INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Mesangial cells (MCs) normally contract in response to specific calcium (Ca²⁺)-mobilizing vasoconstrictor agonists (1), an effect that can be antagonized in a cyclic guanosine monophosphate (cGMP)-dependent manner by nitric oxide (NO) or exogenous nitrovasodilators (1, 4). Endotoxin (lipopolysaccharide, LPS) or a variety of inflammatory cytokines may induce mesangial expression of inducible NO synthase (iNOS) (7), leading to NO synthesis and mesangial relaxation (5). By analogy with vascular smooth muscle, impaired mesangial contraction following LPS exposure is presumed to depend on NO-stimulated increments in cGMP, leading to decreased availability of intracellular calcium (Ca²⁺_i) (6, 12); alternatively, cGMP may diminish Ca²⁺ sensitivity of the contractile apparatus (12). In addition to stimulating the efflux and resequestration of Ca²⁺_i, NO may act in some preparations, via cGMP-mediated or -independent processes, to limit Ca²⁺ influx by augmenting potassium (K⁺) channel-mediated hyperpolarization (13). We sought to explicitly test the hypotheses that LPS could impair agonist-induced Ca²⁺ mobilization in rat MCs to a degree that might explain mesangial relaxation, and that this impairment would depend on iNOS induction and augmented NO synthesis.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Reagents

Escherichia coli LPS (strain 055:B5) was obtained from Difco Laboratories, Detroit, MI; fetal bovine serum (FBS) from HyClone Laboratories, Logan, UT; fura-2 acetoxymethylester (fura-2 AM) and Ca²⁺ standards from Molecular Probes, Eugene, OR; and all other chemicals from Sigma Chemicals, St. Louis, MO.

Cell Culture

Rat MCs were isolated by subculture from glomerular explants, and identified by their morphology on light microscopy and by immunoperoxidase staining for myosin fibrils (14). They were grown on a plastic substratum (25 cm² flasks; GIBCO BRL, Life Technologies, Gaithersburg, MD) in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS, 25 mM glucose, 6 mM L-glutamine, 100 U/ml penicillin G, and 0.1 mg/ml streptomycin. Unless otherwise stated, incubation medium was routinely supplemented with 0.1 mM L-arginine in all experimental groups (which increased the L-arginine concentration from 0.5 mM to 0.6 mM), in order to prevent substrate limitation of iNOS activity. Cells were maintained at 37° C in a 5% CO₂ / 95% O₂ environment, passaged every 5 to 7 d, and used for experiments at 1 to 3 d after confluence, between passages 17 and 42.

Measurement of Intracellular Calcium Concentration

Confluent MCs on eight-well Lab-Tek chambered coverglass slides (Nunc, Inc., Naperville, IL) were incubated overnight (20 to 24 h) in medium (described earlier; 400 µl per well) with or without added combinations of LPS (0.01 to 1.0 mg/ml), N-nitro-L-arginine methyl ester (L-NAME, 0.1 to 1.0 mM), L-arginine (0.1 mM), indomethacin (0.01 mM), and meclofenamate (1 µM). Following incubation, medium was aspirated and frozen (20° C) for subsequent nitrite measurement. Cells were rinsed and then incubated in 4-(2-hydroxyethyl)- 1-piperazine-N'-2-ethanesulfonic acid (HEPES)-buffered saline (HBS: 130 mM NaCl, 5.0 mM KCl, 1.0 mM CaCl₂, 1.0 mM MgSO₄, 10.0 mM HEPES, 10.0 mM dextrose; pH 7.4) with 0.1% bovine serum albumin (BSA) containing 5 µM fura-2 AM for 30 min at room temperature. Cells were then washed twice with fresh buffer and incubated for another 30 min to allow complete hydrolysis of the ester. In selected experiments, all buffers contained L-NAME (1.0 mM) or indomethacin (0.01 mM). The slide was then transferred to the stage of an inverted Nikon Diaphot (Melville, NY) microscope for microspectrofluorimetry, with results being taken from fields containing 60 to 80 cells. The microscope was coupled to an air turbine spectrofluorimeter (Biomedical Instrumentation Group, University of Pennsylvania, Philadelphia, PA) via a fiberoptic light guide. The filter wheel of this ratio fluorimeter contained 340- and 380-nm excitation filters, and was adjusted to rotate at 100 ± 10 Hz. Emitted fluorescence was guided through a 510-nm filter to a photomultiplier tube (PMT), whose current output was converted to a voltage input into an encoding amplifier; the PMT signal was gated to the position of the filter wheel. Amplifier output was then digitized and sampled at 1 Hz with a microcomputer (Lakeshore Technologies, Chicago, IL). Calcium concentration (nM) was calculated with an in vitro calibration curve of known free Ca²⁺ (0 to 1.35 µM) and pentapotassium fura-2 (50 µM) concentrations. The 340    /  380 fluorescence ratio was determined for each calibration standard and plotted as a function of free [Ca²⁺]. Intracellular [Ca²⁺] was calculated from experimental ratios through the equation of Grynkiewicz, using R_max, R_min, and K_d from the calibration plot (15); selected standards were run daily. Ca²⁺ responses were then measured for 20 s to establish a baseline, and for at least 220 s to characterize peak and plateau responses following addition of 1 mg / ml LPS, 0.5 mM bradykinin (BK), or 10 U/ml thrombin; these concentrations were chosen from preliminary dose-response experiments as those resulting in maximal nitrite accumulation (LPS) or in maximal peak calcium mobilization (BK, thrombin).

