A model for the generation of localized transient [Na+] elevations in vascular smooth muscle.

We present a stochastic computational model to study the mechanism of signaling between a source and a target ionic transporter, both localized on the plasma membrane (PM). In general this requires a nanometer-scale cytoplasmic space, or nanodomain, between the PM and a peripheral organelle to reflect ions back towards the PM. Specifically we investigate the coupling between Na(+) entry via the transient receptor potential canonical channel 6 (TRPC6) and the Na(+)/Ca(2+) exchanger (NCX), a process which is essential for reloading the sarcoplasmic reticulum (SR) via the sarco/endoplasmic reticulum Ca(2+)ATPase (SERCA) and maintaining Ca(2+) oscillations in activated vascular smooth muscle. Having previously modeled the flow of Ca(2+) between reverse NCX and SERCA during SR refilling, this quantitative approach now allows us to model the upstream linkage of Na(+) entry through TRPC6 to reversal of NCX. We have implemented a random walk (RW) Monte Carlo (MC) model with simulations mimicking a diffusion process originating at the TRPC6 within PM-SR junctions. The model calculates the average Na(+) in the nanospace and also produces profiles as a function of distance from the source. Our results highlight the necessity of a strategic juxtaposition of the relevant ion translocators as well as other physical structures within the nanospaces to permit adequate Na(+) build-up to initiate NCX reversal and Ca(2+) influx to refill the SR.

Enhancement of Vascular Smooth Muscle Contractility by Alterations of Membranous Architecture.

Plasma membrane (PM) of smooth muscle cells hosts channel molecules regulating the flow of various ions. An intact architecture of PM is essential to orchestrate proper channel functions in order to complete agonist-mediated contraction, which includes Ca2+ release from the sarcoplasmic reticulum (SR) to initiate contraction, and subsequent Ca2+ refilling into SR through PM to sustain muscle contraction. The Junctional Complex (JC), comprising of junctional SR, and its apposing PM and neighboring caveolae, provides a quasi-enclosed microdomain housing receptors as well as ion channels and also restricting ion diffusions into the cytosol so the cell achieves optimal performance. The spatial arrangement of the JC is believed to ensure an uninterrupted Ca2+ cycling route. Full understanding of the functional role of the JC is the key to elucidating the contractile mechanisms of vascular smooth muscle and the physiological function of vessel contraction. The JC can be further divided into two sub-divisions, namely the PM-SR and caveolar regions. Previously, we demonstrated the role of the PM-SR region in the initiation of muscle contraction using pharmacological tools on the inferior vena cava (IVC) of rabbit. In the current study, we further dissected the caveolar region using a cholesterol-disrupting agent to investigate the role of the caveolar region. We conclude that disruption of the caveolar region in rabbit IVC smooth muscle results in augmented muscle contraction in response to adrenergic stimulation and the altered Ca2+ signaling may underlie the augmented contractility. Anat Rec, 302:186-192, 2019. © 2018 Wiley Periodicals, Inc.

Optical Versus Virtual Microscope for Medical Education: A Systematic Review.

Many technological innovations have changed the traditional practice of medical education and clinical practice. Whole slide imaging (WSI) technology provided an unique way of viewing conventional glass slides in histology and pathology laboratories. The WSI technology digitalized glass slide images and made them readily accessible via the Internet using tablets or computers. Users utilized the pan-and-zoom function to view digital images of slides, also referred to as the virtual microscope (VM), simulating use of an optical microscope (OM). Several articles have reported various outcomes on the utility of VM in teaching laboratories. Recently, the Royal College of Physicians and Surgeons of Canada certification examinations for anatomical pathologists ha completely adopted VM for the national licensing examination. To better inform medical educators, there is an urgent need for more structured reviews to draw evidence-based conclusions on the effectiveness of VM and learner's perceptions, in comparison to OM. The current study provides a descriptive summary of published outcomes using the systematic review approach. In conclusion, medical students' performance was improved with adoption of VM into the curriculum and recognized as a preferred learning modality, compared to OM. On the contrary, resident learners' performance was comparable between using OM and VM, with OM being the favored slide-viewing modality.

Inducible nitric oxide synthase depresses cardiac contractile function in Zucker diabetic fatty rats.

