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Significance: It has been reported that children with attention-deficit hyperactivity disorder (ADHD) have impairment in the recognition of angry but not of happy facial expressions, and they show atypical cortical activation patterns in response to facial expressions. However, little is known about neural mechanisms underlying the impaired recognition of facial expressions in school-aged children with ADHD and the effects of acute medication on their processing of facial expressions. Aim: We aimed to investigate the possibility that acute administration of methylphenidate (MPH) affects processing of facial expressions in ADHD children. Approach: We measured the hemodynamic changes in the bilateral temporo-occipital areas of ADHD children observing the happy and angry facial expressions before and 1.5 h after MPH or placebo administration in a randomized, double-blind, placebo-controlled, crossover design study. Results: We found that, regardless of medication, happy expressions induced increased oxyhemoglobin (oxy-Hb) responses in the right inferior occipital region but not in the superior temporal region. For angry expressions, oxy-Hb responses increased after MPH administration, but not after placebo administration, in the left inferior occipital area, whereas there was no significant activation before MPH administration. Conclusions: Our results suggest that (1) ADHD children consistently recruit the right inferior occipital regions to process happy expressions and (2) MPH administration to ADHD children enhances cortical activation in the left inferior occipital regions when they process angry expressions.

Objective: We aimed to clarify the validity of fNIRS measurement for assessing the methylphenidate (MPH) induced neuropharmacological effect in medication-naive children with attention deficit hyperactivity disorder (ADHD) with or without comorbid autism spectrum disorder (ASD), thereby providing the first evidence for differing neurofunctional pathology between the two groups. Methods: We monitored the effects of a single acute clinical dose of MPH on the cortical hemodynamics of 32 medication-naive ADHD children (ADHD with ASD n=11 and ADHD without ASD n=21) performing a go/no-go task before and 1.5 h after MPH or placebo administration, using a randomized, double-blind, placebo-controlled, crossover design. In addition, all subjects’ symptoms were monitored using the Japanese version of the ADHD Rating Scale-IV (ADHDRS- IV-J) before and one month after MPH administration. Results: Medication-naive ADHD children without ASD exhibited reduced right prefrontal activation. MPH medication elicited significant right prefrontal activation except in comparative conditions associated with the placebo effect. Medication-naive ADHD children with ASD exhibited marginal activation before the first day of medication and reduced activation after MPH medication in the right prefrontal area. While the neuropharmacological effects of MPH were different, ADHD-RS-IV-J scores were significantly improved one month after MPH administration in ADHD groups both with and without ASD. Conclusion: Taken together, these results suggest that ADHD with ASD is characterized by a different underlying neurofunctional pathology for inhibition control than that without ASD. To our knowledge, this is the first comparative neuroimaging study between ADHD patients with and without comorbid ASD.

Gender differences in psychiatric disorders are considered to be associated with the serotonergic (5-HTergic) system; however the underlying mechanisms have not been clearly elucidated. In this study, possible involvement of the median raphe nucleus (MRN)-hippocampus 5-HTergic system in gender-specific emotional regulation was investigated, focusing on synaptic plasticity in rats. A behavioral study using a contextual fear conditioning (CFC) paradigm showed that the females exhibited low anxiety-like behavior. Extracellular 5-HT levels in the hippocampus were increased by CFC only in the males. Long-term potentiation (LTP) in the hippocampal CA1 field was suppressed after CFC in the males, which was mimicked by the synaptic response to MRN electrical stimulation. In the MRN, 5-HT immunoreactive cells significantly increased in the females compared with those in the males. Pretreatment with the 5-HT1A receptor agonists tandospirone (10 mg/kg, i.p.) and 8-OH DPAT (3 mg/kg, i.p.) significantly suppressed LTP induction in the males. Synaptic responses to CFC and 5-HT1A receptor interventions were not observed in the females. These results suggest that the metaplastic 5-HTergic mechanism via 5-HT1A receptors in the MRN-hippocampus pathway is a key component for gender-specific emotional regulation and may be a cause of psychiatric disorders associated with vulnerability or resistance to emotional stress. Keywords:: gender difference, serotonin, hippocampus, long-term potentiation, serotonin 5-HT1A receptor

Ketamine, a noncompetitive N-methyl-D-aspartate receptor antagonist, has been used for the treatment of cancer pain as an analgesic adjuvant to opioids. However, ketamine is known to produce psychotomimetic side effects including cognitive impairments under a high-dose situation, presumably as the result of cortical dysfunction. Here, we investigated whether low-dose ketamine was useful as an analgesic adjuvant to morphine for pain control, focusing on frontocortical function.To assess the analgesic effects of ketamine with or without morphine, we performed behavioral and histochemical experiments, using the hot plate test and c-Fos expression analysis in rats. The effect on cortical function was also determined by prepulse inhibition (PPI) of the acoustic startle and evoked potentials in the hippocampal CA1-medial prefrontal cortex (mPFC) synapses as measures of synaptic efficacy.Coadministration of ketamine as a subanalgesic dose significantly enhanced intraperitoneal morphine-induced antinociceptive response, which was measured as the increased reaction latency in the hot plate test. In addition, the noxious thermal stimulus-induced c-Fos expression in the ventrolateral periaqueductal gray matter was significantly suppressed by concomitant ketamine and morphine. In contrast, the subanalgesic dose of ketamine did not impair PPI and synaptic efficacy in the mPFC.The present results indicate that the morphine-induced analgesic effect is enhanced by a concomitant subanalgesic dose of ketamine without affecting cortical function. Our findings possibly support the clinical notion that low-dose ketamine as an analgesic adjuvant has therapeutic potential to reduce opioid dosage, thereby improving the quality of life in cancer pain patients.

The present study elucidated the functional role of modulatory effects of basolateral amygdala (BLA) on synaptic transmission in the rat hippocampus–medial prefrontal cortex (mPFC) pathway, compared with the hippocampal dentate gyrus (DG). Exposure to conditioned fear stress (CFS) or prior BLA activation enhanced tetanus-induced long-term potentiation (LTP) in DG. A similar synaptic response was found by low frequency stimulation (LFS) prior to tetanus. In mPFC, they did not affect LTP, but prior BLA activation, as well as pretreatment with the N-methyl-d-aspartate (NMDA)-receptor antagonist MK-801 (0.1 mg/kg, i.p.), suppressed LFS-primed LTP. This BLA-mediated synaptic pattern was mimicked by synaptic changes observed in the fear extinction process; prior BLA activation suppressed the synaptic potentiation responsible for extinction retrieval and attenuated decreases in fear-related freezing behavior. These data suggest that LFS-primed LTP in mPFC is related to the neural basis of extinction. Extinction-related synaptic potentiation did not occur in a juvenile stress model that exhibited extinction deficit. In addition, LFS-primed LTP was suppressed in this model, which was reversed by the NMDA-receptor agonist d-cycloserine (15 mg/kg, i.p.). These findings suggest that modulatory effects of BLA on synaptic function in the hippocampus–mPFC pathway play a significant role in fear extinction in rats. Keywords:: basolateral amygdala, synaptic potentiation, medial prefrontal cortex, extinction retrieval, low frequency stimulation

