bluuebunny/crossref_metadata_2025 huggingface dataset:
Possible involvement of the α1 isoform of 5′AMP-activated protein kinase in oxidative stress-stimulated glucose transport in skeletal muscle
Authors: Toyoda Taro , Hayashi Tatsuya , Miyamoto Licht , Yonemitsu Shin , Nakano Masako , Tanaka Satsuki , Ebihara Ken , Masuzaki Hiroaki , Hosoda Kiminori , Inoue Gen , Otaka Akira , Sato Kenji , Fushiki Tohru , Nakao Kazuwa
Published: March 2004

Recent studies have suggested that 5′AMP-activated protein kinase (AMPK) is activated in response to metabolic stresses, such as contraction, hypoxia, and the inhibition of oxidative phosphorylation, which leads to insulin-independent glucose transport in skeletal muscle. In the present study, we hypothesized that acute oxidative stress increases the rate of glucose transport via an AMPK-mediated mechanism. When rat epitrochlearis muscles were isolated and incubated in vitro in Krebs buffer containing the oxidative agent H2O2, AMPKα1 activity increased in a time- and dose-dependent manner, whereas AMPKα2 activity remained unchanged. The activation of AMPKα1 was associated with phosphorylation of AMPK Thr172, suggesting that an upstream kinase is involved in the activation process. H2O2-induced AMPKα1 activation was blocked in the presence of the antioxidant N-acetyl-l-cysteine (NAC), and H2O2 significantly increased the ratio of oxidized glutathione to glutathione (GSSG/GSH) concentrations, a sensitive marker of oxidative stress. H2O2 did not cause an increase in the conventional parameters of AMPK activation, such as AMP and AMP/ATP. H2O2 increased 3- O-methyl-d-glucose transport, and this increase was partially, but significantly, blocked in the presence of NAC. Results were similar when the muscles were incubated in a superoxide-generating system using hypoxanthine and xanthine oxidase. Taken together, our data suggest that acute oxidative stress activates AMPKα1 in skeletal muscle via an AMP-independent mechanism and leads to an increase in the rate of glucose transport, at least in part, via an AMPKα1-mediated mechanism.
Nmp4/CIZ regulation of matrix metalloproteinase 13 (MMP-13) response to parathyroid hormone in osteoblasts
Authors: Shah Rita , Alvarez Marta , Jones Daniel R. , Torrungruang Kitti , Watt Andrew J. , Selvamurugan Nagarajan , Partridge Nicola C. , Quinn Cheryl O. , Pavalko Fred M. , Rhodes Simon J. , Bidwell Joseph P.
Published: March 2004

Parathyroid hormone (PTH) regulation of matrix metalloproteinase-13 ( MMP-13) expression in osteoblasts contributes to normal bone turnover. The PTH response region of the rat MMP-13 gene spans nucleotides (nt) −148 to −38 and supports binding of numerous transcription factors, including Runx2, necessary for osteoblast differentiation, c-Fos/c-Jun, and Ets-1. These trans-acting proteins mediate hormone induction via incompletely defined combinatorial interactions. Within this region, adjacent to the distal Runx2 site, is a homopolymeric(dA:dT) element (−119/−110 nt) that conforms to the consensus site for the novel transcription factor nuclear matrix protein-4/cas interacting zinc finger protein (Nmp4/CIZ). This protein regulates bone cell expression of type I collagen and suppresses BMP2-enhanced osteoblast differentiation. The aim of this study was to determine whether Nmp4/CIZ contributes to MMP-13 basal transcription and PTH responsiveness in osteoblasts. Electrophoretic mobility shift analysis confirms Nmp4/CIZ binding within the MMP-13 PTH response region. Mutation of the Nmp4/CIZ element decreases basal activity of an MMP-13 promoter-reporter construct containing the first 1329 nt of the 5′-regulatory region, and overexpression of Nmp4/CIZ protein enhances the activity of the wild-type promoter. The same mutation of the homopolymeric(dA:dT) element enhances the MMP-13 response to PTH and PGE2. Overexpression of Nmp4/CIZ diminishes hormone induction. Mutation of both the homopolymeric(dA:dT) element and the adjacent Runx2 site further augments the PTH response. On the basis of these data and previous studies, we propose that Nmp4/CIZ is a component of a multiprotein assemblage or enhanceosome within the MMP-13 PTH response region and that, within this context, Nmp4/CIZ promotes both basal expression and hormonal synergy.
Pituitary control of lipoprotein and bile acid metabolism in male rats: growth hormone effects are not mediated by prolactin
Authors: Matasconi Manuela , Angelin Bo , Rudling Mats
Published: March 2004

Previous studies have established that growth hormone (GH) has many important effects on the regulation of cholesterol and lipoprotein metabolism. However, human GH (hGH) can also bind to prolactin receptors, eliciting prolactin receptor-mediated effects. In this study, we evaluated whether hGH can exert such responses in currently used animal models and whether prolactin affects lipoprotein and/or hepatic cholesterol metabolism. Normal and hypophysectomized (Hx) male rats were given either hGH or bovine GH, the latter unable to bind to the prolactin receptor. The hormones were continuously infused by use of subcutaneous osmotic mini-pumps for 7 days; blood and livers were collected after euthanasia. Both hormones stimulated hepatic LDL receptor expression and bile acid synthesis to a similar extent and normalized the altered plasma lipoprotein pattern in Hx rats. Prolactin, injected twice daily to Hx male rats, did not exert any effects on the plasma lipoprotein pattern or on cholesterol metabolism. We conclude that previously established effects of hGH on cholesterol metabolism are not mediated by prolactin in male rats.
Cold-induced thermogenesis mediated by GABA in the preoptic area of anesthetized rats
Authors: Osaka Toshimasa
Published: March 2004

