Agonist specific L-type Ca(2+)-current stimulation in ventricular myocytes by a novel steroid-like compound

In cardiac muscle the amplitude of Ca(2+) transients can be increased by enhancing Ca(2+) influx. Among the processes leading to increased Ca(2+) influx, agonists of the L-type Ca(2+)-channel can play an important role. Known pharmacological Ca(2+)-channel agonists act on different binding sites on the channel protein, which may lead not only to enhanced peak currents, but also to distinct changes in other biophysical characteristics of the current. In this study, membrane currents were recorded with the patch-clamp technique in the whole-cell configuration in guinea pig isolated ventricular myocytes in combination with confocal fluorescence Ca(2+) imaging techniques and a variety of pharmacological tools. Testing a new positive inotropic steroid-like compound, we found that it increased the L-type Ca(2+)-current by 2.5-fold by shifting the voltage-dependence of activation by 20.2 mV towards negative potentials. The dose-response relationship revealed two vastly different affinities (EC(50(high-affinity))=4.5+/-1.7 nM, EC(50(low-affinity))=8.0+/-1.1 microM) exhibiting differential pharmacological interactions with three classes of Ca(2+)-current antagonists, suggesting more than one binding site on the channel protein. Therefore, we identified and characterized a novel positive inotropic compound (F90927) as a member of a new class of Ca(2+)-channel agonists exhibiting unique features, which set it apart from other presently known L-type Ca(2+)-channel agonists.

Repetitive TMS over the human oculomotor cortex: comparison of 1-Hz and theta burst stimulation

The aim of the study was to compare the effect duration of two different protocols of repetitive transcranial magnetic stimulation (rTMS) on saccade triggering. In four experiments, two regions (right frontal eye field (FEF) and vertex) were stimulated using a 1-Hz and a theta burst protocol (three 30Hz pulses repeated at intervals of 100ms). The same number of TMS pulses (600 pulses) was applied with stimulation strength of 80% of the resting motor threshold for hand muscles. Following stimulation the subjects repeatedly performed an oculomotor task using a modified overlap paradigm, and saccade latencies were measured over a period of 60min. The results show that both 1-Hz and theta burst stimulation had inhibitory effects on saccade triggering when applied over the FEF, but not over the vertex. One-hertz rTMS significantly increased saccade latencies over a period of about 8min. After theta burst rTMS, this effect lasted up to 30min. Furthermore, the decay of rTMS effects was protocol-specific: After 1-Hz stimulation, saccade latencies returned to a baseline level much faster than after theta burst stimulation. We speculate that these time course differences represent distinct physiological mechanisms of how TMS interacts with brain function.

mRNA expression and binding sites for alpha2-adrenergic receptor subtypes in muscle layers of the ileum and spiral colon of dairy cows

OBJECTIVE: To measure maximum binding capacity (B(max)) and levels of mRNA expression for alpha(2)-adrenergic receptor (AR) subtypes in ileal and colonic muscle layers of healthy dairy cows. SAMPLE POPULATION: Ileal and colonic muscle specimens from 6 freshly slaughtered cows. PROCEDURES: Ileal and colonic muscle layers were obtained by scraping the mucosa and submucosa from full-thickness tissue specimens. Level of mRNA expression for alpha(2)-AR subtypes was measured by real-time reverse transcriptase-PCR analysis and expressed relative to the mean mRNA expression of glyceraldehyde phosphate dehydrogenase, ubiquitin, and 18S ribosomal RNA. Binding studies were performed with tritiated RX821002 ((3)H-RX821002) and subtype-selective ligands as competitors. RESULTS: mRNA expression for alpha(2AD)-, alpha(2B)-, and alpha(2C)-AR subtypes was similar in ileal and colonic muscle layers. The mRNA expression for alpha(2AD)-AR was significantly greater than that for alpha(2B)- and alpha(2C)-AR subtypes, representing 92%, 6%, and 2%, respectively, of the total mRNA. Binding competition of (3)H-RX821002 with BRL44408, imiloxan, and MK-912 was best fitted by a 1-site model. The B(max) of alpha(2AD)- and alpha(2C)-AR sub-types was greater than that of alpha(2B)-AR. The B(max) and level of mRNA expression were only correlated (r = 0.8) for alpha(2AD)-AR. Ratio of B(max) to mRNA expression for alpha(2C)-AR was similar to that for alpha(2B)-AR, but significantly greater than for alpha(2AD)-AR. CONCLUSIONS AND CLINICAL RELEVANCE: Subtypes of alpha(2)-AR in bovine intestinal muscle layers are represented by a mixture of alpha(2AD)- and alpha(2C)-ARs and of alpha(2B)-AR at a lower density. Information provided here may help in clarification of the role of AR subtypes in alpha(2)-adrenergic mechanisms regulating bovine intestinal motility.

