| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Male Reproductive Tract |
Department of Physiology,5 Faculty of Medicine, The University of Hong Kong, Hong Kong, China
Molecular Physiology Laboratory,6 Baker Medical Research Institute, Melbourne, Victoria 8008, Australia
Department of Physiology,7 Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
epididymis, neuropeptides, polypeptide receptors, sperm
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Although AM has specific receptors, it may act through CGRP receptors [12]. The receptor types of AM have been reviewed elsewhere [12]. Clearly, the calcitonin receptor-like receptor (CRLR) accounts for the majority of AM and CGRP binding, and whether it binds to AM or CGRP depends on the type of receptor activity-modifying protein (RAMP) that is present [12]. Considerable evidence also suggests that the putative AM receptor (L1) [13] and the canine orphan receptor RDC1 [14] are not authentic AM receptors. In rat and human epididymides, CGRP was previously shown to stimulate the short-circuit current (Isc) in a dose-dependent manner, and this effect was inhibited by CRGP (8–37) [15]. Together with the finding that AM may act on receptors that also mediate CGRP actions, this suggests that AM may have a physiological action in the epididymis. We have therefore studied AM expression by RIA and solution-hybridization-RNase protection assay in rat epididymis and have characterized the specific binding site for [125I]AM by receptor-binding assay and Scatchard plot analysis. To establish a physiological role of AM in the epididymis, the effect of AM on anion secretion by cultured epididymal cells was investigated. The final objective was to examine expression of the AM receptor (CRLR) and its RAMPs to determine whether RAMP expression correlates with AM/CGRP responsiveness.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Male Sprague-Dawley rats (weight, 250–300 g) were obtained from the Laboratory Animal Unit, Faculty of Medicine, the University of Hong Kong. All procedures had been approved by the Committee on the Use of Live Animals for Teaching and Research (CULATR) of the Faculty of Medicine, the University of Hong Kong, and were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (National Academy of Science).
Materials
Rat AM, rat CGRP, human CGRP (8–37), human AM (22–52), and AM antiserum were purchased from Peninsula (Belmont, CA). Normal rabbit gamma globulin and goat anti-rabbit serum were bought from Second Antibodies Incorporated (Davis, CA). The 125I was from Amersham Pharmacia (Little Chalfont, U.K.). The GF/B filters were from Whatman (Clifton, NJ). Gel filtration standard kit and protein assay reagent were supplied by Bio-Rad (Hercules, CA). All reagents for immunocytochemical study were from the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). For in vitro transcription, a Riboprobe Transcription System kit (Promega, Madison, WI) was used with [32P]uridine triphosphate supplied by Amersham. Both RNase A and RNase T1 were from Sigma (St. Louis, MO).
Tissue Extraction
Whole epididymis or its specific regions (caput, corpus, and cauda) were extracted for AM immunoreactivity by homogenizing in 2 N acetic acid for 1 min, followed by boiling for 10 min. A 50-µl aliquot was taken for protein assay. The homogenate was centrifuged at 18 600 x g for 20 min at 4°C (Beckman J2-21; Fullerton, CA), and the supernatant was lyophilized and stored at -20°C until assay.
Protein Measurement
Fifty microliters of homogenate or BSA standard were boiled with 1 N NaOH for 10 min, and 50 µl of the boiled samples were mixed with 2.5 ml of Bio-Rad protein assay reagent. After 10 min of incubation at room temperature, the samples were measured spectrophotometrically at 595 nm. The ir-AM was expressed as femtomoles per milligram of protein.
Radioimmunoassay
The lyophilized tissue sample was reconstituted in RIA buffer containing 0.1% sodium phosphate (pH 7.4), 0.1% heat-inactivated BSA, 0.05 M sodium chloride, 0.01% sodium azide, and 0.1% Triton X-100. The 125I-labeled AM was prepared from synthetic rat AM in our laboratory by the Chloramine T method and was purified by a Biogel P4 (Bio-Rad) column. Duplicate AM standards (0–500 pg per 100 µl) and samples were incubated at 4°C with 100 µl of AM antiserum and 100 µl of radioactive tracer (8000–10 000 cpm). The antiserum did not cross-react with proAM N-20 peptide. After 18–24 h, 100 µl of normal rabbit gamma globulin (1:300) and goat anti-rabbit serum (1:30) and 100 µl of 10% polyethyleneglycol were added, and the mixture was then incubated at 4°C for another 3 h. After adding 0.4 ml of assay buffer, the bound and free peptides were separated by centrifugation at 1800 x g for 1–2 h. The supernatant was aspirated, and the pellets were counted in a gamma counter (LKB, Turku, Finland). The sensitivity for the assay was 5 pg/tube. The intra- and interassay coefficients of variation were 7% and 10%, respectively.
