Calcium (Ca 2؉ ) release through inositol 1,4,5-trisphosphate receptors (IP 3Rs) regulates the function of virtually every mammalian cell. Unlike ryanodine receptors, which generate local Ca 2؉ events (''sparks'') that transmit signals to the juxtaposed cell membrane, a similar functional architecture has not been reported for IP 3Rs. Here, we have identified spatially fixed, local Ca 2؉ release events (''pulsars'') in vascular endothelial membrane domains that project through the internal elastic lamina to adjacent smooth muscle membranes. Ca 2؉ pulsars are mediated by IP3Rs in the endothelial endoplasmic reticulum of these membrane projections. Elevation of IP 3 by the endothelium-dependent vasodilator, acetylcholine, increased the frequency of Ca 2؉ pulsars, whereas blunting IP3 production, blocking IP3Rs, or depleting endoplasmic reticulum Ca 2؉ inhibited these events. The elementary properties of Ca 2؉ pulsars were distinct from ryanodine-receptor-mediated Ca 2؉ sparks in smooth muscle and from IP3-mediated Ca 2؉ puffs in Xenopus oocytes. The intermediate conductance, Ca 2؉ -sensitive potassium (K Ca3.1) channel also colocalized to the endothelial projections, and blockage of this channel caused an 8-mV depolarization. Inhibition of Ca 2؉ pulsars also depolarized to a similar extent, and blocking K Ca3.1 channels was without effect in the absence of pulsars. Our results support a mechanism of IP 3 signaling in which Ca 2؉ release is spatially restricted to transmit intercellular signals.calcium ͉ endothelium ͉ calcium biosensor ͉ intermediate conductance Ca 2ϩ -sensitive potassium channel ͉ calcium pulsar

Genetically encoded sensor proteins provide unique opportunities to advance the understanding of complex cellular interactions in physiologically relevant contexts; however, previously described sensors have proved to be of limited use to report cell signaling in vivo in mammals. Here, we describe an improved Ca 2؉ sensor, GCaMP2, its inducible expression in the mouse heart, and its use to examine signaling in heart cells in vivo. The high brightness and stability of GCaMP2 enable the measurement of myocyte Ca 2؉ transients in all regions of the beating mouse heart and prolonged pacing and mapping studies in isolated, perfused hearts. Transgene expression is efficiently temporally regulated in cardiomyocyte GCaMP2 mice, allowing recording of in vivo signals 4 weeks after transgene induction. High-resolution imaging of Ca 2؉ waves in GCaMP2-expressing embryos revealed key aspects of electrical conduction in the preseptated heart. At embryonic day (e.d.) 10.5, atrial and ventricular conduction occur rapidly, consistent with the early formation of specialized conduction pathways. However, conduction is markedly slowed through the atrioventricular canal in the e.d. 10.5 heart, forming the basis for an effective atrioventricular delay before development of the AV node, as rapid ventricular activation occurs after activation of the distal AV canal tissue. Consistent with the elimination of the inner AV canal muscle layer at e.d. 13.5, atrioventricular conduction through the canal was abolished at this stage. These studies demonstrate that GCaMP2 will have broad utility in the dissection of numerous complex cellular interactions in mammals, in vivo. atrioventricular node ͉ Ca 2ϩ imaging ͉ genetic sensor ͉ heart development ͉ fluorescent Ca 2ϩ sensor T ransient, highly regulated elevations in cytosolic free Ca 2ϩ underlie numerous cellular processes that enable organ function (1-5). In the mammalian heart, for example, efficient function depends upon the coordinated release and reuptake of Ca 2ϩ ions from intracellular organelles in millions of cells, at rates between 0.5 and 15 Hz throughout life, and even subtle dysfunctions of this process can result in cardiac arrythmias and sudden death. Whereas fluorescent imaging using purpose-designed small Ca 2ϩ -binding indicator molecules has enabled important advances in the understanding of the regulatory processes underlying Ca 2ϩ signaling in single cells (6, 7), these approaches have significant limitations in the context of a complex, multicellular organ such as the beating heart. Thus, difficulties in obtaining an adequate and stable concentration of indicator molecules within cells deep in complex tissues, the incompatibility of loading procedures in the in vivo setting, and the inability to selectively load specific cell lineages constitute substantial experimental constraints on the study of multicellular, processive Ca 2ϩ signaling in complex organ function. Genetically encoded sensors of Ca 2ϩ signaling (7-13) hold great promise in this regard and have been used t...

