The clearance of Ca 2ϩ from nerve terminals is critical for determining the build-up of residual Ca 2ϩ after repetitive presynaptic activity. We found previously that K

Background Asians increasingly seek non-surgical facial esthetic treatments, especially at younger ages. Published recommendations and clinical evidence mostly reference Western populations, but Asians differ from them in terms of attitudes to beauty, structural facial anatomy, and signs and rates of aging. A thorough knowledge of the key esthetic concerns and requirements for the Asian face is required to strategize appropriate facial esthetic treatments with botulinum toxin and hyaluronic acid (HA) fillers.MethodsThe Asian Facial Aesthetics Expert Consensus Group met to develop consensus statements on concepts of facial beauty, key esthetic concerns, facial anatomy, and aging in Southeastern and Eastern Asians, as a prelude to developing consensus opinions on the cosmetic facial use of botulinum toxin and HA fillers in these populations.ResultsBeautiful and esthetically attractive people of all races share similarities in appearance while retaining distinct ethnic features. Asians between the third and sixth decades age well compared with age-matched Caucasians. Younger Asians’ increasing requests for injectable treatments to improve facial shape and three-dimensionality often reflect a desire to correct underlying facial structural deficiencies or weaknesses that detract from ideals of facial beauty.ConclusionsFacial esthetic treatments in Asians are not aimed at Westernization, but rather the optimization of intrinsic Asian ethnic features, or correction of specific underlying structural features that are perceived as deficiencies. Thus, overall facial attractiveness is enhanced while retaining esthetic characteristics of Asian ethnicity. Because Asian patients age differently than Western patients, different management and treatment planning strategies are utilized.Level of Evidence VThis journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to Table of Contents or the online Instructions to Authors www.springer.com/00266.

At the immature calyx of Held, the fast decay phase of a Ca 2ϩ transient induced by tetanic stimulation (TS) was followed by a period of elevated [Ca 2ϩ ] i for tens of seconds, referred to as posttetanic residual calcium (Ca res ). We investigated the source of Ca res and its contribution to posttetanic potentiation (PTP). After TS (100 Hz for 4 s), posttetanic Ca res at the calyx of Held was largely abolished by tetraphenylphosphonium (TPP ϩ ) or Ru360, which inhibit mitochondrial Na ϩ -dependent Ca 2ϩ efflux and Ca 2ϩ uniporter, respectively. Whereas the control PTP lasted longer than Ca res , inhibition of Ca res by TPP ϩ resulted in preferential suppression of the early phase of PTP, the decay time course of which well matched with that of Ca res . TS induced significant increases in release probability (P r ) and the size of the readily releasable pool (RRP), which were estimated from plots of cumulative EPSC amplitudes. TPP ϩ or Ru360 suppressed the posttetanic increase in P r , whereas it had little effect on the increase in RRP size. Moreover, the posttetanic increase in P r , but not in RRP size, showed a linear correlation with the amount of Ca res . In contrast, myosin light chain kinase (MLCK) inhibitors and blebbistatin reduced the posttetanic increase in RRP size with no effect on the increase in P r . Application of TPP ϩ in the presence of MLCK inhibitor peptide caused further suppression of PTP. These findings suggest that Ca res released from mitochondria and activation of MLCK are primarily responsible for the increase in P r and that in the RRP size, respectively.

Glutamatergic synaptic terminals harbor reluctant synaptic vesicles (SVs) that contribute little to synchronous release during action potentials but are release competent when stimulated by sucrose or by direct intracellular application of calcium. It has been noted that the proximity of a release-competent SV to the calcium source is one of the primary factors that differentiate reluctant SVs from fastreleasing ones at the calyx of Held synapse. It has not been known whether reluctant SVs can be converted into fast-releasing ones. Here we show that reluctant SVs are recruited rapidly in an actindependent manner to become fast-releasing SVs once the pool of fast-releasing SVs is depleted by a short depolarization. Recovery of the pool of fast-releasing SVs was accompanied by a parallel reduction in the number of reluctant SVs. Quantitative analysis of the time course of depletion of fast-releasing SVs during high-frequency stimulation revealed that in the early phase of stimulation reluctant SVs are converted rapidly into fast-releasing ones, thereby counteracting short-term depression. During the late phase, however, after reluctant vesicles have been used up, another process of calmodulindependent recruitment of fast-releasing SVs is activated. These results document that reluctant SVs have a role in short-term plasticity and support the hypothesis of positional priming, which posits that reluctant vesicles are converted into fast-releasing ones via relocation closer to Ca 2+ -channels.readily releasable pool | synaptic vesicle dynamics S ynaptic strength is determined by quantal size, the number of release-competent synaptic vesicles (SVs), and their release probability (Pr). The estimate for the number of release-competent SVs, which also is referred to as the "readily releasable pool (RRP) size," depends heavily on the method used for its determination. The RRP size estimated by a cumulative plot of the excitatory postsynaptic currents (EPSC) amplitudes evoked by high-frequency afferent fiber stimulation (RRP cum ) is smaller than that estimated by the application of a hypertonic sucrose solution or presynaptic strong depolarization (1-3). This discrepancy reflects the presence of reluctant SVs, which are scarcely released by an action potential (AP) at glutamatergic synaptic terminals. Consistently, deconvolution analysis of EPSCs evoked by a long depolarizing pulse at the calyx of Held revealed that release-competent SVs can be separated into fast-and slowreleasing SV pools (FRP and SRP, respectively) (4). The FRP vesicles are responsible for phasic release during a high-frequency train of action potentials (APs), whereas the SRP vesicles contribute primarily to asynchronous release, when the intracellular concentration of calcium ions ([Ca 2+ ]) is increased globally during the late period of the train (2). Thus, SRP vesicles can be regarded largely as reluctant SVs at the calyx of Held.Despite the evidence for the presence of reluctant SVs at glutamatergic synapses, their role in short-term plasticity i...

