Methylglyoxal Disrupts Paranodal Axoglial Junctions via Calpain Activation.

Nodes of Ranvier and associated paranodal and juxtaparanodal domains along myelinated axons are essential for normal function of the peripheral and central nervous systems. Disruption of these domains as well as increases in the reactive carbonyl species methylglyoxal are implicated as a pathophysiology common to a wide variety of neurological diseases. Here, using an ex vivo nerve exposure model, we show that increasing methylglyoxal produces paranodal disruption, evidenced by disorganized immunostaining of axoglial cell-adhesion proteins, in both sciatic and optic nerves from wild-type mice. Consistent with previous studies showing that increase of methylglyoxal can alter intracellular calcium homeostasis, we found upregulated activity of the calcium-activated protease calpain in sciatic nerves after methylglyoxal exposure. Methylglyoxal exposure altered clusters of proteins that are known as calpain substrates: ezrin in Schwann cell microvilli at the perinodal area and zonula occludens 1 in Schwann cell autotypic junctions at paranodes. Finally, treatment with the calpain inhibitor calpeptin ameliorated methylglyoxal-evoked ezrin loss and paranodal disruption in both sciatic and optic nerves. Our findings strongly suggest that elevated methylglyoxal levels and subsequent calpain activation contribute to the disruption of specialized axoglial domains along myelinated nerve fibers in neurological diseases.

Neuronal synapse formation induced by microglia and interleukin 10.

Recently, it was found that microglia regulated synaptic remodeling of the developing brain, but their mechanisms have not been well understood. In this study, the action of microglia on neuronal synapse formation was investigated, and the primary target of microglial processes was discovered. When the developing microglia were applied to cultured hippocampal neurons without direct contact, the numbers of dendritic spines and excitatory and inhibitory synapses significantly increased. In order to find out the main factor for synaptic formation, the effects of cytokines released from microglia were examined. When recombinant proteins of cytokines were applied to neuronal culture media, interleukin 10 increased the numbers of dendritic spines in addition to excitatory and inhibitory synapses. Interestingly, without external stimuli, the amount of interleukin 10 released from the intact microglia appeared to be sufficient for the induction of synaptic formation. The neutralizing antibodies of interleukin 10 receptors attenuated the induction of the synaptic formation by microglia. The expression of interleukin 10 receptor was newly found in the hippocampal neurons of early developmental stage. When interleukin 10 receptors on the hippocampal neurons were knocked down with specific shRNA, the induction of synaptic formation by microglia and interleukin 10 disappeared. Pretreatment with lipopolysaccharide inhibited microglia from inducing synaptic formation, and interleukin 1ß antagonized the induction of synaptic formation by interleukin 10. In conclusion, the developing microglia regulated synaptic functions and neuronal development through the interactions of the interleukin 10 released from the microglia with interleukin 10 receptors expressed on the hippocampal neurons.

Suppression of Alzheimer's disease-related phenotypes by geranylgeranylacetone in mice.

Amyloid-ß peptide (Aß) plays an important role in the pathogenesis of Alzheimer's disease (AD). Aß is generated by the secretase-mediated proteolysis of ß-amyloid precursor protein (APP), and cleared by enzyme-mediated degradation and phagocytosis. Transforming growth factor (TGF)-ß1 stimulates this phagocytosis. We recently reported that the APP23 mouse model for AD showed fewer AD-related phenotypes when these animals were crossed with transgenic mice expressing heat shock protein (HSP) 70. We here examined the effect of geranylgeranylacetone, an inducer of HSP70 expression, on the AD-related phenotypes. Repeated oral administration of geranylgeranylacetone to APP23 mice for 9 months not only improved cognitive function but also decreased levels of Aß, Aß plaque deposition and synaptic loss. The treatment also up-regulated the expression of an Aß-degrading enzyme and TGF-ß1 but did not affect the maturation of APP and secretase activities. These outcomes were similar to those observed in APP23 mice genetically modified to overexpress HSP70. Although the repeated oral administration of geranylgeranylacetone did not increase the level of HSP70 in the brain, a single oral administration of geranylgeranylacetone significantly increased the level of HSP70 when Aß was concomitantly injected directly into the hippocampus. Since geranylgeranylacetone has already been approved for use as an anti-ulcer drug and its safety in humans has been confirmed, we propose that this drug be considered as a candidate drug for the prevention of AD.

