Mode of action of piperovatine, an insecticidal piperamide isolated from Piper piscatorum (Piperaceae), against voltage-gated sodium channels.

Piperamides, which are secondary metabolites in the genus Piper, have potent insecticidal activity, and have thus inspired the development of novel insecticides. In this study, piperovatine, a piperamide from Piper piscatorum (Piperaceae), was investigated using a two-electrode voltage clamp to clarify its detailed mode of action against voltage-gated sodium channels, a classic target. In Xenopus oocytes expressing voltage-gated sodium channels from German cockroach (Blattella germanica), piperovatine induced inward currents depending on repetitive openings. For instance, maximal currents were generated with 10 µM piperovatine following 100 trains of depolarizing pulses with frequency 25 Hz. Piperovatine also shifted the half-activation voltage after conditioning pulses from -35 mV to -45 mV. In addition, piperovatine-modified currents were correlated with not only the number of prior conditioning pulses but also the proportion of activated channels. Finally, piperovatine was found to stabilize voltage-gated sodium channels in the fast-inactivated state after opening, and inhibit transition to the slow-inactivated state. These results suggest that piperovatine preferably binds to activated channels and stabilizes voltage sensors at the conformation acquired during depolarization.

Neonatal domoic acid alters in vivo binding of [11C]yohimbine to α2-adrenoceptors in adult rat brain.

RATIONALE: Epilepsy is a debilitating seizure disorder that affects approximately 50 million people. Noradrenaline reduces neuronal excitability, has anticonvulsant effects and is protective against seizure onset. OBJECTIVE: We investigated the role of α2-adrenoceptors in vivo in a neonatal domoic acid (DOM) rat model of epilepsy. METHODS: We injected male Sprague-Dawley rats daily from postnatal day 8-14 with saline or one of two sub-convulsive doses, 20 µg/kg (DOM20) or 60 µg/kg (DOM60) DOM, an AMPA/kainate receptor agonist. The rats were observed in open field, social interaction and forced swim tests at day 50, 75 and 98, respectively. At ~120 days of age, four rats per group were injected and scanned with [11C]yohimbine, an α2-adrenoceptor antagonist, and scanned in a Mediso micro positron emission tomography (PET) scanner to measure α2-adrenoceptor binding. RESULTS: DOM60-treated rats spent more time in the periphery during the open field test and had a significant 26-33 % reduction in [11C]yohimbine binding in the hypothalamus, hippocampus and orbital prefrontal cortex compared to saline-treated rats. On the other hand, DOM20 rats had a significant 34-40 % increase in [11C]yohimbine binding in the hypothalamus, amygdala and entorhinal cortex compared to saline-treated rats, with no obvious behavioural differences. CONCLUSIONS: The current data clearly indicate that low concentrations of DOM given to rats in their second week of life induces long-term changes in α2-adrenoceptor binding in rat brain that may have relevance to the progression of an epilepsy phenotype.

Relaxant effects of a hydroalcoholic extract of Ruta graveolens on isolated rat tracheal rings.

BACKGROUND: Ruta graveolens L. (R. graveolens) is a medicinal plant employed in non-traditional medicines that has various therapeutic properties, including anthelmintic, and vasodilatory actions, among others. We evaluated the trachea-relaxant effects of hydroalcoholic extract of R. graveolens against potassium chloride (KCl)- and carbachol-induced contraction of rat tracheal rings in an isolated organ bath. RESULTS: The results showed that the airway smooth muscle contraction induced by the depolarizing agent (KCl) and cholinergic agonist (carbachol) was markedly reduced by R. graveolens in a concentration-dependent manner, with maximum values of 109 ± 7.9 % and 118 ± 2.6 %, respectively (changes in tension expressed as positive percentages of change in proportion to maximum contraction), at the concentration of 45 µg/mL (half-maximal inhibitory concentration IC50: 35.5 µg/mL and 27.8 µg/mL for KCl- and carbachol-induced contraction, respectively). Additionally, the presence of R. graveolens produced rightward parallel displacement of carbachol dose-response curves and reduced over 35 % of the maximum smooth muscle contraction. CONCLUSIONS: The hydroalcoholic extract of R. graveolens exhibited relaxant activity on rat tracheal rings. The results suggest that the trachea-relaxant effect is mediated by a non-competitive antagonistic mechanism. More detailed studies are needed to identify the target of the inhibition, and to determine more precisely the pharmacological mechanisms involved in the observed biological effects.