Gel Filtration Chromatography
The lyopholized sample was reconstituted in distilled water, and the supernatant, after centrifugation at 18 600 x g for 20 min at 4°C, was acidified with 96% glacial acetic acid to give a final concentration of 1 N acetic acid. The sample was chromatographed on a Biogel P30 (Bio-Rad) column (0.9 x 60 cm), which was eluted with 1 N acetic acid at a flow rate of 1 ml per 10 min for 400 min. These 1-ml fractions were assayed for AM immunoreactivities. The column was calibrated with authentic AM, with blue dextran for void-volume determination, and with the proteins from the gel filtration kit, carbonic anhydrase, and cytochrome c as molecular-weight markers.
Immunocytochemistry
The avidin-biotin histochemical staining procedure was used to localize AM in the epididymis. The rat was perfused transcardially with phosphate-buffered saline and then with fixative containing 4% paraformaldehyde. The whole epididymis was removed, postfixed overnight, and then transferred for 1 day each to 15% and 30% sucrose solution. The frozen tissue was cut with a cryostat (2800 Figocut-E, Reichert-Jung; Leica Instruments GMH, Wetzlar, Germany) at a thickness of 15–20 µm. After the sections were treated for 60 min with 10% methanol and 3% hydrogen peroxide, they were incubated with the primary antibody (1:2000) for AM at 4°C overnight. The avidin-biotin-peroxidase complex in the sections was visualized with diaminobenzidine for 5–10 min.
Solution-Hybridization-RNase Protection Assay
RNA preparation Total RNA was prepared from freshly dissected tissue using Trizol reagent (Gibco-BRL, Life Technologies, Gaithersburg, MD) [16].
Hybridization and quantification The assay for preproAM mRNA was similar to the one used for preproANP mRNA [16]. Plasmids containing preproAM cDNA (613 base pairs [bp] in length) and ß-actin cDNA (387 bp in length) were subcloned into pGEM-4Z and pGEM-3Z, respectively. They were then linearized with restriction enzymes (preproAM: EcoRI for probe synthesis and BamHI for sense RNA standard; ß-actin: HindIII for the probe and EcoRI for the sense RNA standard). The appropriate amount of RNA tissue samples or standards was hybridized with a 32P-labeled riboprobe (100 000 cpm) in hybridization buffer (80% formamide, 40 mM PIPES [pH 6.7], 400 mM NaCl, and 1 mM EDTA) at 45°C for a time ranging from 6 h to overnight after being subjected to 85°C for 5 min. The RNA was digested at 37°C for 30–45 min with RNase A and RNase T1. The RNA hybrids were purified by proteinase K/SDS digestion and precipitated with 1 µg of yeast RNA as carrier after phenol-chloroform extraction. Hybrids were reconstituted in 5 µl of gel loading dye and were separated on a 4% polyacrylamide gel at 160 V and 34 mA for approximately 1 h. The gel was dried and exposed to x-ray film. The hybrid bands on the gel were cut out using the x-ray film as template and the radioactivity counted by a scintillation counter. The epididymal preproAM mRNA content was expressed as femtograms of mRNA per picograms of ß-actin mRNA. The values of preproAM mRNA and ß-actin mRNA were not corrected to their native lengths.
The RNase protection assays of AM-receptor mRNA were carried out using the rat L1, RDC1, and CRLR cDNAs as templates as described previously [17]. The mRNA levels for RAMP1 and RAMP2 were also measured by RNase protection assays [18].