Impaired function of prejunctional adenosine A1 receptors expressed by perivascular sympathetic nerves in DOCA-salt hypertensive rats.

Increased sympathetic nervous system activity contributes to deoxycorticosterone acetate (DOCA)-salt hypertension in rats. ATP and norepinephrine (NE) are coreleased from perivascular sympathetic nerves. NE acts at prejunctional α2-adrenergic receptors (α2ARs) to inhibit NE release, and α2AR function is impaired in DOCA-salt rats. Adenosine, an enzymatic ATP degradation product, acts at prejunctional A1 adenosine receptors (A1Rs) to inhibit NE release. We tested the hypothesis that prejunctional A1R function is impaired in sympathetic nerves supplying mesenteric arteries (MAs) and veins (MVs) of DOCA-salt rats. Electrically evoked NE release and constrictions of blood vessels were studied in vitro with use of amperometry to measure NE oxidation currents and video microscopy, respectively. Immunohistochemical methods were used to localize tyrosine hydroxylase (TH) and A1Rs in perivascular sympathetic nerves. TH and A1Rs colocalized to perivascular sympathetic nerves. Adenosine and N(6)-cyclopentyl-adenosine (CPA, A1R agonist) constricted MVs but not MAs. Adenosine and CPA (0.001-10 µM) inhibited neurogenic constrictions and NE release in MAs and MVs. DOCA-salt arteries were resistant to adenosine and CPA-mediated inhibition of NE release and constriction. The A2A adenosine receptor agonist CGS21680 (C23H29N7O6.HCl.xH2O) (0.001-0.1 µM) did not alter NE oxidation currents. We conclude that there are prejunctional A1Rs in arteries and both pre- and postjunctional A1Rs in veins; thus, adenosine selectively constricts the veins. Prejunctional A1R function is impaired in arteries, but not veins, from DOCA-salt rats. Sympathetic autoreceptor dysfunction is not specific to α2ARs, but there is a more general disruption of prejunctional mechanisms controlling sympathetic neurotransmitter release in DOCA-salt hypertension.

Deletion of Dicer in smooth muscle affects voiding pattern and reduces detrusor contractility and neuroeffector transmission.

MicroRNAs have emerged as important regulators of smooth muscle phenotype and may play important roles in pathogenesis of various smooth muscle related disease states. The aim of this study was to investigate the role of miRNAs for urinary bladder function. We used an inducible and smooth muscle specific Dicer knockout (KO) mouse which resulted in significantly reduced levels of miRNAs, including miR-145, miR-143, miR-22, miR125b-5p and miR-27a, from detrusor preparations without mucosa. Deletion of Dicer resulted in a disturbed micturition pattern in vivo and reduced depolarization-induced pressure development in the isolated detrusor. Furthermore, electrical field stimulation revealed a decreased cholinergic but maintained purinergic component of neurogenic activation in Dicer KO bladder strips. The ultrastructure of detrusor smooth muscle cells was well maintained, and the density of nerve terminals was similar. Western blotting demonstrated reduced contents of calponin and desmin. Smooth muscle α-actin, SM22α and myocardin were unchanged. Activation of strips with exogenous agonists showed that depolarization-induced contraction was preferentially reduced; ATP- and calyculin A-induced contractions were unchanged. Quantitative real time PCR and western blotting demonstrated reduced expression of Cav1.2 (Cacna1c). It is concluded that smooth muscle miRNAs play an important role for detrusor contractility and voiding pattern of unrestrained mice. This is mediated in part via effects on expression of smooth muscle differentiation markers and L-type Ca(2+) channels in the detrusor.

Alterations in sympathetic neuroeffector transmission to mesenteric arteries but not veins in DOCA-salt hypertension.

