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Uterine contractions during labor and preterm labor are influenced by a complex interplay of factors, including hormones and inflammatory mediators. This complexity may contribute to the limited efficacy of current tocolytics for preterm labor, a significant challenge in obstetrics with 15 million cases annually and approximately 1 million resulting deaths worldwide. We have previously shown that the myometrium expresses bitter taste receptors (TAS2Rs) and that their activation leads to uterine relaxation. Here, we investigated whether the selective TAS2R5 agonist phenanthroline can induce relaxation across a spectrum of human uterine contractions and whether the underlying mechanism involves changes in intracellular Ca 2+ signaling. We performed experiments using samples from pregnant women undergoing scheduled cesarean delivery, assessing responses to various inflammatory mediators and oxytocin with and without phenanthroline. Our results showed that phenanthroline concentration-dependently inhibited contractions induced by PGF2α, U46619, 5-HT, endothelin-1 and oxytocin. Furthermore, in hTERT-infected human myometrial cells exposed to uterotonics, phenanthroline effectively suppressed the increase in intracellular Ca 2+ concentration induced by PGF2α, U46619, oxytocin, and endothelin-1. These results suggest that the selective TAS2R5 agonist may not only significantly reduce uterine contractions but also decrease intracellular Ca 2+ levels. This study highlights the potential development of TAS2R5 agonists as a new class of uterine relaxants, providing a novel avenue for improving the management of preterm labor., Competing Interests: Declaration of competing interest All authors declare that there are no conflicts of interest associated with the content of this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Circulating lactate is a fuel source for liver metabolism but may exacerbate metabolic diseases such as nonalcoholic steatohepatitis (NASH). Indeed, haploinsufficiency of lactate transporter monocarboxylate transporter 1 (MCT1) in mice reportedly promotes resistance to hepatic steatosis and inflammation. Here, we used adeno-associated virus (AAV) vectors to deliver thyroxin binding globulin (TBG)-Cre or lecithin-retinol acyltransferase (Lrat)-Cre to MCT1 fl/fl mice on a choline-deficient, high-fat NASH diet to deplete hepatocyte or stellate cell MCT1, respectively. Stellate cell MCT1KO (AAV-Lrat-Cre) attenuated liver type 1 collagen protein expression and caused a downward trend in trichrome staining. MCT1 depletion in cultured human LX2 stellate cells also diminished collagen 1 protein expression. Tetra-ethylenglycol-cholesterol (Chol)-conjugated siRNAs, which enter all hepatic cell types, and hepatocyte-selective tri- N -acetyl galactosamine (GN)-conjugated siRNAs were then used to evaluate MCT1 function in a genetically obese NASH mouse model. MCT1 silencing by Chol-siRNA decreased liver collagen 1 levels, while hepatocyte-selective MCT1 depletion by AAV-TBG-Cre or by GN-siRNA unexpectedly increased collagen 1 and total fibrosis without effect on triglyceride accumulation. These findings demonstrate that stellate cell lactate transporter MCT1 significantly contributes to liver fibrosis through increased collagen 1 protein expression in vitro and in vivo, while hepatocyte MCT1 appears not to be an attractive therapeutic target for NASH., Competing Interests: KM, BY, MK, DE, ME, LL, NR, ET, SH, CD, HW, NM, BB, BM, JR, AK No competing interests declared, MC Reviewing editor, eLife, (© 2023, Min et al.)

Maintenance of fecal continence requires a continuous or basal tone of the internal anal sphincter (IAS). Paradoxically, the basal tone results largely from high-frequency rhythmic contractions of the IAS smooth muscle. However, the cellular and molecular mechanisms that initiate these contractions remain elusive. Here we show that the IAS contains multiple pacemakers. These pacemakers spontaneously generate propagating calcium waves that drive rhythmic contractions and establish the basal tone. These waves are myogenic and act independently of nerve, paracrine or autocrine signals. Using cell-specific gene knockout mice, we further found that TMEM16A Cl - channels in smooth muscle cells (but not in the interstitial cells of Cajal) are indispensable for pacemaking, rhythmic contractions, and basal tone. Our results identify TMEM16A in smooth muscle cells as a critical pacemaker channel that enables the IAS to contract rhythmically and continuously. This study provides cellular and molecular insights into fecal continence., (© 2024. The Author(s).)

Objectives: Nuclear receptor interacting protein 1 (NRIP1) suppresses energy expenditure via repression of nuclear receptors, and its depletion markedly elevates uncoupled respiration in mouse and human adipocytes. We tested whether NRIP1 deficient adipocytes implanted into obese mice would enhance whole body metabolism. Since β-adrenergic signaling through cAMP strongly promotes adipocyte thermogenesis, we tested whether the effects of NRIP1 knock-out (NRIP1KO) require the cAMP pathway., Methods: NRIP1KO adipocytes were implanted in recipient high-fat diet (HFD) fed mice and metabolic cage studies conducted. The Nrip1 gene was disrupted by CRISPR in primary preadipocytes isolated from control vs adipose selective GsαKO (cAdGsαKO) mice prior to differentiation to adipocytes. Protein kinase A inhibitor was also used., Results: Implanting NRIP1KO adipocytes into HFD fed mice enhanced whole-body glucose tolerance by increasing insulin sensitivity, reducing adiposity, and enhancing energy expenditure in the recipients. NRIP1 depletion in both control and GsαKO adipocytes was equally effective in upregulating uncoupling protein 1 (UCP1) and adipocyte beiging, while β-adrenergic signaling by CL 316,243 was abolished in GsαKO adipocytes. Combining NRIP1KO with CL 316,243 treatment synergistically increased Ucp1 gene expression and increased the adipocyte subpopulation responsive to beiging. Estrogen-related receptor α (ERRα) was dispensable for UCP1 upregulation by NRIPKO., Conclusions: The thermogenic effect of NRIP1 depletion in adipocytes causes systemic enhancement of energy expenditure when such adipocytes are implanted into obese mice. Furthermore, NRIP1KO acts independently but cooperatively with the cAMP pathway in mediating its effect on adipocyte beiging., Competing Interests: Declaration of competing interest The authors declare the following competing Interests: M.P.C. and A.G. are inventors of granted US Patent US-8519118-B2, “RIP140 regulation of glucose transport”, and of granted US Patent US-8093223-B2, “RIP140 regulation of diabetes”, related to data in this manuscript on Nrip1/RIP140-depleted mouse and human adipocytes. M.P.C., E.T., and S.M.N. are inventors of US Patent US-20220220461-A1 and PCT WO2022076812A3, “Targeting Nrip1 to Alleviate Metabolic Disease”, related to CRISPR-based depletion of NRIP1 in mouse adipocytes in this manuscript. The University of Massachusetts is the grantee or potential grantee of all the above. M.P.C. declares that he is bound by a confidentiality agreement that prevents him from disclosing a potentially competing interest in this work. S.A.W. is a consultant for Chroma Medicine. The remaining authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Obesity and type 2 diabetes are associated with disturbances in insulin-regulated glucose and lipid fluxes and severe comorbidities including cardiovascular disease and steatohepatitis. Whole body metabolism is regulated by lipid-storing white adipocytes as well as "brown" and "brite/beige" adipocytes that express thermogenic uncoupling protein 1 (UCP1) and secrete factors favorable to metabolic health. Implantation of brown fat into obese mice improves glucose tolerance, but translation to humans has been stymied by low abundance of primary human beige adipocytes. Here we apply methods to greatly expand human adipocyte progenitors from small samples of human subcutaneous adipose tissue and then disrupt the thermogenic suppressor gene NRIP1 by CRISPR. Ribonucleoprotein consisting of Cas9 and sgRNA delivered ex vivo are fully degraded by the human cells following high efficiency NRIP1 depletion without detectable off-target editing. Implantation of such CRISPR-enhanced human or mouse brown-like adipocytes into high fat diet fed mice decreases adiposity and liver triglycerides while enhancing glucose tolerance compared to implantation with unmodified adipocytes. These findings advance a therapeutic strategy to improve metabolic homeostasis through CRISPR-based genetic enhancement of human adipocytes without exposing the recipient to immunogenic Cas9 or delivery vectors., (© 2021. The Author(s).)

