Effect of Hypothalamic-Hypophysary Inhibitory Factor on Mesangial Cell Activation
Abstract We examined the effect of a sodium pump inhibitor isolated from bovine hypothalamus and pituitary tissues on contraction, proliferation, and calcium mobilization in primary cultures of rat mesangial cells. Hypothalamic-hypophysary inhibitory factor (HHIF) inhibited rubidium uptake in a concentration-dependent manner (0.2 U/mL: 56.8±6.3% inhibition). It also induced a concentration- and time-dependent decrease in planar cell surface area. Maximal contraction (25±5% reduction in cell size) was reached at 60 minutes with a concentration of 0.2 U/mL. This effect was inhibited by both verapamil and TMB-8 (10−5 mol/L). HHIF was also observed to increase DNA synthesis (0.2 U/mL: 4361±168 versus 2129±162 cpm per well under control conditions) and cell proliferation (0.2 U/mL: 52 290±1931 versus 10 512±121 cells per well under control conditions). Both effects were also inhibited by verapamil and TMB-8. Moreover, HHIF induced the expression of immediate early genes c-fos and c-jun mRNA. HHIF-induced effects were accompanied by an increase in cytosolic free calcium (203±58 versus 101±2 nmol/L under control conditions), which was inhibited by verapamil and TMB-8. In summary, HHIF induces mesangial cell contraction and proliferation; these effects seem to be mediated by an increase in cytosolic free calcium levels.
The physiological abnormality underlying essential hypertension is a rise in peripheral vascular resistance caused by increased VSMC tone. It is generally assumed that the pathophysiology of essential hypertension involves generalized alterations in membrane transport leading to a relatively decreased renal sodium excretion and expansion of plasma volume.1 One widely discussed theory that reconciles these phenomena affords a pivotal role in the etiology of essential hypertension to a humoral factor able to inhibit Na+,K+-ATPase activity. Plasma volume expansion secondary to an unknown renal tubular defect would increase plasma concentrations of these humoral factors with digitalis-like properties, which would thus become able to inhibit the sodium pump.2 Inhibition of active sodium transport may lead, by several possible pathways, to a rise in [Ca2+]i and subsequent increased vascular tone and blood pressure.3 Hypertension is also associated with abnormalities in vascular smooth muscle growth and glomerulosclerosis. A role for a circulating Na+ transport inhibitor in VSMCs and mesangial cell proliferation has been described.4 5 Recently, Hamlyn et al6 and Mathews et al7 purified from human plasma an endogenous inhibitor that has been characterized as ouabain.
Haupert and Sancho8 have suggested that one endogenous sodium transport inhibitor is synthesized in the hypothalamus; the factor isolated from hypothalamus by Haupert’s laboratory9 has been characterized as a ouabain isomer.10 A low-molecular-weight, nonpeptide, nonlipid Na,K-ATPase inhibitory factor has been isolated and purified from bovine hypothalamus and hypophysis and has been called HHIF.11 This factor has also been extracted from other tissues and human plasma.12 In contrast to ouabain, HHIF inhibits the Ca2+ pump13 and does not have ouabain-like cross-reactivity.11
Among the multiple events associated with proliferation, the first nuclear response is an increased expression of immediate early genes, which are thought to be critically involved in the control of the cell cycle.14 15 The induction of immediate early gene expression is independent of protein synthesis. Among the members of this group are c-fos, c-jun, and early growth response-1 (Egr-1), all of which encode transcription factors.16 The gene products of c-fos and c-jun form the heterodimer activating protein–1 (AP-1), which is a DNA-binding complex that regulates the transcription of other genes.
In the present study we examined the effects of HHIF on contraction, proliferation, expression patterns of immediate early genes, and calcium mobilization of cultured rat mesangial cells.
Collagenase type IA from Clostridium histolyticum, TMB-8, l-glutamine, sodium selenite, transferrin and insulin (human), and fura 2–AM were purchased from Sigma Chemical Co. Penicillin was obtained from Laboratorios Level SA. Streptomycin sulfate was obtained from Antibioticos SA. RPMI 1640, Hanks’ balanced salt solution, trypsin-EDTA solution, and FCS were obtained from Whittaker Laboratories. [3H]Thymidine and 86Rb were purchased from New England Nuclear. Verapamil was a gift of Knoll AG. Wistar rats were purchased from Charles River (Rouan, France). All other reagents were of the highest grade commercially available.