Homicide by Sch from a syringe-like dart ejected by a compound crossbow.

The compound crossbow can be used to eject syringe-like dart loaded with poisonous solution. Succinylcholine (Sch) is a short-acting neuromuscular blocker medically used to achieve complete relaxation of muscle for a good intubation condition. Without the help of an artificial respirator, intramuscular injection of a large dose of Sch can paralyze the respiratory muscle and result in the receiver's death. In this paper, we present the homicide case of a young male killed by Sch from a syringe-like dart ejected by a compound crossbow. The subcutaneous and muscular hemorrhages observed around the entry were more severe than that caused by a medical injection. Additionally, other autopsy results showed the external appearance of a pinhole, general asphyxia signs and pathological findings which were not characteristic. The discovery of a syringe-like dart at the scene is the critical clue and reason for analyzing for Sch, which is commonly used to load syringe-like dart to paralyze and steal dog in the countryside of China.

Relaxant effects of a hydroalcoholic extract of Ruta graveolens on isolated rat tracheal rings

BACKGROUND: Ruta graveolens L. (R. graveolens) is a medicinal plant employed in non-traditional medicines that has various therapeutic properties, including anthelmintic, and vasodilatory actions, among others. We evaluated the trachea-relaxant effects of hydroalcoholic extract of R. graveolens against potassium chloride (KCl)- and carbachol-induced contraction of rat tracheal rings in an isolated organ bath. RESULTS: The results showed that the airway smooth muscle contraction induced by the depolarizing agent (KCl) and cholinergic agonist (carbachol) was markedly reduced by R. graveolens in a concentration-dependent manner, with maximum values of 109 ± 7.9 % and 118 ± 2.6 %, respectively (changes in tension expressed as positive percentages of change in proportion to maximum contraction), at the concentration of 45 µg/mL (half-maximal inhibitory concentration IC50: 35.5 µg/mL and 27.8 µg/mL for KCl- and carbachol-induced contraction, respectively). Additionally, the presence of R. graveolens produced rightward parallel displacement of carbachol dose-response curves and reduced over 35 % of the maximum smooth muscle contraction. CONCLUSIONS: The hydroalcoholic extract of R. graveolens exhibited relaxant activity on rat tracheal rings. The results suggest that the trachea-relaxant effect is mediated by a non-competitive antagonistic mechanism. More detailed studies are needed to identify the target of the inhibition, and to determine more precisely the pharmacological mechanisms involved in the observed biological effects.

Effects of succinylcholine in an organotypic spinal cord-skeletal muscle coculture of embryonic mice.

Intoxication with organophosphorus compounds is an important clinical problem worldwide. Although the core treatments - atropine, oximes and diazepam - are defined, high case fatalities were reported for intoxication with organophosphorus insecticides. In particular the role of oximes is not completely understood since they might benefit only patients poisoned by specific pesticides or patients with moderate poisoning and few randomised trials of such poisoning have been performed. This justifies the need for new in vitro test-systems like cocultures of spinal cord and muscle tissue, which have been recently introduced. However this test-system is not yet fully characterized. In order to estimate the applicability of cocultures of spinal cord and muscle tissue their sensitivity to succinylcholine (di-acetylcholine), a depolarizing muscle relaxant in clinical use, was tested. The test-system evaluated in the current study showed sensitivity to succinylcholine with an EC50 as low as 1µM thereby being close to the EC50 value in adult human patients (2.6µM). Furthermore, action potential activity of spinal ventral horn neurons was not altered by succinylcholine. The latter observations strongly suggest that our preparation well predicts the qualitative and quantitative actions of novel drugs targeting the neuromuscular system in vivo. In summary, cocultures of spinal cord and muscle tissue seem to be a valid test-system for the development and investigation of new oximes. Moreover, practical aspects like transport over long distances to further laboratories, the opportunity to conduct long-term studies and the reduction of animal usage display further advantages of its use.

