Multiple structural elements contribute to the slow kinetics of the Cav3.3 T-type channel

Molecular cloning and expression studies established the existence of three T-type Ca(2+) channel (Ca(v)3) alpha(1) subunits: Ca(v)3.1 (alpha(1G)), Ca(v)3.2 (alpha(1H)), and Ca(v)3.3 (alpha(1I)). Although all three channels are low voltage-activated, they display considerable differences in their kinetics, with Ca(v)3.1 and Ca(v)3.2 channels activating and inactivating much faster than Ca(v)3.3 channels. The goal of the present study was to determine the structural elements that confer the distinctively slow kinetics of Ca(v)3.3 channels. To address this question, a series of chimeric channels between Ca(v)3.1 and Ca(v)3.3 channels were constructed and expressed in Xenopus oocytes. Kinetic analysis showed that the slow activation and inactivation kinetics of the Ca(v)3.3 channel were not completely abolished by substitution with any one portion of the Ca(v)3.1 channel. Likewise, the Ca(v)3.1 channel failed to acquire the slow kinetics by simply adopting one portion of the Ca(v)3.3 channel. These findings suggest that multiple structural elements contribute to the slow kinetics of Ca(v)3.3 channels.     

Veins of the large intestine, stomach and pancreas of the domestic cat and their variability

Most frequently - in 19 cases (63.3%) - the v. colica media was a tributary of a trunk, formed together with the v. mesenterica caudalis, which emptied into the v. mesenterica cranialis. In two cases (6.7%) it emptied independently into the v. mesenterica cranialis. In 25 cases (83.3%) the v. mesenterica caudalis arose from the rectum and, at colon ascendens level, acquired one or more vv. colicae sinistrae. In six cases (19.8%) the v. colica dextra opened into the v. ileocolica and in eight cases (26.7%) it was the initial tributary of an independent v. ileocolica which joined the v. mesenterica cranialis directly. The drainage of venous blood from the stomach is organized as follows: the fundus region is connected to the v. lienalis by means of inter-organ anastomoses. The curvatura minor is drained by means of the v. gastrica sinistra. Venous blood from the region of the curvatura major ventriculi flows partly into the v. lienalis (by way of the vv. gastroepiploicae or small veins) and partly into the v. gastrica sinistra. Drainage of venous blood from the pancreas: a v. pancreaticoduodenalis cranialis was present in 16 cases (53.3%) and a typical v. pancreaticoduodenalis caudalis was found in 12 cases (40.0%). Simultaneous formation of both vv. pancreaticoduodenalis, with a possible incidence of small venous efferents to the v. lienalis, was observed in only nine cases (30.0%). In 21 cases (70%), blood from the region of the flexura duodenojejunalis was drained by the last v. jejunalis; in other cases it drained into the vv. pancreaticoduodenales.(ABSTRACT TRUNCATED AT 250 WORDS)     

Strategies for the cryopreservation of microencapsulated cells

The major challenge in developing cryopreservation protocols for microencapsulated cells is that the relatively large size (300-400 microm) and the fragile semipermeable membrane of microcapsules makes them particularly prone to cryodamage. Rapid-cooling cryopreservation protocols with high DMSO concentrations (3.5M, 25% v/v) resulted in low post-thaw cell viability (<10%), which did not improve with higher concentrations (4.5M, 32% v/v) and longer exposure to DMSO, even though the majority of microcapsules (60-80%) remained intact. Subsequent investigations of slow cooling with a range of DMSO and EG concentrations resulted in a much higher post-thaw cell viability (80-85%), with the majority of the microcapsules remaining intact ( approximately 60%) when DMSO was used at a concentration of 2.8M (20% v/v) and EG at a concentration of 2.7M (15% v/v). The presence of 0.25M sucrose significantly improved post-thaw cell viability upon slow cooling with 2.8M (20% v/v) DMSO, although it had no effect on microcapsule integrity. Multistep exposure and removal of sucrose did not significantly improve either post-thaw cell viability or microcapsule integrity, compared to a single-step protocol. Ficoll 20% (w/v) also did not significantly improve post-thaw cell viability and microcapsule integrity. Hence, the optimal condition for microcapsule cryopreservation developed in this study is slow cooling with 2.8M (20% v/v) DMSO and 0.25M sucrose.     

