Pacemaker role of pericytes in generating synchronized spontaneous Ca2+ transients in the myenteric microvasculature of the guinea-pig gastric antrum

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Pacemaker role of pericytes in generating synchronized spontaneous Ca2+ transients in the myenteric microvasculature of the guinea-pig gastric antrum Hikaru Hashitani a,∗ , Retsu Mitsui a , Shota Masaki a , Dirk F. Van Helden b a b

Department of Cell Physiology, Graduate School of Medical Sciences, Nagoya City University, Nagoya, Japan School of Biomedical Sciences and Pharmacy, University of Newcastle, NSW, Australia

a r t i c l e

i n f o

Article history: Received 15 April 2015 Received in revised form 8 June 2015 Accepted 28 June 2015 Available online xxx Keywords: Pericyte T-type Ca2+ channels Capillaries Ca2+ transients Myenteric microvasculature

a b s t r a c t Properties of spontaneous Ca2+ transients in the myenteric microvasculature of the guinea-pig stomach were investigated. Specifically, we explored the spatio-temporal origin of Ca2+ transients and the role of voltage-dependent Ca2+ channels (VDCCs) in their intercellular synchrony using fluorescence Ca2+ imaging and immunohistochemistry. The microvasculature generated spontaneous Ca2+ transients that were independent of both Ca2+ transients in interstitial cells of Cajal (ICC) and neural activity. Spontaneous Ca2+ transients were highly synchronous along the length of microvasculature, and appeared to be initiated in pericytes and spread to arteriolar smooth muscle cells (SMCs). In most cases, the generation or synchrony of Ca2+ transients was not affected by blockers of L-type VDCCs. In nifedipine-treated preparations, synchronous spontaneous Ca2+ transients were readily blocked by Ni2+ , mibefradil or ML216, blockers for T-type VDCCs. These blockers also suppressed the known T-type VDCC dependent component of ICC Ca2+ transients or slow waves. Spontaneous Ca2+ transients were also suppressed by caffeine, tetracaine or cyclopiazonic acid (CPA). After the blockade of both L- and T-type VDCCs, asynchronous Ca2+ transients were generated in pericytes on precapillary arterioles and/or capillaries but not in arteriolar SMCs, and were abolished by CPA or nominally Ca2+ free solution. Together these data indicate that pericytes in the myenteric microvasculature may act as the origin of synchronous spontaneous Ca2+ transients. Pericyte Ca2+ transients arise from Ca2+ release from the sarco-endoplasmic reticulum and the opening of T-type Ca2+ VDCCs is required for their synchrony and propagation to arteriolar SMCs. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction The microvasculature consisting of precapillary arterioles, capillaries and postcapillary venules plays a principal role in the

Abbreviations: ANO1, anoctamin-1; BSA, bovine serum albumin; CPA, cyclopiazonic acid; FLC, fibroblast-like cells; GI, gastrointestinal; ICC, interstitial cells of Cajal; ICC-MY, myenteric Interstitial cells of Cajal; PDGFR␣, platelet-derived growth factor receptor a; PSS, physiological saline solution; SERCA, sarco-endoplasmic reticulum calcium ATPase; SR/ER, sarco-endoplasmic reticulum; ␣-SMA, a-smooth muscle actin; SK3, small conductance Ca2+ -activated K+ channel 3; SMC, smooth muscle cell; TMEM16A, transmembrane member 16A; VDCC, voltage-dependent Ca2+ channels; vWF, von Willebrand factor. ∗ Corresponding author at: Department of Cell Physiology, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan. Tel.: +81 52 8538131; fax: +81 52 8421538. E-mail address: [email protected] (H. Hashitani).

transport of nutrients to the tissues and removal of tissue excreta. Arterioles control the distribution and rate of blood flow into the tissues to meet demands of individual organs, while transfer of substances predominately occurs across the wall of capillaries. Since capillary filtration and reabsorption is a function of hydrostatic pressure that is determined by arteriolar and venular pressures as well as the ratio of post-to-precapillary resistance, the function of different units of the microvasculature plays a critical role in regulating the microcirculation. However, properties of pericytes and/or smooth muscle cells (i.e. mural cells) in capillaries and venules have been less explored in comparison to those of arteriolar smooth muscle cells (SMCs). We have recently demonstrated that the venules in the submucosal layer of the bladder [1–3], proximal antrum [4] and distal colon [5] develop spontaneous phasic constrictions, suggesting that the venules may more actively contribute to the regulation of the microcirculation than previously thought. Constrictions arise

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through contractions of venular SMCs or pericytes (depending on the vessel or vessel size) through spontaneous SR Ca2+ release opening Ca2+ -activated chloride channels, the resultant depolarization triggering the opening of L-type voltage-dependent Ca2+ channels (VDCCs) and associated Ca2+ influx [1,2,4,5]. In suburothelial venules of the mouse bladder, pericytes, which have a stellate shaped cell body, forming a loose network, exhibited spontaneous Ca2+ transients [2,6], while circumferentially arranged SMCs generated spontaneous Ca2+ transients in other venules. L-type VDCCs also appear to play a critical role in maintaining synchrony amongst the contractile cells of the venules [1,2,4]. The importance of pericytes in contributing to the regulation of capillary blood flow has been recently revealed [7–9]. In several vascular beds, pericytes on precapillary arterioles and/or capillaries have been shown to develop Ca2+ transients, depolarizations and contractile responses upon stimulation with neurotransmitters or humoral substances, e.g., noradrenaline [9], acetylcholine [10], angiotensin-II [11] or endothelin-1 [12]. However, while it is known that venular pericytes exhibit spontaneous electrical, Ca2+ or contractile activity [2,6], this has not been demonstrated for pericytes on precapillary arterioles and capillaries. Moreover, properties of pericytes have been mostly investigated in the cerebral and retinal microvasculature [7–9], while few studies are available for visceral organs [12]. Therefore, their role in regulating the microcirculation of visceral organs remains to be further explored. In the gastrointestinal (GI) tract the myenteric layer, comprised of an extensive nerve plexus and networks of interstitial cells of Cajal (ICC) and PDGFR␣-positive (PDGFR␣+ ) fibroblast-like cells, plays a central role in regulating gastrointestinal (GI) motility. ICC act as pacemaker cells for peristalsis by sending rhythmical propagating depolarizing signals to generate spontaneous phasic contractions of the smooth muscle [13,14]. ICC also receive neural input from both excitatory and inhibitory intrinsic nerves, and thus act as intermediaries of neuromuscular transmission [14]. PDGFR␣+ fibroblast-like cells (FLCs) form a network and generate hyperpolarizing signals either spontaneously or in response to neurally-released purines [14]. Thus, smooth muscle cells are electrically coupled to ICC and PDGFR␣+ cells, forming an integrated unit that is modulated by intrinsic nerves [14]. However, despite the importance of this layer, there have been few studies on the myenteric microcirculation. Previous studies have primarily focused on the functional and morphological characteristics of the microcirculation in the submucosal layer, where another extensive neural plexus is located that plays a critical role in regulating mucosal blood supply [15–17]. To understand the intrinsic regulation of the microcirculation in the myenteric layer, we investigated aspects of the myenteric microvasculature of the guinea-pig gastric antrum by visualizing Ca2+ signalling in pericytes on precapillary arterioles and capillaries, and arteriolar SMCs. We observed highly synchronous spontaneous Ca2+ transients, which were generated in both pericytes on precapillary arterioles and/or capillaries and arteriolar SMCs along the length of the vessels. Therefore, we investigated: (1) the spatio-temporal profile of spontaneous Ca2+ transients in the myenteric microvasculature to identify the origin of spontaneous Ca2+ transients; and (2) mechanism underlying the generation and intercellular synchrony of spontaneous Ca2+ transients, particularly focussing on the involvement of VDCCs.

