Developmental changes in ryanodine- and IP3-sensitive Ca2+ pools in ovine basilar artery

S. M. Nauli, J. M. Williams, S. E. Akopov, L. Zhang, and W. J. Pearce

Department of Physiology, Center for Perinatal Biology, Loma Linda University School of Medicine, Loma Linda, California 92350


    ABSTRACT
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INTRODUCTION
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To explore the hypothesis that cerebrovascular maturation alters ryanodine- and inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pool sizes, we measured total intracellular Ca2+ with 45Ca and the fractions of intracellular Ca2+ released by IP3 and/or caffeine in furaptra-loaded permeabilized basilar arteries from nonpregnant adult and term fetal (139-141 days) sheep. Ca2+ mass (nmol/mg dry weight) was similar in adult (1.60 ± 0.18) and fetal (1.71 ± 0.16) arteries in the pool sensitive to IP3 alone but was significantly lower for adult (0.11 ± 0.01) than for fetal (1.22 ± 0.11) arteries in the pool sensitive to ryanodine alone. The pool sensitive to both ryanodine and IP3 was also smaller in adult (0.14 ± 0.01) than in fetal (0.85 ± 0.08) arteries. Because the Ca2+ fraction in the ryanodine-IP3 pool was small in both adult (5 ± 1%) and fetal (7 ± 4%) arteries, the IP3 and ryanodine pools appear to be separate in these arteries. However, the pool sensitive to neither IP3 nor ryanodine was 10-fold smaller in adult (0.87 ± 0.10) than in fetal (8.78 ± 0.81) arteries, where it accounted for 72% of total intracellular membrane-bound Ca2+. Thus, during basilar artery maturation, intracellular Ca2+ mass plummets in noncontractile pools, decreases modestly in ryanodine-sensitive pools, and remains constant in IP3-sensitive pools. In addition, age-related increases in IP3 efficacy must involve factors other than IP3 pool size alone.

cerebral arteries; cerebrovascular circulation; furaptra; maturation; sarcoplasmic reticulum


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INTRODUCTION
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DISCUSSION
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THE AGONIST-INDUCED Ca2+ release from intracellular organelles that initiates smooth muscle contractile responses is generally mediated by the synthesis and release of the second messenger inositol 1,4,5-trisphosphate (IP3) (34). Ion channel-mediated entry of extracellular Ca2+ can also promote intracellular Ca2+ release via activation of ryanodine-sensitive receptors on the sarcoplasmic reticulum (SR) through a process known as Ca2+-induced Ca2+ release (17). The main intracellular source for both ryanodine- and IP3-sensitive Ca2+ release appears to be the SR (14). As shown in a variety of studies, the relation between receptor activation and Ca2+ release is not constant, which suggests that the coupling between receptor or ion channel activation and Ca2+ release may be influenced or even regulated by physiological and pathophysiological perturbations.

One of the most important physiological processes known to influence vascular contractility is maturation. Neonatal cerebral arteries exhibit similar active stresses, but because of their smaller wall thicknesses they produce less total contractile force than do corresponding adult arteries (8, 31). For multiple agonists, neonatal arteries exhibit greater receptor densities and agonist affinities, greater sensitivity (lower EC50 values), and also altered patterns of Ca2+ mobilization compared with corresponding adult arteries (2, 8, 23). In light of the central but variable role of ryanodine- and IP3-induced Ca2+ release in smooth muscle contractile responses, it is possible that developmental modifications of ryanodine- and IP3-dependent pharmacomechanical coupling may play a role in age-related differences of vascular responses to receptor agonists. Consistent with this hypothesis are previous suggestions that IP3-mediated Ca2+ release is physiologically less important in immature than in mature smooth muscle (1). Such differences may result from a broad variety of maturational changes including smaller intracellular Ca2+ stores, reduced sensitivity of IP3- or ryanodine-sensitive receptors, and/or altered IP3 or ryanodine receptor function in immature compared with mature cerebral arteries. One approach to choose among these possibilities would be to measure IP3- and ryanodine-induced intracellular Ca2+ release in mature and immature arteries. Such measurements have just recently become possible with the use of methods developed in permeabilized vascular preparations (see Fig. 1 of Ref. 12; Ref. 15).

The major goal of the present work was to test the hypothesis that maturation alters the relative sizes of the ryanodine- and IP3-sensitive Ca2+ pools in cerebral arteries. In previous studies, Stauderman et al. (35) divided intracellular Ca2+ stores into three distinct pools including those sensitive to 1) caffeine, 2) IP3, and 3) both caffeine and IP3. In light of recent evidence that other organelles may also contain significant intracellular Ca2+ (10, 39), we have also examined the possible existence of a fourth pool resistant to both caffeine and IP3 but releasable by Ca2+ ionophore. Because our previous observations have indicated that the ovine basilar artery exhibits marked maturational changes in reactivity to multiple agonists that cannot be explained by differences in the receptor apparatus alone (22, 31) and also exhibits marked age-related changes in dependence on extracellular Ca2+ for contraction (2), we chose this model for the present studies. Selection of the basilar artery for these studies was further justified by the well-established observations that arteries >150 µM in diameter, such as the basilar artery, are responsible for one-third or more of total adult cerebrovascular resistance (3) and may play an even more important regulatory role in neonates than in adults (31).


