Ketamine shows, besides its general anaesthetic effect, a potent analgesic effect after spinal administration. We investigated the local anaesthetic-like action of ketamine and its enantiomers in Na+ and K+ channels and their functional consequences in dorsal horn neurones of laminae I-III, which are important neuronal structures for pain transmission receiving most of their primary sensory input from Adelta and C fibres. Combining the patch-clamp recordings in slice preparation with the 'entire soma isolation' method, we studied action of ketamine on Na+ and voltage-activated K+ currents. The changes in repetitive firing behaviour of tonically firing neurones were investigated in current-clamp mode after application of ketamine. Concentration-effect curves for the Na+ peak current revealed for tonic block half-maximal inhibiting concentrations (IC50) of 128 microM and 269 microM for S(+) and R(-)-ketamine, respectively, showing a weak stereoselectivity. The block of Na+ current was use-dependent. The voltage-dependent K+ current (K(DR)) was also sensitive to ketamine with IC50 values of 266 microM and 196 microM for S(+) and R(-)-ketamine, respectively. Rapidly inactivating K+ currents (K(A)) were less sensitive to ketamine. The block of K(DR) channels led to an increase in action potential duration and, as a consequence, to lowering of the discharge frequency in the neurones. We conclude that ketamine blocks Na+ and K(DR) channels in superficial dorsal horn neurones of the lumbar spinal cord at clinically relevant concentrations for local, intrathecal application. Ketamine reduces the excitability of the neurones, which may play an important role in the complex mechanism of its action during spinal anaesthesia.

Extracellular RNA has been shown to induce vascular endothelial growth factor (VEGF)-dependent hyperpermeability in vivo as well as in vitro. Studies were performed to investigate the mechanism of these effects. For permeability studies primary cultures of porcine brain-derived microvascular endothelial cells (BMECs) and for all other analytical studies the human brain endothelial cell line HCMEC/D3 or human umbilical vein endothelial cells (HUVECs) were used. RNA, but not DNA, initiated signaling events by binding of VEGF to neuropilin-1, followed by VEGF-R2 phosphorylation, activation of phospholipase C (PLC), and intracellular release of Ca(2+). Activation of these pathways by RNA also resulted in the release of von Willebrand Factor from Weibel-Palade bodies. Pretreatment of cells with heparinase totally abrogated the RNA-induced permeability changes, whereas RNA together with VEGF completely restored VEGF-R2-mediated hyperpermeability. Although poly:IC increased the interleukin-6 release via activation of toll-like receptor-3 (TLR-3), permeability changes mediated by poly:IC or RNA remained unchanged after blocking TLR-3 or NF-kB activation. These results indicate that extracellular RNA serves an important cofactor function to engage VEGF for VEGF-R2-dependent signal transduction, reminiscent of the coreceptor mechanism mediated by proteoglycans, which might be of relevance for the mobilization and cellular activities of RNA-binding cytokines in general.

cAMP-mediated signaling mechanisms may destabilize or stabilize the endothelial barrier, depending on the origin of endothelial cells. Here, microvascular coronary [coronary endothelial cells (CEC)] and macrovascular aortic endothelial cell (AEC) monolayers with opposite responses to cAMP were analyzed. Macromolecule permeability, isometric force, activation state of contractile machinery [indicated by phosphorylation of regulatory myosin light chains (MLC), activity of MLC kinase, and MLC phosphatase], and dynamic changes of adhesion complex proteins (translocation of VE-cadherin and paxillin) were determined. cAMP signaling was stimulated by the adenosine receptor agonist 5'-N-(ethylcarboxamido)-adenosine (NECA), the beta-adrenoceptor agonist isoproterenol (Iso), or by the adenylyl cyclase activator forskolin (FSK). Permeability was increased in CEC and decreased in AEC on stimulation with NECA, Iso, or FSK. The effects could be inhibited by the PKA inhibitor Rp-8-CPT-cAMPS and imitated by the PKA activator Sp-cAMPS. Under cAMP/PKA-dependent stimulation, isometric force and MLC phosphorylation were reduced in monolayers of either cell type, due to an activation of MLC phosphatase. In CEC but not in AEC, FSK induced delocalization of VE-cadherin and paxillin from cellular adhesion complexes as indicated by cell fractionation and immunofluorescence microscopy. In conclusion, decline in contractile activation and isometric force contribute to cAMP/PKA-mediated stabilization of barrier function in AEC. In CEC, this stabilizing effect is overruled by cAMP-induced disintegration of cell adhesion structures.

