Ontogeny of Local Sarcoplasmic Reticulum Ca2+ Signals in Cerebral Arteries : Ca2+ Sparks as Elementary Physiological Events

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Ontogeny of Local Sarcoplasmic Reticulum Ca2+ Signals in Cerebral Arteries : Ca2+ Sparks as Elementary Physiological Events Maik Gollasch, George C. Wellman, Harm J. Knot, Jonathan H. Jaggar, Deborah H. Damon, Adrian D. Bonev and Mark T. Nelson Circ Res. 1998;83:1104-1114 doi: 10.1161/01.RES.83.11.1104 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1998 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571

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Ontogeny of Local Sarcoplasmic Reticulum Ca21 Signals in Cerebral Arteries Ca21 Sparks as Elementary Physiological Events Maik Gollasch, George C. Wellman, Harm J. Knot, Jonathan H. Jaggar, Deborah H. Damon, Adrian D. Bonev, Mark T. Nelson Abstract—Ca21 release through ryanodine receptors (RyRs) in the sarcoplasmic reticulum is a key element of excitation-contraction coupling in muscle. In arterial smooth muscle, Ca21 release through RyRs activates Ca21-sensitive K1 (KCa) channels to oppose vasoconstriction. Local Ca21 transients (“Ca21 sparks”), apparently caused by opening of clustered RyRs, have been observed in smooth and striated muscle. We explored the fundamental issue of whether RyRs generate Ca21 sparks to regulate arterial smooth muscle tone by examining the function of RyRs during ontogeny of arteries in the brain. In the present study, Ca21 sparks were measured using the fluorescent Ca21 indicator fluo-3 combined with laser scanning confocal microscopy. Diameter and arterial wall [Ca21] measurements obtained from isolated pressurized arteries were also used in this study to provide functional insights. Neonatal arteries (,1 day postnatal), although still proliferative, have the molecular components for excitation-contraction coupling, including functional voltage-dependent Ca21 channels, RyRs, and KCa channels and also constrict to elevations in intravascular pressure. Despite having functional RyRs, Ca21 spark frequency in intact neonatal arteries was '1/100 of adult arteries. In marked contrast to adult arteries, neonatal arteries did not respond to inhibitors of RyRs and KCa channels. These results support the hypothesis that RyRs organize during postnatal development to cause Ca21 sparks, and RyRs must generate Ca21 sparks to regulate the function of the intact tissue. (Circ Res. 1998;83:1104-1114.) Key Words: Ca21 spark n ryanodine receptor n K1 channel n vascular smooth muscle n development ntracellular Ca21 ions regulate a wide variety of cellular processes. It is a widely held belief that many of the actions of Ca21 are not global in nature; rather, they occur in close proximity to Ca21 release sites.1 Local Ca21 transients (“Ca21 sparks”) are thought to be elementary Ca21 signals in heart, skeletal and smooth muscle cells, and possibly neurons.2– 6 Ca21 sparks arise from the activation of ryanodine-sensitive Ca21 release channels (ryanodine receptors [RyRs]) located in the membrane of the sarcoplasmic reticulum (SR). The idea that Ca21 sparks are “the” elementary Ca21 signal of RyRs has been challenged.7–9 Lipp and Niggli9 found that flash photolysis of caged Ca21 caused a homogenous release of Ca21 from cardiac SR, without the detection of Ca21 sparks. This observation suggests that SR Ca21 release can occur via undetectable events, possibly representing the opening of single RyRs. Ca21 sparks therefore may represent the coordinated opening of a cluster of RyRs7 rather than the opening of a single RyR channel. Further, functional RyR1s can be expressed in cultured cells; however, unlike native tissue, these cells do not exhibit Ca21 sparks.10 This finding implies not only that the opening of multiple RyRs is responsible for Ca21 sparks but that additional cellular factors are necessary to organize RyRs into functional Ca21 spark sites.

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This suggestion that Ca21 release events are smaller (eg, through a single RyR) than Ca21 sparks leads to a fundamental question: Do RyRs generate Ca21 sparks to regulate the physiology of the tissue? In arterial smooth muscle, Ca21 release through RyRs causes the activation of Ca21-sensitive K1 (KCa) channels in the sarcolemmal membrane, which appear to be involved in signaling vasodilation.5,11 For example, elevation of intravascular pressure to physiological levels (eg, 60 mm Hg) causes a graded smooth muscle cell membrane potential depolarization to '240 mV, elevation in arterial wall Ca21 to '200 nmol/L, and constriction (“myogenic tone”) of small cerebral arteries.12 Ca21 release through RyRs increases in response to this elevation in Ca21 entry, which in turn activates nearby KCa channels in the sarcolemmal membrane to cause membrane potential hyperpolarization to oppose the pressure-induced depolarization. Recent evidence also suggests that frequency modulation and amplitude modulation of Ca21 sparks and IP3-mediated Ca21 release events play a fundamental role in controlling cell function.13–16 However, it is unclear whether Ca21 sparks are necessary for RyRs to have a significant effect on arterial diameter. In this study, we explored the nature and fundamental functional roles of RyRs by examining their elementary