Receptor-Binding Assays
For AM binding, membranes were prepared by differential centrifugation [19]. Whole epididymis was homogenized in ice-cold, 50 mM Hepes (pH 7.6), comprising 0.25 M sucrose, 10 µg/ml of pepstatin, 0.25 µg/ml each of leupeptin and antipain, 0.1 mg/ml each of benzamidine and bacitracin, and 30 µg/ml of aprotinin. The homogenates were centrifuged at 1500 x g for 20 min at 4°C, and the supernatants were centrifuged at 100 000 x g for 1 h at 4°C. The pellets were resuspended in 10 volumes of the above-mentioned buffer without sucrose and were centrifuged at 100 000 x g for 1 h at 4°C. The final pellets were resuspended, aliquoted, and stored at -70°C until assay. Epididymal membranes (equivalent to 50 µg of protein) were incubated at 4°C for 30 min with 125I-labeled AM (10–300 pM) in 0.3 ml of binding buffer (20 mM Hepes [pH 7.4], 5 mM MgCl2, 10 mM NaCl, 4 mM KCl, 1 mM EDTA, 1 µM phosphoramidon, 0.25 mg/ml of bacitracin, and 0.3% BSA) [19]. After incubation, membranes were filtered through a Brandel cell harvester (Biomedical Research and Development Laboratories, Gaithersburg, MD) using GF/B filter paper that had been soaked overnight in 0.3% polyethylenimine and washed three times with 3 ml of ice-cold, 50 mM Tris-HCl (pH 7.4). Radioactivity retained by the filters was counted by a gamma counter at 75% efficiency. Specific binding was defined as total binding minus nonspecific binding, which was determined in the presence of 500 nM unlabelled rat AM. The ratio of specifically bound to free radioligand concentration was plotted against specific binding (Scatchard plot). The Kd and Bmax values were then obtained from this Scatchard plot.
For CGRP binding, membranes were prepared as previously described [20]. Briefly, tissues were homogenized in 10 volumes of ice-cold buffer containing 10 mM Tris-HCl (pH 7.4), 5 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 1 mg/ml of bacitracin, and 0.1 mg/ml of aprotinin. The homogenates were centrifuged at 1000 x g for 10 min at 4°C. The supernatants were centrifuged at 50 000 x g for 20 min at 4°C, and the pellets were washed twice in buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 2 mM EGTA, 250 mM sucrose, and 0.1 mg/ml of bacitracin. The membranes were then resuspended in the same buffer and stored at -70°C. Protein concentration was assayed as described above. Epididymal membranes (equivalent to 50 µg of protein) were incubated with increasing concentrations of 125I-labeled rat CGRP (10–300 pM) for 60 min at 25°C [20]. Nonspecific binding was determined in the presence of 1 µM, cold rat CGRP. The incubation was stopped by adding 2 ml of cold NaCl (0.9%). The Kd and Bmax values were obtained as described for AM.
In addition, studies regarding the displacement of 125I-labeled AM binding by CGRP receptor (10-12 to 10-6) and of 125I-labeled CGRP binding by AM (10-10 to 10-6) were carried out. Human CGRP (8–37) (a CGRP-receptor antagonist) and human AM (22–52) (an AM-receptor antagonist) were also used in these displacement studies. Specific binding was defined here as the difference in binding in the presence and in the absence of the competitive ligand. All binding assays were carried out in duplicate.
Tissue-Culture Technique and Measurement of Isc
Primary monolayer cultures of rat epididymis were grown on millipore filters by methods described previously [15, 21]. After 4 days of culture, the monolayer became confluent and was ready for the measurement of electrogenic anion secretion using the Isc technique [21]. To measure the Isc, confluent monolayers of the rat epididymal cells (with the basal ends of the cells attached to the filter) were clamped vertically between the two halves of the Using chamber (World Precision Instrument, New Haven, CT). The tissues were short-circuited with the transepithelial potential difference clamped at zero. Responses to AM and CGRP were measured as changes in the Isc at the peak of the response. To study the effect of AM and CGRP, 0.1 µM AM and 0.01 µM CGRP were added to both sides. Because AM had action on the apical side and CGRP on the basolateral side, AM was added to the apical side and CGRP to the basolateral side only in all subsequent studies. To construct a dose-response curve, increasing concentrations of AM (0.02–0.8 µM) were added to the apical side of the epithelium. During experiments in which the CGRP (8–37) antagonist was studied, the antagonist (0.1 µM) was added to both sides before the addition of AM on the apical side and CGRP on the basolateral side. Similarly, 1 µM AM (22–52) (the specific AM-receptor antagonist) was added to both sides before the addition of AM and CGRP.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The ir-AM and preproAM mRNA were detected in the rat epididymis, and the highest expression could be found in the corpus region, followed by the cauda and the caput (Table 1). Figure 1 shows a typical autoradiograph of the electrophoretic separation of the preproAM and ß-actin mRNA hybrids. The radioactivity of each specific band was plotted against picogram standard RNA hybridized to give the linear standard curves. By this means, preproAM mRNA was quantitated and shown to have highest expression in the corpus.