Cardiac contractile dysfunction is a common occurrence in type 2 diabetes. The aim was to examine if inducible nitric oxide synthase (iNOS) causes cardiac dysfunction in Zucker diabetic fatty (ZDF) rats, a model of type 2 diabetes. ZDF and Zucker lean control rats (20 week old) were studied at 6 h after recovery from halothane anaesthesia and surgery that involved insertions of catheters into the iliac arteries, iliac veins and the left ventricle via the right carotid artery. Protein expression and activity of iNOS in the hearts were measured by immunostaining and arginine-citrulline conversion assay, respectively. Both groups had similar baseline left ventricular developed pressure and maximum rate of rise of left ventricular pressure (+dP/dt), but heart rate and rate pressure product were lower in the ZDF than control rats. Dobutamine dose-dependently increased left ventricular developed pressure, +dP/dt, heart rate and rate pressure product in both groups, but the responses were less in the diabetic than control rats. The activity and protein expression of iNOS and nitrotyrosine were higher in the hearts of the diabetic than control rats. Selective inhibition of iNOS by 1400 W (N-3-aminomethyl-benzyl-acetamidine) did not alter responses to dobutamine in the control rats, but augmented the effects of dobutamine on left ventricular developed pressure and rate pressure product in the diabetic rats. The results indicate that activation of iNOS contributed to left ventricular contractile dysfunction in the ZDF rats, and this was partially reversed by selective inhibition of the activity of iNOS.

N-acetylcysteine attenuates PKCbeta2 overexpression and myocardial hypertrophy in streptozotocin-induced diabetic rats.

OBJECTIVE: Oxidative stress-mediated activation of protein kinase C (PKC) beta(2) in the myocardium has been implicated in the development of cardiomyopathy. Overexpression of PKCbeta(2) is associated with increased expression of connective tissue growth factor (CTGF) in myocardium, resulting in myocardial hypertrophy. We hypothesized that chronic treatment with the antioxidant N-acetylcysteine (NAC) would normalize oxidative stress-mediated overexpression of myocardial PKCbeta(2) and CTGF and attenuate the development of myocardial hypertrophy. METHODS: Control and streptozotocin-induced diabetic rats were treated with NAC in drinking water for 8 weeks. At termination rats were surgically prepared for hemodynamic measurement, subsequent to which their hearts were removed to evaluate cardiac performance and histological and biochemical changes. Further, the role of PKCbeta(2) in hyperglycemia-induced cardiomyocyte hypertrophy was tested in cultured neonatal cardiomyocytes. RESULTS: Myocardial hypertrophy, characterized by an increased ratio of ventricle weight to body weight and cardiomyocyte cross-sectional area was found to be higher in untreated diabetic rats. Further, in myocardium, increased levels of 15-F(2t)-isoprostane were accompanied by an increased expression of membrane-bound PKCbeta(2) and CTGF. N-acetylcysteine treatment not only attenuated these changes but also prevented hyperglycemia-induced hypertrophy in cultured neonatal rat cardiomyocytes. CONCLUSIONS: The results suggest that PKCbeta(2) overexpression represents a mechanism causing hyperglycemia-mediated myocardial hypertrophy, which can be prevented by the antioxidant N-acetylcysteine.

A quantitative model for linking Na+/Ca2+ exchanger to SERCA during refilling of the sarcoplasmic reticulum to sustain [Ca2+] oscillations in vascular smooth muscle.

We have developed a quantitative model for the creation of cytoplasmic Ca2+ gradients near the inner surface of the plasma membrane (PM). In particular we simulated the refilling of the sarcoplasmic reticulum (SR) via PM-SR junctions during asynchronous [Ca2+]i oscillations in smooth muscle cells of the rabbit inferior vena cava. We have combined confocal microscopy data on the [Ca2+]i oscillations, force transduction data from cell contraction studies and electron microscopic images to build a basis for computational simulations that model the transport of calcium ions from Na+/Ca2+ exchangers (NCX) on the PM to sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) pumps on the SR as a three-dimensional random walk through the PM-SR junctional cytoplasmic spaces. Electron microscopic ultrastructural images of the smooth muscle cells were elaborated with software algorithms to produce a very clear and dimensionally accurate picture of the PM-SR junctions. From this study, we conclude that it is plausible and possible for enough Ca2+ to pass through the PM-SR junctions to replete the SR during the regenerative Ca2+ release, which underlies agonist induced asynchronous Ca2+ oscillations in vascular smooth muscle.