Recent studies focus on the functional significance of a novel form of synaptic plasticity, low-frequency stimulation (LFS)-induced synaptic potentiation in the hippocampal CA1 area. In the present study, we elucidated dynamic changes in synaptic function in the CA1 field during extinction processes associated with context-dependent fear memory in freely moving rats, with a focus on LFS-induced synaptic plasticity. Synaptic transmission in the CA1 field was transiently depressed during each extinction trial, but synaptic efficacy was gradually enhanced by repeated extinction trials, accompanied by decreases in freezing. On the day following the extinction training, synaptic transmission did not show further changes during extinction retrieval, suggesting that the hippocampal synaptic transmission that underlies extinction processes changes in a phase-dependent manner. The synaptic potentiation produced by extinction training was mimicked by synaptic changes induced by LFS (0.5 Hz) in the group that previously received footshock conditioning. Furthermore, the expression of freezing during re-exposure to footshock box was significantly reduced in the LFS application group in a manner similar to the extinction group. These results suggest that LFS-induced synaptic plasticity may be associated with the extinction processes that underlie context-dependent fear memory. This hypothesis was supported by the fact that synaptic potentiation induced by extinction training did not occur in a juvenile stress model that exhibited extinction deficits. Given the similarity between these electrophysiological and behavioral data, LFS-induced synaptic plasticity may be related to extinction learning, with some aspects of neuronal oscillations, during the acquisition and/or consolidation of extinction memory.

The present study elucidated whether early life stress alters the extracellular signal-regulated kinase (ERK) pathway that underlies fear retrieval and fear extinction based on a contextual fear conditioning paradigm, using a juvenile stress model. Levels of phospho-ERK (pERK), the active form of ERK, increased after fear retrieval in the hippocampal CA1 region but not in the medial prefrontal cortex (mPFC). ERK activation in the CA1 following fear retrieval was not observed in adult rats who received aversive footshock (FS) stimuli during the second postnatal period (2wFS), which exhibited low levels of freezing. In fear extinction, pERK levels in the CA1 were increased by repeated extinction trials, but they were not altered after extinction retrieval. In contrast, pERK levels in the mPFC did not change during extinction training, but were enhanced after extinction retrieval. These findings were compatible in part with electrophysiological data showing that synaptic transmission in the CA1 field and mPFC was enhanced during extinction training and extinction retrieval, respectively. ERK activation in the CA1 and mPFC associated with extinction processes did not occur in rats that received FS stimuli during the third postnatal period (3wFS), which exhibited sustained freezing behavior. The repressed ERK signaling and extinction deficit observed in the 3wFS group were ameliorated by treatment with the partial N-methyl-d-aspartate receptor agonist d-cycloserine. These findings suggest that early postnatal stress induced the downregulation of ERK signaling in distinct brain regions through region-specific regulation, which may lead to increased behavioral abnormalities or emotional vulnerabilities in adulthood.

Thermophysical properties of rare-earth-stabilized zirconia and zirconate pyrochlores, A2Zr2O7 (A = La, Nd, Sm, Gd, Dy, Y), were evaluated by X-ray diffractometry, Raman spectroscopy, and the ultrasound pulse-echo method. Crystallographic analyses elucidated that La2Zr2O7, Nd2Zr2O7, Sm2Zr2O7, and Gd2Zr2O7 had the pyrochlore structure, whereas Dy2Zr2O7 and Y2Zr2O7 had the defect fluorite structure. For lanthanide pyrochlores, the thermal expansion became smaller with increasing ionic radius of A and increasing crystal binding energy. The elastic moduli and Debye temperature evaluated using longitudinal and transverse sound velocities also depend on the ionic radius and binding energy, and hence these values related to mechanical properties increase with the ionic radius of A. On the other hand, Poisson’s ratio was almost comparable among these pyrochlores. In addition, thermophysical properties of actinide pyrochlore are discussed in this study.

In our previous report, we showed that L-arginine induced depolarization of smooth muscle cells of the rat portal vein with an increased contraction. To clarify the ionic mechanism of the membrane depolarization, the effect of L-arginine on the holding current was studied in freshly isolated smooth muscle cells of the rat portal vein. The whole-cell patch-clamp technique was used, with the membrane potential held at -60 mV. In the presence of Na+ in the perfusate, L-arginine 10 mM induced an inward current in about 50% of the cells. In Na+-deficient perfusate, L-arginine 10 mM increased the amplitude of the inward current in a Na+ concentration-dependent manner. BCH, an inhibitor of the Na+-dependent amino acid transporter, ceased the L-arginine-induced current. These results indicate that L-arginine induces an inward current via Na +-dependent mechanisms in rat portal venous smooth muscle cells.

The purpose of the present study was to evaluate the salivary Chromogranin-A (CgA) response to the psychological stress induced by a cognitive test battery. The subjects were 14 healthy volunteers administered the cognitive test battery CogScreen Aeromedical Edition (CogScreen-AE). CogScreen-AE is a test of reaction time and fundamental cognitive ability in the assessment of aeroplane pilots. The subjects were given five batteries of the test (1st approximately 5th) on separate days with 2 week intervals. Saliva samples were collected at 20 min before the test (BASE), immediately before the test (PRE), in the middle of the test (MID), and 5 min after the test (POS) for each subject. The concentration of CgA was determined by enzyme-linked immunosorbent assay. ANOVA revealed a significant by time interaction (BASE, PRE, MID and POS) without a significant effect of battery interaction (1st, 2nd, 3rd, 4th and 5th). The CgA concentration increased at PRE (2.46 +/- 0.24 pmol.mg protein- 1) from BASE (1.19 +/- 0.10 pmol.mg protein- 1). CgA level remained increased in the MID (2.90 +/- 0.26 pmol.mg protein- 1) and remained high in the POS samples (2.81 +/- 0.23 pmol.mg protein- 1). Salivary CgA remained at basal levels during a control study over the same time course without exposure to CogScreen-AE. The changes in salivary CgA secretion as a result of exposure to a cognitive task may indicate psychological stress in humans.

Clonidine, an alpha2-agonist, has been shown to be useful in the treatment of hepatic portal hypertension in cirrhosis. The mechanism has been attributed to a clonidine-induced decrease in sympathetic activity. While clonidine has been shown to stimulate the alpha2-adrenoceptors of blood vessels, there is limited knowledge of the effects of clonidine on the circular muscle of the hepatic portal vein which regulates its blood flow. To investigate clonidine-induced contraction of the circular muscle of the hepatic portal vein and to clarify the possible role of the endothelium in the contraction, we examined the effects of clonidine on the isometric contraction of endothelium-intact and -removed ring preparations of the rat hepatic portal vein. In endothelium-intact preparations, clonidine caused a concentration-dependent increase in the amplitude of contractions. Inhibition of NO synthesis with Nomega-nitro-L-arginine (L-NNA) elevated the resting tone, and increased the amplitude of the clonidine-induced contractions. Inhibition of cyclooxygenase by diclofenac did not change the amplitude of the clonidine-induced contractions observed both in the presence and absence of L-NNA. Application of a single concentration of clonidine induced a clear increase in amplitude of both twitch and tonic contractions. Twitch and tonic contractions induced by clonidine were inhibited by yohimbine. When the endothelium was damaged by sodium deoxycholate, tonic contractions induced by clonidine were completely suppressed, whereas the increase in twitch contractions was not influenced by chemical damage of the endothelium. Neither SKF-96365, a nonselective cation channel blocker, nor superoxide dismutase, a free radical scavenger, in the presence of catalase, changed the tonic contraction induced by clonidine. These results indicate that stimulation of alpha2-adrenoceptors enhanced twitch contractions and induced tonic contractions in the circular muscle of the rat hepatic portal vein, especially in the absence of NO. The latter, but not the former, occurs through an endothelium-dependent pathway.