|
|
Gel Filtration Chromatographic Analysis of Epididymal AM and Immunohistochemical Study
The gel filtration chromatograms of ir-AM in the whole epididymis and in caput, corpus, and cauda regions are depicted in Figure 2. Two ir-AM peaks were seen on all chromatograms. The caput and cauda of the epididymis showed a predominant peak corresponding to that of the authentic rat AM (1–50) and a smaller precursor peak. The opposite was found to be true for the corpus region. As shown in Figure 3, the positive immunostaining of AM could be seen clearly in the epithelial cells of the epididymis surrounding the lumen. The negative control did not show any immunostaining.
|
|
AM Receptor-Binding Sites
The Scatchard plot of AM (Fig. 4A) shows that the Bmax and Kd for AM binding were 400 ± 27 fmol/mg protein and 0.71 ± 0.05 nM, respectively. The binding of 125I-labeled rat AM (1–50) was displaced by unlabelled rat AM (1–50) (50% inhibiting concentration [IC50] = 9.3 ± 0.2 x 10-10 M), human AM (22–52) (IC50 = 6.6 ± 0.1 x 10-9 M), rat CGRP (IC50 = 1.4 ± 0.1 x 10-7 M), and human CGRP-receptor antagonist, CGRP (8–37) (IC50 = 1.1 ± 0.1 x 10-8 M). The 125I-labeled AM competition curve is illustrated in Figure 4B.
|
CGRP Receptor-Binding Sites
The Bmax and Kd of CGRP binding were 75 ± 7.7 fmol/mg protein and 20 ± 1 pM, respectively (Fig. 5A). The binding of 125I-labeled CGRP was displaced by unlabelled rat CGRP (IC50 = 3.3 ± 0.1 x 10-9 M), human CGRP (8–37) (IC50 = 1.0 ± 0.1 x 10-7 M), rat AM (1–50) (IC50 = 3.4 ± 0.2 x 10-7 M), and human AM (22–52) (IC50 = 2.1 ± 0.2 x 10-7 M). The 125I-labeled CGRP competition curve is shown in Figure 5B.
|
Solution Hybridization of CRLR and RAMP RNAs
The RNase-protected bands from L1, RDC1, and CRLR were resolved by polyacrylamide gel electrophoresis (Fig. 6). However, only mRNA encoding CRLR was quantified, because little evidence supports a role of L1 or RDC1 as an AM receptor. The epididymis was shown to express mRNA encoding CRLR at a level of 2.2 ± 0.1 amol/µg RNA after normalization to ß-actin mRNA content. No difference was found among the cauda, corpus, and caput regions (Table 1). Both RAMP1 and RAMP2 mRNAs were found in the epididymis (Fig. 7A). No significant difference was found in their abundance among the three regions of the epididymis (Fig. 7B).
