Single ascending oral dose pharmacokinetics and pharmacodynamics study of EV-077: the specific inhibitor of prostanoid- and isoprostane-induced cellular activation.

PURPOSE: This study was performed to determine the oral pharmacokinetics (PK) of EV-077 and its effects on pharmacodynamic (PD) markers. EV-077 blocks prostanoid-induced and isoprostane-induced cellular activation, and is in development for the treatment of vascular inflammation and associated complications of type-2 diabetes.. METHODS: This single-ascending-dose mono-centre study was randomised, placebo-controlled, and double-blinded within each dose group. Seven EV-077 doses were administered sequentially as an oral solution: 0.0125, 0.125, 0.375, 0.75, 1.25, 1.875 and 2.5 mg/kg body weight. PK, platelet aggregation, bleeding time and safety parameters were measured. Seven to eight healthy male subjects were dosed per group: five to six subjects received EV-077 and two subjects received placebo. RESULTS: Tmax was reached rapidly between 0.5 h and 1.0 h. Both Cmax and AUC increased linearly with the dose. The apparent terminal half-life (t½z) increased with the dose, most likely reflecting the increasing last quantifiable concentration with increasing dose; at 2.5 mg/kg, it was 2.7-6.9 h. Measurement of platelet aggregation showed no effect at 0.0125 mg/kg, and a full and reversible inhibition at doses of 0.125-2.5 mg/kg. The average bleeding time was dose-dependently prolonged, but was always below 9 min. The PK/PD profile showed that at plasma concentrations above 20 ng/ml, EV-077 platelet aggregation was completely inhibited (>90 %). All tested doses were well tolerated. CONCLUSIONS: Orally administered EV-077 was well tolerated, readily absorbed, reached Cmax within 1 h, with a linear PK based on Cmax and AUC. The inhibition of platelet aggregation was complete and reversible at doses of 0.125 mg/kg and higher, and average bleeding time was below 9 min.

Effect of high-fat diet on KKAy and ob/ob mouse liver and adipose tissue corticosterone and 11-dehydrocorticosterone concentrations.

The present study was performed to compare glucocorticoid levels in obese KKA (y) and ob/ob mice with those in normal C57BL/6J mice, and the effect of high-fat diet on glucocorticoids in KKA (y) and ob/ob mice. Liver, mesenteric and epididymal adipose tissue corticosterone and 11-dehydrocorticosterone concentrations as well as circulating corticosterone concentrations were measured. The KKA (y) and ob/ob mice displayed elevated serum corticosterone levels compared to normal mice, 2.0 to 2.8-fold in KKA (y), and 11 to 16-fold in ob/ob mice. Liver corticosterone levels were 3.0 to 5.1 and 6.2 to 8.1-fold, and 11-dehydrocorticosterone levels were 3.4 to 3.6 and 6.7 to 8.2-fold higher in KKA (y) and ob/ob mice compared to normal mice. Mesenteric adipose tissue corticosterone levels were 2.7 to 4.2-fold higher, and 11-dehydrocorticosterone levels were 2 to 4-fold higher in ob/ob than in KKA (y) mice. Epididymal adipose tissue corticosterone levels were 3.0 to 6.2-fold higher, and 11-dehydrocorticosterone levels were 1.8 to 2.0-fold higher in ob/ob than in KKA (y) mice. Circulating, hepatic, and mesenteric and epididymal adipose tissue glucocorticoid concentrations were low in the normal C57BL/6J mouse, high in the ob/ob mouse, and intermediate in the KKA (y) mouse. 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) mRNA levels were doubled in ob/ ob compared to KKA (y) mice in all three tissues. Glucocorticoid concentrations correlated with 11beta-HSD1 mRNA levels. High-fat diet had no effect on the tissue glucocorticoid concentrations.

Tetanus neurotoxin-insensitive vesicle-associated membrane protein localizes to a presynaptic membrane compartment in selected terminal subsets of the rat brain.

Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is a vesicular soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptor (SNARE) that has been implicated in neurite outgrowth. It has previously been reported that TI-VAMP is localised in the somatodendritic compartment of neurons indicating a role in membrane fusion events within dendrites. Using a newly produced monoclonal antibody to TI-VAMP that improves signal/noise immunodetection, we report that TI-VAMP is also present in subsets of axon terminals of the adult rat brain. Four distinctive populations of labelled axon terminals were identified: 1) the hippocampal mossy fibres of the dentate gyrus and of CA3, 2) the striatal peridendritic terminal plexuses in the globus pallidus (GP), substantia nigra pars reticulata (SNr), 3) peridendritic plexuses in the central nucleus of the amygdala, and 4) the primary sensory afferents in the dorsal horn of the spinal cord. The presynaptic localisation of TI-VAMP in these locations was demonstrated by co-localisation with synaptophysin. Ultrastructural studies showed TI-VAMP labelling over synaptic vesicles in the mossy fibres, whereas it was localised in tubulo-vesicular structures and multivesicular bodies in the pyramidal cell dendrites. The presynaptic localisation of TI-VAMP occurred by P15, so relatively late during development. In contrast, dendritic labelling was most prominent during the early post-natal period. Co-localisation with markers of neurotransmitters showed that TI-VAMP-positive terminals are GABAergic in the GP and SNr and glutamatergic in the mossy fibre system and in the dorsal root afferents. Most of these terminals are known to co-localise with neuropeptides. We found met-enkephalin-immunoreactivity in a sizeable fraction of the TI-VAMP positive terminals in the GP, amygdala, and dorsal horn, as well as in a few mossy fibre terminals. The function of TI-VAMP in subsets of mature axon terminals remains to be elucidated; it could participate in the exocytotic molecular machinery and/or be implicated in particular growth properties of the mature axon terminals. Thus, the presence of TI-VAMP in the mossy fibres may correspond to the high degree of plasticity that characterises this pathway throughout adult life.

Selective inhibition of 11beta-hydroxysteroid dehydrogenase type 1 decreases blood glucose concentrations in hyperglycaemic mice.

AIMS/HYPOTHESIS: Current pharmacological treatments for Type II (non-insulin-dependent) diabetes mellitus have various limitations. New treatments are needed to reduce long-term risks for diabetic complications and mortality. We tested a new principle for lowering blood glucose. It is well known that glucocorticoids in excess cause glucose intolerance and insulin resistance. The enzymes 11beta-hydroxysteroid dehydrogenase type 1 and type 2 inter-convert inactive and active glucocorticoids, thereby playing a major role in local modulation of agonist concentration and activation of corticosteroid receptors in target tissues. It has been hypothesized that selective inhibition of 11beta-hydroxysteroid dehydrogenase type 1 decreases excessive hepatic glucose production in hyperglycemia and diabetes. BVT.2733 is a new, small molecule, non-steroidal, isoform-selective inhibitor of mouse 11beta-hydroxysteroid dehydrogenase type 1. The aim of the present study is to test if selective inhibition of 11beta-hydroxysteroid dehydrogenase type 1 lowers blood glucose concentrations in a hyperglycaemic and hyperinsulinaemic mouse model. METHODS: BVT.2733 was given to spontaneously hyperglycaemic KKA(y) mice for 7 days using subcutaneous osmotic mini-pumps. RESULTS: BVT.2733 lowered hepatic PEPCK and glucose-6-phosphatase mRNA, blood glucose and serum insulin concentrations compared with vehicle treated mice. In contrast, hepatic 11beta-hydroxysteroid dehydrogenase type 1 mRNA, liver function marker enzyme expression (aspartate aminotransferase, alanine aminotransferase and alkaline phosphatases), daily food intake and body weight were not altered by the treatment. CONCLUSION/INTERPRETATION: These results suggest that a selective inhibitor of human 11beta-hydroxysteroid dehydrogenase type 1 can become a new approach for lowering blood glucose concentrations in Type II diabetes.

In vitro effects of 4-chloro-3-ethylphenol in skeletal muscle preparations from malignant hyperthermia susceptible and normal swine.