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General preparation. The Loma Linda University Animal Research Committee approved all procedures. Basilar arteries from adult sheep (18-24 mo old) and term (~140 days gestation) fetal sheep were placed in buffer with (in mM) 132 NaCl, 4.2 NaHCO3, 5.9 KCl, 1.4 MgSO4, 1.2 Na2HPO4, 1.6 CaCl2, 11 dextrose, and 10 HEPES (pH 7.36). Segments 3-4 mm long were mounted on wires between a force transducer and a micrometer used to control passive stretch (1). All segments were continuously maintained at 38.5°C (normal ovine core temperature) in normal Krebs solution at optimal stretch, and contractile tensions were continuously digitized and recorded (8). Arterial segments were rotated on the mounting wires several times to destroy the endothelium, and endothelium removal was verified by the absence of a vasodilator response to 1 µM acetylcholine.

Protocol 1: Contractile effects of ryanodine- and caffeine-induced Ca2+ release in intact preparations. Artery segments were first contracted with 122 mM K+ to define maximum contractile capacity, returned to normal buffer for 30 min, and then exposed to 1) 30 µM ryanodine followed by cumulative addition of 30 mM caffeine or 2) addition of 30 mM caffeine alone. These additions of ryanodine and caffeine were conducted at extracellular Ca2+ concentrations of 1.5 mM and 10 nM to assess the role of extracellular Ca2+ in the contractions observed (6).

Protocol 2: Contractile effects of IP3-induced Ca2+ release in permeabilized preparations. Arterial segments were permeabilized with 100 µM beta -escin in Ca2+-free buffer containing (in mM) 125 K-acetate, 5 EGTA, 4 ATP, 4 Mg-acetate, 1 dithiothreitol, 0.01 leupeptin, 1 NaN3, and 10 HEPES at pH 6.9 (1). Intracellular Ca2+ stores were loaded by increasing free Ca2+ to 0.3 µM for 15 min, followed by a 5-min wash in Ca2+-free buffer containing 0.05 mM EGTA. Intracellular Ca2+ release was monitored by recording contractile responses to successive additions of 30 µM IP3 with 15-min Ca2+-free washouts between applications. Multiple lines of evidence indicate that 30 µM is an optimal IP3 concentration for Ca2+ release (1, 15). In control experiments, the effects of 30 µM IP3 were evaluated in permeabilized preparations depleted of Ca2+ by incubation with 10 µM A-23187 as previously described (16). At the end of each experiment, contractile responses to a maximally effective free Ca2+ concentration (10 µM) were obtained.

Protocol 3: Ryanodine-induced Ca2+ transients in intact vascular preparations. Basilar artery segments were loaded with fura 2-AM (5 µM; premixed with 0.01% Pluronic F127) for 4 h at 25°C under protection from light as previously described (2, 45). Loaded segments were mounted in a fluorometer (CAF-110, Japan Spectroscopic) that provided excitation wavelengths of 340 and 380 nm. The emitted fluorescence was filtered at 500 nm, and the ratio of the energies emitted (F340/F380) was calculated automatically as a measure of intracellular Ca2+ and simultaneously recorded with isometric tension. To minimize the influence of autofluorescence or movement artifacts, the F340 and F380 signals were monitored separately and the data were used only when these signals changed as mirror images of one another.

Fura 2-loaded segments were equilibrated for 10 min with 10 µM free Ca2+, 5 mM EGTA, and 5 µM sodium orthovanadate. Orthovanadate was added at this concentration to inhibit Ca2+ uptake by SR Ca2+ pumps and Ca2+ extrusion by plasmalemmal Ca2+ pumps (38) and thus to ensure that ryanodine-induced increases in cytosolic Ca2+ concentration would be stable. To verify that the concentration of vanadate used was optimal, separate artery segments were loaded with fura 2, equilibrated with vanadate, and then contracted with high-potassium physiological salt solution (PSS) containing vanadate. When returned to normal PSS with vanadate these segments failed to relax, and the potassium-induced increase in cytosolic Ca2+ persisted even after 10 min of incubation in normal PSS with vanadate. Thus 5 µM vanadate effectively inhibited Ca2+ transport in our preparations.

To characterize ryanodine-induced release of intracellular Ca2+, we exposed arterial segments, loaded with fura 2 and equilibrated with vanadate, to graded concentrations (0.1-30 µM) of ryanodine while continuously measuring contractile force and the Ca2+ ratio. On completion of these measurements, the minimum fluorescence ratio was obtained by incubation of the preparations in nonfluorescent Ca2+ ionophore Br-A-23187 (10 µM) for 15 min. As shown by Itoh et al. (16), this concentration of ionophore preferentially permeabilizes all intracellular organelles with minimal effects on sarcolemmal permeability.

Protocol 4: IP3-induced Ca2+ transients in permeabilized vascular preparations. Basilar artery segments were loaded with furaptra-AM (5 µM; premixed with 0.01% Pluronic F127) for 3.5 h at 37°C under protection from light. At this temperature furaptra accumulated mainly in the SR (12, 37). The loaded arteries were permeabilized with 150 µM beta -escin for 20 min and then washed in relaxing buffer to remove any cytoplasmic furaptra. Permeabilization was necessary for IP3, which is membrane impermeant, to reach intracellular IP3 receptors (15). After permeabilization, the segments were buffered at either 150 nM or 300 nM free Ca2+. GTP (10 µM) was added as required for IP3-mediated Ca2+ release (37). In separate control experiments, GTP alone had no influence on Ca2+ fluxes from intracellular stores.

