Differential regulation by 1,25-dihydroxyvitamin D3 of calbindin-D9k and calbindin-D28k gene expression in mouse kidney

The mouse kidney is a unique tissue since both vitamin D-dependent calcium binding proteins (calbindin-D9k and calbindin-D28k) are present in the same cells of the distal convoluted tubule. We have used specific complementary DNAs to mouse calbindin-D9k and mouse calbindin-D28k and Northern and slot blot analyses in order to obtain a better understanding of the regulation of two different molecular expressions of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] action in the same cells. Both calbindins were found to be regulated developmentally in a similar manner (an increase in gene expression between birth and 1 week of age, coinciding with nephron differentiation, and a peak at 3 weeks of age). However, the time course of response of the messenger RNA of each calbindin to 1,25(OH)2D3 was markedly different. The peak of induction of renal calbindin-D28k mRNA was at 12 h after a single injection of 1,25(OH)2D3 (200 ng/100 g body wt) to vitamin D-deficient mice, and a decrease was observed at 24 h (similar to the time course of response of other steroid-regulated genes). Interestingly, unlike calbindin-D28k, a delayed response of renal calbindin-D9k mRNA to 1,25(OH)2D3 was observed (the peak of induction was at 24 h after 1,25(OH)2D3 administration). Both genes in mouse kidney did not respond to glucocorticoids, although a dose-dependent decrease (12-86%) of mouse intestinal calbindin-D9k mRNA was observed after dexamethasone treatment, suggesting tissue-specific multiple steroid interactions in the regulation of calbindin gene expression. The finding of a different time course of regulation of each calbindin by 1,25(OH)2D3 suggests that different factors may be regulating the expression of the two different calbindins in mouse kidney and that elucidation of these control mechanisms should provide new insight concerning 1,25(OH)2D3-regulated gene expression.     

Intracellular calcium ion response to glucose in beta-cells of calbindin-D28k nullmutant mice and in betaHC13 cells overexpressing calbindin-D28k

This article describes studies on the glucose-induced responses of intracellular Ca²⁺ concentration ([Ca²⁺]_i), insulin release, and redistribution of calbindin-D_28k, a calcium-binding regulatory protein, in β-cells of pancreatic islets of calbindin-D_28k knockout (KO) and wild-type mice (C57BL6) as well as in βHC-13 control cells and βHC-13 CaBP40 cells (β-cell line overexpressing calbindin-D_28k). Upon increasing the glucose concentration from 2.8 to 30 mM, islets of KO mice showed a significantly greater increase in [Ca²⁺]_i (mean increase in [Ca²⁺]_i, i.e., Δ[Ca²⁺], was 296 nM) compared with wild-type mice (Δ[Ca²⁺]_i=97 nM). βHC-13 CaBP40 cells showed little change in [Ca²⁺]_i upon elevation of glucose from 5.5 to 32.7 mM, whereas βHC-13 control cells exhibited significant increases in [Ca²⁺]_i (Δ[Ca²⁺]_i=510 nM). Similarly, upon addition of 30 mM glucose, the rate of insulin release increased from 25.2 (basal rate) to 145.2 pg/mL/min in βHC-13 control cells, whereas in βHC-13 CaBP40 cells the rate of insulin release was only 27.5 pg/mL/min in high glucose. Thus, levels of calbindin-D_28k in β-cells affect both [Ca²⁺]_i and insulin secretion in response to glucose. The three-dimensional reconstruct of confocal immunofluorescent images showed that glucose caused redistribution of calbindin-D_28k resulting in co-localization in the region of L-type voltage-dependent calcium channels (VDCC). This colocalization may be an important regulatory function concerning Ca²⁺ influx via L-type VDCC and exocytosis of insulin granules.     

