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Effect of Elevated Glucose on Endothelin-Induced Store-Operated and Non-Store-Operated Calcium Influx in Renal Mesangial Cells

Department of Integrative Biology and Pharmacology, University of Texas-Houston Health Science Center, Houston, Texas.

Correspondence to Roger G. O'Neil, The University of Texas-Houston Health Science Center, Department of Integrative Biology, Pharmacology, and Physiology, 6431 Fannin, Houston, TX 77030. Phone : 713-500-6316 ; Fax : 713-500-7444 ; E-mail roneil{at}girch1.med.uth.tmc.edu

	Abstract

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Abstract. Early diabetic nephropathy exhibits renal glomerular hyperfiltration and an increase in renal plasma flow. The hyperfiltration is a dysfunctional state that may arise from a hyperglycemic-induced hypocontractility of glomerular mesangial cells that may be associated with depressed Ca²⁺ signaling events. The present study was designed to determine the effects of acute (minutes) and chronic (days) elevated glucose levels on endothelin-induced calcium signaling with a particular emphasis on the potential influence on stores and store-operated Ca²⁺ influx (SOCI ; also called capacitative calcium entry) in glomerular mesangial cells. Primary cultures of rat mesangial cells were grown in either high (30 mM) or normal (5 mM) glucose-containing media and tested in the presence of either high (30 mM) or normal (5 mM) glucose levels. Intracellular calcium levels were monitored with the calcium-sensitive fluorophore fura-2 before and after treatment with either endothelin-1 (10 nM), to induce typical Ca²⁺ signals, or the endoplasmic reticulum (ER) Ca-ATPase inhibitor thapsagargin (1 µM), to unload ER Ca²⁺ stores. Both acute and chronic exposure to high glucose levels depressed the endothelin-induced calcium signal. However, neither release of Ca²⁺ from stores nor SOCI were depressed by high glucose levels. In contrast, an endothelin-induced calcium entry pathway (likely receptor-operated calcium influx), separate from SOCI, was markedly depressed in the presence of both acute and chronic high glucose levels. The depressant effect of high glucose was rapidly (minutes) reversible upon returning to normal glucose levels. It is concluded that high glucose levels depress endothelin-induced calcium signaling in rat mesangial cells by inhibiting non-SOCI Ca²⁺ entry pathways, namely the receptor-operated Ca²⁺ influx pathway. The glucose-induced alterations in the receptor-operated calcium influx pathway may, in part, contribute to the depressed contractile state of glomerular cells during periods of hyperglycemia.

	Introduction

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Diabetic nephropathy often develops in patients suffering from insulin-dependent diabetes mellitus. A dominant pathophysiologic characteristic is the development of renal glomerular hyperfiltration, which is believed to arise in part from altered responsiveness of glomerular mesangial cells to vasoconstrictor hormones (1,2). There is increasing evidence that the induced hyperfiltration may be related to a combined decreased responsiveness of both the renal afferent arteriole and the glomerular mesangial cells to vasoconstrictor agents (3,4. Recent data point to an important role of hyperglycemia, which accompanies insulin-dependent diabetes mellitus, in this response, which may modulate the contractile responsiveness of the mesangial cells (5) as observed in smooth muscle cells (6). It has been shown by several groups (8,9,10,11), but not all (5,12), that the calcium signals induced by many vasoactive compounds, including angiotensin II (AngII), arginine vasopressin (AVP), norepinephrine, and prostaglandin E₂, are markedly reduced in the presence of chronically elevated glucose levels. Hence, it is becoming increasingly apparent that the reduced calcium signal may be a major contributing factor to the hypocontractility of these cells.

The mechanism by which high glucose levels may depress calcium signals generated by vasoactive peptides is unknown (see Discussion). The generation of Ca²⁺ signals is a complicated process involving several Ca²⁺ transport components for Ca²⁺ influx and Ca²⁺ release from internal stores. Initial studies evaluating the release of Ca²⁺ from stores did not find a major effect of glucose in modulating Ca²⁺ release (10). Recently, Mene and coworkers (11 presented evidence consistent with the view that chronic high glucose levels may modulate a store-operated Ca²⁺ influx pathway (SOCI ; also known as capacitative Ca²⁺ entry), which is activated by Ca²⁺ store depletion 13,14,15). However, the extent to which this pathway or other parallel entry pathways contribute to the change in the glucose-modulated Ca²⁺ signal remains largely unknown.