We studied hypertension-associated changes in prejunctional alpha(2) adrenergic receptor (alpha(2)-AR) function using amperometry to monitor in vitro norepinephrine (NE) measured as oxidation currents. Vasoconstriction was measured using video imaging. NE release was induced by electrical stimulation of sympathetic nerves associated with mesenteric arteries (MA) and veins (MV) of sham and DOCA-salt hypertensive rats. NE oxidation currents were larger in DOCA-salt compared to sham MA; there were no differences between currents in sham and DOCA-salt MV. Increases in NE oxidation currents followed a multi-exponential time course in sham MA. In DOCA-salt MA and sham and DOCA-salt MV, the time course was mono-exponential. Yohimbine (alpha(2)-AR antagonist, 1 microM), caused a mono-exponential increase in NE oxidation currents in sham and DOCA-salt MA. Yohimbine increased NE oxidation currents and constrictions more in sham compared to DOCA-salt MA and compared to MV. UK 14,304 (alpha(2)-AR agonist, 1.0 microM), reduced currents less in DOCA-salt MA and sham and DOCA-salt MV compared to sham MA. Prazosin (alpha(1)-AR antagonist, 0.1 microM) did not alter NE oxidation currents. Prazosin inhibited constrictions more in DOCA-salt compared to sham MA and almost completely blocked constrictions in sham and DOCA-salt MV. Prazosin-resistant constrictions in MA were blocked by the P2 receptor antagonist, PPADS (10 microM). Prejunctional alpha(2)-ARs modify NE concentrations near neuroeffector junctions in MA and MV. alpha(2)-AR function is most prominent in MA and is impaired in DOCA-salt MA but not MV. Purinergic transmission predominates in sham MA. NE is the dominant vasoconstrictor in DOCA-salt MA and sham and DOCA-salt MV.

Prejunctional and peripheral effects of the cannabinoid CB(1) receptor inverse agonist rimonabant (SR 141716).

Rimonabant is an inverse agonist specific for cannabinoid receptors and selective for their cannabinoid-1 (CB(1)) subtype. Although CB(1) receptors are more abundant in the central nervous system, rimonabant has many effects in the periphery, most of which are related to prejunctional modulation of transmitter release from autonomic nerves. However, CB(1) receptors are also expressed in, e.g., adipocytes and endothelial cells. Rimonabant inhibits numerous cardiovascular cannabinoid effects, including the decrease of blood pressure by central and peripheral (cardiac and vascular) sites of action, with the latter often being endothelium dependent. Rimonabant may also antagonize cannabinoid effects in myocardial infarction and in hypotension associated with septic shock or liver cirrhosis. In the gastrointestinal tract, rimonabant counteracts the cannabinoid-induced inhibition of secretion and motility. Although not affecting most cannabinoid effects in the airways, rimonabant counteracts inhibition of smooth-muscle contraction by cannabinoids in urogenital tissues and may interfere with embryo attachment and outgrowth of blastocysts. It inhibits cannabinoid-induced decreases of intraocular pressure. Rimonabant can inhibit proliferation of, maturation of, and energy storage by adipocytes. Among the many cannabinoid effects on hormone secretion, only some are rimonabant sensitive. The effects of rimonabant on the immune system are not fully clear, and it may inhibit or stimulate proliferation in several types of cancer. We conclude that direct effects of rimonabant on adipocytes may contribute to its clinical role in treating obesity. Other peripheral effects, many of which occur prejunctionally, may also contribute to its overall clinical profile and lead to additional indications as well adverse events.

Nitric oxide inhibition and the impact on renal nerve-mediated antinatriuresis and antidiuresis in the anaesthetized rat.

The contribution of nitric oxide (NO) to the antinatriuresis and antidiuresis caused by low-level electrical stimulation of the renal sympathetic nerves (RNS) was investigated in rats anaesthetized with chloralose-urethane. Groups of rats, n= 6, were given i.v. infusions of vehicle, l-NAME (10 microg kg(-1) min(-1)), 1400W (20 microg kg(-1) min(-1)), or S-methyl-thiocitrulline (SMTC) (20 microg kg(-1) min(-1)) to inhibit NO synthesis non-selectively or selectively to block the inducible or neuronal NOS isoforms (iNOS and nNOS, respectively). Following baseline measurements of blood pressure (BP), renal blood flow (RBF), glomerular filtration rate (GFR), urine flow (UV) and sodium excretion (U(Na)V), RNS was performed at 15 V, 2 ms duration with a frequency between 0.5 and 1.0 Hz. RNS did not cause measurable changes in BP, RBF or GFR in any of the groups. In untreated rats, RNS decreased UV and U(Na)V by 40-50% (both P < 0.01), but these excretory responses were prevented in l-NAME-treated rats. In the presence of 1400W i.v., RNS caused reversible reductions in both UV and U(Na)V of 40-50% (both P < 0.01), while in SMTC-treated rats, RNS caused an inconsistent fall in UV, but a significant reduction (P < 0.05) in U(Na)V of 21%. These data demonstrated that the renal nerve-mediated antinatriuresis and antidiuresis was dependent on the presence of NO, generated in part by nNOS. The findings suggest that NO importantly modulates the neural control of fluid reabsorption; the control may be facilitatory at a presynaptic level but inhibitory on tubular reabsorptive processes.

