A new possibility for repairing the anal dysfunction by promoting regeneration of the reflex pathways in the enteric nervous system

Moderate rectal distension elicits recto-rectal reflex contractions and simultaneous recto-internal anal sphincter reflex relaxations that together comprise the defecation reflex. Both reflexes are controlled by 1) pelvic nerves, 2) lumbar colonic nerves, and 3) enteric nervous system. The aim of the present study was to explore a novel approach to repairing the defecation reflex dysfunction by using the plasticity of enteric nervous pathways. Experiments were performed in anesthetized guinea pigs with ethyl carbamate. The rectum 30 mm oral from the anal verge was transected without damage to extrinsic nerves, and subsequent end-to-end one-layer anastomosis was performed. Recovery of the defecation reflex and associated reflex pathways were evaluated. Eight weeks after sectioning of intrinsic reflex nerve pathways in the rectum, the defecation reflex recovered to the control level, accompanied with regeneration of reflex pathways. The 5-HT(4)-receptor agonist mosapride (0.5 and 1.0 mg/kg) significantly (P < 0.01) enhanced the recovered defecation reflex 8 wk after surgery. Two weeks after local treatment with brain-derived neurotrophic factor (BDNF: 10(-6) g/ml) at the rectal anastomotic site, the recto-internal anal sphincter reflex relaxations recovered and some bundles of fine nerve fibers were shown to interconnect the oral and anal ends of the myenteric plexus. These results suggested a possibility for repairing the anal dysfunction by promoting regeneration of the reflex pathways in the enteric nervous system with local application of BDNF.     

In vitro effect of phencyclidine and other psychomotor stimulants on serotonin uptake in human platelets

Phencylidine, ketamine and fluoxetine inhibited serotonin (5-HT) uptake in a non-competitive manner in human blood platelets whereas d- and 1-amphetamine produced a competitive inhibition of 5-HT uptake. Phencyclidine (IC₅₀, 2.5 μM) was one-hundredth as potent as fluoxetine (IC₅₀, 22 νM) but ten times more potent than ketamine (IC₅₀, 25 μM) and d-amphetamine (IC₅₀, 24 μM) and three times more potent than 1-amphetamine (IC₅₀, 80 μM) in inhibition of 5-HT uptake by human blood platelets. The possibility that inhibition of 5-HT may contribute to some of the proposed serotonergic effects of psychomotor stimulants is discussed.     

The binding of 5-hydroxytryptamine to acidic lipids in isobutanol.

Abstract— In order to examine the possibility that acidic lipids can account for the binding of 5-hydroxy [³H]tryptamine (5-HT) to brain tissue, the binding to six acidic lipids was studied using an isobutanol-water partition method. With the exception of the polyphosphoinositides, all the acidic lipids examined bind saturably and with high affinity. The apparent dissociation constants of 5-HT to the acidic lipids were as follows: phosphatidylserine, 0.4 μM; phosphatidic acid, 0.6μM; diphosphoinositide, 0.8 μM; cerebroside sulfate, 1.4 μM; monophosphoinositide, 1.9 μM; and triphosphoinositide, 10 μM. The high affinity of these lipids to 5-HT raises the possibility of some role for them in serotonergic activity.     