Changes in Na + -K + pump activity influence intracellular Na + and K + concentrations. Activation of the electrogenic Na + -K + pump hyperpolarizes plasma membrane and inhibits voltage-dependent Ca 2+ current. Na + -K + pump activity has been shown to be inhibited in the absence of extracellular K + or by cardiac glycosides. Involvement of Na + -K + pump in regulation of cellular functions has been identified by inhibition of responses under inhibition of the Na + -K + pump. In some smooth muscle tissues, responses to β -agonists were inhibited by K + -free medium or ouabain, indicating that responses to β -adrenoceptor agonists are mediated by the Na + -K + pump. However, in other smooth muscle tissues, responses to β -agonists were not inhibited by Na + -K + pump inhibition. The Na + -K + pump plays an important role in the regulation of circulatory function, and the levels of endogenous Na + -K + pump inhibitors have been shown to be elevated in some types of hypertension. Endogenous Na + -K + pump inhibitors possibly affect some of β -adrenoceptor-mediated responses. Regulation of Na + -K + pump may be important in the regulation of smooth muscle activity in normal conditions, as well as under pathophysiological conditions, such as hypertension.

Summary 1. There are known differences in the sensitivity to caffeine between skeletal muscle (soleus) of normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). The present study was performed in order to examine differences in the effects of caffeine on twitch contraction between visceral striated muscle using the outer layer of the oesophagus from WKY rats and stroke-prone SHR (SHRSP). 2. Caffeine, at concentrations ranging from 0.3 to 10 mmol/L, exhibited potentiating effects on twitch contraction in preparations from both WKY rats and SHRSP. The potentiating effect of caffeine was markedly less prominent in preparations from SHRSP compared with preparations from WKY rats. 3. The rate of contraction and relaxation, the time to peak tension and 80% relaxation time were not significantly altered by caffeine at concentrations lower than 3 mmol/L in preparations from either strain. 4. With 10 mmol/L caffeine, the rate of relaxation was markedly reduced and the 80% relaxation time was prolonged, with no significant changes in the rate of contraction, in preparations from WKY rats. These changes were significantly smaller in preparations from SHRSP. 5. The duration of the action potential was greater in preparations from SHRSP than in preparations from WKY rats, although the membrane potential and the amplitude of the action potential were not significantly different between preparations from WKY rats and SHRSP. 6. Caffeine, at 10 mmol/L, prolonged the duration of the action potential in preparations from both strains. The effect of caffeine was not different between preparations from WKY rats and SHRSP. 7. The results of the present study suggest that caffeine augments release of Ca2+ from the sarcoplasmic reticulum (SR) at low concentrations and attenuates Ca2+ re-uptake at 10 mmol/L. Decreased reactivity of SR to caffeine may be a cause of the lesser potentiation of twitch contraction by caffeine in preparations from SHRSP.

The effects of L-arginine on spontaneous contraction of endothelium-denuded longitudinal preparations of the rat portal vein were studied. L-arginine increased the frequency of spontaneous contraction concentration-dependently between 10 microM and 1 mM. Changes in contraction amplitude and duration were not remarkable. D-arginine had a negligible effect on spontaneous contraction. N(omega)-nitro-L-arginine (1 mM) did not affect spontaneous contraction or the response to L-arginine. Addition of N(G)-monomethyl-L-arginine (1 mM), l-lysine (1 mM) or N-ethymaleimide (0.1 mM) increased the frequency of spontaneous contractions and inhibited the effect of L-arginine. Glibenclamide (10 microM) did not affect spontaneous contraction or the response to L-arginine. Spontaneous increase in concentration of intracellular Ca2+, estimated as the ratio of Fura-PE3 fluorescence occurred synchronously with spontaneous contraction. Spontaneous increase in concentration of intracellular Ca2+ occurred more frequently in the presence of L-arginine (1 mM). L-arginine (1 mM) also increased the number of action potential bursts/min in the longitudinal smooth muscle layer. L-arginine (1 mM) also depolarized cell membranes. This study indicates that L-arginine increases the frequency of spontaneous contraction of longitudinal muscle in the rat portal vein by membrane depolarization through mechanisms that do not involve nitric oxide or the inhibition of ATP-sensitive K+ channels.

The present study was undertaken to elucidate the effects of acute and subacute administration of docosahexaenoic acid (DHA) on blood pressure in SHRSP between the ages of 6 and 11 weeks. The continuous administration of DHA for 5 weeks in SHRSP significantly inhibited blood pressure rise as compared with levels in non-treated SHRSP. DHA-treated SHRSP showed significantly decreased urinary epinephrine contents as compared with those in non-treated SHRSP. DHA-treated SHRSP produced a significant decrease in adrenal epinephrine concentration as compared with those in non-treated SHRSP at the age of 11 weeks. DHA-treated SHRSP also demonstrated significant decreases in serum triglycerides, LDL and lipid peroxide levels as compared with levels in non-treated SHRSP. However, a single intraperitoneal administration of DHA did not exert any influence on blood pressure in SHRSP at the age of 11 weeks. These findings suggest that the anti-hypertensive effect induced by a 5-week, continuous administration of DHA appears to be associated with the amelioration of increased sympathoadrenal activity and serum lipid derangement during the developmental stage of hypertension in young SHRSP.The present study clarified that a 5-week, continuous administration of DHA had a protective effect against the development of hypertension in SHRSP.

Endothelium-removed carotid artery strips from stroke-prone spontaneously hypertensive rats spontaneously developed a tonic myogenic contraction. Flufenamic acid reduced the resting tone observed during superfusion with Tyrode's solution, in a concentration-dependent manner. Flufenamic acid also inhibited contractions produced by high-K solutions in a concentration-dependent manner. The resting membrane potential of smooth muscle cells in the artery was around -32 mV, with occasional oscillatory potentials. Flufenamic acid hyperpolarized the membrane in a concentration-dependent manner. The voltage-dependent outward currents recorded in isolated cells with micropipettes filled with high-K+ solution (holding potential, -60 mV) were enhanced by flufenamic acid and inhibited by tetraethylammonium. When the recording micropipette was filled with high Cs to inhibit the K+-current, depolarizing step pulses evoked nifedipine-sensitive inward currents. Flufenamic acid inhibited the inward currents. These results indicate that flufenamic acid inhibits the spontaneous active tone of the carotid artery by inhibiting L-type Ca2+-channels and possibly by membrane hyperpolarization through activation of the voltage-dependent K+-channels.

Effects of chronic treatment of normotensive Wistar rats with N(omega)-nitro-L-arginine methyl ester (L-NAME) on blood pressure and on endothelium-dependent relaxation of the aorta, carotid and iliac arteries were studied. The endothelium-dependent relaxation was compared in arteries from normotensive Wistar Kyoto rats (WKY) and genetically hypertensive rats (stroke-prone spontaneously hypertensive rats, SHRSP). Chronic treatment of normotensive Wistar rats with L-NAME caused an elevation of blood pressure. The elevated blood pressure at 15 weeks of age was significantly higher in these animals than that of untreated Wistar rats, but lower than that of SHRSP. Endothelium-dependent relaxation of the arteries induced by acetylcholine (ACh) was almost abolished by chronic treatment with L-NAME. The remaining small relaxation in arteries from L-NAME-treated rats was completely inhibited by application of L-NAME (10(-4) M). In such preparations, higher concentrations of ACh induced a contraction, which was abolished by removal of the endothelium or by an application of indomethacin (10(-5) M). Endothelium-independent relaxation induced by sodium nitroprusside was similar between preparations from untreated and L-NAME-treated Wistar rats. Endothelium-dependent relaxation was significantly impaired in preparations from SHRSP, when compared with that in those from WKY. However, the impairment was less prominent in preparations from SHRSP than in those from L-NAME-treated rats. These results suggest that the impairment of endothelium-dependent relaxation in the arteries from L-NAME-treated rats is not due to the elevated blood pressure resulting from the chronic treatment, and that impairment of NO synthesis by the endothelium does not play a major role in the initiation of hypertension in SHRSP.