Received June 9, 1998; accepted September 10, 1998. From the Department of Pharmacology, University of Vermont, Burlington. Correspondence to Mark T. Nelson, Department of Pharmacology, University of Vermont, Burlington, VT 05405. E-mail [email protected] © 1998 American Heart Association, Inc. Circulation Research is available at http://www.circresaha.org

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Gollasch et al properties in smooth muscle cells of neonatal and adult cerebral arteries of rat. To gain insights into these issues, we examined the properties of RyRs from the elementary level of Ca21 sparks to their functional effects in intact pressurized arteries. We found that the elementary behavior of RyRs changes during postnatal development of cerebral arteries. Smooth muscle cells in neonatal cerebral arteries, although incompletely differentiated, express functional RyRs, KCa channels, contractile proteins, and voltage-dependent Ca21 channels. Neonatal arteries constrict to increases in transmural pressure, like adult arteries. However, our results indicate that Ca21 sparks develop late in differentiation and that Ca21 sparks are required for RyRs to activate KCa channels and serve as a “brake” on vasoconstriction.

Materials and Methods Adult male and female Sprague-Dawley rats (12 to 14 weeks; '228 g) were euthanized under deep pentobarbital anesthesia (intraperitoneal; 150 mg/kg body weight). Neonatal male and female SpragueDawley rats (1 to 2 days old) were euthanized under deep inhalation anesthesia with methoxyflurane. After decapitation, the brain was removed and quickly transferred to cold (4°C), oxygenated (95% O2; 5% CO2) PSS of the following composition (in mmol/L): NaCl 119, KCl 4.7, NaHCO3 24, KH2PO4 1.2, CaCl2 1.6, MgSO4 1.2, EDTA 0.023, and glucose 11. All experiments were conducted in accordance with the guidelines for the care and use of laboratory animals (NIH publication No. 85-23, 1985) and followed protocols approved by the Institutional Animal Use and Care Committee of the University of Vermont. Animals were supplied by Charles River Laboratories, Inc, St. Constant, Quebec, Canada.

Staining of Arteries for Proliferating Cell Nuclear Antigen Isolated cerebral arteries were fixed in freshly depolymerized paraformaldehyde (2%) in 0.1 mol/L PBS for 2 minutes at room temperature (20°C to 22°C) and then were washed twice in PBS for 5 minutes. Immunoreactivity of proliferating cell nuclear antigen (PCNA) was determined using an anti-PCNA antibody (Clone PC10, DAKO, Carpinteria, Calif) that was coupled to horseradish peroxidase.17 For immunodetection of PCNA, arteries were treated with 3% H2O2 for 5 minutes and then incubated with the antibody to PCNA for 60 minutes at room temperature.

Immunofluorescence Staining Smooth muscle cells from cerebral (basilar) arteries of adult and neonatal rats were isolated as previously described.18,19 Freshly isolated cells seeded onto Cell-tak– coated (Collaborative Biomedical Products) coverslips were fixed in 0.1 mol/L PBS containing 2% or 4% paraformaldehyde for 2 minutes at room temperature (20°C to 22°C). A monoclonal mouse anti-ryanodine receptor (RyR2) antibody20,21 (Clone C3-33, Affinity Bioreagents, Golden, Colo) and a polyclonal rabbit anti-alpha 1C antibody22 were used as primary antibodies to label RyR2 and Ca21 channel alpha 1C subunit, respectively. Cells were incubated overnight with either a 1:100 dilution (in labeling buffer) of stock (1 mg/mL) anti-RyR2 or with a 1:10 dilution of stock (45 mg/mL) anti-alpha 1C antibody solution at 4°C. Cells then were rinsed 4 times with labeling buffer and incubated with a monoclonal secondary antibody (1:200 dilution of 0.6 to 0.7 mg/mL stock solutions) containing either FITC-conjugated goat anti-mouse IgG, FITC-conjugated donkey anti-mouse IgG, FITC-conjugated goat anti-rabbit IgG, or CY5-conjugated goat anti-rabbit IgG (Jackson Laboratories, West Grove, Pa) for 2 hours at room temperature. Cover slips mounted onto slides were viewed with a laser scanning confocal microscope (Biorad MRC 1000). Excitation light of 488 nm for FITC and 647 nm for Cy5-conjugated secondary antibodies was used and emissions measured at 515 to 565 nm (FITC) and 670 to 810 nm (Cy5). Negative control experiments

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were performed with labeling buffer instead of the primary antibodies; in each case, no specific staining was observed (data not shown).