The permeabilized segments were mounted in a fluorometer that was set to excite at wavelengths of 346 and 374 nm. Emitted photons were filtered at 510 nM, and the F346-to-F374 ratio, which quantifies intraorganellar Ca2+ concentration, was continuously recorded in response to IP3 concentrations between 0.1 and 30 µM. The minimum fluorescence ratio was obtained by incubation in Ca2+-free buffer with 10 µM Br-A-23187 for 15 min. Calculations of Ca2+ concentrations were made with standard equations (37) based on an intraorganellar dissociation constant for furaptra of 54 µM as shown previously by in situ calibration (12).

Protocol 5: Relative Ca2+ pool sizes based on IP3 and ryanodine sensitivity in permeabilized vascular preparations. Intracellular organelles were loaded with furaptra-AM as described for protocol 4, after which the artery segments were maintained in 300 nM free Ca2+ and 10 µM GTP. The F346-to-F374 ratio was recorded continuously except during administration of caffeine because millimolar concentrations of caffeine significantly increase the F346-to-F374 ratio through a direct interaction with furaptra (26). Relative pool sizes were determined in two parallel experiments. In the first experiment, the decrease in the F346-to-F374 ratio produced by 30 mM caffeine was taken as a measure of all ryanodine-sensitive Ca2+ pools. Caffeine was used because it produced a rapid release of Ca2+ whereas ryanodine-induced release was typically quite slow. Next, the arteries were exposed to 30 µM IP3 to indicate the size of the remaining IP3-sensitive, ryanodine-resistant Ca2+ pool. In the second experiment, arteries were first exposed to 30 µM IP3, to release all IP3-sensitive stores, and then to 30 mM caffeine, to release the IP3-resistant but ryanodine-sensitive store. On conclusion of both experiments, the arteries were treated with 10 µM Br-A-23187 to determine the size of the pool resistant to both IP3 and ryanodine and establish the minimum fluorescence ratio.

Protocol 6: Measurement of intracellular Ca2+ mass using 45Ca washout. We measured total intracellular Ca2+ mass using 45Ca as described previously (50). Briefly, arteries were contracted for 2 min by exposure to a K+-Krebs-bicarbonate buffer containing 45Ca at 1 × 108 cpm/ml with 3 µM histamine and 10 µM serotonin. The arteries were then relaxed for 18 min in Na+-Krebs-bicarbonate buffer that also contained 45Ca at 1 × 108 cpm/ml. From each basilar artery, we prepared six adjacent artery segments, each of which was treated with a different number of contraction-relaxation cycles. After loading, the segments were washed in sequential wash vials (5 min each) containing ice-cold buffer with (in mM) 2 EGTA, 122.1 NaCl, 5.16 KCl, 2.5 MgSO4, 25.6 NaHCO3, and 11.08 dextrose continuously bubbled with 95% O2-5% CO2 and then dried, weighed, and counted. Cumulative washout curves were constructed, and the size and rate constant for the intracellular pool sizes were determined by fitting the data to
<IT>y=A · e</IT><SUP>(−<IT>k</IT><SUB>1</SUB> · <IT>t</IT>)</SUP><IT>+B · e</IT><SUP>(−<IT>k</IT><SUB>2</SUB> · <IT>t</IT>)</SUP>
where A is mass of the extracellular Ca2+ pool, k1 is the extracellular pool rate constant, t is wash time in minutes, B is mass of all intracellular Ca2+ pools, and k2 is the intracellular pool rate constant.

Values of pool size were plotted against their corresponding number of contraction cycles to indicate the rate of uptake and labeling of the intracellular pools. This relation was then fitted to determine both the steady-state magnitude of the intracellular Ca2+ mass and the number of contraction cycles required to label one-half of that mass using
<IT>Y=</IT>(<IT>C · N</IT>)<IT>/</IT>(<IT>D+N</IT>)
where Y is the measured intracellular Ca2+ mass, C is the maximum steady-state intracellular Ca2+ mass, N is the number of contraction cycles, and D is the number of contraction cycles required to reach half-maximal intracellular Ca2+ mass.