5-HT2A受体在尼可刹米增强新生大鼠延髓脑片呼吸放电中的作用

本文旨在探讨中枢呼吸兴奋剂尼可刹米对新生大鼠基本节律性呼吸的产生和调节的影响及5-HT2A受体在其中的作用.制作新生大鼠离体延髓脑片标本,含面神经后核内侧区(the medial region of the nucleus retrofacialis,mNRF)并保留舌下神经根,灌流改良Kreb'S液(modified Kreb'S solution,MKS),记录舌下神经根呼吸相关节律性放电活动(respiratory-re-lated rhythmic discharge activity,RRDA),观察不同浓度尼可刹米、5-HT2A受体特异激动剂2,5-二甲氧基-4-碘苯基丙烷[1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane,DOI]、5-HT2A受体特异拮抗剂酮舍林(ketanserine)以及联合使用尼可刹米和酮舍林对舌下神经根RRDA的影响.结果显示,尼可刹米在0.5～7μg/mL时对延髓脑片RRDA有兴奋作用,在5 μg/mL时对吸气时程(inspiratory time,TI)、放电积分幅度(integral amplitude,IA)、呼吸周期(respiratory cycle,Re)等呼吸指标综合效果最显著.DOI明显延长TI、增强IA、缩短RC,对RRDA有兴奋作用.酮舍林明显缩短TI、减弱IA、延长RC,对RRDA有抑制作用.联合使用DOI和酮舍林对RRDA无明显作用.酮舍林可完全阻断尼可刹米对RC的作用,部分阻断尼可刹米对IA的作用,对尼可刹米引起的TI变化无明显影响.结果提示,尼可刹米增强新生大鼠离体延髓脑片舌下神经根RRDA,5-HT2A受体可能足尼可刹米作用途径之一.     

Evidence for a complex influence of nicotinic acetylcholine receptors on hippocampal serotonin release

The effects of nicotine on 5-hydroxytryptamine (5-HT) release from serotonergic nerve endings in rat dorsal hippocampal slices were studied. Nicotine (50-500 microM:) caused a concentration-dependent increase in 5-HT release. This effect was antagonised by mecamylamine (0.5 microM:), indicating an action at nicotinic receptors. Nicotine-evoked 5-HT release was not affected by tetrodotoxin (3 microM:), cadmium chloride (0.1 mM:), or the absence of Ca(2+) or Na(+) in the superfusion medium. Unexpectedly, higher concentrations of mecamylamine alone (1-50 microM:) increased 5-HT release. This suggested the presence of inhibitory input to 5-HT neurones and that these inhibitory neurones possess tonically active nicotinic receptors. The effect of mecamylamine (50 microM:) on 5-HT release was reduced by the muscarinic M(1) receptor agonist, McN-A-343 (100 microM:), but pirenzepine (0.005-1 microM:), which blocks M(1) receptors, alone increased 5-HT release. Hippocampal serotonergic neurones are known to possess both excitatory nicotinic receptors and inhibitory M(1) receptors. Although there may be several explanations for our results, one possible explanation is that nicotine stimulates 5-HT release by activating nicotinic heteroreceptors on 5-HT terminals. Mecamylamine (0.5 microM:) antagonises this effect, but higher concentrations increase 5-HT release indirectly by blocking the action of endogenous acetylcholine on nicotinic receptors situated on cholinergic neurones that provide muscarinic inhibitory input to 5-HT neurones.     

Potentiating effect of allatostatin on transmitter quantal secretion in the mouse nerve-muscle synapse

Action of allatostatin on the spontaneous and evoked quantal acetylcholine secretion was studied for the first time in the mouse nerve-muscle synapse. End plate miniature potentials (EPMP) and miniature currents (EPMC) as well as end plate single evoked currents (SEP) were recorded in mouse semidiaphragm. Allatostatin (1 nm–1 μM) produced a dose-dependent increase of the EPMP amplitude (that reached 209% of control at 1 μM of peptide), but without affecting statistically significantly the EPMP frequency and membrane potential of muscle fibers throughout the entire range of its concentrations. The potentiating action of 1 μM peptide on the EPMP amplitude was accompanied by a rise of time of the EPMP ascent and semidrop (by 17 and 13%, respectively). Allatostatin (1 μM) caused a twofold rise of EPMC amplitude, but the time parameters of miniature postsynaptic currents did not change statistically significantly. Amplitude of SEP also increased more than twice under effect of 1 μM peptide, but the SEP quantal composition remained at the control level. On the background of allatostatin there were revealed no rise of the postsynaptic membrane input resistance (on the contrary, it decreased by 25%) and no changes of the EPMC potential-dependent amplitude and of the droptime constant that characterize cholinoreceptor conductivity. The potentiating allatostatin effect on EPMP amplitude was prevented by vesamicol (1 μM), a blocker of transport of acetylcholine into synaptic vesicles. Preliminary treatment of the nerve-muscle preparation with the inhibitor of protein kinase A (PKA) H-89 (50 nM) prevented the allatostatin-evoked EPMC amplitude increment. The obtained data allow us to suggest that allatostatin in the mouse nerve-muscle synapse acts at the presynaptic level by producing an increase of the acetylcholine quantum size due to an intraterminal cascade of reaction with participation of PKA.     

