A function of concentrations of three ketosteroids, [(androstenedione + 17alpha-hydroxyprogesterone)/cortisol], has been shown to provide better discrimination than the concentration of 17alpha-hydroxyprogesterone alone, for the diagnosis of the inherited disease congenital adrenal hyperplasia (CAH). A rapid, direct infusion electrospray ionization tandem mass spectrometry (ESI-MS/MS) method has been developed to measure this ratio in plasma (100 microL). Sensitivity and specificity result from the preparation of mono-Girard T derivatives. Androstenedione, 17alpha-hydroxyprogesterone and cortisol are individually quantitated by isotope dilution. Excellent statistical correlation with radioimmunoassay (RIA) results for the analysis of 17alpha-hydroxyprogesterone is demonstrated. The value of the ratio function of the three ketosteroids is 0.01-0.16 for normal controls (n=26) and 1.83-18.7 for patients with severe CAH (n=4). Profiles of ketosteroids derivatized with Girard T from the plasma of a child with severe CAH, generated by ESI-MS/MS analysis, are shown. Neutral loss scans for 29.5 and 59 Da assist in the identification of the ketosteroids.

The enzymatic actions of the <em>17</em> beta-hydroxysteroid dehydrogenase (<em>17</em> beta HSD) isozymes are crucial in steroid hormone metabolism/physiology. The type 1 isozyme catalyzes the conversion of the biologically inactive C18 steroid, estrone, to the active estrogen, <em>17</em> beta-estradiol, and the enzyme is predominantly expressed in the syncytiotrophoblast of the placenta and the granulosa cells of the ovary. <em>17</em> beta HSD type 2 is highly expressed in placenta, liver, and secretory endometrium and catalyzes the conversion of bioactive estrogens and androgens to biologically inactive <em>17</em>-<em>ketosteroid</em> counterparts. The expression pattern of <em>17</em> beta HSD type 2 protein was determined in human term placenta and fetal liver by immunohistochemical analysis using monoclonal antibodies directed against distinct epitopes of the <em>17</em> beta HSD type 2 protein. In placenta, the protein was detected in the endothelial cells of fetal capillaries, but not in cytotrophoblasts or syncytiotrophoblast. There was dichotomous immunostaining seen among pairs of cotyledonary vessels and chorionic vessels. In the liver, on the other hand, staining was detected in the hepatocytes, but not in the cells lining blood vessels. We conclude that the cell type-specific localization of <em>17</em> beta HSD type 2 is in accord with the proposed physiological role of the enzyme, namely to protect tissues, in this case the fetus, from bioactive estrogen and androgen.

Patients recovering from acute surgical stress often excrete increased <em>17</em>-OH corticosteroids with no change in <em>17</em>-<em>ketosteroids</em>. The explanation for these findings is unclear. In order to investigate possible divergence between cortisol and adrenal androgen metabolism in acute stress, repeated morning cortisol and dehydroepiandrosterone (DHA) measurements were made in patients undergoing ACTH stimulation 48 to 96 hours preoperatively, followed by determinations before and during major surgery, also performed in the morning. Cortisol and DHA are largely metabolized by the liver, so liver blood flow under a constant general anesthetic regimen known not to affect cortisol metabolism was monitored by pre- and intraoperative indocyanine green dye clearance. Results indicated no difference between the cortisol and DHA stimulation resulting from two hours of ACTH stimulation or major surgery, and a small (14.4%) decline in hepatic blood flow during general anesthesia. However, while DHA concentrations remained constant immediately preceding surgery, cortisol concentrations increased by 61% (P less than 0.05). Previous studies have also demonstrated increased concentrations of cortisol before surgical procedures, presumably due to psychological stress. However, this is the first demonstration of a dissociation between concentrations of cortisol and an adrenal androgen due to psychological stress.