decapitation according to procedures approved by The Experimental Animal Committee of Nagoya City University Graduate School of Medical Sciences. The antral region of the stomach on either side of the greater curvature was isolated and immersed in oxygenated physiological saline solution (PSS). The mucosal layer and connective tissue were then removed leaving the underlying smooth muscles layers. 2.2. Intracellular calcium imaging For intracellular calcium imaging, the circular muscle layer was peeled away to expose the myenteric layer attached to the longitudinal muscle layer. Preparations, approximately 8 mm long in the circumferential direction and 5 mm wide, were prepared. Preparations were pinned out on a Sylgard plate (silicone elastomer, Dow Corning Corporation, Midland, MI, USA) at the bottom of the recording chamber (volume, approximately 1 ml), and were superfused with warmed (36 ◦ C) PSS at a constant flow rate (2 ml min−1 ) and equilibrated for 60 min. To visualize Ca2+ transients in the microvasculature, preparations were incubated in low Ca2+ PSS ([Ca2+ ]o = 0.1 mM) containing 0.1–1 ␮M fluo-8 AM (special packaging, Dojindo, Japan) and cremphor EL (0.01%, Sigma) for 10–20 min at 35 ◦ C. Since fluo-8 fluorescence in ICC diminished with time, preparations were loaded with 1 ␮M fluo-4 AM (special packaging, Dojindo, Japan) to specifically investigate ICC Ca2+ transients. Following incubation, the recording chamber was mounted on the stage of an upright epifluorescence microscope (BX51WI, Olympus, Tokyo, Japan) equipped with a back-thinned electron multiplying CCD camera (C9100-13, Hamamatsu Photonics, Hamamatsu, Japan). Preparations were superfused with dyefree PSS, viewed with a water immersion objective (UMPlanFL ×20 or LUMPlanFL ×40, ×60, Olympus) and illuminated at 495 nm. Fluorescence was captured through a barrier filter above 515 nm, and images were obtained every 47–151 ms (frame interval) with an exposure time of 30–70 ms using a micro-photoluminescence measurement system (AQUACOSMOS, Hamamatsu Photonics). Relative amplitudes of Ca2+ transients were expressed as Ft /F0 = (Ft − F0 )/F0 , where Ft is the fluorescence generated by an event, and baseline F0 is the basal fluorescence. 2.3. Intracellular recordings Whole wall antral smooth muscle preparations were prepared for intracellular recording. Preparations approximately 3 mm long and 2 mm wide containing a few GI smooth muscle bundles were pinned out on a Sylgard plate at the bottom of the recording chamber mounted on the stage of an inverted microscope. Preparations were superfused with warmed (36 ◦ C) PSS at a constant flow rate (2 ml min−1 ) and equilibrated for 90 min. GI smooth muscle cells were impaled with glass capillary microelectrodes, filled with 0.5 M KCl (tip resistance, 120–250 M). Membrane potential changes were recorded using a high input impedance amplifier (Axoclamp2B, Axon Instruments, Inc., Foster City, CA, USA) and displayed on a cathode-ray oscilloscope (SS-5702, Iwatsu, Tokyo, Japan). After low-pass filtering (cut off frequency, 1 kHz), membrane potential changes were digitized using a Digidata 1322 interface (Axon Instruments, Inc., Foster City, CA, USA) and stored on a personal computer for later analysis.

2. Methods

2.4. Immunohistochemistry

2.1. Tissue preparation

To expose the myenteric layer, the mucosa, submucosa and circular muscle layers of gastric antral preparations were removed using sharp tweezers and micro-scissors under a dissecting microscope. The residual longitudinal muscle with myenteric layer

Male guinea-pigs weighing 250–300 g, were anesthetized by exposure to sevoflurane vapours and exsanguinated by

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attached was pinned flat and immersed in fixative containing 2% formaldehyde and 15% saturated picric acid in 0.1 M phosphate buffer (pH 7.4) for 5 min. Preparations were then immersed in the same fixative for 4 h at 4 ◦ C. Fixed preparations were immersed in dimethyl sulfoxide to remove picric acid and then washed in PBS. Tissue preparations used for TMEM16A/Ano1 immunohistochemistry were fixed with acetone for 15 min at 4 ◦ C, and were incubated for 30 min with Alexa488-conjugated phalloidin (Molecular Probes) diluted in PBS containing 2% bovine serum albumin at 5 unit/ml. Fixed preparations were incubated with PBS containing 0.3% Triton X-100 for 10 min, incubated with Block Ace (AbD Serotec, USA) for 20 min and incubated with primary antibodies for 4 days at 4 ◦ C. Preparations were incubated with biotinylated anti-rabbit IgG antibody for 30 min and then with fluorescent probe-conjugated streptavidin and/or secondary antibody as well as the nuclear staining reagent Hoechst 33342 (10 ␮g/ml, Molecular Probes) for 2 h. Specimens were examined using a confocal laser scanning microscope (LSM 5 PASCAL, Zeiss). Antibodies used were mouse monoclonal anti-␣ smooth muscle actin (␣-SMA) antibody (1:200, clone 1A4, Sigma), rabbit anti-von Willebrand factor (vWF) antibody (1:400, Abcam), rabbit anti-PGP9.5 antibody (1:500, Ultraclone), rabbit antismall conductance Ca2+ -activated K+ channel 3 (SK3) antibody (1:200, Alomone Labs), biotinylated swine anti-rabbit IgG antibody (1:300, Dako), Alexa488-conjugated streptavidin (10 ␮g/ml, Molecular Probes), Cy3-conjugated streptavidin (4.5 ␮g/ml, Jackson ImmunoResearch) and Cy-3-conjugated goat anti-mouse IgG antibody (2.5 ␮g/ml, Millipore). Primary antibodies were diluted in PBS containing 2% bovine serum albumin (BSA) and 0.3% triton X-100, and other antibodies were diluted in PBS containing 2% BSA. 2.5. Solutions The composition of PSS was (in mM): Na+ , 137.5; K+ , 5.9; Ca2+ , 2.5; Mg2+ , 1.2; HCO3 − , 15.5; H2 PO4 − , 1.2; Cl− , 134 and glucose, 11.5. The pH of PSS was 7.2 when bubbled with 95% O2 and 5% CO2 , and the measured pH of the organ bath solution was approximately 7.4. Drugs used were caffeine, cyclopiazonic acid (CPA), nicardipine, nickel chloride, nifedipine, l-nitro-arginine (LNA), noradrenaline, tetracaine, tetrodotoxin, (from Sigma, St. Louis, MO, USA), mibefradil and ML218 (from Tocris Bioscience, Bristol, UK). Nickel chloride, LNA, noradrenaline and tetrodotoxin were dissolved in distilled water, CPA, nicardipine, tetracaine, mibefradil and ML218 were dissolved in dimethyl sulphoxide, and nifedipine was dissolved in absolute ethanol. Caffeine was directly dissolved in PSS immediately before use. The final concentration of the solvents above in the PSS did not exceed 1:1000. 2.6. Calculations and statistics The following parameters of spontaneous Ca2+ transients were measured: the peak amplitude (Ft /F0 ) measured as the value from the basal Ca2+ level to the peak of Ca2+ transients and the frequency (min−1 ) calculated as an average for over 3 min. For ICC Ca2+ transients, the following parameters were also measured: 1st dF/dt Max (Ft /F0 s−1 ), measured as the maximum slope of the initial rise phase; 2nd dF/dt Max (Ft /F0 s−1 ), measured as the maximum slope of the peak amplitude of the second rise phase. The following parameters of slow waves were measured: resting membrane potential (RMP, mV); peak amplitude (mV), measured as the value from the RMP to the peak of events; 1st dV/dt Max (mV s−1 ), measured as the maximum slope of the initial rise phase; 2nd dV/dt Max (mV s−1 ), measured as the maximum slope of the second rise phase; half-width (s), measured as the time between 50% peak amplitude on the rising and falling phases; frequency