Measurement of Nitrite

The nitrite content of the incubation medium (16) was measured through a chemiluminescent technique involving a glass purge-reflux vessel connected to an NO analyzer (NOA 270B; Sievers Instruments, Boulder, CO). Aliquots of 50 µl of incubation medium were injected into the purge-reflux vessel, where nitrite was reduced to NO by a 1% solution of potassium iodide in glacial acetic acid. NO was then "stripped" from this solution with nitrogen gas. Photon emission from the chemiluminescent reaction of NO with ozone was detected with a PMT, transduced to a voltage signal, and sampled with a microcomputer. Daily calibration with sodium nitrite solutions (0 to 261.5 pmol) yielded linear standard curves; the lowest nonzero standard was 52.3 pmol; the interassay CV was 7.2% at 104.6 pmol (n = 6). The background nitrite content of cell-free medium (0.478 ± 0.077 nmol/400 µl, n = 9) was subtracted from all sample values.

Evaluation of Cytotoxicity

Three indices of cell viability were assessed: morphologic, functional, and biochemical. Cellular morphology under phase-contrast microscopy and basal and agonist-stimulated Ca²⁺ levels measured microspectrofluorimetrically were both routinely assessed in confluent MCs following incubation with LPS (0 to 1 mg/ml) for 20 to 24 h; continued cell viability was additionally noted according to both criteria in MCs incubated with LPS at 1 mg/ml for 48 h, and LPS 3 mg/ml for 24 h. The lactate dehydrogenase (LDH) concentration of the medium was measured with a standard kit (Sigma) and a Beckman DU 640 spectrophotometer (Beckman Instruments, Inc., Schaumburg, IL). Aliquots of 50 µl of incubation medium were introduced into 3 ml cuvettes containing nicotinamide adenine dinucleotide (NAD; 0.43 mM), and the change in absorbance at 340 nm over a period of 3 min at 25° C in a 1 cm light path was measured following addition of pyruvate (0.76 mM) (17). To obtain positive-control LDH release values, confluent rat MCs were incubated in Lab-Tek chambered coverglass slide wells containing 400 µl of HBS (control) or HBS with 1% Triton X-100 for 30 min at 25° C, and buffer was then aspirated for immediate LDH measurement.

Statistical Analysis

Data are expressed as group mean ± SEM, with n referring to the number of wells studied in each group. Group comparisons were made through analysis of variance (ANOVA), with Bonferroni's correction for multiple comparisons; values of p < 0.05 were considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Dose-dependency of LPS Effects

LPS (1 mg/ml) led to rapid increments in [Ca²⁺]_i (peak increase: 200 ± 20 nM [mean ± SE]; n = 6), which were smaller than those occurring with BK (500 µM), and were short-lived (Figure 1: sample traces). Following overnight incubation, LPS (1 mg/ml) caused a small but significant decrease in basal [Ca²⁺]_i (control: 69.9 ± 3.14 nM [mean ± SE], versus LPS: 57.25 ± 2.9 nM; n = 118 to 120, p = 0.0034). LPS at concentrations of 0.1 mg/ml (n = 15) and 0.01 mg/ml (n = 6) had no apparent effect on basal [Ca²⁺]_i.