Endothelin-1-mediated wave-like [Ca2+]i oscillations in intact rabbit inferior vena cava.

Endothelin-1 (ET1) is an endogenous vasoconstrictor released by the vascular system to regulate the contractility of vascular smooth muscle cells (VSMC). It is implicated in the pathogenesis of hypertension and diabetic vasculopathy. In rabbit inferior vena cava (IVC), 10 nM ET1 induces tonic contraction mainly via type A endothelin receptor activation. Using confocal imaging of Fluo-3 loaded in thein situ VSMC within the intact IVC, we found that ET1 elicited [Ca2+]i oscillations with an average frequency of 0.31 +/- 0.01 Hz. These [Ca2+]i oscillations occurred as repetitive Ca2+ waves traveling along the longitudinal axis of the cells with an average velocity of 29 +/- 3 microm/s. The Ca2+ waves were not synchronized between neighboring VSMC nor were they propagated between them. Nifedipine (10 microM) inhibited the tonic contraction by 27.0 +/- 5.0% while SKF96365 (50 microM) abolished the remaining contraction. In a parallel Ca2+ study, nifedipine reduced the frequency of the oscillations to 0.22 +/- 0.01 Hz while SKF96365 abolished the remaining [Ca2+]i oscillations. Subsequent application of 25 mM caffeine elicited no further Ca2+ signal. Thus, we conclude that ET1 stimulates tonic contraction in the rabbit IVC by inducing [Ca2+]i oscillations and that stimulated Ca2+ entry through both the L-type voltage-gated Ca2+ channels and a nifedipine-resistant and SKF96365-sensitive pathway is crucial for the maintenance of [Ca2+]i oscillations and tonic contraction.

Acute intralipid infusion reduces cardiac luminal lipoprotein lipase but recruits additional enzyme from cardiomyocytes.

OBJECTIVE: Lipoprotein lipase (LPL) metabolizes the triglyceride (TG) core of lipoproteins. We evaluated whether circulating lipids can regulate LPL by influencing the transfer of enzyme from the myocyte to the endothelial lumen. METHODS: Acute intralipid (IL, 10% and 20%) infusion was performed in male Wistar rats. After 3 h, insulin resistance was assessed using a euglycemic hyperinsulinemic clamp. Cardiac LPL activity was determined by retrogradely perfusing the hearts with heparin. Immunogold electron microscopy visualized LPL, and heparanase was detected by immunofluorescence. Cardiac myocytes were also isolated, and heparin-releasable LPL activity was measured. RESULTS: IL infusion increased both plasma and cardiac lipids. Circulating basal plasma LPL activity increased for the duration of the infusion. Compared to control (CON) hearts, there was a substantial decrease in heparin-releasable LPL activity at the vascular lumen following 3 h of IL infusion, an effect unrelated to changes in gene and protein expression or whole-body insulin resistance. Although constant perfusion of CON hearts with heparin stripped off most of the luminal bound LPL, hearts from IL-infused animals continued to release excessive amounts of the enzyme, suggesting buildup of LPL within endothelial cells or at the endothelial basolateral surface. Immunogold labeling confirmed this observation and demonstrated robust anti-LPL staining at these sites, only in IL hearts. Perfusing hearts from IL-rats in vitro, in the absence of TG, allowed the accumulated enzyme pool to transfer to the coronary lumen. CONCLUSION: Our data suggest that acute amplification of lipids reduces cardiac luminal LPL but facilitates additional recruitment of cardiomyocyte enzyme. Should this mechanism occur globally, it could contribute towards management of hyperlipidemia.

Calyculin-A disrupts subplasmalemmal junction and recurring Ca2+ waves in vascular smooth muscle.