Bilateral microinjections of GABA (300 mM, 100 nl) or the GABAAreceptor agonist muscimol (100 μM, 100 nl) into the preoptic area (POA) of the hypothalamus increased the rate of whole body O2consumption (V̇o2) and the body core (colonic) temperature of urethane-chloralose-anesthetized, artificially ventilated rats. The most sensitive site was the dorsomedial POA at the level of the anterior commissure. The GABA-induced thermogenesis was accompanied by a tachycardic response and electromyographic (EMG) activity recorded from the femoral or neck muscles. Pretreatment with muscle relaxants (1 mg/kg pancuronium bromide + 4 mg/kg vecuronium bromide iv) prevented GABA-induced EMG activity but had no significant effect on GABA-induced thermogenesis. However, pretreatment with the β-adrenoceptor propranolol (5 mg/kg iv) greatly attenuated the GABA-induced increase in V̇o2and tachycardic responses. Accordingly, the GABA-induced increase in V̇o2reflected mainly nonshivering thermogenesis. On the other hand, cooling of the shaved back of the rat by contact with a plastic bag containing 28°C water also elicited thermogenic, tachycardic, and EMG responses. Bilateral microinjections of the GABAAreceptor antagonist bicuculline (500 μM, 100 nl), but not the vehicle saline, into the POA blocked these skin cooling-induced responses. These results suggest that GABA and GABAAreceptors in the POA mediate cold information arising from the skin for eliciting cold-induced thermogenesis.
Mice with deletion of the mitochondrial glycerol-3-phosphate dehydrogenase gene exhibit a thrifty phenotype: effect of gender
Authors: Alfadda Assim , DosSantos Rosangela A. , Stepanyan Zaruhi , Marrif Husnia , Silva J. Enrique
Published: March 2004

To define the role of mitochondrial glycerol-3-phosphate dehydrogenase (mGPD; EC 1.1.99.5 ) in energy balance and intermediary metabolism, we studied transgenic mice not expressing mGPD (mGPD−/−). These mice had ≈14% lower blood glucose; ≈50% higher serum glycerol; ≈80% higher serum triglycerides; and at thermoneutrality, their energy expenditure (Qo2) was 15% lower than in wild-type (WT) mice. Glycerol-3-phosphate levels and lactate-to-pyruvate ratios were threefold elevated in muscle, but not in liver, of mGPD−/− mice. WT and mGPD−/− mice were then challenged with a high-fat diet, fasting, or food restriction. The high-fat diet caused more weight gain and adiposity in mGPD−/− than in WT female mice, without the genotype differentially affecting Qo2or energy intake. After a 30-h fast, WT female lost 60% more weight than mGPD−/− mice but these latter became more hypothermic. When energy intake was restricted to 50–70% of the ad libitum intake for 10 days, mGPD−/− female mice lost less weight than WT controls, but they had lower Qo2and body temperature. WT and mGPD−/− male mice did not differ significantly in their responses to these challenges. These results show that the lack of mGPD causes significant alterations of intermediary metabolism, which are more pronounced in muscle than liver and lead to a thrifty phenotype that is more marked in females than males. Lower T4-to-T3conversion in mGPD−/− females and a greater reliance of normal females on mGPD to respond to high-fat diets make the lack of the enzyme more consequential in the female gender.
Daily exercise facilitates phase delays of circadian melatonin rhythm in very dim light
Authors: Barger Laura K. , Wright Kenneth P. , Hughes Rod J. , Czeisler Charles A.
Published: March 2004

Shift workers and transmeridian travelers are exposed to abnormal work-rest cycles, inducing a change in the phase relationship between the sleep-wake cycle and the endogenous circadian timing system. Misalignment of circadian phase is associated with sleep disruption and deterioration of alertness and cognitive performance. Exercise has been investigated as a behavioral countermeasure to facilitate circadian adaptation. In contrast to previous studies where results might have been confounded by ambient light exposure, this investigation was conducted under strictly controlled very dim light (standing ∼0.65 lux; angle of gaze) conditions to minimize the phase-resetting effects of light. Eighteen young, fit males completed a 15-day randomized clinical trial in which circadian phase was measured in a constant routine before and after exposure to a week of nightly bouts of exercise or a nonexercise control condition after a 9-h delay in the sleep-wake schedule. Plasma samples collected every 30–60 min were analyzed for melatonin to determine circadian phase. Subjects who completed three 45-min bouts of cycle ergometry each night showed a significantly greater shift in the dim light melatonin onset (DLMO25%), dim light melatonin offset, and midpoint of the melatonin profile compared with nonexercising controls (Student t-test; P < 0.05). The magnitude of phase delay induced by the exercise intervention was significantly dependent on the relative timing of the exercise after the preintervention DLMO25%( r = −0.73, P < 0.05) such that the closer to the DLMO25%, the greater the phase shift. These data suggest that exercise may help to facilitate circadian adaptation to schedules requiring a delay in the sleep-wake cycle.
Tension- and afferent input-associated responses of neuromuscular system of rats to hindlimb unloading and/or tenotomy
Authors: Kawano F. , Ishihara A. , Stevens J. L. , Wang X. D. , Ohshima S. , Horisaka M. , Maeda Y. , Nonaka I. , Ohira Y.
Published: March 2004

