Venkataseshu K. Ganjam

Transcription and Translation of Estrogen Receptor-β in the Male Reproductive Tract of Estrogen Receptor-α Knock-Out and Wild-Type Mice1

Estrogen receptor-α (ERα) has been identified in the male reproductive tract, but the role of estrogen in the male has not been well characterized. In vivo mutations in ERα genes have demonstrated the necessity for ERα-mediated action in male fertility. We asked whether both ERβ messenger RNA and protein were present in the male reproductive tract of wild-type and ERα knock-out (ERα KO) mice, and whether ERβ could compensate for the lack of ERα in infertile male ERα KO mice. Immunohistochemical localization with both N- and C-terminal anti-ERβ antibodies demonstrated that ERβ is present in the Leydig cells of the testes and in the epithelium of both the efferent ductules and the initial segment of the epididymis. RT-PCR amplification was used to confirm ERβ transcription in these tissues. In conclusion, we observed that ERβ messenger RNA and protein continue to be expressed in the Leydig cells, elongated spermatids, efferent ductules, and the initial segment of the epididymides of ERα KO mice, but the pre...

Glucocorticoids and laminitis in the horse

The administration of exogenously administered GCs and syndromes associated with GC excess are both attended by increased risk for the development of laminitis in adult horses. However, there exists substantial controversy as to whether excess GCs cause laminitis de novo. If true, the pathogenesis of laminitis arising from the effects of GC excess is probably different from that associated with diseases of the gastrointestinal tract and endotoxemia. Although a satisfactory explanation for the development of laminitis as a consequence of GC action is currently lacking, numerous possible and plausible theoretical mechanisms do exist. Veterinarians must exert caution with respect to the use of GCs in adult horses. The extent to which individual horses are predisposed to laminitis as a result of GC effect cannot be predicted based on current information. However, the administration of systemic GCs to horses that have been previously affected by laminitis should be used only with extreme caution, and should be accompanied by careful monitoring for further signs of laminitis. The risk of laminitis appears to be greater during treatment using some GCs (especially dexamethasone and triamcinalone) compared with others (prednisone and prednisolone). Whenever possible, to reduce the risk of laminitis, GCs should be administered locally. For example, the risk of GC-associated laminitis is evidently considerably reduced in horses affected with chronic obstructive pulmonary disease (COPD) if GC treatment is administered via inhalation. We have hypothesized that structural changes in the equine hoof that resemble laminitis may arise as a consequence of excess GC effect. Although these changes are not painful per se, and are not associated with inflammation, they could likely predispose affected horses to the development of bona fide laminitis for other reasons. Moreover, the gross morphological appearance of the chronically GC-affected hoof resembles that of a chronically foundered hoof in some respects. Further investigation into the effect of GC on the hoof lamellar interface is clearly needed.

11β-Hydroxysteroid dehydrogenases of the choriocarcinoma cell line JEG-3 and their inhibition by glycyrrhetinic acid and other natural substances

Mineralocorticoid receptor (MR) selectivity for aldosterone is thought to be exerted by enzymes which inactivate competing glucocorticoids before they bind the receptor. Two different 11 beta-hydroxysteroid dehydrogenases (11 beta-HSD) have been described. 11 beta-HSD-1 is NADP(+)-dependent and has a Km in the micromolar range and bidirectional activity. 11 beta-HSD-2 is NAD(+)-dependent, has a Km in the nanomolar range, exhibits only oxidase activity, and colocalizes with the MR in the kidney, so is likely to serve as the gatekeeper for the MR. We have further characterized 11 beta-HSD activity in JEG-3 cells, a cell line derived from a human choriocarcinoma which was reported to have only the high affinity, NAD(+)-dependent 11 beta-HSD-2. We found that the Km for the conversion of corticosterone to 11-dehydrocorticosterone in intact cells and homogenates was about 16 nM. NAD(+)-dependent corticosterone conversion was equal in the nuclear and mitochondrial fractions and less, but significant, in the microsomal fraction. A high affinity, Km = 40 nM, NADP(+)-dependent enzyme was also found in homogenates. The subcellular distribution of this high affinity activity was greatest in the mitochondria, less in the nuclei, and even less, but still significant, in microsomes. Because of its cofactor dependency, high affinity, and different subcellular distribution, we suggest that this enzyme is neither the 11 beta-HSD-1 nor the 11 beta-HSD-2 and have named it 11 beta-HSD-3. Conversion of 11-dehydrocorticosterone to corticosterone did not occur in intact cells or in homogenates incubated with NADH or NADPH. Enzyme activity in intact cells was inhibited by glycyrrhetinic acid, carbenoxolone, progesterone, 5 beta-dihydroprogesterone, and 5 alpha-dihydroprogesterone, but not bile acids.

