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 . 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 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). -encoded, tissue-specific pulsars were distinct from ryanodine--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 ͉ 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 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 , © 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. Ca pulsars differ from Ca2ϩ puffs in that their area and rise time are significantly greater, yet their duration and half-life are significantly less. Fluo-4 values are from 743 pulsars originating from 56 sites recorded in 4 fields, and GCaMP2 values are from 450 pulsars originating from 45 sites recorded in 4 fields. n/a, not applicable.

latency to the next event at the same site (Fig. S1Bb), suggesting their properties were similar to those observed in open arteries that neither of these mechanisms determines the timing of the (Table 1 and Table S1). next event. Calcium pulsars are distinct from IP3R-mediated ‘‘calcium Fig. 2A highlights a pulsar at a hole in the IEL, indicated by puffs’’ previously reported in Xenopus oocytes (15) and cultured the arrow, which lasted Ͻ500 ms (Fig. 2 B and D). Overall, cells (16), which have significantly different kinetic properties pulsars exhibited a mean duration of 270 ms and mean area of and spatial spreads (Table 1). ‘‘Calcium puffs’’ represent the 2 14 ␮m (Table 1). Pulsar properties (amplitude, spatial spread, opening of a small number of IP3Rs that initiates regenerative kinetics, and frequency) were essentially the same when mea- Ca2ϩ waves and global calcium signals and do not have a fixed sured using the calcium-sensitive, fluorescent dye Fluo-4 (Table cellular location (16–18). Distinct from Ca2ϩ pulsars, calcium 1 and Fig. S2C), indicating that calcium-induced GCaMP2 waves were present in individual endothelial cells in the exteri- CELL BIOLOGY conformation changes do not distort the kinetics of pulsars (7). orized endothelium preparation (Fig. S2B and Fig. S4). Calcium Importantly, with GCaMP2 detection, Ca2ϩ elevation induced waves traveled over 12 Ϯ 2 and 9 Ϯ 2 ␮m at a velocity of 48 Ϯ by the calcium ionophore, ionomycin (10 ␮M), was homogenous, 4 and 58 Ϯ 6 ␮m/s, as measured with Fluo-4 (n ϭ 57) and indicating that the calcium biosensor was expressed evenly in the GCaMP2 (n ϭ 17), respectively. endothelium (Fig. S3). Endothelium-specific expression of Calcium pulsars resemble RyR-mediated Ca2ϩ sparks observed GCaMP2 also provided the opportunity to examine Ca2ϩ signals in SM and in that they occur at fixed locations with in pressurized arteries without distortion from SM. Indeed, we respect to the plasma membrane and have similar spatial spreads detected Ca2ϩ pulsars in intact GCaMP2 mesenteric arteries and amplitudes. Notably, however, Ca2ϩ pulsars have slower rise subjected to physiological transmural pressure (80 mmHg). The times and longer durations than Ca2ϩ sparks (Table 1). Treatment pulsars occurred at the IEL holes (Fig. 1C and Movie S2), and with the RyR inhibitor ryanodine, which completely blocks sparks

2ϩ 2ϩ 2ϩ Fig. 3. Ca pulsars originating from IP3-sensitive stores. Removal of extracellular Ca (A) or ryanodine (C) did not affect Ca pulsars. Inhibition of SERCA with 2ϩ CPA (B), of IP3Rs with xestospongin C (D), or of PLC with U73122 (E) decreased Ca pulsars. (F) A data summary of pharmacological experiments targeting the ϩ ϩ source of Ca2 pulsars is given. (n ϭ 6, 5, 4, 6, and 3 arteries for 0 Ca2 , CPA, ryanodine, xestospongin C, and U73122, respectively; *,PϽ 0.05). For A–E, different colors represent F/Fo in ROIs over different pulsar sites in the endothelium. Calcium pulsars were recorded for 2 min, followed by variable incubation times (0 Ca, 5 min; Ry, 35 min; CPA, 15 min; xestospongin C, 40 min; U73122, 15 min) and then 2 min of recording in the drug treatment (see Methods Summary).