Responses of electromyogram (EMG) in soleus muscle and both afferent and efferent neurograms at the fifth lumbar (L5) segmental level of spinal cord were investigated during acute and chronic unloading induced by hindlimb suspension and/or tenotomy in adult rats. The soleus EMG and afferent neurogram decreased 88 and 37%, respectively, relative to those at quadrupedal posture on the floor after acute hindlimb suspension that causes passive shortening of soleus due to ankle plantarflexion. However, the afferent neurogram ( P < 0.05) and soleus EMG ( P > 0.05) recorded on the floor increased after tenotomy of synergists. Furthermore, the afferent input was inhibited when the soleus EMG disappeared after tenotomy of soleus. The afferent neurogram and EMG of the soleus showed correlated responses to a variety of treatments, suggesting that the afferent neurogram recorded at the L5segmental level reflects the neural input associated with the activity level of the soleus predominantly. The level of efferent neurogram decreased after acute hindlimb suspension but was not influenced significantly by tenotomy of synergists and/or soleus itself. The EMG and afferent neurograms remained low up to the 4th day but recovered to the preexperimental levels within 14 days, due to reorganization of sarcomere number and length, as well as the shortening of muscle fiber length and recovery of tension development. It is suggested that the levels of EMG and afferent neurogram associated with antigravity muscle are closely related to the tension development of the muscle.
Effect of photoperiod on the mechanical response of the pregnant rabbit uterus to oxytocin
Authors: Ninomiya-Alarcón J. G. , Hudson R. , Reyes-Guerrero G. , Barrera-Mera B. , Guevara-Guzmán R.
Published: March 2004

We present findings suggesting that photoperiod is important in determining the sensitivity of the late-pregnant rabbit uterus to oxytocin (OT). Longitudinal myometrial strips were taken from term-pregnant and estrous rabbits and mounted in an organ bath for isometric myographic recording at different times during a 16:8-h light-dark cycle (lights on 0600–2200; n = 5/group), and the strength of contractions was registered in response to the application of OT or KCl. Strength of contractions was dose dependent and was up to 200 times greater at doses three to four orders of magnitude lower in tissue taken from pregnant animals during the light phase (0700 and 1300) than during the dark phase (2400 and 0400). Strips from nonpregnant estrous females also showed greater sensitivity and contractile force when taken in the light (0700) than in the dark (0400), although the differences were not significant. Consistent with the influence of photoperiod on uterine sensitivity to OT, strips taken from two groups of pregnant females ( n = 5/group) maintained on a light-dark cycle advanced 12 h showed significantly greater sensitivity and force in response to OT during the new subjective light than during the new subjective dark phase. The photoperiod-dependent contractile response to OT was specific and not simply the result of a change in general mechanical properties of the muscle, because administration of KCl resulted in dose-dependent contractions of similar magnitude in both the light and dark phase. These results are consistent with the fact that rabbits, like other nocturnal mammals, typically give birth during the day.
Renal expression of osmotically responsive cation channel TRPV4 is restricted to water-impermeant nephron segments
Authors: Tian Wei , Salanova Michele , Xu Hongshi , Lindsley Jessie N. , Oyama Terry T. , Anderson Sharon , Bachmann Sebastian , Cohen David M.
Published: March 2004

TRPV4, a nonselective cation channel of the transient receptor potential (TRP) family, is gated by hypotonicity. Expression of TRPV4 mRNA has been detected in the circumventricular organs of the brain responsible for sensing systemic tonicity and in the kidney distal convoluted tubule (DCT), among other sites. No analysis of TRPV4 expression at the protein level has been undertaken and no systematic analysis of expression of this channel has been reported in the kidney. Via RNAse protection assay and immunoblotting, abundant expression of TRPV4 was detected in the cortex, medulla, and papilla. The expression pattern of TRPV4 was characterized in both rat and mouse kidney, which revealed similar patterns of immunoreactivity. TRPV4 expression was absent from the proximal tubule (PT) and descending thin limb (DTL), whereas the strongest expression was observed in the ascending thin limb (ATL). The thick ascending limb (TAL) was strongly positive as was the DCT and connecting tubule. Importantly, the water-permeant cells of the macula densa were unstained. Moderate TRPV4 expression was noted in all collecting duct portions and in papillary epithelium; intercalated cells (type A) exhibited a particularly strong signal. In all positive segments, TRPV4 expression was concentrated at the basolateral membrane. Therefore, TRPV4 is expressed in only those nephron segments that are constitutively (i.e., ATL, TAL, and DCT) or conditionally (i.e., collecting duct) water impermeant and where generation of a substantial transcellular osmotic gradient could be expected. TRPV4 expression is absent from nephron segments exhibiting constitutive water permeability and unregulated apical aquaporin expression (i.e., PT and DTL). These data, although circumstantial, are consistent with a role for TRPV4 in the response to anisotonicity in the mammalian kidney.
Reproducible measurement of voluntary activation of human elbow flexors with motor cortical stimulation
Authors: Todd Gabrielle , Taylor Janet L. , Gandevia S. C.
Published: March 2004

Voluntary activation of muscle is commonly quantified by comparison of the extra force added by motor nerve stimulation during a contraction [superimposed twitch (SIT)] with that produced at rest by the same stimulus (resting twitch). An inability to achieve 100% voluntary activation implies that failure to produce maximal force output from the muscle must have occurred at a site at or above the level of the motoneurons. We have used cortical stimulation to quantify voluntary activation. Here, incomplete activation implies a failure at or above the level of motor cortical output. With cortical stimulation, it is inappropriate to compare extra force evoked during a contraction with the twitch evoked in resting muscle because motor cortical and spinal cord excitability both increase with activity. However, an appropriate “resting twitch” can be estimated. We previously estimated its amplitude by extrapolation of the linear relation between SIT amplitude and voluntary torque calculated from 35 contractions of >50% maximum (Todd G, Taylor JL, and Gandevia SC. J Physiol 551: 661–671, 2003). In this study, we improved the utility of this method to enable evaluation of voluntary activation when it may be changing over time, such as during the development of fatigue, or in patients who may be unable to perform large numbers of contractions. We have reduced the number of contractions required to only three. Estimation of the resting twitch from three contractions was reliable over time with low variability. Furthermore, its reliability and variability were similar to the resting twitch estimated from 30 contractions and to that evoked by conventional motor nerve stimulation.
Contraction-mediated mTOR, p70S6k, and ERK1/2 phosphorylation in aged skeletal muscle
Authors: Parkington Jascha D. , LeBrasseur Nathan K. , Siebert Adam P. , Fielding Roger A.
Published: March 2004

