Testosterone replacement in transgenic sickle cell mice controls priapic activity and upregulates PDE5 expression and eNOS activity in the penis.

Sickle cell disease (SCD)-associated priapism is characterized by decreased nitric oxide (NO) signaling and downregulated phosphodiesterase (PDE)5 protein expression and activity in the penis. Priapism is also associated with testosterone deficiency, but molecular mechanisms underlying testosterone effects in the penis in SCD are not known. Given the critical role of androgens in erection physiology and NO synthase (NOS)/PDE5 expression, we hypothesized that testosterone replacement to eugonadal testosterone levels reduces priapism by reversing impaired endothelial (e)NOS activity and molecular abnormalities involving PDE5. Adult male transgenic Berkeley sickle cell (Sickle) and wild-type (WT) mice were implanted with testosterone pellets, which release 1.2 µg testosterone/day for 21 days, or vehicle. After 21 days, animals underwent erectile function assessment followed by collection of blood for serum testosterone measurements, penes for molecular analysis, and seminal vesicles as testosterone-responsive tissue. Serum testosterone levels were measured by radioimmunoassay; protein expressions of PDE5, α-smooth muscle actin, eNOS and nNOS, and phosphorylation of PDE5 at Ser-92, eNOS at Ser-1177, neuronal (n) NOS at Ser-1412, and Akt at Ser-473 were measured by Western blot in penile tissue. Testosterone treatment reversed downregulated serum testosterone levels and increased (p < 0.05) the weight of seminal vesicles in Sickle mice to levels comparable to that of WT mice, indicating restored testosterone levels in Sickle mice. Testosterone treatment reduced (p < 0.05) prolonged detumescence in Sickle mice and normalized downregulated P-PDE5 (Ser-92), PDE5, P-eNOS (Ser-1177), and P-Akt (Ser-473) protein expressions in the Sickle mouse penis. Testosterone treatment did not affect P-nNOS (Ser-1412), eNOS, nNOS, or α-smooth muscle actin protein expressions in the Sickle mouse penis. In conclusion, in the mouse model of human SCD, increasing testosterone to eugonadal levels reduced priapic activity and reversed impaired Akt/eNOS activity and PDE5 protein expression in the penis.

Endothelial dysfunction in diabetic erectile dysfunction.

Erectile dysfunction (ED) is highly prevalent in diabetes mellitus. Pathophysiological mechanisms underlying diabetes-associated ED are in large part due to endothelial dysfunction, which functionally refers to the inability of the endothelium to produce vasorelaxing messengers and to maintain vasodilation and vascular homeostasis. The precise mechanisms leading to endothelial dysfunction in the diabetic vasculature, including the penis, are not yet fully understood. Hyperglycemia affects endothelial nitric oxide synthase activity and nitric oxide production/bioavailability, nitric oxide-independent relaxing factors, oxidative stress, production and/or action of hormones, growth factors and/or cytokines, and generation and activity of opposing vasoconstrictors. Considering recent advances in the field of vascular biology and diabetes, the emphasis in this review is placed on the mechanisms of hyperglycemia-induced endothelial dysfunction in the pathophysiology of diabetes-associated ED.

Constitutive NOS uncoupling and NADPH oxidase upregulation in the penis of type 2 diabetic men with erectile dysfunction.

Erectile dysfunction (ED) associated with type 2 diabetes mellitus (T2DM) involves dysfunctional nitric oxide (NO) signaling and increased oxidative stress in the penis. However, the mechanisms of endothelial NO synthase (eNOS) and neuronal NO synthase (nNOS) dysregulation, and the sources of oxidative stress, are not well defined, particularly at the human level. The objective of this study was to define whether uncoupled eNOS and nNOS, and NADPH oxidase upregulation, contribute to the pathogenesis of ED in T2DM men. Penile erectile tissue was obtained from 9 T2DM patients with ED who underwent penile prosthesis surgery for ED, and from six control patients without T2DM or ED who underwent penectomy for penile cancer. The dimer-to-monomer protein expression ratio, an indicator of uncoupling for both eNOS and nNOS, total protein expressions of eNOS and nNOS, as well as protein expressions of NADPH oxidase catalytic subunit gp91phox (an enzymatic source of oxidative stress) and 4-hydroxy-2-nonenal [4-HNE] and nitrotyrosine (markers of oxidative stress) were measured by western blot in this tissue. In the erectile tissue of T2DM men, eNOS and nNOS uncoupling and protein expressions of NADPH oxidase subunit gp91phox, 4-HNE- and nitrotyrosine-modified proteins were significantly (p < 0.05) increased compared to control values. Total eNOS and nNOS protein expressions were not significantly different between the groups. In conclusion, mechanisms of T2DM-associated ED in the human penis may involve uncoupled eNOS and nNOS and NADPH oxidase upregulation. Our description of molecular factors contributing to the pathogenesis of T2DM-associated ED at the human level is relevant to advancing clinically therapeutic approaches to restore erectile function in T2DM patients.