Glucose is an adequate energy substrate for the depolarizing action of GABA and glycine in the neonatal rat spinal cord in vitro.

In vitro studies have repeatedly demonstrated that the neurotransmitters γ-aminobutyric acid (GABA) and glycine depolarize immature neurons in many areas of the CNS, including the spinal cord. This widely accepted phenomenon was recently challenged by experiments showing that the depolarizing action of GABA on neonatal hippocampus and neocortex in vitro was prevented by adding energy substrates (ES), such as the ketone body metabolite dl-ß-hydroxybutyric acid (DL-BHB), lactate, or pyruvate to the artificial cerebrospinal fluid (ACSF). It was suggested that GABA-induced depolarizations in vitro might be an artifact due to inadequate energy supply when glucose is the sole energy source, consistent with the energy metabolism of neonatal rat brain being largely dependent on ESs other than glucose. Here we examined the effects of these ESs (DL-BHB, lactate, pyruvate) on inhibitory postsynaptic potentials (IPSPs) recorded from neonatal rat lumbar spinal cord motoneurons (MNs), in vitro. We report that supplementing the ACSF with physiologic concentrations of DL-BHB, lactate, or pyruvate does not alter the reversal potential of IPSPs (E(IPSP)). Only high concentrations of pyruvate hyperpolarized E(IPSP). In addition, the depolarizing action of GABA on primary afferent terminals was not affected by supplementing the ACSF with ES at physiologic concentrations. We conclude that depolarizing IPSPs in immature MNs and the primary afferent depolarizations are not caused by inadequate energy supply. Glucose at its standard concentration appears to be an adequate ES for the neonatal spinal cord in vitro.

Long-term evaluation of organophosphate toxicity and antidotal therapy in co-cultures of spinal cord and muscle tissue.

Victims of nerve agents basically require antidotal treatment. There is need for novel antidotes and for therapeutic procedures that are specifically adapted to these patients. To cope with this challenge, in vitro test systems which are easy to handle and allow for conducting long-term studies would be of great benefit. The present work introduces co-cultures of spinal cord and muscle tissue as ex vivo testing systems meeting these criteria. Cell cultures in which functional neuromuscular synapses formed ex vivo were prepared from embryonic mice. Spontaneous muscle activity was recorded by video microscopy. Muscle contractions involved intact neuromuscular transmission as indicated by the effect of succinylcholine, a muscle relaxant that completely abolished muscle activity. At a concentration of 0.75 µM the nerve agent VX reduced the frequency of spontaneous muscle contractions by about 75%. Subsequent application of obidoxime re-established muscle movements. After 24 h of antidotal treatment, muscle activity approached the level of sham-treated cultures and remained stable over the following week. In summary, co-cultures of spinal cord and muscle tissue are promising tools for evaluating the success of antidotal treatment following organophosphate intoxication over a period of at least seven days.

[Resistance to vecuronium induced muscle relaxation in a patient with mental retardation receiving phenytoin for chronic anticonvulsant therapy].

A 36-year-old woman weighing 31.7 kg with mental retardation was scheduled for dental treatment under general anesthesia. She had undergone anticonvulsant therapy (phenytoin, clonazepam, zonisamide) for years. Standard monitors and bispectral index (BIS) monitor were applied except for an accelomyography. Anesthesia was induced with propofol and vecuronium, and maintained with nitrous oxide in oxygen, with 1.5-2.0% end-tidal concentration of sevoflurane. Forty minutes after induction of anesthesia, spontaneous respiration (SR) started suddenly despite adequate depth of anesthesia (BIS value 35-40). Vecuronium 1 mg was administered and SR stopped immediately. After the event, however, SR started repeatedly and then additional vecuronium was administered every 30-40 minutes to stop SR until the end of the treatment. During the treatment, no factors (hypercapnia, hypoxemia, hyperthermia and so on) to shorten the muscle relaxation were observed. The treatment finished uneventfully She became awake rapidly and extubated. Post-extubation period was also uneventful. In this case, chronic phenytoin therapy induced resistance to vecuronium was highly suspected; however, since clonazepam and zonisamide have elevation effects on blood concentration of phenytoin, they might be also cofactors in resistance to vecuronium. Therefore, patients undergoing chronic anticonvulsant therapy should be paid more attention because they have resistance to neuromuscular blocking drugs.