Ca21 Spark Measurements Freshly isolated adult and neonatal cells were incubated with the Ca21 indicator fluo-3-AM (5 mmol/L) and pluronic acid (0.005%; wt/vol) for 30 minutes at room temperature in Ca21-free Hanks’ solution.5 To examine Ca21 sparks in intact artery segments, basilar arteries (diameter, '100 mm) from neonatal animals were slipped over square glass cannulas (75 mm375 mm310 mm), and secondary branches of posterior cerebral arteries (diameter, 100 to 150 mm) from adult rats were slipped over rectangular glass cannulas (220 mm340 mm310 mm) in a manner similar to that previously described.23,24 Arteries on glass cannulas then were placed into HEPES-PSS containing 10 mmol/L Fluo-3-AM and 0.05% pluronic acid and incubated at 22°C for 60 minutes. After loading with fluo-3-AM, tissues were washed with HEPES-PSS for 30 to 40 minutes at 22°C. The HEPES-PSS had the following composition (in mmol/L): NaCl 135, KCl 5.4, CaCl2 1.8, MgCl2 1, HEPES 10, and glucose 10 (pH 7.4 with NaOH). Smooth muscle cells were imaged using a Noran Oz laser scanning confocal microscope through a No. 1 coverslip using a 603 water immersion lens (numerical aperture51.2; Nikon) attached to a Nikon Diaphot microscope. Images were obtained by illuminating with a krypton/argon laser at 488 nm and recording all emitted light .500 nm. The sampling rate was 60 Hz (1 image every 16.7 ms). Unless indicated, Ca21 sparks were measured in HEPES-PSS at room temperature (20°C to 22°C). For single-cell analysis, fluorescence records were normalized by dividing each image by the average of 8 images obtained during the prestimulus period. Normalized images were filtered subsequently with a 333 median filter using Noran software. Ca21 sparks recorded from 56.3352.8 mm2 areas of intact artery segments were analyzed using custom software written by Dr Adrian Bonev in our laboratory (using IDL 5.0.2; Research Systems, Inc). Baseline fluorescence (Fo) was determined by averaging 10 images without Ca21 spark activity. Fractional fluorescence increases (F/Fo) were determined by dividing an area (1.5431.54 mm2), where a Ca21 spark was present, by Fo. Ca21 sparks were defined as local F/Fo .1.3.

K1 Current Recordings Whole-cell K1 currents in freshly isolated cerebral artery myocytes from neonatal rats were measured using the perforated patch configuration25 of the patch-clamp technique26 at room temperature (20°C to 24°C). The external solution contained (in mmol/L): NaCl 134, KCl 6, MgCl2 1, CaCl2 2, glucose 10, and HEPES 10 (pH 7.4). Patch pipettes (resistance, 3 to 5 MV) were filled with a solution containing (in mmol/L): KAsp 110, KCl 30, NaCl 10, MgCl2 1, HEPES 10, and EGTA 0.05 (pH 7.2). Amphotericin B (Sigma) was dissolved in DMSO and diluted into the pipette solution to give a final concentration of 200 mg/mL. KCa channel activity (NPo: N indicates number of functional channels; Po, open probability) was calculated over 2- to 5-minute intervals as

SO N

tj zj

j51

DY

T,

where tj is the time spent with j51,2,. . . N channels open, N is the maximum number of channels observed, and T is the duration of the recording.

Arterial Wall [Ca21] and Diameter Arterial wall [Ca21] and diameter were measured as previously described.12 Intact isolated distal posterior cerebral arteries of adult rats and intact isolated basilar arteries of neonatal rats were loaded with the ratiometric Ca21-sensitive fluorescent dye Fura-2/AM (2 mmol/L) at room temperature (20°C to 22°C) for 45 minutes. Fura-2–loaded arteries then were mounted in an arteriograph with continuous superfusion (3 to 6 mL/min) of oxygenated PSS at 37°C. After a 20-minute equilibration period, intravascular pressure was

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increased gradually from 2 mm Hg to either 40 mm Hg (neonatal arteries) or 60 mm Hg (adult arteries). Ratio images were obtained at a rate of 0.2 Hz from background-corrected 4-frame–averaged images of the 510640-nm emission from the arteries alternately excited at 340 and 380 nm using the Image-1/FL quantitative ratio imaging software (Universal Imaging Corp). Arterial wall [Ca21] was calculated using the following equation (from Grynkiewicz et al26a): [Ca21]5Kd3b3(R2Rmin)/(Rmax2R). At the end of every experiment, Rmin and Rmax were measured from ionomycin-treated arteries,12 and b was determined. An apparent Kd of 282 nmol/ of Fura-2 for Ca21 was determined previously in this preparation.12

Materials Iberiotoxin (IbTx) was obtained from Peptides International. Fura2/AM, Fluo-3-AM, and pluronic acid were purchased from Molecular Probes. Sodium nitroprusside, dibutyryl-cAMP (db-cAMP), forskolin, and rapamycin were from Sigma. Stock (1 mmol/L) solutions of fluo-3-AM, rapamycin, and Fura-2/AM were made using DMSO as the solvent. Caffeine was from Serva. Ryanodine was obtained from Calbiochem. Nisoldipine was a gift from Dr A. Scriabine of Miles (Bayer) Laboratories (West Haven, Conn). High external K1 solutions were made by isoosmotic substitution of NaCl with KCl in the PSS. The monoclonal mouse anti-RyR2 (Clone 3-33, IgG1) and antiproliferating cell nuclear antigen (Clone PC10) antibodies were from Affinity Bioreagents and DAKO, respectively. All other salts and drugs were obtained from Sigma Chemical Co. All values are given as mean6SEM. The term “n” represents the number of arteries or cells tested. Differences were considered statistically significant at P,0.05 (unpaired Student t test).