Quantitative mRNA analysis of adrenergic receptor subtypes in the intestines of healthy dairy cows and dairy cows with cecal dilatation-dislocation

OBJECTIVE: To investigate the distribution of mRNA coding for 9 adrenoceptor subtypes in the intestines of healthy dairy cows and cows with cecal dilatationdislocation (CDD). SAMPLE POPULATION: Full-thickness specimens of the intestinal wall were obtained from the ileum, cecum, proximal loop of the ascending colon (PLAC), and external loop of the spiral colon (ELSC) of 15 cows with CDD (group 1) and 15 healthy (control) cows (group 2, specimens collected during laparotomy; group 3, specimens collected after slaughter). PROCEDURES: Concentrations of mRNA for 9 adrenoceptor subtypes (alpha(1A), alpha(1B), alpha(1D), alpha(2AD), alpha(2B), alpha(2C), beta(1), beta(2), and beta(3)) were measured by quantitative real-time reverse transcriptase-PCR assay. Results were expressed relative to mRNA expression of a housekeeping gene. RESULTS: Expression of mRNA for alpha(1B)-, alpha(2AD)-, alpha(2B)-, beta(1)-, and beta(2)-adrenoceptors was significantly lower in cows with CDD than in control cows. In the ileum, these receptors all had lower mRNA expression in cows with CDD than in control cows. The same effect was detected in the ELSC for mRNA for alpha(2AD)-, alpha(2B)-, beta(1)-, and beta(2)-adrenoceptors, and in the cecum and PLAC for alpha(2B)- and beta(2)-adrenoceptors. Groups did not differ significantly for alpha(1A)-adrenoceptors. The mRNA expression for alpha(1D)-, alpha(2C)-, and beta(3)-adrenoceptors was extremely low in all groups. CONCLUSIONS AND CLINICAL RELEVANCE: Differences in expression of mRNA coding for adrenoceptors, most pronounced in the ileum and spiral colon, between cows with CDD and control cows support the hypothesis of an implication of adrenergic mechanisms in the pathogenesis of CDD in dairy cows.

Apoptotic neutrophils release macrophage migration inhibitory factor upon stimulation with tumor necrosis factor-alpha

Macrophage migration inhibitory factor (MIF) is an important cytokine involved in the regulation of innate immunity and present at increased levels during inflammatory responses. Here we demonstrate that mature blood and tissue neutrophils constitutively express MIF as a cytosolic protein not associated with azurophil granules. Functionally active MIF, but not proteases stored in azurophil granules, was released from apoptotic neutrophils following short term tumor necrosis factor (TNF)-alpha stimulation in a caspase-dependent manner and prior to any detectable phagocytosis by monocyte-derived macrophages. Moreover, TNF-alpha-mediated MIF release was blocked by glyburide and propenicide, both inhibitors of ATP-binding cassette-type transporters, suggesting that this transporter system is activated during neutrophil apoptosis. Taken together, apoptotic mature neutrophils release MIF upon short term TNF-alpha stimulation. Therefore, apoptosis may not always occur without the induction of pro-inflammatory mechanisms.