Simultaneous determinations of serum concentrations of <em>17</em>OH-progesterone, testosterone, androstenedione, and progesterone, and of urinary excretion of <em>17</em>-<em>ketosteroids</em> and pregnanetriol have been performed at intervals in 31 patients with the C21-hydroxylase form of congenital adrenal hyperplasia. In prepubertal patients there were highly significant correlations between levels of <em>17</em>OH-progesterone and those of testosterone, androstenedione, and progesterone, respectively. Similar correlations were observed in adolescent girls. In adolescent boys rising <em>17</em>OH-progesterone levels were reflected by increasing levels of androstenedione and progesterone, but there was no change in serum testosterone concentrations. Levels of serum <em>17</em>OH-progesterone below 200 ng/dl were uniformly associated with normal serum concentrations of testosterone, androstenedione, and progesterone, and normal urinary <em>17</em>-<em>ketosteroid</em> and pregnanetriol excretion. In contrast, levels above 1000 ng/dl were accompanied by increased levels of the other steroids except in adolescent males; in this group the finding of unchanging serum testosterone concentrations in spite of rising <em>17</em>OH-progesterone levels presumably indicates that testosterone of adrenal origin causes suppression of testicular testosterone production, either through a direct effect upon Leydig cells or via suppression of LH release.

This study was undertaken to determine whether serum dehydroepiandrosterone sulfate (DHEA-S) assays could be used in lieu of urinary <em>17</em>-<em>ketosteroid</em> (<em>17</em>-KS) determinations. To delineate normal values, serum DHEA-S levels were measured in 41 normal women (controls). These averaged 1.78 +/- 0.1 (SE) micrograms/ml and ranged from 0.3 to 2.8 micrograms/ml. Serum DHEA-S and urinary <em>17</em>-KS and creatinine were measured in 71 patients with and without signs of androgen excess who were attending the Reproductive Endocrinology/Infertility Clinic. Serum DHEA-S levels did not correlate with body weight, surface area, or ponderal index, and were not elevated in obese women without androgen excess. Both normal and elevated serum DHEA-S concentrations correlated well with total urinary <em>17</em>-KS when corrected for creatinine (P < .0005). In another group of 26 patients, the 2 urinary <em>17</em>-KS fractions androsterone (A) and dehydroepiandrosterone (DHEA) correlated well with serum DHEA-S levels, but the third urinary <em>17</em>-KS fraction, etiocholanolone (E), did not correlate. Eleven of the 26 patients who had elevated serum DHEA-S concentrations excreted increased amounts of urinary A and DHEA but not E. It is concluded that serum DHEA-S levels are much more conveniently measured than urinary <em>17</em>-KS and should replace the latter because DHEA-S is a better indicator of adrenal "androgen" secretion.

We assessed the hormonal status of adult female volunteers before and during a 3-wk period of weight gain induced by mixed diet overfeeding. Forty-six percent of the 4.3-kg average weight gain experienced by these subjects consisted of lean body mass (LBM) and it is of interest that there were also increases in plasma Somatomedin-C/Insulin-like Growth Factor (SM-C/IGF-1) and testosterone concentrations as well as insulin. We suggest that it was the combined anabolic effect of these three hormones that facilitated the increase in LBM. Of the other assays done, increases were recorded for urinary <em>17</em>-<em>ketosteroids</em>, <em>17</em>-hydroxysteroids, epinephrine, and creatinine, whereas there were no changes in serum cortisol or triiodothyronine (T3), or urine norepinephrine; serum thyroxine (T4) fell slightly. Thus it appears that energy surfeit as well as energy deficit (reported by others) has an effect on blood hormone concentrations.