View larger version (19K): [in this window] [in a new window]   Figure 1.   Sample traces of [Ca2+]i (nM) versus time, showing direct effects of 1.0 mg/ml LPS (A) and of 0.5 mM BK (B and C ) in confluent rat mesangial cells. BK-stimulated responses followed 22 h incubation in the absence (B) or presence (C  ) of 1 mg/ml LPS. Note that LPS incubation impairs BK-induced Ca2+ mobilization and the direct effect of BK exceeds that due to LPS.

Incubation with LPS (1 mg/ml) (in the presence or absence of supplemental L-arginine [vide infra] led to 35 to 60% inhibition of subsequent BK-stimulated peak increments in [Ca²⁺]_i (control: 577 ± 22 nM [mean ± SE], versus LPS: 292 ± 5 nM; n = 89 to 93, p < 0.0001; subgroup data are shown in Tables 1234 and Figures 1 and 2). The LPS-induced impairment of BK-elicited peak [Ca²⁺]_i mobilization was consistently observed through multiple mesangial cell subcultures (mean decrement in peak BK-stimulated [Ca²⁺]_i: Passages 17 through 20: 47%, versus Passages 36 through 42: 50%). Plateau values (220 s after agonist addition) were similarly decreased following LPS (Tables 123; Figure 1). The LPS impairment of BK-induced Ca²⁺ mobilization was routinely associated with increased nitrite accumulation (Figure 3A through D). However, LPS at 0.1 mg/ml failed to decrease BK-induced peak [Ca²⁺]_i (control: 412 ± 33 nM, versus LPS: 361 ± 31 nM; n = 15, p > 0.05), despite a > 2-fold increase in nitrite (control: 0.628 ± 0.144 pmol/400 µl well, versus LPS: 1.542 ± 0.201 pmol/ 400 µl well; n = 4, p < 0.01); BK-stimulated Ca²⁺ mobilization was similarly unaffected by LPS at 0.01 mg/ml (n = 6).

View larger version (45K): [in this window] [in a new window]   Figure 2.   Effects of L-NAME ([A] 1.0 mM) or indomethacin ([B] 0.01 mM) on the inhibitory effect of 24 h incubation with LPS at 1 mg/ml on subsequent BK-stimulated (0.5 mM) peak [Ca2+]i mobilization (nM) in confluent rat mesangial cells. Indomethacin, but not L-NAME, significantly restored BK responsivity to values that did not differ from control.

View larger version (32K): [in this window] [in a new window]   Figure 3.   Effects of substrate supplementation ([A] L-arginine, 0.1 mM), NOS inhibition ([B] L-NAME, 1.0 mM, in the presence of 0.1 mM supplemental L-arginine), or COX inhibition (indomethacin, 0.01 mM, in the presence [C ] or absence [D] of 0.1 mM L-arginine) on LPS-stimulated (1 mg/ml for 24 h) nitrite accumulation in the incubation medium of confluent rat mesangial cells (all results expressed in nmol/well). As shown, supplemental L-arginine augments nitrite accumulation (A), L-NAME inhibits LPS-stimulated nitrite accumulation even in the setting of L-arginine supplementation (B), and indomethacin partially blunts nitrite accumulation (C and D).

No cellular morphologic changes or effects on cell viability were noted under phase-contrast microscopy following incubation with LPS at any dose (0.01 to 1 mg/ml) for up to 48 h, or following 24 h of incubation with LPS at 3 mg/ml. Moreover, although agonist-induced calcium mobilization was impaired by LPS, it was uniformly present. Additionally, LDH release into the incubation medium was assessed as a biochemical marker of cytotoxicity. Incubation of confluent rat MCs with Triton X-100 increased LDH from 2.5 ± 1.8 U/ml (control) to 152.5 ± 28 U/ml (Triton X-100); n = 4, p < 0.02. The increment in LDH concentration of the incubation medium due to exposure to LPS at 1 mg/ml (versus control) for 24 h was 5 U/ml (i.e., 3% of the increment due to Triton X-100; n = 9).