Ledoux et al. PNAS ͉ July 15, 2008 ͉ vol. 105 ͉ no. 28 ͉ 9629 Downloaded by guest on September 25, 2021 IP3R-mediated calcium pulsars in the absence of stimulation sug- gests that endothelial cells tonically produce IP3 under basal conditions. Indeed, inhibition of phospholipase C (PLC) with U73122 or inhibition of calcium uptake by the ER with cyclopia- zonic acid (CPA) significantly reduced calcium pulsar activity (Fig. 3 B, E, and F). Removing external calcium for 5 min did not significantly alter calcium pulsar activity (Fig. 3 A and F). Consistent with these functional observations, no RyR isoforms (RyR1–3) were detected in endothelial cells whereas mRNA for all IP3R isoforms (IP3R1–3) was detected in both the endothelium and the SM (Fig. S5), with IP3R2 giving the strongest signal. Collectively, these results support the concept that Ca2ϩ pulsars are localized 2ϩ Ca release events that are mediated by IP3 activation of IP3Rs in endothelial ER. The effects of PLC inhibition indicate that, in the absence of 2ϩ stimulation, Ca pulsars are stimulated by basal IP3 (Fig. 3 E and F). To increase IP3 production, the endothelial agonist, acetylcholine (ACh) was applied. ACh increased the frequency of calcium pulsars by Ϸ2.4-fold; inhibition of ER calcium uptake (CPA) or PLC activity (U73122) blocked this ACh effect (Fig. S6 and Table S1). ACh-induced increase in Ca2ϩ pulsar fre- quency was due to both the recruitment of new sites and a reduced interval between pulsars at a given site (Fig. S1B). There was also no correlation between pulsar amplitude and latency to Fig. 4. Localization of ER and IP3Rs within IEL holes. Images show immuno- the next event at a given site with ACh (Fig. S1B). ACh also staining for ER calnexin (red) (A–C) and for IP3Rs (red) (D–F) at the level of increased the frequency of Ca2ϩ waves (Ϸ3-fold) and global the IEL (green). (C and F) Superimposed images reveal that ER and IP3R are highly 2ϩ Ϸ concentrated inside distinct IEL holes. (Scale bar, 5 ␮m.) (G) A three-dimensional Ca ( 1.5-fold), consistent with previous reports (19, 20). view along the z axis (2.9 ␮m) shows densities of IP3R-positive fluorescence Our findings indicate that the ER in endothelial cells forms 2ϩ (white) projecting through the depth of the IEL (green). (Scale bar, 1 ␮m.) spatially discrete IP3-sensitive Ca stores localized in and around distinct IEL holes. To assess the distribution of ER at the endothelium–SM interface, we probed for the ER Ca2ϩ-binding ϩ ϩ in SM (1), had no effect on Ca2 pulsars or other Ca2 signals in protein calnexin. Calnexin staining showed that the ER was endothelial cells (Fig. 3 C and F). In contrast, the IP3R inhibitor, distributed in the perinuclear region, along the base of endo- xestospongin C, reduced the frequency of Ca2ϩ pulsars in endo- thelial cells, and most notably within dense plaques correspond- theliumto43Ϯ 7% of control (Fig. 3 D and F). The presence of ing precisely to the positions of holes in the IEL (Fig. 4 B and C).

2ϩ Fig. 5. Ca pulsars hyperpolarizing the endothelium membrane through activation of KCa3.1 channels. (A)(a) CPA (10 ␮M; black bar) depolarizes the endothelial membrane potential, and subsequent addition of ChTX (300 nM; white bar) had no effect. (b) Summary of membrane potential experiments with CPA using microelectrode and perforated patch techniques. Additionally, in microelectrode experiments, CPA exposure was followed by ChTX. Subsequent exposure to 60 mM KCl depolarized the endothelial membrane potential to Ϫ20 Ϯ 2 mV. n ϭ 6 and 4 for microelectrode and perforated patch recordings, respectively. (B)(a) ChTX (300 nM; black bar) depolarizes the endothelial membrane potential, and subsequent addition of CPA (10 ␮M; white bar) had no effect. (b) A summary of microelectrode experiments with ChTX followed by CPA is given. Subsequent exposure to 60 mM KCl depolarized the endothelial membrane potential to Ϫ22 Ϯ 2mV(n ϭ 5; *,PϽ 0.05). All microelectrode experiments were carried out in the presence of paxilline (500 nM) and nitrendipine (100 nM). (C)(a and b) Immunostaining for KCa3.1 (red) at the level of the IEL (green) is shown. (c) The superimposed image reveals distinct densities of KCa3.1 within and around IEL holes. (Scale bar, 5 ␮m.) (d) A three-dimensional view along the z axis (3.1 ␮m) shows densities of KCa3.1-positive fluorescence (white) projecting through the depth of the IEL (green). (Scale bar, 1 ␮m.)