Pregnane-3,<em>17</em> alpha,20-triols bearing unsaturation at delta(7), delta(8), delta(5,7), or delta(5,8) have been tentatively identified as steroid metabolites in Smith-Lemli-Opitz syndrome (SLOS). Starting with <em>17</em> alpha-hydroxypregnenolone diacetate, we have synthesized 13 unsaturated C(21) triols by four different routes in one to four steps. These multifunctional steroids were prepared by a series of regio- and stereoselective transformations chosen to minimize facile olefin isomerization and <em>17</em>-deoxygenation. The results include a study of stereoselectivity in the reduction of <em>17</em> alpha-hydroxy-20-<em>ketosteroids</em>, an alternative method for reducing diethyl azodicarboxylate adducts of delta(5,7) steroids, and an efficient oxidation-isomerization of a delta(5,7) steroid using cholesterol oxidase. The 13 triols and their synthetic precursors were fully characterized by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. The NMR data, together with molecular modeling, indicated unanticipated conformational heterogeneity for two synthetic intermediates, <em>17</em> alpha-hydroxypregna-4,7-diene-3,20-dione and <em>17</em> alpha-hydroxy-5 beta-pregn-7-ene-3,20-dione. The unsaturated C(21) triols are useful as reference standards to study adrenal steroid production in SLOS and to develop methods for pre- and postnatal diagnosis of this congenital disorder.

The <em>17</em>beta-hydroxysteroid dehydrogenases (<em>17</em>beta-HSDs) are involved in the regulation of estrogens and androgens by catalyzing the reduction of <em>17</em>-<em>ketosteroids</em> or the oxidation of <em>17</em>beta hydroxysteroids. The enzyme activities associated with the different <em>17</em>beta-HSD isoforms are widespread in human tissues, not only in classic steroidogenic tissues but also in a large series of peripheral intracrine tissues. Being involved at the end of steroidogenesis, the numerous members of <em>17</em>beta-HSD family constitute interesting therapeutic targets for controlling the concentration of estrogens and androgens. Thus, inhibitors of reductive <em>17</em>beta-HSD isoforms are attractive to block the formation of hydroxysteroids that stimulate estrogeno-sensitive pathologies (breast, ovarian, and endometrium cancers) and androgeno-sensitive pathologies (prostate cancer, benign prostatic hyperplasia, acne, and hirsutism). The inhibitors could be used to block the degradation of estradiol, an attractive strategy for treating osteoporosis and Alzheimer's disease. In addition to their classical use as anti-cancer agents and therapeutic agents, inhibitors of <em>17</em>beta-HSDs are also useful tools to elucidate the role of these enzymes in particular biological systems. The present review article gives a description of novel inhibitors of <em>17</em>beta-HSDs that were published in 2003-2006.

This study is aimed at elucidating the possible protective effects of Nigella sativa oil (NSO) in alleviating the toxicity of chlorpyrifos (CPF) on reproductive performance in male rats. Animals were orally administered with NSO (1 ml/kg/day), CPF (20 mg/kg/day), and NSO + CPF every day for 4 weeks. Results showed that CPF decreased spermatid number, sperm count, daily sperm production, and sperm motility while increased dead sperm and abnormal sperm compared with the control. Also the levels of testosterone, thyroxine levels, steroidogenic enzyme <em>17</em>-<em>ketosteroid</em> reductase, body weight, food intake, and relative weight of reproductive organs were decreased. Thiobarbituric acid reactive substances were increased, while glutathione (GSH) and antioxidant enzymes were decreased in plasma and testes of rats treated with CPF. Histopathological examination of testes showed a decrease in the number of seminiferous tubules, form shrinkage, enlargement of the connective tissue and gametogenic changes in germ cells of rats treated with CPF. NSO alone increased testosterone, semen characteristics, GSH, and antioxidant enzymes and decreased the levels of free radicals. Furthermore, the presence of NSO with CPF alleviates its toxic effects. Our results indicated that NSO can improve semen picture and moderate CPF-induced reproductive toxicity.