The sheep kidney contains a novel unidirectional, high affinity NADP+-dependent 11β-hydroxysteroid dehydrogenase (11β-HSD-3)

Abstract The 11β-hydroxysteroid dehydrogenase (11β-HSD) enzymes convert corticosterone and cortisol to 11-dehydrocorticosterone and cortisone, and are thought to convey extrinsic specificity to the mineralocorticoid receptor by limiting access of the relatively more abundant glucocorticoids to it. Two different 11β-hydroxysteroid dehydrogenases (11β-HSD) have been described and cloned. The liver-type, NADP + -dependent 11β-HSD-1, has an affinity in the micromolar range and bidirectional activity. The NAD + -dependent 11β-HSD-2 has a higher affinity, in the nanomolar range, and exhibits only oxidase activity. 11β-HSD-2, because of its affinity and co-localization with the mineralocorticoid receptor, is likely to serve as the “gatekeeper” for the mineralocorticoid receptor in the kidney. Although the rat kidney expresses both isoforms, only the high-affinity, NAD + -dependent 11β-HSD-2 has been reported in the sheep kidney. We found both 11β-HSD NAD + - and NADP + -dependent activities in sheep kidney to be present. The NAD + -dependent activity exhibited a Km similar to that reported in the literature, 3.85 ± 1.28 nM for corticosterone and 21.3 ± 5.8 for cortisol, was distributed in approximately equal amounts between microsomes and nuclei, and was unidirectional, converting corticosterone to 11-dehydrocorticosterone. The enzyme exhibited prominent substrate inhibition. The NADP + -dependent activity had a Km for corticosterone of 4 ± 1.3 nM and a Km for cortisol of 35.2 ± 2 nM, 100-fold lower than that described for the 11β-HSD-1 in the liver of sheep and other species, and was more prevalent in the microsomes than the nuclei. This enzyme was not inhibited by its substrate. The NAD + -dependent activity was approximately 3–10 times greater than the NADP + -dependent activity when incubated with 5nM corticosterone substrate, but had similar activity when incubated with 100 nM substrate concentrations. CHOP cells (a modified Chinese hamster ovary cell line) transiently transfected with the sheep 11β-HSD-2 plasmid exhibited a marked preference for NAD + as co-factor. Oxidation of corticosterone by transfected cells in the presence of NADP + was present, but minimal; NADP + did not support the metabolism of cortisol, the primary glucocorticoid of sheep. These data suggest the existence of another NADP + -dependent enzyme, 11β-HSD-3, which, because of its high affinity and unidirectional oxidase activity, may play a physiological role in the modulation of glucocorticoid binding to both the mineralocorticoid and glucocorticoid receptors.

The 11β hydroxysteroid dehydrogenase 2 exists as an inactive dimer

The 11beta-hydroxysteroid dehydrogenase types 1 and 2 enzymes (11beta-HSD1 and 11beta-HSD2), modulate glucocorticoid occupation of the mineralocorticoid and glucocorticoid receptors by interconverting corticosterone and cortisol to the inactive metabolites 11-dehydrocorticosterone and cortisone within the target cells. The NAD(+)-dependent 11-HSD 2 in the kidney inactivates corticosterone and cortisol, allowing aldosterone, which is not metabolized, access to the receptor. Studies of the kinetics of 11-HSD 2 activity in the rat kidney have produced inconsistent results. Western blots done in the absence of the reducing agent beta-mercaptoethanol showed two bands with approximate MW of 40 and 80 kDa. When beta-mercaptoethanol was used, only the 40 kDa was detected, indicating that under non-denaturing conditions a significant proportion of the 11beta-HSD 2 exists as a dimer. NAD(+)-dependent conversion of 3H-corticosterone by 20 microg of microsomal protein increased approximately 10 fold with the addition of 5 mM DTT concentration. NADP(+)-dependent activity with 20 microg of microsomal protein was very low and did not change significantly when using DTT. In the presence of DTT, the predominant 11-HSD activity in the rat kidney is NAD(+)-dependent with a K(m) of 15.1 nM, similar to that of the cloned and expressed enzyme. These data suggest that dimerization and subsequent enzyme inactivation occur when protocols promoting oxidation of this protein are used.