Serotonin Inhibits Acetylcholine Release from Rat Striatum Slices: Evidence for a Presynaptic Receptor-Mediated Effect

Rat brain striatum slices were incubated with [3H]choline, perfused with a physiological buffer, and stimulated by perfusion with a K+-enriched buffer for 2 min. The tritium overflow evoked by K+ was decreased by 5-hydroxytryptamine (serotonin, 5-HT) (maximal inhibition 10(-6) M). This effect of 5-HT was mimicked by several agonists (5-methoxytryptamine, N,N-dimethyl-tryptamine, bufotenin) and blocked by serotonergic antagonists (methiothepin, methysergide, cinanserin) but not by haloperidol; methiothepin and methysergide alone slightly increased the K+-evoked overflow of tritium (3H). Inhibition of the tritium release by 5-HT was not suppressed in the presence of tetrodotoxin (TTX) (10(-6) M). These results suggest that 5-HT tonically inhibits acetylcholine (ACh) release from striatal cholinergic neurons by acting on a presynaptic receptor localized on cholinergic terminals.     

Hyperserotonergic phenotype after monoamine oxidase inhibition in larval zebrafish

Serotonin (or 5-hydroxytryptamine; 5-HT) and monoamine oxidase (MAO) are involved in several physiological functions and pathological conditions. We show that the serotonergic system and its development in zebrafish are similar to those of other vertebrates rendering zebrafish a good model to study them. Development of MAO expression followed a similar time course as the 5-HT system. MAO expression and activity were located in or adjacent to serotonergic nuclei and their targets, especially in the ventral hypothalamus. MAO mRNA was detected in the brain from 24 h post-fertilization and histochemical enzyme activity from 42 h post-fertilization. Deprenyl (100 μM) decreased MAO activity 34–74% depending on the age. Inhibition of MAO by deprenyl strongly increased 5-HT but not dopamine and noradrenaline levels. Deprenyl decreased 5-HT-immunoreactivity in serotonergic neurons and induced novel ectopic 5-HT-immunoreactivity neurons in the diencephalon in a manner dependent on 5-HT uptake. Deprenyl administration decreased locomotion, altered vertical positioning and increased heart rate. Treatment with p -chlorophenylalanine normalized 5-HT levels and rescued the behavioral alteration, indicating that the symptoms were 5-HT dependent. These findings suggest that zebrafish MAO resembles mammalian MAO A more than MAO B, metabolizing mainly 5-HT. Applications of this model of hyperserotonergism include pharmacological and genetic screenings.     

In vivo stimulation of ovarian development in the red swamp crayfish,Procambarus clarkii (Girard), by 5-hydroxytryptamine

Summary

The possibility that biogenic amines affect ovarian development in the red swamp crayfish, Procambarus clarkii, was investigated. Females were administered 15 μg/g body weight (bw) of norepinephrine, dopamine, 5-hydroxytryptamine (5-HT) or octopamine on days 1, 5 and 10 and were sacrificed on day 15. Crayfish given 5-HT showed significant increases in ovarian index (30.5%) and oocyte size (34.0%) over the concurrent controls, while norepinephrine, dopamine and octopamine did not significantly affect either the ovarian index or oocyte size. Significantly more labeling by ¹⁴C-leucine of ovarian proteins was found in ovaries of crayfish that were injected with 5-HT in vivo, but when ovarian lobes from crayfish that had not been injected with 5-HT were incubated in vitro with 5-HT added to the incubation medium, no significant change in the level of incorporation of ¹⁴C-leucine into ovarian proteins occurred.