Evaluation of administration of isoflurane at approximately the minimum alveolar concentration on depression of a nociceptive withdrawal reflex evoked by transcutaneous electrical stimulation in ponies

OBJECTIVE: To investigate effects of isoflurane at approximately the minimum alveolar concentration (MAC) on the nociceptive withdrawal reflex (NWR) of the forelimb of ponies as a method for quantifying anesthetic potency. ANIMALS: 7 healthy adult Shetland ponies. PROCEDURE: Individual MAC (iMAC) for isoflurane was determined for each pony. Then, effects of isoflurane administered at 0.85, 0.95, and 1.05 iMAC on the NWR were assessed. At each concentration, the NWR threshold was defined electromyographically for the common digital extensor and deltoid muscles by stimulating the digital nerve; additional electrical stimulations (3, 5, 10, 20, 30, and 40 mA) were delivered, and the evoked activity was recorded and analyzed. After the end of anesthesia, the NWR threshold was assessed in standing ponies. RESULTS: Mean +/- SD MAC of isoflurane was 1.0 +/- 0.2%. The NWR thresholds for both muscles increased significantly in a concentration-dependent manner during anesthesia, whereas they decreased in awake ponies. Significantly higher thresholds were found for the deltoid muscle, compared with thresholds for the common digital extensor muscle, in anesthetized ponies. At each iMAC tested, amplitudes of the reflex responses from both muscles increased as stimulus intensities increased from 3 to 40 mA. A concentration-dependent depression of evoked reflexes with reduction in slopes of the stimulus-response functions was detected. CONCLUSIONS AND CLINICAL RELEVANCE: Anesthetic-induced changes in sensory-motor processing in ponies anesthetized with isoflurane at concentrations of approximately 1.0 MAC can be detected by assessment of NWR. This method will permit comparison of effects of inhaled anesthetics or anesthetic combinations on spinal processing in equids.

Muscle metabolites: functional MR spectroscopy during exercise imposed by tetanic electrical nerve stimulation

Permission from the ethics committee and informed consent were obtained. The purpose of this study was to prospectively evaluate a method developed for the noninvasive assessment of muscle metabolites during exercise. Hydrogen 1 magnetic resonance (MR) spectroscopy peaks were measured during tetanic isometric muscle contraction imposed by supramaximal repetitive nerve stimulation. The kinetics of creatine-phosphocreatine and acetylcarnitine signal changes (P < .001) could be assessed continuously before, during, and after exercise. The control peak (trimethylammonium compounds), which served as an internal reference, did not change. This technique-that is, functional MR spectroscopy-opens the possibility for noninvasive diagnostic muscle metabolite testing in a clinical setting.

One-Hertz transcranial magnetic stimulation over the frontal eye field induces lasting inhibition of saccade triggering

The aim of the study was to examine the effect of low-frequency repetitive transcranial magnetic stimulation on saccade triggering. In five participants, a train of 600 pulses with a frequency of 1 Hz was applied over the right frontal eye field and--as control condition--over the vertex. After repetitive transcranial magnetic stimulation application, oculomotor performance was evaluated with an overlap paradigm. The results show that the repetitive transcranial magnetic stimulation effect was specific for frontal eye field stimulation. Saccade latencies were found to be increased bilaterally for several minutes after the stimulation, and the time course of recovery was different for the ipsilateral and contralateral sides. The results are discussed in the light of possible local and remote repetitive transcranial magnetic stimulation effects on the oculomotor network.

An integrative transcranial magnetic stimulation mapping technique using non-linear curve fitting

The aim of this study is to develop a new simple method for analyzing one-dimensional transcranial magnetic stimulation (TMS) mapping studies in humans. Motor evoked potentials (MEP) were recorded from the abductor pollicis brevis (APB) muscle during stimulation at nine different positions on the scalp along a line passing through the APB hot spot and the vertex. Non-linear curve fitting according to the Levenberg-Marquardt algorithm was performed on the averaged amplitude values obtained at all points to find the best-fitting symmetrical and asymmetrical peak functions. Several peak functions could be fitted to the experimental data. Across all subjects, a symmetric, bell-shaped curve, the complementary error function (erfc) gave the best results. This function is characterized by three parameters giving its amplitude, position, and width. None of the mathematical functions tested with less or more than three parameters fitted better. The amplitude and position parameters of the erfc were highly correlated with the amplitude at the hot spot and with the location of the center of gravity of the TMS curve. In conclusion, non-linear curve fitting is an accurate method for the mathematical characterization of one-dimensional TMS curves. This is the first method that provides information on amplitude, position and width simultaneously.