In mesangial cells, many vasoactive peptides that induce Ca²⁺ signals normally do so through G protein-linked receptors that are coupled to phospholipase C (PLC), resulting in the generation of inositol trisphosphate (IP₃) and diacylglycerol (DAG) (16,17,18,19). The IP₃, in turn, binds to IP₃ receptors on the endoplasmic reticulum (ER) (ER Ca²⁺ release channels) to activate release of Ca²⁺ from the ER stores into the cytoplasm. A second critical component to the calcium signal, however, is induction of Ca²⁺ entry via plasma membrane Ca-permeable channels. The binding of vasoactive peptides to their plasma membrane receptors can directly lead to activation of receptor-operated Ca²⁺ channels ^a through various transduction pathways or, if membrane voltage is altered, to activation of voltage-operated Ca²⁺ channels (20,21). Alternatively, the IP₃^- induced release of Ca²⁺ from the ER can indirectly activate plasma membrane Ca²⁺ entry via SOCI pathways due to unloading (release) of Ca²⁺ from stores (13,14,15). Although SOCI has been shown to reside in mesangial cells 22,23), the mechanism linking the unloading of Ca²⁺ stores to activation of SOCI remains unknown. High glucose levels could potentially modify one or more of these Ca²⁺ transport pathways.

The purpose of the present study was to determine : (1) whether Ca²⁺ signaling induced by the potent vasoactive hormone endothelin-1 (ET) in mesangial cells is modulated by acute elevation in plasma glucose levels ; (2) if modulated, which underlying components of the Ca²⁺ signal are being modulated ; and (3) whether the ET-induced Ca²⁺ signaling components undergo adaptation to prolonged (chronic) exposure to high glucose levels. It was found that ET induces release of Ca²⁺ from stores and activates Ca²⁺ influx through both SOCI pathways and other parallel entry pathways (supposedly receptor-operated Ca²⁺ entry since ET does not activate voltage-operated Ca²⁺ channels in mesangial cells) (24). Both acute and chronic elevation in glucose levels were found to markedly depress the ET-induced Ca²⁺ signal. However, this effect was not due to depression of Ca²⁺ release from stores or inhibition of SOCI, but rather to specific depression of a non-store-operated Ca²⁺ entry pathway, likely a receptor-operated Ca²⁺ entry pathway.

	Materials and Methods

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Primary Cell Culture
Rat mesangial cells were a gift from Hanna E. Abboud (University of Texas Health Science Center, San Antonio, TX). Cells were plated in Waymouth medium containing either 5 or 30 mM glucose, or 5 mM glucose plus 25 mM mannitol as an osmotic control (see Solutions and Chemicals), pH 7.4, in a 37°C tissue culture incubator with a controlled atmosphere of 95% air/5% CO₂ similar to methods used by others (10). The plated cells were then propagated and subcultured every 5 to 7 d. Fully confluent cultures (5 to 7 d) from passages 3 to 16 were used for experimentation.

Measurement of Intracellular Calcium
Confluent primary cultures of mesangial cells were rinsed twice with Dulbecco's phosphate-buffered saline and detached by mild trypsinization (0.05% trypsin plus 0.53 mM ehtylenediaminetetra-acetic acid) for 2 to 3 min. The reaction was stopped by addition of the old medium. The detached cells were rinsed once with isotonic bathing media and resuspended in the fresh isotonic bathing media (see Solutions and Chemicals). Fura-2 was loaded into the cells by incubation of the cells in isotonic bathing media containing 10 µM acetoxymethyl ester of fura-2 (fura-2 AM) for 30 min at 37°C with periodic mild mixing. At the end of the initial 30-min incubation, the mixture of cells and fura 2 AM was diluted 1 : 4 by addition of isotonic bathing media and incubated for another 60 min. The cells were then rinsed with the isotonic bathing media without fura-2 AM, resuspended in the isotonic bathing media, and kept at room temperature until use. Just before use, the cells were rinsed twice, and an aliquot of cells was added to a cuvette containing 2 ml of test solution for measurement of intracellular Ca²⁺. The test solution was isotonic bathing media (310 mosmol/kg H₂O) with or without 5 mM ethyleneglycolbis(ß-aminoethyl ether)-N,N,N',N'-tetra-acetic acid (EGTA) (see Solutions and Chemicals, below).

Intracellular calcium was estimated from the fura-2 fluorescence by excitation at 340 and 380 nm and obtaining the resulting ratio of the emission at 511 nm in the usual manner at 37°C, using a Delta Scan dual excitation fluorometer (Photon Technology International, South Brunswick, NJ) similar to that described before (25). All fluorescence measurements were initiated when the cells were added to the cuvette. Fura-2 fluorescence ratios were converted to intracellular calcium activity ([Ca²⁺]_i) using the formula described by Grynkiewicz et al. (26) as follows :

where R is the ratio at any time, ß is the ratio of the fluorescence emission intensity at 380 nm excitation in Ca²⁺-depleting and Ca²⁺-saturating conditions, K_d is the Ca²⁺ dissociation constant of fura-2 (K_d = 224 nM), R_min is the minimum ratio in Ca²⁺-depleting conditions (addition of 5 mM EGTA), and R_max is the maximum ratio in Ca²⁺-saturating conditions (0.1% Triton X-100 in normal bathing medial containing 1 mM Ca²⁺).

