Introduction

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Airway hyperresponsiveness (AHR) is a defining characteristic of asthma. It is exemplified by increased sensitivity of airways to contractile stimuli and by excessive airway narrowing in vivo. Although various mechanisms have been proposed for AHR (1), enhanced airway smooth muscle (ASM) contractility is a plausible explanation (6). We have chosen to examine a well characterized rat model of AHR out of the several animal models that have been described (6) because we wished to study the behavior of innately hyperresponsive airways. Fisher rat airways are more responsive to methacholine and serotonin (5HT) inhalational challenge in vivo than are the airways of Lewis rats and thus show a hyperresponsiveness that is not agonist specific (9).

The AHR in the Fisher rat has been confirmed to be associated with enhanced airway responses ex vivo. Excised tracheal rings from Fisher rats generate greater isometric tension and Fisher intraparenchymal airways constrict faster and to a narrower diameter than do corresponding Lewis preparations to the same agonists (10). These latter observations, coupled with the lack of receptor specificity, suggest that AHR in the rat is likely associated with ASM that is intrinsically more contractile. Consistent with the notion that altered ASM properties cause exaggerated airway responses in the rat, we have previously demonstrated that the hyperresponsive Fisher ASM cells display enhanced intracellular Ca²⁺ ([Ca²⁺]_i) transients after stimulation by 5HT and bradykinin (13, 14). Consequently, we have focused our attention on some of the possible mechanisms of the differences in Ca²⁺ signaling because the Ca²⁺ transient initiates contraction by activating actomyosin crossbridge cycling. Although smooth muscle contractility can be modulated by Ca²⁺-independent mechanisms, alterations in Ca²⁺ signaling should have important consequences for contractility. We reasoned that as the Ca²⁺ transients in Fisher myocytes are amplified in response to multiple agonists, the mechanisms underlying this response are not receptor specific but occur downstream of agonist-receptor interaction.

In rat ASM, contractile agonists such as 5HT stimulate G-protein-linked plasma membrane receptors and activate phospholipase C (PLC), resulting in the production of inositol (1,4,5)trisphosphate (IP₃). IP₃ releases Ca²⁺ from intracellular stores by opening specific Ca²⁺ channels (IP₃R). Release of [Ca²⁺]_i triggers capacitative entry of extracellular Ca²⁺, which prolongs the elevation of cytosolic Ca²⁺ levels and appears to be important for sustaining smooth muscle contraction (15, 16). Two major pathways of IP₃ metabolism have been described. One route results in the phosphorylation of IP₃ to inositol 1,3,4,5-tetrakisphosphate (IP₄) by IP₃-specific 3-kinase. IP₄ has been implicated in gating Ca²⁺ influx at the plasma membrane of certain cells, but the ubiquity of this signaling pathway is still controversial. The other route leads to the dephosphorylation of the 5-position phosphate by a family of enzymes known as inositol polyphosphate 5-phosphatases; this is the preferred pathway of IP₃ metabolism in many cell types, including ASM (17). The 43-kD 5-phosphatase I and 115-kD 5-phosphatase II appear to be the most active of the 5-phosphatases in hydrolyzing IP₃ and IP₄ (18). A third route of metabolism has been observed in ASM in which a Li⁺-sensitive 1-phosphatase generates inositol 4,5 biphosphate (I(4,5)P₂), but its contribution is negligible compared with the 3-kinase and 5-phosphatase pathways (17). Because IP₃ controls the mobilization of [Ca²⁺]_i, we postulate that the differences in [Ca²⁺]_i mobilization between Fisher and Lewis airway myocytes result from differences in IP₃ availability that are due to differences in IP₃ metabolism. IP₃R sensitivity was also compared between the rat strains to exclude this as an additional source of [Ca²⁺]_i differences.

	Materials and Methods

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Animals

Male Lewis and Fisher 344 rats, age 7 to 9 wk, were purchased from Harlan Sprague Dawley (Indianapolis, IN) and were housed in a conventional facility at McGill University (Montreal, PQ, Canada). The experimental protocols were approved by an institutional animal ethics committee.