Stimulation induced variability of pulse plethysmography does not discriminate responsiveness to intubation

BACKGROUND: Hypnotic depth but not haemodynamic response to painful stimulation can be measured with various EEG-based anaesthesia monitors. We evaluated the variation of pulse plethysmography amplitude induced by an electrical tetanic stimulus (PPG variation) as a potential measure for analgesia and predictor of haemodynamic responsiveness during general anaesthesia. METHODS: Ninety-five patients, ASA I or II, were randomly assigned to five groups [Group 1: bispectral index (BIS) (range) 40-50, effect site remifentanil concentration 1 ng ml(-1);Group 2: BIS 40-50, remifentanil 2 ng ml(-1); Group 3: BIS 40-50, remifentanil 4 ng ml(-1); Group 4: BIS 25-35, remifentanil 2 ng ml(-1); Group 5: BIS 55-65, remifentanil 2 ng ml(-1)]. A 60 mA tetanic stimulus was applied for 5 s on the ulnar nerve. From the digitized pulse oximeter wave recorded on a laptop computer, linear and non-linear parameters of PPG variation during the 60 s period after stimulation were computed. The haemodynamic response to subsequent orotracheal intubation was recorded. The PPG variation was compared between groups and between responders and non-responders to intubation (anova). Variables independently predicting the response were determined by logistic regression. RESULTS: The probability of a response to tracheal intubation was 0.77, 0.47, 0.05, 0.18 and 0.52 in Groups 1-5, respectively (P<0.03). The PPG variability was significantly higher in responders than in non-responders but it did not improve the prediction of the response to tracheal intubation based on BIS level and effect site remifentanil concentration. CONCLUSION: Tetanic stimulation induced PPG variation does not reflect the analgesic state in a wide clinical range of surgical anaesthesia.

Repetitive transcranial magnetic stimulation of the dorsolateral prefrontal cortex affects divided attention immediately after cessation of stimulation

Transcranial magnetic stimulation has evolved into a powerful neuroscientific tool allowing to interfere transiently with specific brain functions. In addition, repetitive TMS (rTMS) has long-term effects (e.g. on mood), probably mediated by neurochemical alterations. While long-term safety of rTMS with regard to cognitive functioning is well established from trials exploring its therapeutic efficacy, little is known on whether rTMS can induce changes in cognitive functioning in a time window ranging from minutes to hours, a time in which neurochemical effects correlated with stimulation have been demonstrated. This study examined effects of rTMS on three measures of executive function in healthy subjects who received one single rTMS session (40 trains of 2 s duration 20 Hz stimuli) at the left dorsolateral prefrontal cortex (DLPFC). Compared to a sham condition one week apart, divided attention performance was significantly impaired about 30-60 min after rTMS, while Stroop-interference and performance in the Wisconsin Card Sorting Test was unaffected after rTMS. Repetitive TMS of the left DLPFC, at stimulation parameters used in therapeutic studies, does not lead to a clinically relevant impairment of executive function after stimulation. However, the significant effect on divided attention suggests that cognitive effects of rTMS are not limited to the of acute stimulation, and may possibly reflect known neurochemical alterations induced by rTMS. Sensitive cognitive measures may be useful to trace those short-term effects of rTMS non-invasively in humans.

Effects of somatosensory stimulation on use-dependent plasticity in chronic stroke

BACKGROUND AND PURPOSE: There is a need to develop strategies to enhance the beneficial effects of motor training, including use-dependent plasticity (UDP), in neurorehabilitation. Peripheral nerve stimulation (PNS) modulates motor cortical excitability in healthy humans and could influence training effects in stroke patients. METHODS: We compared the ability of PNS applied to the (1) arm, (2) leg, and (3) idle time to influence training effects in the paretic hand in 7 chronic stroke patients. The end point measure was the magnitude of UDP. RESULTS: UDP was more prominent with arm stimulation (increased by 22.8%) than with idle time (by 2.9%) or leg stimulation (by 6.4%). CONCLUSIONS: PNS applied to the paretic limb paired with motor training enhances training effects on cortical plasticity in stroke patients.