The 5-HT receptor blocker LY53857 (25 μg/g bw) retarded ovarian development. The 5-HT releaser fenfluramine and the 5-HT potentiator fluoxetine (both 15 μg/g bw) were also used. Crayfish given fenfluramine, fluoxetine, fenfluramine plus 5-HT or fluoxetine plus 5-HT showed significant increases of ovarian index (24.0–102.8%), oocyte size (20.0–87.4%) and in vitro ¹⁴C-leucine labeling of ovarian proteins (30.6–123.6%) over the concurrent controls. The ovaries of crayfish that received the 5-hydroxytryptaminergic neurotoxin 5,6-dihydroxytryptamme (10 μg/g bw) did not show any significant change as compared with the initial control. These findings are consistent with the hypothesis that 5-HT, which is present in the central nervous system of Procambarus clarkii, exerts its stimulatory effect on the ovary of this crayfish indirectly by triggering release of the ovary-stimulating hormone.     

Responses of the rectum and oesophagus of the snail Helix aspersa to purine nucleotides and nucleosides

1. Purine compounds were examined for pharmacological activity in the rectum and oesophagus of the garden snail Helix aspersa.2. In the rectum, adenosine, AMP, ADP and ATP (above 10μM) and acetylcholine (above 1 nM) consistently caused concentration-dependent contractions. The slope of the dose-response curve for ADP in the rectum was significantly steeper than for the other purine compounds. The contractile responses to the nucleotides and acetylcholine, but not adenosine, were selectively potentiated by physostigmine (1μM). Atropine (1 μM) and tubocurarine (30 μM) failed to block the responses to the purines or acetylcholine.3. In the oesophagus, adenosine, AMP, ADP and ATP (above 10 μM) and acetylcholine (above 1 nM) caused concentration-dependent contractions that were antagonised by atropine (l μM). Tubocurarine (30 μM) failed to block the responses to the purine compounds or acetylcholine. Physostigmine (1 μM) potentiated the responses to ADP and acetylcholine but not ATP, AMP or adenosine.4. In both the rectum and the oesophagus, the synthetic analogues of purine compounds inclucling 2-chloroadenosine, α, β -methylene ATP and 2-methylthio ATP were inactive up to a concentration of 100 μM.5. Electrical field stimulation of the rectum and oesophagus produced consistent contractions which were unaffected by atropine (1 μM), tubocurarine (30 μM) or physostigmine (1 μM). These responses were not modulated by any of the purine compounds or their stable analogues.6. The responses obtained appear novel even within known invertebrate purinergic systems, suggesting a differentiation of purinoceptor subtypes in this species. There is evidence in the rectum for AMP, ADP and ATP causing the release of acetylcholine; physostigmine potentiated responses to AMP, ADP and ATP, but not to adenosine. This indicates that activity may be mediated via different types of purinoceptors, perhaps equivalent to the P₁- and P₂-purinoceptors identified in vertebrates.     

T-channels and Na<Superscript>+</Superscript>,Ca<Superscript>2+</Superscript>-exchangers as components of the Ca<Superscript>2+</Superscript>-system of regulation of activity of the heart myocardium of the frog <Emphasis Type="Italic">Rana temporaria</Emphasis>