The presence of more than one dental alloy in the oral cavity often causes pathological galvanic currents and voltage resulting in superficial erosions of the oral mucosa and eventually in the emergence of oral cancer. In the present study the mechanisms of apoptosis of oral mucosa cancer cells in response to electromagnetic fields was investigated. Direct current (DC) electrical fields with field strengths between 2 and 16 V/m, applied for 24 h to UM-SCC-14-C oral mucosa cancer cells, dose-dependently resulted in decreased cell proliferation as evaluated by Ki-67 immunohistochemistry and upregulation of the cyclin-dependent kinase (CDK) inhibitors p21(cip1/waf1) and p27(kip1), which are associated with cell cycle arrest. Electrical field treatment (4 V/m, 24 h) increased apoptosis as evaluated by immunohistochemical analysis of cleaved caspase-3 and poly-(ADP-ribose)-polymerase-1 (PARP-1). Furthermore, robust reactive oxygen species (ROS) generation, increased expression of NADPH oxidase subunits as well as Hsp70 was observed. Electrical field treatment (4 V/m, 24 h) resulted in increased expression of Cu/Zn superoxide dismutase and decreased intracellular concentration of reduced glutathione (GSH), whereas the expression of catalase remained unchanged. Pre-treatment with the free radical scavenger N-acetyl cysteine (NAC) and the superoxide dismutase mimetic EUK-8 abolished caspase-3 and PARP-1 induction, suggesting that apoptosis in oral mucosa cancer cells is initated by ROS generation in response to DC electrical field treatment.

In CMEC exposed to reperfusion conditions, the enhanced Ca2+ overload is due to Ca2+ influx. The influx is not mediated by a Na+-dependent mechanism, but rather is due to activation of the InsP3 receptor of the ER and activation of SOC.

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Heart failure development goes along with a transition from hypertrophic growth to apoptosis induction. In adult cardiomyocytes SMAD proteins are only activated under apoptotic, but not under hypertrophic conditions and are increased at the transition to heart failure. Therefore, SMADs could be candidates that turn the balance from hypertrophic growth to apoptosis resulting in heart failure development. To test this hypothesis we infected isolated rat ventricular cardiomyocytes with adenovirus encoding SMAD4 (AdSMAD4) and investigated the impact of SMAD4 overexpression on the development of apoptosis and hypertrophy under stimulation with phenylephrine (PE). Infection of cardiomyocytes with AdSMAD4 significantly enhanced SMAD-binding activity while apoptosis after 24 and 36 h infection did not rise. But when SMAD4 overexpressing cardiomyocytes were incubated with PE (10 microM), the number of apoptotic cells increased (Ctrl: 94.97 +/- 6.91%; PE: 102.48 +/- 4.78% vs. AdSMAD4 + PE: 118.64 +/- 3.28%). Furthermore expression of caspase 3 as well as bax/bcl2 ratio increased in SMAD4 overexpressing, PE-stimulated cardiomyocytes. In addition, the effects of SMAD4 overexpression on PE-induced hypertrophic growth were analyzed. Protein synthesis 36 h after AdSMAD4 infection was comparable to control cells, whereas the increase in protein synthesis stimulated by phyenylephrine was significantly reduced in SMAD4 overexpressing cells (134.28 +/- 10.02% vs. 100.57 +/- 8.86%). SMAD4 triggers the transition from hypertrophy to apoptosis in ventricular cardiomyocytes. Since SMADs are increased under several pathophysiological conditions in the heart, it can be assumed that it triggers apoptosis induction and therefore contributes to negative remodeling and heart failure progression.

Background The intramuscular injection of ketamine and azaperone was proposed as a suitable anaesthesia for male suckling piglets for surgical castration. However, this can be opposed by massive defensive movements, hypothermia and tachycardia during castration and a long recovery period. The aim of the present study was to test whether the use of S-ketamine and/or a change in the route of application from intramuscular to intranasal could reduce stress responses and the duration of recovery compared to the intramuscular route and the use of racemic ketamine. Seventy-eight healthy, five-day-old male piglets were randomized to six treatment groups in a blinded experimental study, matched by litter and weight. Experimental groups were A (15 mg kg-1 S-ketamine + 2 mg kg-1 azaperone, i.m., surgical castration), B (15 mg kg-1 R/S-ketamine racemate + 2 mg kg-1 azaperone, i.m., surgical castration), C (30 mg kg-1 S-ketamine + 2 mg kg-1 azaperone, i.n., surgical castration), D (15 mg kg-1 R/S-ketamine racemate + 2 mg kg-1 azaperone, i.m.; not castrated), E (positive control group; no anesthesia, surgical castration) and F (negative control group; no anesthesia, not castrated). Results S-ketamine reduced the defensive movement score during castration to a similar extent to racemic ketamine when administered intramuscularly but not via the intranasal route. However, the effects of S-ketamine (both routes) on the increase in cortisol levels and decrease in body temperature were similar to those induced by racemic ketamine. A reduction of the long recovery time known for ketamine-azaperone anaesthesia could not be achieved with S-ketamine in the given dosage, regardless of the route of application. The intranasal administration of ketamine was difficult with the available formulation as the necessary amount exceeded the capacity of the nose cavity. Conclusions Neither the use of S-ketamine nor intranasal administration can be suitable alternatives for the anaesthesia of male suckling piglets for castration.