Effects of Supplemental L-arginine

Incubation with LPS at 1 mg/ml induced a > 6-fold increase in nitrite accumulation in the incubation medium; supplementation with 0.1 mM L-arginine (which increased the L-arginine content of the medium by 20%, from 0.5 mM to 0.6 mM) augmented this effect of LPS, producing a > 9-fold increase in nitrite (Figure 3A). Despite increased nitrite accumulation, L-arginine supplementation was not associated with any augmentation of LPS effects on basal or BK-stimulated Ca²⁺ mobilization (Table 1).

Effect of NOS Inhibition

Incubation with L-NAME (0.1 to 1.0 mM), whether in the presence (Figure 3B; Table 3) or absence (Table 2) of supplemental L-arginine (0.1 mM), did not significantly reduce the effect of LPS on BK-elicited Ca²⁺ mobilization. Indeed, the attenuation of BK-stimulated Ca²⁺ mobilization was not diminished despite partial (41%) inhibition of NO synthesis by 0.1 mM L-NAME and nearly complete suppression of nitrite generation by millimolar L-NAME (Table 3; Figure 3B). Inclusion of L-NAME (1.0 mM) in all buffers during cell loading and Ca²⁺ measurement, as well as during prior incubation with LPS, done in order to prevent possible postincubation NO production, likewise failed to alter the effect of LPS on BK-stimulated Ca²⁺ mobilization (Table 4). The latter experiment was repeated with thrombin (10 U/ml) replacing BK as the calcium-mobilizing agonist (Table 5); again, incubation with LPS (1 mg/ml) profoundly impaired agonist-induced calcium mobilization, and this effect was not prevented by NOS inhibition.

Effects of Cyclooxygenase Inhibition

Following coincubation with indomethacin (0.01 mM) in the presence (Figure 2B; Table 3) or absence (Table 2) of supplemental L-arginine, LPS (1 mg/ml) no longer significantly diminished subsequent BK-stimulated peak [Ca²⁺]_i responses. Likewise, coincubation with indomethacin partly attenuated LPS-induced nitrite accumulation (Figure 3C). Indomethacin similarly reduced LPS-induced impairment of BK-stimulated Ca²⁺ mobilization in the absence of supplemental L-arginine (Table 2), an effect that was again associated with a partial inhibition of LPS-induced nitrite accumulation (Figure 3D). The effect of indomethacin was similar whether it was included throughout the experiment (Table 4) or only during the incubation with LPS. By contrast, addition of indomethacin only during the postincubation period failed to reduce the effect of LPS on BK-induced Ca²⁺ mobilization (Table 4). Coincubation with meclofenamate (1 µM) also reduced LPS- induced impairment of BK-stimulated Ca²⁺ mobilization (control: 323.62 ± 35.14 nM, versus LPS: 144 ± 27.8 nM; n = 8, p < 0.01; control/meclofenamate: 326.37 ± 22.49 nM, versus LPS/meclofenamate: 229.87 ± 26.3 nM; n = 8, p > 0.05). When selected experiments were repeated with thrombin (10 U/ml) instead of BK as the calcium-mobilizing agonist (Table 5), the LPS-induced (1 mg/ml) impairment in Ca²⁺ mobilization was similarly reduced by indomethacin added along with LPS, but not by indomethacin added after LPS.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Administration of LPS to animals (18, 19) or humans (18) causes hypotension, systemic vasodilation, diminished vasopressor responsiveness, and organ failure, in these respects reproducing the salient hemodynamic features of gram-negative sepsis. We (20, 21) and others (18) have demonstrated partial reversal, by inhibitors of the NO-cGMP pathway, of vascular hypocontractility induced by exposure to LPS in vivo or in vitro. Similarly, inhibition of NOS partly restores blood pressure in models of endotoxemic shock (18, 19). The efficacy of these agents is explained by observations that LPS and a variety of inflammatory cytokines induce NOS expression in vascular myocytes (22), renal MCs (4, 7), and other cells (25); this leads to NO synthesis, with accumulation both of the stable breakdown products of NO and of cGMP. Taken together, these data have been interpreted by many as supporting the notion that septic vascular hypocontractility is mediated by LPS-induced NO synthesis, and that pathologic mesangial NO synthesis under such circumstances is of functional renal hemodynamic significance (4, 5, 7, 8, 26). However, the vascular response to NOS inhibition in these intact-vessel and animal protocols is routinely incomplete (18). Furthermore, hypocontractility of MCs induced in vitro by IL-1- is only partly restored by NOS inhibition, which has essentially no effect on tumor necrosis factor- (TNF-)-induced MC contractile dysfunction (8). The experiments in the present study were therefore designed to more explicitly test the hypothesis that LPS interferes with a specific proximate event in contractile signaling, through the use of a well-characterized in vitro system with many mechanistic similarities to vascular smooth muscle, with well-studied molecular responses to cytokine exposure, and with possible relevance to the pathogenesis of septic renal dysfunction.