ASM Cell Culture

First-passage, primary cultures of Fisher and Lewis ASM cells were obtained as previously reported (19). Briefly, cells were isolated from excised tracheas by enzymatic digestion with 0.2% collagenase IV and 0.05% elastase, and were cultured in 1:1 Dulbecco's modified Eagle's medium (DMEM):Ham's F12 supplemented with 10% fetal bovine serum (FBS). When the cells reached confluence, they were subcultured for experimentation. For measurements of Ca²⁺, cells were grown on 25-mm-diameter glass coverslips; otherwise, cells were plated directly onto plastic culture dishes. The cells were confirmed to be smooth muscle by typical morphology, positive immunohistochemical staining with a murine smooth muscle-specific -actin antibody, and the presence of a contractile response to 5HT.

Measurement of [Ca²⁺]_i

[Ca²⁺]_i was measured as previously reported (20). To determine the effect of PLC inhibition on 5HT-mediated Ca²⁺ responses, cells were treated with the PLC blocker U73122 or its inactive analogue U73343.

To compare the sensitivity of the IP₃R between Fisher and Lewis ASM, fura-2-loaded cells were washed with Hanks' buffer without Ca²⁺ and then permeabilized with 200 µM -escin. After removal of -escin, a plastic ring was sealed to the center of the coverslip with vacuum grease and filled with 50 µl of Hanks' buffer without Ca²⁺ to establish a new baseline. For experiments determining the ligand sensitivity of the IP₃R, 25 µl of Hanks' buffer were withdrawn from inside the ring and replaced with 25 µl of IP₃ containing buffer.

Measurement of myo-[³H]Inositol Polyphosphates

The assay of myo-[³H]inositol polyphosphates ([³H]IP) was adapted from a previous report (21). Confluent, first-passage cells were growth arrested with inositol-free DMEM supplemented with 1% FBS and 1 µCi/ml of myo-[³H]inositol (10 to 20 Ci/mmol) for 48 h. To measure the production of total myo-[³H]IP, the cells were preincubated with 10 mM LiCl in Krebs-Hensleit buffer (117 mM NaCl, 4.7 mM KCl, 1.1 mM MgSO₄, 1.2 mM KH₂PO₄, 20 mM NaHCO₃, 2.4 mM CaCl₂, 1 mM glucose, 20 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid [Hepes]) at 37°C for 10 min then challenged with increasing concentrations of 5HT in the presence of LiCl for 10 min at 37°C. Reactions were terminated by the addition of 200 µl of ice-cold 3 M trichloroacetic acid followed by immediate cooling of the dishes on ice. Cells originating from one animal were challenged in triplicate for each test. The cells were scraped off the bottom of the dishes and sonicated. The supernatant was separated from the cellular debris by centrifugation at 4,000 × g for 20 min at 4°C. The supernatant was treated with 0.25:1 vol/vol of 10 mM ethylenediaminetetraacetic acid and then with 1:1 vol/vol of fresh 50% vol/vol trichlorotrifluoroethane/tri-n-octylamine. The aqueous and organic layers were separated by centrifuging at 4,000 × g for 10 min at 4°C. The aqueous layer containing the myo-[³H]IP was neutralized with 0.2:1 vol/vol of 60 mM NaHCO₃ and then assayed for total myo-[³H]IP activity by anion exchange chromatography. Samples were loaded onto columns containing 1 ml formate form AG 1-X8, and myo- [³H]inositol and myo-[³H]glycerophosphoinositide were removed by washing with a solution of 60 mM ammonium formate and 5 mM sodium tetraborate. Myo-[³H]IP were eluted with 12 ml of a solution containing 1 M ammonium formate and 100 mM formic acid. A 2-ml aliquot of the myo-[³H]IP was diluted 1:1 vol/vol with distilled water and quantitated by -counting.