Adenomyosis is a debilitating gynecological disease of the uterus with no medicinal cure. The tissue injury and repair hypothesis for adenomyosis suggests that uterine hyperperistalsis or dysperistalsis plays a pivotal role in establishing adenomyotic lesions. However, specific impairments in uterine peristalsis and the underlying cellular signals for these changes in adenomyosis remain elusive. Here, we report a precision-cut uterine slice preparation that preserves in vivo uterine architecture and generates peristalsis similar to that seen in the whole uterus. We found that uterine peristalsis in neonatal mice at day 14 and adult mice at day 55 presents as bursts with multiple peaks induced by intracellular Ca 2+ oscillations. Using a mouse model of adenomyosis induced by tamoxifen, a selective estrogen receptor modulator, we discovered that uterine peristalsis and Ca 2+ oscillations from adenomyotic uteri on days 14 and 55 become spikes (single peaks) with smaller amplitudes. The peak frequency of Ca 2+ oscillations or peristalsis does not show a difference between control and adenomyotic mice. However, both the estimated force generated by uterine peristalsis and the total Ca 2+ raised by Ca 2+ oscillations are smaller in uteri from adenomyotic mice. Uteri from adenomyotic mice on day 14, but not on day 55, exhibit hyperresponsiveness to oxytocin. Embryo implantations are decreased in adenomyotic adult mice. Our results reveal a mode switch from bursts to spikes (rather than an increased peak frequency) of uterine Ca 2+ oscillations and peristalsis and concurrent hyperresponsiveness to oxytocin in the neonatal stage are two characteristics of adenomyosis. These characteristics may contribute to embryo implantation impairments and decreased fertility in adenomyosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Qu, Lu, Bellve, Lifshitz and ZhuGe.)

Bitter taste receptors (TAS2Rs) and their signaling elements are detected throughout the body, and bitter tastants induce a wide variety of biological responses in tissues and organs outside the mouth. However, the roles of TAS2Rs in these responses remain to be tested and established genetically. Here, we employed the CRISPR/Cas9 gene-editing technique to delete three bitter taste receptors-Tas2r143/Tas2r135/Tas2r126 (i.e., Tas2r triple knockout [TKO]) in mice. The fidelity and effectiveness of the Tas2r deletions were validated genetically at DNA and messenger RNA levels and functionally based on the tasting of TAS2R135 and TAS2R126 agonists. Bitter tastants are known to relax airways completely. However, TAS2R135 or TAS2R126 agonists either failed to induce relaxation of pre-contracted airways in wild-type mice and Tas2r TKO mice or relaxed them dose-dependently, but to the same extent in both types of mice. These results indicate that TAS2Rs are not required for bitter tastant-induced bronchodilation. The Tas2r TKO mice also provide a valuable model to resolve whether TAS2Rs mediate bitter tastant-induced responses in many other extraoral tissues., (© 2021 Wiley Periodicals LLC.)

A persistent basal tone in the internal anal sphincter (IAS) is essential for keeping the anal canal closed and fecal continence; its inhibition via the rectoanal inhibitory reflex (RAIR) is required for successful defecation. However, cellular signals underlying the IAS basal tone remain enigmatic. Here we report the origin and molecular mechanisms of calcium signals that control the IAS basal tone, using a combination approach including a novel IAS slice preparation that retains cell arrangement and architecture as in vivo, 2-photon imaging, and cell-specific gene-modified mice. We found that IAS smooth muscle cells generate two forms of contractions (i.e., phasic and sustained contraction) and Ca 2+ signals (i.e., synchronized Ca 2+ oscillations [SCaOs] and asynchronized Ca 2+ oscillations [ACaOs]) that last for hours. RyRs, TMEM16A, L-type Ca 2+ channels, and gap junctions are required for SCaOs, which account for phasic contraction and 75% of sustained contraction. Nevertheless, only RyRs are required for ACaOs, which contribute 25% of sustained contraction. Nitric oxide, the primary neurotransmitter mediating the RAIR, blocks both types of Ca 2+ signals, leading to IAS's full relaxation. Our results show that the oscillating nature of Ca 2+ signals generates and maintains the basal tone without causing cytotoxicity to IAS. Our study provides insight into fecal continence and normal defecation., (© 2021 Wiley Periodicals LLC.)

Adipocytes deficient in fatty acid synthase (iAdFASNKO) emit signals that mimic cold exposure to enhance the appearance of thermogenic beige adipocytes in mouse inguinal white adipose tissues (iWATs). Both cold exposure and iAdFASNKO upregulate the sympathetic nerve fiber (SNF) modulator Neuregulin 4 (Nrg4), activate SNFs, and require adipocyte cyclic AMP/protein kinase A (cAMP/PKA) signaling for beige adipocyte appearance, as it is blocked by adipocyte Gsα deficiency. Surprisingly, however, in contrast to cold-exposed mice, neither iWAT denervation nor Nrg4 loss attenuated adipocyte browning in iAdFASNKO mice. Single-cell transcriptomic analysis of iWAT stromal cells revealed increased macrophages displaying gene expression signatures of the alternately activated type in iAdFASNKO mice, and their depletion abrogated iWAT beiging. Altogether, these findings reveal that divergent cellular pathways are sufficient to cause adipocyte browning. Importantly, adipocyte signaling to enhance alternatively activated macrophages in iAdFASNKO mice is associated with enhanced adipose thermogenesis independent of the sympathetic neuron involvement this process requires in the cold., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Single-cell sequencing technologies have revealed an unexpectedly broad repertoire of cells required to mediate complex functions in multicellular organisms. Despite the multiple roles of adipose tissue in maintaining systemic metabolic homeostasis, adipocytes are thought to be largely homogenous with only 2 major subtypes recognized in humans so far. Here we report the existence and characteristics of 4 distinct human adipocyte subtypes, and of their respective mesenchymal progenitors. The phenotypes of these distinct adipocyte subtypes are differentially associated with key adipose tissue functions, including thermogenesis, lipid storage, and adipokine secretion. The transcriptomic signature of "brite/beige" thermogenic adipocytes reveals mechanisms for iron accumulation and protection from oxidative stress, necessary for mitochondrial biogenesis and respiration upon activation. Importantly, this signature is enriched in human supraclavicular adipose tissue, confirming that these cells comprise thermogenic depots in vivo, and explain previous findings of a rate-limiting role of iron in adipose tissue browning. The mesenchymal progenitors that give rise to beige/brite adipocytes express a unique set of cytokines and transcriptional regulators involved in immune cell modulation of adipose tissue browning. Unexpectedly, we also find adipocyte subtypes specialized for high-level expression of the adipokines adiponectin or leptin, associated with distinct transcription factors previously implicated in adipocyte differentiation. The finding of a broad adipocyte repertoire derived from a distinct set of mesenchymal progenitors, and of the transcriptional regulators that can control their development, provides a framework for understanding human adipose tissue function and role in metabolic disease., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Presynaptic reuptake, mediated by the dopamine (DA) transporter (DAT), terminates DAergic neurotransmission and constrains extracellular DA levels. Addictive and therapeutic psychostimulants inhibit DA reuptake and multiple DAT coding variants have been reported in patients with neuropsychiatric disorders. These findings underscore that DAT is critical for DA neurotransmission and homeostasis. DAT surface availability is regulated acutely by endocytic trafficking, and considerable effort has been directed toward understanding mechanisms that govern DAT's plasma membrane expression and postendocytic fate. Multiple studies have demonstrated DAT endocytic recycling and enhanced surface delivery in response to various stimuli. Paradoxically, imaging studies have not detected DAT targeting to classic recycling endosomes, suggesting that internalized DAT targets to either degradation or an undefined recycling compartment. Here, we leveraged PRIME ( PR obe I ncorporation M ediated by E nzyme) labeling to couple surface DAT directly to fluorophore, and tracked DAT's postendocytic itinerary in immortalized mesencephalic cells. Following internalization, DAT robustly targeted to retromer-positive endosomes, and DAT/retromer colocalization was observed in male mouse dopaminergic somatodendritic and terminal regions. Short hairpin RNA-mediated Vps35 knockdown revealed that DAT endocytic recycling requires intact retromer. DAT also targeted rab7-positive endosomes with slow, linear kinetics that were unaffected by either accelerating DAT internalization or binding a high-affinity cocaine analog. However, cocaine increased DAT exit from retromer-positive endosomes significantly. Finally, we found that the DAT carboxy-terminal PDZ-binding motif was required for DAT recycling and exit from retromer. These results define the DAT recycling mechanism and provide a unifying explanation for previous, seemingly disparate, DAT endocytic trafficking findings. SIGNIFICANCE STATEMENT The neuronal dopamine (DA) transporter (DAT) recaptures released DA and modulates DAergic neurotransmission, and a number of DAT coding variants have been reported in several DA-related disorders, including infantile parkinsonism, attention-deficit/hyperactivity disorder and autism spectrum disorder. DAT is also competitively inhibited by psychostimulants with high abuse potential. Therefore, mechanisms that acutely affect DAT availability will likely exert significant impact on both normal and pathological DAergic homeostasis. Here, we explore the cellular mechanisms that acutely control DAT surface expression. Our results reveal the intracellular mechanisms that mediate DAT endocytic recycling following constitutive and regulated internalization. In addition to shedding light on this critical process, these findings resolve conflict among multiple, seemingly disparate, previous reports on DAT's postendocytic fate., (Copyright © 2017 the authors 0270-6474/17/379438-15$15.00/0.)