With age, skeletal muscle experiences substantial atrophy and weakness. Although resistance training can increase muscle size and strength, the myogenic response to exercise and the capacity for muscle hypertrophy in older humans and animals is limited. In the present study, we assessed the ability of muscle contractile activity to activate cellular pathways involved in muscle cell growth and myogenesis in adult (Y; 6 mo old) and aged (O; 30 mo old) Fischer 344 × Brown Norway rats. A single bout of rat hindlimb muscle contractile activity was elicited by high-frequency electrical stimulation (HFES) of the sciatic nerve. Plantaris (Pla) and tibialis anterior (TA) muscles were assayed for mammalian target of rapamycin (mTOR), 70-kDa ribosomal protein S6 kinase (p70S6K), and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and total protein either at baseline, immediately after, or 6 h after HFES. mTOR phosphorylation was elevated in Pla (1.3 ± 0.3-fold, P < 0.05) immediately after HFES and to a lesser extent 6 h after HFES (0.6 ± 0.1-fold, P < 0.05) in O rats. Post-HFES, p70S6K phosphorylation increased 1.2 ± 0.3-fold in TA ( P < 0.05) and remained elevated 6 h later (0.6 ± 0.2-fold, P < 0.05) in O rats. ERK phosphorylation was lower in O rats immediately after exercise in both TA (11.1 ± 2.9 vs. 2.1 ± 0.5-fold, P < 0.05) and Pla (6.5 ± 1.5 vs. 1.8 ± 0.5-fold, P < 0.05) and returned to baseline by 6 h in both Y and O rats. Phosphorylation of mTOR, p70S6K, and ERK1/2 are increased in skeletal muscle after a single bout of in situ muscle contractile activity in aged animals, and the response is less than that observed in adult animals. These observations suggest that the anabolic response to a single bout of contraction is attenuated with aging and may help explain the reduced capacity for hypertrophy in aged animals.
Dorsal Spinal Interneurons Forming a Primitive, Cutaneous Sensory Pathway
Authors: Li W.-C. , Soffe S. R. , Roberts Alan
Published: March 2004

In mammals, sensory projection pathways are provided by just three classes of spinal interneuron that develop from the roof-plate. We asked whether similar sensory projection interneurons are present primitively in a developing lower vertebrate where function can be more readily studied. Using an immobilized Xenopus tadpole spinal cord preparation, we define the properties and connections of spinal sensory projection interneurons using whole cell patch recordings from single neurons or pairs, identified by dye filling. Dorsolateral interneurons lie in the tadpole equivalent of the spinal dorsal horn, and have dorsally located dendrites and an ipsilateral ascending axon that projects into the midbrain. These neurons receive direct, mainly AMPA receptor (AMPAR)-mediated, excitation from skin touch sensory neurons. They in turn produce AMPAR and N-methyl-d-aspartate receptor (NMDAR)-mediated excitation of spinal locomotor pattern generator neurons rostrally on the same side of the cord. During swimming, they receive glycinergic modulatory inhibition from ascending interneurons in the spinal locomotor central pattern generator. We conclude that spinal dorsolateral interneurons are a primitive class of excitatory sensory projection neurons activated by ipsilateral cutaneous afferents and carrying excitation ipsilaterally and rostrally as far as the midbrain to initiate or accelerate swimming.
Interlimb Transfer of Novel Inertial Dynamics Is Asymmetrical
Authors: Wang Jinsung , Sainburg Robert L.
Published: March 2004

Mechanisms underlying interlimb transfer of adaptation to visuomotor rotations have recently been explored in depth. However, little data are available regarding interlimb transfer of adaptation to novel inertial dynamics. The present study thus investigated interlimb transfer of dynamics by examining the effect of initial training with one arm on subsequent performance with the other in adaptation to a 1.5-kg mass attached eccentrically to the forearm. Using inverse dynamic analysis, we examined the changes in torque strategies associated with adaptation to the extra mass, and with interlimb transfer of that adaptation. Following initial training with the dominant arm, nondominant arm performance improved substantially in terms of linearity and initial direction control as compared with naïve performance. However, initial training with the nondominant arm had no effect on subsequent performance with the dominant arm. Inverse dynamic analysis revealed that improvements in kinematics were implemented by increasing flexor muscle torques at the elbow to counter load-induced increases in extensor interaction torques as well as increasing flexor muscle torques at the shoulder to counter the extensor actions of elbow muscle torque. Following opposite arm adaptation, the nondominant arm adopted this dynamic strategy early in adaptation. These findings suggest that dominant arm adaptation to novel inertial dynamics leads to information that can be accessed and utilized by the opposite arm controller, but not vice versa. When compared with our previous findings on interlimb transfer of visuomotor rotations, our current findings suggest that adaptations to visuomotor and dynamic transformations are mediated by distinct neural mechanisms.
Sleep-Wake Related Discharge Properties of Basal Forebrain Neurons Recorded With Micropipettes in Head-Fixed Rats
Authors: Lee Maan Gee , Manns Ian D. , Alonso Angel , Jones Barbara E.
Published: March 2004

