Michael Kotlikoff - US grants

Despite the importance of airway smooth muscle excitability in reactive airway disease, very little information is currently available with respect to the precise ionic mechanisms which mediate the neurotransmitter induced excitation of this tissue. Further, the cellular mechansim or mechanisms by which important mediators of bronchoconstriction such as leukotrienes and histamine trigger contraction in airway smooth muscle is not know. This proposal seeks to apply recently developed advances in cell disaggregation and gigaseal recording techniques to isolate specific ion currents in single mammalian airway smooth muscle cells. Ion currents will be identified and studied in isolated airway myocytes from the ferret trachealis muscle by dialyzing the cells with impermeant ions, and using specific channel blocking agents. Specific ion currents will be studied under voltage clamp conditions to determine time-dependent and voltage dependent channel activation and inactivation characteristics. Experiments will be performed to determine if neurotransmitters and bronchoactive agents act by gating these currents. The working hypothesis of this proposal is that cell membrane receptor binding of neurohormonal agents gates an inward calcium current which initiates cell activation. This hypothesis will be examined by resolving whole cell calcium currents at fixed voltage in the presence of varying concentrations of these agents. These studies will thus provide important information about the membrane ionic processes regulating contraction, and the extent to which these processes are important in the response of airway smooth muscle to bronchoconstrictive agents which have been shown to be important mediators of airway hyperreactivity.

This proposal seeks to define the cellular and subcellular processes that regulate calcium influx in human airway smooth muscle (ASM) cells. In airway smooth muscle, agents that are effective at blocking voltage-dependent calcium channels (VDCC), do not inhibit agonist-induced force production in muscle strips, and do not effectively antagonize bronchospasm in vivo. The focus of this proposal will be the identification and characterization of sustained, receptor-activated calcium influx mechanisms in cultured human ASM cells that retain important functional responses to autacoids implicated in asthma and other airway diseases. Measurements of cytosolic calcium using the fluorescent calcium indicator fura-2 will be combined with conventional microelectrode voltage-clamp and patch clamp techniques to examine specific mechanisms of calcium entry and determine the effect of this influx on cytosolic calcium concentration. The hypothesis that sustained calcium influx following receptor-binding occurs via an influx pathway distinct from voltage-activated calcium channels, will be tested by a comparison of the permeability characteristics, pharmacologic sensitivity, and voltage-dependence of sustained, receptor-activated calcium influx and voltage-dependent calcium channels. If receptor-activated calcium influx occurs via VDCC, the cation permeability, antagonist potency, and voltage-dependence of the influx should be predictable from direct measurements of VDCC. A second goal will be to determine the extent to which agonists activate VDCC, and the degree to which activation of these channels contributes to a sustained rise in cytosolic calcium. A direct search for receptor-activated calcium channels distinct from VDCC, which have biophysical properties that match macroscopic agonist-triggered calcium influx, will also be conducted. Finally, the molecular mechanisms by which receptor-binding couples to calcium influx will be determined.

Asthma is a prevalent disorder associated with airway inflammation and reversible bronchospasm. While the pathophysiology of this disorder is still not well understood, cellular mechanisms that mediate contractile and relaxant responses of airway smooth muscle cells play a pivotal role in the disorder. This proposal seeks to further define the role of membrane potassium channels in the neurohormonal responses of airway smooth muscle, and to determine the specific gene products associated with key aspects of airway smooth muscle function. Substantial experimental evidence indicates that large-conductance calcium-activated potassium channels are important hormone targets, whose open probability is modulated by agents that alter smooth muscle tone and that beta-adrenergic agonists act at least partially by opening these channels. Similarly, delayed-rectifier potassium channels appear to be important involved in defining resting electrical and mechanical excitability. The central hypothesis of the proposal is that potassium channels are key determinants of airway smooth muscle excitability, and that specific potassium channel subtypes subserve discrete physiological functions in airway smooth muscle. The roles of these gene products in determining resting tone and electrical activity and coupling hormone binding to specific cellular events will be determined using electrophysiological and molecular biological approaches. The determination of specific channel/receptor coupling processes, the functional role of specific K channel gene products, an the factors that regulate gene expression should enhance our understanding of the role of potassium channels in normal and abnormal airway smooth muscle.