To measure individual myo-[³H]IP species, individual culture dishes were challenged separately with 1 µM 5HT for 4, 6, 8, 10, 15, 20, or 30 s and the triplicates of each time point were pooled. The myo-[³H]IP were extracted and the individual species were separated by reversed-phase, ion-pair high performance liquid chromatography (HPLC) modified from Patthy and coworkers (22) using two Waters model 501 pumps (Waters Corp., Milford, MA) and a gradient controller. A µBondapak C18 guard pak (Waters Corp.) and Nucleosil 5µm C18 250×4.6-mm reversed phase column (Phenomenex, Torrance, CA) were equilibrated for 20 min with solvent A (10 mM tetrabutylammonium hydrogen sulfate [TBAHS], pH 5.0) at a flow rate of 1 ml/min. The sample was loaded onto the column in 100% solvent A and eluted with a gradient consisting of increasing amounts of solvent B (10 mM TBAHS and 35 mM KH₂PO₄ at pH 5.0, plus 30% acetonitrile) in solvent A as follows: 0 min, 0% B; 24 min, 50% B; 49 min, 50% B; 52 min, 100% B; 67 min, 100% B. Fractions were collected every minute and counted. Fractions containing the myo-[³H]IP species of interest were identified using authentic [³H]-labeled standards.

Measurement of IP₃-Specific 5-Phosphatase Activity

IP₃-specific 5-phosphatase activity was measured using a modification of a procedure described in the literature (23). After 48 h of growth arrest, first-passage Lewis and Fisher ASM cells were scraped off the culture dishes in TKM buffer (50 mM Tris, pH 7.4, 25 mM KCl, 5 mM MgCl₂, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol [DTT], 0.03 U/ml aprotinin, 2 µg/ml leupeptin). The buffer and detached intact cells from all the dishes were pooled and the cells were isolated from the buffer by spinning at 500 × g for 6 min at 4°C. The pellet was resuspended with 10 µl/mg protein of TKM and then sonicated for three 30-s intervals with a micro-ultrasonic cell disrupter (Kontes, Vineland, NJ). The suspension was microcentrifuged at 1,000 × g for 15 min at 4°C, and the resulting supernatant was diluted 10 times with TKM. This new volume was further diluted 4.25 times with incubation buffer (IB; 50 mM Hepes/KOH, pH 7.0, 1 mM MgCl₂, 1 mM DTT) and the final solution was ultracentrifuged at 100,000 × g for 60 min at 4°C. The supernatant containing the soluble 5-phosphatase was recovered while the pellet containing the particulate 5-phosphatase was washed, homogenized, and resuspended in the same proportional volumes of TKM and IB as in the supernatant. Both fractions were assayed for 5-phosphatase activity immediately after extraction. Substrate solutions contained 20,000 dpm [³H]IP₃ (20 to 60 Ci/mmol) and varying concentrations of unlabeled IP₃ resulting in final concentrations of 0.83, 2.5, 7.4, 22.2, 66.7, and 200 µM IP₃. The enzyme solution (170 µl of the particulate or soluble fractions) was incubated with 1 mM levamisole for 5 min at 37°C to inhibit alkaline phosphatase (24). Substrate solution (20 µl) was added and the reaction was terminated 10 min later by adding 50 µl of ice-cold 1 N H₃PO₄. Preliminary experiments had demonstrated linear accumulation of inositol 1,4 biphosphate (I(1,4)P₂) in both the particulate and cytosolic fractions of both rat strains over at least 20 min (data not shown). After resting on ice for 30 min, the mixture was neutralized with 50 µl of 1 N NaOH. 5-Phosphatase activity at each concentration of cold IP₃ was assayed in triplicate for cells originating from a single animal.

myo-[³H]inositol 1,4 biphosphate ([³H]IP₂) was detected by anion exchange chromatography as described previously with some minor modifications. After removing trace amounts of [³H]inositol with 16 ml of 60 mM ammonium formate/5 mM disodium tetraborate, [³H]IP₂ was eluted with 20 ml of 400 mM ammonium formate/100 mM formic acid. [³H]IP₃ was subsequently eluted with 12 ml of 1 M ammonium formate/100 mM formic acid.