Bradykinin- and thrombin-induced contraction in MCs depends on receptor binding and G-protein-mediated activation of phospholipase C (PLC), leading to Ca²⁺ mobilization and influx and then to calmodulin-mediated activation of contractile proteins (1). Conversely, either NO or vasodilatory prostaglandins may lead to mesangial relaxation, via pathways that depend on cGMP and cyclic adenosine monophosphate (cAMP), respectively (1, 4). cGMP may act by favoring Ca²⁺ resequestration or efflux (6, 12), or by diminishing the Ca²⁺ sensitivity of the contractile apparatus (12).

LPS (4, 7) or inflammatory cytokines (8) lead, in a tyrosine kinase- or possibly in a nuclear factor-B (NF-B)- dependent manner, to mesangial iNOS expression and nitrite accumulation. Since MCs normally respond to NO, as noted earlier, this induction of NOS may lead to unregulated autocrine relaxation. However, the physiologic consequences of renal iNOS expression are complex, since NOS inhibition leads to altered renal perfusion and to glomerular thrombosis in the setting of experimental endotoxemia (26). Likewise, NOS expression in tubular epithelial cells (25) may directly alter transport function as well as complex autoregulatory, humoral, or feedback mechanisms. LPS or inflammatory cytokines also act in MCs via apparently similar signal-transduction pathways to induce cyclooxygenase-2 (COX-2) (9, 10, 27), suggesting possible contributions by prostanoid mediators to mesangial relaxation, as well as "crosstalk" (10) between these two effector systems. Additionally, endothelin-1 may serve as an intermediate effector, leading to tyrosine kinase-dependent COX induction (27) while limiting iNOS expression (11).

We explored dose-response relationships for both effects of LPS (nitrite production and impairment of agonist-induced Ca²⁺ mobilization) at the upper range of LPS concentrations used in other studies of MCs in vitro, to ensure maximal NOS induction. We noted that MCs appear to be less sensitive to the effects of LPS on nitrite production and cell viability than do other cultured cells such as endothelial cells (28, 29) and macrophages (17), which respond with changes in Ca²⁺ mobilization, free-radical production, and cytotoxicity at various lower doses of LPS. It appears that the concentration of LPS required for in vitro experiments varies according to the cell type being studied, as well as the presence or absence of other cytokines added for synergism. For example, Portoles and colleagues demonstrated a difference in LPS sensitivity between various cell types by studying acute intracellular Ca²⁺ alterations and free-radical formation in endotoxin-treated rat liver endothelial, Kupffer, and parenchymal cells, and found that LPS at 10 µg/ml acutely increased Ca²⁺ in endothelial cells, and increased both Ca²⁺ and free-radical production in Kupffer cells, but had neither effect in hepatic parenchymal cells (30), which required 100 to 200 µg/ml LPS to increase cytosolic free Ca²⁺ (31). Furthermore, we have found that incubation with LPS (1 µg/ml; 18 h) impairs BK-stimulated Ca²⁺ mobilization in bovine aortic endothelial cells (28), and others have found that LPS causes a dose-dependent (0.001 to 1 µg/ ml) impairment of agonist-stimulated Ca²⁺ mobilization and NO release in aortic endothelial cells (29). The sensitivity of macrophages (17) and endothelial cells to the effects of LPS at lower doses contrasts with that of myocytes (22), MCs (4, 7), and some other cell types (25, 30, 31). In fact, published studies of LPS-induced nitrite production in rat MCs (4, 7) and myocytes (22) have routinely used LPS at concentrations of up to 100 µg/ml in order to maximize NO production and nitrite accumulation, without assessing functional endpoints such as agonist-stimulated Ca²⁺ mobilization. Indeed, the protocols of these and other studies often include a combination of cytokines (combinations of interferon-, interleukin-1, and TNF-) and "high-dose" LPS (10 to 100 µg/ml) to further augment induction of NOS, COX-2, and other LPS-stimulated mediators (4, 7, 22). In other studies, stimulation with LPS or cytokines is submaximal, and may be inadequate to permit full assessment of the functional consequences of LPS or cytokine exposure. For example, Shultz and colleagues have repeatedly studied LPS-induced nitrite and cGMP accumulation in rat MCs incubated with LPS at doses of 0.1 to 100 µg/ml for up to 42 h (4, 7), and have noted increased nitrite generation with addition of interferon- (100 U/ml) to high-dose LPS, without significant toxicity (4).

