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Neuronal high-voltage-activated (HVA) Ca(2+) channels are rapidly inactivated by a mechanism that is termed Ca(2+)-dependent inactivation (CDI). In this study we have shown that β-adrenergic receptor (βAR) stimulation inhibits CDI in rat thalamocortical (TC) relay neurons. This effect can be blocked by inhibition of cAMP-dependent protein kinase (PKA) with a cell-permeable inhibitor (myristoylated protein kinase inhibitor-(14-22)-amide) or A-kinase anchor protein (AKAP) St-Ht31 inhibitory peptide, suggesting a critical role of these molecules downstream of the receptor. Moreover, inhibition of protein phosphatases (PP) with okadaic acid revealed the involvement of phosphorylation events in modulation of CDI after βAR stimulation. Double fluorescence immunocytochemistry and pull down experiments further support the idea that modulation of CDI in TC neurons via βAR stimulation requires a protein complex consisting of Ca(V)1.2, PKA and proteins from the AKAP family. All together our data suggest that AKAPs mediate targeting of PKA to L-type Ca(2+) channels allowing their phosphorylation and thereby modulation of CDI.

We demonstrated recently that opioid-induced activation of phospholipase D2 (PLD2) enhances mu- (MOPr) and delta-opioid receptor endocytosis/recycling and thus reduces the development of opioid receptor desensitization and tolerance. However, the mechanistic basis for the PLD2-mediated induction of opioid receptor endocytosis is currently unknown. Here we show that PLD2-generated phosphatidic acid (PA) might play a key role in facilitating the endocytosis of opioid receptors. However, PLD2-derived PA is known to be further converted to diacylglycerol (DAG) by PA phosphohydrolase (PPAP2). In fact, blocking of PA phosphohydrolase activity by propranolol or PPAP2-short interfering RNA (siRNA) transfection significantly attenuated agonist-induced opioid receptor endocytosis. The primary importance of PA-derived DAG in the induction of opioid receptor endocytosis was further supported by the finding that increasing the DAG level by inhibiting the reconversion of DAG into PA with the DAG kinase inhibitor 3-[2-(4-[bis-(4-fluorophenyl)methylene]-1-piperidinyl)ethyl]-2,3-dihydro-2-thioxo-4(1H)quinazolinone (R59949) or the addition of the synthetic cell-permeable DAG analog 1,2-dioctanoyl-sn-glycerol (DOG), further increased the agonist-induced opioid receptor endocytosis. Moreover, the addition of DOG bypasses the PLD2-siRNA- or PPAP2-siRNA-mediated impairment of DAG synthesis and resulted in a restoration of agonist-induced opioid receptor internalization. Further studies established a functional link between PA-derived DAG and the activation of p38 mitogen-activated protein kinase (MAPK) and the subsequent phosphorylation of the Rab5 effector early endosome antigen 1, which has been demonstrated recently to be required for the induction of MOPr endocytosis. Taken together, our results revealed that the regulation of opioid receptor endocytosis by PLD2 involves the conversion of its product PA to DAG resulting in an activation of the p38 MAPK pathway.

Neuronal Ca(2+) channels are rapidly inactivated by a mechanism that is termed Ca(2+)-dependent inactivation (CDI). In this study we investigated the influence of intracellular Ca(2+) release on CDI of high-voltage-activated Ca(2+) channels in rat thalamocortical relay neurons by combining voltage-clamp, Ca(2+) imaging and immunological techniques. Double-pulse protocols revealed CDI, which depended on the length of the conditioning pulses. Caffeine caused a concentration-dependent increase in CDI that was accompanied by an increase in the duration of Ca(2+) transients. Inhibition of ryanodine receptors and endoplasmic Ca(2+) pumps (by thapsigargin or cyclopiazonic acid) resulted in a reduction of CDI. In contrast, inhibition of inositol 1,4,5-tris-phosphate receptors by intracellular application of 2-aminoethoxy diphenyl borate or heparin did not influence CDI. The block of transient receptor potential channels by extracellular application of 2-aminoethoxy diphenyl borate, however, resulted in a significant reduction of CDI. The central role of L-type Ca(2+) channels was emphasized by the near-complete block of CDI by nifedipine, an effect only surpassed when Ca(2+) was replaced by Ba(2+) and chelated by 1,2-bis(o-aminophenoxy)ethane-N,N,N',N',-tetraacetic acid (BAPTA). Trains of action potential-like stimuli induced a strong reduction in high-voltage-activated Ca(2+) current amplitude, which was significantly reduced when intracellular Ca(2+) stores were made inoperative by thapsigargin or Ba(2+)/BAPTA. Western blotting revealed expression of L-type Ca(2+) channels in thalamic and hippocampal tissue but not liver tissue. In summary, these results suggest a cross-signalling between L-type Ca(2+) channels and ryanodine receptors that controls the amount of Ca(2+) influx during neuronal activity.