Data analysis and statistics. All values are means ± SE for the numbers of animals studied. Because we showed previously (31) that the maximum levels of potassium-induced contractile force per unit cross-sectional area (in 107 dyn/cm2) in ovine basilar arteries do not vary significantly between nonpregnant adults (3.63 ± 0.28, n = 37) and term fetal lambs (3.66 ± 0.44, n = 18), we normalized all contractile responses to the maximum contractile response to 122 mM potassium (Kmax). This normalization corrected for age-related differences in smooth muscle mass and facilitated interage comparisons of contractile responses. All dose-response data were fitted to the logistic equation using nonlinear regression to calculate pD2 values (-log ED50). To calculate relative Ca2+ pool sizes, the results were combined from protocols 3, 4, and 5 and analyzed as a series of simultaneous equations using matrix algebra. The four pools defined were the pool releasable by both IP3 and ryanodine, the pool releasable by IP3 but resistant to ryanodine, the pool releasable by ryanodine but resistant to IP3, and the pool releasable by Br-A-23187 but resistant to both IP3 and ryanodine. The sum of all pools was assumed to equal 100% within each experiment. Because the extraction of total vascular Ca2+ was achieved using EGTA buffers of similar composition for determination of both minimum fluorescence ratios and 45Ca content, the total amounts of releasable Ca2+ measured in both the fluorescent measurement and 45Ca washout protocols were considered equal. Correspondingly, absolute pool sizes were calculated as the product of fractional pool size and the estimated total intracellular Ca2+ mass. All comparisons between means were performed using ANOVA with post hoc comparisons via Fisher's protected least significant difference. All data sets were verified to be normally distributed before analysis by ANOVA. All comparisons with negative results had statistical powers of >= 0.8. Unless stated otherwise, n indicates the number of animals (not the number of segments) and statistical significance implies P < 0.05.

Materials. The IP3 (D-myo-inositol 1,4,5-trisphosphate), fura 2-AM, furaptra-AM, and Br-A-23187 used in these studies were obtained from Molecular Probes (Eugene, OR). The tissue solubilizer used (TS-2) was purchased from Research Products International (Mt. Prospect, IL). The 45Ca used was purchased from New England Nuclear (Boston, MA). All other chemicals and reagents were purchased from Sigma (St. Louis, MO) and were of the highest purity available.


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This study used 207 basilar artery preparations obtained from 50 young adult sheep and 51 near-term fetuses. In arteries used for contractility measurements, unstressed baseline diameters averaged 0.62 ± 0.01 mm in adults and 0.53 ± 0.01 mm in fetuses. Maximum contractile tensions obtained in response to 122 mM potassium (Kmax) averaged 5.58 ± 0.56 and 4.42 ± 0.26 g/cm of artery length in adult and fetal basilar arteries, respectively.

Contractile effects of ryanodine- and caffeine-induced Ca2+ release in intact preparations. The transient contractile responses to 30 mM caffeine were significantly less in adult than in fetal basilar arteries (Fig. 1). Responses to 30 µM ryanodine developed slowly, but their peak values were not significantly different from those to caffeine. Addition of caffeine immediately after ryanodine produced only small additional contractions. In artery segments first equilibrated 30 min in PSS buffered at 10 nM Ca2+, ryanodine-induced contractions were not significantly greater than zero.


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Fig. 1.   Contractile responses to ryanodine-induced intracellular Ca2+ release in intact adult and fetal basilar artery segments. Artery segments with loaded Ca2+ stores were contracted with 1) 30 mM caffeine, 2) 30 µM ryanodine followed by 30 mM caffeine, or 3) 30 µM ryanodine alone. All contractions were obtained with extracellular Ca2+ buffered at either 1.6 mM or 10 nM. A: individual traces to illustrate response dynamics. B: comparison of the averaged contractile tensions of each response, normalized to the maximum contractile response to 122 mM K+. Note that pretreatment with ryanodine markedly reduced subsequent responsiveness to caffeine, suggesting that both agonists acted on the same Ca2+ pool. Vertical error bars indicate SE. *Significant differences (P < 0.05) between corresponding adult (n >=  5) and fetal (n >=  7) basilar artery responses.

Contractile effects of IP3-induced Ca2+ release in permeabilized preparations. In permeabilized basilar arteries treated with 10 µM A-23187, administration of 30 µM IP3 failed to produce any changes in contractile force either in adult or fetal arterial preparations, indicating that A-23187 effectively emptied all IP3-releasable Ca2+ stores. In preparations with Ca2+-loaded stores, IP3 evoked contractions that were 20-40% of the maximum contractions evoked by exogenous Ca2+ (Fig. 2A). The IP3 evoked responses were, on average, threefold greater in adult than in fetal arteries. Repeated applications of 30 µM IP3 (without Ca2+ reloading between applications) progressively decreased the magnitudes of the contractile responses, less so in adult than in fetal arteries (Fig. 2B).


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Fig. 2.   Contractile responses to inositol 1,4,5-trisphosphate (IP3)-induced intracellular Ca2+ release in beta -escin-permeabilized adult and fetal basilar artery segments. In permeabilized basilar arteries with loaded Ca2+ stores, additions of 30 µM IP3 in the presence of 10 µM GTP (vertical arrows) evoked greater contraction (normalized to maximum contractile response to 10 µM Ca2+) in adult (36.7 ± 2.0%, n = 7) than in fetal (13.0 ± 1.5%, n = 7) arteries (A). Three consecutive applications of 30 µM IP3 yielded progressively decreasing contractions whose magnitudes were significantly greater in adult than in fetal basilar arteries (B). Vertical error bars indicate SE for n >=  3. *Significant differences (P < 0.05) between corresponding adult and fetal artery responses.