Like many in vitro studies (4, 7, 22, 28), ours used LPS concentrations in excess of those that circulate after LPS administration, suggesting that other mediators generated in vivo may augment LPS responsivity. Extrapolation of results achieved in vitro to in vivo conditions requires consideration of the possible modulatory effects of other mediators generated in the kidney and elsewhere during endotoxemia. As mentioned earlier, cocktails of endotoxin and various cytokines ("cytomix" preparations) intended to more closely approximate possible in vivo conditions by augmenting tissue sensitivity to LPS, lead to leftward shifts in the dose-response relationship for LPS-induced nitrite generation in vitro (4, 22- 25); for example, Gross and colleagues demonstrated time- (2 to 24 h) and dose-dependent nitrite accumulation in rat aortic myocytes with LPS at 1 to 100 µg/ml; addition of interferon- at 50 ng/ml to LPS at 1 µg/ml, 50 µg/ml, or 100 µg/ml (24 h) shifted the LPS-dose-nitrite-generation response curve to the left by 2 log units, resulting in more than a 3-fold increase in nitrite generation induced by LPS at 0.1 mg/ml (from 1.3 mmol/24 h to 4.3 nmol/24 h). Similarly, Wileman and colleagues found that incubation of rat aortic myocytes with LPS induced dose-dependent nitrite accumulation (0.01 to 100 µg/ ml), which was synergistically augmented by the addition of interferon- (100 U/ml) or TNF- (300 U/ml), but not IL-1 (100 U/ml). The synergistic effect of addition of cytokines to LPS in vitro has also been demonstrated in macrophages (17), renal epithelial cells (25), and mesangial cells (4). Whether such an approach would have similarly augmented the LPS-induced impairment of MC Ca²⁺ mobilization observed in our preparation is unknown. Because we were less concerned with doses of specific agents in our in vitro system than with the spectrum of plausible mediators leading to defects in Ca²⁺ mobilization, we restricted our protocols to the use of LPS alone, representing a more focused extension of our previous work with various vascular preparations (19).

In order to ensure that the highest concentration of LPS (1 mg/ml) used in our experiments was not causing cytotoxicity, we assessed morphologic, functional, and biochemical indices of cellular viability following prolonged incubation with LPS, and found no evidence of LPS-induced MC cytotoxicity according to morphologic criteria, preservation of Ca²⁺ homeostasis, or LDH release.