HHIF Extraction and Purification
HHIF was extracted from 1 kg frozen bovine hypothalamus and hypophysis, purified following chromatographic procedures, and physicochemically characterized as previously reported.11 The purified material has chemical and chromatographic characteristics different from any known cardioglycoside.11 12 Each bath of purified material was tested for purity as previously described.11 12 13 In essence, the homogeneous material underwent chromatography in two different chromatographic systems,12 and we again obtained a single, symmetrical peak with an identical spectrum and biological activity as the one of the purified material, suggesting the purity of the final product. This purified factor revealed by mass spectrometric analysis a single unique molecular ion with an accurate mass of 412 277 and mass spectra different from ouabain (unpublished data, 1995). Its ability to inhibit Na,K-ATPase from porcine renal outer medulla has been tested for each batch as previously reported,11 and 1 U was defined as the amount of HHIF required to inhibit the activity of 8 μg purified Na,K-ATPase by 50%.
Mesangial Cell Culture
Glomeruli isolated by successive mechanical sieving (150 and 50 μm) from Wistar rats (150 to 200 g) previously anesthetized with ether were treated with 300 U/mL collagenase, plated in 25-mm2 plastic tissue culture bottles (Nunc), and maintained under previously described conditions.17 18 The culture medium consisted of RPMI 1640 supplemented with 0.1 FCS, l-glutamine (1 mmol/L), penicillin (50 IU/mL), and streptomycin sulfate (0.034 mmol/L) and was buffered with bicarbonate. This culture medium was changed every 2 days. Studies were performed on day 21 or 22. Characterization of culture cells as mesangial cells was confirmed by morphological and functional criteria, strellate aspect, absence of factor VII, contraction with angiotensin II, and absence of growth inhibition with the substitution of l-valine for d-valine, described previously.17 18 Experiments were performed on cultures approaching confluence from the first passage in order to avoid cell dedifferentiation.
Cells were subcultured by treatment with 0.05% trypsin and 0.02% EDTA and plated in 6×4-well plates (Nunc). Cells were incubated with agonists at 37°C over 10 minutes and then pulsed with 1 μCi/mL 86Rb (0.5 mmol/L) during 15 minutes. The cell layers were washed three times with ice-cold culture medium and fixed with 0.5 mL ice-cold 0.1 trichloroacetic acid; acid-insoluble material was solubilized with 0.5 mL of 0.1 mol/L NaOH for 30 minutes at 60°C. After NaOH exposure cells were scraped off and removed; radioactivity was measured on a gamma scintillation counter (Gammamatic 1, Kontron Instruments).
Determination of Planar Surface Area of Mesangial Cells
Direct observation of mesangial cells in plastic flasks was carried out at room temperature under phase contrast with an inverted Nikon photomicroscope with the use of a CCD videocamera (model KP 110, Hitachi Denshi Ltd) and monitor (model VM-921E, Hitachi). With the use of an on-line videoprinter (Sony UP-910) serial photographs of the cells were taken before and after the addition of test substances. Five to 10 cells were analyzed per photograph. In each experimental block all the cells were from a single culture. Experiments were done in triplicate. Planar surface was determined by computerized image analysis with an IBAS II image-analyzer system (Kontron Instruments). Actual areas were calculated after correction for microscope and photographic magnification.