Hormonal control of Leydig cell differentiation.

Leydig cell progenitors contain significant concentrations of androgen receptors. When the metabolism of DHT to 3 alpha-DIOL is blocked, DHT stimulates testosterone production by Leydig cell progenitors, most probably via an androgen receptor dependent mechanism. Rapid metabolism by 3 alpha-HSD may limit the potency of exogenous DHT to stimulate differentiation of Leydig cell progenitors in vitro. Insulin-like growth factor-I enhances androgen production by purified immature Leydig cells. The elevated sensitivity of immature Leydig cells versus adult Leydig cells to IGF-I stimulation indicates that this peptide hormone has a role in their differentiation during puberty.

Tissue-specific dysregulation of cortisol metabolism in equine laminitis

REASONS FOR PERFORMING STUDY The role of glucocorticoids (GCs) in the pathogenesis of laminitis is incompletely understood. Local tissue activity of GC is regulated by the steroid converting enzyme, 11beta-hydroxysteroid dehydrogenase-1 (11beta-HSD-1). Changes in integumentary (skin and hoof lamellar) 11beta-HSD activity occurring during laminitis could affect the extent to which GCs are involved in its development. HYPOTHESIS That changes in integumentary 11beta-HSD-1 activity associated with the laminitic condition would lead to elevated local tissue levels of GCs, which could subsequently contribute, through paracrine and autocrine mechanisms, to the further development of laminitis; and that similar changes in 11beta-HSD-1 activity would be evident in both skin and hoof lamellar tissue. METHODS Activity of 11beta-HSD-1 was determined in skin and hoof lamellar tissue specimens obtained from normal and laminitic horses using a radiometric assay. Skin samples were obtained from 10 normal horses and from 10 horses before and after induction of acute laminitis following administration of starch via nasogastric tube. Hoof lamellar samples were obtained from 10 normal horses, 10 horses following induction of acute laminitis and 4 chronically-foundered horses. Bidirectional 11beta-HSD-1 activity was measured in both skin and lamellar tissues. RESULTS 11-ketoreductase activity exceeded 11beta-dehydrogenase activity in both skin and lamellar tissues. Cutaneous activity was higher than lamellar 11beta-HSD-1 activity in all groups. Both ketoreductase and dehydrogenase activity increased in skin and lamellae following experimental induction of acute laminitis, but the increase in ketoreductase activity was substantially greater than that for dehydrogenase in the lamellae. Induction of acute laminitis was attended by increases of 227 and 220% in cutaneous dehydrogenase and ketoreductase activity, respectively, and 173 and 398% in lamellar dehydrogenase and ketoreductase activity, respectively (P<0.05). CONCLUSIONS The 11-ketoreductase moiety of 11beta-HSD-1 plays a role in equine skin and hoof lamellae regarding the regulation of local glucocorticoid activity. Increased 11-ketoreductase activity will lead to increased local tissue GC activity by virtue of conversion of cortisone to cortisol. POTENTIAL RELEVANCE The laminitic condition is attended by integumentary biochemical changes that enhance the local concentration of cortisol, especially in the hoof lamellar interface. Through multiple and diverse actions, increased local GC activity contributes to the pathogenesis and morbidity associated with laminitis. Pharmacological manipulation of 11beta-HSD-1 deserves further investigation regarding the prevention and treatment of laminitis.