We found that LPS acutely increased MC Ca²⁺_i, in accord with previous reports of acute Ca²⁺ mobilization by LPS or lipid A (the active component of LPS) in a variety of other cells, including renal epithelial cells, hepatocytes, macrophages, Kupffer cells, platelets, brain microglial cells, and endothelial cells (30). Others have noted increased (hepatocytes, lymphocytes, skeletal muscle, and rat aorta), decreased (hepatocytes, ventricular myocytes), or unchanged (lymphocytes) basal Ca²⁺ and/or impaired agonist-stimulated Ca²⁺ mobilization (hepatocytes, lymphocytes) in cells or tissues isolated from experimental animals or human patients late in the course of sepsis or endotoxemia (32). As noted earlier, we (28) and others (29) have also found unchanged basal Ca²⁺ and impaired agonist-stimulated Ca²⁺ mobilization in vascular endothelial cells following prolonged incubation with LPS (1 µg/ml) in vitro. Our data now reveal that prolonged incubation of confluent rat MCs in the presence of 1.0 mg/ml LPS, but not lower concentrations of this agent, decreased subsequent agonist-induced Ca²⁺ mobilization, in addition to causing a small but significant decrease in basal Ca²⁺_i. These data support the hypothesis that LPS impairs vasoconstrictor-agonist-induced Ca²⁺ mobilization in rat MCs.

In accord with prior studies, we confirmed that prolonged incubation of confluent MCs in culture with LPS led to a > 6-fold increase in nitrite accumulation (4, 7). Even this large increment in nitrite underestimated the magnitude of iNOS induction, since there was a much larger (> 9-fold) increase in nitrite following supplementation of the incubation medium with 0.6 mM rather than 0.5 mM L-arginine. This increased NO synthesis following arginine supplementation was not, however, associated with any further impairment of the response to BK. Likewise, lower doses of LPS, which failed to alter subsequent responses to BK, still led to significant increments in NO synthesis. Since we had hypothesized that NO production mediates the LPS-induced decrement in agonist-induced Ca²⁺ mobilization, we assessed the effect of L-NAME at a concentration (1.0 mM) shown previously to inhibit iNOS-mediated effects in both intact vessels (hypocontractility) and cultured cells (nitrite and cGMP accumulation), and confirmed, through nitrite measurement, a nearly complete suppression of LPS-induced NO synthesis in our preparation. Surprisingly, this nearly complete inhibition of pathologic NO synthesis failed to significantly restore agonist-induced Ca²⁺ mobilization in these cells. Collectively, these results suggest that NO neither acted directly to inhibit agonist-induced Ca²⁺ mobilization in our studies nor made major indirect contributions to this effect, leading us to explore other, NO-independent, mechanisms.

We then used indomethacin to infer contributions of COX-2 products to effects of LPS on MC Ca²⁺ mobilization. Morrison and colleagues had noted previously that 0.01 mM indomethacin results in nearly complete inhibition of cytokine-induced prostaglandin E₂ (PGE₂) production by MCs, PGE₂ being the major COX-2 product in these cells (9, 10, 35). Meclofenamate (a COX inhibitor chemically unrelated to indomethacin) was also used to repeat selected studies in order to assess the possible contribution of indomethacin-specific effects on Ca²⁺ homeostasis. Overnight incubation of MCs with indomethacin, but not meclofenamate, increased BK- (and thrombin-) stimulated Ca²⁺ mobilization in both control and LPS-exposed MCs; meclofenamate acted only to preserve BK-stimulated Ca²⁺ mobilization in LPS-exposed MCs.