Earlier we have shown that regulation of rhythm and strength of the frog heart contractions, mediated by transmitters of the autonomic nervous system, is of the Ca²⁺-dependent character. In the present work, we studied chronoand inotropic effect of verapamil—an inhibitor of Ca²⁺-channels of the L-type, of nickel chloride-an inhibitor of Ca²⁺—channels of the T-type and of Na⁺,Ca²⁺exchangers as well as of adrenaline and acetylcholine (ACh) after nickel chloride. It has been found that the intracardially administered NiCh₂ at a dose of 0.01 μg/kg produced a sharp fall of amplitude of action potential (AP) and an almost twofold deceleration of heart rate (HR). The intracardiac administration of NiCh₂ (0.01 μg/kg) on the background of action of verapamil (6 mg/kg, i/m) led as soon as after 3 min to even more prominent HR deceleration and to further fall of the AP amplitude by more than 50% as compared with norm. An intracardiac administration of adrenaline (0.5 mg/kg) partly restored the cardiac activity. However, preservation of the myocardium electrical activity in such animals was brief and its duration did not exceed several minutes. Administration of Ni²⁺ on the background of acetylcholine (3.6 mg/kg) led to almost complete cessation of cardiac activity. As soon as 3 min after injection of this agent the HR decreased to 2 contractions/min. On electrograms (EG), the 10-fold fall of the AP amplitude was recorded. To elucidate role of extraand intracellular Ca²⁺ in regulation of strength of heart contractions, isometric contraction of myocardium preparations was studied in response to action of NiCl₂ (10–200 μM), verapamil (70 μM), adrenaline (5 μM), and acetylcholine (0.2 μM) after NiCl₂. It has been found that Ni²⁺ causes a dose-dependent increase of the muscle contraction amplitude. Minimal change of the contraction amplitude (on average, by 14.9% as compared with control) was recorded at a Ni²⁺ concentration of 100 μM. An increase of Ni²⁺ in the sample to 200 μM increased the cardiac contraction strength, on average, by 41%. The negative inotropic action of verapamil was essentially reduced by 100 μM Ni²⁺. Adrenaline added to the sample after Ni²⁺ produced stimulating effect on the cardiac muscle, with an almost twofold rise of the contraction amplitude. ACh (0.2 μM) decreased the cardiac contraction amplitude, on average, by 56.3%, whereas Ni²⁺ (200 μM) administered after ACh not only restored, but also stimulated partly the myocardial work. Within several parts of percent there was an increase of such isometric contraction parameters as amplitude of the effort developed by muscle, maximal rate, maximal acceleration, time of semirise and semifall. The obtained experimental results indicate that the functional activity of the frog pacemaker and contractile cardiomyocytes is regulated by Ca²⁺-dependent mechanisms. Structure of these mechanisms includes the potential-controlled Land T-channels of the plasma membrane as well as Na⁺,Ca²-exchangers characteristic exclusively of contractile cardiomyocytes. The existence of these differences seems to be due to the cardiomyocyte morphological peculiarities that appeared in evolution at the stage of the functional cell specialization.     

Potentiation of the carotid artery occlusion reflex by a cholinergic system in the posterior hypothalamic nucleus

Bilateral occlusion of the common carotid arteries of urethane-anesthetized rats evoked a pressor response of 14 ± 1 mm Hg. Injection into the lateral cerebral ventricle of neostigmine (0.2–1.0 μg) or physostigmine (10–15 μg) caused a dose-dependent increase in basal blood pressure and in the magnitude of the carotid artery occlusion (CAO) pressor reflex. Neostigmine (1 μg) and physostigmine (15 μg) caused nearly maximal and approximately equal degrees of cholinesterase inhibition in several brain regions. The recovery of the cardiovascular parameters and of brain cholinesterase activity was significantly faster following physostigmine compared to neostigmine. Prior intracerebroventricular injection of atropine (0.3 μg) or hemicholinium-3 (20 μg) prevented the increases in basal pressure and the CAO pressor response. Potentiation of the CAO reflex also followed injection of physostigmine or neostigmine into the posterior hypothalamic nucleus and of injection of physostigmine intravenously. Injection of atropine bilaterally into the posterior hypothalamic nucleus prior to intravenous injection of physostigmine prevented the potentiation of the CAO reflex but not the increase in basal blood pressure. These results indicate that acetylcholine in the posterior hypothalamic nucleus serves as a neurotransmitter in a pathway which can potentiate the pressor response to carotid artery occlusion and thus modulate baroreceptor reflexes.     