Mouse calbindin-D(9k) gene expression in the uterus during late pregnancy and lactation

Calbindin-D(9k) (CaBP-9k) is a cytosolic calcium binding protein mainly expressed in duodenum, placenta and uterus. In order to understand the expression pattern and regulation of uterine CaBP-9k gene, the expression of CaBP-9k mRNA and its regulation by estrogen (E2) and progesterone (P4) were investigated in the mouse uterus during late pregnancy (from day 12 to 18) and lactation. The expression levels of uterine CaBP-9k, estrogen receptor alpha (ERalpha) and progesterone receptor (PR) mRNAs were measured by Northern blot analysis. The expression levels of mouse uterine CaBP-9k mRNA gradually increased from pregnancy day 16 (P16), peaked at P18 (6.0-fold vs. P12) and declined at birth and during lactation. The expression levels of ERalpha and PR mRNAs indicated a similar fluctuation as CaBP-9k mRNA, suggesting the role of sex steroids/receptors in the regulation of CaBP-9k gene. To investigate effect of steroid hormone on CaBP-9k mRNA expression, three groups of animals were injected (s.c) with steroid hormone antagonists (RU486, tamoxifen, and ICI182780), respectively. RU486, a P4 antagonist, induced a significant decrease in CaBP-9k mRNA expression at 48 (3.2-fold) and 72 h (3.8-fold). However, tamoxifen and ICI182780, E2 antagonists, had no effect on CaBP-9k mRNA expression. Combined treatment with RU486 and ICI182780 did not further decrease the expression level of CaBP-9k mRNA when compared with RU486 treatment at 48 and 72 h. In addition, the treatment with RU40555, a glucocorticoid/progesterone antagonist, resulted in a decrease at 48 and 72 h following treatment. These results indicate that E2 is not likely involved in the regulation of CaBP-9k gene in the mouse uterus during late pregnancy and lactation. In conclusion, the present results suggest that P4, not E2 is a key regulator of CaBP-9k mRNA expression during late pregnancy and lactation.     

Redistribution of calbindin-D28k in chick intestine in response to calcium transport.

Vitamin D and its hormonally active metabolite 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] are known to alter several parameters associated with stimulated intestinal Ca2+ transport: levels of calbindin-D28K, tubulin, and endosomal-lysosomal organelles containing Ca2+, and calbindin-D28K. In the present study the as yet unexamined relationship among Ca2+ transport, calbindin-D28K, and microtubules was studied by immunofluorescence microscopy. In vitamin D3-treated or 1,25-(OH)2D3-treated chicks, in the absence of Ca2+ transport, immunofluorescence microscopy of intestinal tissue fixed at 25 C indicated a colocalization of calbindin-D28K and tubulin along epithelial cell brush border and basal-lateral membranes. Initiation of in situ Ca2+ absorption for 10, 20, or 30 min before tissue fixation resulted first in increased punctate calbindin-D28K staining and then in a progressive decrease in intestinal cell- and microtubule-associated calbindin-D28K, with a concomitant increase in calbindin-D28K labeling in the villus core. When intestinal tissue from 1,25-(OH)2D3-treated chicks was chilled to 4 C before fixation (a procedure shown by others to cause microtubule depolymerization), evaluation by immunofluorescence microscopy revealed diffuse cytoplasmic staining of both the immunoreactive tubulin and its associated calbindin-D28K. These results indicate the possible involvement of calbindin-D28K with tubulin during the process of Ca2+ transport and the secretion of the calbindin-D28K as a consequence of the overall transport process. Electron microscopy with immunogold labeling revealed intestinal epithelial calbindin-D28K to be localized inside of small vesicles and lysosome-like structures, with sparse cytoplasmic labeling. Subsequent electron microscopic analysis of intestinal epithelial microtubules prepared by polymerization and depolymerization revealed immunogold labeling in coprecipitated vesicular remnants, with consistently light staining of filaments traversing segments of the microtubules. In biochemical studies, isolation of intestinal microtubules or tubulin by three distinct procedures revealed increasing levels of associated calbindin-D28K as a function of time after 1,25-(OH)2D3 repletion of vitamin D-deficient chicks. Addition of calbindin-D28K to intestinal microtubules isolated from vitamin D-deficient chicks exhibited saturable binding when exogenous calbindin-D28K reached levels comparable to those present in vitamin D-replete chick intestine. Collectively, these results suggest that calbindin-D28K is predominantly located in membrane-delimited vesicles, with a very minor component associated with filamentous elements that can be isolated with tubulin and microtubules. Additionally, calbindin-D28K is dynamically involved in Ca2+ transport in the intestine.     