Preterm birth (PTB) is the leading cause of neonatal mortality and morbidity, with few prevention and treatment options. Uterine contraction is a central feature of PTB, so gaining new insights into the mechanisms of this contraction and consequently identifying novel targets for tocolytics are essential for more successful management of PTB. Here we report that myometrial cells from human and mouse express bitter taste receptors (TAS2Rs) and their canonical signaling components ( i.e., G-protein gustducin and phospholipase C β2). Bitter tastants can completely relax myometrium precontracted by different uterotonics. In isolated single mouse myometrial cells, a phenotypical bitter tastant (chloroquine, ChQ) reverses the rise in intracellular Ca 2+ concentration ([Ca 2+ ] i ) and cell shortening induced by uterotonics, and this reversal effect is inhibited by pertussis toxin and by genetic deletion of α-gustducin. In human myometrial cells, knockdown of TAS2R14 but not TAS2R10 inhibits ChQ's reversal effect on an oxytocin-induced rise in [Ca 2+ ] i Finally, ChQ prevents mouse PTBs induced by bacterial endotoxin LPS or progesterone receptor antagonist mifepristone more often than current commonly used tocolytics, and this prevention is largely lost in α-gustducin-knockout mice. Collectively, our results reveal that activation of the canonical TAS2R signaling system in myometrial cells produces profound relaxation of myometrium precontracted by a broad spectrum of contractile agonists, and that targeting TAS2Rs is an attractive approach to developing effective tocolytics for PTB management.-Zheng, K., Lu, P., Delpapa, E., Bellve, K., Deng, R., Condon, J. C., Fogarty, K., Lifshitz, L. M., Simas, T. A. M., Shi, F., ZhuGe, R. Bitter taste receptors as targets for tocolytics in preterm labor therapy., (© FASEB.)

Bitter taste receptors (TAS2Rs or T2Rs) belong to the superfamily of seven-transmembrane G protein-coupled receptors, which are the targets of >50% of drugs currently on the market. Canonically, T2Rs are located in taste buds of the tongue, where they initiate bitter taste perception. However, accumulating evidence indicates that T2Rs are widely expressed throughout the body and mediate diverse nontasting roles through various specialized mechanisms. It has also become apparent that T2Rs and their polymorphisms are associated with human disorders. In this review, we summarize the physiological and pathophysiological roles that extraoral T2Rs play in processes as diverse as innate immunity and reproduction, and the major challenges in this emerging field., (© 2017 Lu et al.)

siRNAs are a new class of therapeutic modalities with promising clinical efficacy that requires modification or formulation for delivery to the tissue and cell of interest. Conjugation of siRNAs to lipophilic groups supports efficient cellular uptake by a mechanism that is not well characterized. Here we study the mechanism of internalization of asymmetric, chemically stabilized, cholesterol-modified siRNAs (sd-rxRNAs ® ) that efficiently enter cells and tissues without the need for formulation. We demonstrate that uptake is rapid with significant membrane association within minutes of exposure followed by the formation of vesicular structures and internalization. Furthermore, sd-rxRNAs are internalized by a specific class of early endosomes and show preferential association with epidermal growth factor (EGF) but not transferrin (Tf) trafficking pathways as shown by live cell TIRF and structured illumination microscopy (SIM). In fixed cells, we observe ∼25% of sd-rxRNA co-localizing with EGF and <5% with Tf, which is indicative of selective endosomal sorting. Likewise, preferential sd-rxRNA co-localization was demonstrated with EEA1 but not RBSN-containing endosomes, consistent with preferential EGF-like trafficking through EEA1-containing endosomes. sd-rxRNA cellular uptake is a two-step process, with rapid membrane association followed by internalization through a selective, saturable subset of the endocytic process. However, the mechanistic role of EEA1 is not yet known. This method of visualization can be used to better understand the kinetics and mechanisms of hydrophobic siRNA cellular uptake and will assist in further optimization of these types of compounds for therapeutic intervention., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Smooth muscle sphincters exhibit basal tone and control passage of contents through organs such as the gastrointestinal tract; loss of this tone leads to disorders such as faecal incontinence. However, the molecular mechanisms underlying this tone remain unknown. Here, we show that deletion of myosin light-chain kinases (MLCK) in the smooth muscle cells from internal anal sphincter (IAS-SMCs) abolishes basal tone, impairing defecation. Pharmacological regulation of ryanodine receptors (RyRs), L-type voltage-dependent Ca(2+) channels (VDCCs) or TMEM16A Ca(2+)-activated Cl(-) channels significantly changes global cytosolic Ca(2+) concentration ([Ca(2+)]i) and the tone. TMEM16A deletion in IAS-SMCs abolishes the effects of modulators for TMEM16A or VDCCs on a RyR-mediated rise in global [Ca(2+)]i and impairs the tone and defecation. Hence, MLCK activation in IAS-SMCs caused by a global rise in [Ca(2+)]i via a RyR-TMEM16A-VDCC signalling module sets the basal tone. Targeting this module may lead to new treatments for diseases like faecal incontinence.

Aims/hypothesis: During pregnancy, adipose tissue (AT) must expand to support the growing fetus and the future nutritional needs of the offspring. Limited expandability of AT is associated with insulin resistance, attributed to ectopic lipid deposition. This study aimed to investigate human AT expandability during pregnancy and its role in the pathogenesis of gestational diabetes mellitus (GDM)., Methods: This cross-sectional study of omental (OM) and subcutaneous (SQ) AT collected at Caesarean delivery included 11 pregnant and three non-pregnant women with normal glucose tolerance (NGT), five with GDM, three with type 2 diabetes mellitus. Adipocyte size, capillary density, collagen content and capillary growth were measured. Affymetrix arrays and real-time PCR studies of gene expression were performed., Results: Mean OM adipocyte size was greater in women with GDM than in those with NGT (p = 0.004). Mean OM and SQ capillary density was lower in GDM compared with NGT (p = 0.015). Capillary growth did not differ significantly between groups. The most differentially expressed AT transcript when comparing non-pregnant and pregnant women corresponded to the IGF binding protein (IGFBP)-5, the expression levels of which was found by subsequent quantitative real-time PCR to be lower in women with GDM vs women with NGT (p < 0.0001)., Conclusions/interpretation: The relative OM adipocyte hypertrophy and decreased OM and SQ capillary density are consistent with impaired AT expandability in GDM. The induction of adipose tissue IGFBP5 in pregnancy and its decrease in GDM point to the importance of the IGF-1 signalling pathway in AT expansion in pregnancy and GDM susceptibility.

Changes in adipose tissue mass must be accompanied by parallel changes in microcirculation. Investigating the mechanisms that regulate adipose tissue angiogenesis could lead to better understanding of adipose tissue function and reveal new potential therapeutic strategies. Angiogenesis is defined as the formation of new capillaries from existing microvessels. This process can be recapitulated in vitro, by incubation of tissue in extracellular matrix components in the presence of pro-angiogenic factors. Here, we describe a method to study angiogenesis from adipose tissue fragments obtained from mouse and human tissue. This assay can be used to define effects of diverse factors added in vitro, as well as the role of endogenously produced factors on angiogenesis. We also describe approaches to quantify angiogenic potential for the purpose of enabling comparisons between subjects, thus providing information on the role of physiological conditions of the donor on adipose tissue angiogenic potential., (© 2014 Elsevier Inc. All rights reserved.)