Treatment Groups
The control glucose group of mesangial cells was cultured in control media containing 5 mM glucose for 5 to 7 d (chronic) and then tested (acute) in the presence of 5 mM glucose (chronic : acute treatment ; 5G : 5G). The chronic high glucose group was cultured in media containing 30 mM glucose for 5 to 7 d and tested in 30 mM glucose (30G : 30G), while the chronic group's osmolarity control was similarly cultured in media containing 5 mM glucose + 25 mM mannitol and tested in 5 mM glucose + 25 mM mannitol (5G + 25M : 5G + 25M). The acute high glucose group of mesangial cells was cultured in media containing 5 mM glucose and tested in 30 mM glucose (5G : 30G), while the acute group's osmolarity control was cultured in media containing 5 mM glucose and tested in media containing 5 mM glucose + 25 mM mannitol (5G : 5G + 25M). To test whether the effects of chronic high glucose were reversible, a final group of cells was cultured in 30 mM glucose and tested in 5 mM glucose (30G : 5G).

Solutions and Chemicals
The tissue culture medium used for all studies was Waymouth medium, containing 16% heat-inactivated fetal bovine serum, penicillin (100 U/ml), streptomycin (100 µg/ml), gentamicin (25 µg/ml), human transferrin (10 µg/ml), hydrocortisone (5 x 10 to 8 M), and insulin (5 µg/ml), 25 mM NaHCO₃, pH 7.4. The following experimental solutions were used for measurements of intracellular calcium. The isotonic bathing solution was composed of (in mM) : 140 NaCl, 4.2 KCl, 0.4 Na₂-HPO₄, 0.5 NaH₂PO₄, 0.3 MgCl₂, 0.4 MgSO₄, 1 CaCl₂-H₂O, 20 Hepes, pH 7.4, at 37°C. Just before use, 0.2% bovine serum albumin (BSA) and 5 or 30 mM glucose, or 5 mM glucose plus 25 mM mannitol were added to the solution for each of the treatment groups as outlined above (see Treatment Groups). The osmolalities of all solutions were determined using a vapor-pressure osmometer (Wescor, Logan, UT). In some studies, the bathing media Ca²⁺ levels were reduced to nominally zero by the removal of Ca²⁺ and addition of 5 mM EGTA to the bathing media (pH 7.4).

The chemicals and drugs used in the study were as follows. Fura-2 free acid and fura-2 AM were obtained from Calbiochem (La Jolla, Ca). Tissue culture supplements were obtained from Life Technologies (Grand Island, NY) and JRH Biosciences (Lenexa, KS). Thapsigargin (TG), ET, and all other reagents were obtained from Sigma Chemical Co. (St. Louis, MO).

Statistical Analyses
Data are expressed as mean values ± SEM ; n is the number of replicates. Significance was determined with the paired or unpaired t test as appropriate, or, for multiple comparison, ANOVA and Student-Neumann-Keuls post hoc test as appropriate. A value of P < 0.05 was considered significant.

	Results

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Ca²⁺ Signaling in Renal Mesangial Cells
To determine the basal Ca²⁺ signaling components in mesangial cells, culture cells were treated with either the potent vasoconstrictor hormone ET (10 nM) to induce a normal Ca²⁺ signal, or the ER Ca²⁺ pump inhibitor TG (1 µM) to release Ca²⁺ from internal ER stores.

Figure 1A (top trace) depicts a typical example of ET-induced Ca²⁺ signaling for cells cultured and tested in the presence of normal glucose levels (5 mM). Addition of ET (10 nM) produces an immediate rise in intracellular Ca²⁺ from a basal level of near 140 nM to a peak value of more than 550 nM, followed by relaxation of the intracellular Ca²⁺ levels to a pseudoplateau greater than that observed during the basal resting conditions. On average, the ET-induced peak response was 575 ± 76 nM (Table 1A, 5G : 5G group).

View larger version (15K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Representative traces showing the effects of endothelin-1 (ET) (A) and thapsigargin (TG) (B) on intracellular Ca2+ levels in the presence of normal glucose levels (5 mM). Studies were done in the presence of normal extracellular Ca2+ levels (+Ca2+, 1 mM) and nominally zero Ca2+ levels (-Ca2+).