This proposal seeks to further define the cellular and subcellular processes that regulate calcium influx in airway smooth muscle. Previous studies have characterized receptor-activated calcium influx in human airway smooth muscle cells following exposure to bronchoconstrictive agents such as acetylcholine, histamine, and bradykinin. The focus of this proposal will be to more completely characterize the conductances associated with this calcium influx in an effort to determine the channel protein responsible for mediating sustained, agonist-induced contraction in airway smooth muscle, and to determine the molecular mechanisms by which calcium channel proteins are activated during this process. Single- cell, simultaneous measurements of calcium and current will be made using the nystatin modification of the whole-cell patch-clamp technique in order to directly examine the conductances that underlie sustained increases in cytosolic calcium during agonist exposure. Simultaneous measurements of force and cytosolic calcium in tissue strips will allow the correlation of single-cell and single-channel measurements with physiologic bronchoconstrictive responses. Mechanisms by which voltage-dependent calcium channels are modulated by agonist/receptor binding will also be examined, since this linkage is known to occur. These studies should provide specific information about the mechanisms by which exposure to bronchoconstrictive agents results in sustained increases in cytosolic calcium and attendant force production. Pharmacological regulation of these channel proteins may prove to be an efficient and specific method of bronchodilation.

This proposal seeks to extend our study of nonselective cation channels and calcium-activated chloride channels in airway smooth muscle cells. Experiments will determine the degree to which these channels are activated by inflammatory mediators associated with asthma, and the processes linking receptor binding and channel activation. Numerous spasmogens are released in asthma and the diversity of mediators produced during airway inflammation suggests that the identification of common excitation/contraction coupling pathways may lead to effective strategies to limit bronchospasm. The degree to which inflammatory compounds, known to be released during asthmatic bronchoconstriction, activate non-selective cation channels and calcium-activated chloride channels in airway smooth muscle is currently unknown. Our recent studies have identified mechanisms by which acetylcholine activates ICat and ICI(Ca). The current reapplication seeks to extend these studies on the molecular processes linking receptor coupling to channel activation, and to gain clinically relevant information about the link between persistent airway inflammation and the activation of these ion channels. Our central hypothesis is that nonselective cation channels and calcium release channels are activated by inflammatory mediators, and that this activation results in a sustained increased in the calcium permeability and sustained depolarization of the myocyte. We proposed to determine the extent to which inflammatory substances activate these channels in airway smooth muscle cells, determine the post-receptor mechanisms associated with receptor/channel coupling, and determine the role of specific calcium release processes in channel activation, including the role of quantal calcium release events (calcium sparks) in the activation of ICI(Ca) channels.

This project seeks to examine specific excitation-contraction coupling mechanisms in isolated bladder smooth muscle cells, and to determine if these processes are associated with the contractile dysfunction associated with chronic obstruction of the urinary bladder. Two important coupling process that have been implicated in this dysfunction will be examined: non-selective cation channels and calcium-induced calcium release (CICR). Cation channels that are activated by neurotransmitters and may be calcium permeant will be identified using simultaneous single-cell, patch-clamp methods and fura 2 calcium measurements. ATP acting on purinergic receptors plays an important role in urinary bladder function. The ligand- gated cation channels mediating purinergic excitatory currents in bladder smooth muscle cells will be identified and correlated with the properties of recently cloned P2X receptor/channels. The calcium permeation, biophysical and pharmacological properties of these channels in rabbit detrusor will be determined. CICR appears to be an important component of excitation/contraction coupling in urinary bladder myocytes. The ability of calcium currents and cation currents to release calcium from sarcoplasmic reticulum will be determine using simultaneous measurements of intracellular calcium and current in voltage-clamped myocytes. The expression of P2X genes will also be determined. These processes will be compared in cells dissociated from normal, decompensated, and reversed urinary bladders to determine the role of non-selective cation channels and CICR in post obstructive urinary bladder dysfunction. The extent to which CICR, non-selective cation channels or downstream calcium release/uptake processes are altered in decompensated bladders will shed light on the processes underlying bladders dysfunction associated with urinary outflow obstruction.