The effects of NG-nitro-L-arginine (L-NNA) on blood pressure of various strains of spontaneously hypertensive rats were studied. Blood pressure of the rats was higher in the order of WKY, SHR, SHRSP, M-SHRSP. L-NNA caused an elevation of the blood pressure, which was greatest in SHR and smallest in WKY and M-SHRSP. Endothelium-dependent relaxation of aortae by acetylcholine was greatest in preparations from WKY and it decreased as the blood pressure of rats increased. Phenylephrine (higher than 10(-6) mg/kg) caused an elevation of the blood pressure, which was greatest in SHR and smallest in M-SHRSP. It was suggested that L-NNA elevated blood pressure by inhibiting the basal or flow-induced release of nitric oxide from the endothelium that is causing a reduction in vascular smooth muscle tone. The smaller effect of L-NNA in WKY was due to weak smooth muscle tone, while the smaller effect in SHRSP and M-SHRSP is due to impaired function of endothelium.

The role of nitric oxide in the electrical and mechanical activities of the rat portal vein was examined in circular muscle preparations with intact endothelium that were isolated from the longitudinal muscle layer. In contrast to the longitudinal muscle preparation, the circular muscle preparation did not show spontaneous phasic contraction. Inhibition of nitric oxide synthesis by Nomega-nitro-L-arginine (L-NNA) induced a tonic contraction. The contraction was inhibited by L-arginine, sodium nitroprusside or nifedipine. L-NNA did not induce contraction in endothelium-damaged preparations. The membrane potential of smooth muscle cells recorded in endothelium-intact preparations showed sporadic action potentials. L-NNA increased the frequency of action potentials without changing the resting membrane potential. The action potentials were inhibited by nifedipine. In the presence of L-NNA, sodium nitroprusside decreased the frequency of the action potentials without changing the resting membrane potential. These results indicated that contraction of rat portal vein circular muscles is inhibited tonically by nitric oxide, at least partly through inhibition of electrical activity.

Effects of isoproterenol on contraction and membrane potential of gastric smooth muscle were studied in stroke prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar Kyoto rats (WKY). Circular muscle preparation from the gastric fundus developed tonic contraction by re administration of Ca2+ to a nominally Ca2+-free solution. The contraction was inhibited by nifedipine or nicardipine. Isoproterenol induced relaxation when it was applied to the Ca2+-induced contraction. The amplitude of isoproterenol induced relaxation was concentration-dependent. Propranolol 10(-6) M abolished the relaxation induced by isoproterenol 10(-7) M. In the preparation from SHRSP, the amplitude of isoproterenol induced relaxation was smaller than that from WKY between 3 x 10(-9) and 10(-7) M. Forskolin, an adenylate cyclase activator, induced concentration-dependent relaxation. There was no difference in the relaxation induced by forskolin between preparations from WKY and SHRSP. Dibutilyl cyclic AMP, a membrane permeable analogue of cyclic AMP, also induced similar relaxation in preparations from WKY and SHRSP. Resting membrane potential of smooth muscle cell was not different between preparations from WKY and SHRSP. Isoproterenol hyperpolarized the membrane concentration-dependently. Isoproterenol-induced hyperpolarization in the preparation from SHRSP was smaller than that from WKY between 10(-8) and 10(-6) M. When the membrane was depolarized by Tyrode's solution containing 40 mM K+, isoproterenol-induced hyperpolarization was almost abolished. In this condition, the isoproterenol-induced relaxation was inhibited partly, however, there was no difference in the amplitude of relaxation between preparations from WKY and SHRSP. Therefore, isoproterenol-induced hyperpolarization contributed at least partly to the relaxation. Forskolin hyperpolarized the membrane by the same amplitude in the preparations from WKY and SHRSP. These results indicate that a decrease in hyperpolarization may contribute to the decreased relaxation by isoproterenol in the preparation from SHRSP.

1. The effects of cyclopiazonic acid (CPA), a selective inhibitor of sarcoplasmic reticulum (SR) Ca2+-ATPase, on twitch contraction and on the resting state of tension and intracellular Ca2+ level ([Ca2+]i) of the oesophageal striated muscle of stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar Kyoto rats (WKY) were compared. 2. CPA (10 micronM) augmented the twitch contraction of oesophageal striated muscle preparations from both SHRSP and WKY, reducing the rate of relaxation (-dT/dt), and thus resulting in the prolongation of the time to 80% relaxation. The effect was significantly smaller in the SHRSP preparations. 3. In the resting state, CPA caused a sustained elevation of [Ca2+]i. The elevation was greater in the WKY preparations. Tension development accompanied by the elevation was observed in WKY preparations, but not in SHRSP preparations. 4. The sustained elevation of [Ca2+]i induced by CPA was eliminated by the removal of extracellular Ca2+. Both the elevated [Ca2+]i and tension in the preparations from WKY were reduced by flufenamic acid (100 micronM), mefenamic acid (100 micronM), lanthanum (La3+, 100 micronM), gadolinium (Gd3+, 100 micronM) and SK&F 96365 (100 micronM) but not by verapamil (10 micronM). 5. Thapsigargin (3 micronM), another SR Ca2+-ATPase inhibitor, produced similar effects on basal tension to those of CPA, although it reduced the amplitude of twitch contraction. 6. These results suggest that in the rat oesophageal striated muscle, (1) CPA extends the sequestrating time of Ca2+ into the SR, (2) CPA induces a Ca2+ influx mediated through verapamil-insensitive pathways, possibly nonselective cation channels, and (3) the mechanism of [Ca2+](i) modulation due to CPA-sensitive SR Ca2+-ATPase is deteriorated in the oesophageal striated muscle from SHRSP as compared with WKY preparations.