Measurement of [Ca2+]i
Measurement of [Ca2+]i was performed as previously reported.19 Fluorescence was measured at 37°C with a fluorescence spectrophotometer provided with a thermostatically controlled cuvette holder (Perkin-Elmer LS50) at an emission wavelength of 510 nm. An excitation wavelength of 340 nm was chosen for monitoring of the Ca2+-induced shift in fura 2 fluorescence as determined by previous calibration determinations. Autofluorescence, as measured in cells under similar conditions except that they were not loaded with fura 2, was found to be less than 10% of the total fluorescence determined in fura 2–loaded cells in all the experiments. [Ca2+]i was calculated as described by Olivera and López-Novoa.19
Cell proliferation was measured by [3H]thymidine uptake into DNA and by measurement of viable cell numbers. For this purpose cells were subcultured by treatment with 0.05% trypsin and 0.02% EDTA and plated in 6×4-well plates. Mesangial cells were rendered quiescent by removal of the serum-containing growth medium. Cells were incubated for 2 days with similar culture medium except that it contained a low amount (0.005) of FCS. During this time the cells were observed to incorporate a very low amount of [methyl-3H]thymidine, indicating the quiescent state. At this point the cells were reactivated by exposure to culture medium containing the substances to be tested. The conditions tested included cell exposure to quiescent medium alone (basal conditions); 0.2, 0.02, and 0.002 U/mL HHIF; 10−5 mol/L verapamil; 10−5 mol/L TMB-8; and medium containing 0.1 FCS as a positive control. After 18 hours of incubation the cells were pulsed for 6 hours with 1 μCi/mL [methyl-3H]thymidine. Cell layers were washed with ice-cold phosphate-buffered saline and fixed with 1 mL ice-cold 0.1 trichloroacetic acid for 15 minutes; acid-insoluble material was solubilized with 1 mL of 0.1 mol/L NaOH for 30 minutes at 60°C. After NaOH exposure radioactivity was determined on a scintillation counter (Betamatic IV, Kontron Instruments).
We also checked the number of viable cells in each well using a commercial modification (Cell Titter 96, Promega) of the method of Hansen et al.20 In brief, cells subcultured to subconfluence in 24-well plates were rendered quiescent as described above. Cells were then incubated for 24 hours with culture medium and the substances described above. The medium was removed and 1 mL fresh incubation medium was added. Then, 150 μL of a solution of tetrazolium bromide salt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5 mg/mL] was added to each well. The plate was returned to the incubator (Ultima; Revco Scientific, Inc) for 4 hours, after which 1 mL of the solubilization solution containing sodium dodecyl sulfate and N,N-dimethyl formamide, pH 4.7, was added. After 1 hour at room temperature the contents of the well were mixed thoroughly with a Pasteur pipette to obtain a uniform color. The contents of the wells were then transferred to a cuvette and read at 570 nm on a spectrophotometer (Shimadzu UV-120-02). Optical density at 570 nm was proportional to the number of viable cells in each well.5
c-fos and c-jun Gene Expression
Mesangial cells were rendered quiescent by removal of the serum-containing growth medium and incubation for 3 days with similar culture medium without FCS. Cells were treated with 0.2 U/mL HHIF, and cells treated with 0.1 FCS were used as positive controls. After 30 minutes of stimulation mRNA was extracted from 5×105 cells by NP-40 lysis.21 The RNA (1 to 2 μg) was subjected to a reverse transcription reaction with the use of oligo(dT) primers (Promega). cDNA was amplified by PCR with oligonucleotides derived from two adjacent exons of the actin gene, c-fos gene, and c-jun gene with the use of a thermal cycler (Dual Cycler, Linus) and amplitaq DNA polymerase.22 Oligonucleotides were synthesized at the Department of Microbiology and Genetics, University of Salamanca, and their sequences were as follows: FOS 1: 5′-CAGCCGACTCCTTCTCCAG-3′; FOS 2: 5′-GCCACGGAGGAGACCAGAGT-3′; JUN 1: 5′-GACCTTCTACGACGATGC-3′; JUN 2: 5′-CAGCGCCAGCTACTGAGGC-3′; ACT 1: 5′-AGGCCAACCGCGAGAAGATGACC-3′; and ACT 2: 5′-GAAGTCCAGGGCGACGTAGCAC-3′.
The amplification conditions consisted of denaturing at 94°C for 2 minutes, annealing at 60°C for 2 minutes, and extension at 72°C for 2 minutes. The number of cycles was 25. PCR products were size-fractionated by agarose gel electrophoresis. After staining with ethidium bromide, DNA bands were visualized with a UV transilluminator (Vilber Lourmat TF-35M).
In proliferation studies each value is the mean of three wells. Data were obtained as the average of 4 to 12 values. Comparisons between means of multiple groups were analyzed by one-way ANOVA and Scheffé’s multiple comparisons test. Time course studies were analyzed by two-way ANOVA.