Mechanistic link between erectile dysfunction and systemic endothelial dysfunction in type 2 diabetic rats.

Men with type 2 diabetes mellitus (T2DM) and erectile dysfunction (ED) have greater risk of cardiovascular events than T2DM men without ED, suggesting ED as a predictor of cardiovascular events in diabetic men. However, molecular mechanisms underlying endothelial dysfunction in the diabetic penis explaining these clinical observations are not known. We evaluated whether the temporal relationship between ED and endothelial dysfunction in the systemic vasculature in T2DM involves earlier redox imbalance and endothelial nitric oxidase synthase (eNOS) dysfunction in the penis than in the systemic vasculature, such as the carotid artery. Rats were rendered T2DM by high-fat diet for 2 weeks, followed by an injection with low-dose streptozotocin. After 3 weeks, erectile function (intracavernosal pressure) was measured and penes and carotid arteries were collected for molecular analyses of eNOS uncoupling, protein S-glutathionylation, oxidative stress (4-hydroxy-2-nonenal, 4-HNE), protein expression of NADPH oxidase subunit gp91(phox) , endothelium-dependent vasodilation in the carotid artery, and non-adrenergic, non-cholinergic (NANC)-mediated cavernosal relaxation. Erectile response to electrical stimulation of the cavernous nerve and NANC-mediated cavernosal relaxation was decreased (p < 0.05), while relaxation of the carotid artery to acetylcholine was not impaired in T2DM rats. eNOS monomerization, protein expressions of 4-HNE and gp91(phox) , and protein S-glutathionylation, were increased (p < 0.05) in the penis, but not in the carotid artery, of T2DM compared to non-diabetic rats. In conclusion, redox imbalance, increased oxidative stress by NADPH oxidase, and eNOS uncoupling, occur early in T2DM in the penis, but not in the carotid artery. These molecular changes contribute to T2DM ED, while vascular function in the systemic vasculature remains preserved.

Comparisons of brain, uterus, and liver mRNA expression for cytochrome p450s, DNA methyltransferase-1, and catechol-o-methyltransferase in prepubertal female Sprague-Dawley rats exposed to a mixture of aryl hydrocarbon receptor agonists.

Non-ortho polychlorinated biphenyls (PCBs), polychlorinated dibenzodioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs) are ubiquitous environmental contaminants that exert their toxicity mostly through activation of the aryl-hydrocarbon receptor (AhR), and are referred to as AhR agonists. The objective was to study, by real time reverse-transcriptase-polymerase chain reaction (RT-PCR), the effects of postnatal exposure to a reconstituted mixture of AhR agonists present in breast milk (3 non-ortho PCBs, 6 PCDDs, and 7 PCDFs, referred to here-in-after as AhRM) on mRNA expression of estrogen receptor (ERalpha), enzymes involved with the metabolism of estrogens [catechol-o-methyltransferase (Comt), cytochrome P450 (Cyp)1A1, 1B1 and 2B1], and DNA methyltransferase-1 (Dnmt1), in brain areas, liver and uterus of immature female rats. Neonates were exposed by gavage during postnatal day (PND) 1-20 with dosages equivalent to 1, 10, 100, and 1000 times the estimated average human exposure level, and were sacrificed at PND 21. None of the end points were affected in uterine cross-sections, or in samples of uterine tissue layers collected by laser capture microdissection. At 1000x, the AhRM reduced Dnmt1 mRNA abundance to 28% and 32% of control in the liver and hypothalamus, respectively. In the brain, Cyp1A1 was increased (409%) but ERalpha was reduced (66%). Similarly, mRNA abundance for Comt isoforms was reduced in the liver (45%) and brain areas (55-70%). AhRM at 100x, the lowest effective dose, exerted a 220% increase in brain cortex Comt [membrane bound (Mb)], a 219% increase in hepatic Cyp1B1, and a 63% decrease in hepatic Comt (soluble (S)+Mb). These results support the possibility that early exposure to environmental contaminants could lead to effects mediated by changes in DNA methylation and/or estrogen metabolism and signaling.

Light-emitting properties of recombinant semi-synthetic aequorins and recombinant fluorescein-conjugated aequorin for measuring cellular calcium.