Measurement of Type I and Type II 5-Phosphatase Expression

Particulate and soluble 5-phosphatases were crudely purified as described and compared by Western blot analysis. Each sample lane contained either 5 µg protein for blotting with anti-5-phosphatase I or 100 µg protein for blotting with anti-5-phosphatase II. The samples were loaded onto a precast Novex 4 to 12% triglycine gel and run at 130 mV with a buffer containing Tris base, glycine, and 0.1% sodium dodecyl sulfate in a Novex electrophoresis system (Helixx Technologies Inc., Scarborough, ON, Canada). The proteins were transferred onto a nitrocellulose membrane using a 250 mA current for 2 h at 4°C in a transfer solution containing NaHCO₃, Na₂CO₃ and 80% vol/vol methanol. The membrane was subsequently blocked overnight at 4°C with a solution containing 7% skim milk and 1% FBS in TTBS (NaCl, Tris base, 0.1% Tween 20) and then was incubated with either polyclonal anti-5-phosphatase I raised to the N terminal (1:10,000, generously donated by Dr. P. W. Majerus, Washington University School of Medicine, St. Louis, MO) or polyclonal anti-C-terminal anti-5-phosphatase II (1:20) (25) for 2 h at room temperature. After washing, the membranes were incubated with horseradish peroxidase-conjugated donkey antirabbit immunoglobulin (Ig)G (1:1,000) for 1 h at room temperature. Protein bands were developed with the EC200+ detection kit.

Reagents

Elastase IV, collagenase IV, -escin, IP₃, trichlorotrifluoroethane, tri-n-octylamine, TBAHS, levamisole, and general biochemical reagents were purchased from Sigma Chemical Company (Oakville, ON, Canada). Trypsin was purchased from Worthington (Freehold, NJ). DMEM and FBS were obtained from GIBCO (Burlington, ON, Canada). Fura-2AM and pluronic acid were supplied by Molecular Probes (Eugene, OR). U73122 and U73343 were obtained from Biomol Research (Plymouth Meeting, PA). [³H]IP₃ was supplied by Mandel (Guelph, ON, Canada). Myo-[³H]inositol and other [³H]IP standards, horseradish peroxidase-conjugated donkey antirabbit IgG, and the EC200+ detection kit were obtained from Amersham Life Sciences (Oakville, ON, Canada). AG 1-X8 anion exchange resin was supplied by Biorad Laboratories (Mississauga, ON, Canada). Novex gradient triglycine electrophoresis gels were purchased from Helixx Technologies Inc. (Scarborough, ON, Canada).

Statistical Analysis

Concentration-response curves were compared with analysis of variance, whereas single comparisons were tested with the Student's t test. P < 0.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Effect of PLC Inhibition on 5HT-Stimulated Ca²⁺ Mobilization

Fisher cells were treated with the PLC inhibitor U73122 or its inactive analogue U73343 to verify that 5HT stimulated PLC-dependent Ca²⁺ mobilization in rat ASM. When cells were pretreated with 50 µM of U73343 followed by stimulation with 1 µM 5HT, [Ca²⁺]_i rose significantly from 107.0 ± 13.6 to 351.5 ± 28.7 nM (P < 0.001). Pretreatment of cells with 50 µM U73122 completely abolished Ca²⁺ responses to 1 µM 5HT (baseline: 103.5 ± 10.4 nM; 1 µM 5HT: 107.7 ± 21.9 nM), confirming that 5HT mobilization of [Ca²⁺]_i in these cells was dependent on PLC.

Comparison of 5HT-Stimulated PLC Activity

As 5HT appeared to mobilize Ca²⁺ exclusively via PLC activation in rat ASM, we investigated whether the strain-related differences in Ca²⁺ mobilization arose from differences in PLC activity by comparing the accumulation of total [³H]IP in Lewis and Fisher ASM cells in response to increasing concentrations of 5HT. 5HT elicited a concentration-dependent increase in [³H]IP accumulation in both strains, but there was no difference in the maximal response between the strains (Lewis: 514.2 ± 76.2% baseline levels; Fisher: 451.7 ± 98.1% baseline). The EC₅₀ (log M) values for 5HT in Lewis (6.5 ± 0.3) and Fisher (5.9 ± 0.2) ASM were also similar.