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(min−1 ) which was calculated as an average for over 3–5 min of recordings. Measured values were expressed as mean ± standard deviation (n = number of preparations as well as animals (N), unless otherwise specified). Statistical significance was tested using paired t-test, and considered significant if p < 0.05. The synchrony of Ca2+ transients amongst pericytes was analyzed using the cross-correlation function of Clampfit 10 software (Axon Instruments, Molecular Devices, Union City, CA, USA). 3. Results 3.1. Spontaneous Ca2+ transients in the myenteric microvasculature Near synchronous spontaneous Ca2+ transients were observed in pericytes and arteriolar SMCs (henceforth grouped by the term mural cells) in the myenteric microvasculature of the guinea-pig stomach. Arteriolar Ca2+ transients were associated with slowly developing, weak vasoconstriction only in relatively larger arterioles with diameters of approximately 20–30 ␮m. Pericytes and SMCs in fine arterioles also exhibited near synchronous spontaneous Ca2+ transients, but changes in vascular diameter were not evident. The contractility of the myenteric arterioles was further investigated by examining the effects of LNA on vasoconstriction. In 7 control arterioles, in which the outer edges of the arteriolar wall were readily detectable, spontaneous Ca2+ transients resulted in the reduction of arteriolar diameter to 88.2 ± 5.2% of the resting diameter (i.e. quiescent period before the Ca2+ transients, n = 7). In 5 arterioles, which had been exposed to l-nitro arginine (100 ␮M), spontaneous Ca2+ transients were associated with larger reductions of arteriolar diameter to 80.8 ± 3.3% (n = 5, p < 0.05) of the resting values (Supplementary Video 1). Therefore, under the present experimental conditions, myenteric arterioles appear to be exposed to substantial release of endogenous nitric oxide, which suppresses their contractility. Supplementary Video 1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ceca.2015.06.012 3.2. Role of L-type VDCCs in generating Ca2+ transients of the microvasculature Fluorescence recordings from targeted cells or regions of interest (ROI) were used to investigate the spatio-temporal profile of spontaneous Ca2+ transients in the myenteric microvasculature. However, such measurements were made difficult under control conditions (i.e. PSS) due to spontaneous contractions of gastric longitudinal smooth muscle. Therefore, the effects of blockers of L-type VDCCs on the generation and synchrony of spontaneous Ca2+ transients were examined, as these are known to considerably reduce gastric contractions. L-type Ca2+ channel antagonists were generally ineffective in inhibiting the spontaneous Ca2+ transients with nifedipine (5 ␮M) inhibiting transients in 3 of 12 microvessels (n = 12) and nicardipine (1 ␮M) none out of 3 microvessels (n = 3). The inhibition was reversible, as in the 3 preparations that were inhibited by nifedipine, the spontaneous Ca2+ transients recovered upon return to PSS. These findings differ to previous reports of spontaneous Ca2+ transients in venules of the bladder or gastric submucosa [1,2,4] where blockade of L-type VDCCs disrupted near synchronous Ca2+ transients in the mural cell syncytium in all cases. In contrast, Ca2+ transients in gastric smooth muscle and associated contractions were greatly diminished in all 18 preparations. Therefore, all subsequent experiments were carried out in the

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presence of nifedipine (5 ␮M) to minimize movement artefacts due to contractions of the longitudinal smooth muscle. 3.3. Distinct types of cells in the myenteric layer generating spontaneous Ca2+ transients Besides spontaneous Ca2+ transients in the microvasculature, ICC-MY generated prominent spontaneous Ca2+ transients that were near synchronous within the cluster or network of ICC-MY. These cells were clearly distinguishable from the microvasulature (Fig. 1). ICC-MY Ca2+ transients were periodically generated with a frequency of 3.7 ± 0.52 min−1 , and had an amplitude of 1.0 ± 0.38 Ft /F0 and a half-width of 4.8 ± 1.3 s (n = 27). In 8 preparations, in which Ca2+ transients in ICC-MY (Fig. 1A–C) and microvessel mural cells (Fig. 1A, B and D) were simultaneously recorded in a field of view, no correlation was observed between the two cell groups (Fig. 1A–D; Supplementary Video 2). Supplementary Video 2 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ceca.2015.06.012 The third population of cells, presumably fibroblast-like cells (FLCs), which were distributed either along microvasculature or in the interstitial space, and were clearly distinguishable from pericytes or smooth muscle cells of the microvasculature, also generated spontaneous Ca2+ transients (Fig. 1E–G; Supplementary Video 3). FLCs had round or square shaped cell bodies of 13.5 ± 2.1 ␮m × 21 ± 2.5 ␮m, and generated asynchronous Ca2+ transients that had a frequency of 2.1 ± 0.41 min−1 , an amplitude of 1.3 ± 0.34 Ft /F0 and a half-width of 9.5 ± 2.9 s (n = 7). FLC Ca2+ transients occurred independently from ICC or Ca2+ transients in the microvasculature. Supplementary Video 3 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ceca.2015.06.012 3.4. Identification of various cell types in the myenteric layer Immunoreactivity for ␣-smooth muscle actin (␣-SMA) revealed the microvasculature amongst residual gastric smooth muscle bundles in the myenteric layer of the gastric antrum (Fig. 2A). The oval shaped cell bodies of ␣-SMA-positive mural cells were clearly visualized. Immunoreactivity for von Willebrand factor (vWF), an endothelial marker, discriminated ␣-SMA-positive microvasculature from gastric smooth muscle bundles (Fig. 2A and B). Double immunostaining with an antibody for TMEM16A/Ano1 Ca2+ -activated Cl− channels and phalloidin, a marker for actin filaments, revealed an extensive network of ICC-MY that were immnoreactive for TMEM16A/Ano1 but not phalloidin (Fig. 2C and D). In contrast, the microvasculature and gastric smooth muscle bundles were positive for phalloidin staining but not TMEM16A/Ano1 (Fig. 2C). The adjacent localization indicated that the microvasculature was distributed in the layer where ICC-MY were located. Fibroblast-like cells (FLCs) that were immunoreactive for small conductance Ca2+ -activated K+ channels 3 (SK3) were preferentially distributed around the microvasculature (Fig. 2E and F). Arterioles with a larger diameter that were covered by densely packed, circumferentially-arranged vascular SMCs (Fig. 2F) and residual gastric smooth muscle bundles were stained by ␣-SMA (Fig. 2F) but not SK3 (Fig. 2E and F). 3.5. Lack of neural modulation of microvessel Ca2+ transients In 5 preparations (N = 5) where mural cells of the exhibited spontaneous Ca2+ transients (Fig. 3A and C), electrical field stimulation (EFS, 10 Hz for 1 s, pulse width 100 ␮s) evoked Ca2+ transients in a network of nerve fibres but failed to trigger mural cell Ca2+ transients (Fig. 3A and B; Supplementary Video 4). In another