Differences in the acetylcholine (ACh)-induced endothelium-dependent relaxation and hyperpolarization of the mesenteric arteries of Wistar Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP) were studied. Relaxation was impaired in preparations from SHRSP and tendency to reverse the relaxation was observed at high concentrations of ACh in these preparations. Relaxation was partly blocked by NG-nitro-L-arginine (L-NOARG, 100 μM) and, in the presence of L-NOARG, tendency to reverse the relaxation was observed in response to higher concentrations of ACh, even in preparations from WKY. The relaxation remaining in the presence of L-NOARG was also smaller in preparations from SHRSP. The tendency to reverse the relaxation observed at higher concentrations of ACh in preparations from SHRSP or WKY in the presence of L-NOARG were abolished by indomethacin (10 μM). Elevating the K+ concentration of the incubation medium decreased relaxation in the presence of both indomethacin and L-NOARG. Relaxation in the presence of L-NOARG and indomethacin was reduced by the application of both apamin (5 μM) and charybdotoxin (0.1 μM). This suggests that the relaxation induced by ACh is brought about by both endothelium-derived relaxing factor (EDRF, nitric oxide (NO)) and hyperpolarizing factor (EDHF), which activates Ca2+-sensitive K+ channels. Electrophysiological measurement revealed that ACh induced endothelium-dependent hyperpolarization of the smooth muscle of both preparations in the presence of L-NOARG and indomethacin; the hyperpolarization being smaller in the preparation from SHRSP than that from WKY. These results suggest that the release of both NO and EDHF is reduced in preparations from SHRSP. In addition, indomethacin-sensitive endothelium-derived contracting factor (EDCF) is released from both preparations; the release being increased in preparations from SHRSP. British Journal of Pharmacology (1999) 126, 709–716; doi:10.1038/sj.bjp.0702355

The effects of chronic treatment of stroke-prone spontaneously hypertensive rats (SHRSP) with carvedilol, an antihypertensive agent which has both alpha- and beta-adrenoceptor-blocking actions, on membrane potential and relaxation of mesenteric resistant artery were studied. Five-week old SHRSP were treated with carvedilol for three months. At 16 weeks, the resting membrane potential of arteries from carvedilol-treated SHRSP was more negative than that of arteries from untreated SHRSP. The magnitude of acetylcholine-induced hyperpolarization in arteries from carvedilol-treated SHRSP was not different from that of arteries from untreated SHRSP. In the presence of noradrenaline, the membrane potential of arteries from carvedilol-treated SHRSP was more negative than that of arteries from untreated SHRSP. The membrane potential of arteries from carvedilol-treated SHRSP in the presence of noradrenaline and acetylcholine was more negative than that of arteries from untreated SHRSP. The acetylcholine-induced relaxation in noradrenaline-precontracted preparations from carvedilol-treated SHRSP was greater than that in preparations from untreated SHRSP and was smaller than that in preparations from Wistar Kyoto rats. Scanning electronmicroscopy showed that carvedilol-treatment decreased the structural abnormalities of the endothelium of arteries from SHRSP. These results indicate that chronic carvedilol treatment made the membrane potential of smooth muscle more negative and improved endothelial function in the mesenteric artery of SHRSP, which may contribute to the antihypertensive effect of carvedilol.

Contractile sensitivity to Ca2+ was studied in rat portal vein circular muscle isolated from normotensive Wistar Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP). There was no difference in resting membrane potential between WKY and SHRSP. Elevation of extracellular K+ concentration induced similar increase in tension in preparations from WKY and SHRSP. High K(+)-depolarized preparation developed tension increase when extracellular Ca2+ concentration was increased. The sensitivity to extracellular Ca2+ was similar in preparations from WKY and SHRSP. Permeabilized preparation contracted when free Ca2+ concentration was increased. Sensitivity of permeabilized preparation to Ca2+ was similar in preparations from WKY and SHRSP. These results indicate that regulation of contraction by Ca2+ was similar between WKY and SHRSP. Since there was no change in high K-induced contraction in intact preparations or Ca-induced contraction in permeabilized preparations, there seemed to be no difference in Ca2+ influx via voltage-dependent channels in portal vein circular muscle from WKY and SHRSP.

Control of the active tone by endothelium in aortae from various strains of spontaneously hypertensive rats was studied. The active tone was negligibly observed in endothelium-intact preparation. The application of N(G)-nitro-L-arginine (L-NNA, 10(-4) M) induced slowly developed active tone in the preparations from hypertensive rats but no active tone was induced in the preparation from normotensive Wistar Kyoto rats (WKY). The developed tension was stronger in preparations from rats with higher blood pressure as observed in endothelium denuded preparations. The developed active tone in the presence of L-NNA was greater than that observed in endothelium denuded preparations. The active tone was abolished by the removal of extracellular Ca2+ or by the application of Ca-antagonists. L-arginine counteracted the effects of L-NNA and depressed the developed active tone in the presence of the latter drug. The application of indomethacin (10(-5) M) depressed the active tone of the preparations from SHRSP by 25.5+/-5.2%. Increasing extracellular K+ concentration or application of tetraethylammonium (TEA) could not be used to observe the effect of endothelium-derived factors on the active tone, because of their strong contractile effect. Simultaneous application of apamin and charybdotoxin induced an elevation of tension which was often associated with spontaneous tension oscillation. It is concluded that the active tone, which is smooth muscle origin, is depressed by endothelium-derived nitric oxide (NO) strongly and potentiated by a product of arachidonic acid cascade through cyclooxygenase pathway. The involvement of endothelium-derived hyperpolarizing factor (EDHF) in the depressing effect of endothelium is thought to be small.

Eugenol (> or = 0.1 mM) inhibited the contractions induced by various stimulants, such as 90 mM extracellular K+ solution ([K+]0), histamine and noradrenaline in the rabbit ear artery. Inhibitory actions of eugenol occurred in a concentration-dependent manner, however, eugenol more dominantly inhibited the histamine-induced contraction than those induced by either 90 mM [K+]0 solution or noradrenaline. Removal of both endothelium and adventitia did not change the inhibitory actions of eugenol on the 90 mM [K+]0- and noradrenaline-induced contractions, however, attenuated those on the histamine-induced contraction. Chlorphenylamine abolished the histamine-induced contraction, but neither cimetidine, ranitidine nor thioperamide modified the eugenol actions on the contractions induced by histamine. Pretreatment with nitric oxide syntheses inhibitor NG-nitro-L-arginine (LNNA; 100 microM), but not soluble guanylate cyclase inhibitor methylene blue (MB; 10 microM), prevented endothelium/adventitia-dependent augmentation of the eugenol-induced relaxation on the histamine-induced contraction. When an intact tissue, but not an endothelium/adventitia-denuded tissue, was placed at the up-stream close to the other denuded preparation (test preparation), histamine-induced contraction observed in the test preparation tended to be augmented. Similarly, eugenol-induced relaxation was also augmented by the same treatment. Eugenol (0.3 mM) inhibited the excitatory junction potentials (EJPs) without hyperpolarization of the membrane. However, a high concentration of eugenol (1 mM) slightly hyperpolarized the membrane (ca. 5 mV). No transient enhancement of amplitude of EJP was recorded. These results suggest that eugenol may inhibit the histamine-induced muscle contraction directly, and the inhibition is augmented by the adventitia and endothelium partly by vasoactive substances, which were released from the adventitia/endothelium-derived substances in rabbit ear artery.

Oscillatory contractions of arteries in vitro are frequently induced by receptor stimulation with agonists. In certain arteries, the membrane potential and cytoplasmic intracellular free Ca2+ concentration in smooth muscle cells oscillate in the presence of agonists. The oscillatory changes are related to the Ca2+ release from store sites as well as Ca2+ influx. Gap junctions between smooth muscle cells play important roles in the synchronization of Ca2+ concentration in arterial tissues. Some of the agonist-induced oscillatory contractions are dependent on endothelium. Endothelial cells or endothelium-derived substances may modulate the tension oscillation of smooth muscle cells. In arteries from hypertensive animals, oscillatory contraction has been induced by agonists more frequently than that from normotensive animals. Abnormalities in membrane excitability of smooth muscle cells, gap junctions and endothelium may influence the tension oscillation in arteries from hypertensive rats.

