of nitric oxide synthesis l-NAME (10^− 4 M) did not alter the arterial effects of endothelin-1. Therefore, angiotensin II may potentiate the constriction to endothelin-1 in normal cerebral arteries by activating AT₁ receptors. The observed cerebrovascular increased response to endothelin-1 after ischemia–reperfusion might be related in part to activation of AT₁ receptors under this condition.     

Biopharmaceutical classification of drugs using intrinsic dissolution rate (IDR) and rat intestinal permeability

The solubility and dissolution rate of active ingredients are of major importance in preformulation studies of pharmaceutical dosage forms. In the present study, passively absorbed drugs are classified based on their intrinsic dissolution rate (IDR) and their intestinal permeabilities. IDR was determined by measuring the dissolution of a non-disintegrating disk of drug, and effective intestinal permeability of tested drugs in rat jejunum was determined using single perfusion technique. The obtained intrinsic dissolution rate values were in the range of 0.035–56.8 mg/min/cm² for tested drugs. The minimum and maximum intestinal permeabilities in rat intestine were determined to be 1.6 × 10⁻⁵ and 2 × 10⁻⁴ cm/s, respectively. Four classes of drugs were defined: Category I: P_eff,rat > 5 × 10⁻⁵ (cm/s) or P_eff,human > 4.7 × 10⁻⁵ (cm/s), IDR > 1(mg/min/cm²), Category II: P_eff,rat > 5 × 10⁻⁵ (cm/s) or P_eff,human > 4.7 × 10⁻⁵ (cm/s), IDR < 1(mg/min/cm²), Category III: P_eff,rat < 5 × 10⁻⁵ (cm/s) or P_eff,human < 4.7 × 10⁻⁵ (cm/s), IDR > 1 (mg/min/cm²) and Category IV: P_eff,rat < 5 × 10⁻⁵ (cm/s) or P_eff,human < 4.7 × 10⁻⁵ (cm/s), IDR < 1(mg/min/cm²). According to the results obtained and proposed classification of drugs, it is concluded that drugs could be categorized correctly based on their IDR and intestinal permeability values.     

17β-Nitro-5α-androstan-3α-ol and its 3β-methyl derivative: Neurosteroid analogs with potent anticonvulsant and anxiolytic activities

Many 17-substituted androstan-3α-ol analogs act as positive allosteric modulators of GABA_A receptors and exert anticonvulsant and anxiolytic-like activity actions in animal models. The endogenous neurosteroid allopregnanolone (17β-acetyl; 1) is among the most potent of these. Here we demonstrate that 3α-hydroxy-17β-nitro-5α-androstane (2b) and its 3β-methyl analog (3α-hydroxy-3β-methyl-17β-nitro-5α-androstane; 2c) modulate GABA_A receptors as assessed by [³⁵S]t-butylbicyclo-phosphorothionate and [³H]flunitrazepam binding with potencies equivalent to or greater than 1. These compounds also had potencies equivalent to or greater than 1 in the pentylenetetrazol and 6 Hz seizure models in the mouse. Furthermore, 2b exhibited anxiolytic-like activity in the elevated zero maze. The 3β-hydroxy, 3α-desmethyl analog (2a) was devoid of activity on GABA_A receptors in vitro but had moderate activity in the seizure models, possibly as a result of epimerization in vivo at the 3-position. This conclusion was supported by the lack of in vivo activity of the 3β-hydroxy, 3α-methyl analog (2d), which is not expected to undergo epimerization. We conclude that nitro can serve as a bioisostere for acetyl at the 17β-position of 5α-androstan-3α-ol, such that the nitro analog fully retains the bioactivity of the endogenous neurosteroid at GABA_A receptors.     

Electrochemical behavior and determination of rutin on a pyridinium-based ionic liquid modified carbon paste electrode

In this paper the electrochemical behavior of rutin on a pyridinium-typed ionic liquid modified carbon paste electrode (IL-CPE) was investigated and further used for rutin sample determination. The IL-CPE showed strong electrocatalytic effects to the oxidation of rutin. In phosphate buffer solution (PBS, pH 2.5; 0.1 M) a pair of well-defined cyclic voltammetric redox peaks of rutin appeared with the redox peak located at 512 mV (Epa) and 448 mV (Epc) (vs. SCE), respectively. The redox peak current was increased about 27.5 times more than that on traditional carbon paste electrode (CPE). The electrochemical parameters of rutin on the IL-CPE were calculated with the results of the charge transfer coefficient (α), the number of electron transfer (n) and the electrode reaction rate constant (k_s) as 0.53, 1.80 and 2.39 s⁻¹, respectively. The cathodic peak currents increased linearly with the concentration of rutin in the range from 5.0 × 10⁻⁷ to 1.0 × 10⁻⁴ M with the detection limit as 3.58 × 10⁻⁷ M (3σ). The relative standard deviation (RSD) of 10 successive detection of 5.0 × 10⁻⁵ M rutin was 4.2%. The method was successfully applied to the determination of rutin content in tablets samples with good recovery. The modified electrode showed good stability and reproducibility without the influence of the coexisting substances.     