This proposal seeks to extend our study of nonselective cation channels and calcium-activated chloride channels in airway smooth muscle cells. Experiments will determine the degree to which these channels are activated by inflammatory mediators associated with asthma, and the processes linking receptor binding and channel activation. Numerous spasmogens are released in asthma and the diversity of mediators produced during airway inflammation suggests that the identification of common excitation/contraction coupling pathways may lead to effective strategies to limit bronchospasm. The degree to which inflammatory compounds, known to be released during asthmatic bronchoconstriction, activate non-selective cation channels and calcium-activated chloride channels in airway smooth muscle is currently unknown. Our recent studies have identified mechanisms by which acetylcholine activates ICat and ICI(Ca). The current reapplication seeks to extend these studies on the molecular processes linking receptor coupling to channel activation, and to gain clinically relevant information about the link between persistent airway inflammation and the activation of these ion channels. Our central hypothesis is that nonselective cation channels and calcium release channels are activated by inflammatory mediators, and that this activation results in a sustained increased in the calcium permeability and sustained depolarization of the myocyte. We proposed to determine the extent to which inflammatory substances activate these channels in airway smooth muscle cells, determine the post-receptor mechanisms associated with receptor/channel coupling, and determine the role of specific calcium release processes in channel activation, including the role of quantal calcium release events (calcium sparks) in the activation of ICI(Ca) channels.

DESCRIPTION (Adapted from the Applicant's Abstract): This project seeks to examine specific excitation-contraction coupling mechanisms in bladder smooth muscle cells, and to determine whether these processes are associated with the contractile dysfunction known to occur with chronic obstruction of the urinary bladder. Particular focus will be on the molecular mechanisms underlying calcium-induced calcium release (CICR) in bladder smooth muscle cells, using laser scanning confocal microscopy and patch clamp methods. Calcium-induced calcium release, or the release of intracellular calcium ions by the influx of calcium across the cell membrane, has been shown to play an important role in excitation-contraction coupling in bladder myocytes. Evidence suggests that impairment of this process is associated with the adaptive changes that occur in bladder smooth muscle during outlet obstruction. We will determine the biophysical characteristics of CICR in normal urinary bladder myocytes and compare that with observations in myocytes from obstructed bladders. Knockout mice with targeted deletions of ryanodine receptors will be generated and the CICR process examined to determine the role of specific intracellular calcium channels and associated modulatory proteins in excitation-contraction coupling of bladder smooth muscle.

DESCRIPTION (provided by applicant): The objectives of this proposal are to determine the molecular processes underlying spontaneous Ca2+ release events in smooth muscle cells. Spontaneous and triggered release of Ca2+ ions from the sarcoplasmic reticulum of smooth muscle cells is a key event associated with contraction of the smooth muscle and bronchoconstriction of the airways. In recent years it has become clear that this Ca2+ release process is quite complicated, with two major intracellular Ca2+ release. Ryanodine receptors comprise one family of Ca2+ release channels; the function of these channels in skeletal and cardiac muscle is well established, but their role in the contraction of smooth muscle is less clear. A prominent feature of the electrical activity of smooth muscle cells in the airways is the appearance of spontaneous depolarizing and spontaneous hyperpolarizing currents. These currents arise from the opening of calcium-sensitive membrane channels following a brief release of calcium from the sarcoplasmic reticulum. This calcium release has been shown to arise from the spontaneous gating of ryanodine receptors, and the Ca2+ release that occurs have been termed Ca2+ sparks. These sparks arise from specific areas within smooth muscle cells, analogous to their appearance at the triads of striated muscle. Release of Ca2+ is also propagated as waves throughout the cell cytoplasm by the gating of ryanodine receptors. The underlying structures and channels mediating and regulating spontaneous Ca2+ release and propagated Ca2+ waves will be determined in this proposal. Mice have been generated lacking specific elements of ryanodine receptor complex and the properties of the Ca2+ release in myocytes from these cells will be determined as a way of identifying the specific role of individual proteins in this complex process. Moreover, the effect of this deletion on airway function will be determined, in order to assess the importance of this process in the complex control of airway tone.