Antinociceptive activity of atranorin in mice orofacial nociception tests.

Physicochemical characterization and antinociceptive and anti-inflammatory activities of atranorin (AT) extracted from Cladina kalbii Ahti in formalin- and capsaicin-induced orofacial pain and anti-inflammatory tests in rodents were studied. Physicochemical characterization showed that AT has the general formula C19H18O8. Male Swiss mice were pretreated with AT (100, 200, and 400 mg/kg, i.p.), morphine (3 mg/kg, i.p.), or vehicle (0.9% saline with two drops of 0.2% Tween 80) before formalin (20 microl, 2%) or capsaicin (20 microl, 2.5 microg) were injected into the right vibrissa. Our results showed that i.p. treatment with AT displayed marked inhibitory effects in different orofacial pain tests in mice. AT (400 mg/kg, i.p.) was effective in reducing the nociceptive face-rubbing behavioural response in both phases of the formalin test, which was also naloxone-sensitive. Additionally, AT produced a significant antinociceptive effect at all doses in the capsaicin test. Such results were unlikely to be provoked by motor abnormality, since AT-treated mice exhibited no performance alteration on the rota rod apparatus. AT exhibited significant anti-inflammatory activity in the acute model of inflammation (leukocyte migration to the peritoneal cavity), carrageenan- and arachidonic acid-induced hind paw edema in rats. Additionally, AT exhibited a dose-dependent antioxidant activity in vitro, as assessed by total radical-trapping antioxidant parameter and total antioxidant reactivity assays. All these findings suggest that AT might represent an important tool for the management of orofacial pain and/or inflammatory disorders.

Depolarizing stimuli cause persistent and selective loss of orexin in rat hypothalamic slice culture.

A hypothalamic neuropeptide orexin (hypocretin) is a critical regulator of physiological processes including sleep/wakefulness and feeding. Using organotypic slice culture of rat hypothalamus, we found that exposure to elevated extracellular concentration of K(+) (+10-30 mM) for 24-72h led to a substantial decrease in the number of neurons immunoreactive for orexin and a co-existing neuropeptide dynorphin-A. In contrast, the same treatment affected neither the number of melanin-concentrating hormone-immunoreactive neurons nor the number of total neurons. A substantial decrease of orexin-immunoreactive neurons was also induced by 72h treatment with 1-10 microM veratridine, a Na(+) channel activator. The effect of elevated K(+) was only partially reversible, and that of veratridine was virtually irreversible, although the decrease in orexin immunoreactivity was not associated with signs of cell damage assessed by propidium iodide uptake and Hoechst 33342 nuclear staining. In addition, the level of preproorexin mRNA did not decrease during treatment with elevated K(+) or veratridine. After treatment with elevated K(+) and veratridine, c-Fos immunoreactivity appeared in orexin-immunoreactive neurons but not in melanin-concentrating hormone-immunoreactive neurons, suggesting selective excitation of orexin neurons. However, the amount of orexin released extracellularly was paradoxically decreased by treatment with elevated K(+) and veratridine. Overall, these characteristics of orexin neurons may be taken into consideration to understand the behaviors of these neurons under physiological and pathophysiological conditions.

Synaptic transmission and plasticity in an active cortical network.

BACKGROUND: The cerebral cortex is permanently active during both awake and sleep states. This ongoing cortical activity has an impact on synaptic transmission and short-term plasticity. An activity pattern generated by the cortical network is a slow rhythmic activity that alternates up (active) and down (silent) states, a pattern occurring during slow wave sleep, anesthesia and even in vitro. Here we have studied 1) how network activity affects short term synaptic plasticity and, 2) how synaptic transmission varies in up versus down states. METHODOLOGY/PRINCIPAL FINDINGS: Intracellular recordings obtained from cortex in vitro and in vivo were used to record synaptic potentials, while presynaptic activation was achieved either with electrical or natural stimulation. Repetitive activation of layer 4 to layer 2/3 synaptic connections from ferret visual cortex slices displayed synaptic augmentation that was larger and longer lasting in active than in silent slices. Paired-pulse facilitation was also significantly larger in an active network and it persisted for longer intervals (up to 200 ms) than in silent slices. Intracortical synaptic potentials occurring during up states in vitro increased their amplitude while paired-pulse facilitation disappeared. Both intracortical and thalamocortical synaptic potentials were also significantly larger in up than in down states in the cat visual cortex in vivo. These enhanced synaptic potentials did not further facilitate when pairs of stimuli were given, thus paired-pulse facilitation during up states in vivo was virtually absent. Visually induced synaptic responses displayed larger amplitudes when occurring during up versus down states. This was further tested in rat barrel cortex, where a sensory activated synaptic potential was also larger in up states. CONCLUSIONS/SIGNIFICANCE: These results imply that synaptic transmission in an active cortical network is more secure and efficient due to larger amplitude of synaptic potentials and lesser short term plasticity.