The protein binding of methotrexate by the serum of normal subjects

Summary The protein binding of methotrexate by serum from eight normal volunteers was assessed by continuous ultrafiltration at pH 7.4 and 37°C. Methotrexate concentrations were measured by radioimmunoassay and the data analysed by the method of Scatchard. The major binding protein was albumin which bound 87.3% of the drug in serum. Analysis of the Scatchard plots indicated two distinct groups of binding sites. Class I was found to have 0.16±0.05 (S D) binding sites with an intrinsic association constant of 71.15±35.98 (S D)×10⁴ M^–1: Class II had 2.01±0.93 (S D) binding sites and an affinity of 0.18±0.15×10⁴ M^–1. No great change in the percentage of methotrexate bound occurred until the total concentration of the drug exceeded 50 µMol 1^–1.     

In vitro characterisation of BF227 binding to α-synuclein/Lewy bodies

Amyloid-β (Aβ) plaques are a pathological hallmark of Alzheimer's disease and a current target for positron emission tomography (PET) imaging agents. Whilst [¹¹C]-PiB is currently the most widely used PET ligand in clinic, a novel family of benzoxazole compounds have shown promise as Aβ imaging agents; particularly BF227. We characterised the in vitro binding of [¹⁸F]-BF227 toward α-synuclein to address its selectivity for Aβ pathology, to establish whether [¹⁸F]-BF227 binds to α-synuclein/Lewy bodies, in addition to Aβ plaques. In vitro [¹⁸F]-BF227 saturation studies were conducted with 200 nM α-synuclein or Aβ₁₋₄₂ fibrils or 100 μg of Alzheimer's disease, pure dementia with Lewy bodies or control brain homogenates. Non-specific binding was established with PiB (1 μM). In vitro binding studies indicated that [¹⁸F]-BF227 binds with high affinity to two binding sites on Aβ₁₋₄₂ fibrils (K_D1 = 1.31 and K_D2 = 80 nM, respectively) and to one class of binding sites on α-synuclein fibrils (K_D = 9.63nM). [¹⁸F]-BF227 bound to Aβ-containing Alzheimer's disease brain (K_D = 25 ± 0.5 nM), but failed to bind to Aβ-free dementia with Lewy bodies or age-matched control homogenates. Moreover, BF227 labelled both Aβ plaques and Lewy bodies in immunohistochemical/fluorescence analysis of human Alzheimer's disease and Parkinson's disease brain sections, respectively. This study suggests that [¹⁸F]-BF227 is not Aβ-selective. Evaluation of BF227 as a potential biomarker for Parkinson's disease is warranted.     

Passive permeability of dihexadecyl phosphate vesicles altered by aliphatic alcohols and ω-diols

Alcohols act as anaesthetics only up to a certain chain length, beyond which their biological activity disappears. Although the molecular nature of general target sites remains unknown, presently available data support the hypothesis that this ‘cut-off” in anaesthetic activity could be due to a corresponding cut-off in the absorption of long-chain alcohols into lipid-bilayer portions of nerve membranes. To test this hypothesis, we developed a method based on leakage of Ru(bpy)₃²⁺ ions across the membrane of dihexadecylphosphate (DHP) vesicles induced by aliphatic alcohols (C₁ to C₁₈) and some of their ω-diol. The permeant effects of aliphatic linear alcohols expressed as PD₅₀ values rise to a maximum for n-dodecanol (PD₅₀=2×10⁻³m 1⁻¹). Dodecanol was found to be the alcohol which presents the greatest anaesthetic potency among the series of linear aliphatic alcohols (cut-off anaesthetic effect). The results are discussed in terms of the structural physicochemical and geometrical characteristics of the permeating alchohols.     

Antimicrobial and cancer cell growth inhibitory activities of 3β-acetoxy-17β-( -prolyl)amino-5α-androstane in vitro

The in vitro activity of the steroidal amide 3β-acetoxy-17β-( -prolyl)amino-5α-androstane against 179 Gram-positive clinical isolates was examined. The minimum bactericidal concentration (MBC)/MIC ratios were ≤2 for 73% of methicillin-resistant Staphylococcus aureus, 59% of vancomycin-resistant Enterococcus spp. and 88% of penicillin-resistant Streptococcus pneumoniae. The androstane derivative was bactericidal for a variety of other Gram-positive genera, including Nocardia, Corynebacterium and Listeria. Variation in MICs is pH 6–8 media was slight. The frequency of occurrence of bacterial spontaneous mutations to resistance ranged from 10⁻⁶ to 10⁻⁹. Kill curve analysis confirmed the bactericidal nature of the steroidal amide, and demonstrated that killing was time dependent but not concentration dependent for all organisms. The ability of 3β-acetoxy-17β-( -prolyl)amino-5α-androstane to inhibit human cancer cell growth was also evaluated. The concentration required to inhibit 50% of cell growth (GI₅₀) was <2.5 mg/l for all cell lines examined. In single-dose murine toxicity evaluations, the androstane derivative was non-toxic at doses up to 400 mg/kg.     