15 kinds of recombinant semi-synthetic aequorins and a recombinant fluorescein-conjugated aequorin were prepared and their properties in Ca(2+)-triggered luminescence were studied. The semi-synthetic aequorins showed a wide range of Ca(2+)-sensitivity. The luminescence intensity of a high-sensitivity type (hcp-aequorin) was greater than 10(4)-times that of a low-sensitivity type (n-aequorin) at pCa 6.0-6.5. The fluorescein-conjugated aequorin exhibited fluorescence in addition to the Ca(2+)-triggered luminescence, thus it can be used to visualize the diffusion and distribution of aequorin in cells. The data obtained, particularly the Ca(2+)-sensitivity curves, are useful in selecting a suitable semi-synthetic aequorin for an experiment.

Selective amplification of luteinizing hormone by adenosine in rat luteal cells.

Adenosine amplification of LH-stimulated cAMP accumulation in rat luteal cells is rapid and dependent on mitochondrial ATP production. The objective of the present studies was to determine if this effect of adenosine is specific for LH and to gain information on the mechanism of the ATP-dependent amplification of LH action in rat luteal cells. Adenosine significantly amplified maximum cAMP accumulation in response to LH, isoproterenol, forskolin, and cholera toxin. However, amplification of this response by adenosine was significantly greater for LH than for the other agonists. The relative order of amplification by adenosine was LH greater than isoproterenol greater than forskolin greater than cholera toxin; the relative magnitudes of amplification by adenosine were 1, 0.6, 0.2, and 0.2, respectively. Neither LH, isoproterenol, forskolin, nor cholera toxin had any effect on cellular levels of ATP, and adenosine produced a similar rate of increase and maximal levels of ATP in the presence of all agonists. Ionomycin, a calcium ionophore, inhibited LH- and cholera toxin-stimulated cAMP accumulation and produced a dose-dependent depletion of ATP. Adenosine reversed the inhibitory effect of ionomycin on LH-stimulated cAMP accumulation and cellular levels of ATP. However, adenosine did not reverse the inhibitory effect of ionomycin on cholera toxin-stimulated cAMP accumulation, although its effects on cellular ATP levels were identical to those on LH. Thus, the selective amplification of LH by adenosine is not merely a substrate effect on adenylate cyclase activity. The nature of adenylate cyclase activation by cholera toxin and forskolin and the weak amplification by adenosine of these agonists compared to that of LH indicate that the site of the ATP-dependent action of adenosine appears to be before or on the G-protein of adenylate cyclase. We suggest that adenosine, by an ATP-dependent process, either increases the availability of functional LH receptors or increases coupling between the LH receptor and adenylate cyclase.

The antigonadotropic actions of prostaglandin F2 alpha and phorbol ester are mediated by separate processes in rat luteal cells.

Protein kinase-C (PKC) has been suggested as a possible mediator of the antigonadotropic action of prostaglandin F2 alpha (PGF2 alpha) in luteal cells. To examine this possibility, we evaluated the effects of phorbol ester [12-O-tetradecanoylphorbol-13-acetate (TPA)] in relation to those of PGF2 alpha on cAMP accumulation and ATP levels as well as on the subcellular distribution of PKC activity in rat luteal cell cultures. Treatment of luteal cells for 1 h with TPA or PGF2 alpha produced a dose-dependent inhibition of LH-stimulated cAMP accumulation. Maximal inhibition produced by PGF2 alpha was about 35% greater than that produced by TPA. Moreover, PGF2 alpha produced a further inhibition of LH action when the cells were maximally inhibited by TPA. Staurosporine, a PKC inhibitor, reversed inhibition of LH-dependent cAMP accumulation produced by TPA, but had no effect on the response to PGF2 alpha. Furthermore, cells in which PKC was persistently activated by prolonged TPA treatment lost their responsiveness to additional TPA, but continued to show inhibition of cAMP accumulation by PGF2 alpha. TPA also produced a dose-dependent decrease in cell levels of ATP in contrast to PGF2 alpha. Finally, TPA produced a rapid redistribution of PKC activity from the cytosolic to the particulate fraction, whereas PGF2 alpha produced only a slight redistribution. We conclude that the acute antigonadotropic action of PGF2 alpha in rat luteal cells occurs via mechanisms other than phorbol ester-sensitive PKC activation.

Inhibition of protein synthesis and hormone-sensitive steroidogenesis in response to hydrogen peroxide in rat luteal cells.