4 preparations (N = 4) where mural cells of the microvasculature exhibited spontaneous Ca2+ transients, TTX (1 ␮M) failed to block these transients (Fig. 3D). These findings indicate that the Ca2+ transients neither rely on neural activity nor voltage-gated Na+ channels. Supplementary Video 4 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ceca.2015.06.012 Consistent with the above findings, double immunostaining for ␣-SMA and PGP9.5, a neuronal marker, revealed the presence of arterioles close to the myenteric plexus in a single plane image taken by confocal laser microscopy (Fig. 3E). Notably, immunostaining with PGP9.5, a neuronal marker, while revealing nerves of the myenteric plexus indicated that there were no perivascular nerve fibres in the myenteric microvasculature (Fig. 3E). In contrast, perivascular nerve fibres with many varicosities were detected along the intramuscular microvasculature (Fig. 3F). 3.6. Spontaneous Ca2+ transients in arteriolar SMCs and pericytes In 70 preparations taken from 64 guinea-pigs, spontaneous Ca2+ transients in mural cells of the myenteric microvasculature were subdivided into those of that occurred in the SMCs of arterioles and those in pericytes. We were not successful in visualizing the entire microvascular network, presumably because sections of the network were embedded in gastric smooth muscle or covered by non-vascular elements in the myenteric layer. Therefore, unlike a published study on the microvasculature in the ureteric microvasculature [12], clear distinction between precapillary arterioles and capillaries was not possible in our study. As a consequence, we leave definitive description open referring to these as precapillary arterioles/capillaries. In 57 arterioles (N = 41), vessel walls were covered by densely packed, circumferentially arranged SMCs and had a diameter of 22.7 ± 5.9 ␮m (Fig. 4A and B). In contrast, 51 precapillary arterioles/capillaries (N = 39) had a diameter of 8.8 ± 1.6 ␮m and pericytes that had an oval shaped cell body of 6.8 ± 1.5 ␮m × 15.5 ± 3.0 ␮m formed a sparse line along the vessels (n = 42; Fig. 4D and E). SMCs in 57 arterioles generated spontaneous near synchronous Ca2+ transients at a frequency of 2.9 ± 1.2 min−1 , and had an amplitude of 1.0 ± 0.43 Ft /F0 and a half-width of 4.1 ± 3.3 s (Fig. 4A–C). Pericytes generated spontaneous near synchronous Ca2+ transients with a frequency of 3.1 ± 0.94 min−1 , and had an amplitude of 1.2 ± 0.49 Ft /F0 and a half-width of 3.9 ± 2.1 s (Fig. 4D–F; Supplementary Video 5). Supplementary Video 5 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ceca.2015.06.012 In 19 preparations (N = 19), where arterioles and precapillary arterioles/capillaries branching from the arterioles were visualized within a field of view, the SMCs on arterioles and pericytes on the precapillary arterioles/capillaries generated spontaneous Ca2+ transients. Careful examination revealed that 67 out of 139 Ca2+ transients generated in 23 precapillary arteriolar/capillary pericytes preceded arteriolar SMC Ca2+ transients (Fig. 5A–D; Supplementary Videos 1 & 6), while a time delay was not evident between pericyte-coated branches and arteriolar SMCs for 57 Ca2+ transients, indicating that both activities initiated at an interval less than the sampling rate of approximately 100 ms. The reverse where Ca2+ transients in SMCs preceded activity in pericytes was not observed. Fifteen Ca2+ transients in precapillary arteriolar/capillary pericytes terminated at the branch point with the arteriolar SMCs failing to generate Ca2+ transients (Fig. 5E–H; Supplementary Video 7). These observations indicate that spontaneous Ca2+ transients first generate in pericytes on precapillary arterioles or capillaries, which then entrain Ca2+ transients in the arteriolar SMCs.

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Fig. 1. Distinct types of cells generating spontaneous Ca2+ transients in myenteric layer of the guinea-pig stomach. In the myenteric layer of an antral preparation, ICC-MY (red and blue traces in A) generated spontaneous Ca2+ transients that were synchronous within a network of ICC (A and C). Arrowheads in magenta indicate cell bodies of ICC-MY in (C). Arteriolar SMCs (black and magenta traces in A) also generated spontaneous Ca2+ transients that were synchronous along the length of the vessel (A and D). During a quiescent period, neither ICC nor mural cells in the microvasculature exhibited Ca2+ fluorescence (B). The arrowhead in yellow indicates the arteriole (D). Note that arteriolar Ca2+ transients were generated independently from those of ICC-MY (A). In the myenteric layer of another antral preparation, fibroblast-like cells (FLCs) surrounding a larger arteriole developed spontaneous Ca2+ transients (E). FLC Ca2+ transients were generated in individual FLCs and were not synchronous amongst FLCs (F and G). Arrowheads in yellow indicate three FLCs and the arrowhead in magenda indicates FLC generating Ca2+ transients (F and G). Note that the larger arteriole remained quiescent (F and G). The scale bar in (G) applies to all the images. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Supplementary Videos 6 and 7 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ceca. 2015.06.012 3.7. Role of T-type VDCCs in generating synchronous microvessel Ca2+ transients The basis for generation of the synchronous Ca2+ transients was further examined by applying known blockers of T-type VDCCs (i.e. nickel chloride, mibefradil, ML218). Nickel chloride (10 ␮M) (n = 8; Fig. 6A), mibefradil (1 ␮M) (n = 6; Fig. 6B) and ML218 (1 ␮M) (n = 13; Fig. 6C) all prevented the generation of synchronous spontaneous Ca2+ transients. Since T-type VDCCs are low-voltage activated, the effects of increasing extracellular potassium concentrations ([K+ ]o )

and resultant depolarizations on Ca2+ transients in the microvasculature were also examined in 8 preparations. Increasing [K+ ]o from 5.9 mM to 10.6 mM prevented the generation of microvessel Ca2+ transients (n = 4; Fig. 6D) or reduced their amplitude (n = 4). Further increases in [K+ ]o to 15.3 mM (n = 2) or 20 mM (n = 2) abolished the residual Ca2+ transients.

3.8. Effects of T-type Ca2+ VDCCs on slow waves and ICC Ca2+ transients The specificity of the blockers was further verified by examining their effects on the known T-type VDCC dependent component of ICC Ca2+ transients or slow waves.