Regulation of Phenobarbital-Inducible Cytochrome-P450 2B1/2 mRNA by Lovastatin and Oxysterols in Primary Cultures of Adult Rat Hepatocytes

Lovastatin (LOVA) is a potent inhibitor of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase widely used in clinical practice. We treated primary cultures of adult rat hepatocytes, maintained in a minimal, serum-free medium on Matrigel, a reconstituted basement membrane, with this drug, and found that the amounts of P450 2B2 mRNA detected on Northern blots were increased at the same doses (10⁻⁵ to 3 × 10⁻⁵ M) required for induction of HMG-CoA reductase mRNA, a gene known to be under oxysterol regulatory control. LOVA treatment produced selective effects increasing also the mRNA levels for P450s 2C6, 2C7, 3A1, and 4A1 but not for 1A1, 2A1/2, or NADPH-cytochrome P450 oxidoreductase. LOVA treatment increased the induction of 2B1/2 mRNA in cells cotreated with either phenobarbital (PB; 10⁻⁴ M) or clotrimazole (CTZ; 10⁻⁵ M), or of 3A1 mRNA in cells cotreated with PB (2 × 10⁻³ M), but not dexamethasone (10⁻⁵ M). LOVA treatment did not potentiate the induction of 1A1 or 4A1 mRNA in cells cotreated with β-naphthoflavone (10⁻⁵ M) or ciprofibrate (10⁻⁴ M), respectively. In contrast to the potentiation of 2B1/2 mRNA induction produced by treatments with LOVA in combination with PB or CTZ, cotreatment of hepatocytes with PB and CTZ did not result in increased induction relative to that seen in cells treated with either agent alone. Treatment of hepatocyte cultures with either mevalonate (3 × 10⁻⁴ to 3 × 10⁻³ M), the immediate product of HMG-CoA reductase, or 25-hydroxycholesterol (10⁻⁶ to 10⁻⁵ M), a model oxysterol, resulted in dose-dependent suppression of 2B1/2 mRNA induction in cells treated with PB-like inducers. Taken together, our results demonstrate that LOVA is a unique inducer of P450 mRNA in cultured rat hepatocytes and implicate oxysterols as potential intracellular modulators of 2B1/2 induction. We conclude that endogenous metabolic factors including those related to cholesterol biosynthesis are critical in induction of liver cytochromes P450 2B1 and 2B2 by PB and "PB-like" agents.     

Prostaglandin F2α Binding to Bovine Ocular and Synthetic Melanins in Vitro

Abstract: The binding of ³H-prostaglandin F_2α to bovine iris and synthetic melanin was studied in vitro using a ligand binding assay. Prostaglandin F_2α was reversibly bound to both types of melanin. The binding was saturable and the Scatchard analysis revealed the existence of at least two binding sites with the corresponding K_D values of 3.71 nM and 1.99 μM for natural and 4.99 nM and 0.19 μM for synthetic melanin, respectively. The high affinity B_max values were 3.4 nM/g for natural and 2.5 nM/g for synthetic melanin. The dissociation of prostaglandin F_2α from melanin after dilution of the complex was rapid and uniform.     

Prejunctional α-adrenoceptors regulate nitrergic neurotransmission in the rabbit urethra

We evaluated the effects of prejunctional α-adrenoceptors on nitric oxide (NO)-mediated urethral relaxation in rabbits using a muscle bath technique and high-performance liquid chromatography coupled with a microdialysis procedure. The amount of NO₂⁻/NO₃⁻ released during electrical field stimulation was measured by an NO₂⁻/NO₃⁻ analyzer based on the Griess method. Pretreatment with phenylephrine (0.01 μM) and yohimbine (0.1–10 μM) significantly reduced the relaxation responses induced by electrical field stimulation. In contrast, pretreatment with clonidine (0.01 μM) and prazosin (0.01–1 μM) enhanced the relaxation responses. Cys-NO-induced relaxations of rabbit urethral smooth muscle were not affected by pretreatment with α-adrenoceptor agonists and antagonists. The amount of NO₂⁻/NO₃⁻ released by electrical field stimulation increased after pretreatment with clonidine (0.01 μM) and prazosin (0.01–1 μM), but decreased after pretreatment with phenylephrine (0.01 μM) and yohimbine (0.1–10 μM). The results suggest that the release of NO from nitrergic nerves in the rabbit urethra is reduced and increased by stimulation of prejunctional α₁- and α₂-adrenoceptors, respectively.