Previous studies have shown that the guinea pig inferior mesenteric artery receives spinal sensory vasodilatory innervation, which can be activated by colon distention and electrical nerve stimulation. In the present study, we investigated the hypotheses that nitric oxide synthase (NOS) is present in guinea pig primary sensory neurons in the dorsal root ganglion (DRG) and in nerve fibers surrounding the mesenteric arteries, and that nitric oxide (NO) is a sensory neurotransmitter in the inferior mesenteric artery in vitro. Double-labeling immunohistochemistry showed that neuronal NOS-IR was found in 12% of cells of guinea pig thoracic and lumbar DRGs; in 95.1% of these cells it was colocalized with substance P (SP), and SP immunoreactivity (SP-IR) was present in 23% of cells of the same DRGs. Neuronal NOS-like immunoreactivity was localized in nerve fibers surrounding guinea pig mesenteric artery and 25% of them were double stained with SP-IR. Endothelium-denuded inferior mesenteric artery preparations in vitro were incubated with guanethidine (30 microns, 30 min) and pre-contracted with methoxamine (30 microns). The NO donors, sodium nitroprusside (1 micron) and L-nitrosocysteine (300 microns), produced 91.0 +/- 5.5 and 90.4 +/- 9.6% vasodilatation of total vasodilatation in the vessel segments, respectively, which was capsaicin- or tetrodotoxin-insensitive. Repetitive electrical field stimulation of the preparations produced a frequency-dependent vasodilatation which was reduced by pretreatment with capsaicin or by tetrodotoxin (10 microns). The NOS inhibitor N omega-nitro-L-arginine (L-NNA) (100 microns, 30 min) diminished the nerve-evoked vasodilatation from 41.8 +/- 8.4 to 21.4 +/- 9.7% at 2 Hz and from 50.8 +/- 5.6 to 19.0 +/- 7.3% at 15 Hz (P < 0.05), whereas NG-nitro-L-arginine methyl ester (L-NAME, 100 microns-1 mM) did not significantly inhibit the relaxation. The stereo isomer nitro-D-arginine (D-NNA, 100 microns, 30 min) was ineffective. These findings suggest that NO is a neurotransmitter released from primary sensory nerves which mediates vasodilation in vitro.

The membrane hyperpolarization produced by endothelium-derived hyperpolarizing factor (EDHF) is one of the components in the endothelium-dependent relaxations. Different subtype of endothelium receptor is involved in the release of EDHF from that for nitric oxide. Although EDHF has not been identified, it may be related to cytochrome P450. The responses mediated by EDHF have been studied by observation of membrane potential or relaxation which is resistant to inhibitors of nitric oxide synthase and cyclooxygenase. The responses are dependent on potassium gradient across cell membrane, therefore, the response is mediated by potassium channels. The EDHF-mediated hyperpolarization or relaxation is sensitive to charybdotoxin plus apamin, inhibitors of calcium activated potassium channels. The EDHF-mediated response is more remarkable in small resistant arteries than large arteries, therefore, EDHF may play an important role in the regulation of vascular resistance. The EDHF-mediated response is decreased in hypertensive or aged animals. Understanding of EDHF-mediated response will promote development of a new strategy for the treatment of pathophysiological conditions.

Transglutaminase 2 (TG2) is a multifunctional protein that contributes to inflammatory disease when aberrantly expressed. Although macrophages express TG2, the factor stimulating TG2 expression remains poorly characterized in these cells. In the present study, we examined the effects of the stress-related catecholamines adrenaline and noradrenaline on macrophage expression of TG2 in RAW264.7 murine macrophages and murine bone marrow-derived macrophages. Treatment with adrenaline markedly increased TG2 mRNA expression and increased TG2 protein levels. While the β2-adrenoceptor-selective antagonist ICI 118,551 completely blocked adrenaline-induced TG2 mRNA expression, the β2-adrenoceptor specific agonist salmeterol increased TG2 expression. Noradrenaline also increased TG2 mRNA expression at higher doses than the effective doses of adrenaline. The effect of adrenaline on TG2 mRNA expression was mimicked by treatment with the membrane-permeable cAMP analog 8-Br-cAMP. Thus, increased intracellular cAMP following stimulation of β2-adrenoceptors appeared to be responsible for adrenaline-induced TG2 expression. Because stress events activate the sympathetic nervous system and result in secretion of the catecholamines, adrenoceptor-mediated increase in macrophage TG2 expression might be associated with stress-related inflammatory disorders.

The aim of this study was to evaluate saliva as a potential monitoring medium for procainamide (PA) and its metabolite, N-acetylprocainamide (NAPA). Saliva concentrations of PA and NAPA were determined both in single and repeated oral administration of PA in four healthy subjects. PA and NAPA were detected both in serum and saliva after 500 mg of single oral administration of PA. After single oral administration, serum and saliva concentrations of PA and NAPA reached peak levels at about 1 h and declined thereafter. The mean half-lives of PA were 2.35 h in serum and 1.28 h in saliva. The mean half-lives of NAPA were 5.29 h in serum and 5.01 h in saliva. In this study, PA and NAPA concentrations in saliva were nearly twice as high as those in serum upon chronic oral administration as well as those in a single oral dose of PA. Significant correlation coefficients were observed between serum and saliva concentrations of PA (r = 0.78, p0.001, n = 21) and NAPA (r = 0.76, p0.001, n = 21) in single oral administration of PA. Significant correlation coefficients were also observed between serum and saliva concentrations of PA (r = 0.89, p0.001, n = 17) and NAPA (r = 0.87, p0.001, n = 19) after repeated oral administration of PA. The saliva-to-serum ratios of PA and NAPA maintained nearly constant at 1 h after oral administration. It would appear from this study that saliva is a suitable medium for monitoring PA and NAPA concentration regarding acetylator status.

Differences in alpha(2)-adrenoceptor-induced relaxation of the aorta between stroke-prone spontaneously hypertensive rats (SHRSP) and control normotensive Wistar Kyoto rats (WKY) were studied. Changes in the tension of ring preparations of the aortas were measured isometrically. Relaxation was observed in the preparations precontracted in the presence of ONO-11113, a thromboxane A(2) analogue. The alpha(2)-agonist clonidine and UK-14304 induced dose-dependent relaxation in both the WKY and SHRSP preparations. The relaxation was impaired in the SHRSP preparation. A modified sandwich experiment showed that the relaxing substance from the SHRSP endothelium was decreased. Acetylcholine (ACh) also induced dose-dependent relaxation, and the relaxation was impaired in the SHRSP preparations. alpha(2)-Agonists induced a greater degree of impairment in the relaxation than did ACh. The relaxation induced by alpha(2)-agonists and by ACh was blocked by N G-nitro-L-arginine (L-NNA). Indomethacin improved the relaxation induced by ACh but not that induced by alpha(2)-agonists in the SHRSP aortas. These results suggest that the impairment of relaxation by alpha(2)-agonists in SHRSP is not caused by the increase in the release of endothelium-derived contracting factor (EDCF) but by the reduction in the release of nitric oxide (NO). Alteration of the alpha(2)-adrenoceptors and/or the intracellular mechanism through which NO is synthesized by stimulation of the alpha(2)-adrenoceptors may be the cause of the reduction in relaxation.