Rationale: Asthma is a chronic inflammatory disorder with a characteristic of airway hyperresponsiveness (AHR). Ca(2+)-activated Cl(-) [Cl((Ca))] channels are inferred to be involved in AHR, yet their molecular nature and the cell type they act within to mediate this response remain unknown., Objectives: Transmembrane protein 16A (TMEM16A) and TMEM16B are Cl((Ca)) channels, and activation of Cl((Ca)) channels in airway smooth muscle (ASM) contributes to agonist-induced airway contraction. We hypothesized that Tmem16a and/or Tmem16b encode Cl((Ca)) channels in ASM and mediate AHR., Methods: We assessed the expression of the TMEM16 family, and the effects of niflumic acid and benzbromarone on AHR and airway contraction, in an ovalbumin-sensitized mouse model of chronic asthma. We also cloned TMEM16A from ASM and examined the Cl(-) currents it produced in HEK293 cells. We further studied the impacts of TMEM16A deletion on Ca(2+) agonist-induced cell shortening, and on Cl((Ca)) currents activated by Ca(2+) sparks (localized, short-lived Ca(2+) transients due to the opening of ryanodine receptors) in mouse ASM cells., Measurements and Main Results: TMEM16A, but not TMEM16B, is expressed in ASM cells and its expression in these cells is up-regulated in ovalbumin-sensitized mice. Niflumic acid and benzbromarone prevent AHR and contraction evoked by methacholine in ovalbumin-sensitized mice. TMEM16A produces Cl((Ca)) currents with kinetics similar to native Cl((Ca)) currents. TMEM16A deletion renders Ca(2+) sparks unable to activate Cl((Ca)) currents, and weakens caffeine- and methacholine-induced cell shortening., Conclusions: Tmem16a encodes Cl((Ca)) channels in ASM and contributes to Ca(2+) agonist-induced contraction. In addition, up-regulation of TMEM16A and its augmented activation contribute to AHR in an ovalbumin-sensitized mouse model of chronic asthma. TMEM16A may represent a potential therapeutic target for asthma.

Bronchodilators are a standard medicine for treating airway obstructive diseases, and β2 adrenergic receptor agonists have been the most commonly used bronchodilators since their discovery. Strikingly, activation of G-protein-coupled bitter taste receptors (TAS2Rs) in airway smooth muscle (ASM) causes a stronger bronchodilation in vitro and in vivo than β2 agonists, implying that new and better bronchodilators could be developed. A critical step towards realizing this potential is to understand the mechanisms underlying this bronchodilation, which remain ill-defined. An influential hypothesis argues that bitter tastants generate localized Ca(2+) signals, as revealed in cultured ASM cells, to activate large-conductance Ca(2+)-activated K(+) channels, which in turn hyperpolarize the membrane, leading to relaxation. Here we report that in mouse primary ASM cells bitter tastants neither evoke localized Ca(2+) events nor alter spontaneous local Ca(2+) transients. Interestingly, they increase global intracellular [Ca(2+)]i, although to a much lower level than bronchoconstrictors. We show that these Ca(2+) changes in cells at rest are mediated via activation of the canonical bitter taste signaling cascade (i.e., TAS2R-gustducin-phospholipase Cβ [PLCβ]- inositol 1,4,5-triphosphate receptor [IP3R]), and are not sufficient to impact airway contractility. But activation of TAS2Rs fully reverses the increase in [Ca(2+)]i induced by bronchoconstrictors, and this lowering of the [Ca(2+)]i is necessary for bitter tastant-induced ASM cell relaxation. We further show that bitter tastants inhibit L-type voltage-dependent Ca(2+) channels (VDCCs), resulting in reversal in [Ca(2+)]i, and this inhibition can be prevented by pertussis toxin and G-protein βγ subunit inhibitors, but not by the blockers of PLCβ and IP3R. Together, we suggest that TAS2R stimulation activates two opposing Ca(2+) signaling pathways via Gβγ to increase [Ca(2+)]i at rest while blocking activated L-type VDCCs to induce bronchodilation of contracted ASM. We propose that the large decrease in [Ca(2+)]i caused by effective tastant bronchodilators provides an efficient cell-based screening method for identifying potent dilators from among the many thousands of available bitter tastants., Competing Interests: The authors have declared that no competing interests exist.

Cell surface receptors and other proteins internalize through diverse mechanisms at the plasma membrane and are sorted to different destinations. Different subpopulations of early endosomes have been described, raising the question of whether different internalization mechanisms deliver cargo into different subsets of early endosomes. To address this fundamental question, we developed a microscopy platform to detect the precise position of endosomes relative to the plasma membrane during the uptake of ligands. Axial resolution is maximized by concurrently applied total internal reflection fluorescence and epifluorescence-structured light. We found that transferrin receptors are delivered selectively from clathrin-coated pits on the plasma membrane into a specific subpopulation of endosomes enriched in the multivalent Rab GTPase and phosphoinositide-binding protein Rabenosyn-5. Depletion of Rabenosyn-5, but not of other early endosomal proteins such as early endosome antigen 1, resulted in impaired transferrin uptake and lysosomal degradation of transferrin receptors. These studies reveal a critical role for Rabenosyn-5 in determining the fate of transferrin receptors internalized by clathrin-mediated endocytosis and, more broadly, a mechanism whereby the delivery of cargo from the plasma membrane into specific early endosome subpopulations is required for its appropriate intracellular traffic.

Dopaminergic signaling and plasticity are essential to numerous CNS functions and pathologies, including movement, cognition, and addiction. The amphetamine- and cocaine-sensitive dopamine (DA) transporter (DAT) tightly controls extracellular DA concentrations and half-life. DAT function and surface expression are not static but are dynamically modulated by membrane trafficking. We recently demonstrated that the DAT C terminus encodes a PKC-sensitive internalization signal that also suppresses basal DAT endocytosis. However, the cellular machinery governing regulated DAT trafficking is not well defined. In work presented here, we identified the Ras-like GTPase, Rin (for Ras-like in neurons) (Rit2), as a protein that interacts with the DAT C-terminal endocytic signal. Yeast two-hybrid, GST pull down and FRET studies establish that DAT and Rin directly interact, and colocalization studies reveal that DAT/Rin associations occur primarily in lipid raft microdomains. Coimmunoprecipitations demonstrate that PKC activation regulates Rin association with DAT. Perturbation of Rin function with GTPase mutants and shRNA-mediated Rin knockdown reveals that Rin is critical for PKC-mediated DAT internalization and functional downregulation. These results establish that Rin is a DAT-interacting protein that is required for PKC-regulated DAT trafficking. Moreover, this work suggests that Rin participates in regulated endocytosis.

Short-lived, localized Ca(2+) events mediate Ca(2+) signaling with high efficiency and great fidelity largely as a result of the close proximity between Ca(2+)-permeable ion channels and their molecular targets. However, in most cases, direct evidence of the spatial relationship between these two types of molecules is lacking, and, thus, mechanistic understanding of local Ca(2+) signaling is incomplete. In this study, we use an integrated approach to tackling this issue on a prototypical local Ca(2+) signaling system composed of Ca(2+) sparks resulting from the opening of ryanodine receptors (RYRs) and spontaneous transient outward currents (STOCs) caused by the opening of Ca(2+)-activated K(+) (BK) channels in airway smooth muscle. Biophysical analyses of STOCs and Ca(2+) sparks acquired at 333 Hz demonstrate that these two events are associated closely in time, and approximately eight RYRs open to give rise to a Ca(2+) spark, which activates ∼15 BK channels to generate a STOC at 0 mV. Dual immunocytochemistry and 3-D deconvolution at high spatial resolution reveal that both RYRs and BK channels form clusters and RYR1 and RYR2 (but not RYR3) localize near the membrane. Using the spatial relationship between RYRs and BK channels, the spatial-temporal profile of [Ca(2+)] resulting from Ca(2+) sparks, and the kinetic model of BK channels, we estimate that an average Ca(2+) spark caused by the opening of a cluster of RYR1 or RYR2 acts on BK channels from two to three clusters that are randomly distributed within an ∼600-nm radius of RYRs. With this spatial organization of RYRs and BK channels, we are able to model BK channel currents with the same salient features as those observed in STOCs across a range of physiological membrane potentials. Thus, this study provides a mechanistic understanding of the activation of STOCs by Ca(2+) sparks using explicit knowledge of the spatial relationship between RYRs (the Ca(2+) source) and BK channels (the Ca(2+) target).