To assess the fraction of the Ca²⁺ signal that may be due to Ca²⁺ release from internal stores, the study was repeated with extracellular Ca²⁺ levels reduced to nominally zero levels (-Ca²⁺) by the addition of the Ca²⁺ chelator EGTA. Removal of extracellular Ca²⁺ appeared to modestly depress the basal Ca²⁺ levels, although on average the change was not significantly different from the control values with extracellular Ca²⁺ present (Table 1). As shown by the example in Figure 1A (bottom trace), in the absence of extracellular Ca, ET produced a depressed Ca²⁺ signal whose peak value averaged only 239 ± 73 nM (Table 1A, 5G : 5G group). The Ca²⁺ signal reflects release of Ca²⁺ from internal Ca²⁺ stores. The difference between the peak Ca²⁺ signal in the presence and absence of extracellular Ca²⁺ will primarily delineate initial influx of Ca²⁺ across the plasma membrane. Hence, based on these results, it follows that although part of the ET-induced rise in intracellular Ca²⁺ is due to release of Ca²⁺ from stores, a major fraction of the rise in intracellular Ca²⁺ must be due to activation of Ca²⁺ influx across the plasma membrane.

Calcium Stores and SOCI in Mesangial Cells
The potential involvement of SOCI in Ca²⁺ signaling in mesangial cells was evaluated in a parallel series of experiments by assessing the effects of TG, a specific inhibitor of the ER Ca²⁺-ATPase, to unload ER Ca²⁺ stores. A concentration of 1 µM TG was shown to maximally empty internal stores (data not shown). As shown by the example in Figure 1B (top trace), addition of TG induces a rise in intracellular Ca²⁺ to a peak value within 1 min that then relaxes to a new pseudoplateau value modestly greater than that observed in the basal state. For all cells, the peak Ca²⁺ value averaged near 470 nM (Table 1B, 5G : 5G group). Repeating the experiments in the absence of extracellular Ca²⁺ (-Ca²⁺) resulted in a depressed signal (Figure 1B, bottom trace) that averaged near 235 nM for all cells (Table 1), reflecting release of Ca²⁺ from stores. Hence, the unloading of stores by TG in the presence of extracellular Ca²⁺ induces Ca²⁺ influx, which accounts for the enhanced peak Ca²⁺ signal compared to that observed in the absence of extracellular Ca. This induced Ca²⁺ influx reflects typical SOCI (14). It follows that Ca²⁺ influx via SOCI must also be a component of the ET-induced Ca²⁺ influx because ET also causes release of Ca²⁺ from ER stores. Furthermore, because the ET-induced Ca²⁺ peak in the presence of extracellular Ca²⁺ (575 nM) tends to be greater than that observed for the TG-induced peak (470 nM), although not significantly greater, the ET-induced Ca²⁺ peak likely reflects the contributions of additional Ca influx pathways stimulated by ET (see below).

Acute High Glucose Effects on ET-Induced Ca²⁺ Signaling Pathways
To test the effects of elevated glucose on the ET-induced influx and release of Ca²⁺, mesangial cells were cultured in varying concentrations of glucose or glucose plus mannitol (osmotic control) for 5 to 7 d before use (see Materials and Methods). To determine the acute affects of high glucose levels, cells were cultured in normal glucose (5 mM) and tested with ET in high glucose (30 mM) (5G : 30G group). As shown by the example in Figure 2A, with acute high glucose levels the ET-induced peak Ca²⁺ signal in the presence of extracellular Ca only reached a value of approximately 350 nM, well below that observed in the presence of normal glucose levels (compare to Figure 1A). In all cases, the acute high glucose group (5G : 30G) demonstrated a significantly depressed ET-induced Ca²⁺ signal compared to that observed for the normal glucose group (5G : 5G). The ET-induced peak Ca²⁺ signal averaged 335 nM (change = 188 ± 42) with acute high glucose (5G : 30G group) compared to 575 nM (change = 445 ± 61) observed for the normal glucose control group (5G : 5G group) (see Table 1A and Figure 3A).

View larger version (15K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Representative traces showing the effects of ET and TG on intracellular Ca2+ levels in the presence of acute high glucose levels (30 mM). Studies were done in the presence of normal extracellular Ca2+ levels (+Ca2+, 1 mM) and nominally zero Ca2+ levels (-Ca2+).

View larger version (22K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Summary of the acute effects of ET on intracellular Ca2+ levels in the presence of acute levels of 5 mM glucose, 30 mM glucose, or 5 mM glucose + 25 mM mannitol (osmotic control). Studies were done in the presence of normal extracellular Ca2+ levels (+Ca2+, 1 mM) and nominally zero Ca2+ levels (-Ca2+).

In a parallel series of studies, the effect of acute high glucose levels on ET-induced release of Ca from Ca stores was also assessed in cells in which the extracellular Ca was removed. A representative trace showing the ET-induced release of Ca from internal stores in cells exposed to acute high glucose levels (5G : 30G group) in the absence of extracellular Ca (-Ca) is shown in Figure 2A. In the absence of extracellular Ca, the observed changes in intracellular Ca levels for the acute high glucose conditions are very similar to that observed for cells exposed to normal glucose levels (5G : 5G, compare with Figure 1A). On average, the change in intracellular Ca levels between the two groups was not significantly different. In the absence of extracellular Ca²⁺ (-Ca), the ET-induced release of Ca²⁺ from internal stores produced a change in intracellular Ca of 156 ± 67 nM Ca for the normal glucose group and a change of 122 ± 19 for the acute high glucose group (Figure 3B).