Serum markers of lamellar basement membrane degradation and lamellar histopathological changes in horses affected with laminitis

In order better to evaluate the extent to which degradation of the lamellar basement membrane (LBM) by matrix metalloproteinases (MMP) occurs in equine laminitis, we determined the concentration of type IV collagen and laminin in normal and laminitic horses, using specific immunoassays. Blood samples were obtained from both the jugular and the cephalic veins of horses (n = 10) before and after the induction of acute alimentary laminitis by carbohydrate overload. Jugular and cephalic venous blood samples were also obtained from horses affected with naturally occurring laminitis (n = 16) and nonlaminitic controls (n = 8). The serum collagen IV concentration was not changed following the induction of laminitis in the experimental group. Serum collagen IV concentration was increased in jugular venous blood obtained from cases of naturally occurring laminitis (mean +/- s.e. 218.04 +/- 18.59 ng/ml) compared with nonlaminitic controls (157.50 +/- 10.93 ng/ml) (P<0.05). Serum collagen IV concentration was also increased in jugular venous blood obtained from severely laminitic horses (219.50 +/- 18.18 ng/ml) compared with nonlaminitic controls (157.50 +/- 10.93 ng/ml) (P<0.05). A difference in serum concentration of collagen IV was not identified based on chronicity of naturally occurring laminitis. Serum laminin concentration did not differ between laminitic and nonlaminitic horses. Differences in serum laminin concentration were not identified based on sampling location (jugular or cephalic vein), severity of laminitic pain, or chronicity of spontaneous laminitis. In conclusion, the circulating concentration of collagen IV was increased in horses affected with naturally occurring laminitis. The potential role for serum collagen IV assay for characterisation of equine laminitis warrants further investigation.

Endogenous testosterone attenuates neointima formation after moderate coronary balloon injury in male swine

AIMS Previous studies from our laboratory have demonstrated that testosterone increases coronary smooth muscle protein kinase C delta (PKC delta) both in vivo and in vitro and inhibits coronary smooth muscle proliferation by inducing G(0)/G(1) cell cycle arrest in a PKC delta-dependent manner. The purpose of the present study was to determine whether endogenous testosterone limits coronary neointima (NI) formation in a porcine model of post-angioplasty restenosis. METHODS AND RESULTS Sexually mature, male Yucatan miniature swine were either left intact (IM), castrated (CM), or castrated with testosterone replacement (CMT; Androgel, 10 mg/day). Angioplasty was performed in both the left anterior descending and left circumflex coronary arteries with balloon catheter overinflation to induce either moderate (1.25-1.3 x diameter; 3 x 30 s) or severe (1.4x diameter; 3 x 30 s) injury, and animals were allowed to recover for either 10 or 28 days. Injured coronary sections were dissected, fixed, stained (Verheoff-Van Gieson, Ki67, PKC delta, p27), and analysed. Vessels without internal elastic laminal rupture were excluded. Following moderate injury, intimal area, intima-to-media ratio (I/M), and I/M normalized to rupture index (RI) were increased in CM compared with IM and CMT. RI, medial area, and intimal/medial thickness (IMT) were not different between groups. NI formation was inversely related to serum testosterone concentration. Conversely, following severe injury, there were no significant differences between the groups. Testosterone inhibited proliferation and stimulated PKC delta and p27(kip1) expression during NI formation (10 days post-injury). CONCLUSION These findings demonstrate that endogenous testosterone limits coronary NI formation in male swine and provides support for a protective role for testosterone in coronary vasculoproliferative diseases, such as restenosis and atherosclerosis.

Chronic mineralocorticoid excess and cardiovascular remodeling

Chronic mineralocorticoid (MC) excess, whether due to elevated plasma aldosterone (ALDO) or deoxycorticosterone (DOC), is associated with a perivascular fibrosis of systemic and coronary arterioles. This remodeling of resistance vessels contributes to the appearance of hypertension. Chronic MC excess is also accompanied by cardiac myocyte necrosis, secondary to myocardial potassium depletion, and a subsequent reparative fibrosis that appears in the normotensive, nonhypertrophied right and hypertensive, hypertrophied left ventricles. Fibrosis contributes to the appearance of ventricular arrhythmias and dysfunction. Herein, clinical and experimental evidence linking chronic, inappropriate (relative to dietary sodium) elevations in circulating ALDO and DOC with these reactive and reparative forms of fibrous tissue formation in the heart and other tissues is presented.