Reversal of the effects of centrally-administered morphine on colonic motility in dogs by the benzodiazepine receptor antagonist RO 15-1788

The effects of intravenous (i.v.) and intracerebroventricular (i.c.v.) administration of morphine on jejunal and colonic motility were investigated in conscious dogs chronically prepared with strain gage transducers and compared to those of i.c.v. DAGO, a highly selective opiate mu agonist. Morphine i.v. (100 micrograms/kg) and i.c.v. (10 micrograms/kg) administered 3 hrs after a meal stimulated colonic motility for 3-5 hrs and induced a phase 3 on the jejunum, which appeared after a 15-60 min delay following i.c.v. administration. These effects were reproduced by DAGO administration at doses of 2 micrograms/kg i.v. and 0.2 micrograms/kg i.c.v. The effects of i.v., but not those of i.c.v., morphine and DAGO on jejunal and colonic motility were blocked by a previous administration of naloxone (100 micrograms/kg i.v.). The colonic stimulation but not the jejunal phase 3 induced by i.c.v. morphine and DAGO were blocked by RO 15-1788 (1 mg/kg i.v.), a selective benzodiazepine antagonist. The colonic stimulation induced by i.v. morphine or DAGO was not modify by i.v. RO 15-1788. It is concluded that i.c.v. administration of mu agonist involved benzodiazepine but not opiate receptors to stimulate colonic motility in dogs.     

Effect of neuropeptide Y on the hypothalamic-pituitary-adrenal axis in the dog

There is increasing evidence that neuropeptide Y (NPY) affects the release of pituitary hormones, including adrenocorticotropic hormone (ACTH). The present study was designed to clarify the mechanism by which NPY activates the hypothalamic-pituitary-adrenal (HPA) axis in the dog. Mongrel dogs were equipped with a chronic cannula allowing intra-third (i.t.v.) or intra-lateral (i.l.v.) cerebroventricular administration. A 1.19 nmol, i.t.v. dose of NPY produced as great an ACTH and cortisol response as did equimolar ovine corticotropin releasing factor (CRF). This action of NPY was dose-dependent and shared by peptide YY (PYY) and pancreatic polypeptide (PP), other members of the PP family peptide. Intravenously (i.v.) administered NPY (1.19-11.9 nmol) was much less potent than i.v. CRF in stimulating ACTH and cortisol secretion. However, i.v. NPY significantly increased plasma ACTH and cortisol concentrations, raising the possibility that NPY may modulate the activity of corticotrophs. We have next investigated the possible relationship between NPY and CRF on the HPA axis. Pretreatment with a novel CRF antagonist, alpha-helical CRF9-41 (130.9 nmol i.t.v. or 261.8 nmol i.v.), partly but significantly attenuated the ACTH and cortisol responses to i.t.v. NPY (1.19 nmol). Furthermore, adding a subthreshold dose of i.t.v. NPY (0.119 nmol) to i.t.v. CRF (1.19 nmol) or i.v. NPY (2.38 nmol) to i.v. CRF (0.595 nmol) resulted in the potentiation of CRF-induced ACTH secretion. These results indicate that NPY may activate the HPA axis in concert with CRF probably at hypothalamic and/or pituitary levels. The present findings that NPY evokes ACTH secretion and potentiates the effectiveness of CRF as a secretagogue, together with high concentrations of NPY in the hypothalamus and pituitary portal blood, suggest that NPY is involved in the multihormonal control of ACTH release.     

I-II loop structural determinants in the gating and surface expression of low voltage-activated calcium channels

The intracellular loops that interlink the four transmembrane domains of Ca(2+)- and Na(+)-channels (Ca(v), Na(v)) have critical roles in numerous forms of channel regulation. In particular, the intracellular loop that joins repeats I and II (I-II loop) in high voltage-activated (HVA) Ca(2+) channels possesses the binding site for Ca(v)beta subunits and plays significant roles in channel function, including trafficking the alpha(1) subunits of HVA channels to the plasma membrane and channel gating. Although there is considerable divergence in the primary sequence of the I-II loop of Ca(v)1/Ca(v)2 HVA channels and Ca(v)3 LVA/T-type channels, evidence for a regulatory role of the I-II loop in T-channel function has recently emerged for Ca(v)3.2 channels. In order to provide a comprehensive view of the role this intracellular region may play in the gating and surface expression in Ca(v)3 channels, we have performed a structure-function analysis of the I-II loop in Ca(v)3.1 and Ca(v)3.3 channels using selective deletion mutants. Here we show the first 60 amino acids of the loop (post IS6) are involved in Ca(v)3.1 and Ca(v)3.3 channel gating and kinetics, which establishes a conserved property of this locus for all Ca(v)3 channels. In contrast to findings in Ca(v)3.2, deletion of the central region of the I-II loop in Ca(v)3.1 and Ca(v)3.3 yielded a modest increase (+30%) and a reduction (-30%) in current density and surface expression, respectively. These experiments enrich our understanding of the structural determinants involved in Ca(v)3 function by highlighting the unique role played by the intracellular I-II loop in Ca(v)3.2 channel trafficking, and illustrating the prominent role of the gating brake in setting the slow and distinctive slow activation kinetics of Ca(v)3.3.     