Endometrial and cervical cancers, uterine myoma, and endometriosis are very common uterine diseases. Worldwide, more than 800,000 women are affected annually by gynecological cancers, as a result of which, more than 360,000 die. During their reproductive age, about 70% of women develop uterine myomas and 10-15% suffer from endometriosis. Uterine diseases are associated with aberrant inflammatory responses and concomitant increased production of prostaglandins (PG). They are also related to decreased differentiation, due to low levels of protective progesterone and retinoic acid, and to enhanced proliferation, due to high local concentrations of estrogens. The pathogenesis of these diseases can thus be attributed to disturbed PG, estrogen, and retinoid metabolism and actions. Five human members of the aldo-keto reductase 1B (AKR1B) and 1C (AKR1C) superfamilies, i.e., AKR1B1, AKR1B10, AKR1C1, AKR1C2, and AKR1C3, have roles in these processes and can thus be implicated in uterine diseases. AKR1B1 and AKR1C3 catalyze the formation of PGF2α, which stimulates cell proliferation. AKR1C3 converts PGD2 to 9α,11β-PGF2, and thus counteracts the formation of 15-deoxy-PGJ2, which can activate pro-apoptotic peroxisome-proliferator-activated receptor γ. AKR1B10 catalyzes the reduction of retinal to retinol, and thus lessens the formation of retinoic acid, with potential pro-differentiating actions. The AKR1C1-AKR1C3 enzymes also act as <em>17</em>-keto- and 20-<em>ketosteroid</em> reductases to varying extents, and are implicated in increased estradiol and decreased progesterone levels. This review comprises an introduction to uterine diseases and AKR1B and AKR1C enzymes, followed by an overview of the current literature on the AKR1B and AKR1C expression in the uterus and in uterine diseases. The potential implications of the AKR1B and AKR1C enzymes in the pathophysiologies are then discussed, followed by conclusions and future perspectives.

A 45-year-old man presented with gynecomastia, hypertension and a large left adrenal mass. Further evaluation revealed elevated serum concentrations of estrogen, estrone sulfate, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, deoxycorticosterone, and aldosterone and increased 24-hour urinary <em>17</em>-<em>ketosteroid</em> and free cortisol excretion. Removal of a 10 kg adrenocortical carcinoma led to normalization of the hormone concentrations and partial resolution of the gynecomastia. There was no clinical evidence of metastases. Incubation of tumor slices demonstrated that the tumor had an active aromatase and sulfotransferase. We estimated that about half the serum estrone arose from peripheral conversion of androstenedione. Feminizing adrenocortical carcinomas are rare and this case is unusual given the lack of clinical metastases and the probable dual source of estrogen from tumor as well as from the peripheral conversion of tumor-derived androgens.

The hypogonadal (hpg) mouse has a congenital deficiency in gonadotrophin-releasing hormone and the gonads consequently lack exposure to endogenous gonadotrophins during development. To determine the effect of FSH on Leydig cell function in these animals adult hpg mice were injected twice daily with FSH (2 micrograms injections) or LH (40 ng injections, the presumed LH contamination of FSH used). Following FSH treatment there was a clear stimulation of the seminiferous epithelium and in animals injected with FSH plus [3H]thymidine, the incorporation of label was largely confined to the germ cells with no apparent uptake by the Sertoli cells. In FSH-treated testes the Leydig cells contained numerous large lipid droplets, similar to the unstimulated hpg testis. There was no evidence of the interstitial hyperplasia which is observed following injection of high doses of LH (2 micrograms twice daily). There was no change in basal androgen content of the testis in vivo following FSH treatment but injection of a maximal dose of human chorionic gonadotrophin (hCG), 1 h before death, markedly increased testicular androgen content only in the FSH-treated group. Testicular androgen production in vitro was significantly increased following FSH treatment both under basal conditions (FSH-treated, <em>17</em>.4 pmol/testis; control, 1.46 pmol/testis) and during stimulation by hCG (FSH-treated, 940 pmol/testis; control, 81 pmol/testis). Associated with the increased androgen production following FSH treatment there were significant increases in the activities of three steroidogenic enzymes; cholesterol side-chain cleavage (186-fold increase over control), <em>17</em> alpha-hydroxylase (103-fold increase) and <em>17</em>-<em>ketosteroid</em> reductase (<em>17</em>7-fold increase).(ABSTRACT TRUNCATED AT 250 WORDS)