Amino acid release at the spinomedullary junction after inflammation of the TMJ region in male and female rats.

Temporomandibular joint (TMJ) disorders are painful conditions that are more prevalent in women than men. This study tested the hypothesis that acute inflammation of the TMJ region evoked sex-related changes in amino acid transmitter concentrations at the trigeminal subnucleus/upper cervical cord (Vc/C2) junction, the major terminal zone for TMJ sensory afferents. Microdialysis samples were collected in male, intact and ovariectomized (OvX) female rats after injection of mustard oil into the TMJ region (TMJ-MO) under barbiturate anesthesia. Males displayed increases in glutamate, aspartate and serine at 5 min and secondary increases 40-45 min after TMJ-MO. Intact and OvX females given low dose estrogen (LE2) displayed increases in glutamate, aspartate and serine at 5 min but no secondary increase at 40 min, while OvX females given high dose estrogen (HE2) revealed no increases after TMJ-MO. Glycine increased 20 min after TMJ-MO in males and cycling females, but not in OvX rats. Perfusion of high potassium through the probe evoked similar increases in glutamate, aspartate and glycine in all groups. In separate experiments, perfusion of the glutamate-aspartate reuptake inhibitor, L-trans-2,4-pyrrolidine dicarboxylate (PDC), through the probe caused a prompt elevation in glutamate that was significantly greater in HE2 than LE2 females or males. These results suggested sex hormone status affects glutamatergic neurotransmission at the Vc/C2 junction by acting, in part, through modulation of glutamate reuptake. Altered amino acid transmitter release and/or availability at the Vc/C2 junction may contribute to differential processing of sensory input from the TMJ region in males and females.

Increase of insulin secretion by ginsenoside Rh2 to lower plasma glucose in Wistar rats.

1. The aim of the present study was to clarify the role of ginsenoside Rh2 as the active compound in Panax ginseng root for lowering plasma glucose in animals. 2. Plasma glucose was assessed using the glucose oxidase method. Changes in the levels of insulin and C-peptide in plasma were measured by ELISA using commercially available kits. 3. After intravenous injection into fasting Wistar rats for 60 min, ginsenoside Rh2 (0.1-1.0 mg/kg) decreased plasma glucose in a dose-dependent manner. In parallel with the decrease in plasma glucose, increases in plasma insulin levels, as well as plasma C-peptide, were observed in rats receiving the same treatment. These effects of Rh2 were reversed by atropine (0.1-1.0 mg/kg), but not affected by the ganglionic nicotinic antagonists pentolinium or hexamethonium (both at 7.5 mg/kg). 4. Disruption of synaptically available acetylcholine (ACh) using an inhibitor of choline uptake (hemicholinium-3; 1-10 microg/kg) or an inhibitor of vesicular ACh transport (vesamicol; 1.5-3.5 mg/kg) abolished the actions of Rh2. In addition, physostigmine (0.1-0.5 mg/kg), at a concentration sufficient to inhibit acetylcholinesterase, enhanced the actions of the ginsenoside Rh2. Thus, mediation of the effects of Rh2 to enhance insulin secretion by ACh released from nerve terminals can be considered. 5. Blockade of the increase in plasma insulin and the plasma glucose-lowering action of Rh2 by 4-diphenylacetoxy-N-methylpiperdine methiodide (4-DAMP; 5-10 microg/kg) indicates the participation of muscarinic M(3) receptors. Increases in plasma C-peptide level induced by Rh2 were also sensitive to 4-DAMP. 6. The results of the present study suggest that ginsenoside Rh2 has the ability to increase insulin secretion as a result of the release of ACh from nerve terminals that then stimulates muscarinic M(3) receptors in pancreatic cells. This finding shows the mechanism for the plasma glucose-lowering action of ginsenoside Rh2, that is one of the major principles contained in P. ginseng root. Thus, ginsenoside Rh2 may be applied as an adjuvant for the management of diabetes.