BACKGROUND AND OBJECTIVE: The in vitro contracture test with halothane and caffeine is the current gold standard for diagnosis of malignant hyperthermia. This test has a sensitivity of 99.0% but a specificity of only 93.6%. Therefore, an alternative drug is desirable which distinguishes between malignant hyperthermia-susceptible and malignant hyperthermia-normal subjects with a higher specificity and sensitivity. METHODS: 4-chloro-3-ethylphenol has recently been shown to trigger Ca2+-induced Ca2+-release in skeletal muscle terminal cisternae and to increase the myoplasmic free Ca2+ concentration in skeletal muscle fibres. The purpose of this study was to investigate the ability of 4-chloro-3-ethylphenol to distinguish between malignant hyperthermia-susceptible and malignant hyperthermia-normal porcine muscle specimen in the in vitro contracture test. Ten malignant hyperthermia-susceptible and 14 malignant hyperthermia-normal swine were anaesthetized and muscle biopsies were taken. For the in vitro contracture test muscle specimens were exposed to cumulative concentrations of 4-chloro-3-ethylphenol (12.5 to 200 micromol L(-1)). RESULTS: 4-chloro-3-ethylphenol produced contractures in a concentration-dependent manner in the malignant hyperthermia-susceptible muscle bundles. In contrast, cumulative 4-chloro-3-ethylphenol did not generate contractures in malignant hyperthermia-normal specimens. Contractures were significantly greater (P < 0.05) in the malignant hyperthermia-susceptible compared to the malignant hyperthermia-normal preparations in all 4-chloro-3-ethylphenol concentration steps from 50 micromol L(-1) to 200 micromol L(-1). There was no overlap between the two groups above a concentration of 75 micromol L(-1) in cumulative 4-chloro-3-ethylphenol in vitro contracture tests. CONCLUSIONS: It remains to be verified whether an in vitro contracture test with 4-chloro-3-ethylphenol can also discriminate between malignant hyperthermia-susceptible and malignant hyperthermia-normal humans. Since no prior tested agent revealed a clear differentiation in contracture development without overlap, the 4-chloro-3-ethylphenol test might be a promising new approach to the diagnosis of malignant hyperthermia.

A common exocytotic mechanism mediates axonal and dendritic outgrowth.

Outgrowth of the dendrites and the axon is the basis of the establishment of the neuronal shape, and it requires addition of new membrane to both growing processes. It is not yet clear whether one or two exocytotic pathways are responsible for the respective outgrowth of axons and dendrites. We have previously shown that tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) defines a novel network of tubulovesicular structures present both at the leading edge of elongating dendrites and axons of immature hippocampal neurons developing in primary culture and that TI-VAMP is an essential protein for neurite outgrowth in PC12 cells. Here we show that the expression of the N-terminal domain of TI-VAMP inhibits the outgrowth of both dendrites and axons in neurons in primary culture. This effect is more prominent at the earliest stages of the development of neurons in vitro. Expression of the N-terminal domain deleted form of TI-VAMP has the opposite effect. This constitutively active form of TI-VAMP localizes as the endogenous protein, particularly concentrating at the leading edge of growing axons. Our results suggest that a common exocytotic mechanism that relies on TI-VAMP mediates both axonal and dendritic outgrowth in developing neurons.

Distinct functional properties of the TAP subunits coordinate the nucleotide-dependent transport cycle.

BACKGROUND: The transporter associated with antigen processing (TAP) consists of two polypeptides, TAP1 and TAP2. TAP delivers peptides into the ER and forms a "loading complex" with MHC class I molecules and accessory proteins. Our previous experiments indicated that nucleotide binding to TAP plays a critical role in the uptake of peptide and the release of assembled class I molecules. To investigate whether the conserved nucleotide binding domains (NBDs) of TAP1 and TAP2 are functionally equivalent, we created TAP variants in which only one of the two ATP binding sites was mutated. RESULTS: Mutations in the NBDs had no apparent effect on the formation of the loading complex. However, both NBDs had to be functional for peptide uptake and transport. TAP1 binds ATP much more efficiently than does TAP2, while the binding of ADP by the two chains is essentially equivalent. Peptide-mediated release of MHC class I molecules from TAP was blocked only when the NBD of TAP1 was disrupted. A different NBD mutation that does not affect nucleotide binding has strikingly different effects on peptide transport activity depending on whether it is present in TAP1 or TAP2. CONCLUSIONS: Our findings indicate that ATP binding to TAP1 is the initial step in energizing the transport process and support the view that ATP hydrolysis at one TAP chain induces ATP binding at the other chain; this leads to an alternating and interdependent catalysis of both NBDs. Furthermore, our data suggest that the peptide-mediated undocking of MHC class I is linked to the transport cycle of TAP by conformational signals arising predominantly from TAP1.