Chronic High Glucose Effects on ET-Induced Ca²⁺ Signaling Pathways
Figure 4A shows typical traces demonstrating the effects of ET on Ca²⁺ signaling in cells chronically cultured in, and then tested in, high glucose media (30G : 30G). As demonstrated by the representative example shown in Figure 4A, in cells treated with chronic high glucose levels the ET-induced Ca²⁺ signal was markedly depressed in the presence of extracellular Ca²⁺, with the peak Ca²⁺ signal approaching a value of only 250 nM. On average, the ET-induced Ca²⁺ peak was only 313 ± 84 nM (Table 2A, 30G : 30G group), significantly below the peak of 575 ± 76 nM observed for the normal glucose group (Table 2A, 5G : 5G group). This depressant affect of chronic high glucose was not due to alterations in bathing media osmolality, as the peak Ca²⁺ signal was not depressed in cells in which the chronic high glucose was substituted for mannitol (Figure 5A, 5G + 25M : 5G + 25 M group). Hence, chronic high glucose levels markedly depress the ET-induced Ca signal in these cells, similar to that observed for the affects of acute high glucose levels.

View larger version (14K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Representative traces showing the effects of ET and TG on intracellular Ca2+ levels in the chronic glucose group. The mesangial cells were cultured in high glucose levels (30 mM) and tested with high glucose levels present (30 mM). Studies were done in the presence of normal extracellular Ca2+ levels (+Ca2+, 1 mM) and nominally zero Ca2+ levels (-Ca2+).

View larger version (22K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Summary of the chronic effects of ET on intracellular Ca2+ levels in the presence of 5 mM glucose, 30 mM glucose, or 5 mM glucose + 25 mM mannitol (osmotic control). Studies were done in the presence of normal extracellular Ca2+ levels (+Ca2+, 1 mM) and nominally zero Ca2+ levels (-Ca2+).

Finally, the affects of chronic high glucose levels on ET-induced release of Ca from internal stores was also assessed. A representative example showing the ET-induced release of Ca from stores in cells in which extracellular Ca²⁺ was first removed (-Ca) is shown in Figure 4A. Although there was a tendency for the Et-induced change in intracellular Ca to be depressed in the chronic high glucose group from that observed in the normal glucose group, the change was not significantly different (see Figure 5B and Table 2A). On average, in the absence of extracellular Ca the ET-induced change in intracellular Ca levels averaged 156 ± 67 nM for the normal glucose group (5G : 5G) and 61 ± 15 nM for the chronic high glucose group (30G : 30G).

Acute High Glucose Effects on Ca²⁺ Stores and SOCI
To further extend our assessment of the influence of high glucose levels on Ca stores and SOCI, the effects of treating mesangial cells with TG to completely release Ca from TG-sensitive ER Ca stores was studied. The difference in the rise in [Ca²⁺]_i after the addition of TG with and without extracellular calcium would, by definition, represent SOCI (13,14,15).

As shown by the summary data in Figure 6, A and B, the TG-induced peak changes in intracellular Ca²⁺ levels in the presence or absence of extracellular Ca²⁺ were not affected by acute high glucose levels. In the presence of extracellular Ca, the TG-induced rise (change) in intracellular Ca²⁺ levels average near 300 nM for both the acute high glucose group (5G : 30G group) and the normal glucose group (5G : 5G group). Similarly, in the absence of extracellular Ca²⁺ (-Ca²⁺), the TG-induced rise in intracellular Ca²⁺ levels averaged near 130 nM for both the acute high glucose group (5G : 30G group) and the normal glucose group (5G : 5G group). Hence, acute high glucose levels do not appear to alter the Ca ER stores or release of Ca from these stores.

View larger version (25K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Summary of the acute effects of TG on intracellular Ca2+ levels in the presence of acute levels of 5 mM glucose, 30 mM glucose, or 5 mM glucose + 25 mM mannitol (osmotic control). Studies were done in the presence of normal extracellular Ca2+ levels (+Ca2+, 1 mM) and nominally zero Ca2+ levels (-Ca2+).