DESCRIPTION (provided by applicant): The aim of this proposal is to develop, validate, and utilize new transgenic mouse models for the study of the cellular and molecular details of neuromuscular regulation of organs of the genitourinary tract. The complex, multicellular nature of GU organs is a significant barrier to the understanding of the cellular interactions that are critical to GU organ physiology, pathophysiology, and transformation. Gain-of-function and loss-of-function transgenesis in the mouse has emerged as perhaps the single most informative process in determining the in vivo function of individual genes in mammals, and over the next two decades one of the major efforts of biology will be the functional annotation of the mammalian genome through selective alterations of the mouse genome. Despite the increasing use of these methods to probe cellular and organ function, and the increasing availability of mice in which recombinase recognition sequences have been inserted for specific genes so that the genes can be selectively inactivated, few of these approaches have been designed to target cells of the GU. This PAR proposal seeks to apply methods of transgenesis and recombinant protein engineering to provide tools for the study of cells of the GU system, and to use these mice to examine aspects of GU function. Separate transgenic lines will be created in which specific cellular lineages express: 1) a ligand -activated Cre recombinase transgene (Cre-ER (T2)) in which Cre recombinase is under cell specific and temporal control for conditional mutagenesis and Ca2+ -sensitive fluorescence studies; 2) a bicistronic construct consisting of enhanced Green Fluorescent Protein (eGFP) and Cre recombinase for conditional mutagenesis, fate mapping, and cell sorting for single cell or gene expression studies; and 3) GFP - based Ca2+ -sensitive fluorescent proteins (G-CaMPS1.6x and C-CaMPS1.3) for lineage specific studies of cell function and signaling. All lines of mice will be characterized and made available to the scientific community to accelerate the use of conditional mutagenesis, fate mapping, and in-vivo imaging techniques in the study of cells of the GU tract. We will use the mice to address important questions relating to neuromuscular transmission and the development and function of Interstitial Cells of Cajal (ICC) in the urogenital tract.