Ca2+ sparks are short lived and localized Ca2+ transients resulting from the opening of ryanodine receptors in sarcoplasmic reticulum. These events relax certain types of smooth muscle by activating big conductance Ca2+-activated K+ channels to produce spontaneous transient outward currents (STOCs) and the resultant closure of voltage-dependent Ca2+ channels. But in many smooth muscles from a variety of organs, Ca2+ sparks can additionally activate Ca2+-activated Cl(-) channels to generate spontaneous transient inward current (STICs). To date, the physiological roles of Ca2+ sparks in this latter group of smooth muscle remain elusive. Here, we show that in airway smooth muscle, Ca2+ sparks under physiological conditions, activating STOCs and STICs, induce biphasic membrane potential transients (BiMPTs), leading to membrane potential oscillations. Paradoxically, BiMPTs stabilize the membrane potential by clamping it within a negative range and prevent the generation of action potentials. Moreover, blocking either Ca2+ sparks or hyperpolarization components of BiMPTs activates voltage-dependent Ca2+ channels, resulting in an increase in global [Ca2+](i) and cell contraction. Therefore, Ca2+ sparks in smooth muscle presenting both STICs and STOCs act as a stabilizer of membrane potential, and altering the balance can profoundly alter the status of excitability and contractility. These results reveal a novel mechanism underlying the control of excitability and contractility in smooth muscle.

A central concept in the physiology of neurosecretion is that a rise in cytosolic [Ca(2+)] in the vicinity of plasmalemmal Ca(2+) channels due to Ca(2+) influx elicits exocytosis. Here, we examine the effect on spontaneous exocytosis of a rise in focal cytosolic [Ca(2+)] in the vicinity of ryanodine receptors (RYRs) due to release from internal stores in the form of Ca(2+) syntillas. Ca(2+) syntillas are focal cytosolic transients mediated by RYRs, which we first found in hypothalamic magnocellular neuronal terminals. (scintilla, Latin for spark; found in nerve terminals, normally synaptic structures.) We have also observed Ca(2+) syntillas in mouse adrenal chromaffin cells. Here, we examine the effect of Ca(2+) syntillas on exocytosis in chromaffin cells. In such a study on elicited exocytosis, there are two sources of Ca(2+): one due to influx from the cell exterior through voltage-gated Ca(2+) channels, and that due to release from intracellular stores. To eliminate complications arising from Ca(2+) influx, we have examined spontaneous exocytosis where influx is not activated. We report here that decreasing syntillas leads to an increase in spontaneous exocytosis measured amperometrically. Two independent lines of experimentation each lead to this conclusion. In one case, release from stores was blocked by ryanodine; in another, stores were partially emptied using thapsigargin plus caffeine, after which syntillas were decreased. We conclude that Ca(2+) syntillas act to inhibit spontaneous exocytosis, and we propose a simple model to account quantitatively for this action of syntillas.

The biological function of receptors is determined by their appropriate trafficking through the endosomal pathway. Following internalization, the transferrin (Tf) receptor quantitatively recycles to the plasma membrane, whereas the epidermal growth factor (EGF) receptor undergoes degradation. To determine how Tf and EGF engage these two different pathways we imaged their binding and early endocytic pathway in live cells using total internal reflection fluorescence microscopy (TIRF-M). We find that EGF and Tf bind to distinct plasma membrane regions and are incorporated into different endocytic vesicles. After internalization, both EGF-enriched and Tf-enriched vesicles interact with endosomes containing early endosome antigen 1 (EEA1). EGF is incorporated and retained in these endosomes, while Tf-containing vesicles rapidly dissociate and move to a juxtanuclear compartment. Endocytic vesicles carrying EGF recruit more Rab5 GTPase than those carrying Tf, which, by strengthening their association with EEA1-enriched endosomes, may provide a mechanism for the observed cargo-specific sorting. These results reveal pre-endocytic sorting of Tf and EGF, a specialized role for EEA1-enriched endosomes in EGF trafficking, and a potential mechanism for cargo-specified sorting of endocytic vesicles by these endosomes.

Ca(2+) sparks are highly localized, transient releases of Ca(2+) from sarcoplasmic reticulum through ryanodine receptors (RyRs). In smooth muscle, Ca(2+) sparks trigger spontaneous transient outward currents (STOCs) by opening nearby clusters of large-conductance Ca(2+)-activated K(+) channels, and also gate Ca(2+)-activated Cl(-) (Cl((Ca))) channels to induce spontaneous transient inward currents (STICs). While the molecular mechanisms underlying the activation of STOCs by Ca(2+) sparks is well understood, little information is available on how Ca(2+) sparks activate STICs. In the present study, we investigated the spatial organization of RyRs and Cl((Ca)) channels in spark sites in airway myocytes from mouse. Ca(2+) sparks and STICs were simultaneously recorded, respectively, with high-speed, widefield digital microscopy and whole-cell patch-clamp. An image-based approach was applied to measure the Ca(2+) current underlying a Ca(2+) spark (I(Ca(spark))), with an appropriate correction for endogenous fixed Ca(2+) buffer, which was characterized by flash photolysis of NPEGTA. We found that I(Ca(spark)) rises to a peak in 9 ms and decays with a single exponential with a time constant of 12 ms, suggesting that Ca(2+) sparks result from the nonsimultaneous opening and closure of multiple RyRs. The onset of the STIC lags the onset of the I(Ca(spark)) by less than 3 ms, and its rising phase matches the duration of the I(Ca(spark)). We further determined that Cl((Ca)) channels on average are exposed to a [Ca(2+)] of 2.4 microM or greater during Ca(2+) sparks. The area of the plasma membrane reaching this level is <600 nm in radius, as revealed by the spatiotemporal profile of [Ca(2+)] produced by a reaction-diffusion simulation with measured I(Ca(spark)). Finally we estimated that the number of Cl((Ca)) channels localized in Ca(2+) spark sites could account for all the Cl((Ca)) channels in the entire cell. Taken together these results lead us to propose a model in which RyRs and Cl((Ca)) channels in Ca(2+) spark sites localize near to each other, and, moreover, Cl((Ca)) channels concentrate in an area with a radius of approximately 600 nm, where their density reaches as high as 300 channels/microm(2). This model reveals that Cl((Ca)) channels are tightly controlled by Ca(2+) sparks via local Ca(2+) signaling.

Caffeine-activated, large-conductance, nonselective cation channels (LCCs) have been found in the plasma membrane of isolated cardiac myocytes in several species. However, little is known about the effects of opening these channels. To examine such effects and to further understand the caffeine-activation mechanism, we carried out studies using whole-cell patch-clamp techniques with freshly isolated cardiac myocytes from rats and mice. Unlike previous studies, thapsigargin was used so that both the effect of opening LCCs and the action of caffeine were independent of Ca(2+) release from intracellular stores. These Ca(2+)-permeable LCCs were found in a majority of the cells from atria and ventricles, with a conductance of approximately 370 pS in rat atria. Caffeine and all its direct metabolic products (theophylline, theobromine, and paraxanthine) activated the channel, while isocaffeine did not. Although they share some similarities with ryanodine receptors (RyRs, the openings of which give rise to Ca(2+) sparks), LCCs also showed some different characteristics. With simultaneous Ca(2+) imaging and current recording, the localized fluorescence increase due to Ca(2+) entry through a single opening of an LCC (SCCaFT) was detected. When membrane potential, instead of current, was recorded, SCCaFT-like fluorescence transients (indicating single LCC openings) were found to accompany membrane depolarizations. To our knowledge, this is the first report directly linking membrane potential changes to a single opening of an ion channel. Moreover, these events in cardiac cells suggest a possible additional mechanism by which caffeine and theophylline contribute to the generation of cardiac arrhythmias.