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Fig. 2. Identification of microvasculature in the myenteric layer of guinea pig gastric antrum. Residual gastric smooth muscle bundles and the microvasculature were visualized by immunohistochemistry using an antibody to ␣-smooth muscle actin (␣-SMA) (red in A). Immunoreactivity for von Willebrand factor (vWF), an endothelial marker was used to discriminate the ␣-SMA-positive microvasculature from gastric smooth muscle (green in A and B). The network of ICC-MY was demonstrated by its immunoreactivity for TMEM16A/Ano1 (red) but not phalloidin staining (green; C and D), while the microvasculature and gastric smooth muscle bundles were positive for phalloidin staining but not TMEM16A/Ano1 immunohistochemistry (C). An arteriole with a larger diameter was surrounded by cells immunopositive for small conductance Ca2+ -activated K+ channel 3 (SK3, green) (E and F). The arteriole was covered by circumferentially arranged vascular SMCs (red in F). The scale bar in (C) also applies to (D). The scale bar in (F) also applies to (A), (B) and (E). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Spontaneous Ca2+ transients recorded from ICC-MY consisted of two distinct components; the first rapid component that is likely to be caused by generation of the T-type VDCC-dependent rising phase of driving potentials, and the second slower component that

may correspond to the plateau phase of this potential (see [18,19]). Nickel chloride (100 ␮M) dramatically slowed the rising slope of the first component (n = 6; Fig. 7A). Mibefradil (10 ␮M; n = 7; Fig. 7B) or ML218 ([20]; 1 ␮M; n = 7; Fig. 7C) also slowed the rising slope of

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Fig. 3. Lack of neural modulation of spontaneous Ca2+ transients in the myenteric microvasculature. Electrical field stimulation (EFS, 10 Hz for 1 s, pulse width 100 ␮s) evoked Ca2+ transients in a myenteric network of nerve fibres (arrowheads in magenta) but failed to trigger Ca2+ transients in the myenteric microvasculature (A and B), these occurring spontaneously independent of EFS (A and C). Arrowheads in yellow indicate the arteriole and pericyte-coated branch in (C). The scale bar in (A) also applies to (B) and (C). Synchronous Ca2+ transients persisted in a preparation treated with TTX (1 ␮M) for 20 min (D). Application of the neuronal marker PGP9.5 demonstrated the myenteric plexus (green) and that there were no nerves on the adjacent ␣-SMA-positive arteriole (arrow, red, E). In contrast, perivascular varicose nerve fibres were detected along an intramuscular arteriole (arrow, green) within the muscle layer (red, F). Scale bar in (F) also applies to (E). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

the first component of ICC Ca2+ transients. The effects of the three T-type VDCC blockers on the parameters of ICC Ca2+ transients are summarized in Table 1. Slow waves consisted of the two distinct components; the initial component resulting from electrotonic spread of ICC-MY driving potentials, and the secondary component generated in

intramuscular ICC [13,21]. Nickel chloride (100 ␮M) greatly slowed the rising slope of the initial component of slow waves (n = 5; Fig. 7D). Mibefradil (10 ␮M; n = 4; Fig. 7E) or ML218 (1 ␮M; n = 6; Fig. 7F) also slowed the rising slope of the 1st components of slow waves. The effects of the blockers for T-type VDCC on the parameters of slow waves are summarized in Table 2.

Table 1 Effects of T-type Ca2+ channel blockers on ICC-MY Ca2+ transients. Blockers

Frequency (min−1 )

Control (n = 6) Ni (10 ␮M) Control (n = 7) Mibefradil (10 ␮M) Control (n = 7) ML218 (1 ␮M)

3.7 4.5 3.5 4.2 3.8 3.5

± ± ± ± ± ±

0.45 0.60* 0.55 0.82* 0.63 0.63

Amplitude (Ft /F0 ) 0.78 0.54 1.0 0.65 1.1 0.81

± ± ± ± ± ±

0.30 0.19* 0.42 0.36* 0.41 0.39*

Half-width (s) 4.8 3.5 5.0 3.6 4.5 3.7

± ± ± ± ± ±

0.92 1.14* 1.3 1.9* 1.7 1.4*

1st dF/dt Max (Ft /F0 s−1 ) 2.8 0.42 2.1 0.49 1.9 0.35

± ± ± ± ± ±

1.3 0.09* 1.3 0.22* 0.49 0.15*

2nd dF/dt Max (Ft /F0 s−1 ) 1.0 0.80 1.2 0.84 1.4 1.2

± ± ± ± ± ±

0.53 0.47* 0.43 0.52* 0.77 0.75*

Data shown are mean ± s.d. * Significantly different from control values (p < 0.05).

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Fig. 4. Spontaneous Ca2+ transients in arteriolar smooth muscle cells and pericytes. In the myenteric layer of an antral preparation, circumferentially-arranged arteriolar SMCs generated spontaneous Ca2+ transients that were synchronous along the length of the vessel (A–C). (C) Spontaneous Ca2+ transients (black, red and blue traces) recorded from arteriolar SMCs at locations indicated by yellow dotted circles in (A) and (B). In the myenteric layer of another antral preparation, pericytes in a precapillary arteriole and branching capillaries generated spontaneous Ca2+ transients that were synchronous along the branch of the vessel (D–F). (F) Spontaneous Ca2+ transients (black, red and blue traces) recorded from pericytes indicated by yellow dotted circles in (D) and (E). The scale bar in (E) applies to all the images. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 2 Effects of T-type Ca2+ channel blockers on slow waves. Blockers

RMP (mV)

Frequency (min−1 )

Control (n = 5) Ni (10 ␮M) Control (n = 4) Mibefradil (10 ␮M) Control (n = 6) ML218 (1 ␮M)

−62.7 −62.6 −63.7 −63.3 −63.3 −63.0

3.5 5.6 3.6 4.8 2.9 3.2

± ± ± ± ± ±

1.2 1.1 2.1 1.9 0.78 0.84

± ± ± ± ± ±

0.36 1.1* 0.96 0.76* 0.37 0.37

Amplitude (mV) 27.9 24.8 33.2 33.0 25.3 25.3

± ± ± ± ± ±

2.2 4.8 5.5 6.7 4.3 3.9

Half-width (s) 3.2 3.2 3.7 3.7 3.1 3.2

± ± ± ± ± ±

0.47 0.53 0.38 1.9 0.38 0.27

1st dF/dt Max (mV s−1 ) 29 3.1 25 16 23 12

± ± ± ± ± ±

9.5 2.1* 6.7 4.9* 8.7 6.8*

2nd dF/dt Max (mV s−1 ) 42 21 51 33 24 14

± ± ± ± ± ±

21 16* 24 12* 15 9.3*

Data shown are mean ± s.d. * Significantly different from control values (p < 0.05).

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Fig. 5. Temporal relationship between Ca2+ transients in pericytes and arteriolar smooth muscle cells. Sequential fluo-8 fluorescent images of a spontaneous Ca2+ transient activity taken every 58 ms demonstrated that Ca2+ transients were initiated in pericytes of precapillary arterioles/capillaries (yellow dotted circle), and then spread to arteriolar SMCs (sky blue dotted circle; A–C). (D) Pericyte Ca2+ transients (black) preceded arteriolar Ca2+ transients (red). Sequential fluo-8 fluorescent images of spontaneous Ca2+ transients taken every 100 ms demonstrated that Ca2+ transients initiated in the lower branch of the precapillary arteriole/capillary (yellow dotted circle) triggered Ca2+ transients in an arteriole (sky blue dotted circle) and upper branch (yellow dotted circles; E). A pericyte Ca2+ transient (red) that preceded Ca2+ transients in other regions (black and blue; G). In the same microvasculature, sequential fluo-8 fluorescent images of a spontaneous Ca2+ transient taken every 100 ms demonstrated that this Ca2+ transient initiated in the lower branch failed to spread to the arteriole (F). A pericyte Ca2+ transient (red) that was not followed by Ca2+ transients in other regions (black and blue; H). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.9. Role of intracellular Ca2+ stores in generating Ca2+ transients in the microvasculature A role of intracellular Ca2+ release from SR/ER Ca2+ stores in generation of microvessel mural cell Ca2+ transients was examined. Application of CPA (10 ␮M), an inhibitor of the SR/ER Ca2+ ATPase (SERCA), prevented the generation of Ca2+ transients in pericytes and SMCs, while causing a small rise in basal Ca2+ levels (n = 15; Fig. 8A). Caffeine (1 mM; n = 14; Fig. 8C), which is reported to inhibit IP3 receptors [22,23], or tetracaine (100 ␮M; n = 10; Fig. 8B), an inhibitor of ryanodine-receptors, abolished microvessel-associated Ca2+ transients without affecting basal Ca2+ levels. Switching from