[unreadable] DESCRIPTION (provided by applicant): Postobstructive urinary tract dysfunction is a disorder with extremely high morbidity and devastating social and psychological consequence, resulting in a complex of symptoms including incontinence, increased frequency of urination, and an inappropriate sense of urinary urgency, all of which appear to result from abnormal contractile activity of the smooth muscle lining the bladder. In this proposal we seek to understand the generation of rhythmic or phasic contractile activity in the normal and postobstructed urinary bladder in vivo. Although the study of the cellular signals underlying phasic activity is extraordinarily difficult in vivo, it is important to understand this activity in the context of neural and hormonal inputs that play an important role in the regulation of contractile activity. We have recently developed methods to monitor intracellular free Ca2+ in vivo through the development of transgenic mice expressing a novel high signal-noise, genetically encoded Ca2+ indicator. GCaMP2, the Ca2+ indicator, is stable at physiological temperatures and approaches the brightness of GFP, enabling sustained, high resolution recording of cellular Ca2+ transients in the living mouse. Mice in which this molecule is highly expressed in urinary bladder smooth muscle have been created and will be used to visualize cellular Ca2+ signals in vivo. We will use these mice to more fully understand the dysfunction associated with urinary tract outlet obstruction. The basis of abnormal contractile activity in postobstructive mice that develop spontaneous bladder overactivity will be studied to understand the cellular and molecular basis for abnormal bladder contractions. We hypothesize that bladder hyperactivity occurs secondary to the development of abnormal pacemaker activity and anomalous Ca2+ waves associated with dysregulated Ca2+ release from the sarcoplasmic reticulum (SR) of smooth muscle or Interstitial Cells of Cajal (ICC). To test this hypothesis, we will image phasic Ca2+ signals in smooth muscle cells under normal conditions, seek to understand the cellular and molecular basis for spontaneous activity, determine the extent to which this pattern is disturbed by sustained outflow obstruction, and test whether abnormal activity arises due to alterations in SR Ca2+ release in muscle or changes in the activity of pacemaker cells in the urinary bladder. These findings should provide a clearer understanding of smooth muscle dysfunction associated with urinary tract outflow obstruction. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): This exploratory grant seeks to develop a novel and promising system for intracellular imaging. We have identified RNA aptamers that bind to and markedly reduce the fluorescence of Green Fluorescent Proteins. The aptamer structures identified bind to GFP, eGFP, YFP, and CFP with high affinity, but selectively inhibit the fluorescence emission of GFP and eGFP (KD = 14 nM), largely through a decrease in the molar extinction coefficient. Here we propose to exploit fluorescence quenching RNA aptamers as a flexible, genetically encodable molecular detection system. We will optimize genetic constructs for the expression of nuclear and cytoplasmic RNA aptamers (Aim 1). The fluorescent protein binding structure will be utilized as an allosterically regulated reporter component of a bivalent RNA by linkage to a second detector domain (Aim 2); binding of a target molecule to the detector domain within the RNA aptamer will resulting in unbinding of the fluorescence quenching RNA aptamer from GFP and the induction of a robust fluorescent signal. Such a system could provide a novel approach to in vitro and in vivo cellular imaging, and could also be adapted to rapid biohazard detection. Biochemical signaling within cells and between cells underlies normal and abnormal function. Recent advances in optical imaging and genetic specification have revolutionized our understanding of complex physiological processes such as development and the response to disease. This proposal seeks to develop a flexible and broadly applicable method for the construction of genetically encoded optical sensors that will report on the concentration, location, and structure of specific molecules in live cells, thereby markedly expanding the tools for understanding complex cellular responses. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Genetically encoded Ca2+ sensors hold great promise for the dissection of complex physiology in vivo. The ability to make molecular scale measurements in real time in mammals, and to determine lineage-specific signaling events by genetic specification, provides unprecedented experimental power to determine the complex cell-cell communications that underlie normal organ function, and the dysfunction that attends and is the hallmark of disease. A number of laboratories have developed circularly permutated EGFP-Calmodulin/M13 fusion proteins to understand several complex biological processes in vivo, and these tools have begun to provide a novel window on heart development, heart repair, and endothelial cell signaling. While these studies demonstrate the feasibility of real-time, in vivo imaging at the molecular scale in mammals using genetically encoded Ca2+ indicators, limitations of current molecules prevent their comprehensive exploitation. These limitations include less than optimal signal/noise characteristics, a nonlinear Ca2+ response, the limited spectral range of effective probes, and their non-ratiometric nature. The overall goal is to develop improved genetically encoded Ca2+ sensors (GECIs) through the determination of the structural basis of Ca2+ -dependent fluorescence, the development of multiwavelength indicators that provide the ability to quantify Ca2+ signals in vivo, and the creation of red- shifted GECIs that enable studies of cell-cell signaling in vivo. The effort represents an extension of an ongoing collaboration between the laboratories of Dr. Michael Kotlikoff, who has significant experience with the design and function of GECIs, Dr. Holger Sondermann, who is an expert in protein structure and function, and Dr. Warren Zipfel, a biophysicist with expertise in fluorescence photophysics. These studies will address several significant limitations of current molecules and extend the range of studies for which such molecules will be useful. Emphasis will be placed on the optimization of developed sensors for in vivo performance, as determined by their expression in transgenic mice. PUBLIC HEALTH RELEVANCE: This project will produce novel molecules that can be used in vitro in cell systems and in vivo in animals to determine cellular function in the context of disease or organ repair. The proposal will produce novel proteins that will track the function of cells at the molecular level.