Effect of HHIF on 86Rb Uptake
HHIF decreased 86Rb uptake by mesangial cells in a concentration-dependent manner. Inhibition with the highest concentration of HHIF used (0.2 U/mL) was stronger than that with 10−4 mol/L ouabain, a submaximal concentration used to compare the effect of HHIF with a well-known Na+-pump inhibitor (Table⇓).
Effect of HHIF on Mesangial Planar Cell Surface Area
After 60 minutes of incubation HHIF decreased mesangial planar cell surface area. This effect was concentration- and time-dependent (Figs 1⇓ and 2⇓). By contrast, no significant changes were observed at that time in cells incubated under control conditions.
Preincubation of mesangial cells with the calcium antagonist verapamil (10−5 mol/L), a voltage-dependent calcium channel inhibitor, significantly blunted the contractile response to HHIF (0.2 U/mL). In addition, TMB-8 (10−5 mol/L), an inhibitor of Ca2+ release from the endoplasmic reticulum, also blunted the response to HHIF (Fig 2⇑).
Effect of HHIF on [Ca2+]i
HHIF induced a slow and progressive increase in [Ca2+]i from resting levels in a concentration-dependent manner (Fig 3⇓). The lowest concentration used in our experiments (0.002 U/mL) did not produce any change in [Ca2+]i, but higher concentrations (0.02 and 0.2 U/mL) induced a statistically significant increase in [Ca2+]i. This increase was time dependent, with a maximal increase in [Ca2+]i of approximately twofold the basal values at 10 minutes (Fig 3⇓). Verapamil (10−5 mol/L) and TMB-8 (10−5 mol/L) blunted the response to HHIF (0.2 U/mL) (Fig 4⇓).
Effect of HHIF on Cellular Proliferation
HHIF also stimulated [3H]thymidine incorporation into DNA. This effect was significant at concentrations of 0.2 and 0.02 U/mL (Fig 5A⇓). Verapamil (10−5 mol/L) abolished HHIF (0.02 and 0.2 U/mL)–stimulated thymidine incorporation into DNA (Fig 6⇓). TMB-8 (10−5 mol/L) also blocked the HHIF-induced effect on thymidine incorporation into DNA (Fig 6⇓).
A dose-dependent increase in the number of viable cells after 24 hours of incubation in the presence of HHIF was observed (Fig 5B⇑). This effect was partially inhibited by verapamil (10−5 mol/L) and TMB-8 (10−5 mol/L) (Fig 7⇓).
c-fos and c-jun Expression
Fig 8⇓ shows representative agarose gel electrophoresis of amplification products by reverse transcriptase–PCR of c-fos, c-jun, and actin genes. In cells treated for 30 minutes with 0.1 FCS, cDNA amplification gave a band of approximately 195 bp, which is consistent with the calculated size of the c-fos gene (Fig 8A⇓, lane 2). Treatment with 0.2 U/mL HHIF induced the expression of c-fos mRNA (Fig 8A⇓, lane 3); this expression was not observed in quiescent cells (Fig 8A⇓, lane 1). A band of approximately 310 bp, corresponding to the calculated size of the actin gene, appeared with all treatments (Fig 8⇓).
The results obtained in the present work suggest that the endogenous sodium pump inhibitor obtained from hypothalamus (HHIF) is able to inhibit 86Rb uptake by mesangial cells. HHIF was also able to decrease the mesangial cell planar surface area, an event that represents cell contraction.23 Moreover, HHIF increased DNA synthesis, assessed by [3H]thymidine incorporation into DNA, and cell proliferation, measured as the increase in the number of cells per well. As similar effects have been previously reported for ouabain,5 the specific inhibitor of the Na,K-ATPase, it can be suggested that the effects of HHIF are mediated by its ability to inhibit the sodium pump.
HHIF also induced the expression of the immediate early response genes c-fos and c-jun mRNA, an event closely associated with cell proliferation.14 A common mechanism mediating both mesangial cell contraction and proliferation as well as c-fos and c-jun mRNA expression is the increase in [Ca2+]i.24 25 Our data reveal that HHIF induces a slow and sustained increase in [Ca2+]i.