In vitro alpha-adrenoceptor autoradiography of the urethra and urinary bladder of the female pig, cat, guinea-pig and rat.

OBJECTIVE: The aim of this study was to investigate the distribution of alpha1- and alpha2-adrenoceptors in the urethra and urinary bladder of the female pig, cat, guinea-pig and rat. MATERIALS AND METHODS: The binding distributions of an alpha1-adrenoceptor ligand (3H-prazosin) and an alpha2-adrenoceptor ligand (3H-rauwolscine) were determined using in vitro autoradiography. Autoradiograms were analysed by combining computer-based image analysis and light microscopy. RESULTS: In the pig, guinea-pig and rat urethra 3H-prazosin binding was highest in the muscle layer. In the cat urethra 3H-prazosin binding could not be analysed due to a negative chemography artefact. In the pig, cat and guinea-pig urethra 3H-rauwolscine binding was highest in the urothelium, followed by the sub-mucosa, with low levels in muscle. Little 3H-rauwolscine binding was observed in the rat urethra. In the urinary bladder of all species 3H-prazosin binding was low. In the rat bladder, binding was higher in the trigone than in the dome. In the pig, cat and guinea-pig bladder 3H-rauwolscine binding was highest in the mucosa. with little binding in muscle or lamina propria. In the rat bladder, there was little binding and no regional differences. CONCLUSIONS: Alpha1-adrenoceptors were predominantly located in urethral smooth muscle, indicating their contractile importance in maintaining continence. Alpha2-Adrenoceptors were present in the urethral submucosa and bladder mucosa, but not in muscle, suggesting a role in regulation of blood flow, urethral lubrication and tumescence, but not in contraction.

Selectivity of oxymetazoline for urethral pressure vs blood pressure in the anaesthetized female rabbit.

OBJECTIVE: The aim of this study was to test alpha-adrenergic reference agonists for tissue selectivity in the urethra and to pharmacologically characterize the functional alpha-adrenoceptor type of the female rabbit urethra in vivo. MATERIAL AND METHODS: The effect of alpha-adrenergic agonists and antagonists on the urethral pressure was compared with that on blood pressure and heart rate measured simultaneously in the anaesthetized female rabbit. RESULTS: Oxymetazoline, NS-49, phenylephrine and phenylpropanolamine enhanced the urethral pressure in a dose-dependent manner. Phenylephrine and phenylpropanolamine also enhanced the blood pressure with significantly lower ED50 (dose that gives half of the maximal enhancing effect) values than for the urethral pressure. This was in contrast to oxymetazoline and NS-49. The ED50 values for oxymetazoline on urethral pressure, and systolic and diastolic blood pressure were 0.00067, 0.0030 and 0.0020 mg/kg, respectively. The ED50 values for NS-49 on urethral pressure, and systolic and diastolic blood pressure were 0.019, 0.21 and 0.18 mg/kg, respectively. Clonidine and UK 14,304 had no effect on urethral or blood pressure. The oxymetazoline-evoked increase in urethral pressure was inhibited by WB-4101 with an ID50 (dose that gives half of the inhibitory effect) significantly lower than that for rauwolscine. CONCLUSIONS: The results suggest that in the female rabbit in vivo activation of alpha1-adrenoceptors increased the urethral pressure. Phenylephrine and phenylpropanolamine, in contrast to oxymetazoline and NS-49, selectively enhanced blood pressure as compared with urethral pressure. Provided that the present results also have validity in humans, it would seem possible to develop urethra-selective drugs for treatment of stress incontinence with few or no cardiovascular side-effects.

Role of tetanus neurotoxin insensitive vesicle-associated membrane protein (TI-VAMP) in vesicular transport mediating neurite outgrowth.

How vesicular transport participates in neurite outgrowth is still poorly understood. Neurite outgrowth is not sensitive to tetanus neurotoxin thus does not involve synaptobrevin-mediated vesicular transport to the plasma membrane of neurons. Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is a vesicle-SNARE (soluble N-ethylmaleimide-sensitive fusion protein [NSF] attachment protein [SNAP] receptor), involved in transport to the apical plasma membrane in epithelial cells, a tetanus neurotoxin-resistant pathway. Here we show that TI-VAMP is essential for vesicular transport-mediating neurite outgrowth in staurosporine-differentiated PC12 cells. The NH(2)-terminal domain, which precedes the SNARE motif of TI-VAMP, inhibits the association of TI-VAMP with synaptosome-associated protein of 25 kD (SNAP25). Expression of this domain inhibits neurite outgrowth as potently as Botulinum neurotoxin E, which cleaves SNAP25. In contrast, expression of the NH(2)-terminal deletion mutant of TI-VAMP increases SNARE complex formation and strongly stimulates neurite outgrowth. These results provide the first functional evidence for the role of TI-VAMP in neurite outgrowth and point to its NH(2)-terminal domain as a key regulator in this process.

