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Functional Architecture of Inositol 1,4,5-Trisphosphate Signaling in Restricted Spaces of Myoendothelial Projections

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Functional Architecture of Inositol 1,4,5-Trisphosphate Signaling in Restricted Spaces of Myoendothelial Projections Functional architecture of inositol 1,4,5-trisphosphate signaling in restricted spaces of myoendothelial projections Jonathan Ledoux*, Mark S. Taylor†, Adrian D. Bonev*, Rachael M. Hannah*, Viktoriya Solodushko†, Bo Shui‡, Yvonne Tallini‡, Michael I. Kotlikoff‡, and Mark T. Nelson*§ *Department of Pharmacology, College of Medicine, University of Vermont, Burlington, VT 05405; †Department of Physiology, College of Medicine, University of South Alabama, Mobile, AL 36688; and ‡Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853 Edited by David E. Clapham, Harvard Medical School, Boston, MA, and approved May 6, 2008 (received for review February 28, 2008) -Calcium (Ca2؉) release through inositol 1,4,5-trisphosphate recep- epoxyeicosatrienoic acids, and direct electrical communica tors (IP3Rs) regulates the function of virtually every mammalian tion via gap junctions (2, 3). Engagement of the EDHF cell. Unlike ryanodine receptors, which generate local Ca2؉ events functionality, regardless of its specific identity, depends on the (‘‘sparks’’) that transmit signals to the juxtaposed cell membrane, activation of calcium-sensitive, small-conductance (KCa2.3) a similar functional architecture has not been reported for IP3Rs. and intermediate-conductance (KCa3.1) potassium channels in .(Here, we have identified spatially fixed, local Ca2؉ release events the vascular endothelium (3 (‘‘pulsars’’) in vascular endothelial membrane domains that project Although Ca2ϩ waves and oscillations have been reported in through the internal elastic lamina to adjacent smooth muscle native endothelial preparations (4–6), organized patterns of 2؉ 2ϩ membranes. Ca pulsars are mediated by IP3Rs in the endothelial Ca signaling have not been identified, despite the unique endoplasmic reticulum of these membrane projections. Elevation polarity of the vascular endothelium and known functional 2ϩ of IP3 by the endothelium-dependent vasodilator, acetylcholine, connections between endothelial and SM cells. To explore Ca 2؉ increased the frequency of Ca pulsars, whereas blunting IP3 signaling in endothelial cells in the arterial wall, we engineered CELL BIOLOGY production, blocking IP3Rs, or depleting endoplasmic reticulum a mouse that expresses a calcium biosensor (GCaMP2) exclu- Ca2؉ inhibited these events. The elementary properties of Ca2؉ sively in the endothelium (7). Gene-encoded, tissue-specific pulsars were distinct from ryanodine-receptor-mediated Ca2؉ expression of calcium sensors permits the examination of Ca2ϩ 2؉ sparks in smooth muscle and from IP3-mediated Ca puffs in dynamics in multicellular preparations without prolonged peri- Xenopus oocytes. The intermediate conductance, Ca2؉-sensitive ods of dye loading and confounding influence of fluorescence potassium (KCa3.1) channel also colocalized to the endothelial from multiple cell types (8). projections, and blockage of this channel caused an 8-mV depo- larization. Inhibition of Ca2؉ pulsars also depolarized to a similar Results extent, and blocking KCa3.1 channels was without effect in the The vascular endothelium is separated from SM cells in the absence of pulsars. Our results support a mechanism of IP3 signal- arterial wall by the internal elastic lamina (IEL). The IEL ing in which Ca2؉ release is spatially restricted to transmit inter- contains numerous holes through which endothelial membrane cellular signals. projections penetrate, allowing direct contact and communica- tion between endothelial and SM membranes through myoen- calcium ͉ endothelium ͉ calcium biosensor ͉ intermediate conductance dothelial gap junctions (9–13). To allow visualization of calcium Ca2ϩ-sensitive potassium channel ͉ calcium pulsar signals while preserving the organization of this endothelial– IEL–SM structure, we exteriorized the endothelial surface of he location and patterning of intracellular calcium (Ca2ϩ) third-order mouse mesenteric artery segments by making a Tsignals encode information that regulates distinct aspects of longitudinal incision and pinning the rectangular preparation. cell function. Two major intracellular calcium release channels Individual endothelial cells with an average visible surface area Ϯ ␮ 2 in the sarco-/endoplasmic reticulum (SR/ER) of mammalian of 737 35 m were readily distinguishable (Fig. 1A). Black cells—inositol 1,4,5-trisphosphate receptors (IP Rs) and ryano- holes in the autofluorescence of the IEL, representing projection 3 points of the endothelial membrane to the SM membrane and dine receptors (RyRs)—have the potential to generate modu- Ϯ lated and localized calcium signals. Elementary release events sites of connecting gap junctions (9, 11–13), also were seen [5.6 ␮ 2 Ϯ termed ‘‘calcium sparks’’ correspond to the activation of a 0.4 holes/1,000 m , with a mean surface