The effect of vecuronium is enhanced by a large rather than a modest dose of gentamicin as compared with no preoperative gentamicin.

UNLABELLED: We compared the effect of two doses of gentamicin versus no gentamicin (NG) given before surgery on the neuromuscular relaxant effect of vecuronium. Seventy patients (intraabdominal procedures) were randomly allocated to receive preoperative large-dose (4 mg/kg) gentamicin (LD), a modest dose (1.2 mg/kg) of gentamicin (MD), or NG. No more than one dose of gentamicin was given before the vecuronium administration. Serum gentamicin levels, the time for 25% recovery of the first twitch in the train-of-four after a bolus of vecuronium, and the time from cessation of the vecuronium infusion to extubation of the trachea were estimated. Serum gentamicin levels were higher (P < 0.001) for LD than MD. The time for 25% recovery of the first twitch after the vecuronium bolus was slightly longer with LD than MD (P = 0.06) and longer in LD than NG (P = 0.001) (42.9 +/- 23.6 min versus 36.2 +/- 17 min and 27.4 +/- 9 min, respectively). The time to extubation was similar with LD and MD and longer for LD than NG (P = 0.008) (34.7 +/- 19.2 min versus 27.4 +/- 19.3 min and 19.4 +/- 10.1 min, respectively). The differences in these times were insignificant between MD and NG. Gentamicin administered as a LD rather than MD enhanced the neuromuscular blockade of vecuronium as compared with NG given before surgery. IMPLICATIONS: We demonstrated that the neuromuscular relaxant effect of vecuronium is enhanced by a large (4 mg/kg) rather than a modest (1.2 mg/kg) dose of gentamicin as compared with no gentamicin given before surgery.

Dissociation of human thalamic and cortical SEP gating as revealed by intrathalamic recordings under muscle relaxation.

'Gating' refers to a reduction of cortical somatosensory evoked potentials (SEP) under multiple simultaneous afferent inputs. This study used the opportunity for intrathalamic recordings in patients with movement disorders to clarify to what extent cortical SEP gating is preceded by thalamic gating. Recordings were performed in 10 patients, narcotised by intravenous propofol when receiving implantation of a therapeutic deep brain stimulator system. SEP were elicited by an 8.1-Hz median nerve stimulation at twice motor threshold and were recorded simultaneously from both intrathalamic and scalp electrodes before and after the application of the depolarising muscle blocker succinylcholine which eliminated both the background muscular tone and the repetitive muscle twitches caused by the median nerve stimulation. Peripheral compound action potentials recorded at the upper arm remained unchanged after complete muscle relaxation, proving a continuously effective nerve stimulation. In contrast, the primary cortical SEP component (N20) was significantly increased under succinylcholine (+17%). This cortical release from gating was not paralleled, however, by an increased thalamic response; rather, the primary thalamic response (P16) showed a slight (-9%) but highly significant amplitude reduction. As the recordings were performed in narcotised patients, any potentially variable attentional bias on part of the subjects can be excluded as confounding factor when comparing the two experimental conditions with vs. without reafferent somatosensory inflow. Thus, given the high signal-to-noise ratio of intrathalamically recorded SEP, the present study shows a distinct thalamo-cortical dissociation with the primary somatosensory cortex representing the predominant level exhibiting SEP gating.

Release of acetylcholine by Die-Huang-Wan to enhance insulin secretion for lowering plasma glucose in Wistar rats.