Nucleotide binding by TAP mediates association with peptide and release of assembled MHC class I molecules.

BACKGROUND: Newly synthesised peptide-receptive major histocompatibility complex (MHC) class I molecules form a transient loading complex in the endoplasmic reticulum with the transporter associated with antigen processing (TAP) and a set of accessory proteins. Binding of peptide to the MHC class I molecule is necessary for dissociation of the MHC class I molecule from the complex with TAP, but other components of the complex might also be involved. To investigate the role of TAP in this process, mutations that block nucleotide binding were introduced into the ATP-binding site of TAP. RESULTS: Mutant TAP formed apparently normal loading complexes with MHC class I molecules and accessory components, but had no nucleotide-binding or peptide-transport activity. Nevertheless, whereas wild-type loading complexes in detergent lysates could be dissociated by addition of peptides that bind MHC class I molecules, mutant complexes could not be dissociated in this way. Depletion of nucleotide diphosphates or triphosphates from wild-type lysates blocked peptide-mediated dissociation of MHC class I molecules, which could be reversed by readdition of nucleotide diphosphates or triphosphates. Complexes between mutant TAP and MHC class I molecules remained associated in vivo until they were degraded. Disruption of nucleotide binding also eliminated TAP's peptide-binding activity. CONCLUSIONS: Peptide-mediated dissociation of the MHC class I molecule from the loading complex depends on conformational signals arising from TAP. Integrity of the nucleotide-binding site is required not only for transmission of this conformational signal to the loading complex, but also for binding of peptide to TAP. Thus, the dynamic activity of the loading complex is synchronised with the nucleotide-mediated peptide-binding and transport cycle of TAP.

Characterisation of the functional alpha-adrenoceptor subtype in the isolated female pig urethra.

The aim of the present study is to characterise the contraction-mediating functional alpha-adrenoceptor of the female pig urethra. Alpha-adrenoceptor reference agonists were used to contract the isolated female pig urethra. The relative intrinsic activity was noradrenaline (1.0), phenylephrine (0.91), methoxamine (0.74), (+/-)-3'-(2-amino-1-hydroxyethyl)-4'-fluoromethane-sulfonanilid e hydrochloride (NS-49) (0.68), oxymetazoline (0.60), dopamine (0.50), clonidine (0.43), midodrine (0.32), ephedrine (0.30), 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK 14,304) (0.11), and phenylpropanolamine (0.11). The 21 competitive antagonists used caused parallel rightward shifts in the alpha-adrenoceptor agonist concentration-response curves, giving linear Schild-plots with slopes not significantly different from unity, suggesting that contraction was mediated by a single receptor. The antagonist pK(B) values calculated were R(-)-tamsulosin (9.68), risperidone (9.19), 2-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-4,4-dimethyl-1,3(2H,4H)-+ ++isoquinolinedione (AR-C 239) (9.09), 2-([2,6-dimethoxyphenoxyethyl]aminomethyl)-1,4-benzodioxane (WB-4101) (8.87), N-[3-[4-(2-methoxyphenyl)-1-piperazinyl]propyl]-3-methyl-4-oxo-2-phenyl- 4H-1-benzopyran-8-carboxamide monomethanesulfonate (Rec 15/2739/3) (8.81), 5-methylurapidil (8.59), prazosin (8.57), benoxathian (8.56), S(+)-tamsulosin (8.27), indoramin (8.11), doxazosin (7.96), alfuzosine (7.82), phentolamine (7.70), terazosin (7.52), spiperone (7.48), oxymetazoline (7.40), 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]deca ne-7,9-dione dihydrochloride (BMY 7378) (7.05), corynanthine (6.98), rauwolscine (6.40), yohimbine (6.22), and N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-alpha,alpha-dime thyl-1H-indole-3-ethanamine hydrochloride (RS 17053) (6.07). Correlation of subtype-selective antagonist pK(B) values was best with published values for the alpha1a/1A-adrenoceptor subtype. Therefore, the present results suggest that contraction of the female pig urethra is caused by activation of the alpha1A-adrenoceptor.