5HT-Stimulated Formation of Inositol Polyphosphates

Agonist-induced IP₃ levels are the net outcome of PLC-mediated IP₃ production and of 3-kinase- and 5-phosphatase-mediated degradation. Measurements of total [³H]IP accumulation provides an index of PLC activity but it does not provide information on the magnitude of the IP₃ transient nor on the rate of IP₃ degradation. To assess these latter parameters, the time course of the formation and degradation of IP₃, IP₄, and Ins (1,4)P₂ after 5HT stimulation in each rat strain was evaluated. Cultured ASM cells radiolabeled with [³H]inositol were stimulated with 1.0 µM 5HT, and [³H]IP₂, [³H]IP₃, and [³H]IP₄ were separated by reversed-phase, ion-pair HPLC. In Lewis cells, neither [³H]IP₃ nor [³H]IP₄ were detectably elevated above baseline during the first 30 s after 5HT treatment (Figure 1, upper panel). [³H]IP₂, conversely, steadily increased to 166.9 ± 22.5% above baseline levels at 30 s after 5HT stimulation. In Fisher cells (Figure 1, lower panel), [³H]IP₃ peaked at 122.9 ± 5.7% above baseline levels 6 s after 5HT application. [³H]IP₂ rose steadily to 216.8 ± 16.9% baseline levels by 30 s after stimulation. There was no change in [³H]IP₄ levels in Fisher cells during the first 30 s after agonist administration, whereas IP₃ was undetectable in cells from either rat strain at these times. These latter results are consistent with IP metabolism in bovine ASM, which showed increased IP₄ and IP₃ only after 1 min of carbachol stimulation (26). A strain comparison of [³H]IP₃ levels during the first 10 s of 5HT stimulation is shown in the inset of the lower panel of Figure 1. At 6 s, the [³H]IP₃ transient was significantly higher (P < 0.05) in Fisher than in Lewis extracts.

View larger version (19K): [in this window] [in a new window]   Figure 1.   Production of specific IP isomers. Cells were prelabeled with myo-[3H]inositol for 48 h and challenged with 1 µM 5HT. [3H]inositol (1,2)P2, [3H]inositol (1,4,5)P3, and [3H]inositol (1,3,4,5)P4 were separated with reversed-phase, ion-pair HPLC. Lewis, upper panel (n = 5 animals, each sample run in triplicate); Fisher, lower panel (n = 6). The inositol species are identified in the upper panel for Lewis using open symbols, whereas the corresponding closed symbols are used for Fisher in the lower panel. Inset: Comparison of [3H]inositol (1,4,5)P3 production between Lewis (open squares) and Fisher cells (closed squares). *P < 0.05, Fisher versus Lewis.

IP₃-Specific 5-Phosphatase Activity

The lack of [³H]IP₃ accumulation yet abundant [³H]IP₂ production in Lewis ASM cells indicated that [³H]IP₃ had been produced but perhaps was metabolized too rapidly for the transient to be detectable. Conversely, the detectable [³H]IP₃ transient in Fisher myocytes suggested the possibility that [³H]IP₃ was not metabolized as rapidly. We therefore investigated whether IP₃-specific 5-phosphatase activity differed in Fisher and Lewis ASM cells. IP₃-specific 3-kinase activity was not examined because [³H]IP₄ levels remained relatively stable throughout the period of 5HT stimulation in both rat strains.

The kinetics (Vmax and Michaelis constant [K_m] for IP₃) of P₃-specific 5-phosphatase activity in the particulate versus cytosolic fractions from Lewis and Fisher ASM were compared. A representative Eadie-Hofstee plot of enzyme activities in the particulate fraction is shown in Figure 2. The Vmax for particulate 5-phosphatase activity was significantly lower in Fisher (15.1 ± 2.0 pmol IP₃ hydrolyzed/min/µg protein) compared with Lewis ASM (22.9 ± 1.7 pmol IP₃ hydrolyzed/min/µg protein; P < 0.05) (Figure 3, upper panels). In addition, the K_m of the enzyme for IP₃ of the particulate activity (Figure 3, lower panels) was also significantly lower in cells from Fisher (5.9 ± 1.1 µM) than from Lewis rats (10.8 ± 1.7 µM; P < 0.05). The Vmax (Fisher: 7.7 ± 0.9 pmol IP₃ hydrolyzed/min/µg protein; Lewis: 7.9 ± 0.7 pmol IP₃ hydrolyzed/min/µg protein) and the K_m (Fisher: 7.4 ± 1.2 µM; Lewis: 6.6 ± 0.7 µM) of soluble 5-phosphatase activity were similar between the two strains of rat (Figure 3, right panels). The Vmax of the soluble activity was significantly lower than the Vmax of the particulate activity from either strain (P < 0.001). However, the K_m of the soluble activity was similar to that of the particulate activity in Fisher ASM.