New Determinant for the CaVbeta2 subunit modulation of the CaV1.2 calcium channel

Ca(v)beta subunits support voltage gating of Ca(v)1.2 calcium channels and play important role in excitation-contraction coupling. The common central membrane-associated guanylate kinase (MAGUK) region of Ca(v)beta binds to the alpha-interaction domain (AID) and the IQ motif of the pore-forming alpha(1C) subunit, but these two interactions do not explain why the cardiac Ca(v)beta(2) subunit splice variants differentially modulate inactivation of Ca(2+) currents (I(Ca)). Previously we described beta(2Deltag), a functionally active splice variant of human Ca(v)beta(2) lacking MAGUK. By deletion analysis of beta(2Deltag), we have now identified a 41-amino acid C-terminal essential determinant (beta(2)CED) that stimulates I(Ca) in the absence of Ca(v)beta subunits and conveys a +20-mV shift in the peak of the I(Ca)-voltage relationship. The beta(2)CED is targeted by alpha(1C) to the plasma membrane, forms a complex with alpha(1C) but does not bind to AID. Electrophysiology and binding studies point to the calmodulin-interacting LA/IQ region in the alpha(1C) subunit C terminus as a functionally relevant beta(2)CED binding site. The beta(2)CED interacts with LA/IQ in a Ca(2+)- and calmodulin-independent manner and need LA, but not IQ, to activate the channel. Deletion/mutation analyses indicated that each of the three Ca(v)beta(2)/alpha(1C) interactions is sufficient to support I(Ca). However, beta(2)CED does not support Ca(2+)-dependent inactivation, suggesting that interactions of MAGUK with AID and IQ are crucial for Ca(2+)-induced inactivation. The beta(2)CED is conserved only in Ca(v)beta(2) subunits. Thus, beta(2)CED constitutes a previously unknown integrative part of the multifactorial mechanism of Ca(v)beta(2)-subunit differential modulation of the Ca(v)1.2 calcium channel that in beta(2Deltag) occurs without MAGUK.     

Distribution,Disease Level and Virulence Variation of Bremia lactucae on Lactuca sativa in the Czech Republic in the Period 1999–2011

We report the distribution and disease level of downy mildew on lettuce (Lactuca sativa) and virulence variation in populations of its causal agent (Bremia lactucae) in the Czech Republic during the period 1999–2011. Disease incidence was not high; among a total of 92 different localities surveyed, 43.4% of them were infected by lettuce downy mildew at least once during the whole period. However, among individual years, differences were found in disease incidence that ranged from 4.8% (2009) to 66.7% (2004). A total of 128 isolates of B. lactucae collected from infected leaf samples in 35 different localities during the surveying period were included in the virulence analysis. Virulence was examined on a set of 19 differential genotypes of Lactuca sativa and Lactuca serriola (EU‐A test set). Isolates exhibited quite a broad variation in virulence to individual Lactuca differential genotypes. Eighteen of 19 virulence factors (v‐factors) tested were present in the samples. The most frequently detected factors were v1–4, v5/8, v6, v7, v10–14, v16, v36 and v38; factor v17 was not found. The most pronounced temporal shift was recorded for factors v36 and v38 whose frequency increased during the studied period. V‐factors 15, 17, 18 and 37 were present in low frequencies in a pathogen population, and their corresponding gene (Dm15) or resistance factors (R17, R18 and R37) may have the best potential for resistance breeding in the Czech Republic. Broad diversity of v‐phenotypes (63 different ones) was identified during the study period. The numbers of v‐factors per v‐phenotype (resp. isolate) varied within a range of 5–15. Within the 128 analysed isolates, only 9 v‐phenotypes were recorded repeatedly (three or more times). Possible reasons of recorded virulence variation are discussed.     