AP180 maintains the distribution of synaptic and vesicle proteins in the nerve terminal and indirectly regulates the efficacy of Ca2+-triggered exocytosis.

AP180 plays an important role in clathrin-mediated endocytosis of synaptic vesicles (SVs) and has also been implicated in retrieving SV proteins. In Drosophila, deletion of its homologue, Like-AP180 (LAP), has been shown to increase the size of SVs but decrease the number of SVs and transmitter release. However, it remains elusive whether a reduction in the total vesicle pool directly affects transmitter release. Further, it is unknown whether the lap mutation also affects vesicle protein retrieval and synaptic protein localization and, if so, how it might affect exocytosis. Using a combination of electrophysiology, optical imaging, electron microscopy, and immunocytochemistry, we have further characterized the lap mutant and hereby show that LAP plays additional roles in maintaining both normal synaptic transmission and protein distribution at synapses. While increasing the rate of spontaneous vesicle fusion, the lap mutation dramatically reduces impulse-evoked transmitter release at steps downstream of calcium entry and vesicle docking. Notably, lap mutations disrupt calcium coupling to exocytosis and reduce calcium cooperativity. These results suggest a primary defect in calcium sensors on the vesicles or on the release machinery. Consistent with this hypothesis, three vesicle proteins critical for calcium-mediated exocytosis, synaptotagmin I, cysteine-string protein, and neuronal synaptobrevin, are all mislocalized to the extrasynaptic axonal regions along with Dap160, an active zone marker (nc82), and glutamate receptors in the mutant. These results suggest that AP180 is required for either recycling vesicle proteins and/or maintaining the distribution of both vesicle and synaptic proteins in the nerve terminal.

Traditional oriental herbal medicine, Bakumondo-to, suppresses vagal neuro-effector transmission in guinea pig trachea.

OBJECTIVE: Bakumondo-to (Maimendong tang) is a traditional oriental herbal medicine that has been used as an antitussive agent. We previously demonstrated that Bakumondo-to attenuates airway hyperresponsiveness induced by ozone. However, the mechanism(s) responsible for this effect remains unclear. In the present study, we examined the mechanism whereby Bakumondo-to inhibits ozone-induced airway hyperresponsiveness. First, we examined the effect of Bakumondo-to on prostanoids production, which are key mediators to airway hyperresponsiveness after ozone exposure. Second, we studied its effects on the vagal neuroeffector transmission, because vagal nerve is likely to play an important role in airway hyperresponsiveness after ozone. METHODS: We measured the effects of Bakumondo-to on the concentrations of prostanoids in bronchoalveolar lavage fluid before and after ozone. We evaluated the effects of Bakumondo-to on the contraction of guinea pig tracheal smooth muscle evoked by electrical field stimulation (EFS) or the exogenous application of acetylcholine (ACh). Isometric tension of tracheal strips was measured in the presence of indomethacin (10(-6) M) and of guanethidine (10(-6) M). RESULTS: Ozone caused significant increase in prostaglandin E2 (PGE2) and thromboxane B2 (TXB2); however, Bakumondo-to did not affect the increase in these prostanoids. Bakumondo-to (0.01 mg/mL-1 mg/mL) significantly suppressed the contraction evoked by EFS, but did not affect the ACh-evoked contraction, indicating that Bakumondo-to suppressed tracheal smooth muscle contraction pre-junctionally. CONCLUSION: These results suggest that the mechanism by which Bakumondo-to inhibits airway hyperresponsiveness depends on inhibiting the release of acetylcholine from vagal nerve terminals.

Neuroeffector mechanisms involved in the regulation of dog splenic arterial tone.

It has been recognized that sympathetic neurons release several transmitters but mainly adenosine 5'-triphosphate (ATP), noradrenaline, and neuropeptide Y (NPY). Recently, we reported that periarterial nerve electrical stimulation (PNS) produced biphasic vasoconstrictions consisting of an initial transient, predominantly P2X-purinoceptor-mediated constriction followed by a prolonged, alpha(1)-adrenoceptor-mediated one in canine isolated splenic arteries. In this article, we tried to analyze the effects of several selective key drugs that influence the PNS-induced responses, and we functionally showed sympathetic transmitter releasing mechanisms by pharmacological analysis using purinergic, adrenergic, and NPYergic agonists and antagonists.