Estrogen modulates expression of putative housekeeping genes in the mouse uterus

Estrogens regulate gene expression and cell proliferation in target tissues. In studies of estrogen-regulated gene expression, identification of appropriate housekeeping genes (HKGs), reference genes whose expression is not altered by treatment, is difficult. The goal of this study was to define HKGs unaltered by estrogen in the mouse uterus. Ovariectomized C57BL6 mice were dosed with 20 micrograms/kg ethinylestradiol and the uterus was collected at 6, 24, and 72 h later to bracket the biphasic time course of estrogen action in the rodent uterus. RNA was isolated, cDNA synthesized and equal amounts of cDNA were added to real-time PCR reactions. The expression of seven out of nine putative HKGs was altered by estrogen in the mouse uterus. Estrogen induced four gene expression profiles, expression of: (1) Actb and Hsp90ab1 were up-regulated early, (2) B2m and Gusb were up-regulated late, (3) Gapdh, Hprt1, and Ppia were up regulated at all time points, and (4) Rpl13a and 18srRNA were unaltered. This highlights the need to empirically determine the appropriate HKG for each experimental condition. Based on these results, we suggest using Rpl13a or 18srRNA as HKGs for xenoestrogen studies in the mouse uterus and as good candidates to test under different experimental conditions.     

Proteomic identification and immunolocalization of increased renal calbindin-D28k expression in OVE26 diabetic mice

Diabetic nephropathy is a common diabetic complication that is associated with alterations in the expression of several renal proteins and abnormal calcium homeostasis. We performed proteomic analysis to screen for global changes of renal protein expression in diabetic kidney. Proteins extracted from the whole kidney of 120-day-old OVE26 (a transgenic model of Type 1 diabetes) and FVB (non-diabetic background strain) mice were separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and visualized by SYPRO Ruby staining (n = 5 in each group). Quantitative intensity analysis revealed 41 differentially expressed proteins, of which 30 were identified by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) followed by peptide mass fingerprinting. One of the altered proteins with the greatest magnitude of change was the calcium-binding protein, calbindin-D28k, whose expression was increased 6.7-fold in diabetic kidney. We confirmed the increase in calbindin-D28k expression in diabetic kidney by Western blot analysis. Immunohistochemical study demonstrated that calbindin-D28k expression was markedly increased in tubular epithelial cells of distal convoluted tubules (DCT), collecting ducts (CD), and proximal convoluted tubules (PCT) in diabetic kidney. Calbindin-D28k plays a critical role in maintaining calcium homeostasis. The elevation in renal calbindin-D28k expression in our model may indicate a compensatory mechanism to overcome hypercalciuria in diabetes.     

Identification of sequence elements in mouse calbindin-D28k gene that confer 1,25-dihydroxyvitamin D3- and butyrate-inducible responses.

We have examined the 5' flanking region of the mouse calbindin-D28k gene and identified a 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-responsive element by deletion mutant analysis of the native promoter as well as by studies with a heterologous thymidine kinase (TK) promoter. The segment between residues -200 and -169 was found to confer a dose-dependent 1,25-(OH)2D3 responsiveness through the TK promoter in Ros 17/2.8 cells as well as in CV-1 cells cotransfected with pAV-hVDR (human vitamin D receptor expression vector). This region contains sequences homologous to the rat osteocalcin vitamin D response element (VDRE). Incubation of this element with nuclear extracts from 1,25-(OH)2D3-treated Ros 17/2.8 cells or from 1,25-(OH)2D3-treated COS cells that had been transfected with pAV-hVDR resulted in a specific protein-DNA interaction. In addition to 1,25-(OH)2D3, sodium butyrate, a differentiating agent, has also been found to modulate expression of calbindin-D28k. Deletion analysis of the mouse calbindin-D28k promoter as well as studies with a heterologous TK promoter resulted in identification of a butyrate-responsive element between -180 and -150 that was found to bind specifically to nuclear factors from butyrate-treated Ros 17/2.8 cells. This butyrate-responsive element may represent a genetic element acted upon by enhancer binding proteins. In summary, the 5' flanking region of the mouse calbindin-D28k gene contains responsive elements that interact with nuclear factors and may mediate, at least in part, the enhanced expression of this gene by 1,25-(OH)2D3 and butyrate.     