Ventricular tachyarrhythmias are the main cause of sudden death in patients after myocardial infarction. Here we show that transplantation of embryonic cardiomyocytes (eCMs) in myocardial infarcts protects against the induction of ventricular tachycardia (VT) in mice. Engraftment of eCMs, but not skeletal myoblasts (SMs), bone marrow cells or cardiac myofibroblasts, markedly decreased the incidence of VT induced by in vivo pacing. eCM engraftment results in improved electrical coupling between the surrounding myocardium and the infarct region, and Ca2+ signals from engrafted eCMs expressing a genetically encoded Ca2+ indicator could be entrained during sinoatrial cardiac activation in vivo. eCM grafts also increased conduction velocity and decreased the incidence of conduction block within the infarct. VT protection is critically dependent on expression of the gap-junction protein connexin 43 (Cx43; also known as Gja1): SMs genetically engineered to express Cx43 conferred a similar protection to that of eCMs against induced VT. Thus, engraftment of Cx43-expressing myocytes has the potential to reduce life-threatening post-infarct arrhythmias through the augmentation of intercellular coupling, suggesting autologous strategies for cardiac cell-based therapy.

In the epithelium of the lower airways, a cell type of unknown function has been termed "brush cell" because of a distinctive ultrastructural feature, an apical tuft of microvilli. Morphologically similar cells in the nose have been identified as solitary chemosensory cells responding to taste stimuli and triggering trigeminal reflexes. Here we show that brush cells of the mouse trachea express the receptors (Tas2R105, Tas2R108), the downstream signaling molecules (α-gustducin, phospholipase C β2 ) of bitter taste transduction, the synthesis and packaging machinery for acetylcholine, and are addressed by vagal sensory nerve fibers carrying nicotinic acetylcholine receptors. Tracheal application of an nAChR agonist caused a reduction in breathing frequency. Similarly, cycloheximide, a Tas2R108 agonist, evoked a drop in respiratory rate, being sensitive to nicotinic receptor blockade and epithelium removal. This identifies brush cells as cholinergic sensors of the chemical composition of the lower airway luminal microenvironment that are directly linked to the regulation of respiration.airway sensory innervation | respiratory epithelium | jugular-nodose ganglion | bitter-tasting substances

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The peripheral nervous system has complex and intricate ramifications throughout many target organ systems. To date this system has not been effectively labeled by genetic markers, due largely to inadequate transcriptional specification by minimum promoter constructs. Here we describe transgenic mice in which enhanced green fluorescent protein (eGFP) is expressed under the control of endogenous choline acetyltransferase (ChAT) transcriptional regulatory elements, by knock-in of eGFP within a bacterial artificial chromosome (BAC) spanning the ChAT locus and expression of this construct as a transgene. eGFP is expressed in ChAT(BAC)-eGFP mice in central and peripheral cholinergic neurons, including cell bodies and processes of the somatic motor, somatic sensory, and parasympathetic nervous system in gastrointestinal, respiratory, urogenital, cardiovascular, and other peripheral organ systems. Individual epithelial cells and a subset of lymphocytes within the gastrointestinal and airway mucosa are also labeled, indicating genetic evidence of acetylcholine biosynthesis. Central and peripheral neurons were observed as early as 10.5 days postcoitus in the developing mouse embryo. ChAT(BAC)-eGFP mice allow excellent visualization of all cholinergic elements of the peripheral nervous system, including the submucosal enteric plexus, preganglionic autonomic nerves, and skeletal, cardiac, and smooth muscle neuromuscular junctions. These mice should be useful for in vivo studies of cholinergic neurotransmission and neuromuscular coupling. Moreover, this genetic strategy allows the selective expression and conditional inactivation of genes of interest in cholinergic nerves of the central nervous system and peripheral nervous system.