|
|
Effect of AM on Isc in Monolayer Cells of the Epididymis
Addition of AM to the apical side of the epididymis caused a dose-dependent increase in Isc (inwardly directed), with the maximal response obtained at 0.4 µM AM (Fig. 8A). The CGRP applied to the basolateral side elicited a greater increase in Isc of shorter duration (Fig. 8B). The AM-induced increase in Isc was inhibited by the addition of 1 mM diphenylamine-2 carboxylate, a known chloride-channel blocker, or by the removal of chloride from the bathing solution (results not shown). The AM (22–52), an AM-receptor antagonist, completely abolished the AM-induced Isc but did not affect the CGRP-induced Isc (Fig. 8C). Similarly, CGRP (8–37), a CGRP-receptor antagonist, reduced most of the CGRP-induced Isc but did not affect the AM-induced Isc (Fig. 8D).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
We have shown that ir-AM is present in the epididymis at an approximately 7-fold lower concentration than in the adrenal gland, although the preproAM mRNA levels in these tissues are similar. This discrepancy between the peptide contents and mRNA levels suggests that AM may be actively secreted by the epididymis, as has been shown in other tissues [3]. The corpus region has higher levels of AM and preproAM mRNA than the caput and the cauda. However, the gel filtration study demonstrated that the major peak in the corpus is the precursor, with an estimated molecular weight of approximately 12 000, whereas in the other two regions, the major peak is the authentic AM. The decrease in the ratio of the biologically active form to the precursor suggests that AM may be more actively secreted here than in the caput and cauda regions and that AM may play a more important role in the corpus region. Correlated with this, the same region has the highest preproAM mRNA level, which indicates the greatest rate of AM synthesis. This finding contrasts with that reported for epididymal CGRP, which is only found in the cauda region [15].
Our receptor-binding study also shows that specific binding sites exist in the rat epididymis. The displacement studies show that at least some of these AM-binding sites can also bind CGRP. This is similar to the highly vascularized tissues such as the lung and the liver [19], where the distribution of AM-binding sites is similar to that of CGRP receptors. The Bmax and Kd values for AM and CGRP reveal that the maximal binding of AM is approximately 5-fold higher than that of CGRP and that the binding affinity for CGRP is much greater than that for AM. These results suggest that, by virtue of their differences in maximum numbers of binding sites and in binding affinities, they may function under different physiological situations or respond to different stimuli. It is pertinent to recall that AM is present in all three regions, whereas CGRP is only found in the cauda region [15].
The presence of specific binding sites is further supported by data from the RNase protection assay, which demonstrated mRNAs encoding CRLR receptors. We have also measured mRNA for the putative AM receptors, L1 and RDC1 (see Fig. 6), and although RDC1 is present, L1 is not, which is consistent with this not being an AM receptor [23]. According to the excellent work of McLatchie et al. [24], CRLR can function as a CGRP receptor in the presence of RAMP1 or as an AM receptor in the presence of RAMP2 or RAMP3. It was later proposed that a functional AM receptor is composed of at least three proteins: the ligand-binding protein (CRLR), an accessory protein (RAMP2), and a coupling protein for signal transduction (RCP) [25, 26]. The same has also been proposed for the functional CGRP receptor, except that RAMP2 is replaced by RAMP1 [26]. Our results on RAMP mRNA contents also demonstrate that both RAMP1 and RAMP2 exist in the epididymis. The RAMPs can therefore function at the plasma membrane to modify the specificity of CRLR to that of CGRP or AM, depending on which member of the RAMPs is expressed [26]. It is a pity that we could not study the receptor binding in different regions of the epididymis because of the availability of tissues. However, no difference was found in the receptor mRNA levels among the three regions, and because RAMP1 and RAMP2 are similarly expressed in all these regions, the binding characteristics of the receptors are probably similar. Presumably, CRLR can associate with either RAMP1 or RAMP2 to produce distinct receptors with high affinity for either CGRP or AM. However, at this stage, we cannot determine whether CGRP and AM receptors have distinct cellular localization patterns.
The Isc experiments demonstrate that AM acts on the apical side to increase anion secretion via a chloride channel. The AM had previously been shown to increase Isc for chloride secretion in the rat colon, and the effective doses were the same order of magnitude as those in the present study [27]. It was shown in the present experiments that AM acts specifically on the apical side (i.e., the luminal side), whereas CGRP is effective only on the basolateral side. The CGRP is more potent at increasing the Isc, probably because it has a higher affinity for its receptors than AM has for its own. However, its effect is transient, in contrast to that of AM, suggesting a difference in signaling pathways. The present study using specific AM- and CGRP-receptor antagonists suggests that AM acted on specific AM receptors that are distinct from the CGRP receptors. The epididymis has only one layer of epithelial cells, and in the monolayer culture, the basal ends attach to the millipore filter. The RAMP1 may go to the basolateral side of the cell by protein trafficking and RAMP2 to the apical side, with the result that CRLR on the apical side becomes AM specific and that on the basolateral side CGRP specific. Alternatively, there may be co-expression with different RAMPs in different epithelial cells, so that some will respond only to AM and others only to CGRP. However, again, RAMP1 must be on the basolateral side and RAMP2 on the apical end.