View larger version (17K): [in this window] [in a new window]   Figure 2.   Representative Eadie-Hofstee plot of IP3-specific 5-phosphatase kinetics in particulate fractions from Fisher (closed circles) and Lewis (open circles) ASM cells. Unstimulated cells were fractionated into particulate and cytosolic compartments, each of which was incubated with [3H]IP3 and increasing concentrations of unlabeled IP3. 5-Phosphatase activity was assessed by production of [3H]inositol (1,4)P2. Vmax = y-intercept, Km = slope.

View larger version (19K): [in this window] [in a new window]   Figure 3.   Kinetics of IP3-specific 5-phosphatase activity in particulate and cytosolic compartments of ASM homogenates. Km, upper panels; Vmax, lower panels; particulate fraction, left panels; soluble fraction, right panels; Lewis, open bars (n = 5 animals, each sample run in duplicate); Fisher, solid bars (n = 5). *P < 0.05.

Expression of Type I and Type II 5-Phosphatases

The protein expression of 5-phosphatase I and 5-phosphatase II isozymes was compared in the particulate and cytosolic fractions of Lewis and Fisher ASM cells by Western blot analysis. The left panels in Figure 4 illustrate the differences in band density of particulate type I (Figure 4A) and II isozymes (Figure 4B) between the two strains of rat. Although 5-phosphatase I was originally purified as a 43-kD enzyme, the protein predicted by cloning studies suggests a slightly larger molecular mass (27, 28). Indeed, we detected an immunoreactive protein migrating at approximately 49 kD (Figure 4A, left panel). Densitometric analysis reproducibly demonstrated that the expression of 5-phosphatase I in the particulate fraction of Fisher ASM extracts was significantly lower than in Lewis extracts (n = 4, Fisher = 36.9 ± 11.2% of Lewis density; P < 0.05). No 5-phosphatase I was detected in the cytosolic fraction of ASM extracts from either Lewis or Fisher rats, which is consistent with the enzyme's predominantly membrane localization (29).

View larger version (27K): [in this window] [in a new window]   Figure 4.   5-Phosphatase expression in the particulate fraction of Fisher and Lewis ASM cell homogenates. (Upper panels) Type I 5-phosphatase. (Left) Representative immunoblot of type I 5-phosphatase. A total of 5 µg of protein was loaded in each lane. Molecular weight markers are indicated left of the blot, and the band of interest is indicated with an arrow right of the blot. (Right) Type I 5-phosphatase levels averaged from extracts from four different rats in each strain (each extract run in duplicate), expressed as % optical density of Lewis fractions (P < 0.005). (Lower panels) Type II 5-phosphatase. (Left) Representative immunoblot of type I 5-phosphatase. A total of 100 µg of protein was loaded in each lane. Molecular weight markers are indicated left of the blot, and the band of interest is indicated with an arrow right of the blot. (Right) Type II 5-phosphatase levels averaged from three different rats in each strain (each extract run in duplicate), expressed as % optical density of Lewis fractions (P < 0.05). Lewis, open bars; Fisher, solid bars.

5-Phosphatase II protein expression was also reproducibly elevated in the particulate fraction of Lewis extracts as shown by increased levels of the 115-kD polypeptide (n = 3, Fisher = 52.6 ± 17.1% of Lewis density; P < 0.05) (Figure 4B, right panel). In contrast, the soluble fraction of both Fisher and Lewis cell extracts demonstrated a variety of immunoreactive proteins (data not shown), but the significance of these bands is uncertain. However, at least three distinct isoforms have been proposed to be encoded by the 5-phosphatase II gene, and in addition, this 5-phosphatase appears extremely sensitive to proteolytic cleavage in the cytosolic fraction of the cell (32).