A 28-year-old male pseudohermaphrodite with gynaecomastia was raised as a female until the age of <em>17</em> years, at which time he developed masculine features (deepening of the voice, development of facial hair, male distribution of body hair and male body habitus) and assumed a male gender role. He had a small phallus with perineal urethra, absence of labioscrotal fusion, presence of vaginal pouch and undescended testes. The testicular biopsy showed hyalinization of the tubular basement membrane, lack of spermatogenesis and hyperplastic Leydig cells. Baseline peripheral plasma studies showed androstenedione concentrations ten times normal, low testosterone, elevated oestrone and elevated gonadotrophins. The in vitro incubation of testicular tissue showed no significant conversion of androstenedione to testosterone. However, two types of peripheral tissues, skin fibroblasts and erythrocytes, had a normal conversion, as did the body overall as measured by the technique of androstenedione constant infusion. These studies demonstrate that the <em>17</em>-<em>ketosteroid</em> reductase deficiency of the patient was limited to the testes.

A 58-year-old postmenopausal woman with high plasma testosterone levels and virilization, as demonstrated by hirsutism and alopecia, is presented. Urinary <em>17</em>-<em>ketosteroids</em> and <em>17</em>-hydroxycorticosteroids as well as the computed axial tomography scan of the adrenal glands were normal. Although no pelvic mass was detected by sonography or pelvic examination, the patient was found to have small pure Leydig cell tumour of the left ovary. Following total abdominal hysterectomy and bilateral salpingo-oophorectomy, the patient had regression of the hirsutism, and the plasma testosterone dropped to normal level.

Lactophoricin (LPcin-I) is a 23-amino acid peptide that corresponds to the carboxyterminal 113-135 region of component-3 of proteose peptone (PP3), a minor phosphoglycoprotein found in bovine milk. It has been reported that lactophoricin has antibacterial activity and a cationic amphipathic helical structure, but its shorter analogous peptide (LPcin-II), a <em>17</em>-amino acid peptide, corresponding to the 119-135 region of PP3 does not display antibacterial activity. LPcin-I and LPcin-II have similar charge ratios and identical hydrophobic/hydrophilic sectors, according to their helical wheel projection patterns, and both peptides show cationic amphipathic helical folding and interact with membranes. However, it is known that only LPcin-I incorporates into planar lipidic bilayers to form voltage-dependent channels. In this study, the authors cloned and expressed the two recombinant peptides as <em>ketosteroid</em> isomerase (KSI) fusion proteins inclusion bodies in Escherichia coli. These peptides were subjected to NMR structural studies to explore their structure-activity relationships. Fusion proteins were purified by Ni-NTA affinity chromatography under denaturing conditions, and recombinant LPcin-I and LPcin-II were released from fusion by CNBr cleavage. Final purifications of LPcin-I and LPcin-II were achieved by preparative reversed-phase high performance liquid chromatography. Using these methods, we obtained several tens of milligrams of uniformly and selectively (15)N labeled peptides per liter of growth, which was sufficient for solid-state NMR spectroscopy. Peptides were identified by tris-tricine polyacrylamide gel electrophoresis and HSQC spectra. Initial structural data were obtained by solution NMR spectroscopy and compared in membrane-like environments.