Stimulation of calbindin-D(9k) mRNA expression in the rat uterus by octyl-phenol,nonylphenol and bisphenol

Quantification of estrogen-induced changes in the expression levels of endogenous genes such as pS2 and vitellogenin could be an assay to detect estrogenicity of chemicals. Considering its regulation by estrogen, in the present study, we hypothesize that the calbindin-D(9k) (CaBP-9k) gene has the possibility as a biomarker for estrogenic response of the environmental estrogens. We analyzed the time- and dose-dependent CaBP-9k mRNA expression in the immature rats by 3-day injection of 17beta-estradiol (E2) and alkylphenol acid [octyl-phenol (OP) and nonylphenol (NP)] and bisphenol A(BPA)) which are environmentally persistent and reported to have some estrogenic activity in experimental test systems. The expression of CaBP-9k mRNA was compared with uterotropic response of the compounds. A significant increase in CaBP-9k mRNA expression was observed when treated with 1000 mg/kg body weight (BW) per day of OP (18-fold versus control), NP (17-fold versus control) and BPA (6-fold versus control) for 3 days in dot blot assays. Using Northern blot analysis, a more dramatic increase of CaBP-9k mRNA expression was observed when treated with 1000 mg/kg BW per day of OP (132-fold versus control) and NP (113-fold versus control) for 3 days. Treatment with 10 and 100 mg/kg BW per day of NP and 100 mg/kg BW per day of OP for 3 days induced a small but significant increase in CaBP-9k mRNA expression. As expected, a single dose of E2 (40 microg/kg BW per day) for 3 days induced a significant increase in CaBP-9k mRNA expression as revealed by dot (15-fold versus control) or Northern blot assay (102-fold versus control). In a time response experiment using Northern blot assay, a significant increase in CaBP-9k mRNA expression was observed as early as 3 h, peaked at 6 h and continued until 72 h after treatment with 1000 mg/kg BW per day of OP, NP, and 48 h after treatment with 1000 mg/kg BW per day of BPA. A similar time-dependent response was observed when assessed by dot blot assay. Uterotropic response of the compounds was determined and compared with CaBP-9k mRNA expression. The alkylphenolic compounds induced a significant increase in the uterine wet weight at 1000 mg/kg BW per day of OP and NP, not BPA. A strong correlation between in vivo uterotropic assay and CaBP-9k mRNA expression assay was observed. In order to investigate the possible mechanisms by which the compounds regulate CaBP-9k mRNA expression, we studied the effect of the compound on the ERalpha mRNA level using total RNA from the treated rats. The alkylphenolic compounds as well as E2 stimulate the expression of ERalpha mRNA in a similar pattern to that of CaBP-9k mRNA in terms of dose- and time-dependent response. Strong regulation of CaBP-9k mRNA expression by E2 and the environmental estrogens and its correlation with in vivo uterotropic assay suggest that CaBP-9k gene can be used as a biomarker gene for assaying estrogenicity of putative estrogenic compounds.     

Expression of calbindin-D(28k) in a pancreatic islet beta-cell line protects against cytokine-induced apoptosis and necrosis.

Cytokines produced by immune system cells that infiltrate pancreatic islets are candidate mediators of islet beta-cell destruction in autoimmune (type 1) diabetes mellitus. Because the calcium binding protein, calbindin-D(28k), can prevent apoptotic cell death in different cell types, we investigated the possibility that calbindin-D(28k) may prevent cytokine-mediated islet beta-cell destruction. Using the expression vector BSRalpha, rat calbindin-D(28k) was stably expressed in the pancreatic islet beta-cell line, betaTC-3. Calbindin-D(28k) expression resulted in increased cell survival in the presence of the cytotoxic combination of the cytokines IL-1beta (30 U/ml), TNFalpha (10(3) U/ml), and interferon gamma (10(3) U/ml). The greatest protection was observed in the betaTC-3 cell clone expressing the highest concentration of calbindin-D(28k). Apoptotic cell death was detected by annexin V staining and by the TdT-mediated dUTP-X nick end labeling assay in vector-transfected betaTC-3 cells incubated with cytokines (14-15% apoptotic cells). The number of apoptotic cells was significantly decreased in calbindin-D(28k)-overexpressing betaTC-3 cells incubated with cytokines (5-6% apoptotic cells). To address the mechanism of the antiapoptotic effects of calbindin, studies were done to examine whether calbindin inhibits free radical formation. The stimulatory effects of the cytokines on lipid hydroperoxide, nitric oxide, and peroxynitrite production were significantly decreased in the calbindin-D(28k)-expressing betaTC-3 cells. Our findings indicate that calbindin-D(28k), by inhibiting free radical formation, can protect against cytokine-mediated apoptosis and destruction of beta-cells. These findings suggest that calbindin-D(28k) may be an important regulator of cell death that can protect pancreatic islet beta-cells from autoimmune destruction in type 1 diabetes.     