The basal forebrain has been shown to play an important role in cortical activation of wake and paradoxical sleep (PS), yet has also been posited to play a role in slow wave sleep (SWS). In an effort to determine whether these different roles may be fulfilled by different cell groups, including cholinergic and GABAergic cells, we recorded from 123 units in waking-sleeping, head-fixed rats using micropipettes to allow juxtacellular labeling. Functional sets of intermingled cell groups emerged as units whose discharge was as follows: 1) maximum in active wake (aW) and positively or not correlated with EEG gamma activity, while positively correlated with nuchal EMG activity, and thus potentially facilitatory for waking and behavioral arousal (12%); 2) maximum in SWS or SWS-PS and positively correlated with delta EEG activity, while not or negatively correlated with EMG activity, and thus potentially promotive for sleep with cortical slow wave activity and/or accompanying behavioral changes (16%); 3) maximum in PS or PS and aW and positively correlated with gamma and theta EEG activity, while negatively or not correlated with EMG activity, and thus potentially promotive for cortical activation during PS or PS and W (62%); and 4) equivalent across all states and thus not involved in state regulation (∼10%). Units of each group also manifested different firing patterns typified as slow tonic (19.5%), fast tonic (32.5%), or fast phasic (48%), including rhythmic bursting (6%). Through these diverse cell groups, the basal forebrain has the capacity to modulate cortical activity, behavior, and/or related physiological processes across the sleep-waking cycle and thereby regulate the sleep-wake state of the animal.
Rhythmic Motor Activity in Thin Transverse Slice Preparations of the Fetal Rat Spinal Cord
Authors: Nakayama Kiyomi , Nishimaru Hiroshi , Kudo Norio
Published: March 2004

Networks generating locomotor-like rhythmic motor activity are formed during the last week of the fetal period in the rat spinal cord. We investigated the coordinated rhythmic motor activity induced in transverse slice preparations of the lumbar spinal cord taken from fetal rats as early as embryonic day (E) 16.5. In slices as thin as 100 μm, bath-application of 5-hydroxytryptamine (5-HT) induced rhythmic [Ca2+]i elevations in motoneurons labeled with Calcium Green-1 dextran. The rhythmic [Ca2+]i elevations were similar in frequency to that in the intact lumbar spinal cord, although there was no temporal correlation between the activity in the left and right sides of 100-μm slices. Such rhythmic [Ca2+]i elevations were observed in the slices taken from all lumbar segments. Moreover, the rhythmic activity was abolished by simultaneous blockade of glutamate, glycine, and GABAA receptors, indicating that synaptic transmission mediated by these receptors is important for the generation of the rhythm in these slices. Synchronous rhythmic activity between the left-right sides was found in slices thicker than 200 μm taken from any segmental level of the lumbar spinal cord. In these preparations, commissural neurons were activated synchronously with ipsilateral motoneurons. These results indicate that the neuronal networks sufficient to generate coordinated rhythmic activity are contained in one-half of a single lumbar segment at E16.5. Such spinal cord slices are a promising experimental model to investigate the neuronal mechanisms and the development of rhythm generation in the spinal cord.
Blockade of the Angiotensin II Type 1 Receptor Stabilizes Atherosclerotic Plaques in Humans by Inhibiting Prostaglandin E
2
–Dependent Matrix Metalloproteinase Activity
Authors: Cipollone Francesco , Fazia Maria , Iezzi Annalisa , Pini Barbara , Cuccurullo Chiara , Zucchelli Mirco , de Cesare Domenico , Ucchino Sante , Spigonardo Francesco , De Luca Mariella , Muraro Raffaella , Bei Roberto , Bucci Marco , Cuccurullo Franco , Mezzetti Andrea
Published: March 2004

Background—

Clinical trials have demonstrated that agents that inhibit the angiotensin II pathway confer benefit beyond the reduction of blood pressure alone. However, the molecular mechanism underlying this effect has yet to be investigated. Recently, we have demonstrated enhanced expression of inducible cyclooxygenase (COX) and prostaglandin (PG)E
2
-dependent synthase (COX-2/mPGES-1) in human symptomatic plaques and provided evidence that it is associated with metalloproteinase (MMP)-induced plaque rupture. Thus, the aim of this study was to characterize the effect of the angiotensin II type 1 (AT
1
) receptor antagonist irbesartan on the inflammatory infiltration and expression of COX-2/mPGES-1 and MMPs in human carotid plaques.

Methods and Results—

Seventy patients with symptomatic carotid artery stenosis were randomized to irbesartan (300 mg/d) or chlorthalidone (50 mg/d) for 4 months before endarterectomy. Plaques were subjected to analysis of COX-1, COX-2, mPGES-1, MMP-2, and MMP-9, angiotensin II, AT
1
, AT
2
, and collagen content by immunocytochemistry, Western blot, and reverse-transcriptase polymerase chain reaction, whereas zymography was used to detect MMP activity. Immunohistochemistry was also used to identify CD68+ macrophages, CD3+ T lymphocytes, smooth muscle cells (SMCs), and HLA-DR+ inflammatory cells. Plaques from the irbesartan group had fewer (
P
<0.0001) macrophages, T lymphocytes, and HLA-DR+ cells; less (
P
<0.0001) immunoreactivity for COX-2/mPGES-1 and MMPs; reduced (
P
<0.0001) gelatinolytic activity; and increased (
P
<0.0001) collagen content. It is worth noting that COX-2/mPGES-1 inhibition was observed after incubation in vitro with irbesartan but not with the selective AT
2
blockade PD123,319.