Endocytosis of the mu-opioid receptor (MOPr) has been shown to play a protective role against the development of tolerance to opioid drugs by facilitating receptor reactivation and recycling. It has been further demonstrated, that the opioid-mediated and ADP-ribosylation factor (ARF)-dependent activation of phospholipase D2 (PLD2) is a prerequisite for MOPr endocytosis. In this study, we investigated which particular ARF protein is involved in opioid-mediated PLD2 activation and what are the mechanisms of ARF function in MOPr trafficking and signaling. By coexpressing the MOPr and dominant negative or constitutively active ARF mutants in human embryonic kidney (HEK) 293 cells and primary cultured cortical neurons as well as by using siRNA technology, we identified the ARF6 protein to be involved in the regulation of MOPr endocytosis. We also found that expression of an effector domain mutant of ARF6, which is incapable of activating PLD, blocked agonist-induced endocytosis suggesting that ARF6 function in MOPr trafficking is PLD2-mediated. Analogously, opioid-mediated activation of PLD2 is blocked in the presence of dominant negative ARF6 mutants. Finally, we also showed that ARF6 protein influences the recycling/reactivation of internalized MOPr and thus modulates agonist-induced MOPr desensitization. Together, these results provide evidence that ARF6 protein regulates MOPr trafficking and signaling via PLD2 activation and hence affects the development of opioid receptor desensitization and tolerance.

In this study we present the experimental and mathematical model for a precise assessment of isolated blood vessels dynamic response under a sudden change of blood pressure. Only the end points within the time interval of the considered dynamic response of the blood vessel, or so-called "alternate steady states" of the processes, were usually considered in various studies. These studies do not provide an insight how the process variables change between these alternate steady states. Isolated blood vessels (rat abdominal aorta) were used to determine how the process dynamics can be described in detailed quantitative terms by mathematical parameters. The experimental model and mathematical procedures presented in this study describe precisely (at a high sensitivity level) the time history of the pressure and the diameter change in between alternate steady states, when an abrupt change of blood pressure occurs at the vessel outlet. Also, the experimental model and mathematical procedures were used to determine changes in the stress-strain law, caused by the action of L-arginine. The presented experimental design and mathematical model can be used for assessment of isolated blood vessel dynamic responses under different stimuli, such as drug effects, electrostimulation etc.

It has been suggested that the specific burst firing patterns of thalamic neurons reflect differential expression of low-voltage-activated (LVA) Ca(2+) channel subtypes and their splice variants. By combining electrophysiological, molecular biological, immunological, and computational modeling techniques we here show that diverging LVA Ca(2+) currents of thalamocortical relay (TC) and GABAergic interneurons of the dLGN correlate with a differential expression of LVA Ca(2+) channel splice variations and isoforms (alpha1G-a in TC; alpha1G-bc and alpha1I in interneurons). Implementation of the observed LVA Ca(2+) current differences into a TC neuron model changed the burst firing from TC-like to interneuron-like. We conclude that alternative splicing of the alpha1G isoform in dLGN TC and interneurons, and the exclusive expression of the alpha1I isoform in interneurons play a prominent role in setting the different LVA Ca(2+) current properties of TC and interneurons, which critically contribute to the diverging burst firing behavior of these neurons.