Hydrogen peroxide (H2O2) is generated in the corpus luteum at functional luteal regression and produces rapid antigonadotropic effects in rat luteal cells. However, the mechanism by which peroxide interrupts LH- and cAMP-sensitive progesterone synthesis is unknown. The post-cAMP site of H2O2 action is due to the reduced cholesterol availability in mitochondria, and this process is well known to be dependent on protein synthesis. Therefore, we examined whether H2O2 may interfere with protein and RNA synthesis, and whether such responses may be associated with inhibition of steroidogenesis. Incorporation of radiolabeled amino acids into luteal proteins was inhibited in response to H2O2 in a time- and dose-dependent manner, and these doses are similar to those that inhibit progesterone synthesis, shown earlier in the identical paradigm. The inhibitory effect of H2O2 on amino acid incorporation was not due to increased protein degradation, impaired transport of amino acids, or depletion of cellular ATP levels. H2O2 also inhibited RNA synthesis, increased RNA degradation, and impaired the efficiency of mRNA as a translation template. The time course for the inhibitory effect of H2O2 on protein and RNA synthesis was very rapid and coincident with inhibition of steroidogenesis. Inhibition of protein and RNA synthesis and steroidogenesis were reversed by preincubation of cells with the cell-permeable metal chelator o-phenanthroline, which implicates metal-dependent radical generation as the probable mediator of these actions of H2O2. We conclude that the target of the post-cAMP site of peroxide-induced inhibition of cAMP-dependent steroidogenesis is the inhibition of rapidly inducible proteins that are known to mediate translocation of cholesterol within mitochondria, where it is used as a substrate for pregnenolone synthesis.

Functional differentiation of placental syncytiotrophoblasts during baboon pregnancy: developmental expression of chorionic somatomammotropin messenger ribonucleic acid and protein levels.

The objective of the present study was to determine whether, in addition to the onset of chorionic somatomammotropin (CS) production previously shown to result from the morphological differentiation of cytotrophoblasts into syncytiotrophoblasts, there is a further developmental increase in the capacity of syncytiotrophoblasts to produce CS with advancing stages of baboon pregnancy. Placentas were obtained from baboons in early (days 48-62), mid (days 97-110), and late (days 161-175) gestation (term = 184 days), and CS messenger ribonucleic acid (mRNA) and protein levels were determined in a syncytiotrophoblast-rich cell fraction isolated by Percoll gradient centrifugation. CS mRNA levels in syncytiotrophoblasts, expressed as a ratio of beta-actin, exhibited a progressive increase from early (0.04 +/- 0.04 relative arbitrary units) to mid (2.37 +/- 0.33; P < 0.001) to late (3.66 +/- 0.39; P < 0.05) gestation. Levels of the 22-kDa CS protein were very low on days 48-55 (0.83 +/- 0.09 arbitrary units), increased 10-fold (P < 0.001) on days 57-60 (8.11 +/- 0.68), and increased (P < 0.001) to a maximum of 14.58 +/- 0.58 near term. CS mRNA levels in whole placental villous tissue increased (P < 0.05) between early (0.89 +/- 0.48) and mid (2.97 +/- 0.47) gestation, then remained constant. CS protein exhibited a similar increase (P < 0.001) in villous tissue between early (2.32 +/- 0.40) and mid (6.07 +/- 0.24) gestation, then remained constant. The increase in mRNA and protein levels of CS in the placenta was accompanied by a progressive (P < 0.001) rise in serum CS. We conclude that in addition to the morphological differentiation of cytotrophoblasts into syncytiotrophoblasts that has been well established to result in the onset of CS biosynthesis, villous syncytiotrophoblasts undergo functional/biochemical differentiation thereafter, manifested as an increase in the capacity for the synthesis of CS.

Metal chelators reverse the action of hydrogen peroxide in rat luteal cells.

In rat luteal cells hydrogen peroxide (H2O2) interferes with the functional coupling of the luteinizing hormone (LH) receptor and blocks cAMP-dependent progesterone production. To test if this action of H2O2 is dependent on the generation of hydroxyl radicals, the effects of metal chelators and hydroxyl radical scavengers were evaluated. The heavy metal chelator o-phenanthroline prevented H2O2 inhibition of LH-sensitive cAMP and progesterone accumulation and depletion of ATP. Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) partially reversed inhibition of cAMP accumulation by H2O2 and completely prevented H2O2-induced ATP depletion, but had no effect on H2O2 inhibition of progesterone synthesis. Three other heavy metal chelators, deferoxamine, bathocuproinedisulfonic acid (BA) and penicillamine, as well as hydroxyl radical scavengers ethanol, thiourea and N-(2-mercaptopropionyl)glycine (MPG), had no effect on the luteolytic actions of H2O2. Differential effects of the chelators were probably due to differences in their cell permeability and subcellular compartmentalization. We conclude that metal chelators block the luteolytic actions of H2O2 by a mechanism probably linked to inhibition of hydroxyl radical generation.