Results Neonatal Myocytes Are Proliferative and Have Voltage-Dependent Ca21 Channels and Ryanodine Receptors It is well known that smooth muscle cells in neonatal arteries are still proliferative and incompletely differentiated,27 as indicated by the presence of PCNA (n56 arteries; Figure 1A). PCNA is an indicator for cells in S-phase,28,29 and as expected, smooth muscle cells in intact adult arteries did not stain positive for PCNA (n56 arteries; data not shown). Smooth muscle cells isolated from neonatal (,1 day postnatal) arteries are smaller (membrane capacitance: adult, 10.8 pF19; neonate, 3.860.2 pF; n56 cells), and less elongated than adult cells. However, 2 key components of excitation-contraction (E-C) coupling in muscle, voltage-dependent Ca21 channels and RyRs, appear to be present in smooth muscle cells isolated from both neonatal (,1 day postnatal) and adult arteries. The cell membranes of both adult and neonatal myocytes stained positive for the alpha 1C subunit of the voltage-dependent (L-type) Ca21 channel (Figure 1B; also see Figures 2 and 3 for functional evidence of Ca21 channels in neonatal arteries).22,30 To provide qualitative evidence for RyRs, isolated smooth muscle cells were stained with a monoclonal anti-RyR2 antibody (Figure 1C). Adult cells had distinct staining along the cell membrane, whereas the neonatal cells had much more diffuse staining (n530 for both adult and neonatal cells). These results support the existence of RyRs in both adult and neonatal cells and are consistent with the subplasma membrane location of RyRs,31 and hence Ca21 sparks, in adult cells. The presence of RyRs in neonatal myocytes suggested the possibility that these cells should also exhibit Ca21 sparks.

Isolated Smooth Muscle Cells From Neonatal Arteries Do Not Exhibit Ca21 Sparks In adult myocytes, Ca21 sparks can be observed readily and occur near the cell membrane (Figure 4A), consistent with the proposed activation of nearby plasmalemmal KCa channels.5 Although neonatal myocytes have RyRs (Figure 1C), Ca21 sparks were not observed in smooth muscle cells isolated from neonatal cerebral arteries bathed in PSS (Figure 4B; n555 cells; 10 s of scanning per cell). In an attempt to enhance the probability of visualizing Ca21 sparks in neonatal cells, several agents were used that previously have been shown to increase Ca21 spark frequency in isolated adult myocytes. For example, activators of adenylyl cyclase (eg, forskolin, db-cAMP) or sodium nitroprusside (SNP), a nitrovasodilator that increases cGMP levels, have been shown to increase Ca21 spark frequency in adult cells by '3-fold.16 However, Ca21 sparks were not observed in neonatal cells bathed in forskolin (10 mmol/L), db-cAMP (250 mmol/L), or SNP (10 mmol/L) (n552 cells). Increasing Ca21 influx through Ca21 channels by either membrane depolarization (by elevating external K1 to 60 mmol/L) or application of the Ca21 channel agonist, Bay K 8644 (1 mmol/L), which significantly increases Ca21 spark frequency in adult myocytes,5 did not cause Ca21 sparks in neonatal cells (n534 cells). In addition, Ca21 sparks were not observed in neonatal cells bathed in high (60 mmol/L) K1 in the presence of forskolin (n518 cells). Rapamycin (10 mmol/L), which affects the FK-506 binding protein and prolongs cardiac muscle Ca21 sparks,32 also did not cause Ca21 sparks in neonatal cells (n525 cells). Because protein kinase C activators decrease Ca21 spark frequency in adult myocytes,18 the effects of an inhibitor of PKC (calphostin C, 300 nmol/L) were examined on cells from neonatal arteries, with no Ca21 sparks being observed (n521 cells). To summarize, a total of 336 smooth muscle cells from neonatal cerebral arteries were examined, without 1 spark being detected. In contrast, Ca21 sparks could be observed in the majority of smooth muscle cells isolated from adult arteries during 10 s of scanning, as has been shown previously5,16,18 (Figure 4), with an apparent frequency of 0.1260.03 Hz per cell (n560). Based on the Ca21 spark frequency in adult myocytes and taking into account cell size differences, at least 200 Ca21 sparks should have been observed in neonatal cells over the observation period. These results suggest the possibility that RyRs in neonatal myocytes are incapable of generating Ca21 sparks.