Effects of reboxetine on sympathetic neuroeffector transmission in rabbit carotid artery.

The effect of reboxetine on sympathetic neuroeffector transmission in rabbit isolated carotid artery was examined. Reboxetine (10-8-3 x 10-6 M) and cocaine (10-6-3 x 10-5 M), but not desipramine (10-8-3 x 10-7 M), increased contractions evoked by electrical field stimulation. At higher concentrations, reboxetine (10-4 M), cocaine (3 x 10-4 M), and desipramine (3 x 10-7-10-5 M) inhibited the neurogenic contractions. The enhancement seen with reboxetine and cocaine was partially reversible, whereas the inhibition was readily reversible. Reboxetine (10-7 M) and cocaine (10-5 M) prevented the inhibitory action of bretylium (10-6 M). Reboxetine (10-8-10-5 M), desipramine (10-7-10-4 M), and cocaine (10-6-10-5 M) increased the stimulation-evoked [3H]norepinephrine release. Pargyline (5 x 10-4 M) augmented the facilitatory effect of reboxetine (3 x 10-9-10-6 M) and cocaine (10-7-3 x 10-5 M). Reboxetine (10-8-10-6 M), desipramine (10-8-10-6 M), and cocaine (3 x 10-8-10-5 M) reduced the [3H]norepinephrine (10-8 M) uptake. Reboxetine (10-7 M) and cocaine (10-5-2 x 10-4 M) enhanced the contractions evoked by phenylephrine and norepinephrine. Higher concentrations of reboxetine antagonized the contractions. Reboxetine (10-5-6 x 10-5 M) antagonized the contractions evoked by potassium. The contractions evoked by tyramine (3 x 10-6-10-3 M) was reduced by reboxetine (3 x 10-8-10-6 M) and by cocaine (10-7-10-5 M). We conclude that reboxetine inhibits the membrane amine pump (uptake-1) in the terminals of postganglionic adrenergic neurons in a cocaine-like manner.

Fe2+ decreases the taurine-induced Cl- current in acutely dissociated rat hippocampal neurons.

The effects of ferrous ions (Fe(2+)) on taurine-induced Cl(-) current (I(tau)) recorded from single neurons, which was freshly isolated from the rat hippocampal CA1 area, were studied with conventional whole-cell recording under voltage-clamp conditions. Using standard pharmacological approaches, we found that the currents gated by concentrations of taurine (

Lack of evidence for peripheral alpha(1)- adrenoceptor blockade during long-term treatment of heart failure with carvedilol.

OBJECTIVES: The purpose of this study was to determine whether carvedilol's alpha(1)-adrenoceptor antagonism persists during long-term therapy of patients with congestive heart failure (CHF). BACKGROUND: Carvedilol and metoprolol differ in that carvedilol also antagonizes beta(2)- and alpha(1)-adrenoceptors. We hypothesized that in contrast to metoprolol, carvedilol would increase calf vascular conductance (CVC), blunt neurally mediated vasoconstriction and attenuate neuroeffector transfer function gain. METHODS: We randomized 36 patients with CHF (age 55 +/- 1 years, ejection fraction 19 +/- 1%, means +/- SE) to either drug. Blood pressure (BP), heart rate, muscle sympathetic nerve activity (MSNA) and CVC were assessed before and after four months of treatment. The variability of BP and MSNA was determined using fast Fourier transformation. RESULTS: Paired data were obtained in 23 (carvedilol, 13; metoprolol, 10) subjects. Both beta-blockers decreased heart rate, but neither affected mean BP or CVC (carvedilol: 0.016 +/- 0.002 to 0.018 +/- 0.003 U; metoprolol: 0.020 +/- 0.002 to 0.020 +/- 0.004 U). Isometric handgrip exercise (30% of maximum) increased heart rate, mean BP and MSNA. The calf vasoconstrictor response to handgrip exercise was not affected by carvedilol (from 16 +/- 6 resistance U to 25 +/- 10 resistance U, NS). The gain of the transfer of oscillations in MSNA into BP under resting conditions was not attenuated by carvedilol. CONCLUSIONS: Carvedilol did not increase CVC, blunt the calf vasoconstrictor response to handgrip or attenuate the gain of the neuroeffector transfer function, indicating the absence of functionally important peripheral alpha(1)-adrenoceptor antagonism during long-term treatment of CHF.