The effect of phenylpropanolamine on the urethral pressure and heart rate is retained after repeated short-term administration in the unanaesthetized, conscious dog.

The present study was performed to test whether high dose agonist (phenylpropanolamine) administration twice a day causes desensitization of urethral alpha-adrenoceptors in vivo. Urethral pressure was measured on five consecutive days of phenylpropanolamine treatment of the unanaesthetized, conscious dog, and the method is described in detail. Phenylpropanolamine hydrochloride proper (75 mg, 5.1-6.1 mg kg-1), and a sustained-release formulation, both significantly increased urethral pressure and decreased heart rate. Interruption of administration for two to three days did not alter the effect. The present results suggest that the effect of phenylpropanolamine was retained, and that the alpha-adrenoceptors of dog urethra did not desensitize after repeated administration of the alpha-adrenoceptor agonist phenylpropanolamine twice a day.

Presynaptic alpha 2A-adrenoceptors regulate the 3H-noradrenaline secretion in the guinea-pig urethra.

The subtype of the functional presynaptic autoreceptor in adrenergic nerves of the male guinea-pig urethra was pharmacologically characterised. The urethra was incubated with 3H-noradrenaline and superfused with Tyrode solution in vitro and the fractional secretion of 3H-noradrenaline evoked by 300 electrical shocks at 5 Hz was measured. alpha-Adrenoceptor antagonists enhanced the 3H-noradrenaline secretion. The effects of BRL 44408 ((+/-)-2-[(4,5-dihydro-1H-imidazol-2yl)methyl]-2,3- dihydro-1-methyl-1H-isoindole), BRL 41992 ((-)-1,2-dimethyl-2,3,9,13b-tetrahydro-1H-dibenzo[c,f]imidazo[1,5- a] azepine), and WB-4101 (2-([2,6-dimethoxyphenoxyethyl]aminomethyl)-1,4- benzodioxane) were tested. The rank order of apparent EC50 values was BRL 44408 < BRL 41992 < WB-4101, and correlated best with constants for the alpha 2A-C10/alpha 2A adrenoceptor subtype. The results suggest that 3H-noradrenaline secretion in guinea-pig urethra is regulated by a presynaptic adrenoceptor of the alpha 2A subtype.

Classification of the presynaptic muscarinic receptor subtype that regulates 3H-acetylcholine secretion in the guinea pig urinary bladder in vitro.

The experiments were done to investigate the presence and subtype of functionally presynaptic muscarinic receptors in cholinergic nerves of the guinea pig urinary bladder. Bladder strips were incubated with 3H-choline and superfused with Tyrode's solution containing eserine. Secreted 3H-acetylcholine was separated from 3H-choline. The electrically evoked 3H-acetylcholine secretion increased with the stimulation frequency. 3H-Acetylcholine secretion was enhanced by muscarinic antagonists, was depressed by carbachol and by alpha adrenoceptor agonists but was not influenced by drugs acting at beta adrenoceptors or purinoceptors. The rank order for the enhancing effect of muscarinic antagonist EC50 values was propantheline < atropine < methylatropine < N-desethyloxybutynin < UH-AH 37 < benzhexol < AQ-RA 741 < 4-DAMP < procyclidine < emepronium < secoverine < oxybutynin < tropicamide < promethazine < himbacine < hexahydrosiladifenidol < methoctramine = pirenzepine < dicyclomine < AF-DX 116, and the EC50 values correlated best with constants for the M4/m4 muscarinic receptor subtype. The enhancing effect of atropine was counteracted by carbachol; the effects of atropine and emepronium were not additive. The 3H-acetylcholine secretion was also enhanced by forskolin, 3-isobutyl-1-methylxanthine, 8-bromo cyclic AMP and dibutyryl cyclic AMP. The combined effects of atropine and forskolin were additive. These results suggest that the 3H-acetylcholine secretion in the guinea pig urinary bladder is regulated by a presynaptic muscarinic autoreceptor of the M4 subtype that is not coupled to adenylate cyclase.