Excitation-contraction coupling (E-C coupling) in phenylephrine(PE)-stimulated rabbit inferior vena cava (IVC) depends on the generation of asynchronous recurring Ca2+ waves in the in situ vascular smooth muscle cells (VSMC). Previous studies by our group have implicated a putative non-selective cationic store-operated channel and the reverse-mode Na+-Ca2+ exchange in refilling of the intracellular Ca2+ store via the sarco/endoplasmic reticulum ATPase (SERCA) and the maintenance of the recurring Ca2+ waves. We hypothesize that for the proper functioning of these three Ca2+ translocators in the process of SR refilling, the plasma membrane (PM) and the underlying superficial sarcoplasmic reticulum (SR) form specialized PM-SR junctions, which are essential for the maintenance of the recurring Ca2+ waves. In order to test this hypothesis, calyculin-A, a serine/threonine phosphatase inhibitor that has been demonstrated to result in the disruption of the PM-SR junctions was used. In the control rabbit IVC, electron microscopy of the in situ VSMC indicates that 14.2+/-0.7% of the PM is closely apposed by the prominent superficial SR network, forming numerous flattened PM-SR junctional cytoplasmic spaces. In the control IVC stimulation with 5 microM PE resulted in sustained recurring Ca2+ waves with a frequency of 0.42+/-0.02 Hz. In calyculin-A treated rabbit IVC, a concentration-dependent dissociation of the superficial SR and loss of PM-SR junctions was observed. This progressive loss of the PM-SR junctions occurs over the same concentration range as the inhibition of PE-induced recurring Ca2+ waves. These findings offer support for the hypothesis that the presence of the PM-SR junctions is required for the generation of asynchronous recurring Ca2+ waves, which underlie excitation-contraction coupling in the VSMC of the rabbit IVC.

Rearrangement of the close contact between the mitochondria and the sarcoplasmic reticulum in airway smooth muscle.

The mitochondria and the sarcoplasmic reticulum (SR) are two major intracellular calcium-storing organelles that exhibit close functional interaction with each other. Close spatial association is believed to be important for their functional interaction. In this study, we have characterized the spatial relationship between the SR and the mitochondria in porcine tracheal smooth muscle cells (TSMC) under different conditions. By examining the cross-section of unstimulated TSMC with electron microscopy, we found that 99.4 +/- 0.5% of the mitochondria seen on random cross-sections were situated within 30 nm of the SR and that 82.2 +/- 6.7% of the mitochondria were completely enveloped by the SR network. Overall, 48.0 +/- 3.5% of the mitochondrial outer membrane was within 30 nm with the SR. After stimulation of the TSMC with acetylcholine (ACh) or 80 mM [K(+)] solution 97.0 +/- 2.1% and 98.6 +/- 1.4% of the mitochondria observed were situated within 30 nm of the SR, respectively. However, the proportion of the mitochondria that was completely enveloped by the SR was significantly reduced to 12.2 +/- 5.9% in ACh-stimulated cells and 9.7 +/- 6.6% in 80 mM [K(+)] stimulated cells. The percentage of mitochondrial membrane closely associated with the SR was correspondingly lower at 10.1 +/- 1.0% during ACh stimulation and 10.8 +/- 0.9% during 80 mM [K(+)] stimulation. During smooth muscle cell stimulation, the SR appears to unwrap from the mitochondria and extend into the cytoplasm while maintaining close contact with the mitochondria over a smaller area. Such static and dynamic components of the close spatial association between the mitochondria and the SR may serve as a structural basis for the selective and efficient Ca(2+) trafficking between the two organelles in TSMC.

Organellar junctions promote targeted Ca2+ signaling in smooth muscle: why two membranes are better than one.

Numerous cellular processes are regulated by fluctuations in the concentration of a single cation, Ca(2+). To accomplish this feat, cells have developed mechanisms that target Ca(2+) signals to specific effectors in both space, by strategically localizing effectors and ion-transporting molecules, and time, by encoding the regulation of the frequency of Ca(2+) oscillations. With an emphasis on smooth muscle, we have analyzed how the interaction of Ca(2+) transporters located on closely apposing membranes of the plasma membrane, sarcoplasmic reticulum and mitochondria provides the structural foundation for site-specific and time-specific Ca(2+) signaling. These junctional membrane complexes can either control the concentration of Ca(2+) in the microdomain that surrounds an effector molecule or deliver Ca(2+) from the translocator on one membrane to a second translocator on the opposing membrane without significant diffusion into the bulk cytosol, an event we term 'linked Ca(2+) transport'.

Agonist-induced mitochondrial Ca2+ transients in smooth muscle.