Sensitivity of Ca²⁺ Release to IP₃

Although we could not distinguish which 5-phosphatase isozyme was more important in regulating the Ca²⁺ signals in the Fisher and Lewis ASM cells, we nonetheless wished to determine the relevance of 5-phosphatase activity to the differences in Ca²⁺ responses between the two strains of rat. Because IP₃-mediated Ca²⁺ release from intracellular stores also depends on the density, sensitivity, isoform, and allosteric regulation (33) of the IP₃R, we compared Ca²⁺ release to exogenous IP₃ as a measure of the sensitivity and responsiveness of the IP₃R to its ligand. Potential regulation of this response by the 5-phosphatases was suppressed by administering saturating concentrations of substrate. Cells were permeabilized with -escin and Ca²⁺ was released from intracellular stores with increasing concentrations of exogenous IP₃ (Figure 5). Baseline [Ca²⁺]_i after cell permeabilization was similar between Lewis and Fisher ASM (149.3 ± 8.4 and 157.3 ± 8.0 nM, respectively) (Figure 5, inset). Increasing IP₃ from 0.01 to 1.0 mM caused similar concentration-dependent increases in [Ca²⁺]_i in ASM from both strains of rat. These results suggest that the IP₃R itself does not appear to play any role in the [Ca²⁺]_i differences between the rat strains.

View larger version (16K): [in this window] [in a new window]   Figure 5.   Sensitivity of [Ca2+]i release to inositol (1,4,5)P3. Cells permeabilized with -escin were stimulated with exogenous Inositol (1,4,5)P3. Changes in [Ca2+]i were monitored spectrofluorometrically and are represented as nM increase above baseline. Inset depicts baseline [Ca2+]i after permeabilization. Lewis, open circles (n = 5 to 7 samples from three rats); Fisher, closed circles (n = 5 to 6).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we sought to elucidate the mechanism underlying exaggerated Ca²⁺ mobilization by 5HT in Fisher ASM cells compared with Lewis ASM cells. Our results suggest that higher IP₃ levels account for the differences in evoked Ca²⁺ responses between the strains. We postulated that differences in the metabolism of IP₃ rather than its production account for the differences in IP₃ transients and Ca²⁺ mobilization between Fisher and Lewis ASM cells. Our present data support this postulate through the finding of lower activity and protein expression of the two IP₃-specific 5-phosphatase isozymes in Fisher cells, which likely determine the rate of degradation of IP₃ in ASM cells.

Higher agonist generation of IP₃ in ASM has been reported in allergic and maturational animal models of AHR (8, 23), and an underlying mechanism has been explored in the maturational model. These investigators attributed the higher IP₃ transients in the ASM of hyperresponsive, immature rabbits to lower IP₃ 3-kinase activity (23). However, detailed analysis of agonist-triggered IP metabolism in bovine ASM revealed that > 85% of IP₃ degradation proceeds through the 5-phosphatase pathway (17), and indeed, even in the maturational studies, the Vmax of the 5-phosphatase was at least two orders of magnitude higher than the Vmax of the 3-kinase, irrespective of age. Although the 3-kinase does have a lower K_m for the substrate, the differences in affinity between the two enzymes are likely irrelevant when IP₃ levels rapidly rise and saturate the enzymes after receptor activation. In addition, its lower K_m indicates that the 3-kinase would saturate more easily, which would further limit its ability to compete with the 5-phosphatase for substrate. These observations, as well as our present analyses using rat myocytes, indicate that the 5-phosphatase dominates IP₃ metabolism in ASM. Furthermore, the delayed formation of IP₄ in ASM (26) indicates delayed 3-kinase activity, which is incompatible with the immediate and rapid metabolism of the IP₃ transient by this pathway. We therefore investigated whether the greater accumulation of IP₃ in Fisher ASM cells was specifically due to lower metabolic activity of the 5-phosphatase. Although the K_m and Vmax of IP₃-specific 5-phosphatase activity were similar in the cytosolic fractions of cell extracts from both rat strains, both the Vmax and K_m were significantly lower in the particulate fraction of Fisher extracts than in Lewis extracts. We believe that differences in the particulate compartment alone could account for the differences in the IP₃ transient between Fisher and Lewis myocytes because previous reports have localized the majority of IP₃-specific 5-phosphatase activity (70 to > 90%) to membranes (29). The membrane therefore appears to be the primary site of IP₃ inactivation, consistent with a strategic location for the enzyme designed to tightly regulate this second messenger (34).