9630 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0801963105 Ledoux et al. Downloaded by guest on September 25, 2021 2ϩ Fig. 6. Model of endothelial Ca pulsars. IP3R-dense ER stores follow portions of the endothelial cell membrane that evaginate through holes in the IEL and interface with underlying SM cell membranes. Repetitive localized Ca2ϩ events (pulsars) originate from these deep Ca2ϩ stores that are regionally delimited to CELL BIOLOGY 2ϩ the myoendothelial junction and the base of the endothelial cell. These ongoing dynamic Ca signals are driven by constitutive IP3 production and are inherently dependent on the level of endothelial stimulation. The left detail depicts a single endothelial projection through the IEL. KCa3.1 channels in the plasma membranes of these endothelial projections are in very close proximity to Ca2ϩ pulsars, eliciting persistent Ca2ϩ-dependent hyperpolarization of the membrane potential at the myoendothelial junctions. The right detail illustrates the endothelial influence on the SM membrane potential at the myoendothelial interaction 2ϩ site where Ca pulsars would activate KCa3.1 channels and hyperpolarize the endothelial membrane. This hyperpolarization can be transmitted to the SM ϩ through gap junction channels or by activation of SM Kir channels by K ions released by endothelial KCa3.1 channels. Membrane hyperpolarization promotes relaxation of SM through a decrease in voltage-dependent open probability.

Subsequent probing revealed a similar distribution of IP3Rs, endothelial membrane potential in the presence of ChTx (Fig. which were highly concentrated in both large and small IEL holes 5B). These results suggest that Ca2ϩ pulsars, and not global Ca2ϩ, (Fig. 4 E and F). Multiple images captured through the depth of directly activate KCa3.1 channels in the endothelium, and lend the IEL from the bottom surface of the endothelium to the top support to the concept that the KCa3.1 channel-dependent surface of the first SM cells revealed the contiguous presence of component of endothelial membrane potential is regulated by 2ϩ IP3Rs in the holes through the depth of the lamina (Fig. 4G). Ca pulsars. These findings suggest the existence of an IP3R-dense, inner- membrane ER structure that protrudes with the endothelial cell Discussion plasma membrane through the IEL and interfaces with SM cell Our results support the concept of a IP3R signaling structure membranes at the myoendothelial junction. with a profound functional bearing on endothelial–SM intercel- Calcium-sensitive potassium (KCa2.3 and KCa3.1) channels lular communication (Fig. 6). In a process reminiscent of may be key targets of discrete calcium signals in the endothelium. neuronal projections to target cells, vascular endothelial cells Importantly, KCa3.1 channels are in close proximity to IEL holes send projections through the IEL that contact SM cells. IP3Rs (11) and appear to localize specifically within endothelial pro- localized to these membrane projections mediate local Ca2ϩ jections traversing the holes (Fig. 5C). To explore the possible release events (pulsars), establishing a mechanism for Ca2ϩ- 2ϩ communication of Ca pulsars to KCa3.1 channels, we measured dependent signaling from endothelial cells to SM cells. One membrane potentials of endothelial cells in intact arteries. target of calcium pulsars appears to be KCa3.1 channels that are Blocking KCa3.1 channels with charybdotoxin (ChTX) depolar- colocalized to endothelial projections (Fig. 5C) (11). Activation izes this preparation by Ϸ8 mV (Fig. 5B) (21). Blocking KCa2.3 of endothelial KCa3.1 channels (and KCa2.3 channels) is a channels with apamin produces a considerably smaller effect (Ϸ3 common denominator of the various EDHF mechanisms that mV) on endothelial membrane potential (21). CPA, which communicate dilating influences from endothelial cells to SM blocks calcium uptake into the ER, effectively inhibited calcium (3). Prominent among these mechanisms is direct electrical pulsars (Fig. 3 B and F and Fig. S6 B and C) and caused a communication via myoendothelial gap junctions and direct transient (Ͻ15 min) increase in global calcium (27 Ϯ 6%) (22), activation of inward rectifier potassium channels on SM by presumably through the depletion of ER calcium. On the basis released potassium ions (Fig. 6) (2, 3, 23). Thus, a minimal of these disparate effects, we predicted that CPA should cause intercellular functional unit is likely composed of endothelial endothelial membrane potential hyperpolarization if global cal- IP3Rs, KCa3.1 channels, and gap-junction-forming cium activates KCa3.1 channels and should cause depolarization and inward rectifier potassium channels (Kir2.1) and voltage- if localized calcium pulsars activate KCa3.1 channels. CPA dependent calcium channels (Cav1.2) on the SM (Fig. 6). Other caused a Ϸ10-mV depolarization (Fig. 5Ab) but had no effect endothelial calcium-dependent processes (e.g., endothelial nitric when KCa3.1 channels were blocked (Fig. 5B). CPA did not affect oxide synthase and phospholipase A2) also may be activated by