Beta2-adrenoceptor-mediated prejunctional facilitation and postjunctional inhibition of sympathetic neuroeffector transmission in the guinea pig vas deferens.

This study examines the role of prejunctional and postjunctional beta-adrenoceptors in the modulation of sympathetic cotransmission in the guinea pig vas deferens. The prejunctional involvement of beta-adrenoceptors was evaluated by testing the effects of several agonists and antagonists on the nerve stimulation-evoked overflow of ATP and norepinephrine (NE) from the "in vitro" vas deferens. The nonsubtype-selective beta-adrenoceptor agonist isoproterenol and the beta2-subtype-selective agonist clenbuterol increased, to a similar degree, the overflow of ATP and NE, while the beta1-subtype-selective agonist xamoterol and the beta3-subtype-selective agonist BRL 37 344 had no effect. Pretreatment with ICI 118, 551, a beta2-subtype-selective antagonist, abolished the facilitation of cotransmitter release by isoproterenol and clenbuterol, while the beta1-subtype-selective antagonist atenolol had no effect. Activation of beta-adrenoceptors by either isoproterenol or clenbuterol, but not by xamoterol and BRL 37 344, reduced the amplitude of contractions evoked by exogenously applied ATP. Pretreatment with propranolol or ICI 118, 551, but not atenolol, prevented these inhibitory effects. Isoproterenol in lower concentrations produced dose-dependent reduction of the purinergic but not the adrenergic phase of nerve stimulation-induced contraction of the guinea pig vas deferens. When applied in concentrations greater than 1 microM, isoproterenol, but not clenbuterol, actually produced a concentration-dependent facilitation of contractions evoked by both nerve stimulation and exogenously applied ATP. Antagonists of alpha-adrenoceptors blocked these facilitatory effects. Together, these results demonstrate that beta2-adrenoceptors can influence sympathetic neuroeffector transmission both prejunctionally, where they facilitate equally well the release of sympathetic cotransmitters and postjunctionally, where they inhibit smooth muscle contractions evoked by ATP.

Effects of cannabinoids on sympathetic and parasympathetic neuroeffector transmission in the rabbit heart.

Cannabinoids elicit marked cardiovascular responses. It is not clear how peripheral effects on the autonomic nervous system contribute to these responses. The aim of the present study was to characterize the peripheral actions of cannabinoids on the autonomic innervation of the heart. Experiments were carried out on pithed rabbits. In the first series of experiments, postganglionic sympathetic cardioaccelerator fibers were stimulated electrically. The synthetic cannabinoid receptor agonists WIN55212-2 (0.005, 0.05, 0.5, and 1.5 mg kg(-1) i.v.) and CP55940 (0.003, 0.03, 0.3, and 1 mg kg(-1) i.v.) dose dependently inhibited the electrically evoked cardioacceleration. The inhibition by WIN55212-2 (0.5 mg kg(-1) i.v.) was prevented by the CB(1) cannabinoid receptor antagonist SR141716A (0.5 mg kg(-1) i.v.). WIN55212-2 (0.5 mg kg(-1) i.v.) did not change the increase in heart rate evoked by injection of isoprenaline. In the second series of experiments, preganglionic vagal fibers were stimulated electrically. WIN55212-2 (0.005, 0.05, and 0.5 mg kg(-1) i.v.) and CP55940 (0.003, 0.03, and 0.3 mg kg(-1) i.v.) dose dependently inhibited the stimulation-evoked decrease in heart rate. The inhibition produced by WIN55212-2 (0.005, 0.05, and 0.5 mg kg(-1) i.v.) was antagonized by SR141716A (0.5 mg kg(-1) i.v.). The results indicate that cannabinoids, by activating CB(1) cannabinoid receptors, inhibit sympathetic and vagal neuroeffector transmission in the heart. The mechanism of the sympathoinhibition is probably presynaptic inhibition of noradrenaline release from postganglionic sympathetic neurons. The mechanism of the inhibition of vagal activity was not clarified: cannabinoids may have an inhibitory action on both pre- and postganglionic vagal neurons.

