Haruo Kobayashi

Inhibition of Neurotransmission by Peptides Containing the Synaptic Protein Interaction Site of N-Type Ca2+ Channels

N-type Ca2+ channels bind directly to the synaptic core complex of VAMP/synaptobrevin, syntaxin, and SNAP-25. Peptides containing the synaptic protein interaction (synprint) site caused dissociation of N-type Ca2+ channels from the synaptic core complex. Introduction of synprint peptides into presynaptic superior cervical ganglion neurons reversibly inhibited synaptic transmission. Fast EPSPs due to synchronous transmitter release were inhibited, while late EPSPs arising from asynchronous release following a train of action potentials were increased and paired-pulse facilitation was increased. The corresponding peptides from L-type Ca2+ channels had no effect, and the N-type peptides had no effect on Ca2+ currents through N-type Ca2+ channels. These results are consistent with the hypothesis that binding of the synaptic core complex to presynaptic N-type Ca2+ channels is required for Ca2+ influx to elicit rapid, synchronous neurotransmitter release.

Myosin II is involved in transmitter release at synapses formed between rat sympathetic neurons in culture

The presynaptic function of myosin II was studied at cholinergic synapses formed between rat superior cervical ganglion neurons in culture. Immunofluorescent staining showed that myosin II was colocalized with synaptophysin at the presynaptic nerve terminals. Antimyosin II antibody introduced into presynaptic neurons inhibited synaptic transmission. Transmission was also inhibited in a dose-dependent manner by two inhibitors of myosin light chain kinase: a peptide, SM-1, and an organic inhibitor, wortmannin. The inhibition produced by these agents was dependent on presynaptic activity. Extracellularly applied wortmannin also blocked synaptic transmission, but its effects were slower in onset. Wortmannin also decreased postsynaptic potentials and post-tetanic potentiation in intact superior cervical ganglia. These results suggest a model in which myosin light chain kinase phosphorylates myosin, and the resultant change in actin-myosin interactions is involved in neurotransmitter release.

Effects of arsenic on cholinergic parameters in brain in vitro

The effects of sodium arsenite (arsenite) on the cholinergic system in the brain of the mouse were investigated in vitro and compared with those of N-ethylmaleimide (NEM) and iodoacetate, both of which are alkylating sulfhydryl reagents. Arsenite, at concentrations greater than 10(-4) M, inhibited depolarized and nondepolarized release of acetylcholine (ACh) from cerebral slices, the synthesis of ACh in the slices, high-affinity uptake of choline into synaptosomes and activity of acetylcholinesterase (AChE). On the other hand, arsenite potentiated dose-dependently the activity of choline acetyltransferase (ChAT). N-Ethylmaleimide and iodoacetate showed inhibitory effects similar to those of arsenite. However, some exceptions were that N-ethylmaleimide did not have any effect on the nondepolarized release of ACh while iodoacetate had no effect on high affinity uptake of choline and activity of AChE. In contrast to arsenite, N-ethylmaleimide and iodoacetate inhibited the activity of ChAT. Neither of arsenite, N-ethylmaleimide nor iodoacetate showed any effect on the binding of [3H]quinuclidinyl benzilate to muscarinic ACh receptors. Although arsenite is thought to inhibit the cholinergic system in brain in vivo, its potentiating effect on ChAT and inhibition of AChE may reduce this harmful effect.

Presynaptic inhibition by noradrenaline of the EPSC evoked in neonatal rat sympathetic preganglionic neurons

Visually identified and electrophysiologically characterized sympathetic preganglionic neurons (SPNs) were recorded using the whole-cell voltage clamp technique in slices of neonatal rat spinal cord. Monosynaptic excitatory postsynaptic currents (EPSCs) were evoked by electrical stimulation of the nucleus intercalatus in the presence of strychnine (5 microM) and bicuculline (10 microM). These EPSCs were abolished by the antagonist of AMPA-type glutamate receptors, 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX; 10 microM). Bath applied noradrenaline (NA; 0.5-50 microM) dose-dependently and reversibly decreased by up to around 60% the amplitude of the EPSC, without affecting the holding current. The EPSC depression by NA was not accompanied by a change in EPSC reversal potential (around +5 mV), nor were inward currents generated by pressure application of glutamate affected by NA application. A comparable degree of EPSC depression was also seen with the alpha2-adrenoceptor agonist clonidine (5 microM), and the alpha2A-agonist oxymetazoline (5 microM), while the alpha1-agonist phenylephrine (100 microM) caused only a 22% depression. The EPSC depression caused by NA (10 microM) was completely antagonized by either the alpha-antagonist phentolamine (10 microM) or the alpha2-antagonist idazoxan (2 microM). Conversely, the beta-adrenoceptor antagonist popranolol (5 microM), and the alpha1-, alpha2B- and alpha2C-antagonist prazosin (2 microM) were without effect. These results indicate that activation of presynaptic alpha2A-adrenoceptors on inputs to SPNs decreases glutamate release.