Ca21 Spark Frequency Is Very Low in Intact Neonatal Arteries The single cell results suggest that RyRs may organize during development to form “Ca21 spark sites.” If so, it may be possible to detect a small number of spark sites, if a larger sample of neonatal cells were examined. We recently have developed a method to measure Ca21 sparks in intact cerebral arteries.24 With this approach, a significant number of cells can be examined simultaneously in an intact arterial wall. Measurement of Ca21 sparks in intact tissue also minimizes concerns about the condition of isolated cells after exposure to digestive enzymes.24,33 Approximately 1200 cells were examined in 6 different neonatal arteries over '1800 s. A very small number of Ca21 sparks (4)

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Figure 1. Staining of smooth muscle cells from neonatal arteries for PCNA, L-type Ca21 channels, and RyRs. A, Many smooth muscle cells in neonatal cerebral arteries stain positive (darkly stained nuclei) for PCNA. Immunoreactivity of PCNA was determined in crosssections of neonatal arteries using an anti-PCNA antibody coupled with horseradish peroxidase. B, Single smooth muscle cells from adult and neonatal arteries stained for L-type voltage-dependent Ca21 channels. Isolated vascular smooth muscle cells were incubated with a polyclonal rabbit anti-alpha 1C antibody generated against the amino acid sequence 799 to 817 (EEEEKERKKLARTASPEKK) of the cytoplasmic linker between repeat II and III of the alpha 1C subunit of the L-type voltage-dependent Ca21 channel. C, Adult and neonatal cells stained with a monoclonal anti-RyR2 antibody. Adult cells had distinct staining along the cell membrane, whereas staining in neonatal cells was more diffuse.

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Ca21 Sparks as Elementary Physiological Events Figure 2. Ryanodine increases arterial wall Ca21 and causes vasoconstriction in adult but not neonatal cerebral arteries. Original recordings of the simultaneous measurement of arterial wall [Ca21] and diameter in isolated pressurized adult (A) and neonatal (B) cerebral arteries loaded with Fura-2/AM. The stable levels of arterial wall [Ca21] and constriction at elevated intravascular pressure are indicated by horizontal dotted lines. In arteries isolated from adult animals, ryanodine (RYA; 10 mmol/L) increased arterial wall [Ca21] and caused vasoconstriction. The effects of ryanodine and IbTx (100 nmol/L) on diameter and Ca21 levels were not additive in arteries from adults. In arteries isolated from neonatal animals, ryanodine had no effect. Increased extracellular K1 (60 mmol/L) increased arterial wall [Ca21] and constricted both adult and neonatal cerebral arteries, whereas inhibitors of L-type Ca21 channels [nimodipine (Nim; 10 nmol/L) or nisoldipine (Nisol; 100 nmol/L)] decreased arterial wall [Ca21] and maximally dilated both adult and neonatal cerebral arteries. Lower panel represents intravascular pressure (P, mm Hg).

were detected in these arteries bathed in PSS. In the same 6 arteries, membrane depolarization by elevating the K1 concentration in the PSS from 6 to 30 mmol/L increased the number of Ca21 sparks observed from 4 to 23. In sharp contrast, '2000 sparks would have been observed in smooth muscle cells in 6 intact adult cerebral arteries bathed in 30 mmol/L K1 over the same scan duration and scan areas (based on a Ca21 spark frequency of 1.2060.28 Hz in a 56.3352.8–mm area; n56; Figure 5B; see also Jaggar et al24). These results suggest a '100-fold difference in Ca21 spark frequency between neonatal and adult arteries. Ca21 spark frequency increased to adult levels over the first 3 weeks of postnatal development (spark frequency at 1 week, 0.5161.2 Hz; at 3 weeks, 1.3062.6 Hz; 30 mmol/L extracellular K1; n54). The fractional increase in fluorescence during a Ca21 spark and t1/2 of decay, however, were similar

(P.0.05) in intact neonatal and adult arteries in 30 mmol/L K1 [F/Fo: neonatal, 1.6260.06 (n535 sparks); adult, 1.6960.02 (n5135 sparks); t1/2: neonatal, 58.4614.5 ms, (n56 sparks); adult, 46.764.3 ms (n514 sparks)]. These single cell and intact artery data indicate that Ca21 sparks are very rare in neonatal (,1 day postnatal) arteries, even under conditions that would greatly increase their frequency (eg, membrane depolarization).

Caffeine Causes Ca21 Transients in Neonatal and Adult Myocytes The existence of Ca21 sparks, albeit at very low frequency, as well as that of RyR staining suggests that RyRs are present in neonatal arterial myocytes. A common feature of RyR1 and RyR2 is their ability to be activated by caffeine, which increases their open-state probability.34 To Figure 3. IbTx increases arterial wall Ca21 and causes vasoconstriction in adult but not in neonatal cerebral arteries. Original recordings of the simultaneous measurement of arterial wall [Ca21] and diameter in isolated pressurized adult (A) and neonatal (B) cerebral arteries loaded with Fura-2/AM. The stable levels of arterial wall [Ca21] and constriction at elevated intravascular pressure are indicated by horizontal dotted lines. In arteries isolated from adult animals, IbTx (100 nmol/L) increased arterial wall [Ca21] and caused vasoconstriction. The effects of IbTx and ryanodine (10 mmol/L) on diameter and Ca21 levels were not additive in arteries from adults. In arteries isolated from neonatal animals, IbTx had no effect. Increased extracellular K1 (60 mmol/L) increased arterial wall [Ca21] and constricted both adult and neonatal cerebral arteries, whereas inhibitors of L-type Ca21 channels (Nim, 10 nmol/L or Nisol, 100 nmol/L) decreased arterial wall [Ca21] and maximally dilated both adult and neonatal cerebral arteries. See Figure 2 legend for expansions to abbreviations.