Previous studies indicate that boutons from the same axon exhibit distinct Ca 2ϩ dynamics depending on the postsynaptic targets. /Ca 2ϩ exchanger (mitoNCX). In contrast, blockers of mitoNCX had no effect on the Ca res at small MFBs. Post-tetanic Ca res has been proposed as a mechanism for post-tetanic potentiation (PTP). We examined mitochondrial involvement in PTP at mossy fiber synapses on hilar mossy cells (MF3 MC synapse) and on hilar interneurons (MF3 HI synapse), which are presumably innervated via large and small MFBs, respectively. Consistent with the differential contribution of mitochondria to Ca res at large and small MFBs, mitoNCX blockers significantly reduced the PTP at the MF3 MC synapse, but not at the MF3 HI synapse. In contrast, protein kinase C (PKC) inhibitors significantly reduced the PTP at MF3 HI synapse, but not at the MF3 MC synapse. These results indicate that mitochondria-and PKC-dependent PTP are expressed at distinct hilar mossy fiber synapses depending on postsynaptic targets.

We have shown previously that cardiac G protein-gated inwardly rectifying K ؉ (GIRK) channels are inhibited by Gq protein-coupled receptors (GqPCRs) via phosphatidylinositol 4,5-bisphosphate (PIP2) depletion in a receptor-specific manner. To investigate the mechanism of receptor specificity, we examined whether the activation of GqPCRs induces localized PIP2 depletion. When we applied endothelin-1 to the bath, GIRK channel activities recorded in cell-attached patches were not changed, implying that PIP2 signal is not diffusible but is a localized signal. To test this possibility, we directly measured lateral diffusion by introducing fluorescence-labeled phosphoinositides to a small area of the membrane with patch pipettes. After pipettes were attached, phosphatidylinositol 4-monophosphate or phosphatidylinositol diffused rapidly to the entire membrane, whereas PIP2 was confined to the membrane patch inside the pipette. The confinement of PIP2 was disrupted after cytochalasin D treatment, suggesting that the cytoskeleton is responsible for the low mobility of PIP2. The diffusion coefficient (D) of PIP2 in the plasma membrane measured with the fluorescence recovery after photobleaching technique was 0.00039 m 2 ͞s (n ‫؍‬ 6), which is markedly lower than D of phosphatidylinositol (5.8 m 2 ͞s, n ‫؍‬ 5). Simulation of PIP2 concentration profiles by the diffusion model confirms that when D is small, the kinetics of PIP2 depletion at different distances from phospholipase C becomes similar to the characteristic kinetics of GIRK inhibition by different agonists. These results imply that PIP2 depletion is localized adjacent to GqPCRs because of its low mobility, and that spatial proximity of GqPCR and the target protein underlies the receptor specificity of PIP2-mediated signaling. P rotein-lipid interactions have been increasingly appreciated in recent years. In particular, phosphatidylinositol 4,5-bisphosphate (PIP 2 ) binds a wide variety of cellular proteins, including cytoskeletal proteins and ion channels, and evidence that the binding is essential for their functions is accumulating (1, 2). Because many proteins are known to be regulated by PIP 2 , the question arises as to how a particular protein, among many others, is selectively regulated by PIP 2 changes generated by specific signals. In fact, the same question has been asked for many years in the context of the signaling role of Ca 2ϩ , i.e., ''how are so many different Ca 2ϩ -dependent reactions selectively choreographed within a cell?'' The results of numerous studies undertaken to answer this question have led to the idea that the secret lies in localization, i.e., cells have many means of generating intracellular Ca 2ϩ signals, and the spatial proximities of these signals to the molecular targets of Ca 2ϩ determine the specificity of Ca 2ϩ action (3). However, it is not known whether this idea can be applied to PIP 2 -dependent signaling.Because experimental methods of measuring or visualizing changes in PIP 2 during signaling pathway stimulation remain ...