<em>17</em>beta-Hydroxysteroid dehydrogenases (<em>17</em>HSDs) catalyze the interconversions between high-activity <em>17</em>beta-hydroxysteroids and low-activity <em>17</em>-<em>ketosteroids</em>. Several distinct <em>17</em>HSD isoenzymes have been characterized. They have unique tissue distribution patterns suggesting a specific function for each of the isoenzymes in modifying sex steroid hormone activity. The activities of <em>17</em>HSDs are essential for gonadal sex steroid biosynthesis and they are also involved in the modulation of steroid hormone action in peripheral tissues. <em>17</em>HSD type 1 (<em>17</em>HSD1) is needed for estradiol biosynthesis in ovarian granulosa cells and it is also expressed in breast tissue, thus increasing locally estradiol concentration. <em>17</em>HSD type 2 (<em>17</em>HSD2) is another <em>17</em>HSD enzyme involved in estrogen metabolism. The type 2 enzyme has an opposite activity catalyzing estradiol to estrone, thereby reducing the exposure of tissues to estrogen action. Preliminary data suggest that <em>17</em>HSD2 may predominate in human non-malignant breast epithelial cells, while <em>17</em>HSD type 1 activity prevails in malignant cells. Determination of the three-dimensional structure of human <em>17</em>HSD1 has led to an atomic level description of the estradiol binding pocket of the enzyme and an understanding of its mechanism of action, and the molecular basis for the estrogen-specificity of the enzyme. Deprivation of an estrogen response by using specific <em>17</em>HSD1 inhibitors is a tempting approach to treat estrogen-dependent breast cancer.

The glans clitoris is a target organ that is responsive to androgenic stimuli and enlarges throughout life. The size of the glans clitoris can be quantitated by determining the clitoral index (CI), which is the product of the sagittal and transverse diameters of the glans. Four hundred ten patients, ranging in age from <em>17</em> to 35 years, were examined. Ninety-five percent of 249 normal women had a CI less than 35 mm2. Of 85 patients with clitoromegaly (CI greater than 35 mm2) in addition to at least 1 other clinical sign of excess adrogenic stimulation, 53 (62%) had abnormally high values for either or both total serum testosterone and <em>17</em>-<em>ketosteroid</em> levels. The CI is a useful bioassay for the clinical recognition of excess androgenic stimulation.

OBJECTIVE

Girls with precocious pubarche (PP) are at increased risk for ovarian dysfunction, hyperinsulinism and dyslipidaemia in adolescence, in particular when PP is preceded by reduced fetal growth. However, it is not known whether PP girls still have adrenal hyperandrogenism after puberty and if so, which fraction of PP girls develops so-called functional adrenal hyperandrogenism (FAH), an entity characterized by ACTH-dependent <em>17</em>-<em>ketosteroid</em> excess.

METHODS

Data were longitudinally collected from 47 girls with PP: at birth (weight for gestational age), at diagnosis of PP (age 6.7+/- 1.1 years) and in adolescence (age 15.0+/-1.9 years).

METHODS

Serum dehydroepiandrosterone sulphate (DHEAS) and androstenedione were measured at PP diagnosis, as well as the <em>17</em>-hydroxyprogesterone (<em>17</em>-OHP) response to ACTH; postpubertal evaluation included assessment of adrenal and ovarian function, and of insulin responses to a glucose load. PP girls were considered to have FAH in adolescence if both DHEA and androstenedione responses to ACTH were excessive >> 1500 ng/dl and>> 350 ng/dl, respectively).

RESULTS

At diagnosis of PP, girls had high DHEAS and androstenedione levels, as well as high <em>17</em>-OHP responses to ACTH. In adolescence, PP girls had a normal BMI, presented with mild hirsutism and had high baseline and post-ACTH concentrations of most adrenal androgens, low SHBG levels and tended to have hyperinsulinemia and to present biological signs of ovarian hyperandrogenism. More than a third of the PP cohort developed FAH in adolescence. Neither baseline DHEAS, androstenedione, nor post-ACTH <em>17</em>-OHP values at diagnosis of PP predicted the development of FAH in adolescence. In PP girls, only a low weight at birth was found to be significantly associated with subsequent FAH.