PSS to nominally Ca2+ free solution reduced basal Ca2+ levels and prevented pericyte and SMC-associated Ca2+ transients (n = 9; Fig. 8D). Upon the re-addition of extracellular Ca2+ , basal Ca2+ levels returned to the original levels and spontaneous Ca2+ transients were restored. 3.10. Properties of asynchronous Ca2+ transients in pericytes In 4 preparations (N = 4), exposed to nifedipine (5 ␮M), synchronous Ca2+ transients were generated in pericyte cell networks (Fig. 9A–C), with the cross-correlation function for pericyte Ca2+ transients showing a prominent peak near time zero (Fig. 9D).

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Fig. 6. Effects of T-type VDCC blockers on spontaneous Ca2+ transients in the microvasculature. Application of NiCl2 (10 ␮M) prevented the generation of spontaneous Ca2+ transients in the myenteric microvasculature (A). In another preparation, mibefradil (10 ␮M) abolished spontaneous Ca2+ transient in the myenteric microvasculature (B). Scale bar in (B) also applies to (A). ML218 (1 ␮M) also blocked the generation of microvasculature spontaneous Ca2+ transients (C). In a different preparation, increasing [K+ ]o from 5.9 mM to 10.9 mM suppressed spontaneous Ca2+ transients in the microvasculature leaving small Ca2+ fluctuations (D). Scale bar in (D) also applies to (C).

Subsequent application of ML218 (1 ␮M) interrupted the synchronicity leaving asynchronous Ca2+ transients (n = 4, Fig. 9C and E). The lack of synchrony amongst pericyte Ca2+ transients was evident by the absence of a prominent peak near the time zero in their cross-correlation analysis (Fig. 9F). In all 10 preparations (N = 10) that were exposed to both nifedipine (5 ␮M) and ML218 (1 ␮M) applied together, asynchronous Ca2+ transients appeared in pericytes, while arteriolar SMCs remained quiescent (Fig. 9G–I, Supplementary Video 8). Asynchronous Ca2+ transients were readily abolished by CPA (10 ␮M; n = 5; Fig. 9J), which again caused a rise in basal Ca2+ levels, or by switching from normal PSS to nominally Ca2+ free solution, this associated with a reduction in basal Ca2+ levels (n = 7). Supplementary Video 8 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ceca.2015.06.012 4. Discussion The present study investigated the spatio-temporal profile of synchronized spontaneous Ca2+ transients in the microvasculature of the gastric myenteric layer. The generation of these Ca2+

transients depended on Ca2+ release from the SR/ER of pericytes. In contrast, their synchrony involved T-type VDCCs, presumably through activation of VDCCs facilitating intercellular entrainment of asynchronus Ca2+ transients across the syncytium of pericytes and/or smooth muscle cells. Spontaneous Ca2+ transients in the myenteric microvasculature often first initiated in precapillary arteriolar/capillary pericytes followed by generation of Ca2+ transients in SMCs of parent arterioles. 4.1. Spontaneous Ca2+ transients in myenteric microvasculature mural cells arise independent of interstitial cells and nerves In the myenteric layer of the gastrointestinal tract, an extensive network of ICC-MY generate spontaneous Ca2+ transients [24,25] and associated pacemaker potentials that subserve a role in generation of electrical slow waves in the musculature [13]. Comparison of spontaneous Ca2+ transients in ICC-MY to microvessel-associated Ca2+ transients indicated there was no temporal correlation. Thus Ca2+ transients in pericytes or arteriolar SMCs occur independently from ICC-MY activity. Furthermore, pericytes or SMCs of the myenteric microvasculature, or perivascular interstitial cells were not

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Fig. 7. Effects of T-type VDCC blockers on ICC Ca2+ transients and slow waves. ICC-MY within an ICC network generated spontaneous Ca2+ transients consisting of two distinct components under control conditions (A–C). NiCl2 (100 ␮M; A), mibefradil (10 ␮M; B) and ML218 (1 ␮M; C) all slowed the rising phase of the initial component and reduced the ICC-MY Ca2+ transients. Slow waves recorded from circular smooth muscle had two distinct components (D–F). NiCl2 (100 ␮M; D), mibefradil (10 ␮M; E) and ML218 (1 ␮M; F) all slowed the rising phase of the initial component. Different preparations were used for A–F. Traces in D–F represent averaged traces of 10–20 slow waves.

immunoreactive for antibodies against Kit receptor, a marker for ICC. Therefore, neither ICC nor Kit-positive interstitial cells act as pacemaker cells for spontaneous Ca2+ transients in the myenteric microvasculature. This is consistent with the finding that Kit-positive cells were not detected in mouse portal vein [26] or suburothelial venules in the rat [1] and mouse [2] bladder, tissues that develop spontaneous activity. Moreover, no functional disturbance was reported in portal vein of W/Wv mutant mice that display regional and subtype specific deficiency of ICC and associated dysfunctions in the GI tract [26]. Spontaneous Ca2+ transients in the myenteric microvasculature were not affected by TTX or electrical field stimulation (EFS). PGP9.5-positive perivascular nerve fibres were not found along the myenteric microvasculature, whereas perivascular nerve fibres with many varicosities were identified adjacent to the intramuscular microvasculature. This is in contrast to the microvasculature in the submucosal layer of the GI tract where both arterioles and venules are functionally innervated by sympathetic nerves [4,5,15]. Notably, 3-D imaging with a technique termed “vessel painting” has revealed that the periganglionic capillary network is in close contact with ganglionic cells as well as glial cells in the myenteric layer of mouse small intestine [27]. Therefore, the myenteric microvasculature is likely to provide the primary microcirculation for the myenteric nerve plexus. The fact that it is not neurally controlled suggests that local metabolic products or paracrine substances released from endothelium or perivascular cells are key to local control of this microcirculatory system. 4.2. Generation of synchronous spontaneous Ca2+ transients in myenteric microvasculature mural cells most often involves T-type but not L-type Ca2+ channels Blockade of L-type VDCCs did not disrupt the generation or synchrony of Ca2+ transients in precapillary arteriolar/capillary pericytes or arteriolar SMCs in 15 out of 18 myenteric preparations. In addition, the myenteric microvasculature was capable of