DESCRIPTION (provided by applicant): Advances in genetically encoded sensor and effector proteins have introduced unprecedented opportunities for the study of complex biological processes in vivo that provide an opportunity to rapidly advance knowledge critical to the understanding and treatment of cardiovascular and cardiopulmonary diseases. These optical tools have not been widely exploited due to the lack of available transgenic mouse lines expressing these reagents in relevant tissues. Such genetic reagents could be easily and efficiently deployed for the study of NHLBI relevant biology and disease, transforming the capacity of NHLBI scientists. We propose the creation of purpose -designed mouse lines that express Ca2+ sensors and Optogenetic cell activators in NHLBI relevant tissues. The Genetic Resource for Optical Signaling will consist of 50 mouse lines designed for combinatorial crosses enabling the co-expression of sensors and effectors, or red and green Ca2+ sensors in interacting lineages. This strategy will enable the combination of two synergistic technologies (Ca2+ sensors and optogenetic proteins) through simple, high-yield crosses that constrain expression of the transgene to cells of interest. As such, the resource will enable NHLBI researchers to conduct experiments not otherwise possible in a cost effective and highly efficient manner, accelerating the use of optical genetic tools and markedly accelerating scientific discovery. The Genetic Resource for Optical Signaling (GROS) will create and validate the lines, communicate their availability, and permanently bank them for distribution to the scientific community following the funding period. RELEVANCE: We will create a resource that advances cardiovascular and cardiopulmonary science consisting of lines of mice expressing 3rd generation optical sensors and cell activators in lineages of relevance to NHLBI science. These mice will be fully characterized and made available to the community, creating permanently available reagents that will markedly enhance the capabilities of hundreds of scientists. (End of Abstract)

DESCRIPTION (provided by applicant): There is increasing evidence that recovery of function following a heart attack is markedly influenced by revascularization of the infarcted region, which influences the extent of injury, the degree of fibrosis within the infarct, post injury remodeling, and the incidence of arrhythmia. Despite the importance of revascularization, the processes underlying angiogenesis and/or vasculogenesis remain poorly understood, including the degree to which precursor cells are involved in new vessel formation, the origin of these putative precursors (e.g. bone marrow or resident adventitial cells), and the signals that govern processes such as homing, fate, and endothelial cell (EC) mitosis. This proposal examines fundamental aspects of post-infarction revascularization in vivo, exploiting recent advances in the understanding and genetic specification of vascular precursors. Our central hypothesis is that infarction triggers the expansion and differentiation of endogenous vascular precursors that are induced through key heterotypic cell-cell interactions within the perivascular niche. The proposed experiments will identify the degree to which these precursors drive revascularization, determine their developmental origin, and establish the role of the expression of c-kit, SCF, Sca1, and SDF-1?/CXCR4 signaling in this process. Defined precursor populations and terminally differentiated vascular cells (endothelial and smooth muscle) will be genetically tagged and the lineage of post-infarct nascent vascular cells examined to determine the role of angiogenesis and vasculogenesis. The role of c-kit and its ligand stem cell factor (SCF), as well as other receptor tyrosine kinases, will be examined in conditionally deleted (SCF) mice. Heterotypic signaling functions of Sca1 cells that home to infarcts and are used for cell therapy, and the importance of cardiac and marrow CXCR4 signaling will be determined in knockout or conditionally inactivated mice. These experiments will establish a conceptual framework for the understanding of ischemic revascularization of the heart, a critical step in the development of therapeutic strategies directed toward enhancing vascular repair.