Total internal reflection fluorescence (TIRF) microscopy reveals highly mobile structures containing enhanced green fluorescent protein-tagged glucose transporter 4 (GLUT4) within a zone about 100 nm beneath the plasma membrane of 3T3-L1 adipocytes. We developed a computer program (Fusion Assistant) that enables direct analysis of the docking/fusion kinetics of hundreds of exocytic fusion events. Insulin stimulation increases the fusion frequency of exocytic GLUT4 vesicles by approximately 4-fold, increasing GLUT4 content in the plasma membrane. Remarkably, insulin signaling modulates the kinetics of the fusion process, decreasing the vesicle tethering/docking duration prior to membrane fusion. In contrast, the kinetics of GLUT4 molecules spreading out in the plasma membrane from exocytic fusion sites is unchanged by insulin. As GLUT4 accumulates in the plasma membrane, it is also immobilized in punctate structures on the cell surface. A previous report suggested these structures are exocytic fusion sites (Lizunov et al., J. Cell Biol. 169:481-489, 2005). However, two-color TIRF microscopy using fluorescent proteins fused to clathrin light chain or GLUT4 reveals these structures are clathrin-coated patches. Taken together, these data show that insulin signaling accelerates the transition from docking of GLUT4-containing vesicles to their fusion with the plasma membrane and promotes GLUT4 accumulation in clathrin-based endocytic structures on the plasma membrane.

Cell-substrate contacts, called focal adhesions (FAs), are dynamic in rapidly moving cells. We show that supervillin (SV)--a peripheral membrane protein that binds myosin II and F-actin in such cells--negatively regulates stress fibers, FAs, and cell-substrate adhesion. The major FA regulatory sequence within SV (SV342-571) binds to the LIM domains of two proteins in the zyxin family, thyroid receptor-interacting protein 6 (TRIP6) and lipoma-preferred partner (LPP), but not to zyxin itself. SV and TRIP6 colocalize within large FAs, where TRIP6 may help recruit SV. RNAi-mediated decreases in either protein increase cell adhesion to fibronectin. TRIP6 partially rescues SV effects on stress fibers and FAs, apparently by mislocating SV away from FAs. Thus, SV interactions with TRIP6 at FAs promote loss of FA structure and function. SV and TRIP6 binding partners suggest several specific mechanisms through which the SV-TRIP6 interaction may regulate FA maturation and/or disassembly.

Ca2+ stores were studied in a preparation of freshly dissociated terminals from hypothalamic magnocellular neurons. Depolarization from a holding level of -80 mV in the absence of extracellular Ca2+ elicited Ca2+ release from intraterminal stores, a ryanodine-sensitive process designated as voltage-induced Ca2+ release (VICaR). The release took one of two forms: an increase in the frequency but not the quantal size of Ca2+ syntillas, which are brief, focal Ca2+ transients, or an increase in global [Ca2+]. The present study provides evidence that the sensors of membrane potential for VICaR are dihydropyridine receptors (DHPRs). First, over the range of -80 to -60 mV, in which there was no detectable voltage-gated inward Ca2+ current, syntilla frequency was increased e-fold per 8.4 mV of depolarization, a value consistent with the voltage sensitivity of DHPR-mediated VICaR in skeletal muscle. Second, VICaR was blocked by the dihydropyridine antagonist nifedipine, which immobilizes the gating charge of DHPRs but not by Cd2+ or FPL 64176 (methyl 2,5 dimethyl-4[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylate), a non-dihydropyridine agonist specific for L-type Ca2+ channels, having no effect on gating charge movement. At 0 mV, the IC50 for nifedipine blockade of VICaR in the form of syntillas was 214 nM in the absence of extracellular Ca2+. Third, type 1 ryanodine receptors, the type to which DHPRs are coupled in skeletal muscle, were detected immunohistochemically at the plasma membrane of the terminals. VICaR may constitute a new link between neuronal activity, as signaled by depolarization, and a rise in intraterminal Ca2+.

Clathrin assembly at the plasma membrane is a fundamental process required for endocytosis. In cultured cells, most of the clathrin is localized to large patches that display little lateral mobility. The functional role of these regions is not clear, and it has been thought that they may represent artifacts of cell adhesion of cultured cells. Here we have analyzed clathrin organization in primary adipose cells isolated from mice, which are nonadherent and fully differentiated. The majority of clathrin on the plasma membrane of these cells (>60%) was found in large clathrin patches that displayed virtually no lateral mobility and persisted for many minutes, and a smaller amount was found in small spots that appeared and disappeared rapidly. Direct visualization of transferrin revealed that it bound onto large arrays of clathrin, internalizing through vesicles that emerge from these domains. High resolution imaging (50 images/s) revealed fluorescence intensity fluctuations consistent with the formation and detachment of coated vesicles from within large patches. These results reveal that large clathrin assemblies are active regions of endocytosis in mammalian cells and highlight the importance of understanding the mechanistic basis for this organization.

Spontaneous, short-lived, focal cytosolic Ca2+ transients were found for the first time and characterized in freshly dissociated chromaffin cells from mouse. Produced by release of Ca2+ from intracellular stores and mediated by type 2 and perhaps type 3 ryanodine receptors (RyRs), these transients are quantitatively similar in magnitude and duration to Ca2+ syntillas in terminals of hypothalamic neurons, suggesting that Ca2+ syntillas are found in a variety of excitable, exocytotic cells. However, unlike hypothalamic nerve terminals, chromaffin cells do not display syntilla activation by depolarization of the plasma membrane, nor do they have type 1 RyRs. It is widely thought that focal Ca2+ transients cause "spontaneous" exocytosis, although there is no direct evidence for this view. Hence, we monitored catecholamine release amperometrically while simultaneously imaging Ca2+ syntillas, the first such simultaneous measurements. Syntillas failed to produce exocytotic events; and, conversely, spontaneous exocytotic events were not preceded by syntillas. Therefore, we suggest that a spontaneous syntilla, at least in chromaffin cells, releases Ca2+ into a cytosolic microdomain distinct from the microdomains containing docked, primed vesicles. Ryanodine (100 microM) reduced the frequency of Ca2+ syntillas by an order of magnitude but did not alter the frequency of spontaneous amperometric events, suggesting that syntillas are not involved in steps preparatory to spontaneous exocytosis. Surprisingly, ryanodine also increased the total charge of individual amperometric events by 27%, indicating that intracellular Ca2+ stores can regulate quantal size.

Speckles are nuclear bodies that contain pre-mRNA splicing factors and polyadenylated RNA. Because nuclear poly(A) RNA consists of both mRNA transcripts and nucleus-restricted RNAs, we tested whether poly(A) RNA in speckles is dynamic or rather an immobile, perhaps structural, component. Fluorescein-labeled oligo(dT) was introduced into HeLa cells stably expressing a red fluorescent protein chimera of the splicing factor SC35 and allowed to hybridize. Fluorescence correlation spectroscopy (FCS) showed that the mobility of the tagged poly(A) RNA was virtually identical in both speckles and at random nucleoplasmic sites. This same result was observed in photoactivation-tracking studies in which caged fluorescein-labeled oligo(dT) was used as hybridization probe, and the rate of movement away from either a speckle or nucleoplasmic site was monitored using digital imaging microscopy after photoactivation. Furthermore, the tagged poly(A) RNA was observed to rapidly distribute throughout the entire nucleoplasm and other speckles, regardless of whether the tracking observations were initiated in a speckle or the nucleoplasm. Finally, in both FCS and photoactivation-tracking studies, a temperature reduction from 37 to 22 degrees C had no discernible effect on the behavior of poly(A) RNA in either speckles or the nucleoplasm, strongly suggesting that its movement in and out of speckles does not require metabolic energy.

Ca(2+) sparks are highly localized Ca(2+) transients caused by Ca(2+) release from sarcoplasmic reticulum through ryanodine receptors (RyR). In smooth muscle, Ca(2+) sparks activate nearby large-conductance, Ca(2+)-sensitive K(+) (BK) channels to generate spontaneous transient outward currents (STOC). The properties of individual sites that give rise to Ca(2+) sparks have not been examined systematically. We have characterized individual sites in amphibian gastric smooth muscle cells with simultaneous high-speed imaging of Ca(2+) sparks using wide-field digital microscopy and patch-clamp recording of STOC in whole cell mode. We used a signal mass approach to measure the total Ca(2+) released at a site and to estimate the Ca(2+) current flowing through RyR [I(Ca(spark))]. The variance between spark sites was significantly greater than the intrasite variance for the following parameters: Ca(2+) signal mass, I(Ca(spark)), STOC amplitude, and 5-ms isochronic STOC amplitude. Sites that failed to generate STOC did so consistently, while those at the remaining sites generated STOC without failure, allowing the sites to be divided into STOC-generating and STOC-less sites. We also determined the average number of spark sites, which was 42/cell at a minimum and more likely on the order of at least 400/cell. We conclude that 1) spark sites differ in the number of RyR, BK channels, and coupling ratio of RyR-BK channels, and 2) there are numerous Ca(2+) spark-generating sites in smooth muscle cells. The implications of these findings for the organization of the spark microdomain are explored.