Noncytoplasmic and filamentous appearance of calbindin-D28k and tubulin in double, indirect immunofluorescent staining of embryonic chick tissue.

Double indirect immunofluorescent labeling of embryonic chick tissue was undertaken for the vitamin D-induced calcium binding protein, calbindin-D28k, and microtubules. Immunoreactivities for both calbindin-D28k and tubulin were found to exhibit a filamentous staining pattern in mesonephros, metanephros, intestine, and brain. In the intestine, staining was absent at 19 days, while immunolabeling of tubulin became evident at 20 days, and both antigens were present in 21-day tissue. In intestinal epithelium, as well as in 10-day metanephros, it was strikingly evident that cells either stained for both antigens or were negative for both calbindin-D28k and tubulin. In 11-12-day metanephros, an increased number of cells with both immunoreactivities were found. In 15-17-day brain, tubulin was evident within all cells but stained most intensely in Purkinje cells which were also positive for calbindin-D28k. Mesonephros from 4-5-day embryos revealed immunolabeling of both tubulin and the calcium binding protein. A statistical analysis of the various cell types revealed that the vast majority contained either both antigens or neither of the immunoreactivities. Of the more than 600 cells scored, none were found to be positive for calbindin-D28k, while at the same time negative for tubulin. It is concluded that calbindin-D28k exhibits a noncytoplasmic distribution in all tissues tested and that the filamentous appearance may reflect localization of the antigen in tubulo-vesicular organelles associated with cytoskeleton.     

Estrogen induces CCN5 expression in the rat uterus in vivo

Estrogen plays an important role in the normal physiology as well as various pathologies of the uterus. Given the nature of uterine remodeling during the reproductive cycle and pregnancy, we sought to determine whether CCN5, a gene that we have shown to be important in smooth muscle cell proliferation and migration, is an estrogen-induced gene in the uterus. In the present study, we demonstrate that levels of CCN5 mRNA and protein expression were 5-fold higher in uteri from proestrous females relative to metestrous females, a finding consistent with estrogen induction of the CCN5 gene. Ovariectomized rats treated with exogenous estrogen or estrogen and progesterone exhibited 4- to 8-fold higher levels of CCN5 mRNA and protein than animals treated with either progesterone or vehicle alone. Analysis of rat uterine sections using immunohistochemistry demonstrates CCN5 localization throughout the uterus, including the endometrium and endometrial glands as well as the myometrium. Thus, our data indicate that CCN5 is positively regulated by estrogen in the rat uterus and suggests that this gene may play an important role in maintaining normal uterine physiology.     

Extracellular calcium modulates vitamin D-dependent calbindin-D28K gene expression in chick kidney cells

The effect of extracellular calcium ion (Ca2+) concentration on 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-induction of vitamin D-dependent calcium-binding protein (calbindin-D28K) and its mRNA levels was examined in primary chick kidney cells in vitro. When exposed to normal medium Ca2+ (1.0 mM), 1,25-(OH)2D3 increased calbindin-D28K mRNA, as measured by Northern analysis, by 4-10 fold over basal levels by 12 to 24 h after addition of hormone. In the presence of 0.5 mM Ca2+, 1,25-(OH)2D3 induced calbindin-D28K mRNA by only 2 fold, whereas, when cells were exposed to 2 mM Ca2+, the induction was 10-15 fold. This calcium modulation of 1,25-(OH)2D3 induction was also observed at the level of calbindin-D28K protein concentrations as measured by radioimmunoassay. The alterations in medium Ca2+ were not associated with any change in the rate of total RNA or protein synthesis. These studies suggest that both Ca2+ and 1,25-(OH)2D3 participate in the regulation of calbindin-D28K gene expression in the kidney.     