Subtype classification of presynaptic alpha 2-adrenoceptors.

1. Presynaptic alpha 2-adrenoceptors in a few tissues have recently been pharmacologically classified in functional studies. 2. Autoreceptors are of alpha 2A-subtype in rabbit occipito-parietal cortex, rat cerebral cortex, vas deferens, submandibular gland, kidney, guinea-pig ileum submucosal arterioles and urethra. 3. Heteroreceptors are of alpha 2A-subtype in rat cerebral cortex, vas deferens, guinea-pig ileum submucosal plexus and Auerbach's plexus. 4. In rat atria autoreceptors have been shown to be of alpha 2B-subtype. 5. Classification is done mainly with alpha 2A-adrenoceptor-selective oxymetazoline, WB 4101 and BRL 44408, and the alpha 2B-adrenoceptor-selective prazosin, AR-C 239, chlorpromazine and BRL 41992. 6. With four alpha 2-adrenoceptor subtypes to consider, a larger number of subtype-selective compounds may have to be characterized in the classification in the many tissues, where presynaptic alpha 2-adrenoceptors are found.

Subtype classification of the presynaptic alpha-adrenoceptors which regulate [3H]-noradrenaline secretion in guinea-pig isolated urethra.

1. The following experiments were carried out to investigate the presence and type of functional presynaptic receptors in adrenergic nerves of the guinea-pig urethra. 2. The urethra from male guinea-pigs was incubated with [3H]-noradrenaline and superfused with Tyrode solution in vitro. The fractional secretion of [3H]-noradrenaline evoked by 300 electrical pulses was measured. 3. The [3H]-noradrenaline secretion was positively frequency-dependent, yielding a half-maximal secretion at 8 +/- 5 Hz. Stimulation was usually applied at 5 Hz. 4. The [3H]-noradrenaline secretion was not altered by noradrenaline (1 or 100 microM), norephedrine (1 microM), isoprenaline (0.1 microM), 5-hydroxytryptamine (10 microM), oxotremorine (10 microM), adenosine (0.2 mM), propranolol (1 microM), atropine (1 microM) or 8-phenyltheophylline (10 microM). 5. The [3H]-noradrenaline secretion was enhanced by clonidine (3 microM), chlorpromazine (10 microM), metitepine (1 microM), 4-aminopyridine (0.5 mM), tetraethylammonium (2 mM), 3-isobutyl-1-methylxanthine (4 mM), 8-bromo cyclic AMP (1 mM) and forskolin (25 microM). 6. The alpha-adrenoceptor antagonists rauwolscine, yohimbine, phentolamine, prazosin and AR-C 239 maximally enhanced the [3H]-noradrenaline secretion to about 300% of control. The partial alpha-adrenoceptor agonist oxymetazoline maximally enhanced the secretion to about 200% of control. The order of apparent EC50 values was rauwolscine less than yohimbine less than phentolamine less than oxymetazoline less than prazosin less than AR-C 239.7. The enhancing effects of yohimbine (1 microM) with tetraethylammonium (2mM), 8-bromo cyclic AMP (1 mM), or forskolin (25,microM) were additive, but not those of yohimbine (1 microM) with prazosin (10 microM), 4-aminopyridine (0.5 mM), or 3-isobutyl-1-methylxanthine (4 mM).8. These results suggest that the [3H]-noradrenaline secretion in the guinea-pig urethra is regulated by presynaptic alpha2A-adrenoceptors which may, in a cyclic AMP-independent manner, be coupled to a 4-aminopyridine-sensitive potassium channel. The secretion is not influenced by compounds acting at beta-adrenoceptors, muscarinic cholinoceptors or adenosine receptors.

A new H-oxime restores rat diaphragm contractility after esterase inhibition in vitro.

Acetylcholine esterase inhibitors block cholinergic neurotransmission. This blockade can be reversed by oximes. However, a universally effective esterase reactivator does not exist. A new H-oxime, HLö 7, was tested on rat diaphragm strips. Electrically evoked contractions were blocked by di-2-propyl fluorophosphate (DFP), tabun, sarin and soman. Whereas pralidoxime, obidoxime and HI 6 reversed the blockade induced by three of these organophosphorus compounds, HLö 7 restored the contractions after short blockade induced by all four organophosphorus compounds tested.