Hippocampal Serotonin 5-HT1A Receptor Enhances Acetylcholine Release in Conscious Rats

Abstract: We investigated changes in the extracellular levels of acetylcholine (ACh) following local application of serotonergic agents to the dorsal hippocampus of freely moving rats by means of perfusion using a microdialysis technique. Perfusion of serotonin (5-HT; 10 μM, for 30 min at a rate of 3 μl/min), dissolved in Ringer's solution containing 10 μM eserine, showed no marked effect on the extracellular levels of ACh. 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 20 μM), a 5-HT_1A agonist, increased ACh levels, whereas 7-trifluoromethyl-4-(4-methyl-1 -piperazinyl)-pymoto[1,2-a]quinoxaline (CGS-12066B; 100 μM), a 5-HT_1B agonist, decreased it. Clomipramine (2 μM), an uptake inhibitor of 5-HT, had no effect on ACh levels. Following perfusion of 1-(2-methoxyphenyl)-4-[4- (2-phthalimido)butyl]piperazine (NAN-190; 10 μM), which is a selective 5-HT_1A antagonist, the effect of 8-OH-DPAT was totally abolished, whereas CGS-12066B decreased extracellular ACh levels. 5-HT, as well as Clomipramine, had a decreasing effect on ACh levels after pretreatment with NAN-190. These results indicate that the 5-HT_1A receptor, which exists in the dorsal hippocampus, enhances the spontaneous ACh release, and that the mechanism of serotonergic modulation of ACh release partly depends on both the stimulatory control via the 5-HT_1A receptor and the suppressive one via the 5-HT_1B receptor in the dorsal hippocampus of rats.     

Effect of acetylcholine on the tail artery reaction in rats

The response to acetylcholine (10(-5) g/ml) was studied on the rat tail artery perfused with Krebs buffer. Perfusion with acetylcholine produced vasodilation (by 69%) in arteries pre-constricted with transmural nerve stimulation. Atropine (10(-6) g/ml) blocked more than 95% of this response. Acetylcholine had a vasodilating effect on arteries pre-constricted with norepinephrine.     

Innervation of holothurian body wall muscle: inhibitory effects and localization of 5-HT

We investigated innervation to body wall muscles as well as distribution of 5-HT (serotonin) and its effects on longitudinal muscles of body wall (LMBW) of the sea cucumber Apostichopus japonicus. With serial sections we found neural branches and fibers extending from hyponeural part of radial nerve towards LMBW and circular muscles of body wall. With the aqueous aldehyde (Faglu) method yellow fluorescence indicating indolamines was observed in LMBW and in the mesentery connecting LMBW to the body wall. With indirect immunohistochemistry 5-HT-like immunoreactivity was observed in LMBW and in mesentery. These results strongly suggested that both LMBW and mesentery contained 5-HT. The effects of monoamine neurotransmitters were studied in LMBW. Putative neurotransmitters tested were 5-HT, adrenaline, noradrenaline, dopamine, and DOPA at the concentration of 10(-6) M. The application of 5-HT caused no contraction or relaxation, but it inhibited the contraction induced by 10(-6)-10(-5) M acetylcholine (ACh). Catecholamines were ineffective by themselves and had no effects on the contraction induced by ACh. The present histological, histochemical, and pharmacological studies strongly suggested that holothurian LMBW was innervated by inhibitory serotonergic neurons of the hyponeural nervous system.     

Presynaptic Control of the Synthesis and Release of Dopamine from Striatal Synaptosomes: A Comparison Between the Effects of 5-Hydroxytryptamine, Acetylcholine, and Glutamate