generating near synchronous Ca2+ transients in the 70 preparations studied that had been pre-treated with nifedipine. This was in marked contrast to spontaneous Ca2+ transients in mural cells (i.e. pericytes or SMCs) of venules in the rat bladder, mouse bladder and the submucosa of the rat stomach, where L-type VDCCs play a critical role in their synchrony [1,2,4]. In freshly isolated retinal pericytes of the rat, inward currents arising from the opening of L-type VDCCs were demonstrated [28]. However, it was reported that L-type VDCCs have a minimal role in establishing the basal Ca2+ concentration of capillary pericytes, while L-type VDCCs in mural cells in the proximal retinal microvasculature exhibit significant basal activity [29]. Thus, in terms of the involvement of L-type VDCCs, the properties of Ca2+ transients in the microvasculature seem to vary amongst vascular beds or may exhibit regional differences even within a microvascular network (see also [30]). Importantly, we found that the near synchronous mural cell Ca2+ transients in the myenteric microvasculature were abolished by blockers of T-type VDCCs used at concentrations comparable to those used to completely block Cav 3.1 T-type VDCCs [20,31]. The blockers for T-type VDCCs also effectively suppressed the known T-type VDCC dependent component of slow waves or ICC-MY Ca2+ transients in the same preparations [18,19,32,33], suggesting that these blockers may selectively inhibit T-type VDCCs in the present experimental conditions. T-type VDCCs are low threshold channels with channels opening even at membrane potentials as negative as −70 mV [34]. A role for T-type VDCCs suggests that the membrane potential of myenteric microvasculature mural cells is relatively hyperpolarized. This is consistent with the finding that mildly elevated [K+ ]o prevented the generation of synchronous Ca2+ transients in these vessels. T-type Ca2+ channels are functionally expressed in arterial and arteriolar SMCs and contribute to vascular tone in several vascular beds [35–38]. In cerebral arteries, nifedipine-resistant, mibefradil-sensitive vascular tone was increased with decreasing vessel size, suggesting that T-type VDCCs play a larger role in smaller arteries [37]. A study using freshly isolated vascular SMCs

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Fig. 8. Role of intracellular Ca2+ stores and extracellular Ca2+ release in generating spontaneous Ca2+ transients in the microvasculature. CPA (10 ␮M; A), tetracaine (100 ␮M; B) and caffeine (1 mM; C) abolished Ca2+ transients in the myenteric microvasculature. Scale bar in (C) also applies to (B). Nominally Ca2+ free PSS had the same action, which was reversed upon return to control PSS (D). CPA and nominally Ca2+ -free solution had the additional actions of slightly increasing (A) and reducing (D) basal Ca2+ levels respectively. Different preparations were used for A–D.

demonstrated that the fraction of T-type VDCC currents increase dramatically along the lower branches of the mesenteric artery, rising to almost 100% in submucosal arterioles [39]. In the present study we found that spontaneous Ca2+ transients in mural cells in myenteric microvessels with a diameter of less than 30 ␮m predominately rely on T-type VDCCs with only a minority of vessels (3 of 18) dependent on L-type VDCCs. It should be noted that L-type VDCC inhibition has a larger effect on myogenic tone in cerebral arteries at high pressure where the arterial smooth muscle is depolarized, while T-type VDCC blockade exerts a greater effect at low pressure where the smooth muscle is at more negative potentials [38]. Since we studied the microvasculature without transmural pressure or blood flow, relative contributions of L-type and T-type VDCCs to spontaneous Ca2+ transients may be different in more physiological conditions. Kotecha & Hill [40] demonstrated that increasing the intraluminal pressure in skeletal muscle arterioles induces progressive membrane depolarization. In their study, the membrane potential was depolarized from −55 mV at zero pressure to −50 mV at 30 mmHg that is equivalent to precapillary arteriolar pressure. Therefore, it is reasonable to assume that substantial T-type VDCC currents are

available even in pressurized arterioles. Furthermore, conducted vasoconstriction in rat mesenteric arterioles (<40 ␮m) in vivo was shown to exclusively rely on T-type VDCCs [41]. 4.3. Generation of spontaneous Ca2+ transients in myenteric microvasculature mural cells depends on SR/ER Ca2+ stores While the blockade of T-type VDCCs prevented the generation of near synchronous Ca2+ transients in the myenteric microvasculature, Ca2+ transients were also readily abolished by CPA, caffeine or tetracaine, suggesting a primary role of SR/ER Ca2+ release. Therefore a unifying hypothesis that could underlie pericyte pacemaking and synchronization is that it arises through the cycling of Ca2+ stores. This has been proposed in lymphatics [42,43], arteries and arterioles [44,45] and in the distal gastric antrum [46]. The mechanism requires intercellular electrical connectivity, which pericytes can readily achieve through direct coupling by gap junctions to each other and/or to the endothelium [47], the latter acting as a low resistance path for electrical transmission [48]. The mechanism also requires that membrane depolarization be coupled to Ca2+ store release [42,43]. In the case of myenteric pericytes or arteriolar SMCs

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Fig. 9. Properties of asynchronous Ca2+ transients in pericytes. In a myenteric precapillary arteriole/capillary, pericytes generated spontaneous Ca2+ transients (A and B). A cross-correlation function for three pericytes generating near synchronous spontaneous Ca2+ transients (yellow dotted circles in C) showed a prominent peak at time zero (D). ML218 (1 ␮M) prevented the generation of synchronous Ca2+ transients leaving asynchronous Ca2+ transients (E). A cross-correlation function applied for the same cells in ML218 did not show a prominent peak at time zero (F). In another pericyte-coated microvessel, which had been treated with nifedipine (5 ␮M) and ML218 (1 ␮M), pericytes generated asynchronous Ca2+ transients (arrows in H and I). During a quiescent period, no pericyte exhibited Ca2+ transients (G). CPA (10 ␮M) increased basal Ca2+ levels and abolished the asynchrnous Ca2+ transients (J). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

this can be achieved by T-type VDCCs that when activated depolarize the membrane and let in Ca2+ causing activation of store Ca2+ release through Ca2+ -induced Ca2+ release (CICR). Another key linking factor is that the store Ca2+ release can cause depolarization by the opening of Ca2+ -activated Cl− or cation channels. Indeed, pericytes in the descending vasa recta and retina have Ca2+ -activated Cl− currents associated with cyclic oscillations in cytosolic Ca2+ concentrations [11,28]. ICC in the GI tract are known to express TMEM16A/Ano1 Ca2+ -activated Cl− channels [49,50]. In the present study, TMEM16A/Ano1 immunofluorescence was detected in ICC-MY but not in the myenteric microvasculature of the guinea-pig gastric antrum. In mouse small intestine, pericytes associated with the microvasculature near the myenteric plexus

were shown to express cation permeable, maxi-anion channels, but were not immunoreactive for Ano1 [51]. SR-ER Ca2+ stores have been shown to function through InsP3 receptor-operated Ca2+ release in ICC of the GI tract [52,53], lymphatic SMCs [43,54] and suburothelial venular pericytes [1,2]. In comparison, some blood vessels and interstitial cells of the urethra have Ca2+ stores that function through both ryanodine- and InsP3 receptors [45,55]. In the myenteric microvasculature, caffeine and tetracaine blocked spontaneous Ca2+ transients in pericytes, suggesting that both InsP3 - and ryanodine-receptors may be involved in the generation of these Ca2+ transients [22,23]. SR-ER store Ca2+ release may be the primary event in generating spontaneous Ca2+ transients in pericytes, given CPA-induced inhibition of SERCA