Activation of the purified erythrocyte plasma membrane Ca2+- ATPase by organic solvents

In this report it is shown that organic solvents mimic the stimulatory effects of calmodulin and acidic phospholipids on the erythrocyte plasma membrane Ca2+-ATPase. The solvents used were dimethyl sulfoxide (20%, v/v), glycerol (20% v/v), ethylene glycol (20%, v/v) and polyethylene glycol (Mr 6000-8000) (10%, w/v). These solvents increased both the affinity for Ca2+ and the turnover number of the enzyme. The increase in Ca2+ affinity is additive to that achieved with calmodulin. The calcium cooperativity observed in the presence of calmodulin disappears after the addition of dimethyl sulfoxide to the medium. The present data support the proposal that activation of the erythrocyte plasma membrane Ca2+-ATPase is promoted by hydrophobic interactions along the enzyme molecule.     

The effect of ethanol on the kinetics of lipase-mediated enantioselective esterification of 4-methyloctanoic acid and the hydrolysis of its ethyl ester.

The Novozym 435(R) catalyzed esterification and hydrolysis reactions of 4-methyloctanoic acid (ethyl ester) were investigated. In both the hydrolysis and esterification reactions, the increase of ethanol concentration led to an increase in enantiomeric ratio (E). For hydrolysis of the ethyl ester, the E-value increased from 5.5 [0% (v/v) EtOH] up to 12 [20% (v/v) EtOH]. In case of esterification, the E-value was already 16 [14% (v/v) EtOH] and rose to 57 [73% (v/v) EtOH]. When combining these results of esterification and hydrolysis, an enantiomeric ratio of 350 can be estimated for the sequential kinetic resolution of 4-methyloctanoic acid. In this way, enantiopure 4-methyloctanoic acid could be obtained after two consecutive reaction steps.     

Effect of ionic liquid on the kinetics of peroxidase catalysis

The effect of a water-miscible ionic liquid, 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4]), on the horseradish peroxidase (HRP)-catalyzed oxidation of 2-methoxyphenol (guaiacol) with hydrogen peroxide (H2O2) was investigated. HRP maintains its high activity in the aqueous mixtures containing various concentrations of the ionic liquid and even in 90% (v/v) ionic liquid. In order to minimize the effect of solution viscosity on the kinetic constants of HRP catalysis, the enzymatic reactions in the subsequent kinetic study were performed in water-ionic liquid mixtures containing 25% (v/v) ionic liquid at maximum. As the concentration of [BMIM][BF4] increased for the oxidation of guaiacol by HRP, the K(m) value increased with a slight decrease in the k(cat) value: The K(m) value increased from 2.8 mM in 100% (v/v) water to 22.5 mM in 25% (v/v) ionic liquid, indicating that ionic liquid significantly weakens the binding affinity of guaiacol to HRP.     

Origin of the v. portae and variability of its tributaries in laboratory animals. II. The guinea pig (Cavia aperea f. porcellus)

The authors investigated the origin of the v. portae and variability of its main tributaries in 30 adult guinea pigs (Cavia aperea f. porcellus). The venous bed was visualized by means of blue-dyed latex. The v. portae was formed the most frequently - in 17 cases (56.7%) - by the confluence of three veins - the v. mesenterica cranialis, the v. lienalis and the v. gastroepiploica dextra; less often it was formed from four or five tributaries and in one case only it was formed by union of the v. mesenterica cranialis and the v. lienalis. The v. gastrica sinistra was a tributary of the v. lienalis in 24 cases (80.0%). It arose on the ventral and the dorsal surface of the stomach, in the region adjacent to the curvatura minor ventriculi. In 21 cases (70.0%) it was joined by an independent v. cardiaca. The v. lienalis originated in the hilus lienis, usually as a result of the union of two venous trunks. Inter-organ anastomoses were found in 26 cases (86.7%); in 10 cases (33.3%) they occurred simultaneously between the spleen and the stomach and between the spleen and the pancreas, while in 16 cases (53.3%) they connected the superior pole of the spleen with the stomach. An independent v. gastroepiploica dextra was present in 19 cases (63.3%) as a tributary of the v. portae. In 22 cases it was joined by the v. pylorica. A v. pancreaticoduodenalis cranialis was present in 27 cases (90.0%). In 18 cases (60.0%) it united with the v. gastroepiploica dextra to form a common trunk, the v. gastroduodenalis, which joined the v. portae; in two cases (6.7%) it was an independent tributary of the v. portae. In six cases it was connected with the v. lienalis and in three cases (10.0%) it was replaced by a few vv. pancreaticae emptying into the v. lienalis. A typical v. pancreaticoduodenalis caudalis, as a tributary of the a. mesenterica cranialis, was found in 19 cases (63.3%). In two cases, together with the v. pancreaticoduodenalis cranialis, it formed a single trunk (the v. pancreaticoduodenalis communis). A comparison of the v. portae and its tributaries in the guinea pig and the cat has so far not brought to light any significant morphological differences relative to their different zoological classification and their different modes of life.     