We investigated the role of mitochondria (MT) in calcium signaling in a culture of rat aortic smooth muscle cells. We used targeted aequorin to selectively measure [Ca2+] in this organelle. Our results reveal that smooth muscle cell stimulation with agonists causes a large, transient increase in mitochondrial [Ca2+] ([Ca2+]m). This large transient can be blocked with inhibitors of the sarco-endoplasmic reticulum Ca2+-ATPase, suggesting a close relationship between the sarcoplasmic reticulum (SR) and the mitochondria. FCCP completely abolished the response to agonists, and targeted mitochondrial GFP revealed a vast tubular network of MT in these cells. When added before stimulation with ATP, IP3 inhibitors partially blocked the ATP-induced rise in mitochondrial Ca2+ release. The role of the Na+/Ca2+ exchanger (NCX) was examined by removing extracellular Na+. This procedure prevented the decrease in the [Ca2+]m transient normally seen on removal of extracellular Ca2+. We propose a functional linkage of MT and SR dependent on a narrow junctional space between the two organelles in which Ca2+ diffusion is restricted. Approximately half of the mitochondria appear to be associated with the superficial SR, which communicates with the extracellular space via NCX.

Inhibition of iNOS augments cardiovascular action of noradrenaline in streptozotocin-induced diabetes.

OBJECTIVE: The aim was to determine if inducible nitric oxide synthase (iNOS) contributes to depressed cardiovascular function at the acute phase of streptozotocin-induced diabetes. METHODS: Male Wistar rats were injected with streptozotocin [60 mg/kg, intravenously (i.v.)] or the vehicle (0.9% NaCl) and were studied 3 weeks later. RESULTS: The diabetic and control rats had similar mean arterial pressure (MAP) and total peripheral resistance (TPR). Noradrenaline (NA) increased in vivo left ventricular contractility (LV +dP/dt), MAP and TPR in both groups; however, the responses were markedly less in the diabetic than control rats. Acute administration of 1400W (selective inhibitor of iNOS; 3 mg/kg followed by 3 mg/kg/h, i.v.) did not alter responses to NA in the control rats, but augmented the influence of NA on MAP, TPR and LV +dP/dt in the diabetic rats. At this time, reverse transcription-polymerase chain reaction (RT-PCR) products (RNA) of iNOS were present in the hearts of the diabetic but not control rats. The activity of iNOS was threefold higher in the hearts of the diabetic rats relative to the controls, and the increase was inhibited by 1400W. Furthermore, immunostaining (proteins) of iNOS and nitrotyrosine (NT; marker of peroxynitrite) were identified in the hearts of the diabetic but not control rats. In contrast, the RT-PCR products of eNOS, activity of eNOS and immunostaining of eNOS were of similar intensity in the hearts of both groups. CONCLUSIONS: Activation of iNOS contributes to depressed cardiovascular contractile function to NA at the acute phase of streptozotocin-induced diabetes. Selective inhibition of iNOS partially restored cardiovascular responses to NA.

Relationship between the sarcoplasmic reticulum and the plasma membrane.

Ionic interactions between the plasma membrane (PM) and the sarcoplasmic reticulum (SR) play a crucial role in smooth muscle activation and homeostasis. The most common form of Ca2+ signalling seen in vascular smooth musde of conduit arteries and capacitance veins consists of repetitive asynchronous Ca2+ waves. In the inferior vena cava of the rabbit these waves are initiated by Ca2+ release via InsP3 receptors (InsP3R) and propagated by regenerative Ca2+ release. Maintenance of the [Ca2+] oscillations is dependent on Ca2+ entry through the Na+/Ca2+-exchanger (NCX) which is driven in the reverse mode by Na+ entry through non-specific cation channels. The latter are also responsible for depolarization and activation of voltage-gated Ca2+ channels. The sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA) in the sheet-like junctional SR is responsible for refilling and completing the cycle. Under resting conditions the interaction between the superficial SR and the NCX is reversed with Ca2+ release channels supplying Ca2+ to the NCX in the PM to be extruded in exchange of extracellular Na+. It is proposed that the above Ca2+ transport between the SR lumen and the extracellular space takes place at PM-SR junctions across a narrow junctional space.

Ca2+ signaling in smooth muscle: TRPC6, NCX and LNats in nanodomains.