Conclusions—

This study demonstrates that irbesartan decreases inflammation and inhibits COX-2/mPGES-1 expression in plaque macrophages, and this effect may in turn contribute to plaque stabilization by inhibition of MMP-induced plaque rupture.
Sinoatrial Node Dysfunction and Early Unexpected Death of Mice With a Defect of
klotho
Gene Expression
Authors: Takeshita Kyosuke , Fujimori Toshihiko , Kurotaki Yoko , Honjo Haruo , Tsujikawa Hiroshi , Yasui Kenji , Lee Jong-Kook , Kamiya Kaichiro , Kitaichi Kiyoyuki , Yamamoto Koji , Ito Masafumi , Kondo Takahisa , Iino Shigeo , Inden Yasuya , Hirai Makoto , Murohara Toyoaki , Kodama Itsuo , Nabeshima Yo-ichi
Published: March 2004

Background—

Homozygous mutant mice with a defect of
klotho
gene expression (
kl/kl
) show multiple age-related disorders and premature death from unknown causes.

Methods and Results—

The
kl/kl
mice subjected to 20-hour restraint stress showed a high rate (20/30) of sudden death, which was associated with sinoatrial node dysfunction (conduction block or arrest). Heart rate and plasma norepinephrine of
kl/kl
mice, unlike those of wild-type (WT) mice, failed to increase during the stress. Intrinsic heart rate after pharmacological blockade of autonomic nerves in
kl/kl
mice was significantly lower than that in WT mice (380±33 versus 470±44 bpm; n=7). The sinus node recovery time after an overdrive pacing (600 bpm, 30 seconds) in
kl/kl
mice was significantly longer than in WT mice (392±37 versus 233±24 ms; n=6). In isolated sinoatrial node preparations, the positive chronotropic effect of isoproterenol was significantly less, whereas the negative chronotropic effect of acetylcholine was significantly greater in
kl/kl
than in WT mice. There was no degenerative structural change in the sinoatrial node of
kl/kl
mice. The precise localization of
klotho
was analyzed in newly prepared
klotho
-null mice with a reporter gene system (

kl
−geo

). Homozygous

kl
−geo

mice showed characteristic age-associated phenotypes that were almost identical to those of
kl/kl
mice. In the

kl
−geo

mice,
klotho
expression was recognized exclusively in the sinoatrial node region in the heart in addition to parathyroid, kidney, and choroid plexus.

Conclusions—

In the heart,
klotho
is expressed solely at the sinoatrial node.
klotho
gene expression is essential for the sinoatrial node to function as a dependable pacemaker under conditions of stress.
Constitutive Cardiac Overexpression of Sarcoplasmic/Endoplasmic Reticulum Ca
2+
-ATPase Delays Myocardial Failure After Myocardial Infarction in Rats at a Cost of Increased Acute Arrhythmias
Authors: Chen Ying , Escoubet Brigitte , Prunier Fabrice , Amour Julien , Simonides Warner S. , Vivien Benoît , Lenoir Christophe , Heimburger Michèle , Choqueux Christine , Gellen Barnabas , Riou Bruno , Michel Jean-Baptiste , Franz Wolfgang M. , Mercadier Jean-Jacques
Published: March 2004

Background—

Heart failure often complicates myocardial infarction (MI), and sarcoplasmic/endoplasmic reticulum Ca
2+
-ATPase (SERCA2a) is underexpressed in the failing myocardium. We examined the effect of preexisting cardiac SERCA2a protein overexpression on rat survival and left ventricular (LV) remodeling after MI.

Methods and Results—

Baseline myocardial SERCA2a expression was 37% higher in transgenic (TG) rats than in their wild-type (WT) controls, consistent with enhanced myocardial function. The mortality rate of TG rats during the 24 hours after surgical MI was higher than that of WT rats (71% versus 35%,
P
<0.001), associated with a higher frequency of ventricular arrhythmias, and was normalized by lidocaine treatment. The increased acute-phase mortality in TG rats was not accompanied by increased 6-month mortality. Function of the noninfarcted myocardium, as assessed by tissue Doppler imaging, was higher in TG rats than in WT rats for up to 1 month after MI, a beneficial effect no longer observed at 3 months. LV remodeling and global function were similar in TG and WT rats. No difference in papillary muscle function was found at 6 months.

Conclusions—

Constitutive cardiac SERCA2a overexpression has a transient beneficial effect on remote myocardium function in rat MI, with no improvement in LV global function or prevention of LV remodeling and failure. This benefit is associated with a higher risk of acute mortality, which is prevented by lidocaine treatment.
AT
1
Receptor Mediated Augmentation of Intrarenal Angiotensinogen in Angiotensin II-Dependent Hypertension
Authors: Kobori Hiroyuki , Prieto-Carrasquero Minolfa C. , Ozawa Yuri , Navar L. Gabriel
Published: March 2004

Angiotensin (Ang) II-infused hypertensive rats exhibit increases in renal angiotensinogen mRNA and protein, as well as urinary angiotensinogen excretion in association with increased intrarenal Ang II content. The present study was performed to determine if the augmentation of intrarenal angiotensinogen requires activation of Ang II type 1 (AT
1
) receptors. Male Sprague-Dawley rats (200 to 220 g) were divided into 3 groups: sham surgery (n=10), subcutaneous infusion of Ang II (80 ng/min, n=11), and Ang II infusion plus AT
1
blocker (ARB), olmesartan (5 mg/d, n=12). Ang II infusion progressively increased systolic blood pressure (SBP) compared with sham (178±8 mm Hg versus119±4 at day 11). ARB treatment prevented hypertension (113±6 at day 11). Twenty-four-hour urine collections were taken at day 12, and plasma and tissue samples were harvested at day 13. The Ang II+ARB group had a significant increase in plasma Ang II compared with Ang II and sham groups (365±46 fmol/mL versus 76±9 and 45±14, respectively). Nevertheless, ARB treatment markedly limited the enhancement of kidney Ang II by Ang II infusion (65±17 fmol/g in sham, 606±147 in Ang II group, and 288±28 in Ang II+ARB group). Ang II infusion significantly increased kidney angiotensinogen compared with sham (1.69±0.21 densitometric units versus 1.00±0.17). This change was reflected by increased angiotensinogen immunostaining in proximal tubules. ARB treatment prevented this increase (1.14±0.12). Urinary angiotensinogen excretion rates were enhanced 4.7× in Ang II group (4.67±0.41 densitometric units versus 1.00±0.21) but ARB treatment prevented the augmentation of urinary angiotensinogen (0.96±0.23). These data demonstrate that augmentation of intrarenal angiotensinogen in Ang II-infused rats is AT
1
-dependent and provide further evidence that urinary angiotensinogen is closely linked to intrarenal Ang II in Ang II-dependent hypertension.
Sphingosine-1-Phosphate Inhibits C-Type Natriuretic Peptide Activation of Guanylyl Cyclase B (GC-B/NPR-B)
Authors: Abbey-Hosch Sarah E. , Cody Alyssa N. , Potter Lincoln R.
Published: March 2004