CONCLUSIONS

These longitudinal findings in girls with PP point to the possibility of an endocrine sequence of prenatal onset: low weight at birth, PP in childhood and adrenal hyperandrogenism in adolescence. The pathophysiological mechanisms underpinning this newly recognized sequence remain to be identified.

<em>17</em>beta-Hydroxysteroid dehydrogenase/<em>17</em>-<em>ketosteroid</em> reductases (<em>17</em>HSD/KSR) play a key role in regulating steroid receptor occupancy in normal tissues and tumors. Although <em>17</em>HSD/KSR activity has been detected in ovarian epithelial tumors, our understanding of which isoforms are present and their potential for steroid metabolism is limited. In this investigation, <em>17</em>HSD/KSR activity from a series of ovarian epithelial tumors was assayed in cytosol and microsomes under conditions which differentiate between isoforms. Inhibition studies were used to further characterize the steroid specificities of isoforms in the two subcellular fractions. Activity varied widely between tumors of the same histopathologic classification. The highest levels of activity were observed in mucinous tumors. Michaelis constants, maximum velocities, estradiol-<em>17</em>beta/testosterone (E(2)/T) activity ratios and inhibition patterns were consistent with a predominance of microsomal <em>17</em>HSD/KSR2 and cytosolic <em>17</em>HSD/KSR5, isoforms reactive with both E(2) and T, with evidence of estrogenic <em>17</em>HSD/KSR1 in cytosol from some samples. In tumors where activity and mRNA expression were both characterized, Northern blots, PCR and sequence analysis indicated <em>17</em>HSD/KSR5 was the predominant isoform. The presence of <em>17</em>HSD/KSR5, which also has both 3alpha-HSD/KSR and 20alphaHSD/KSR activity, and <em>17</em>HSD/KSR2 which also has 20alpha-HSD activity, could influence not only estrogen and androgen binding but progesterone receptor occupancy, as well, in receptor-containing tumors.

We recently demonstrated that a <em>17</em>-<em>ketosteroid</em>, epiandrosterone, attenuates L-type Ca(2+) currents (I(Ca-L)) in cardiac myocytes and inhibits myocardial contractility. Because <em>17</em>-<em>ketosteroids</em> are known to inhibit glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, and to reduce intracellular NADPH levels, we hypothesized that inhibition of G6PD could be a novel signaling mechanism which inhibit I(Ca-L) and, therefore, cardiac contractile function. We tested this idea by examining myocardial function in isolated hearts and Ca(2+) channel activity in isolated cardiac myocytes. Myocardial function was tested in Langendorff perfused hearts and I(Ca-L) were recorded in the whole-cell patch configuration by applying double pulses from a holding potential of -80 mV and then normalized to the peak amplitudes of control currents. 6-Aminonicotinamide, a competitive inhibitor of G6PD, increased pCO(2) and decreased pH. Additionally, 6-aminonicotinamide inhibited G6PD activity, reduced NADPH levels, attenuated peak I(Ca-L) amplitudes, and decreased left ventricular developed pressure and ±dp/dt. Finally, dialyzing NADPH into cells from the patch pipette solution attenuated the suppression of I(Ca-L) by 6-aminonicotinamide. Likewise, in G6PD-deficient mice, G6PD insufficiency in the heart decreased GSH-to-GSSG ratio, superoxide, cholesterol and acetyl CoA. In these mice, M-mode echocardiographic findings showed increased diastolic volume and end-diastolic diameter without changes in the fraction shortening. Taken together, these findings suggest that inhibiting G6PD activity and reducing NADPH levels alters metabolism and leads to inhibition of L-type Ca(2+) channel activity. Notably, this pathway may be involved in modulating myocardial contractility under physiological and pathophysiological conditions during which the pentose phosphate pathway-derived NADPH redox is modulated (e.g., ischemia-reperfusion and heart failure).