It is generally accepted that the endothelium regulates vascular tone independent of the activity of the sympathetic nervous system. Here, we tested the hypothesis that the activation of sympathetic nerves engages the endothelium to oppose vasoconstriction. Local inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) signals ("pulsars") in or near endothelial projections to vascular smooth muscle (VSM) were measured in an en face mouse mesenteric artery preparation. Electrical field stimulation of sympathetic nerves induced an increase in endothelial cell (EC) Ca(2+) pulsars, recruiting new pulsar sites without affecting activity at existing sites. This increase in Ca(2+) pulsars was blocked by bath application of the α-adrenergic receptor antagonist prazosin or by TTX but was unaffected by directly picospritzing the α-adrenergic receptor agonist phenylephrine onto the vascular endothelium, indicating that nerve-derived norepinephrine acted through α-adrenergic receptors on smooth muscle cells. Moreover, EC Ca(2+) signaling was not blocked by inhibitors of purinergic receptors, ryanodine receptors, or voltage-dependent Ca(2+) channels, suggesting a role for IP(3), rather than Ca(2+), in VSM-to-endothelium communication. Block of intermediate-conductance Ca(2+)-sensitive K(+) channels, which have been shown to colocalize with IP(3) receptors in endothelial projections to VSM, enhanced nerve-evoked constriction. Collectively, our results support the concept of a transcellular negative feedback module whereby sympathetic nerve stimulation elevates EC Ca(2+) signals to oppose vasoconstriction.

Abstract-To study endothelial cell (EC)-specific Ca 2ϩ signaling in vivo we engineered transgenic mice in which the Ca 2ϩ sensor GCaMP2 is placed under control of endogenous connexin40 (Cx40) transcription regulatory elements within a bacterial artificial chromosome (BAC), resulting in high sensor expression in arterial ECs, atrial myocytes, and cardiac Purkinje fibers. High signal/noise Ca 2ϩ signals were obtained in Cx40 BAC -GCaMP2 mice within the ventricular Purkinje cell network in vitro and in ECs of cremaster muscle arterioles in vivo. Microiontophoresis of acetylcholine (ACh) onto arterioles triggered a transient increase in EC Ca 2ϩ fluorescence that propagated along the arteriole with an initial velocity of Ϸ116 m/s (nϭ28) and decayed over distances up to 974 m. The local rise in EC Ca 2ϩ was followed (delay, 830Ϯ60 ms; nϭ8) by vasodilation that conducted rapidly (mm/s), bidirectionally, and into branches for distances exceeding 1 mm. At intermediate distances (300 to 600 m), rapidly-conducted vasodilation occurred without changing EC Ca 2ϩ , and additional dilation occurred after arrival of a Ca 2ϩ wave. In contrast, focal delivery of sodium nitroprusside evoked similar local dilations without Ca 2ϩ signaling or conduction. We conclude that in vivo responses to ACh in arterioles consists of 2 phases: (1) a rapidly-conducted vasodilation initiated by a local rise in EC Ca 2ϩ but independent of EC Ca 2ϩ signaling at remote sites; and (2) a slower complementary dilation associated with a Ca 2ϩ wave that propagates along the endothelium. 2ϩ signaling is implicated in regulating the resistance microvasculature. [1][2][3][4][5] Conducted electrical signals travel for millimeters along the vessel wall, 6 mediate coordinated vasomotor responses to localized stimuli, 7-9 and involve distinct Ca 2ϩ signals within smooth muscle (SM) and ECs. For example, arteriolar dilation in response to the endothelium-dependent vasodilator acetylcholine (ACh) involves an increase in ECs 2 and decrease in SM 10 Ca 2ϩ . Although recent intravital studies have demonstrated a crucial role for the endothelium in conducted vasodilation, 9 the extent to which Ca 2ϩ signals are transmitted along the arteriolar wall and the mechanisms underlying Ca 2ϩ transmission are controversial. Some studies indicate that EC Ca 2ϩ responses to dilatory stimuli are localized, 1,2 whereas others indicate that Ca 2ϩ signals can travel along the endothelium for a millimeter or more. 5,11 The majority of information concerning microvascular Ca 2ϩ signaling has been derived from isolated vessels studied in vitro, 4,5,12 largely because of the difficulty of selectively loading ECs or SM cells with Ca 2ϩ sensitive dyes in vivo. 1,10 A fundamental limitation to isolated arterioles is their disconnection from networks in which they normally reside. The extent to which manipulation of arterioles to obtain dye loading and the loss of physiological parameters (eg, pressure or flow) alter Ca 2ϩ signaling is unknown. Therefore, alternative approaches are necessa...