The inhibition of Na,K-ATPase may lead to elevated [Ca2+]i through several possible mechanisms. First, inhibition of the sodium pump may result in slight membrane depolarization26 and consequently increased Ca2+ influx through activated voltage-gated Ca2+ channels.27 This hypothesis is in agreement with our findings pointing to the inhibition of an HHIF-induced increase in [Ca2+]i and cell contraction by the voltage-gated Ca2+ channel blocker verapamil. Okada et al28 and Meyer-Lehnert et al29 have also reported that ouabain induces a sustained increase in [Ca2+]i in VSMCs that can be inhibited by verapamil, suggesting that transmembrane Ca2+ influx through activated voltage-gated Ca2+ channels would play a role in the increase in [Ca2+]i. Our findings are also consistent with those of Goto and colleagues,30 who showed that a digitalis-like factor from human urine enhances 45Ca uptake in VSMCs. However, other studies31 have suggested that partial inhibition of Na,K-ATPase in smooth muscle cells (and presumably in mesangial cells) is unable to induce any substantial degree of cell depolarization.
According to our results, a second hypothesis would be that HHIF might also release calcium from the endoplasmic reticulum. This suggestion is supported by the fact that the inhibition of intracellular calcium release by TMB-8, an endoplasmic calcium release blocker, inhibits HHIF-induced increases in [Ca2+]i and abolishes the cell proliferation and contraction induced by HHIF. It is noteworthy that both blockers, verapamil and TMB-8, significantly inhibit the effect of HHIF on mesangial cells. This could be accounted for by the need for a threshold value in [Ca2+]i to be reached in order for the cells to be activated. The source of this calcium could be either the extracellular medium or the intracellular stores. Therefore, blocker-induced inhibition of calcium mobilization leads to a loss of cellular ability to respond to HHIF. Along this line, it has been recently reported32 that the persistent Ca2+ influx that follows activation of mesangial cells by vasoconstrictors should be attributed to an inward current sensitive to the initial release of internally stored Ca2+. Thus, the inositol 1,4,5-trisphosphate–sensitive Ca2+ pool may serve as a “trigger” for a sustained response, thus initiating a cascade that would amplify the signaling mechanism.
Third, sodium pump inhibition would lead to a rise in cytosolic sodium, a decrease in the transmembrane Na+ gradient, and thus an inhibition of Na+-Ca2+ exchange.30 33 Inhibition of Na+-Ca2+ exchange would induce an increase in [Ca2+]i or an accumulation of Ca2+ in the sarcoplasmic reticulum.31 The presence of Na+-Ca2+ exchange in the plasma membrane of mesangial cells34 35 is consistent with a role of the latter mechanism in an HHIF-induced increase in [Ca2+]i.
Finally, we have recently reported that HHIF is able to inhibit plasma membrane Ca2+-ATPase.12 As this ATPase is involved in the control of [Ca2+]i by pumping out Ca2+ from the cytosol, Ca2+ pump inhibition could also be a mechanism through which HHIF would be able to increase [Ca2+]i. This hypothesis is consistent with the findings of Goto et al30 showing that a digitalis-like factor from human urine inhibits 45Ca efflux in VSMCs.
In summary, the present study demonstrates that a sodium pump inhibitor obtained from bovine hypothalamus and hypophysis (HHIF), which is different from ouabain, induces mesangial cell activation, including contraction and proliferation. These effects seem to be mediated at least in part by an increase in [Ca2+]i. However, the discrepancy in threshold doses for the different effects of HHIF on mesangial cells suggests that the mechanism of action of this substance is highly complex and probably not all the effects observed are mediated exclusively by the increase in [Ca2+]i.
Selected Abbreviations and Acronyms
|[Ca2+]i||=||cytosolic free calcium concentration|
|FCS||=||fetal calf serum|
|HHIF||=||hypothalamic-hypophysary inhibitory factor|
|PCR||=||polymerase chain reaction|
|TMB-8||=||3,4,5-trimethoxybenzoic acid 8-(diethylamine)octyl ester|
|VSMC||=||vascular smooth muscle cell|
The present study was partially supported by the “Comisión Interministerial de Investigación Científica y Técnica,” Spain (grant SAF 92/0039). We thank Dr Rogelio Gonzalez-Sarmiento for his advice with PCR, Dr José J. García-Marín for his helpful comments on the manuscript, and Nicolas Skinner for his help with the English translation.
- Received January 26, 1995.
- Revision received March 14, 1995.
- Accepted August 18, 1995.
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