Background— Dimethyl lithospermate B (dmLSB) is an extract of Danshen, a traditional Chinese herbal remedy, which slows inactivation of I Na , leading to increased inward current during the early phases of the action potential (AP). We hypothesized that this action would be antiarrhythmic in the setting of Brugada syndrome. Methods and Results— The Brugada syndrome phenotype was created in canine arterially perfused right ventricular wedge preparations with the use of either terfenadine or verapamil to inhibit I Na and I Ca or pinacidil to activate I K-ATP . AP recordings were simultaneously recorded from epicardial and endocardial sites together with an ECG. Terfenadine, verapamil, and pinacidil each induced all-or-none repolarization at some epicardial sites but not others, leading to ST-segment elevation as well as an increase in both epicardial and transmural dispersions of repolarization (EDR and TDR, respectively) from 12.9±9.6 to 107.0±54.8 ms and from 22.4±8.1 to 82.2±37.4 ms, respectively ( P <0.05; n=9). Under these conditions, phase 2 reentry developed as the epicardial AP dome propagated from sites where it was maintained to sites at which it was lost, generating closely coupled extrasystoles and ventricular tachycardia and fibrillation. Addition of dmLSB (10 μmol/L) to the coronary perfusate restored the epicardial AP dome, reduced EDR and TDR to 12.4±18.1 and 24.4±26.7 ms, respectively ( P <0.05; n=9), and abolished phase 2 reentry-induced extrasystoles and ventricular tachycardia and fibrillation in 9 of 9 preparations. Conclusions— Our data suggest that dmLSB is effective in eliminating the arrhythmogenic substrate responsible for the Brugada syndrome and that it deserves further study as a pharmacological adjunct to implanted cardioverter/defibrillator usage.

This investigation used a patch clamp technique to test the hypothesis that protein kinase G (PKG) contributes to the phosphorylation and activation of ATP-sensitive K ؉ (K ATP ) channels in rabbit ventricular myocytes. Nitric oxide donors and PKG activators facilitated pinacidilinduced K ATP channel activities in a concentration-dependent manner, and a selective PKG inhibitor abrogated these effects. In contrast, neither a selective protein kinase A (PKA) activator nor inhibitor had any effect on K ATP channels at concentrations up to 100 and 10 M, respectively. Exogenous PKG, in the presence of both cGMP and ATP, increased channel activity, while the catalytic subunit of PKA had no effect. PKG activity was prevented by heat inactivation, replacing ATP with adenosine 5-O-(thiotriphosphate) (a nonhydrolyzable analog of ATP), removing Mg 2؉ from the internal solution, applying a PKG inhibitor, or by adding exogenous protein phosphatase 2A. The effects of cGMP analogs and PKG were observed under conditions in which PKA was repressed by a selective PKA inhibitor. The results suggest that K ATP channels are regulated by a PKG-signaling pathway that acts via PKG-dependent phosphorylation. This mechanism may, at least in part, contribute to a signaling pathway that induces ischemic preconditioning in rabbit ventricular myocytes.Protein phosphorylation is a putative effector mechanism in the infarct size-limiting effect of ischemic preconditioning (1), a phenomenon whereby a brief period of ischemia and reperfusion can protect the heart against subsequent prolonged ischemia and reperfusion injury (2). Indeed, it was shown that phosphorylation levels decrease during ischemia in nonpreconditioned hearts, whereas they increase during ischemia in preconditioned hearts (3). A number of cellular components, including cytoskeletal and stress proteins, have been proposed as potential phosphorylation targets in the ischemic preconditioned heart (4 -6).ATP-sensitive K ϩ channels (K ATP channels) 1 in sarcolemma and mitochondria are also modulated by phosphorylation (7, 8).The majority of the studies on the contribution of phosphorylation to K ATP channel activity to the cardioprotective effects of ischemic preconditioning have centered on the role of protein kinase C. Protein kinase C may act as a link between one or more receptor-mediated pathways and increased K ATP channel activity and thus lead to ischemic preconditioning (9). The release of endogenous substances, such as adenosine, bradykinin, nitric oxide (NO), and prostacyclin (10 -12), has been proposed as a potential mechanism of ischemic preconditioning. These substances increase cGMP via direct stimulation of myocardial cells or via the endothelium. It was reported that the cGMP levels in preconditioned hearts are higher than in nonpreconditioned hearts (13-15). Since cGMP can induce protein phosphorylation via protein kinase G (PKG) activation, the involvement of PKG-dependent phosphorylation in ischemic preconditioning is expected. To date, however, the role of PKG ...