C-type natriuretic peptide (CNP) binds and activates the transmembrane guanylyl cyclase B receptor (NPR-B), which decreases vascular tone and inhibits cell proliferation and migration. In contrast, the bioactive lipid sphingosine-1-phosphate (S1P) elicits the opposite physiological effects. Here, we demonstrate a potent acute inhibitory effect of S1P on NPR-B activity in NIH3T3 fibroblasts and A10 vascular smooth muscle cells. In fibroblasts, S1P reduced CNP-dependent cGMP elevations to the same levels as 10% fetal bovine serum, the most potent NPR-B desensitizing agent known. The reduction was dose-dependent (IC
50
=0.08 μmol/L) and due to decreased NPR-B activity because CNP-dependent guanylyl cyclase activities were markedly diminished in membranes prepared from S1P-treated cells. Similarly, in A10 cells, S1P inhibition was rapid (t
1/2
=2 to 5 minutes), dose-dependent (IC
50
=0.3 μmol/L S1P), and mediated by a cell surface receptor. The mechanism of the S1P-dependent desensitization in A10 cells did not require NPR-B degradation or protein kinase C activation, but did require elevated calcium concentrations because a nonspecific calcium ionophore also inhibited NPR-B and an intracellular calcium chelator blocked a significant portion of the S1P response. These are the first data demonstrating cross-talk between the natriuretic peptide and S1P signaling systems. They suggest that the effects of S1P on vascular disease and wound healing may be mediated in part through inhibition of NPR-B.
α-2 Adrenergic Transmission and Human Baroreflex Regulation
Authors: Tank Jens , Diedrich Andre , Szczech Elke , Luft Friedrich C. , Jordan Jens
Published: March 2004

We observed earlier that central α-2 adrenoceptor stimulation in mice greatly augments parasympathetic tone. To test the effects in humans, we assessed autonomic vasomotor tone and baroreflex regulation in 9 normal young adults on 2 occasions, once with and once without clonidine. We determined heart rate (HR), beat-by-beat blood pressure (BP), and muscle sympathetic nerve activity. HR variability was analyzed in the time and frequency domain. Pharmacological baroreflex slopes were determined using incremental phenylephrine and nitroprusside infusions. Clonidine lowered resting BP (122±4/73±3 versus 100±7/55±3 mm Hg,
P
<0.01), muscle sympathetic nerve activity (18±3 versus 4±2 bursts/min,
P
<0.01), and HR (62±3 versus 56±3 bpm,
P
<0.05). The baroreflex heart rate curve was reset to much lower HR values and showed no saturation at low HR. HR variability profoundly increased during clonidine plus phenylephrine (total power: 3224±843 versus 8943±2329 ms
2
,
P
<0.05). High-frequency power was 1451±520 at baseline and 6720±2475 ms
2
during baroreceptor loading (
P
<0.05). The low-frequency/high-frequency ratio decreased (1.94±0.41 versus 0.69±0.10,
P
<0.05). In contrast, clonidine reduced resting sympathetic vasomotor tone and shifted the operating point of the sympathetic baroreflex to a flat part of the sympathetic baroreflex curve. The shift decreased the ability of the baroreflex to withdraw sympathetic vasomotor tone during baroreflex loading. These baroreflex changes were associated with a moderate increase in phenylephrine responsiveness. We conclude that α-2 adrenoceptor stimulation has a differential effect on baroreflex HR and vasomotor regulation. α-2 Adrenoceptor stimulation greatly augments baroreflex-mediated bradycardia, most likely by parasympathetic activation.
Prolyl Oligopeptidase Is Involved in Release of the Antifibrotic Peptide Ac-SDKP
Authors: Cavasin Maria A. , Rhaleb Nour-Eddine , Yang Xiao-Ping , Carretero Oscar A.
Published: March 2004