Ryanodine-induced Ca2+ transients in intact vascular preparations. Ryanodine evoked dose-dependent increases in cytosolic Ca2+. Because these increases occurred in the presence of 10 nM extracellular Ca2+, they indicate the release of intraorganellar Ca2+ (Fig. 3A). After treatment with Br-A-23187, Ca2+ concentrations attained relatively stable plateau values, indicating that 5 µM orthovanadate effectively blocked Ca2+ transport and that sarcolemmal permeability was unaffected by Br-A-23187. The ability of ryanodine to release Ca2+ was significantly less in adult than in fetal arteries (Fig. 3B). pD2 values for ryanodine were not significantly different in adult and fetal preparations.


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Fig. 3.   Ryanodine-induced transients in cytosolic Ca2+ in intact basilar arteries. In the presence of 5 µM vanadate and 10 nM extracellular Ca2+, ryanodine evoked a concentration-dependent release of Ca2+. All intraorganellar Ca2+ remaining after ryanodine was released by 10 µM Br-A-23187, after which Ca2+ attained a stable plateau (A). These plateau values decreased by only 0.58 ± 0.22 and 0.64 ± 0.18%/min in adult and fetal basilar arteries, respectively, indicating that 5 µM vanadate effectively blocked Ca2+ reuptake by sarcoplasmic reticulum Ca2+ pumps and Ca2+ extrusion by plasmalemmal Ca2+ pumps. pD2 values for the adult and fetal concentration-response relations did not differ significantly, but the Ca2+ fraction released by ryanodine was significantly smaller in adult than in fetal arteries (B). The vertical error bars indicate SE for 5 adults and 6 fetuses. F340/F380, ratio of energies emitted at excitation wavelengths of 340 and 380 nm.

IP3-induced Ca2+ transients in permeabilized vascular preparations. To validate furaptra compartmentalization within the SR, we compared the dynamics of fluorescence generated during permeabilization with beta -escin after furaptra loading at both 22 and 37°C (12). In both adult and fetal arteries loaded at 22°C, beta -escin decreased fluorescence because of leakage of dye from the cytoplasm into the Ca2+-free medium. In arteries loaded at 37°C, permeabilization induced little change in fluorescence, indicating that most dye was sequestrated within intracellular organelles. These experiments also confirmed that very high concentrations of beta -escin (>= 1.5 mM) were required to destroy intracellular ultrastructure (40). Thus, for reasons articulated by Golovina and Blaustein (12), the intraorganellar Ca2+ concentrations indicated in our furaptra preparations most probably reflected SR calcium, with small contributions possible from the nucleus or nuclear envelope (12, 37).

Baseline intraorganellar Ca2+ concentrations were significantly greater in adult than fetal arteries, and exposure of these preparations to IP3 evoked concentration-dependent declines in the F346-to-F374 ratio, indicating release of intraorganellar Ca2+ (Fig. 4A). When normalized to total Ca2+ released, IP3-induced Ca2+ release was significantly greater in adult compared with fetal arteries at all IP3 concentrations (Fig. 4B). IP3 sensitivity and total Ca2+ release were not significantly different at ambient Ca2+ concentrations of 150 or 300 nM in either age group. At 150 nM, the sizes of the IP3-releasable pools were 63.0% and 13.3% of the ionophore-releasable Ca2+ in adult and fetal arteries, respectively. At 300 nM Ca2+, the corresponding values were 57.8% and 21.1%, respectively (Fig. 4C). In contrast, pD2 values for IP3 were not significantly different in adult and fetal preparations for either level of ambient Ca2+ used.


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Fig. 4.   IP3-induced changes in intraorganellar furaptra fluorescence in beta -escin-permeabilized adult and fetal basilar artery segments. Baseline organellar Ca2+ concentrations averaged 140.7 ± 12.7 and 62.3 ± 9.3 µM in adult and fetal arteries, respectively (n = 7 each, P < 0.01). After permeabilization, administration of IP3 in the presence of 10 µM GTP yielded concentration-dependent decreases in fluorescence. Ca2+ remaining after IP3 treatment was released with 10 µM Br-A23187 (A). 346/374, Ratio of furaptra fluorescence measured at wavelengths of 346 and 374 nm. When normalized to the total amount of Ca2+ released, the magnitudes of IP3-induced Ca2+ release were significantly greater in adult than in fetal basilar arteries, regardless of ambient Ca2+ concentration (B and C). pD2 values for the adult and fetal release curves (range: 6.00-6.18) did not vary significantly with age or ambient Ca2+ concentration (C). Vertical error bars indicate SE for n >=  5. * Significant age-related differences (P < 0.05).

Ca2+ distribution based on IP3 and ryanodine sensitivity in permeabilized vascular preparations. When arteries were exposed first to 30 µM IP3 (Fig. 5, left), the Ca2+ fraction released was significantly greater in adult (63.8%) than fetal (19.4%) arteries, but the fraction released by subsequent 30 mM caffeine was significantly less in adult (4.0%) than fetal (8.5%) arteries. The remaining Ca2+ released by 10 µM Br-A-23187 was then significantly less in adult (32.2%) than in fetal (72.1%) arteries. When arteries were exposed first to caffeine (Fig. 5, right) the Ca2+ released was significantly smaller in adult (8.8%) than in fetal (15.0%) arteries and subsequent release by IP3 was significantly greater in adult (59.4%) than in fetal (13.2%) arteries. Br-A-23187 then released significantly less Ca2+ in adult (31.8%) than in fetal (71.9%) arteries.