area of 4.43 0.09 ␮ 2 spatially fixed cluster of RyRs in the SR membrane. The m ; supporting information (SI) Fig. S1Aa] (14). organization of RyRs in SR elements permits intimate commu- In unstimulated arteries from GCaMP2-expressing mice, we nication with the closely juxtaposed plasma membrane. In detected calcium spark-like events in proximity to the holes in the IEL (Figs. 1 and 2 and Movie S1). The majority of these smooth muscle (SM), calcium sparks activate calcium-sensitive 2ϩ large-conductance potassium channels in the plasma membrane, events, which we have termed ‘‘Ca pulsars,’’ occurred in or causing a transient hyperpolarizing current that decreases vaso- constriction (1). No such local calcium signaling architecture Author contributions: J.L., M.S.T., A.D.B., and M.T.N. designed research; J.L., A.D.B., R.M.H., with analogous functional significance has been reported for and V.S. performed research; A.D.B., B.S., Y.T., and M.I.K. contributed new reagents/ IP3Rs. analytic tools; J.L., M.S.T., A.D.B., and M.T.N. analyzed data; and J.L., M.S.T., and M.T.N. Elevation of Ca2ϩ in the vascular endothelium is thought to wrote the article. play a major role in transmitting vasoregulatory signals to The authors declare no conflict of interest. adjacent SM cells in the arterial wall. Calcium-dependent This article is a PNAS Direct Submission. endothelial signals include nitric oxide (NO), prostaglandins §To whom correspondence should be addressed. E-mail: [email protected]. (PGs), and a mechanistically diverse non-NO, non-PG func- This article contains supporting information online at www.pnas.org/cgi/content/full/ tionality termed endothelial-derived hyperpolarizing factor 0801963105/DCSupplemental. (EDHF), which is variably attributable to potassium ions, © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0801963105 PNAS ͉ July 15, 2008 ͉ vol. 105 ͉ no. 28 ͉ 9627–9632 Downloaded by guest on September 25, 2021 Fig. 1. Ca2ϩ pulsars colocalized with IEL holes. (A)(a) IEL autofluorescence shows the presence of ‘‘holes’’ in the IEL. (b) Initiation sites of Ca2ϩ pulsars from the composite image correspond to holes in the IEL (red arrows). The yellow arrows indicate pulsar sites not associated with detectable IEL holes. (Scale bar, 10 ␮m.) (c) Histogram illustrating the distance between Ca2ϩ pulsar initiation sites and IEL holes in endothelium (n ϭ 357 pulsar sites). (B) Time course of a three-dimensional Ca2ϩ pulsar originating from within an IEL hole (white circle) shown in the leftmost image. (Scale bar, 5 ␮m.) (C)Ca2ϩ pulsars from a pressurized artery (80 mmHg) expressing GCaMP2. (Ca) An endothelial cell and its nucleus are outlined (dotted lines), with the initiation sites (Cb and Cc) indicated by red arrows. (Scale bar, 10 ␮m.) See also Movie S1 and Movie S2. within 2 ␮m of holes in the IEL (Fig. 1Ac). Fig. 1A shows a field Calcium pulsars occurred repetitively at the same site (Fig. 2), in which 21 of 27 pulsar sites (78%) localized to detectable holes with a mean time of 9.8 Ϯ 1.0 s between pulsars at a given site in the IEL. Fig. 1B illustrates a single pulsar occurring in a single (Fig. S1Ba). The interval between pulsars may be determined by hole. There was no correlation between the size of the hole and local calcium depletion of the involved ER element or by the the amplitude and frequency of the associated Ca2ϩ pulsars (Fig. amount of calcium released during an event. However, there was S1 Ab and Ac). no correlation between the amplitude of a Ca2ϩ pulsar and the Fig. 2. Kinetics and repetitive occurrences of Ca2ϩ pulsars. (A) Average of 10 images of a field of endothelial cells from mesenteric arteries of a GCaMP2-expressing mouse. The red arrow indicates the initiation site of Ca2ϩ pulsars shown in B and C. (Scale bar, 10 ␮m.) (B) Life span of a Ca2ϩ pulsar is shown. The field of view corresponds to the green square in A (Scale bar, 10 ␮m.) (C) Repetitive occurrence of Ca2ϩ pulsars at one site expressed as a line-scan analysis along the yellow line in A. (Scale bar, 5 s.) (D) Representative traces illustrating Ca2ϩ pulsar kinetics originating from two different sites (red and blue lines). See also Movie S1. 9628 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0801963105 Ledoux et al. Downloaded by guest on September 25, 2021 Table 1. Comparison of Ca2؉ sparks from vascular SM cells, Ca2؉ puffs from Xenopus oocytes, and Ca2؉ pulsars from mesenteric endothelium Ca2ϩ pulsars Parameters Ca2ϩ sparks (26) Ca2ϩ puffs Fluo-4 GCaMP2 Amplitude, F/Fo 2.0 50–500 nM (27) 1.77 Ϯ 0.10 1.70 Ϯ 0.02 Area, ␮m2 13.6 2–4 (16, 27) 15.9 Ϯ 0.6 14.1 Ϯ 0.5 Rise time, ms 22.7 Ͻ100 (28) 212 Ϯ 11 163 Ϯ 6 Duration, ms n/a 1,000 (16, 27) 257 Ϯ 12 269 Ϯ 6 t1/2, ms 55.9 375 (15) 146 Ϯ 9 168 Ϯ 5 Frequency, Hz n/a n/a 0.10 Ϯ 0.02 0.08 Ϯ 0.02 Descriptive parameters of Ca2ϩ signals in terms of peak amplitude, area measured at 50% of the amplitude of the peak, rise time as measured during 10–90% 2ϩ of the signal, duration as measured from 50% of the signal before and after the peak, and the half-time for decay (t1/2) of the signal.
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