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Figure 4. Lack of Ca21 sparks in single smooth muscle cells from cerebral arteries of neonatal (,1 day postnatal) rats. A, 3-D plot of a Ca21 spark in an adult myocyte occurring in close proximity to the plasma membrane. B and C, Series of 9 consecutive images (16.7 ms apart) illustrating a Ca21 spark in an adult myocyte (B) and no detectable change in Ca21 in a neonatal myocyte (C). The mean peak increase in fluorescence (F/Fo) of Ca21 sparks in adult myocytes is '1.7, with a mean spatial spread on the order of 2.5 mm5 and a t1/2 of decay of '50 ms.5,18,24 Although Ca21 sparks were routinely observed during 10-s scans in cells isolated from adult cerebral arteries (ie, 72 sparks from 60 cells), no Ca21 sparks were detected in smooth muscle cells isolated from neonatal arteries.

explore the functionality of RyRs in neonatal myocytes, the effects of caffeine were examined. Ca21 sparks were not observed in isolated neonatal cells, even in the continued presence of 0.3 mmol/L (n545 cells) or 1.0 mmol/L

(n512 cells) caffeine. In contrast, superfusion of caffeine at 0.3 mmol/L increased the frequency of Ca21 sparks in single myocytes isolated from adult arteries '4-fold, from 0.1260.03 Hz to 0.5460.11 Hz, respectively (n560 cells).

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Figure 5. Ca21 sparks in intact arteries of adult and neonatal rats. Upper panels illustrate average fluorescence (mean, 100 consecutive images) from part of a scan area obtained from a neonatal (left panel) and an adult (right panel) cerebral artery bathed in 30 mmol/L extracellular K1. The location of Ca21 sparks occurring during the 10-s scans (60 images/s) are indicated by the labeled boxes (1.54 mm31.54 mm). F/Fo versus time for the corresponding boxes are illustrated in the lower panels along with an additional spark site located within the scan area of the adult artery. A total of 10 Ca21 sparks were recorded during the 10-s scan of the adult artery, with only 1 Ca21 spark recorded from the 10-s scan of the neonatal artery. Approximately 20 different 56.3 mm352.8 mm–scan areas were examined in each artery, corresponding to '200 different cells per artery. In arteries from neonatal rats, the majority of areas scanned had no Ca21 sparks. Unlike the neonatal arteries, virtually all areas of the adult arteries exhibited Ca21 sparks.

To increase further the activation of RyRs, caffeine was applied rapidly at a higher concentration (10 mmol/L). This concentration of caffeine, as previously has been shown,16,18 causes a typical whole-cell global Ca21 transient in adult myocytes because of the rapid activation of a significant number of RyRs (Figure 6). Such caffeine-induced Ca21 transients have been used routinely as a measure of SR Ca21 content in muscle cells.16,18,35 Rapid application of caffeine (10 mmol/L) to neonatal myocytes caused a global Ca21 transient, with an amplitude similar (P.0.05) to adult myocytes (F/Fo57.760.5 in adult versus 8.360.8 in neonatal cells; n512; Figure 6). These results indicate the presence of functional, caffeine-sensitive RyRs in neonatal cells. Furthermore, this result and the similar Ca21 spark amplitudes in neonatal and adult arteries also suggest that differences in SR Ca21 load are not responsible for the observed differences in Ca21 spark frequency.

Ca21 Sparks Are Required for the Regulation of Arterial Wall Ca21 and Diameter by Ryanodine Receptors The existence of functional RyRs in (adult) cells with Ca21 sparks and in (neonatal) cells with a very low spark frequency provided the opportunity to probe the physiological role of Ca21 sparks. In other words, is RyR activity in the absence of

Ca21 sparks sufficient to regulate tissue function? Elevation of intravascular pressure (eg, from 10 to 40 or 60 mm Hg) has been shown to cause an elevation of global Ca21 (from 120 to 200 nmol/L) and constriction (by 30%) of small cerebral arteries from adult animals5,11,12,35 (Figures 2A and 3A). Elevation of intravascular pressure from 10 to 40 mm Hg also increased arterial wall Ca21 from 121618 nmol/L (n53) to 195619 nmol/L (n515) and constricted neonatal arteries from 211610 mm to 16868 mm (n514; Figures 2B and 3B). In both cases, the elevation in arterial wall Ca21 and the vasoconstriction were reversed by inhibitors of L-type, voltage-dependent Ca21 channels such as nisoldipine (100 nmol/L; Figures 2 and 3).11,36,37 These results suggest that the mechanisms that cause pressure-induced elevations in Ca21 and vasoconstriction, including the contractile proteins, are present and functional in neonatal myocytes. Furthermore, L-type Ca21 channels are present (see also Figure 1B) and function to regulate the diameter of neonatal arteries. In sharp contrast to adult arteries, neonatal arteries did not respond to inhibitors of Ca21 sparks (ryanodine) and inhibitors of KCa channels (IbTx), which cause a profound elevation of arterial wall Ca21 and vasoconstriction of adult arteries (Figures 2, 3, and 7) (see also Nelson et al5 and Knot et al35 for more data on adult arteries). Ryanodine (10 mmol/L),

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Figure 6. Caffeine induces similar Ca21 transients in both neonatal and adult myocytes. A, Time course of fluorescence changes during a bolus addition of caffeine (10 mmol/L) in an adult and neonatal arterial myocyte. B, Averaged peak fluorescence changes in adult and neonatal arterial myocytes caused by 10 mmol/L caffeine (n512).