Following the recent observation of localized cytosolic subplasmalemmal [Na+] elevations (LNats) in rat aortic smooth muscle cells, we discuss here the current evidence for the structural and molecular roles of cytosolic nanodomains at close junctions of the plasma membrane (PM) and sarcoplasmic reticulum (SR) in the generation of LNats. These junctions, the loss of which might contribute to vascular aging and disease, provide a platform for ion metabolism signalplexes and the interaction of localized Na+ and Ca2+ gradients. We moreover suggest the existence in the junctions of a Na+ diffusional barrier as a necessary condition for the generation of LNats. LNats are likely a fundamental feature of near membrane ion signaling in many cell types, and their discovery offers new possibilities for elucidating the mechanism, function and pathogenesis of Na+ and Ca2+ signaling nanodomains.

Effective stimulation of growth in MCF-7 human breast cancer cells by inhibition of syntaxin18 by external guide sequence and ribonuclease P.

Syntaxin18 (Stx18) is an endoplasmic reticulum (ER)-membrane bound SNARE protein involved in membrane trafficking between the ER and Golgi as well as in phagocytosis. Stx18 has also been shown to physically interact with proteins involved in the cell cycle and apoptosis. These findings suggest the possible role of Stx18 in regulating cell growth. In this study, we used theoretically designed external guide sequence molecule which utilizes RNase P to cleave Stx18 mRNA and down-regulate Stx18 levels in MCF-7 human breast cancer cells. We showed that down-regulation of Stx18 leads to significant enhancement of growth in MCF-7 cells. Consistent with this finding was the observation that over-expression of Stx18 using the CMV promoter led to suppression of cell growth. Over-expressing Stx18 had no effect on c-myc mRNA expression and half-life, suggesting that the mechanism does not involve control at the transcriptional and post-transcriptional level of the c-myc gene. Finally, we showed that Stx18 is over-expressed in clinical human breast cancer. Overall, this study showed that Stx18 plays a role in the growth of human breast cancer cells and provided the basis for further investigation in determining whether it can be used as a prognostic marker and as a molecular target in the treatment of breast cancer.

Acetylcholine-induced asynchronous calcium waves in intact human bronchial muscle bundle.

Calcium (Ca2+) is an important activator of the contractile machinery in airway smooth muscle (ASM). While agonist-induced Ca2+ signals are well characterized in animal ASM, little is known about what occurs in adult human ASM. In this study, we examined the Ca2+ signal elicited by acetylcholine (ACh) in smooth muscle cells of the intact human bronchial muscle strips obtained from fresh surgical specimens in relation to muscle contraction. We found that ACh induces repetitive Ca2+ waves that spread along the longitudinal axis of individual cells in the intact human bronchial smooth muscle strips. These Ca2+ waves display no apparent synchronization between neighboring cells, and their generation precedes force development. Comparison of the ACh concentration dependence of tissue contraction and selected parameters of the asynchronous Ca2+ waves (ACW) reveals that the graded force generation by ACh-stimulated human bronchial muscle strips is achieved by differential recruitment of cells to initiate Ca2+ waves and by enhancement of the frequency of ACW once the cells are recruited. Furthermore, pharmacologic characterization shows that the ACW are produced by repetitive cycles of SR Ca2+ release via ryanodine-sensitive channels followed by SR Ca2+ reuptake by sarco(endo)plasmic reticulum Ca2+ ATPase. Extracellular Ca2+ entry involving receptor-operated channels/store-operated channels, reverse-mode Na+/Ca2+ exchange, and to a lesser extent L-type voltage-gated Ca2+ channels is required to maintain the ACW. These findings for the first time demonstrate the occurrence and the role of ACW in excitation-contraction coupling in adult human ASM.

Mechanism of ACh-induced asynchronous calcium waves and tonic contraction in porcine tracheal muscle bundle.