The lower Vmax suggested that there is quantitatively less 5-phosphatase in Fisher cell membranes, whereas the nearly twofold difference in K_m indicates that different isozymes are present or expressed at different levels in the two rat strains. To validate this, we compared the protein expression of two 5-phosphatase isozymes. The IP 5-phosphatases have been classified into four subfamilies according to the specific substrates hydrolyzed (35). Only the type I and type II 5-phosphatases hydrolyze IP₃. The type I subfamily to date consists of only one enzyme 43 to 49 kD (5-phosphatase I) that is predominantly membrane located via farnesylation of the C-terminal CAAX motif (13, 29, 34). Several type II isozymes have been well described and include the 75-kD 5-phosphatase synaptojanin and OCRL, the product of the gene mutated in Lowe's oculocerebrorenal syndrome (36).The latter 5-phosphatase predominantly hydrolyzes phosphatidyl inositol (4,5)P₂, and although synaptojanin metabolizes IP₃, the kinetics for this reaction have not been reported (18, 37, 38). The 75-kD type II isoenzyme (designated 5-phosphatase II) (29, 39) is clearly an active IP₃-specific 5-phosphatase that is expressed in lung (25). Recently, it was shown that this isoform may result from proteolysis of a 115-kD enzyme that is predominantly membrane associated (25). We detected both 5-phosphatase I and II in the particulate fractions of Fisher and Lewis extracts, but only the 5-phosphatase II isoform in the cytosolic fractions. Cytosolic IP₃-specific 5-phosphatase activity in both rat strains is therefore solely dependent on 5-phosphatase II. In the particulate compartment, levels of both isozymes were higher in Lewis than in Fisher rats. This was consistent with the hypothesis that the higher Vmax in Lewis rats is due to higher protein levels, whereas the interstrain differences in K_m arise from differential isozyme expression. Unfortunately, the relative expression of each isozyme in either strain could not be determined from our present data.

An alternative possibility is that allosteric regulation of 5-phosphatase activity differs between the two rat strains. 5-Phosphatase type I activity is enhanced either by complex formation with phosphorylated pleckstrin in human platelet cytosol or after binding to the scaffolding protein 14-3-3 (40, 41). Both of these modulations increase the Vmax without altering the K_m of the enzyme for IP₃. However, our findings likely reflect the unregulated kinetics of the 5-phosphatases because enzyme activity was assayed in crude lysates in which the normal structural architecture necessary for complex formation may be absent.

We next investigated whether the different [Ca²⁺]_i signals in Fisher and Lewis airway myocytes were associated with differential 5-phosphatase activity, and if so, whether this could be the sole mechanism for the different Ca²⁺ signals or whether there were additional strain-related differences in the function of the IP₃R. The latter hypothesis was rejected based on the similar Ca²⁺ responses of permeabilized Fisher and Lewis myocytes to excess exogenous IP₃ at concentrations that saturated 5-phosphatase activity. The different 5-phosphatase activities thus appeared to be the sole mechanism accounting for the interstrain differences in Ca²⁺ release. Several studies support the plausibility of this argument. Various molecular manipulations of the 5-phosphatase I in situ have shown it capable of modulating Ca²⁺ signaling at the level of IP₃-mediated Ca²⁺ release (34, 42, 43).

We previously reported that enhanced Ca²⁺ mobilization appears to be related to the greater contractility of Fisher ASM that is in turn associated with AHR. In the present study, we observed interstrain differences in the dynamics of the IP₃ transient as well as lower activity and expression of membrane-associated 5-phosphatases in Fisher than in Lewis airway myocytes. We propose that heterogeneous expression of the IP₃-specific 5-phosphatases governs the differences in [Ca²⁺]_i mobilization in Fisher and Lewis ASM cells and consequently may be a determinant of AHR.