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Fig. 5.   Relative ryanodine- and IP3-sensitive pool sizes in ovine adult and fetal basilar arteries. A: representative traces of furaptra fluorescence ratios in response to 30 µM IP3, 30 mM caffeine, and 10 µM Br-A23187 added sequentially in the presence of 10 µM GTP in permeabilized arteries. Note that the relative pool sizes were determined by 2 parallel experiments differing only in the order of IP3 and caffeine exposures. The dotted lines join the traces where the recording was stopped after caffeine addition to eliminate initial fluorescent artifacts typical of caffeine. Caffeine was washed from the arteries before resumption of recording. B: the averaged ability of IP3, caffeine, and Br-A23187 in different orders of exposure to release intraorganellar Ca2+. Ca2+ release is expressed as percentage of the maximum Br-A-23187-induced release. Vertical error bars indicate SE for 5 experiments/group. *Significant differences (P < 0.05) between corresponding adult and fetal artery responses.

The Ca2+ release results were used to define a series of linear equations with four unknowns, one for each Ca2+ pool. These equations assumed only that Br-A-23187 released all membrane-bound intracellular Ca2+, that pool sizes were consistent within a given age group, and that agonist affinities were uniform across all receptors of a given type. The estimated sizes of the pool sensitive to both ryanodine and IP3 were quite small (range: 5-7% of total releasable Ca2+), as were the sizes of the pool sensitive to ryanodine only (range: 4-10% of total releasable Ca2+) (Fig. 6). The pool sensitive to IP3 only varied dramatically with age and was fourfold larger in adult than in fetal arteries, whereas the pool resistant to both ryanodine and IP3 was twofold smaller in adult than in fetal arteries.


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Fig. 6.   Relative ryanodine- and IP3-sensitive pool sizes in ovine adult and fetal basilar arteries. The relative distribution of Ca2+ among the Ca2+ pools sensitive to IP3 only, sensitive to ryanodine only (Ry), sensitive to both IP3 and ryanodine (IP3+Ry), and resistant to both IP3 and ryanodine but sensitive to Br-A-23187 (Ry/IP3-Resistant) is shown for adult (A) and fetal (B) arteries. For each age group, the total area within the square is defined as 100% of the intracellular Ca2+ and the areas represented by the different pools directly reflect relative pool size. These values were calculated from the responses shown in Fig. 5, and they indicate that the Ry and Ry/IP3-Resistant pools were significantly smaller in adult than fetal arteries, whereas the IP3-sensitive pools were significantly larger in adult than fetal arteries. The IP3+Ry pool was small but of a similar size in both age groups.

Total Ca2+ mass and pool size based on 45Ca uptake and washout. Repeated cycles of contraction and relaxation rapidly labeled all intracellular Ca2+ pools (Fig. 7A). Half-maximal labeling was achieved with 0.9 and 1.2 cycles in adult and fetal arteries, respectively (P = not significant; Fig. 7B). Adult basilar arteries contained significantly less Ca2+ mass on a dry weight basis (2.7 nmol/mg) than did fetal basilar arteries (12.2 nmol/mg). In contrast, the rate constant of washout of 45Ca was similar in adult and fetal arteries and averaged 0.016 ± 0.001 and 0.013 ± 0.002 s-1, respectively.


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Fig. 7.   Estimates of Ca2+ mass in ovine adult and fetal basilar arteries. The relations between Ca2+ content and the number of contraction cycles (A) were used to obtain estimates of the maximum Ca2+ content and the number of contraction cycles required to reach half-maximal Ca2+ content (Kc) (B). Although values of Kc did not vary significantly with age, maximum Ca2+ content was significantly smaller in adult than fetal basilar arteries. Vertical error bars indicate SE for 6 adults and 7 fetuses. *Significant difference at P < 0.05.

To estimate the Ca2+ mass in each pool, the relative pool size (Fig. 6) was multiplied by the intracellular Ca2+ mass (Fig. 7B). The resulting absolute size of the pool sensitive to IP3 only was similar in the adult (1.6 nmol/mg) and fetus (1.7 nmol/mg) (Fig. 8). All other pools, however, were significantly smaller in adult than fetal arteries. The pool sensitive to ryanodine only averaged 0.11 and 1.22 nmol/mg in adult and fetal arteries, respectively. Corresponding values for the pool sensitive to both ryanodine and IP3 were 0.14 and 0.85 nmol/mg. The most dramatic age-related difference in pool size was obtained for the pool resistant to both IP3 and ryanodine; here, the masses averaged 0.87 and 8.78 nmol/mg for adult and fetal basilar arteries, respectively.


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Fig. 8.   Relative ryanodine- and IP3-sensitive pool sizes in ovine adult and fetal basilar arteries. Ca2+ pool sizes were calculated as the product of fractional pool size (Fig. 6) and total intracellular Ca2+ content (Fig. 7). The absolute size of the IP3 pool was similar in adult and fetal arteries, but all other pools were significantly smaller in adult than in fetal basilar arteries. Values are means and SE for 11 adults and 12 fetuses. *Significant differences at P < 0.05.