Stimulation of the tracheal muscle bundle by acetylcholine (ACh) results in the generation of asynchronous repetitive Ca2+ waves (ACW) in intact tracheal smooth muscle (TSM) cells. We showed previously that ACW underlie cholinergic excitation-contraction coupling in porcine TSM and that Ca2+ entry through the L-type voltage-gated Ca2+ channel (VGCC) contributes partially to maintenance of the ACW. However, the mechanism of the ACW remains undefined. In this study, we pharmacologically characterized the mechanism of ACh-induced ACW in the intact porcine tracheal muscle bundle. We found that inhibition of receptor-operated channels/store-operated channels (ROC/SOC) by SKF-96365 completely abolished the nifedipine-insensitive component of ACh-mediated ACW and tonic contraction. Blockade of Na+/Ca2+ exchange with KB-R7943 or 2',4'-dichlorobenzamil or removal of extracellular Na+ resulted in nearly complete inhibition of the nifedipine-insensitive component of ACh-mediated ACW and tonic contraction. Inhibition of the sarco(endo)plasmic reticulum Ca2+-ATPase by cyclopiazonic acid abolished the ongoing ACW. Application of 2-aminoethoxydiphenyl borate (2-APB) or xestospongin C to inhibit the inositol 1,4,5-trisphosphate-sensitive sarcoplasmic reticulum (SR) Ca2+ release channels produced no effect on ACh-mediated ACW and tonic contraction. However, pretreatment with caffeine or ryanodine inhibited ACh-induced ACW. Furthermore, application of procaine or tetracaine prevented the generation and abolished the ongoing ACh-mediated ACW and tonic contraction. Collectively, these results indicate that the ACh-stimulated ACW in porcine TSM are produced by repetitive cycles of Ca2+ release from SR through 2-APB- and xestospongin C-insensitive Ca2+ release channels, and plasmalemmal Ca2+ entry involving reverse-mode Na+/Ca2+ exchange, ROC/SOC, and L-type VGCC is required to refill the SR via SERCA to support the ongoing ACW.

Store-operated Ca2+ entry modulates sarcoplasmic reticulum Ca2+ loading in neonatal rabbit cardiac ventricular myocytes.

Store-operated Ca2+ entry (SOCE), which is Ca2+ entry triggered by the depletion of intracellular Ca2+ stores, has been observed in many cell types, but only recently has it been suggested to occur in cardiomyocytes. In the present study, we have demonstrated SOCE-dependent sarcoplasmic reticulum (SR) Ca2+ loading (load(SR)) that was not altered by inhibition of L-type Ca2+ channels, reverse mode Na+/Ca2+ exchange (NCX), or nonselective cation channels. In contrast, lowering the extracellular [Ca2+] to 0 mM or adding either 0.5 mM Zn2+ or the putative store-operated channel (SOC) inhibitor SKF-96365 (100 microM) inhibited load(SR) at rest. Interestingly, inhibition of forward mode NCX with 30 microM KB-R7943 stimulated SOCE significantly and resulted in enhanced load(SR). In addition, manipulation of the extracellular and intracellular Na+ concentrations further demonstrated the modulatory role of NCX in SOCE-mediated SR Ca2+ loading. Although there is little knowledge of SOCE in cardiomyocytes, the present results suggest that this mechanism, together with NCX, may play an important role in SR Ca2+ homeostasis. The data reported herein also imply the presence of microdomains unique to the neonatal cardiomyocyte. These findings may be of particular importance during open heart surgery in neonates, in which uncontrolled SOCE could lead to SR Ca2+ overload and arrhythmogenesis.

The critical component to establish in vitro BBB model: Pericyte.

The blood-brain barrier (BBB), a highly regulated membranous barrier of brain capillaries, consists of an intricate network of tight junctions (TJs) that segregate the central nervous system (CNS) from systemic blood circulation and maintain a delicate homeostasis of the CNS environment. While endothelial cells (ECs) of brain capillaries are clearly the principal cellular element of BBB, the formation and regulation of intact BBB structure appear to require the interactions of endothelial cells with other cellular components. Astrocytes, one of the major non-neural cells in the brain, associate closely and interact with capillary endothelial cells during the angiogenesis and BBB development. Current in vitro cellular models for the study of BBB functions often incorporate astrocytes with endothelial cells. However, another foremost cell type, CNS pericyte, which intimately embraces brain capillary endothelium, attracts relatively little attention for its role in developing the in vitro BBB system. This review will analyze the critical functions of pericytes in angiogenesis in various systems and discuss the relevance of these functions in mediating the development, maintenance, and regulation of BBB. The author will also discuss the functional role of actin in both ECs and pericytes, and further elaborate the molecular mechanisms of BBB permeability regulation that involves the transduction pathway-mediated actin remodeling process. Finally, the rationale of incorporating pericytes for establishing a better in vitro BBB model will be emphasized.