Adaptive evolution of the vertebrate skeletal muscle sodium channel

Tetrodotoxin (TTX) is a highly potent neurotoxin that blocks the action potential by selectively binding to voltage-gated sodium channels (Na(v)). The skeletal muscle Na(v) (Na(v)1.4) channels in most pufferfish species and certain North American garter snakes are resistant to TTX, whereas in most mammals they are TTX-sensitive. It still remains unclear as to whether the difference in this sensitivity among the various vertebrate species can be associated with adaptive evolution. In this study, we investigated the adaptive evolution of the vertebrate Na(v)1.4 channels. By means of the CODEML program of the PAML 4.3 package, the lineages of both garter snakes and pufferfishes were denoted to be under positive selection. The positively selected sites identified in the p-loop regions indicated their involvement in Na(v)1.4 channel sensitivity to TTX. Most of these sites were located in the intracellular regions of the Na(v)1.4 channel, thereby implying the possible association of these regions with the regulation of voltage-sensor movement.     

Modulation of the K(v)4.3 channel by syntaxin 1A

The SNARE protein syntaxin 1A (Syn1A) is known to inhibit delayed rectifier K(+) channels of the K(v)1 and K(v)2 families with heterogeneous effects on their gating properties. In this study, we explored whether Syn1A could directly modulate K(v)4.3, a rapidly inactivating K(v) channel with important roles in neuroendocrine cells and cardiac myocytes. Immunoprecipitation studies in HEK293 cells coexpressing Syn1A and K(v)4.3 revealed a direct interaction with increased trafficking to the plasma membrane without a change in channel synthesis. Paradoxically, Syn1A inhibited K(v)4.3 current density. In particular, Syn1A produced a left-shift in steady-state inactivation of K(v)4.3 without affecting either voltage dependence of activation or gating kinetics, a pattern distinct from other K(v) channels. Combined with our previous reports, our results further verify the notion that the mechanisms involved in Syn1A-K(v) interactions vary significantly between K(v) channels, thus providing a wide scope for Syn1A modulation of exocytosis and membrane excitability.     

Structural and energetic determinants of apo calmodulin binding to the IQ motif of the Na(V)1.2 voltage-dependent sodium channel

The neuronal voltage-dependent sodium channel (Na(v)1.2), essential for generation and propagation of action potentials, is regulated by calmodulin (CaM) binding to the IQ motif in its α subunit. A peptide (Na(v)1.2(IQp), KRKQEEVSAIVIQRAYRRYLLKQKVKK) representing the IQ motif had higher affinity for apo CaM than (Ca(2+))(4)-CaM. Association was mediated solely by the C-domain of CaM. A solution structure (2KXW.pdb) of apo (13)C,(15)N-CaM C-domain bound to Na(v)1.2(IQp) was determined with NMR. The region of Na(v)1.2(IQp) bound to CaM was helical; R1902, an Na(v)1.2 residue implicated in familial autism, did not contact CaM. The apo C-domain of CaM in this complex shares features of the same domain bound to myosin V IQ motifs (2IX7) and bound to an SK channel peptide (1G4Y) that does not contain an IQ motif. Thermodynamic and structural studies of CaM-Na(v)1.2(IQp) interactions show that apo and (Ca(2+))(4)-CaM adopt distinct conformations that both permit tight association with Na(v)1.2(IQp) during gating.     