Cholinergic system of brain tissue in rats poisoned with the organophosphate, 0,0-dimethyl 0-(2,2-dichlorovinyl) phosphate

The cholinergic system of the brain was investigated in rats acutely poisoned with the organophosphate, 0,0-dimethyl 0-(2,2-dichlorovinyl) phosphate (DDVP), (6 mg/kg, sc, with saline as a control). The amounts of three fractions of acetylcholine (ACh)--free (extraterminal), labile-bound (intraterminal/cytoplasmic), and stable-bound (intraterminal/vesicular)--increased in the rats over a period of 5 to 60 min after injection of DDVP, showing peaks which were 2.45, 1.82, and 1.4 times as high as the respective control amounts. No difference was seen in the amount of any fraction of ACh between treated and control rats killed 3 and 24 hr after injection. Acetylcholinesterase (AChE) activity decreased to between 12 and 43% of the control over a period of 5 to 180 min and recovered almost completely within 24 hr after injection. No appreciable changes were seen in either spontaneous or potassium-induced ACh release in brain tissue slices obtained from rats treated with DDVP. ACh synthesis in slices was suppressed significantly 20 min, but not 24 hr, after injection of DDVP. In the brain crude synaptosomal preparation, high-affinity choline uptake, which is generally thought to be a rate-limiting step for ACh synthesis, was suppressed 20 min after DDVP. No appreciable changes were seen in high-affinity choline uptake at 24 hr low-affinity choline uptake, and choline acetyltransferase activity after injection of DDVP. These results suggest that ACh synthesis and high-affinity choline uptake may be in a suppressed state when ACh concentration, especially intraterminal ACh, is increased and AChE activity is decreased in the brain cholinergic system of rats poisoned with DDVP. The increase in the intraterminal ACh may be due to an inhibition of AChE activity at this site and/or a re-uptake of ACh in the synaptic cleft, not to an inhibition of ACh release or an increase in ACh synthesis.

Effect of methylmercury on brain acetylcholine concentration and turnover in mice

Effects of methylmercury chloride (MMC) on regional acetylcholine (ACh) concentrations and turnover in the mouse brain were studied and compared with those of 3′-chloro-4-stilbazole (CS) and of hemicholinium-3 (HC-3). A long-term treatment with MMC (5 mg Hg/kg/day) induced nervous signs and decreased ACh in striatum and cerebral cortex, and conversion ratios of [14C]ACh in cerebellum, striatum, and cerebral cortex of mice. A single administration of CS (200 mg/kg) decreased ACh in the cortex and the conversion ratios in the three brain regions. An intracerebral injection of HC-3 (100 μg/kg) also decreased ACh and the conversion ratios in striatum and cortex. It is postulated that ACh concentration and ACh turnover rate in brain of methylmercury-poisoned animals may be reduced.

Peptidergic and nitrergic inhibitory neurotransmissions in the hamster jejunum: regulation of vasoactive intestinal peptide release by nitric oxide

Regulation of vasoactive intestinal peptide (VIP) release by nitric oxide (NO) was investigated in the hamster jejunum. Electrical field stimulation and applied NO (3-100 microM) evoked biphasic hyperpolarizations consisting of an initial transient hyperpolarizing component followed by a second more slowly developing component (late component). The NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (200 microM) abolished the biphasic inhibitory junction potential evoked by electrical field stimulation. The NO scavenger oxyhemoglobin (50 microM) and the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ; 10 microM) abolished both components of the inhibitory junction potentials and the NO-induced hyperpolarizations. VIP(6-28) (1 microM), which abolished VIP (3 microM)-induced hyperpolarizations, also inhibited the late components of the inhibitory junction potentials and the NO-induced hyperpolarizations. ODQ inhibited VIP release and cAMP production by electrical field stimulation and NO application. N(6)-2,0-Dibutyryladenosine 3,5-cyclic monophosphate (0.1-3 mM) caused a membrane hyperpolarization. These results suggest that NO may stimulate VIP release from enteric nerves in the hamster jejunum. In addition, we propose that NO and NO-stimulated VIP contribute to the early and late components of the inhibitory junction potentials, respectively, in the circular smooth muscle cells of the hamster jejunum.