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RESULTS
DISCUSSION
REFERENCES

The distribution of Ca2+ within vascular smooth muscle is highly heterogeneous and dynamic and varies with artery type, size, and location (20, 29). Studies of this distribution have identified multiple Ca2+ pools, the most important being those sensitive to IP3 or ryanodine. The relative size and independence of these pools remain uncertain, however, as some studies suggest that IP3 and ryanodine release a common Ca2+ pool (30) whereas others suggest that IP3 and ryanodine release functionally and spatially distinct Ca2+ stores (18) (43). In addition, the impact of vascular maturation on these stores is uncertain, although it is clear that Ca2+ distribution and handling are considerably different in the contractile and synthetic smooth muscle phenotypes (21, 44) and that contractility changes considerably during postnatal maturation (31). In relation to these issues, the present results offer two main findings. First, there are at least four functionally independent calcium stores in fetal and adult basilar arteries. Second, the relative sizes of each of these stores change significantly with postnatal development.

Maturation and cerebrovascular intracellular Ca2+ mass. An essential step for measurement of absolute Ca2+ pool size was determination of total intracellular Ca2+ mass. The nonlinear tracer kinetic approach we used enabled estimates of mass in adult arteries (2.7 nmol/mg dry wt) consistent with published values (1-3 nmol/mg dry wt) (32) and also provided the first known estimates of fetal basilar Ca2+ content. From contractility measurements, Nakanishi et al. (27) concluded that intracellular Ca2+ content must be much smaller in adult than fetal mesenteric arteries. Brunette (5) also reported that accelerated skeletal development in late fetal and early postnatal life is supported by increased active transport of Ca2+ across the placenta, which more than doubles tissue Ca2+ during late gestation to values markedly greater than in adults. Against this background, our observation that basilar Ca2+ content averaged more than fourfold less in adult than in fetal basilar arteries appears reasonable and consistent with published evidence.

IP3-sensitive Ca2+ pool. Relative to total releasable Ca2+, the IP3-sensitive pool accounted for a much larger Ca2+ fraction in adult (64%) than in fetal (19%) arteries even though mass within the IP3-sensitive pool was equivalent in adult and fetal arteries. Because pD2 values for IP3-induced Ca2+ release agreed with published values (15) but did not vary with age, the IP3 receptors involved were probably of the same type and phosphorylation state in both age groups (41). Given that IP3 receptor density changes little during ovine cerebrovascular maturation (49), the ability of IP3 to release Ca2+ appears unaffected by maturation in ovine basilar arteries. Thus age-related changes in IP3-induced Ca2+ release cannot explain the observed age-related differences in the coupling between IP3 concentration and contractile response (Fig. 2). Because IP3-induced Ca2+ release was similar at 150 and 300 nM Ca2+ (Fig. 4), differences in sensitivity to ambient Ca2+ concentration within the low physiological range also cannot explain age-related differences in the contractile efficacy of IP3 (15, 19).

If sensitivity to IP3 and the mass of Ca2+ released by IP3 are similar in adult and fetal basilar arteries, then differences in coupling between IP3 and contraction must involve some component downstream from Ca2+ release. This downstream component cannot be myofilament Ca2+ sensitivity, because this is significantly less in adult than in fetal arteries, particularly during agonist-stimulated contractions (1). Alternatively, Ca2+ release geometry may be more favorable for myofilament activation in adult than in fetal basilar arteries. Consistent with this possibility, smooth muscle cells have very different dimensions and volumes in adult and fetal ovine cerebral arteries (7). In addition, if IP3-induced Ca2+ release is similar but depletion of the IP3-sensitive pool is slower in adult than in fetal arteries (Fig. 2), the fetal SR must be less able to resequester released Ca2+. Correspondingly, Ca2+ transport protein (sarcoplasmic or endoplasmic reticulum Ca2+-ATPase, SERCA) abundance in cardiac SR is greater in adult than in fetal sheep (25).

Ryanodine-sensitive Ca2+ pool. Ryanodine produced concentration-dependent increases in cytosolic Ca2+ with similar pD2 values in both age groups (Fig. 3), suggesting that the receptor involved was probably the same in both adult and fetal arteries. Because ryanodine released Ca2+ slowly (Fig. 1) and can hyperpolarize the plasmalemma through activation of Ca2+-sensitive potassium channels (17), we also used caffeine to release Ca2+ from the ryanodine-sensitive pool. Caffeine and ryanodine released Ca2+ from a common pool (Fig. 1; Ref. 20), although they activate the ryanodine receptor differently (48). Nonspecific effects of caffeine on adenosine receptors (4), phosphodiesterase (42), ion channels (13), or IP3 receptors (24) should have been negligible at the caffeine concentration we used, particularly because caffeine was thoroughly washed out before subsequent treatments. Consistent with this view, the pool size estimates obtained with ryanodine (Fig. 3) and caffeine (Fig. 5) experiments were quite similar.