The feasibility of determining localized Ca(2+) influx using only wide-field fluorescence images was explored by imaging (using fluo-3) single channel Ca(2+) fluorescence transients (SCCaFTs), due to Ca(2+) entry through single openings of Ca(2+)-permeable ion channels, while recording unitary channel currents. Since the image obtained with wide-field optics is an integration of both in-focus and out-of-focus light, the total fluorescence increase (DeltaF(total) or "signal mass") associated with a SCCaFT can be measured directly from the image by adding together the fluorescence increase due to Ca(2+) influx in all of the pixels. The assumptions necessary for obtaining the signal mass from confocal linescan images are not required. Two- and three-dimensional imaging was used to show that DeltaF(total) is essentially independent of the position of the channel with respect to the focal plane of the microscope. The relationship between Ca(2+) influx and DeltaF(total) was obtained using SCCaFTs from plasma membrane caffeine-activated cation channels when Ca(2+) was the only charge carrier of the inward current. This relationship was found to be linear, with the value of the slope (or converting factor) affected by the particular imaging system set-up, the experimental conditions, and the properties of the fluorescent indicator, including its binding capacity with respect to other cellular buffers. The converting factor was used to estimate the Ca(2+) current passing through caffeine-activated channels in near physiological saline and to estimate the endogenous buffer binding capacity. In addition, it allowed a more accurate estimate of the Ca(2+) current underlying Ca(2+) sparks resulting from Ca(2+) release from intracellular stores via ryanodine receptors in the same preparation.

Recently, it has become possible to record the localized fluorescence transient associated with the opening of a single plasma membrane Ca(2+) permeable ion channel using Ca(2+) indicators like fluo-3. These Single Channel Ca(2+) Fluorescence Transients (SCCaFTs) share some of the characteristics of such elementary events as Ca(2+) sparks and Ca(2+) puffs caused by Ca(2+) release from intracellular stores (due to the opening of ryanodine receptors and IP(3) receptors, respectively). In contrast to intracellular Ca(2+) release events, SCCaFTs can be observed while simultaneously recording the unitary channel currents using patch-clamp techniques to verify the channel openings. Imaging SCCaFTs provides a way to examine localized Ca(2+) handling in the vicinity of a channel with a known Ca(2+) influx, to obtain the Ca(2+) current passing through plasma membrane cation channels in near physiological solutions, to localize Ca(2+) permeable ion channels on the plasma membrane, and to estimate the Ca(2+) currents underlying those elementary events where the Ca(2+) currents cannot be recorded. Here we review studies of these fluorescence transients associated with caffeine-activated channels, L-type Ca(2+) channels, and stretch-activated channels. For the L-type Ca(2+) channel, SCCaFTs have been termed sparklets. In addition, we discuss how SCCaFTs have been used to estimate Ca(2+) currents using the rate of rise of the fluorescence transient as well as the signal mass associated with the total fluorescence increase.

Localized, brief Ca2+ transients (Ca2+ syntillas) caused by release from intracellular stores were found in isolated nerve terminals from magnocellular hypothalamic neurons and examined quantitatively using a signal mass approach to Ca2+ imaging. Ca2+ syntillas (scintilla, L., spark, from a synaptic structure, a nerve terminal) are caused by release of approximately 250,000 Ca ions on average by a Ca2+ flux lasting on the order of tens of milliseconds and occur spontaneously at a membrane potential of -80 mV. Syntillas are unaffected by removal of extracellular Ca2+, are mediated by ryanodine receptors (RyRs) and are increased in frequency, in the absence of extracellular Ca2+, by physiological levels of depolarization. This represents the first direct demonstration of mobilization of Ca2+ from intracellular stores in neurons by depolarization without Ca2+ influx. The regulation of syntillas by depolarization provides a new link between neuronal activity and cytosolic [Ca2+] in nerve terminals.

Stretch-activated channels (SACs) have been found in smooth muscle and are thought to be involved in myogenic responses. Although SACs have been shown to be Ca(2+) permeable when Ca(2+) is the only charge carrier, it has not been clearly demonstrated that significant Ca(2+) passes through SACs in physiological solutions. By imaging at high temporal and spatial resolution the single-channel Ca(2+) fluorescence transient (SCCaFT) arising from Ca(2+) entry through a single SAC opening, we provide direct evidence that significant Ca(2+) can indeed pass through SACs and increase the local [Ca(2+)]. Results were obtained under conditions where the only source of Ca(2+) was the physiological salt solution in the patch pipette containing 2 mM Ca(2+). Single smooth muscle cells were loaded with fluo-3 acetoxymethyl ester, and the fluorescence was recorded by using a wide-field digital imaging microscope while SAC currents were simultaneously recorded from cell-attached patches. Fluorescence increases at the cell-attached patch were clearly visualized before the simultaneous global Ca(2+) increase that occurred because of Ca(2+) influx through voltage-gated Ca(2+) channels when the membrane was depolarized by inward SAC current. From measurements of total fluorescence ("signal mass") we determined that about 18% of the SAC current is carried by Ca(2+) at membrane potentials more negative than the resting level. This would translate into at least a 0.35-pA unitary Ca(2+) current at the resting potential. Such Ca(2+) currents passing through SACs are sufficient to activate large-conductance Ca(2+)-activated K(+) channels and, as shown previously, to trigger Ca(2+) release from intracellular stores.

Agonist-induced translocation of RhoA and the spatio-temporal change in myosin regulatory light chain (MLC20) phosphorylation in smooth muscle was clarified at the single cell level. We expressed green fluorescent protein-tagged RhoA in the differentiated tracheal smooth muscle cells and visualized the translocation of RhoA in a living cell with three-dimensional digital imaging analysis. The stimulation of the cells by carbachol initiated the translocation of green fluorescent protein-tagged wild type RhoA to the plasma membrane within a minute. The change in MLC20 phosphorylation level after carbachol stimulation was monitored by using phospho-Ser-19-specific antibody recognizing the phosphorylated MLC20 in single cells. Cells expressing the dominant negative form (T19N) of RhoA significantly suppressed sustained MLC20 phosphorylation during the prolonged phase (>300 s), whereas the maximum phosphorylation level (reached at 10 s after stimulation) of these cells was not significantly different from the control cells. The kinetics of RhoA translocation was consistent with that of sustained myosin phosphorylation, suggesting the involvement of a RhoA pathway. Carbachol stimulation increased myosin phosphorylation within a minute both at the cortical and the central region. On the other hand, during prolonged phase, myosin phosphorylation was sustained at the cortical region of the cells but not at the central fibers. A myosin light chain kinase-specific inhibitor, ML-9, diminished myosin phosphorylation at the central region of the cells after the stimulation but not at the cortical area. On the other hand, Y-27632, a Rho kinase-specific inhibitor, diminished myosin phosphorylation at the cortical region but not the central region. The results clearly show that the myosin light chain kinase pathway and the Rho pathway distinctly change myosin phosphorylation in smooth muscle cells in both a temporal and spatial manner.

We recorded Ca2+ sparks and spontaneous transient outward currents (STOCs) simultaneously in smooth muscle cells using whole-cell patch recording and a unique, high-speed widefield digital imaging system to monitor fluo-3 fluorescence in both two and three dimensions (2D and 3D). In 2D imaging, the correlation between the amplitude of a spark and its corresponding STOC was a weak one, and 27 % of the sparks failed to cause STOCs. The STOCless sparks were not significantly different in amplitude from those that caused STOCs. Three-dimensional imaging disclosed that STOCless sparks were located close to the cell surface, and on average their apparent distance from the cell surface was not significantly different from the sparks that cause STOCs. Statistical evaluation of spark clusters disclosed that there were regions of the cell where the probability of spark occurrence was high and others where it was quite low.