Chronic High Glucose Effects on Ca²⁺ Stores and SOCI
Figure 4B shows an example of the effects of TG for a chronic high glucose group with and without Ca²⁺. The signal is very similar to that observed for the normal glucose group (compare to Figure 1B). Indeed, the peak Ca²⁺ value after TG treatment in the presence of extracellular Ca²⁺ averaged 470 ± 60 nM for the normal glucose group and 478 ± 54 nM for the chronic high glucose group (Table 2B), convincingly demonstrating that the SOCI was not affected by chronic high glucose treatment (Figure 7). Similarly, in the absence of extracellular Ca²⁺, the peak Ca²⁺ values did not differ between the two groups even though there was a tendency for the mean change to be smaller in the presence of chronic glucose (Table 2). (Note that this lack of a chronic affect is fully consistent with that observed for the effects of acute high glucose levels on Ca²⁺ release ; see Table 1.) These results provide strong evidence that neither chronic high glucose levels nor acute high glucose levels alter intracellular calcium stores and the release of calcium from these stores.

View larger version (26K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. Summary of the chronic effects of TG on intracellular Ca2+ levels in the presence of chronic levels of 5 mM glucose, 30 mM glucose, or 5 mM glucose + 25 mM mannitol (osmotic control). Studies were done in the presence of normal extracellular Ca2+ levels (+Ca2+, 1 mM) and nominally zero Ca2+ levels (-Ca2+).

Do Ca²⁺ Signals Adapt to High Glucose Levels ?
The determine whether the Ca²⁺ signaling pathways adapt to chronic high glucose levels, cells were cultured in high glucose media (30 mM) for 5 to 7 d, but then tested in normal glucose media (5 mM) (30G : 5G group). Figure 8, A and B, shows typical traces for ET-induced and TG-induced Ca²⁺ signaling. In Figure 8A, the ET-induced Ca²⁺ signal in the presence of extracellular Ca²⁺ approaches 650 nM, equal to or exceeding the Ca²⁺ signal in cells cultured and tested in normal glucose media (compare to Figure 1A). Similarly, the TG-induced Ca²⁺ signal appears equivalent to that observed for cells cultured and tested in normal glucose media (compare to Figure 1B) and shows no evidence of being depressed (Figure 1B). Therefore, both acute and chronic elevations of glucose depress the ET-induced Ca²⁺ influx, but this depression is readily and rapidly reversed upon restoration of normal glucose levels, demonstrating little or no adaptation of the Ca²⁺ signaling pathways to high glucose, at least not over the 5- to 7-d treatment period.

View larger version (16K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 8. Representative traces showing the effects of ET and TG on intracellular Ca2+ levels in the presence of normal glucose levels (5 mM) after chronic exposure to 30 mM glucose. Studies were done in the presence of normal extracellular Ca2+ levels (+Ca2+, 1 mM) and nominally zero Ca2+ levels (-Ca2+).

	Discussion

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Effects of Acute and Chronic High Glucose Levels on ET-Induced Calcium Signaling
The present study demonstrates for the first time that acute elevation in glucose levels can depress the ET-induced Ca²⁺ signaling in rat mesangial cells. It was also shown that this effect is similar to that observed for chronic elevation in glucose levels for several days. Others have also shown that the Ca²⁺ signals induced by other vasoactive peptides was similarly depressed in the presence of chronic elevated glucose levels (8,9,11,22). The present study demonstrates that this affect is not due to alterations in Ca²⁺ release from Ca²⁺ stores, but appears to arise as a result of a specific inhibition of Ca²⁺ influx (see below) across the plasma membrane, findings consistent with those recently reported by Mene and coworkers during chronic elevation of glucose levels (11). The effect is not related to an osmotic influence of the high glucose because substitution of glucose with an equivalent amount of mannitol provides a normal Ca²⁺ signal. Furthermore, because acute and chronic elevations in glucose depress the Ca²⁺ influx, this effect is likely related to an elevated uptake of glucose into the cells, possibly due to de novo synthesis of DAG (27) as discussed below. Indeed, glucose influx may also be responsible for depressing Ca²⁺ influx in vascular smooth muscle cells (7). The implications of this effect of glucose are broad, demonstrating that a glucose-dependent depression of the Ca²⁺ signal may, in part, contribute to the hypocontractile response of mesangial cells to vasoactive peptides during periods of elevated plasma glucose levels, such as that observed in diabetes mellitus (1). The resulting hypocontractility may, in turn, lead to glomerular hyperfiltration, as observed in diabetes mellitus even when the plasma concentration of vasoactive agents are elevated above normal (1).