The ryanodine-sensitive pool contained a smaller fraction of total Ca2+ and less mass in adult compared with fetal basilar arteries (Figs. 6 and 8). Correspondingly, magnitudes and rates of ryanodine- and caffeine-induced contractions were significantly less in adult than fetal basilar arteries (Fig. 1), although some of this difference might be accounted for by reduced myofilament Ca2+ sensitivity in adult arteries (1). Ryanodine-induced contractions required extracellular Ca2+, as also recently reported for ovine middle cerebral artery (22). Thus ryanodine-induced calcium release stimulated Ca2+ entry either directly or through store-operated Ca2+ entry, as recently reported in renal smooth muscle (9). Whereas the small ryanodine-sensitive pool in adult arteries might easily be depleted and thus be likely to stimulate capacitative Ca2+ entry, this is less likely in fetal arteries, where the ryanodine-sensitive pool was markedly larger and thus harder to deplete. Age-related differences in ryanodine-sensitive pool size further suggest that the capacity for Ca2+-induced Ca2+ release should be less in adult than fetal basilar arteries. Undoubtedly, these age-related differences in ryanodine-sensitive Ca2+ release are species dependent, as ryanodine receptors are completely nonfunctional at birth in rat cerebral arteries (11) and are absent in proliferating cells of the rat aorta (44).

Overlap between IP3- and ryanodine-sensitive Ca2+ pools. Despite reported overlap between ryanodine- and IP3-sensitive pools (19, 30), other evidence suggests that these pools are functionally distinct (18, 43). More IP3 receptors are found in central than in peripheral locations (47). Only ryanodine-sensitive Ca2+ pools near the sarcolemma mediate Ca2+-induced Ca2+ release (19), Ca2+ spark formation (17), and superficial buffer barrier function (19). Consistent with this functional specialization, caffeine failed to deplete the IP3-sensitive pool and IP3 did not deplete the caffeine-sensitive pool (Fig. 5) in ovine basilar arteries. Furthermore, the pool sensitive to both IP3 and ryanodine averaged only 5-7% of total releasable Ca2+ regardless of age (Fig. 6). These data suggest functional separation between the IP3- and ryanodine-sensitive Ca2+ pools in the ovine basilar artery. Consistent with the smaller ryanodine-induced contractions in adult arteries (Fig. 1), the pool sensitive to both IP3 and ryanodine was smaller in adult (0.14 nmol/mg) than in fetal (0.85 nmol/mg) basilar arteries. This latter difference reflects a trend for less mass in adult than fetal artery Ca2+ pools.

IP3- and ryanodine-resistant Ca2+ pool. Ca2+ mass in the pools insensitive to IP3 and ryanodine was 10-fold smaller in adult than in fetal arteries (Fig. 8) but accounted for a major fraction of membrane-bound Ca2+ in both age groups (Fig. 6). The extra fetal Ca2+ was probably not within the mitochondria (39), golgi (46), or nucleus (10) and instead was most probably located within the endoplasmic reticulum (ER), which contains most second messenger Ca2+ in noncontractile cells (33). Consistent with this possibility, ER volume is generally smaller in mature than in immature arteries (27), which, in turn, reflects less synthetic activity and more smooth muscle cells with a contractile phenotype in adult compared with fetal arteries (28). Alternatively, this large fetal Ca2+ store may simply be an SR fraction without functional IP3 or ryanodine receptors. In either case, vascular maturation and differentiation appear to be associated with loss of approximately two-thirds of all fetal vascular Ca2+, most of which is lost from a membrane-bound Ca2+ pool insensitive to either IP3 or ryanodine. A major consequence of this large fetal noncontractile pool was that it dramatically reduced the fraction of cell Ca2+ in the IP3-sensitive pool, even though the mass of IP3-releasable Ca2+ was the same in fetal and adult arteries.

Functional implications. Similar to values reported for other preparations (12, 37), organellar Ca2+ concentrations averaged 141 and 62 µM in adult and fetal basilar arteries, respectively. These concentrations, combined with the observation that total organellar Ca2+ mass was much smaller in adult than fetal arteries, predicts that total organellar volume must be dramatically smaller in adult than fetal basilar arteries. The large fetal organellar volume, which possibly includes ER or SR without functional IP3 or ryanodine receptors, appears to attenuate the ability of IP3-released Ca2+ to induce contraction, even though the Ca2+ mass released is similar to that in the adult. Restriction of diffusion or compartmentalization (20, 36) may limit Ca2+ activation of contractile proteins less in mature than in immature arteries. Conversely, ryanodine-evoked Ca2+ release appears less effectively coupled to contraction in adult than in fetal arteries, perhaps because of a larger ryanodine-sensitive pool in fetal arteries. The larger fetal ryanodine-sensitive pool further suggests that Ca2+-induced Ca2+ release and Ca2+ spark formation may be enhanced in fetal arteries, but this has not been found in other preparations (11), suggesting that an immature vascular morphology, which includes a large organellar volume, may also influence the distribution and function of Ca2+ released from ryanodine-sensitive pools. More importantly, the present data indicate that in the ovine basilar artery Ca2+ is distributed among four functionally independent stores, with little overlap between the IP3- and ryanodine-releasable pools. With maturation, intracellular Ca2+ mass falls dramatically in noncontractile pools, decreases modestly in ryanodine-sensitive pools, and remains relatively constant in IP3- sensitive pools, clearly indicating that postnatal development has a dramatic impact on both the function and distribution of Ca2+ in ovine basilar arteries.


    FOOTNOTES

Address for reprint requests and other correspondence: W. J. Pearce, Center for Perinatal Biology, Loma Linda Univ. School of Medicine, Loma Linda, CA 92350 (E-mail: wpearce{at}som.llu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 22 August 2000; accepted in final form 12 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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