Examination of the solvent perturbation technique as a method to identify enzyme catalytic groups

The present study was undertaken for the purpose of evaluating the solvent perturbation technique as a method to identify enzyme catalytic residues. For establishment of expected directions and sizes of pKa perturbations for different types of acids in different classes of solvents, a study of the pKa of a series of acids in mixed solvent systems was carried out. Consistent with previous findings, the presence of organic solvents (25% v/v) increased the pKa values of neutral acids while it decreased or did not change the pKa values of cationic acids. The size of the perturbation observed was dependent on the nature of the organic solvent and on the polarity of the neutral form of the acid. The solvent perturbation studies were then extended to the catalytic aspartate residue of yeast hexokinase. The pKa of this residue was determined from the MgATP V/K profile measured in the presence and absence of organic solvents (25% v/v). While dimethylformamide and methanol induced small but perhaps significant increases in the observed pKa, dimethyl sulfoxide and propylene glycol did not. The pKa values, from the MgATP V/K profiles measured in the presence of fully saturating glucose, were not significantly increased by the organic solvents. The pKi vs. pH profile for the competitive inhibitor lyxose was also measured in the presence and absence of organic solvents. While methanol (25% v/v), dimethylformamide (25% v/v), and dioxane (17.5% v/v) induced a large increase in the pKa, propylene glycol and dimethyl sulfoxide (25% v/v) did not. The results from this investigation indicate that the solvent perturbation technique should not be relied upon indiscriminately.     

Fibroblast growth factor homologous factor 1B binds to the C terminus of the tetrodotoxin-resistant sodium channel rNav1.9a (NaN)

In this study we demonstrate a direct interaction between a cytosolic fibroblast growth factor family member and a sodium channel. A yeast two-hybrid screen for proteins that associate with the cytoplasmic domains of the tetrodotoxin-resistant sodium channel rNa(v)1.9a (NaN) led to the identification of fibroblast growth factor homologous factor 1B (FHF1B), a member of the fibroblast growth factor family, as an interacting partner of rNa(v)1.9a. FHF1B selectively interacts with the C-terminal region but not the other four intracellular segments of rNa(v)1.9a. FHF1B binds directly to the C-terminal polypeptide of rNa(v)1.9a both in vitro and in mammalian cell lines. The N-terminal 5-77 amino acid residues of FHF1B are essential for binding to rNa(v)1.9a. FHF1B did not interact with C termini of two other sodium channels hNa(v)1.7a (hNaNE) and rNa(v)1.8a (SNS), which share 50% similarity to the C-terminal polypeptide of rNa(v)1.9a. FHF1B is the first growth factor found to bind specifically to a sodium channel. Although the functional significance of this interaction is not clear, FHF1B may affect the rNa(v)1.9a channel directly or by recruiting other proteins to the channel complex. Alternatively, it is possible that rNa(v)1.9a may help deliver this factor to the cell membrane, where it exerts its function.     

Pre- and postsynaptic mechanisms in the clonidine- and oxymetazoline-induced inhibition of gastric motility in the rat

The inhibitory effect of clonidine (non-selective alpha2-adrenoceptor agonist) and oxymetazoline (alpha2A-adrenoceptor selective agonist) was compared on basal and stimulated gastric motor activity (gastric tone and contractions) using the balloon method in the rat. It was shown that oxymetazoline (0.2-1.7 micromol/kg, i.v.) decreased the basal motility, while clonidine (1.9-3.8 micromol/kg, i.v.) failed to affect it. When motility was stimulated centrally by insulin (5 IU/rat, i.v.), both clonidine (1.9-3.8 micromol/kg, i.v.) and oxymetazoline (0.1-3.4 micromol/kg, i.v.) inhibited the gastric motor activity. However, while the effect of clonidine was antagonized by the non-selective alpha2-adrenoceptor antagonist yohimbine (5 micromol/kg, i.v.) and the alpha2A-adrenoceptor selective antagonist BRL 44408 (3 micromol/kg, i.v.), the effect of oxymetazoline was only partially affected. Prazosin (alpha1- and alpha2B-adrenoceptor antagonist, 0.07-0.28 micromol/kg, i.v.) also failed to reverse the effect of oxymetazoline. Furthermore, when gastric motility was stimulated peripherally by activation of postsynaptic cholinergic muscarinic receptors by the combination of carbachol (0.14 micromol/kg, i.v.) and hexamethonium (37 micromol/kg, i.v.), clonidine (3.8 micromol/kg, i.v.) failed to affect the increased motor activity, however, oxymetazoline (0.8-3.4 micromol/kg, i.v.) exerted a pronounced inhibition. These results suggest that different mechanisms may be involved in the inhibitory effect of clonidine and oxymetazoline; while clonidine reduces the gastric motility by activation of presynaptic alpha2-adrenoceptors, postsynaptic component in the effect of oxymetazoline has also been raised.