The influence of blood pressure and age of spontaneously hypertensive rats on endothelium-dependent tension oscillation of aortic preparation were studied. Rats with different blood pressures, normotensive Wistar Kyoto rats (WKY), spontaneously hypertensive rats (SHR), stroke-prone SHR (SHRSP) and malignant type of SHRSP (M-SHRSP), were used. The effects of antihypertensive treatment of SHRSP on the tension oscillation were also studied. High doses of noradrenaline induced tension oscillations in endothelium-intact preparations of all strains of young rats. The rate of the occurrence of the tension oscillation decreased age-dependently. The decrease was faster when the blood pressure of the rats was higher. Application of acetylcholine in the presence of noradrenaline induced a relaxation and tension oscillations, both of which were negatively dependent on age and blood pressure. Antihypertensive treatment of hypertensive rats with hydralazine or captopril prevented a decrease in incidence of the tension oscillation. These influences of age and blood pressure as well as antihypertensive treatments on the tension oscillation resembled those on the endothelium-dependent relaxation and are thought to be brought about by functional changes of the endothelium.

The influences of endothelium on the basal tone of aortae from various strains of spontaneously hypertensive rats with different blood pressure (SHR, SHRSP, M-SHRSP) were studied. Endothelium-intact preparations of aortae from spontaneously hypertensive rats exhibited spontaneous active tone, which was greater in the order of SHRSHRSPM-SHRSP. The active tone of the M-SHRSP preparations was about 40% of high-K(+)-induced contraction, while that of normotensive WKY was less than 5%. The active tone was enhanced by the removal of endothelium. The active tone was sensitive to extracellular Ca2+ and abolished by verapamil. The application of N(G)-monomethyl-L-arginine caused the increase in the active tone which was counteracted by L-arginine. These results indicate that the active tone of smooth muscle increases as the blood pressure of the rat increases, and that endothelium attenuates the active tone by releasing nitric oxide (NO) spontaneously. It was also demonstrated that the attenuating action of endothelium was impaired depending on the blood pressure level.

Differences in the influences of endothelium-derived nitric oxside (NO) on alpha-agonists-induced contraction in the aortae of spontaneously hypertensive and normotensive rats were studied by blocking NO synthesis with NG-nitro-L-arginine (L-NNA). L-NNA potentiated the contraction induced by noradrenaline. The potentiation was smaller in the preparation from stroke-prone spontaneously hypertensive rats (SHRSP) than in the preparation from Wistar Kyoto rats (WKY). Similar potentiation was observed in the contraction induced by phenylephrine; the potentiation was also smaller in the preparation from SHRSP. alpha 2-agonists, clonidine and UK-14304 induced dose-dependent contraction only in the presence of L-NNA. The dose-response curves for alpha 2-agonists in SHRSP aorta were different from those in WKY aorta; the maximum tension was observed at the concentration of 10(-6) M in the preparation from WKY, while the contraction further increased up to 10(-4) M in the preparation from WKY. Noradrenaline, clonidine and UK-14304 but not phenylephrine induced relaxation which was blocked by L-NNA. The relaxation was impaired in the preparation from SHRSP in greater extent than that by acetylcholine. It is suggested that basic or noradrenaline-stimulated NO release from endothelium decreased in the preparation from SHRSP and that alpha 2-adrenoceptor of both the endothelium and smooth muscle may be altered in the preparation from SHRSP.

Fear extinction-based exposure treatment is an important component of psychotherapy for anxiety disorders such as posttraumatic stress disorder (PTSD). Recent studies have focused on pharmacological approaches combined with exposure therapy to augment extinction. In this study, we elucidated the therapeutic potential of the serotonin 1A (5-HT(1A) ) receptor agonist tandospirone compared with the effects of the N-methyl-D-aspartate partial agonist D-cycloserine (DCS), focusing on the possible involvement of dopaminergic mechanisms. We used a rat model of juvenile stress [aversive footshock (FS)] exposure during the third postnatal week (3wFS). The 3wFS group exhibited extinction deficit reflected in sustained fear-related behavior and synaptic dysfunction in the hippocampal CA1 field and medial prefrontal cortex (mPFC), which are responsible for extinction processes. Tandospirone administration (5 mg/kg, i.p.) before and after the extinction trials ameliorated both the behavioral deficit and synaptic dysfunction, i.e., synaptic efficacy in the CA1 field and mPFC associated with extinction training and retrieval, respectively, was potentiated in the tandospirone-treated 3wFS group. Extracellular dopamine release in the mPFC was increased by extinction retrieval in the non-FS control group. This facilitation was not observed in the 3wFS group; however, tandospirone treatment increased cortical dopamine levels after extinction retrieval. DCS (15 mg/kg, i.p.) also ameliorated the extinction deficit in the 3wFS group, but impaired extinction in the non-FS control group. These results suggest that tandospirone has therapeutic potential for enhancing synaptic efficacy associated with extinction processes by involving dopaminergic mechanisms. Pharmacological agents that target cortical dopaminergic systems may provide new insights into the development of therapeutic treatments of anxiety disorders, including PTSD.

Serotonin (5-HT)(1A) receptors play a critical role in the 5-HTergic mechanism associated with fear memory. Previously we showed that adult rats exposed to early postnatal stress, i.e. footshock (FS) stress experienced during the second week (PND 14-18, 2W-FS), exhibited low levels of fear expression. The present study explored whether aversive stress exposure in the second and/or the third week (PND 21-25, 3W-FS) affects the function of cortical 5-HT(1A) receptors, using in vivo and in vitro experiments. A 5-HT(1A) receptor agonist, 8-OH-DPAT (0.5 mg/kg, i.p.), slightly decreased the evoked potential in the mPFC in Non-FS control and 3W-FS group. In contrast, the evoked potential increased after 8-OH-DPAT in the 2W-FS group. The in vitro experiment using patch-clamp recording showed that application of 8-OH-DPAT (10 microM) elicited membrane hyperpolarization of pyramidal neurons in the mPFC in the Non-FS and 3W-FS groups, whereas no changes in membrane potential were observed in the 2W-FS group. These results suggest that synaptic facilitation induced by 8-OH-DPAT resulted from functional changes in cortical 5-HT(1A) receptors. Thus, aversive stress exposure during the second postnatal period appears to cause persistent changes mediated via 5-HT(1A) receptors, presumably involving signal transduction regulating the development of synaptic connectivity underlying fear circuits.

Two kinds of outward K+ currents were examined in single smooth muscle cells from pregnant rat uterus, using whole-cell voltage clamp. The first and faster component was more sensitive to 4-aminopyridine (4-AP), whereas the second and slower (delayed) component was more sensitive to tetraethylammonium (TEA). A possible third K+ component (Ca activated K+ current) was not recorded as the pipette solution included EGTA. Forskolin inhibited the outward current in a concentration-dependent manner (50% inhibition occurred at about 30 microM); it affected the delayed component rather than the fast component. 8-Bromo-cAMP did not alter the outward current. In addition, inhibitors of protein kinase A and GDP-beta S and GTP-gamma S did not affect the forskolin-induced inhibition. These results indicate that forskolin inhibition of the delayed component of the outward current is independent of cAMP generation in the pregnant rat myometrial cells. Therefore, forskolin seems to directly inhibit specific K+ channels, as was reported for several other cell types.