The effects of 5-HT and glutamate on dopamine synthesis and release by striatal synaptosomes were investigated and compared with the action of acetylcholine, which acts presynaptically on this system. 5-HT inhibited (28%) synthesis of [¹⁴C]dopamine from L-[U-¹⁴C]tyrosine, at 10-⁵M and above. This contrasts with the action of acetylcholine, which stimulated [¹⁴C]-dopamine synthesis by 24% at 10-⁴ M. Tissue levels of GABA were unaffected by either 5-HT or acetylcholine up to concentrations of 10-⁴ M. The inhibitory action of 5-HT (5 × 10^?5 M and 2 × 10^?4 M) on [¹⁹C]dopamine synthesis was completely abolished by methysergide (2 × 10^?6 M). Higher concentrations of methysergide (10^?4 M) or cyproheptadine (10^?5 M) inhibited [¹⁴C]dopamine synthesis by 28% and 25%, respectively, when added alone to synaptosomes. However, only methysergide prevented the further inhibition of synthesis caused by 5-HT. At concentrations of 2 × 10^?5 M and above, 5-HT stimulated [¹⁴C]dopamine release. This releasing action differed from that of acetylcholine, which occurred at lower concentrations (e.g., 10^?6 M). Methysergide (up to 10^?4 M) or cyproheptadine (2 × 10^?4 M) did not reduce the 5-HT (5 × 10^?5 M)-induced release of [¹⁴C]dopamine, but methysergide (10^?4 M) showed a potentiation (49%) of this increased release. The stimulatory effects of 5-HT (2 × 10^?5 M) and K⁺ (56 mM) on [¹⁴C]dopamine release were additive, indicating that two separate mechanisms were involved. However, when both agents were present the stimulatory effect of K⁺ (56 mM) on [¹⁴C]dopamine synthesis was not seen above the inhibitory effect of 5-HT. Glutamate (0.1-5 mM) did not affect [⁴C]dopamine release or its synthesis from L-[U-¹⁴C]tyrosine. It is concluded that 5-HT modulates the synthesis of dopamine in striatal nerve terminals through a presynaptic receptor mechanism, an action antagonised by methysergide. The releasing action of 5-HT apparently occurs through a separate mechanism which is also distinct from that involved in the response to K⁺ depolarisation.     

Serotonin-Elicited Amplification of Adenylate Cyclase Activity in Hippocampal Membranes from Adult Rat

The activity of the adenylate cyclase located in membranes prepared from hippocampus of adult rat can be stimulated by serotonin (5-HT) (Ka = 4 X 10(-7) M). The maximal effect is obtained with 10 microM 5-HT. Freezing of the tissue decreases the 5-HT stimulation; this stimulation is optimal in the presence of 82.5 mM Tris-maleate buffer (pH 7.4) and 50 microM GTP. The adenylate cyclase activity of membranes prepared from cortex, hypothalamus, and colliculi of adult rats is not significantly stimulated by 5-HT. Dopamine (DA) also stimulates adenylate cyclase located in hippocampal membranes; its effect can be blocked by haloperidol (10(-6) M), which fails to inhibit 5-HT stimulation. Moreover, p-chlorophenylalanine treatment for 2 weeks or selective lesion of 5-HT axons afferent to the hippocampus increases the Vmax of 5-HT stimulation, but fails to change that of DA stimulation. The 5-HT stimulation can be inhibited by metergoline, spiroperidol, and pizotyline (10(-6) M), but not by the same concentrations of mianserin, ketanserine, alprenolol, phenoxybenzamine, and mepyramine. The 5-HT stimulation of adenylate cyclase of hippocampal membranes can be mimicked by tryptamine, 5-methoxytryptamine, bufotenine, and to a lesser extent by LSD; N-methyltryptamine, N-methyltryptophan, and 5-hydroxytryptophan are inactive. Studies with kainic acid suggest that the 5-HT recognition site (5-HT1) linked to adenylate cyclase is located on the membrane of intrinsic hippocampal neurons.     

Properties of the membrane-bound acetylcholine receptor from cow brain nucleus caudatus

A new procedure for isolation of the membrane-bound acetylcholine receptor from cow brain N. caudatus is proposed. The procedure described includes tissue disruption by homogenization and ultrasonication, isolation of a crude membrane fraction and subsequent washing of the membranes with 1 M NaCl and 0,6 M KCl. The receptor yield is 40 nmoles per 1 g of tissue. The receptor is of a muscarine type. The K alpha values for the complexes with acetylcholine (3,8.10(-5) M), D,L-muscarine (3,7.10(-5) M), pilocarpine (3,7.10(-5) M), methylfurmethide (3,7.10(-5) M), atropine (2,5.10(-8) M and 1,2.10(-9) M) and platyphyllin (2,3.10(-7) M) were determined. The atropine binding within the pH range of 5 to 8 is weakly dependent on concentrations of Ca2+, Mg2+ and EDTA and EGTA sodium salts as well as on pH. Atropine binding is inhibited by high concentrations of NaCl. It is concluded that N. caudatus from cow brain is a convenient source for isolation of the muscarine acetylcholine receptor.     