IbTx (100 nmol/L), and the combination had no effect on arterial wall Ca21 and diameter of pressurized neonatal arteries with tone (n511), whereas these agents increased arterial wall Ca21 by '50 nmol/L and constricted adult arteries by '50 mm. Membrane depolarization with high K1 or activation of RyRs with caffeine (10 mmol/L) caused a similar elevation of arterial wall Ca21 and constriction in neonatal and adult arteries (Figure 7). Ryanodine (10 mmol/L) blocked the effects of 10 mmol/L caffeine in arteries from both adults and neonates confirming the functional presence of RyRs in both tissue types. Therefore, it appears that the presence of caffeine-sensitive RyRs in neonatal myocytes is not sufficient for regulation of KCa channels and arterial diameter, even when the 2 major activators of RyRs (cytoplasmic and SR Ca21) are similar in neonatal and adult myocytes. This result is consistent with the idea that RyRs generate Ca21 sparks to regulate KCa channels, and ultimately arterial diameter.

Smooth Muscle Cells From Neonatal Arteries Have KCa Channels The lack of effect of ryanodine or IbTx on arterial wall Ca21 and diameter may reflect a lack (or low frequency) of Ca21 sparks and/or the absence of KCa channels. To examine KCa channels, K1 currents were measured by the whole cell perforated patch-clamp technique in single cells isolated from neonatal arteries. Membrane capacitance of these cells (3.860.2 pF; n56) was lower than that of adult cells ('11 pF),19 consistent with a smaller surface area. Currents through

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Figure 7. Summary of the effects of ryanodine, IbTx, caffeine, and high K1 on arterial wall Ca21 and diameter in neonatal and adult cerebral arteries. Changes in vascular wall [Ca21] (top) and diameter (bottom) were measured simultaneously in isolated pressurized (adult560 mm Hg, n512; neonatal540 mm Hg, n511) cerebral arteries. RyA 10 mmol/L; IbTx 100 nmol/L; caffeine (CAF) 10 mmol/L; and HIK (extracellular K1) 60 mmol/L.

single KCa channels were clearly discernible in these isolated neonatal cells (Figure 8A). The channels had the characteristic single channel conductance (12064 pS, 220 to 20 mV; n56; Figure 8B) and voltage-dependence of KCa channels as well as sensitivity to block by IbTx (NPo50.021960.006, control versus NPo50.002460.006 in the presence of 100 nmol/L IbTx; holding potential (Vh)530 mV; n56). KCa channel currents [spontaneous transient outward currents (STOCs)] caused by Ca21 sparks were not observed in neonatal cells (Vh5240 to 220 mV), consistent with a lack of Ca21 sparks in these cells. These results indicate that neonatal arterial myocytes have functional KCa channels. However, these results do not address the issue of whether the density or location of KCa channels is appropriate for STOC generation, if Ca21 sparks were to occur.

Discussion Developmental Changes in Ryanodine Receptors That Cause Ca21 Sparks: Implications for the Nature of Ca21 Sparks At birth, cerebral arteries possess voltage-dependent Ca21 channels, RyRs, and KCa channels as well as the ability to constrict to pressure. Smooth muscle cells from both neonatal and adult arteries have RyRs (Figure 1C) and respond robustly to the RyR-activator, caffeine (Figures 6 and 7).35,38 Yet, Ca21 spark frequency was extremely low in

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Figure 8. KCa channels are present in myocytes from cerebral arteries of neonatal animals. A, Consecutive records of single KCa channel openings before (control) and after application of IbTx (100 nmol/L; 10 minutes) recorded using the whole-cell perforated patch configuration of the patch-clamp technique (Vh530 mV). STOCs were not observed in neonatal cells (n56). B, Relationship between single KCa channel current amplitude and voltage (n56). [K1]o56 mmol/L, [K1]i5140 mmol/L.