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abolished asynchronous Ca2+ transients recorded from pericytes when L- and T-type VDCCs were blocked. Extracellular Ca2+ influx via a pathway other than L- and T-type VDCCS is also required to maintain the cytosolic Ca2+ oscillator in pericytes, as both synchronous as well as asynchronous Ca2+ transients were abolished by nominally Ca2+ free solution. 4.4. Myenteric pericytes pace arteriolar Ca2+ transients and resultant arteriolar vasomotion An interesting finding of this study was that spontaneous Ca2+ transients often first initiated in precapillary arteriolar/capillary pericytes followed by generation of Ca2+ transients in SMCs of parent arterioles. We found that 49% of Ca2+ transients first generated in precapillary arteriolar/capillary pericytes and spread to the SMCs of arterioles with a time delay of approximately 100 ms or greater whereas in 41% the activity developed in time periods less than 100 ms and, due to the sampling rate of our system, temporal distinction between pericyte and SMC activation was not possible. In the remaining 10%, pericyte Ca2+ transients sometimes failed to spread to arterioles. Active pericyte-coated microvessels that initiated spontaneous Ca2+ transients did not generally maintain their dominance switching from one to another with time. Therefore, it is also possible that some Ca2+ transients that occurred apparently simultaneously in SMC-coated arterioles and pericyte-coated branches may have been initiated by pericytes located outside the field of view. The finding that SMC Ca2+ transients never preceded pericyte Ca2+ transients led us to the thesis that pericytes were the dominant pacemaker. Interestingly, the cerebral microvasculature imaged in vivo showed that capillary dilatation in response to the excitation of vasodilatory neurons preceded arteriolar dilatation, suggesting that hyperpolarizing signals initiated in capillaries may spread to arterioles [9]. Studies on the retinal microvasculature indicate that the axial electrotonic voltage transmission is highly efficient, particularly in capillaries, but significant voltage dissipation occurs at branch points [56]. Therefore, it is reasonable to assume that pericytes may act as pacemaker cells generating spontaneous Ca2+ transients and associated depolarizing signals that entrain arteriolar SMCs to develop synchronous Ca2+ transients in mural cells of the microvasculature. The interpretation that pericytes act as the pacemaker relates also to parallels from our studies of lymphatic and gastric pacemaking [42,43,46], where pacemaking occurs through coupled oscillator-based entrainment of the Ca2+ store release-refill cycle (i.e. the Ca2+ clock) across cells. This has also been proposed for arterial/arteriolar vasomotion [44,45]. 4.5. Role of myenteric pericytes Synchronous spontaneous Ca2+ transients in the myenteric microvasculature caused no measurable constriction in ␣-actin positive capillary or precapillary arterioles. While most of our experiments were carried out in preparations that had been treated with blockers for L-type VDCCs, modest constrictions were observed in larger arterioles surrounded by circumferentially oriented SMCs even in the absence of L-type VDCC blockade. Since the LNA-treated myenteric arterioles exhibited more vigorous constrictions associated with spontaneous Ca2+ transients, it appears that an excessive release of endogenous nitric oxide from the endothelium, myenteric nerves or interstitial cells suppress arteriolar contractility, presumably by reducing Ca2+ sensitivity of contractile proteins. Although we do not yet know the mechanisms underlying the up-regulation of NO production, the lack of luminal pressure as well as flow and/or non-physiological high oxygen pressure (see below) may play a role. Synchronized spontaneous Ca2+ transients in the myenteric microvasculature may play a fundamental role in regulating

vascular resistance in upstream SMC-coated arterioles where spontaneous vasomotion developed. Despite the lack of a detectable reduction in vascular diameter in pericyte-coated microvessels, spontaneous Ca2+ transients in pericytes may contribute to vascular permeability by changing pericyte and/or endothelial morphology, the latter possibly through increasing Ca2+ levels in the endothelium via gap junctions between these two cell types [47]. Bicarbonate buffered PSS gassed with a mixture of 95% oxygen and 5% carbon dioxide is most commonly used for in vitro experiments at least for smooth muscle research. Nevertheless, since oxygen pressure in the microcirculation is in the range 20–40 mm Hg [57], present experimental conditions are non-physiological. Another shortcoming of our experiments was an inability to perfuse these small vessels. These issues may change the frequency and magnitude of the contractions but are unlikely to change the underlying mechanisms, especially given that vasomotion (i.e. near synchronous rhythmic vessel constrictions) is also commonly observed in vivo [58–60]. Interestingly Bouskela et al. [60] investigated the effects of PO2 finding that increasing superfusate PO2 caused inhibition of vasomotion but the constrictions were reestablished by blocking potassium channels, suggesting that the underlying activity (i.e. rhythmic Ca2+ transients) persisted in high PO2 conditions but could no longer cause vasomotion. In conclusion we present evidence that synchronous spontaneous Ca2+ transients in pericytes on myenteric precapillary/capillary pericytes may pace Ca2+ transients in arteriolar SMCs and resultant vasomotion in these parent arterioles. The Ca2+ transients in these myenteric pericytes and smooth muscle cells arise through Ca2+ release from SR/ER Ca2+ stores with entrainment and hence synchronization predominantly mediated by T-type Ca2+ channels. Conflict of interest No competing interests. Authors’ contribution H.H. and D.V.H. involved in conception and design of the experiments. H.H., R.M., S.M. and D.V.H. involved in collection, analysis and interpretation of data. H.H., R.M. and D.V.H. involved in drafting the article or revising it critically for important intellectual content. Acknowledgements This study was supported by Grant-in-Aid for Scientific Research (B) (No. 22390304) and Grant-in-Aid for Challenging Exploratory Research (No. 21659377) from The Japan Society for Promotion of the Science (JSPS) to H.H. References [1] H. Hashitani, H. Takano, K. Fujita, R. Mitsui, H. Suzuki, Functional properties of suburothelial microvessels in the rat bladder, J. Urol. 185 (2011) 2382–2391. [2] H. Hashitani, R. Mitsui, Y. Shimizu, R. Higashi, K. Nakamura, Functional and morphological properties of pericytes in suburothelial venules of the mouse bladder, Br. J. Pharmacol. 167 (2012) 1723–1736. [3] Y. Shimizu, S. Mochizuki, R. Mitsui, H. Hashitani, Neurohumoral regulation of spontaneous constrictions in suburothelial venules of the rat urinary bladder, Vascul. Pharmacol. 60 (2014) 84–94. [4] R. Mitsui, H. Hashitani, Functional properties of submucosal venules in the rat stomach, Pflugers Arch. 467 (2015) 1327–1342. [5] R. Mitsui, S. Miyamoto, H. Takano, H. Hashitani, Properties of submucosal venules in the rat distal colon, Br. J. Pharmacol. 170 (2013) 968–977. [6] R. Mitsui, H. Hashitani, Immunohistochemical characteristics of suburothelial microvasculature in the mouse bladder, Histochem. Cell Biol. 140 (2013) 189–200. [7] C.M. Peppiatt, C. Howarth, P. Mobbs, D. Attwell, Bidirectional control of CNS capillary diameter by pericytes, Nature 443 (2006) 700–704.

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Please cite this article in press as: H. Hashitani, et al., Pacemaker role of pericytes in generating synchronized spontaneous Ca2+ transients in the myenteric microvasculature of the guinea-pig gastric antrum, Cell Calcium (2015), http://dx.doi.org/10.1016/j.ceca.2015.06.012