Spontaneously developed tension (active tone) and intracellular Ca2+ level of aortae from spontaneously hypertensive rats (SHR), stroke-prone SHR (SHRSP), malignant SHRSP (M-SHRSP) and control Wistar Kyoto rats (WKY) were compared. Systolic blood pressure of WKY, SHR, SHRSP and M-SHRSP was 130 mmHg, 200 mmHg, 250 mmHg and 260 mmHg, respectively. Preparations from all strains of spontaneously hypertensive rats exhibited active tone which was abolished by the removal of extracellular Ca2+ or by the application of verapamil. The active tone was greater in the order of aortae from SHR < SHRSP < M-SHRSP. Intracellular Ca2+ level measured by Fura-2 method decreased by the removal of extracellular Ca2+. The degree of the decrease was greater as the blood pressure of the rats increased, indicating the greater elevation of intracellular Ca2+ level in the pressure of extracellular Ca2+. A correlation was obtained between the active tone, intracellular Ca2+ level and blood pressure. Thus, it was demonstrated that the development of the active tone is brought about by the changes in Ca2+ influx of smooth muscle cell membrane and the degree of the change is positively related to the degree of hypertension.

The non-competitive N-methyl-D-aspartate NMDA receptor antagonist ketamine, a dissociative anesthetic capable of inducing analgesia, is known to have psychotomimetic actions, but the detailed mechanisms remain unclear because of its complex properties. The present study elucidated neural mechanisms of the effect of ketamine, at doses that exert psychotomimetic effects without anesthetic and analgesic effects, by evaluating cortical synaptic responses in vivo. Systemic administration (i.p.) of low (1 and 5 mg/kg), subanesthetic (25 mg/kg) and anesthetic (100 mg/kg) doses of ketamine dose-dependently decreased hippocampal stimulation-evoked potential in the medial prefrontal cortex (mPFC) in freely moving rats. The behavioral analysis assessed by prepulse inhibition (PPI) of acoustic startle response showed that ketamine (5 and 25 mg/kg, i.p.) produced PPI deficit. Thus, the psychotomimetic effects observed in ketamine-treated groups (5 and 25 mg/kg, i.p.) are associated with the induction of synaptic depression in the hippocampus-mPFC neural pathway. Based on these results, we further examined the underlying mechanisms of the ketamine-induced synaptic depression under anesthesia. Ketamine (5 and 25 mg/kg, i.p.) caused increases in dialysate dopamine in the mPFC in anesthetized rats. Moreover, the ketamine-induced decreases in the evoked potential, at the dose 5 mg/kg which has no anesthetic and analgesic effects, were indeed absent in dopamine-lesioned rats pretreated with 6-hydroxydopamine (6-OHDA; 150 μg/rat, i.c.v.). Ketamine (5 mg/kg, i.p.)-induced synaptic depression was blocked by pretreatment with dopamine D1 receptor antagonist SCH 23390 (10 μg/rat, i.c.v.) but not dopamine D2 receptor antagonist haloperidol (1.5 mg/kg, i.p.), suggesting that dopaminergic modulation mediated via D1 receptors are involved in the synaptic effects of ketamine. Furthermore, ketamine (5 mg/kg, i.p.)-induced synaptic depression was prevented also by GABAA receptor antagonist bicuculline (0.2 or 2 μg/rat, i.c.v.). These findings suggest that ketamine at the dose that exerts psychotomimetic symptoms depresses hippocampus-mPFC synaptic transmission through mechanisms involving dopaminergic modulation mediated via D1 receptors, which may lead to a net augmentation of synaptic inhibition mediated via GABAA receptors.

The effect of sodium vanadate on action potential and twitch contraction of guinea pig ureter was studied and compared with that of ouabain, elevated K+, low temperature, and a Ca agonist (BAY K 8644), which can be expected to exert certain comparative effects. Sodium vanadate markedly potentiated twitch contraction. Potentiation by vanadate was associated with marked prolongation of relaxation time. Sodium vanadate caused only slight depolarization of the membrane but marked changes in action potential. The duration of action potential was prolonged and the number of oscillatory spike potentials increased. These effects were different from those of other treatments. It is concluded that prolongation of action potential and the increase in the number of spikes are the main cause of potentiation of twitch contraction by sodium vanadate. In addition, inhibition of Ca pump activity of the smooth muscle membrane system by vanadate might also be involved in potentiation of twitch contraction.

The effects of chronic treatment with SQ29852, an angiotensin-converting enzyme inhibitor, on spontaneous smooth muscle tone and endothelium-dependent relaxation of aorta from stroke-prone spontaneously hypertensive rats (SHRSP) were studied and compared with those of captopril. Endothelium-removed aorta from 16-week-old SHRSP exhibited a high amplitude of spontaneously developed active tension (active tone), whereas no active tone was observed in the preparation from control normotensive Wistar-Kyoto (WKY) rats. Treatment with SQ29852 or captopril at age 5-16 weeks prevented the development of hypertension. No active tone could be detected in the preparation from SQ29852-treated SHRSP. Endothelium-dependent relaxation was markedly reduced in the preparation from nontreated SHRSP compared with WKY rats. Treatment with SQ29852 prevented the impairment of endothelium-dependent relaxation. It was also shown that norepinephrine-induced contraction was markedly depressed in endothelium-intact aorta from SQ29852-treated rats. The effects of SQ29852 were more prominent than those of hydralazine when blood pressure was maintained at similar levels. It was suggested that SQ29852 exerts an action on both vascular smooth muscle and endothelium that is mediated by the inhibition of angiotension-converting enzyme in addition to indirect actions of SQ29852 that are brought about by blood pressure lowering.

The influences of low temperature and vanadate, both of which have been known to inhibit the Ca-pump activity of membranous systems of smooth muscle cells, on the relaxation of mesenteric artery of stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar-Kyoto rats (WKY) were studied. Relaxation was induced by the removal of extracellular Ca in high-K-depolarized preparations, so that the involvement of receptor kinetics and changes in membrane potential could be excluded. Relaxation was significantly slower in the preparation from SHRSP than in that from SHRSP. Low temperature prolonged relaxation in preparations from both SHRSP and WKY. No difference in the effect of low temperature was observed between the preparations, which showed Q10 values between 2.8 and 3.5 for 50 and 80% relaxation time. Sodium vanadate also prolonged the time course of relaxation in both preparations. No difference in the effect of sodium vanadate was observed between the preparations from WKY and SHRSP. These results suggest that Ca-pump activity is involved in the relaxation of K-depolarized smooth muscle of mesenteric arteries from both WKY and SHRSP. However, the difference in the pump activity may not contribute greatly to the difference in the relaxation of K-depolarized preparations by the removal of extracellular Ca.

1. The time courses of the relaxation, induced by removal of extracellular Ca2+, of K-depolarized mesenteric artery preparations from stroke prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto rats (WKY) were compared. 2. The time course of the decline in extracellular Ca2+ was estimated from the time course of the relaxation and the concentration-response curve of K+-depolarized preparations to Ca2+. The time course of the decline in the intracellular free Ca2+ concentration was also estimated from the reported relation between Ca2+ concentration and the contraction of skinned vascular smooth muscle. 3. The time course of relaxation was exponential, the curve being made up of three components. The time course was slower in preparations from SHRSP, especially the first component of the relaxation curve. 4. The time courses of the decline in the intracellular and extracellular Ca2+ concentrations were also exponential, being made up of three components and were also slower in the preparation made from SHRSP. 5. The wall and muscle layer of the mesenteric arteries used in the present experiments were significantly thicker in the SHRSP preparations. 6. Calculation of the half relaxation time, based on the diffusion of Ca2+ across the blood vessel wall, suggested that the slower relaxation in preparations from SHRSP is due largely to the thicker muscle layer, although differences in Ca2+ sequestration by the smooth muscle cells may also be involved.

Mechanical properties of stabilized zirconia and zirconate pyrochlore were evaluated by experiment, MD simulation and ab initio calculation.