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a ubiquitous tetrapeptide hydrolyzed almost exclusively by angiotensin-converting enzyme (ACE). Chronic treatment with Ac-SDKP decreases cardiac and renal fibrosis and inflammatory cell infiltration in hypertensive rats. However, very little is known about endogenous synthesis of Ac-SDKP, except that thymosin-β
4
may be the most likely precursor. Two enzymes are potentially able to release Ac-SDKP from thymosin-β
4
: prolyl oligopeptidase (POP) and endoproteinase asp-N. POP is widely present and active in several tissues and biological fluids, whereas endoproteinase asp-N appears to be lacking in mammals. Therefore, we hypothesized that POP is the main enzyme involved in synthesizing the antifibrotic peptide Ac-SDKP. We investigated in vitro and in vivo production of Ac-SDKP. Using kidney cortex homogenates, we observed that Ac-SDKP was generated in a time-dependent manner in the presence of exogenous thymosin-β
4
, and this generation was significantly inhibited by several POP inhibitors (POPi), Z-prolyl-prolinal, Fmoc-prolyl-pyrrolidine-2-nitrile, and S17092. Long-term administration of S17092 in rats significantly decreased endogenous levels of Ac-SDKP in the plasma (from 1.76±0.2 to 1.01±0.1 nM), heart (from 2.31±0.21 to 0.83±0.09 pmol/mg protein), and kidneys (from 5.62±0.34 to 2.86±0.76 pmol/mg protein). As expected, ACE inhibitors significantly increased endogenous levels of Ac-SDKP in the plasma, heart, and kidney, whereas coadministration of POPi prevented this increase. We concluded that POP is the main enzyme responsible for synthesis of the antifibrotic peptide Ac-SDKP.
Pulmonary Hypertension in Transgenic Mice Expressing a Dominant-Negative BMPRII Gene in Smooth Muscle
Authors: West James , Fagan Karen , Steudel Wolfgang , Fouty Brian , Lane Kirk , Harral Julie , Hoedt-Miller Marloes , Tada Yuji , Ozimek John , Tuder Rubin , Rodman David M.
Published: March 2004

Bone morphogenetic peptides (BMPs), a family of cytokines critical to normal development, were recently implicated in the pathogenesis of familial pulmonary arterial hypertension. The type-II receptor (BMPRII) is required for recognition of all BMPs, and targeted deletion of BMPRII in mice results in fetal lethality before gastrulation. To overcome this limitation and study the role of BMP signaling in postnatal vascular disease, we constructed a smooth muscle–specific transgenic mouse expressing a dominant-negative BMPRII under control of the tetracycline gene switch (SM22-tet-BMPRII
delx4+
mice). When the mutation was activated after birth, mice developed increased pulmonary artery pressure, RV/LV+S ratio, and pulmonary arterial muscularization with no increase in systemic arterial pressure. Studies with SM22-tet-BMPRII
delx4+
mice support the hypothesis that loss of BMPRII signaling in smooth muscle is sufficient to produce the pulmonary hypertensive phenotype.
Ultrasound Perfusion Imaging in Acute Middle Cerebral Artery Infarction Predicts Outcome
Authors: Seidel Günter , Meyer-Wiethe Karsten , Berdien Grit , Hollstein Dirk , Toth Daniel , Aach Til
Published: March 2004

Background and Purpose—

Initial reports indicate that transcranial harmonic imaging after ultrasound contrast agent bolus injection (BHI) can detect cerebral perfusion deficits in acute ischemic stroke. We evaluated parametric images of the bolus washout kinetics.

Methods—

Twenty-three patients with acute internal carotid artery infarction were investigated with perfusion harmonic imaging after SonoVue bolus injection ≤40 hour after the onset of symptoms. The findings were compared with those of cranial computed tomography (CCT) and clinical course 4 months after stroke.

Results—

Images of pixel-wise peak intensity (PPI) and time to peak intensity could be calculated for all patients. Spearman rank correlations of
r
=0.772 (
P
<0.001) and
r
=0.572 (
P
=0.008) between area of PPI signal decrease and area of infarction in the follow-up CCT as well as outcome after 4 months were obtained, respectively.

Conclusions—

In the early phase of acute ischemic stroke, BHI after SonoVue bolus injection is a useful ultrasound tool for analyzing cerebral perfusion deficits at the patient’s bedside. BHI data correlate with the definite area of infarction and outcome after 4 months.
Sample Size Calculations in Acute Stroke Trials: A Systematic Review of Their Reporting, Characteristics, and Relationship With Outcome
Authors: Weaver Chris S. , Leonardi-Bee Jo , Bath-Hextall Fiona J. , Bath Philip M.W.
Published: March 2004

Background and Purpose—

Only a few randomized controlled trials in acute stroke have shown a treatment-related benefit. Inadequate trial design, especially low sample size, may partly explain this failure. We investigated sample size calculations (SSCs) in a systematic review of acute stroke trials.

Methods—

Full reports of nonconfounded randomized controlled trials that recruited patients within 1 week of stroke onset and were published before the end of 2001 were identified from the Cochrane Library and other bibliographic databases. Information on the SSC and outcome event rates was collected for each trial.

Results—

Of 189 identified trial reports, 57 (30%) reported ≥1 components of the SSC, phase II 14/129 (11%) versus phase III 43/60 (72%) (
P
<0.001), with 32 (56%) giving all the required parameters. Significance (α) was mentioned in 54 (96%) reports; 53 used a significance level of α=0.05. And 55 (98%) reports gave the power (1−β) of the study (median [25th and 75th percentile] 0.80 [0.80, 0.90]). The anticipated percentage of control subjects having a primary outcome event was given in 24 (42%) articles: case fatality 21.8% (11.8%, 23.5%, n=4) and combined death or disability/dependency 55.5% (44.5%, 66.3%, n=20); 25 studies used other outcomes and 8 studies gave insufficient information. Four of the 22 trials achieved a control rate within 5% of their prediction. 49 (86%) reports gave the anticipated treatment effect; case fatality: anticipated 9.5% (1.1%, 12.5%, n=6), achieved −0.3% (−4.1%, +2.4%); combined death or disability/dependency: anticipated 13.0% (10.0%, 16.0%, n=25), achieved 1.8% (−0.5%, +5.4%). The median calculated sample size was 600 (198, 995, n=54).

Conclusions—

Too few trial publications report the assumptions underlying their SSC. Most trials were underpowered, ie, power <0.90, used inappropriate assumptions for event rates, and were grossly overoptimistic in their expectation of treatment effect. These deficiencies will together have resulted in trials being far too small and reduced their chance of being able to detect real treatment effects.