Effects of a selective neuropeptide Y Y2 receptor antagonist, BIIE0246, on Y2 receptors at peripheral neuroeffector junctions.

1. This study investigated the effects of BIIE0246, a novel neuropeptide Y (NPY) Y2 receptor antagonist, on the inhibition of cholinergic neuroeffector transmission in rat heart and guinea-pig trachea and purinergic neuroeffector transmission in guinea-pig vas deferens produced by the NPY Y2 receptor agonist, N-acetyl [Leu28,31] NPY 24-36. 2. In pentobarbitone anaesthetized rats, supramaximal stimulation every 30 s, of the vagus nerve innervating the heart, increased pulse interval by approximately 100 ms. This response was attenuated by intravenous administration of N-acetyl [Leu28,31] NPY 24-36 (10 nmol x kg(-1)). 3. Transmural stimulation of segments of guinea-pig trachea at 1 min intervals with 5 s trains of stimuli at 0.5, 5, 10, 20 and 40 Hz evoked contractions which were reduced in force by N-acetyl [Leu28,31] NPY 24-36 (2 microM). 4. In guinea-pig vasa deferentia, the amplitude of excitatory junction potentials evoked by trains of 20 stimuli at 1 Hz was reduced in the presence of N-acetyl [Leu28,31] NPY 24-36 (1 microM). 5. In all preparations BIIE0246 attenuated the inhibitory effect of N-acetyl [Leu28,31] NPY 24-36 but had no effect when applied alone. 6. The findings support the view that the nerve terminals of postganglionic parasympathetic and sympathetic neurones possess neuropeptide Y Y2 receptors which, when activated, reduce neurotransmitter release.

Separate modulation of neuropeptide Y1 receptor on purinergic and on adrenergic neuroeffector transmission in canine splenic artery.

Our previous study demonstrated that the vasoconstrictor responses to trains of up to 10 pulses at 1 Hz of stimulation appeared to be purinergic monophasic, whereas a longer train of 30 pulses induced a biphasic vasoconstriction consisting of an initial, transient purinergic constriction followed by a prolonged adrenergic response. Neuropeptide Y (NPY) at doses of 0.01 and 0.1 microM produced a dose-dependent inhibition on the monophasic and biphasic vasoconstrictor responses to nerve stimulation. The treatment with Leu31 Pro34 neuropeptide Y (LP-NPY) (0.03 microM) did not affect the monophasic responses to short pulse trains of stimulation. However, LP-NPY markedly potentiated the second phase response to 30 pulse trains of stimulation, although it did not modify the first one. The LP-NPY-induced potentiation was abolished by BIBP 3226 (1 microM), a selective NPY Y1 receptor antagonist. The results indicate that the activation of NPY Y1 receptors may enhance the prolonged adrenergic vasoconstriction but not the transient purinergic response in the canine splenic artery.

Neuroeffector transmission to different layers of smooth muscle in the rat penile bulb.

1. Using intracellular recording techniques, two distinct layers of smooth muscle were identified in the rat penile bulb. The inner muscle layer (parenchyma) exhibited spontaneous action potentials, while the outer sheet (sac) was electrically quiescent. 2. In the parenchyma, transmural stimulation initiated non-adrenergic, non-cholinergic (NANC) inhibitory junction potentials (IJPs) which were abolished by Nomeganitro-L-arginine (LNA) or 1H-[1,2, 4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). The amplitude of IJPs was reduced by ouabain, dinitrophenol or decreasing the extracellular potassium concentration ([K+]o) but not by several K+ channel blockers. 3. The parenchyma also received an excitatory innervation mediated by alpha-adrenoceptors which caused a contraction that was not associated with a membrane potential change. 4. In the sac, transmural stimulation initiated two component excitatory junction potentials (EJPs) mediated by alpha-adrenoceptors and associated action potentials. The initial component was more dramatically suppressed than the secondary component by caffeine, ryanodine or cyclopiazonic acid (CPA). Lowering of the extracellular chloride concentration ([Cl-]o) selectively inhibited the rapid component of EJPs, while niflumic acid was less potent. 5. These results suggest that IJPs in the parenchyma result from the release of NO which stimulates sodium pump activity following the activation of guanylate cyclase. In the sac, the activation of alpha-adrenoceptors initiates EJPs by releasing Ca2+ from intracellular stores which activates Ca2+-activated channels.