neonatal myocytes, even under conditions to enhance RyR open probability (eg, membrane depolarization and caffeine). Based on caffeine-induced Ca21 transients, SR Ca21 load seemed similar in neonatal and adult myocytes; therefore, Ca21 spark amplitude should be similar, as

observed. There are several possible explanations for the observed low frequency of Ca21 sparks in neonatal arteries: (1) If cytoplasmic Ca21, which activates RyRs, was much lower in neonatal myocytes, this might explain some of the difference in Ca21 spark frequency. However, cytoplasmic Ca21 was similar in smooth muscle cells of neonatal and adult arteries (Figures 2 and 3), arguing against this possibility. (2) If RyR open time is much shorter in the neonatal than adult myocytes, then amplitudes of many Ca21 sparks might fall below the detection limit. However, the amplitudes of the Ca21 sparks observed in the neonatal arteries were no different from those in adult arteries. (3) If RyR density were much lower (,1/100) in neonatal myocytes than in adult myocytes, then Ca21 spark frequency also would be much lower. However, neonatal myocytes stained positively for an antibody to RyRs, and the rise time and amplitudes of the caffeine-induced Ca21 transients were similar in neonatal and adult myocytes. (4) If the coordinated opening of several tightly clustered RyRs is required to cause a Ca21 spark, then it is possible that RyRs in neonatal myocytes are not clustered in a manner to permit Ca21 sparks to occur (Figure 9). In this case, the unitary efflux of Ca21 through a single RyR would be too low to be detected, and the coordinated opening of a tightly clustered group of RyRs causes a Ca21 spark. There are several lines of evidence from both cardiac and skeletal muscle of a Ca21 spark (“the elementary event”) being made up of several smaller events (“quarks” or “fundamental events”).7 Figure 9 illustrates a model for our results based on the latter hypothesis. Specifically, we propose that during development (.1 day postnatal), RyRs cluster in terminal SR plaques to cause a Ca21 spark that activates KCa channels. The appearance of Ca21 sparks occurs in development after the appearance of RyRs, Ca21 channels, and KCa channels. It appears as if the molecular components for E-C coupling are first expressed, and then spatially organized to perform their specific functions.

Figure 9. Proposed model illustrating the organization of RyR in neonatal and adult arteries. This model proposes that during development (.1 day postnatal), RyRs cluster in terminal SR plaques to cause a Ca21 spark to activate KCa channels. The appearance of Ca21 sparks occurs in development after the appearance of RyRs, Ca21 channels, and KCa channels, to cause a membrane potential (Vm) hyperpolarization, which closes voltage-dependent calcium channels (VDCCs).

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Gollasch et al

Open Probability of KCa Channels Is Very Low in the Absence of Ca21 Sparks KCa channels are both voltage- and Ca21-sensitive.39 Our approach (whole-cell perforated patch-clamp technique) enabled the measurement of the whole-cell activity (NPo) of single KCa channels. In the absence of Ca21 sparks, whole-cell KCa channel NPo was extremely low in both adult myocytes (0.005; 0 mV)18 and neonatal (0.02; 30 mV; Figure 8) myocytes. Given their Ca21 dependence and voltage dependence, whole-cell KCa channel NPo in both adult and neonatal myocytes would be in the order of 1024 to 1022 under physiological conditions (240 mV; 200 nmol/L global Ca21; see Porter et al16 for a detailed discussion of this issue). However, the frequency of Ca21 sparks ('1 spark/s per cell), measured at physiological membrane potentials and arterial wall Ca21, elevate the whole-cell NPo to between 0.1 and 1.0, which would contribute significantly to the cell’s membrane potential, given the input resistance of smooth muscle cells and the relatively large single KCa channel conductance.16,37,40 Regardless of the uncertainties, KCa channels would not contribute significantly to the membrane conductance of arterial myocytes without a Ca21 spark frequency of .1021 Hz/cell. These considerations also support the idea that the apparent Ca21 spark frequency observed in neonatal myocytes (,1022 Hz/cell) would not cause sufficient KCa channel activity to regulate the membrane potential of smooth muscle cells in intact neonatal arteries.

Ca21 Sparks Are Elementary Physiological Events Neonatal arteries possess the molecular components for E-C coupling and its negative feedback regulation (ie, L-type, voltage-dependent Ca21 channels, RyRs, KCa channels). As expected, blockers of L-type Ca21 channels lower arterial wall Ca21 and dilate neonatal arteries (Figures 2 and 3). Contrary to expectation, blockers of RyRs (ryanodine) and KCa channels (IbTx) had no effect on arterial wall Ca21 and diameter of pressurized neonatal arteries with tone, even though these arteries have RyRs and KCa channels. The missing feature of E-C coupling in neonatal arteries is Ca21 sparks. In contrast, adult arteries have a much higher spark frequency (.100-fold) than neonatal arteries and respond robustly to inhibitors of RyRs and KCa channels.5,11,35 Therefore, these results provide unique support for the idea (Figure 9) that RyRs must generate Ca21 sparks to regulate arterial function.

Acknowledgments This work was supported by NIH grants HL44455 and HL51728, NSF grants BIR-9601683 and IBN-9631416, and fellowships from the NIH (F32HL09920 to G.C.W.), AHA (to J.H.J.), and a Feodor Lynen fellowship from the Alexander von Humboldt-Stiftung (to M.G.). We thank Drs J. Brayden, L.F. Santana, D. Welsh, G. Perez, T. Firth, T. Heppner, and G. Herrera for comments on the manuscript. We also thank Dr Hannelore Hasse for providing the L-type Ca21 channel antibody, and Dr Doug Taajtes and Greg Hendricks for their assistance with immunohistochemistry.

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