A novel muscarinic receptor antagonist AF-DX 116 differentially blocks slow inhibitory and slow excitatory postsynaptic potentials in the rabbit sympathetic ganglia

Muscarinic, slow postsynaptic potentials (s-epsp and s-ipsp) in the rabbit superior cervical ganglia were shown to be differentially depressed by a novel cardioselective M2-type antagonist AF-DX 116: it antagonized the s-ipsp with IC50 value of 1.5 X 10(-7) M, which is 16-fold more potent in depressing the s-ipsp than the s-epsp. A hyperpolarizing component in the biphasic potential changes induced by a muscarinic agonist, methacholine, was selectively eliminated by this antagonist. AF-DX 116 was thus shown to be an useful tool for discriminating the M2-type muscarinic responses from those of M1-type in the nervous system.

An electrophysiological study of excitatory purinergic neuromuscular transmission in longitudinal smooth muscle of chicken anterior mesenteric artery

1 The object of the present study was to clarify the neurotransmitters controlling membrane responses to electrical field stimulation (EFS) in the longitudinal smooth muscle cells of the chicken anterior mesenteric artery. 2 EFS (5 pulses at 20u2003Hz) evoked a depolarization of amplitude 19.7±2.1u2003mV, total duration 29.6±3.1u2003s and latency 413.0±67.8u2003ms. This depolarization was tetrodotoxin (TTX)‐sensitive and its amplitude was partially decreased by atropine (0.5u2003μM); however, its duration was shortened by further addition of prazosin (10u2003μM). 3 Atropine/prazosin‐resistant component was blocked by the nonspecific purinergic antagonist, suramin, in a dose‐dependent manner, indicating that this component is mediated by the neurotransmitter adenosine 5′‐triphosphate (ATP). 4 Neither desensitization nor blocking of P2X receptor with its putative receptor agonist α,β‐methylene ATP (α,β‐MeATP, 1u2003μM) and its antagonist pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonic (PPADS, up to 50u2003μM), had significant effect on the purinergic depolarization. In contrast, either desensitization or blocking of P2Y receptor with its putative agonist 2‐methylthioATP (2‐MeSATP, 1u2003μM) and its antagonist Cibacron blue F3GA (CBF3GA, 10u2003μM) abolished the purinergic depolarization, indicating that this response is mediated through P2Y but not P2X receptor. 5 The purinergic depolarization was inhibited by pertussis toxin (PTX, 600u2003ngu2003ml−1). Furthermore, it was significantly inhibited by a phospholipase C (PLC) inhibitor, U‐73122 (10u2003μM), indicating that the receptors involved in mediating the purinergic depolarization are linked to a PTX‐sensitive G‐protein, which is involved in a PLC‐mediated signaling pathway. 6 Data of the present study suggest that the EFS‐induced excitatory membrane response occurring in the longitudinal smooth muscle of the chicken anterior mesenteric artery is mainly purinergic in nature and is mediated via P2Y purinoceptors.

Protein kinase C activators mimic the M2-muscarinic receptor-mediated effects on the action potential in isolated sympathetic neurons of rabbits.

Protein kinase C activators 1,2-oleoylacetylglycerol (OAG, 0.5-50 microM), a synthetic diacylglycerol analog, and phorbol-12,13-dibutyrate (Pb(Bu)2, 0.016-1.6 microM) depressed the calcium (Ca)-dependent components of action potentials in isolated superior cervical ganglion cells of rabbits. Similar depressions were elicited when the M2-muscarinic receptors were activated. This muscarinic modification of the action potential was obscured after the perfusion with protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7, 50 microM). It seems that protein kinase C is an intermediator between the M2-muscarinic receptors and the Ca channels regulating the firing rate of the postganglionic cells.