Although coincubation with indomethacin or meclofenamate largely prevented the effect of LPS on subsequent agonist-induced Ca²⁺ mobilization in MCs, our data do not suggest that PGE₂ mediates this effect. Since we removed the incubation medium and replaced it with five successive exchanges of buffered saline prior to measuring agonist-induced Ca²⁺ mobilization, it is unlikely that accumulated bioactive prostanoids were present at the time of Ca²⁺ measurements. Likewise, since there was no effect of indomethacin added after LPS incubation, although it had similarly blocked the effects of LPS whether present only during LPS exposure or throughout the entire protocol (i.e., during exposure and in all buffers used subsequently), we concluded that the effect of indomethacin did not depend on COX inhibition during Ca²⁺ measurements, but rather on a cellular consequence of COX activation that had occurred during the overnight incubation with LPS. Regulatory effects of PGE₂ (via cAMP or otherwise) on LPS-induced gene transcription or posttranscriptional events may be important in mediating the observed effects of LPS on cellular Ca²⁺ homeostasis (and iNOS induction and NO production), with the result that inhibition of COX can prevent these effects of LPS only when the COX inhibitor is present throughout the period of LPS exposure. This is consistent with the findings of Chaudry and colleagues that basal Ca²⁺ was unchanged and agonist-stimulated Ca²⁺ decreased in lymphocytes from septic animals, that systemic administration of indomethacin (throughout endotoxemia or sepsis) restored cellular Ca²⁺ mobilization, and that incubation of lymphocytes from control rats with PGE₂ in vitro similarly impaired agonist-stimulated Ca²⁺ mobilization (34). Indeed, the protective effect observed with indomethacin might not even depend on limiting prostanoid synthesis, but might rather be an indirect effect, resulting from the inhibition of byproducts such as the reactive oxygen species generated during COX activity (9, 30, 36) (mesangial oxygen-free-radical production has been shown to cause mesangial hypocontractility [36]). Furthermore, LPS- or cytokine-induced free-radical synthesis plays an important role in regulating iNOS and COX-2 synthesis by MCs at both the transcriptional and posttranscriptional levels, and COX activity may therefore act to augment COX-2 and iNOS induction and function (9). Also noteworthy is that heme oxygenase (HOX-1) is simultaneously induced by LPS or cytokines in MCs, and provides protection against oxidant stress by generating bilirubin (an antioxidant) from heme. It is particularly interesting to note the inhibitory effect of PGE₂, the major product of interleukin-1 (IL-1)-induced COX-2 in rat MCs, on HOX-1 induction by the same stimulus (35). Thus, COX-2 activity induced by IL-1 in these cells not only increases free-radical generation, but also ultimately blunts concomitant antioxidant activity.

Coincubation with indomethacin also resulted in partial inhibition of LPS-induced nitrite accumulation, suggesting an interaction between the COX and NO pathways that differs fundamentally from that described previously in MCs (10). Tetsuka and coworkers had noted increased nitrite generation by IL-1-treated MCs when they were coincubated with indomethacin, due apparently to withdrawal of an inhibitory effect of induced PGE₂ on iNOS activity. Our data do not preclude such an interaction, but rather suggest that an opposing interaction might occur at steps unique to LPS- but not IL-1-mediated signal transduction, perhaps limiting secondary induction of cytokine synthesis. Aeberhard and colleagues have reported dose-dependent inhibition of iNOS gene expression and nitrite production by several nonsteroidal antiinflammatory drugs (NSAIDs) (including indomethacin) in rat pulmonary alveolar macrophages stimulated with a combination of LPS (100 ng/ml) and interferon- (500 U/ml) for 8 h, in the absence of cytotoxicity (as assessed by LDH release) (17). The similarity of these results to our findings in rat MCs is reinforced by the fact that in Aeberhard and colleagues' study, NSAIDs were significantly less effective when added after 6 h of LPS/ interferon exposure than when they were present throughout the incubation. Further dissection of this interaction would require assessment of iNOS and COX-2 expression, PGE₂ synthesis, and cytokine induction. However, whatever the mechanism underlying inhibition of nitrite accumulation by indomethacin, it is unlikely to have contributed to the effect on Ca²⁺ mobilization observed in our study, given the ineffectiveness of more complete iNOS inhibition with L-NAME.

In summary, we hypothesized that LPS-induced expression of iNOS in MCs would diminish subsequent agonist-induced cellular Ca²⁺ mobilization via a direct effect of NO. We confirmed, for two distinct Ca²⁺-mobilizing vasoconstrictor agonists, that incubation with LPS significantly impaired agonist-induced Ca²⁺ mobilization, to a degree that appears sufficient to contribute to mesangial relaxation. However, impaired Ca²⁺ mobilization was dissociated from LPS-induced increments in NO production, suggesting that any possible contributions of NO to this effect of LPS on MCs must be at sites distal to the mobilization of Ca²⁺ by contractile agonists (i.e., through pharmacomechanical coupling, with diminished Ca²⁺ sensitivity of the contractile apparatus) (12). Moreover, COX inhibition during (but not after) incubation with LPS significantly normalized agonist-induced Ca²⁺ mobilization, perhaps by preventing an indirect effect of prostaglandins or by limiting the generation of reactive oxygen species that would normally accompany LPS-induced COX activity. Whether such mechanisms obtain in other contractile cells, or contribute to organ dysfunction in sepsis, remains to be determined.