Evaluating predictors of appropriate implantable cardioverter-defibrillator (ICD) therapy in patients with idiopathic dilated cardiomyopathy (IDC) may be helpful in developing risk stratification strategies for these patients. Fifty-four patients with IDC underwent ICD implantation and were followed up. Twenty-three patients (42%) had a class I indication for ICD implantation; the remaining patients underwent implantation for multiple risk factors for sudden death including left ventricular dysfunction, nonsustained ventricular tachycardia, syncope, or positive electrophysiologic study results. Clinical, electrocardiographic, and electrophysiologic data were collected. Appropriate ICD therapy was defined as an antitachycardia pacing therapy or shock for tachyarrhythmia determined to be either ventricular tachycardia or ventricular fibrillation. Appropriate ICD therapy was observed in 23 patients (42%). There was a significant difference in use of beta-blocker therapy between patients who did and did not have appropriate ICD therapy (p <0.0003). Cox regression analysis identified the following univariate predictors (p <0.1): class I indication (p <0.005) and lack of use of beta-blocker therapy (p <0.0007). In multivariate analysis, only lack of beta-blocker use (relative risk 0.15, 95% confidence intervals 0.05 to 0.45; p <0.0007) was identified as a predictor of appropriate ICD therapy. Of the patients who received ICD therapy, only 4 (17%) were taking beta blockers, whereas 21 of the 31 patients (68%) who did not receive ICD therapy were treated with beta blockers (p <0.0003). In patients with IDC selected for ICD implantation, the most consistent predictor of appropriate ICD therapy was lack of beta-blocker use. Attempts should be made to administer beta blockers to these patients, if tolerated.

Introduction: Although premature beats originating in areas of ischemia have been shown to be important in initiating ventricular fibrillation (VF), the participation of the ischemic zone in maintenance of VF has not been investigated., Methods and Results: Ten normal dogs underwent induction of two separate episodes of VF, before and 10 minutes after left anterior descending coronary artery ligation. Ischemic VF was allowed to occur spontaneously or was induced by burst pacing after 10 minutes of ischemia. Unipolar epicardial electrograms were recorded using an 8 x 14 plaque electrode array (interelectrode distance 2.5 mm) placed over the anterior wall. Activation during VF was characterized by VF cycle length (CL) and wavefront organization based on linking analysis of epicardial activation directions at adjacent sites. Individual plaque sites were separated into regions based on electrogram morphology during ischemia: R1 = no ischemia; R2 = mild-to-moderate ischemia (minor ST elevation and QRS widening); and R3 = severe ischemia (marked ST elevation and QRS widening). Percent conduction block was calculated based on the percent of cycles during which sites were not activated during VF. There were no significant differences noted in mean CL or mean percent conduction block in the peri-ischemic region R1 compared to the same region under nonischemic (control) conditions. During ischemia, the mean CL was noted to increase in R2 from 111+/-14 msec (control) to 128+/-29 msec (ischemia) and in R3 from 113+/-14 msec (control) to 150+/-42 msec (ischemia) (P < 0.05). The percentage conduction block in R2 increased from 6%+/-11% (control) to 14%+/-16% (ischemia) and in R3 from 4%+/-6% (control) to 44%+/-21% (ischemia) (P < 0.05). Linking analysis revealed no significant changes in VF organization at distances of 2.5 mm in regions R1 and R2 under both control and ischemic conditions. Premature beats initiating fibrillation originated at the border between the normal and mildly ischemic zones., Conclusions: (1) Some VF characteristics are altered in ischemic regions including a longer VFCL and greater percentage of functional block. (2) VF characteristics are unchanged in immediately adjacent nonischemic myocardium. (3) Although the ischemic zone may be involved in the initiation of VF and has unique activation characteristics during VF, it does not affect VF characteristics in the adjacent nonischemic zone, suggesting that it may not play a major role in VF maintenance.