We have recently observed that Die-Huang-Wan has an ability to stimulate the secretion of insulin to decrease the plasma glucose levels in normal rats. In the present study, this effect of Die-Huang-Wan was reversed by the general muscarinic antagonists atropine and scopolamine, but not affected by the ganglionic nicotinic antagonist pentolinium or hexamethonium. Moreover, disruption of synaptically available acetylcholine using an inhibitor of choline uptake, hemicholinium-3, or vesicular acetylcholine transport, vesamicol, abolished the actions induced by Die-Huang-Wan. Mediation of acetylcholine released from nerve terminals by this product can thus be considered. Also, physostigmine at concentration sufficient to inhibit acetylcholinesterase enhanced the effect of Die-Huang-Wan. Blockade of the increase of plasma insulin and plasma glucose lowering action of Die-Huang-Wan by 4-diphenylacetoxy-N-methylpiperdine methiodide (4-DAMP) indicated the mediation of muscarinic M3 receptors. The results suggest that Die-Huang-Wan may enhance the release of acetylcholine from nerve terminals to stimulate the muscarinic M3 receptors for augmenting insulin release to produce plasma glucose lowering action.

Effects of phytoestrogen genistein on delayed after depolarization and triggered activity induced by ouabain in guinea pig papillary muscles.

The purpose of this study was to investigate the effects of phytoestrogen genistein (GST) on delayed after depolarization (DAD) and triggered activity (TA) induced by ouabain in guinea pig papillary muscles. Action potentials (APs) were recorded from the guinea pig papillary muscles with standard glass microelectrode technique. The results are as follows: (1) DAD and TA induced by ouabain (1 micromol/L) were markedly inhibited by pretreatment with GST (10, 50, 100 micromol/L) in a concentration-dependent manner. (2) NG-nitro-L-arginine methyl ester (L-NAME, 1 mmol/L), an NO synthase inhibitor, failed to affect the above effects of GST. (3) 5 micromol/L 17beta-estradiol (E2) or 10 micromol/L GST alone showed no effects on DAD and TA, whereas GST combined with E(2) at the same doses exerted significant inhibitory effects on DAD and TA. Since GST is known to reduce the calcium influx, it is suggested that GST might have antiarrhythmic effects, possibly by reducing calcium influx. The antiarrhythmic effects of GST may contribute to its cardioprotective action.

Sympathetic modulation of muscle spindle afferent sensitivity to stretch in rabbit jaw closing muscles.

Previous reports showed that sympathetic stimulation affects the activity of muscle spindle afferents (MSAs). The aim of the present work is to study the characteristics of sympathetic modulation of MSA response to stretch: (i) on the dynamic and static components of the stretch response, and (ii) on group Ia and II MSAs to evaluate potentially different effects. In anaesthetised rabbits, the peripheral stump of the cervical sympathetic nerve (CSN) was stimulated at 10 impulses s(-1) for 45-90 s. The responses of single MSAs to trapezoidal displacement of the mandible were recorded from the mesencephalic trigeminal nucleus. The following characteristic parameters were determined from averaged trapezoidal responses: initial frequency (IF), peak frequency at the end of the ramp (PF), and static index (SI). From these, other parameters were derived: dynamic index (DI = PF - SI), dynamic difference (DD = PF - IF) and static difference (SD = SI - IF). The effects of CSN stimulation were also evaluated during changes in the state of intrafusal muscle fibre contraction induced by succinylcholine and curare. In a population of 124 MSAs, 106 units (85.4 %) were affected by sympathetic stimulation. In general, while changes in resting discharge varied among different units (Ia vs. II) and experimental conditions (curarised vs. non-curarised), ranging from enhancement to strong depression of firing, the amplitude of the response to muscle stretches consistently decreased. This was confirmed and detailed in a quantitative analysis performed on 49 muscle spindle afferents. In both the non-curarised (23 units) and curarised (26 units) condition, stimulation of the CSN reduced the response amplitude in terms of DD and SD, but hardly affected DI. The effects were equally present in both Ia and II units; they were shown to be independent from gamma drive and intrafusal muscle tone and not secondary to muscle hypoxia. Sympathetic action on the resting discharge (IF) was less consistent. In the non-curarised condition, IF decreased in most Ia units, while in II units decreases and increases occurred equally often. In the curarised condition, IF in group II units mostly increased. The results have important functional implications on the control of motor function in a state of 'high' sympathetic activity, like excessive stress, as well as in certain pathological conditions such as sympathetically maintained pain.