Ledoux et al. PNAS ͉ July 15, 2008 ͉ vol. 105 ͉ no. 28 ͉ 9631 Downloaded by guest on September 25, 2021 calcium pulsars or differentially activated by other calcium Revolution TL acquisition software (Andor Technology). Bound Ca2ϩ was signals (global Ca2ϩ or waves). We propose that the Ca2ϩ pulsar detected by exciting at 488 nm with a solid-state laser and collecting emitted ϩ is a fundamental endothelial Ca2 signal whose activity is finely fluorescence above 510 nm. Fractional fluorescence (F/Fo) was evaluated by regulated by physiological agents (i.e., agonists and flow) that dividing the fluorescence of a region of interest (ROI) in the collected image modulate intracellular levels of IP and Ca2ϩ. Given their by an average fluorescence of 50 images without activity from the same ROI 3 using custom-designed software (A.D.B., unpublished data). The endothelial unique, restricted localization to points of contact with vascular 2ϩ cell surface was measured by automated analysis of the area enclosed by a SM, endothelial Ca pulsars likely encode a variety of bidirec- freehand ROI drawn around the outline of individual endothelial cells. Global tional endothelium–SM signals (4, 13). Consequently, disruption Ca2ϩ levels were measured over the entire area of a cell, defined by the of this IP3R signaling process may be a hallmark of endothelial freehand ROI outline. Ca2ϩ pulsars were analyzed by using an ROI defined by dysfunction observed in virtually all cardiovascular diseases. a5ϫ 5 pixel box positioned at a point corresponding to peak pulsar ampli- tude. Line-scan analysis was performed offline. In preparations from wild-type Methods Summary mice (i.e., non-GCaMP2-expressing), endothelial cells were preferentially Procedures. Animal procedures used in this study are in accord with loaded with Fluo-4 (10 ␮M) for 45 min at 30°C in the presence of pluronic acid institutional guidelines and were approved by the Institutional Animal Care (2.5 ␮g/ml) before imaging, as previously described (21). The field of view was and Use committee of the University of Vermont. Mesenteric arteries (Ϸ125 Ϸ115 ϫ 137 ␮m, corresponding to Ϸ25–30 partial and whole cells and 13 active ␮m in diameter) were freshly harvested from 3- to 4-month-old C57BL6- or cells per field. There were 89 Ϯ 7 holes per field, and generally between 10 and GCaMP2-expressing mice. We used bacterial artificial chromosome transgen- 28 active sites were identified in each field. esis (7, 24), expressing the circularly permutated, calcium sensor, G-CaMP2, under the control of the connexin40 (Cx40) promoter; Cx40 is expressed in Solutions, Pressurized Arteries, Immunohistochemistry, Membrane Potential vascular endothelium but not vascular SM (25). Adult female mice were Recording, Reverse-Transcriptase PCR, and Statistics. These methods are euthanized by intraperitoneal injection of sodium pentobarbital (150 mg/kg) described in SI Text. followed by a thoracotomy. Third-order mesenteric arteries from mice were cleaned of connective tissue, cut longitudinally, and pinned to a Sylgard block ACKNOWLEDGMENTS. We thank David Hill-Eubanks, Gayathri Krishnamoor- with the endothelium facing up. thy, Ismail Laher, and Stephen V. Straub for comments and J. Brayden for help with the microelectrode recordings. This work was supported by National 2؉ 2ϩ Endothelial Ca Imaging. Ca imaging was performed with a Revolution Institutes of Health Grants HL44455, DK53832, DK65947, and HL77378 (to Andor confocal system (Andor Technology) with an electron-multiplying CCD M.T.N.) and HL45239, DK65992, and DK58795 (to M.I.K); the Canadian Insti- camera on an upright Nikon microscope with a ϫ60, water-dipping objective tutes for Health Research (to J.L.); and the Totman Trust for Medical Research (NA 1.0). Images were acquired at 15–30 frames per second with Andor (to M.T.N.).

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