Expression of calbindin-D28K in motoneuron hybrid cells after retroviral infection with calbindin-D28K cDNA prevents amyotrophic lateral sclerosis IgG-mediated cytotoxicity.

Calbindin-D28K and/or parvalbumin appear to influence the selective vulnerability of motoneurons in amyotrophic lateral sclerosis (ALS). Their immunoreactivity is undetectable in motoneurons readily damaged in human ALS, and in differentiated motoneuron hybrid cells [ventral spinal cord (VSC 4.1 cells)] that undergo calcium-dependent apoptotic cell death in the presence of ALS immunoglobulins. To provide additional evidence for the role of calcium-binding proteins in motoneuron vulnerability, VSC 4.1 cells were infected with a retrovirus carrying calbindin-D28K cDNA under the control of the promoter of the phosphoglycerate kinase gene. Differentiated calbindin-D28K cDNA-infected cells expressed high calbindin-D28K and demonstrated increased resistance to ALS IgG-mediated toxicity. Treatment with calbindin-D28K antisense oligodeoxynucleotides, which significantly decreased calbindin-D28K expression, rendered these cells vulnerable again to ALS IgG toxicity.     

Specific reduction of calcium-binding protein (28-kilodalton calbindin-D) gene expression in aging and neurodegenerative diseases.

The present studies establish that there are specific, significant decreases in the neuronal calcium-binding protein (28-kDa calbindin-D) gene expression in aging and in neurodegenerative diseases. The specificity of the changes observed in calbindin mRNA levels was tested by reprobing blots with calmodulin, cyclophilin, and B-actin cDNAs. Gross brain regions of the aging rat exhibited specific, significant decreases (60-80%) in calbindin mRNA and protein levels in the cerebellum, corpus striatum, and brain-stem region but not in the cerebral cortex or hippocampus. Discrete areas of the aging human brain exhibited significant decreases (50-88%) in calbindin protein and mRNA in the cerebellum, corpus striatum, and nucleus basalis but not in the neocortex, hippocampus, amygdala, locus ceruleus, or nucleus raphe dorsalis. Comparison of diseased human brain tissue with age- and sex-matched controls yielded significant decreases (60-88%) in calbindin protein and mRNA in the substantia nigra (Parkinson disease), in the corpus striatum (Huntington disease), in the nucleus basalis (Alzheimer disease), and in the hippocampus and nucleus raphe dorsalis (Parkinson, Huntington, and Alzheimer diseases) but not in the cerebellum, neocortex, amygdala, or locus ceruleus. Since calbindin gene expression decreased specifically in brain areas known to be particularly affected in aging and in each of the neurodegenerative diseases, these findings suggest that decreased calbindin gene expression may lead to a failure of calcium buffering or intraneuronal calcium homeostasis, which contributes to calcium-mediated cytotoxic events during aging and in the pathogenesis of neurodegenerative diseases.     

Estradiol rapidly inhibits soluble guanylyl cyclase expression in rat uterus

Previous reports that investigated the regulation of the NO/soluble guanylyl cyclase (sGC)/cGMP pathway by estrogenic compounds have focused primarily on the levels of NO, NO-producing enzymes, and cGMP in various tissues. In this study, we demonstrate that 17beta-estradiol (E2) regulates the alpha(1) and beta(1) subunits of the NO receptor, sGC, at the mRNA and protein levels in rat uterus. Using real-time quantitative PCR, we found that within 1 h of in vivo E2 administration to rats, sGC mRNA levels begin to diminish. After 3 h, there is a maximal diminution of sGC mRNA expression (sGC alpha(1) 10% and sGC beta(1) 33% of untreated). This effect was blocked by the estrogen receptor antagonist, ICI 182,780, indicating that estrogen receptor is required. The effect of E2 also was observed in vitro with incubations of uterine tissue, indicating that the response does not depend on the secondary release of other hormones or factors from other tissues. Puromycin did not block the effect, suggesting the effects occur because of preexisting factors in uterine tissues and do not require new protein synthesis. Using immunoblot analysis, we found that sGC protein levels also were reduced by E2 over a similar time course as the sGC mRNA. We conclude that sGC plays a vital role in the NO/sGC/cGMP regulatory pathway during conditions of elevated estrogen levels in the rat uterus as a result of the reduction of sGC expression.