Co-expression of the 5-HT3B serotonin receptor subunit alters the biophysics of the 5-HT3 receptor

Homomeric complexes of 5-HT(3A) receptor subunits form a ligand-gated ion channel. This assembly does not fully reproduce the biophysical and pharmacological properties of native 5-HT(3) receptors which might contain the recently cloned 5-HT(3B) receptor subunit. In the present study, heteromeric assemblies containing human 5-HT(3A) and 5-HT(3B) subunits were expressed in HEK 293 cells to detail the functional diversity of 5-HT(3) receptors. We designed patch-clamp experiments with homomeric (5-HT(3A)) and heteromeric (5-HT(3AB)) receptors to emphasize the kinetics of channel activation and desensitization. Co-expression of the 5-HT(3B) receptor subunit reduced the sensitivity for 5-HT (5-HT(3A) receptor: EC(50) 3 micro M, Hill coefficient 1.8; 5-HT(3AB) receptor: EC(50) 25 micro M, Hill coefficient 0.9) and markedly altered receptor desensitization. Kinetic modeling suggested that homomeric receptors, but not heteromeric receptors, desensitize via an agonist-induced open-channel block. Furthermore, heteromeric 5-HT(3AB) receptor assemblies recovered much faster from desensitization than homomeric 5-HT(3A) receptor assemblies. Unexpectedly, the specific 5-HT(3) receptor agonist mCPBG induced an open-channel block at both homomeric and heteromeric receptors. Because receptor desensitization and resensitization massively affect amplitude, duration, and frequency of synaptic signaling, these findings are evidence in favor of a pivotal role of subunit composition of 5-HT(3) receptors in serotonergic transmission.     

Roles of glutamatergic and serotonergic mechanisms in reflex control of the external urethral sphincter in urethane-anesthetized female rats

This study was conducted to examine reflex mechanisms that mediate urinary bladder and external urethral sphincter (EUS) coordination in urethane-anesthetized female Sprague-Dawley rats. We investigated the properties of EUS reflexes elicited by electrical stimulation of pelvic nerve afferent axons (pelvic-EUS reflex). The changes in the reflexes induced by bladder distension and administration of agonists or antagonists for glutamatergic or serotonergic receptors were examined. The reflexes consisted of an early response (ER, 18- to 22-ms latency) and a late, long-duration (>100-ms latency) response (LR), which consisted of bursts of activity at 20- to 160-ms interburst intervals. In a few experiments, a reflex with an intermediate (40- to 70-ms) latency was also identified. With the bladder empty, the ER, but not the LR, was detected in the majority of experiments. The LR was markedly enhanced when the bladder was distended. The ER remained, but the LR was abolished, after spinal cord transection at T8-T9. The ER and LR were significantly decreased 75 and 35%, respectively, by the N-methyl-D-aspartate receptor antagonist MK-801 (0.3 mg/kg iv), but only decreased 18 and 14%, respectively, by the alpha-amino-5-methylisoxazole-4-propionate receptor antagonist LY-215490 (3 mg/kg iv). The serotonin (5-HT1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (1 mg/kg iv) enhanced spontaneous EUS activity and the pelvic-EUS reflex. WAY-100635 (0.1-1 mg/kg iv), a 5-HT1A antagonist, reversed the effect of 8-hydroxy-2-(di-n-propylamino)-tetralin and suppressed EUS activity and the pelvic-EUS reflex. These results indicate that glutamatergic and serotonergic mechanisms are important in the reflex pathways underlying bladder- sphincter coordination in rats.