The hypocontractile response of mesangial cells to vasoactive substances has been shown for cells from diabetic animals as well as for cells from nondiabetic animals, but where the extracellular glucose levels were chronically elevated above normal (5,12). It has been suggested that the contractile elements themselves may undergo rearrangement and lead to an altered ability of these cells to contract (12). However, there is accumulating evidence that Ca²⁺ signaling induced by vasoactive agents, in general, is depressed in cells exposed to elevated glucose levels and that this may contribute to the hypocontractile responsiveness of these cells. Indeed, it has been shown that vasoactive substances, notably AngII, noradrenaline, prostaglandin (PGF2a), and arginine vasopressin (AVP), elicit significantly reduced [Ca²⁺]_i transients in high glucose (22 mM) media compared to normal (5.5 mM) conditions (8,9,10). Indeed, the present study demonstrates that high glucose levels similarly depress the ET-induced [Ca²⁺]_i transients. Furthermore, the present study shows that the effect of high glucose was to depress the ET-induced calcium entry into the cells, a finding similar to that observed by Mene and coworkers for the actions of AngII (11). However, although these later investigators attributed the affects of high glucose on calcium influx to inhibition of SOCI, the results of the present study using ET convincingly demonstrate that the depressant action of elevated glucose levels are not due to inhibition of SOCI, but rather are due to inhibition of an apparent receptor-operated Ca²⁺ influx pathway operating in parallel to the SOCI pathway (see below).

It should be noted, however, that not all reports support the concept of a glucose-dependent Ca²⁺ signal in mesangial cells. In studies from one group, it was reported that Ca²⁺ signals induced by vasoactive peptides (AngII, ET, AVP) were not altered in the presence of elevated glucose levels (5,10), although the cells were shown to have a hypocontractile response. The reason for this difference from that observed in the current studies and those of others, as heretofore mentioned, is not known, but could be related to the culture passage of the cells used in the studies (culture passage 15 to 40 versus 3 to 15 in the current study) or the higher concentrations of vasoactive substances used (e.g., 100 nM ET versus 10 nM ET in the present study). However, despite these possible factors, responses of cultured mesangial cells to vasoactive peptides are known to be highly variable among individual cells, even if the cells are from the same plating as shown by Kanamori et al. (9) for AngII. We have also noted a reduced but variable responsiveness of cultured mesangial cells to AngII (data not shown). Hence, caution must be exercised in interpreting the quantitative response of cultured mesangial cells to any given stimuli.

Effects of High Glucose on Calcium Release from Internal Calcium Stores
The transduction pathways underlying the effects of glucose on calcium signaling are not well defined. Early studies in rat mesangial cells have focused on a possible role of either the "polyol pathway" or the DAG/protein kinase C pathway in modulating the PLC/IP₃ pathway. It follows from this assessment that elevated glucose levels should lead to a depressant effect of vasoactive peptides on the production of IP₃ and, in turn, on the resulting changes in [Ca²⁺]_i. However, such an affect should be associated with a decrease in the release of calcium from stores and a consequent decrease in SOCI. Our data show that there were no differences in ET-induced Ca²⁺ release from internal stores among normal, acute, and chronic high glucose groups. Likewise, Mene and coworkers (11) have shown that the addition of a Ca²⁺ ionophore to release Ca²⁺ from internal stores did not affect the amount of releasable Ca²⁺ in cells cultured in chronic high glucose growth conditions. This implicates a glucose affect either on a Ca²⁺ entry pathway that is not linked to the release of Ca²⁺ from internal stores, such as receptor-operated Ca²⁺ entry, or an effect on a signaling component distal to the release of Ca²⁺ from stores.

Effects of High Glucose on Store-Operated and Non-Store-Operated Calcium Influx
Which Ca²⁺ influx pathway is inhibited by elevated glucose levels ? A recent report from Mene and coworkers (11) implicated a role for SOCI in this process. However, to more fully address this issue, the present study focused on assessing the effects of glucose on both SOCI and parallel entry pathways activated by ET, supposedly receptor-operated Ca²⁺ influx. It should be noted that these earlier workers measured the effect of glucose on SOCI by first completely removing all extracellular Ca²⁺ to deplete internal stores, then monitoring the changes in intracellular Ca²⁺ levels upon restoration of extracellular Ca²⁺ levels to normal. This technique would, by definition, include contributions to intracellular Ca²⁺ levels from all active Ca²⁺ influx pathways. In the current studies, we attempted to refine this process by monitoring the changes in intracellular Ca²⁺ after addition of TG to unload stores, but where extracellular Ca²⁺ levels were always maintained at normal levels. In this way, the changes in intracellular Ca²⁺ levels should primarily reflect contributions from SOCI. By this approach, we were able to demonstrate that the affect of high glucose levels on Ca²⁺ influx is not due to alterations in SOCI. Indeed, SOCI was not altered by either acute or chronic high glucose groups (Figures 6B and 7B). This is in keeping with the observations that the ability to release Ca²⁺ from internal stores and, hence, the signal to release Ca, were not affected by high glucose. It follows that neither the PLC/IP₃ pathway linked to the ER, the release of calcium from internal ER stores, nor the SOCI appear to be responsible for the reduction in calcium influx in the presence of elevated levels of glucose.