Ca(2+) sparks are highly localized cytosolic Ca(2+) transients caused by a release of Ca(2+) from the sarcoplasmic reticulum via ryanodine receptors (RyRs); they are the elementary events underlying global changes in Ca(2+) in skeletal and cardiac muscle. In smooth muscle and some neurons, Ca(2+) sparks activate large conductance Ca(2+)-activated K(+) channels (BK channels) in the spark microdomain, causing spontaneous transient outward currents (STOCs) that regulate membrane potential and, hence, voltage-gated channels. Using the fluorescent Ca(2+) indicator fluo-3 and a high speed widefield digital imaging system, it was possible to capture the total increase in fluorescence (i.e., the signal mass) during a spark in smooth muscle cells, which is the first time such a direct approach has been used in any system. The signal mass is proportional to the total quantity of Ca(2+) released into the cytosol, and its rate of rise is proportional to the Ca(2+) current flowing through the RyRs during a spark (I(Ca(spark))). Thus, Ca(2+) currents through RyRs can be monitored inside the cell under physiological conditions. Since the magnitude of I(Ca(spark)) in different sparks varies more than fivefold, Ca(2+) sparks appear to be caused by the concerted opening of a number of RyRs. Sparks with the same underlying Ca(2+) current cause STOCs, whose amplitudes vary more than threefold, a finding that is best explained by variability in coupling ratio (i.e., the ratio of RyRs to BK channels in the spark microdomain). The time course of STOC decay is approximated by a single exponential that is independent of the magnitude of signal mass and has a time constant close to the value of the mean open time of the BK channels, suggesting that STOC decay reflects BK channel kinetics, rather than the time course of [Ca(2+)] decline at the membrane. Computer simulations were carried out to determine the spatiotemporal distribution of the Ca(2+) concentration resulting from the measured range of I(Ca(spark)). At the onset of a spark, the Ca(2+) concentration within 200 nm of the release site reaches a plateau or exceeds the [Ca(2+)](EC50) for the BK channels rapidly in comparison to the rate of rise of STOCs. These findings suggest a model in which the BK channels lie close to the release site and are exposed to a saturating [Ca(2+)] with the rise and fall of the STOCs determined by BK channel kinetics. The mechanism of signaling between RyRs and BK channels may provide a model for Ca(2+) action on a variety of molecular targets within cellular microdomains.

Discrete localized fluorescence transients due to openings of a single plasma membrane Ca(2+) permeable cation channel were recorded using wide-field digital imaging microscopy with fluo-3 as the Ca(2+) indicator. These transients were obtained while simultaneously recording the unitary channel currents using the whole-cell current-recording configuration of the patch-clamp technique. This cation channel in smooth muscle cells is opened by caffeine (Guerrero, A., F.S. Fay, and J.J. Singer. 1994. J. Gen. Physiol. 104:375-394). The localized fluorescence transients appeared to occur at random locations on the cell membrane, with the duration of the rising phase matching the duration of the channel opening. Moreover, these transients were only observed in the presence of sufficient extracellular Ca(2+), suggesting that they are due to Ca(2+) influx from the bathing solution. The fluorescence transient is characterized by an initial fast rising phase when the channel opens, followed by a slower rising phase during prolonged openings. When the channel closes there is an immediate fast falling phase followed by a slower falling phase. Computer simulations of the underlying events were used to interpret the time course of the transients. The rapid phases are mainly due to the establishment or removal of Ca(2+) and Ca(2+)-bound fluo-3 gradients near the channel when the channel opens or closes, while the slow phases are due to the diffusion of Ca(2+) and Ca(2+)-bound fluo-3 into the cytoplasm. Transients due to short channel openings have a "Ca(2+) spark-like" appearance, suggesting that the rising and early falling components of sparks (due to openings of ryanodine receptors) reflect the fast phases of the fluorescence change. The results presented here suggest methods to determine the relationship between the fluorescence transient and the underlying Ca(2+) current, to study intracellular localized Ca(2+) handling as might occur from single Ca(2+) channel openings, and to localize Ca(2+) permeable ion channels on the plasma membrane.

To localize activated protein kinase C (PKC) in smooth muscle cells, an antibody directed to the catalytic site of the enzyme was used to assess PKC distribution by immunofluorescence techniques in gastric smooth muscle cells isolated from Bufo marinus. An antibody to vinculin was used to delineate the cell membrane. High-resolution three-dimensional images of immunofluorescence were obtained from a series of images collected through focus with a digital imaging microscope. Cells were untreated or treated with agents that increase PKC activity (10 microM carbachol for 1 min, 1 microM phorbol 12-myristate 13-acetate (PMA) for 10 min), or have no effect on PKC activity (1 micrometer 4-alpha phorbol, 12,13-didecanoate (4-alpha PMA)). In unstimulated cells, activated PKC and vinculin were located and organized at the cell surface. Cell cytosol labeling for activated PKC was sparse and diffuse and was absent for vinculin. After treatment with carbachol, which stimulates contraction and PKC activity, in addition to the membrane localization, the activated PKC exhibited a pronounced cytosolic fibrillar distribution and an increased total fluorescence intensity relative to vinculin. The distributions of activated PKC observed after PMA but not 4-alpha PMA were similar to those observed with carbachol. Our results indicate that in resting cells there is a pool of activated PKC near the cell membrane, and that after stimulation activated PKC is no longer membrane-confined, but is present throughout the cytosol. Active PKC appears to associate with contractile filaments, supporting a possible role in modulation of contraction.

To further the understanding of oxidative effects on inflammation injury to muscle fiber structure, fluorescent imaging analysis of human striated muscle tissues from a variety of inflammatory or postinflammatory etiologies was undertaken in a search for accumulated coproporphyrin, a red autofluorescent byproduct of heme biosynthesis that would theoretically be formed under oxidative insult. Using a differential excitation method of in situ analysis, porphyrin autofluorescence was detected in intact fibers within the context of the yellow autofluorescent subsarcolemmal lipofuscin granules. Relative measurements of porphyrin concentration in the granules from different patients indicated that the acute/subacute inflammatory specimens grouped significantly higher than the more chronic inflammatory and nonpathological specimens. Myoglobin was also found to be associated with the granules. Myoglobin heme iron could potentially serve as a Fenton reagent for the intracellular generation of hydroxyl radicals, which are responsible for the oxidation of the porphyrinogens. High-performance liquid chromatography analysis of extracted dense particles revealed coproporphyrin as the sole porphyrin present. The observation of coproporphyrin within lipofuscin granules, previously unreported, suggests that lipofuscin accumulation in striated muscle may begin under conditions of acute oxidative stress, as marked by the oxidation of extramitochondrial porphyrinogens that are immediately incorporated into the granules.

The spatial relation between mitochondria and endoplasmic reticulum (ER) in living HeLa cells was analyzed at high resolution in three dimensions with two differently colored, specifically targeted green fluorescent proteins. Numerous close contacts were observed between these organelles, and mitochondria in situ formed a largely interconnected, dynamic network. A Ca2+-sensitive photoprotein targeted to the outer face of the inner mitochondrial membrane showed that, upon opening of the inositol 1,4,5-triphosphate (IP3)-gated channels of the ER, the mitochondrial surface was exposed to a higher concentration of Ca2+ than was the bulk cytosol. These results emphasize the importance of cell architecture and the distribution of organelles in regulation of Ca2+ signaling.

The transport of mRNAs into developing dendrites and axons may be a basic mechanism to localize cytoskeletal proteins to growth cones and influence microfilament organization. Using isoform-specific antibodies and probes for in situ hybridization, we observed distinct localization patterns for beta- and gamma-actin within cultured cerebrocortical neurons. beta-Actin protein was highly enriched within growth cones and filopodia, in contrast to gamma-actin protein, which was distributed uniformly throughout the cell. beta-Actin protein also was shown to be peripherally localized after transfection of beta-actin cDNA bearing an epitope tag. beta-Actin mRNAs were localized more frequently to neuronal processes and growth cones, unlike gamma-actin mRNAs, which were restricted to the cell body. The rapid localization of beta-actin mRNA, but not gamma-actin mRNA, into processes and growth cones could be induced by dibutyryl cAMP treatment. Using high-resolution in situ hybridization and image-processing methods, we showed that the distribution of beta-actin mRNA within growth cones was statistically nonrandom and demonstrated an association with microtubules. beta-Actin mRNAs were detected within minor neurites, axonal processes, and growth cones in the form of spatially distinct granules that colocalized with translational components. Ultrastructural analysis revealed polyribosomes within growth cones that colocalized with cytoskeletal filaments. The transport of beta-actin mRNA into developing neurites may be a sequence-specific mechanism to synthesize cytoskeletal proteins directly within processes and growth cones and would provide an additional means to deliver cytoskeletal proteins over long distances.