It is apparent, then, that two receptors bind AM in the epididymis. One is the specific AM receptor (a complex of CRLR and RAMP2) with high affinity for AM and low affinity for CGRP. The other is the CGRP1 receptor (a complex of CRLR and RAMP1) with high affinity for CGRP and low affinity for AM. The binding of AM to its specific receptor can be displaced by high levels of CGRP and the binding to the CGRP1 receptor by low levels of CGRP. The binding of AM to the low-affinity CGRP receptor is not revealed in the present binding experiment. No action of AM occurs on the basolateral side, because the low affinity of the CGRP receptor for AM would require a much higher level of AM for its action. The same applies to the lack of action by CGRP on the luminal side.
In conclusion, the addition of AM to the apical side stimulated electrogenic anion secretion in the epididymis by acting on the apical side of the epithelium. Together with the presence of AM and preproAM mRNA and AM receptor and RAMP mRNAs, this suggests that AM may be released into the epididymal lumen to act in a paracrine or autocrine fashion to stimulate anion transport and that AM may play a role during sperm maturation in the epididymis by altering the ionic environment. This may provide an additional regulation of fluid secretion to that of CGRP in the caput and corpus regions. The AM in the luminal fluid also may have a direct effect on sperm maturation and sperm motility. The high level of AM and preproAM mRNA and the lower active peptide:precursor ratio suggests that AM may be more important in the corpus region.
|
FOOTNOTES |
|---|
2 Correspondence: Fai Tang, Department of Physiology, Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Poktulam, Hong Kong, China. FAX: 852 2855 9730; ftang{at}hkucc.hku.hk
3 Current address: C.K. Life Sciences Ltd., 2 Dai Fu Street, Tai Po Industrial Estate, Tai Po, New Territories, Hong Kong, China
4 Current address: Cryptome Research Pty. Ltd., P.O. Box 6492, St. Kilda Road Central, Melbourne, Victoria 8008, Australia
Received: 3 September 2002.
First decision: 26 September 2002.
Accepted: 30 December 2002.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  Y.-Y. Li, L. Li, I. S.-S. Hwang, F. Tang, and W.-S. O Coexpression of Adrenomedullin and Its Receptors in the Reproductive System of the Rat: Effects on Steroid Secretion in Rat Ovary Biol Reprod, August 1, 2008; 79(2): 200 - 208. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-F. Chan, W.-S. O, and F. Tang Adrenomedullin in the Rat Testis. I: Its Production, Actions on Testosterone Secretion, Regulation by Human Chorionic Gonadotropin, and Its Interaction with Endothelin 1 in the Leydig Cell Biol Reprod, April 1, 2008; 78(4): 773 - 779. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-Y. Li, W.-S. O, and F. Tang Effect of Aging on the Expression of Adrenomedullin and Its Receptor Component Proteins in the Male Reproductive System of the Rat J. Gerontol. A Biol. Sci. Med. Sci., December 1, 2007; 62(12): 1346 - 1351. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. S. S. Hwang, M. L. Fung, E. C. Liong, G. L. Tipoe, and F. Tang Age-Related Changes in Adrenomedullin Expression and Hypoxia-Inducible Factor-1 Activity in the Rat Lung and Their Responses to Hypoxia J. Gerontol. A Biol. Sci. Med. Sci., January 1, 2007; 62(1): 41 - 49. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-Y. Li, I. S.-S. Hwang, W.-S. O, and F. Tang Adrenomedullin Peptide: Gene Expression of Adrenomedullin, its Receptors and Receptor Activity Modifying Proteins, and Receptor Binding in Rat Testis--Actions on Testosterone Secretion Biol Reprod, August 1, 2006; 75(2): 183 - 188. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Mittra and J.-P. Bourreau Gs and Gi coupling of adrenomedullin in adult rat ventricular myocytes Am J Physiol Heart Circ Physiol, May 1, 2006; 290(5): H1842 - H1847. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