Because SOCI is not altered by elevated glucose levels, the effect of glucose on ET-induced calcium influx must, by definition, reflect changes in a Ca²⁺ entry pathway parallel to SOCI. This parallel pathway is not likely a voltage-operated Ca²⁺ entry pathway because ET does not appear to activate voltage-operated Ca²⁺ channels (24). Hence, this parallel pathway likely reflects a receptor-operated Ca entry pathway that is activated by ET binding to its receptor. However, the effects of ET on Ca signaling are likely to be complex because the ET receptor coupled to PLC (16) will generate IP₃, which, in turn, will lead to release of Ca²⁺ from stores and activation of SOCI. Consequently, since the present study also demonstrates that the ET-induced Ca²⁺ signal appears to be greater than that induced by maximally releasing Ca²⁺ from internal stores with TG, an additional component of Ca²⁺ influx must be activated by ET. It is this newly identified receptor-operated Ca²⁺ influx pathway that is the component of the Ca²⁺ signal that appears to be depressed in the presence of elevated glucose levels with little or no affect on SOCI as discussed above (compare Figure 3A with Figure 6A). Hence, it follows that the dominant affect of elevated glucose levels on Ca²⁺ influx is to depress receptor-operated Ca²⁺ influx in mesangial cells.

Reversibility of Glucose Effects on the Ca²⁺ Signal
In the early stages of diabetes, the renal pathology is associated with a decreased afferent arteriolar resistance and hypocontractility of mesangial cells. This leads to raised intraglomerular pressure, an important factor contributing to the pathogenesis of fractional mesangial expansion and progressive glomerulosclerosis (1,28). This state is generally accompained by a chronic elevation in plasma glucose levels that may contribute to the hypocontractility. Although it is generally believed that chronic exposure of cells to elevated glucose leads to part of the pathology, the present studies demonstrate that the effect of elevated glucose levels on the Ca²⁺ signal is primarily an acute phenomenon that does not require chronic exposure to elevated glucose levels. It was found that both chronic (days) and acute (minutes) exposure of mesangial cells to elevated glucose levels (30 mM) depressed the ET-induced Ca²⁺ signal by a similar amount. Most importantly, however, was the demonstration that when cells were first chronically exposed to high glucose levels for several days, subsequent acute reduction of the glucose levels to normal (5 mM) for several minutes immediately restores the ET-induced Ca²⁺ signal to normal. Hence, the effect of elevated glucose levels on the Ca²⁺ signal is an acute effect that is readily reversible with no apparent adaptation of the Ca²⁺ signaling components to chronically elevated glucose levels for up to 1 wk. It may be that prolonged chronic exposure to elevated glucose for several weeks may have additional effects (29).

Mechanism of High Glucose Effects on Ca²⁺ Signaling
It would appear from these studies that the effect of elevated glucose on Ca²⁺ signaling in mesangial cells is not a generalized phenomenon. The elevated glucose levels do not appear to affect either the Ca²⁺ stores (or release from stores) or the SOCI. The depressant effect of glucose on Ca²⁺ signaling appears to be solely on a newly unmasked receptor-operated Ca²⁺ influx pathway, supposedly a receptor-operated Ca²⁺ channel. The mechanism by which this occurs is currently unknown. It is not likely due to a downregulation of the ET receptor because the ET-induced release of Ca²⁺ from stores does not appear to be affected by high glucose levels, particularly during acute exposure to high glucose levels (Figure 3B). A lack of an effect of glucose on receptor levels would be in keeping with the finding of Amiri and Garcia (29) that treatment of mesangial cells with high glucose levels for up to 14 d did not affect AngII receptor levels (longer exposures depressed receptor levels). Hence, the effect of elevated glucose levels must be on an event downstream from the ET receptor. Recent studies point to a possible role of DAG, synthesized de novo from glucose, and protein kinase C redistribution in this process (27,30). Other studies have presented evidence for a role of advanced glycation end products, generated in the presence of high glucose levels, which may themselves bind to and alter Ca²⁺ signaling components in mesangial cells (31). Exactly how these downstream events may modulate Ca²⁺ signaling components in mesangial cells is currently not well understood. Additional studies are needed to elucidate the various components of the signal transduction pathways in regulating the Ca²⁺ transport events and the mechanism by which these pathways are modulated in the presence of altered glucose levels.

	Acknowledgments

 
This study was supported by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-40545) to Dr. O'Neil. The rat mesangial cells were a generous gift from Dr. Hanna E. Abboud, Division of Nephrology, University of Texas Health Science Center (San Antonio, TX). We thank Dr. Min Zhang for helpful discussions and critical rerading of the manuscript and Lijun Leng and Liliana Harris for technical assistance.

	Footnotes

 
^a The term "receptor-operated Ca²⁺ channel" is used here in a broad sense to reflect Ca²⁺ channels that may be directly linked to the ET receptor or indirectly linked to the receptor through a receptor-activated transduction pathway.

	References

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