Effects of postnatal exposure to mixtures of non-ortho-PCBs, PCDDs, and PCDFs in prepubertal female rats.

There are concerns that postnatal exposure to organochlorines present in breast milk could lead to adverse health effects. We reconstituted four mixtures of aryl-hydrocarbon receptor (AhR) agonists (3 non-ortho polychlorinated biphenyls [PCBs], 6 polychlorinated dibenzodioxins [PCDDs], 7 polychlorinated dibenzofurans [PCDFs], or all 16 chemicals together [referred to as AhRM]) based on their concentrations in breast milk, and examined their effects following exposure by gavage from day 1 until day 20 of age. Female neonates received dosages of AhRM equivalent to 1, 10, 100, or 1000 times the amount consumed by an infant over the first 24 days of life. Other groups received the PCBs, the PCDDs, or the PCDFs at the 1000x level. All rats were sacrificed at 21 days of age. Changes in ethoxyresorufin-o-deethylase hepatic activity, thymus and body weights, and serum thyroxin were linked to the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxic equivalents (TEQ) of the four mixtures (1000x-AhRM > PCDDs > PCBs > PCDFs). To test for AhRM antiestrogenicity, two additional groups received 1.5 microg/kg of 17alpha-ethynyl estradiol (EE) with or without the 1000x-AhRM. The AhRM had no effect on uterine weight or EE-stimulated uterine growth. The actions of the combined EE and AhRM treatments suggest additive effects in decreasing pentoxyresorufin-o-deethylase activity and spleen weight, but nonadditive/antagonistic effects on adrenal weight and serum thyroxin. In conclusion, (1) 10x-AhRM had no detectable effects, (2) TEQ values relate to observed toxicities, even when testing complex mixtures of AhR agonists, and (3) indications of tissue-specific additive and nonadditive/antagonistic effects, but no synergism, were observed when doses of AhRM were increased, or combined with EE.

eNOS-uncoupling in age-related erectile dysfunction.

Aging is associated with ED. Although age-related ED is attributed largely to increased oxidative stress and endothelial dysfunction in the penis, the molecular mechanisms underlying this effect are not fully defined. We evaluated whether endothelial nitric oxide synthase (eNOS) uncoupling in the aged rat penis is a contributing mechanism. Correlatively, we evaluated the effect of replacement with eNOS cofactor tetrahydrobiopterin (BH(4)) on erectile function in the aged rats. Male Fischer 344 'young' (4-month-old) and 'aged' (19-month-old) rats were treated with a BH(4) precursor sepiapterin (10 mg/kg intraperitoneally) or vehicle for 4 days. After 1-day washout, erectile function was assessed in response to electrical stimulation of the cavernous nerve. Endothelial dysfunction (eNOS uncoupling) and oxidative stress (thiobarbituric acid reactive substances, TBARS) were measured by conducting western blot in penes samples. Erectile response was significantly reduced in aged rats, whereas eNOS uncoupling and TBARS production were significantly increased in the aged rat penis compared with young rats. Sepiapterin significantly improved erectile response in aged rats and prevented increase in TBARS production, but did not affect eNOS uncoupling in the penis of aged rats. These findings suggest that aging induces eNOS uncoupling in the penis, resulting in increased oxidative stress and ED.

Semi-synthetic aequorins with improved sensitivity to Ca2+ ions.

Thirty-seven coelenterazine analogues were synthesized and incorporated into apo-aequorin, yielding 30 semi-synthetic aequorins that have the capacity to emit a significant amount of light in the presence of Ca2+. The properties of resultant photoproteins were investigated. The most prominent feature of those photoproteins was the wide range in their sensitivities to Ca2+ concentration. The relative intensity of Ca2+-triggered luminescence of the photoproteins ranged from 0.01 to 190 when compared with natural aequorin (relative intensity 1.0) at pCa 6 for the cases where the relative intensity is less than 1 and at pCa 7 for the cases where the relative intensity is higher than 1. Eight of the semi-synthetic aequorins belonged to the class of e-aequorin. With two of those photoproteins, the degree of dependence of the luminescence intensity ratio I400/I465 on pCa was greater than that with e-aequorin, suggesting that these two photoproteins are possibly superior to e-aequorin in measuring Ca2+ concentration by the ratio method.

Semi-synthetic aequorin. An improved tool for the measurement of calcium ion concentration.

The photoprotein aequorin isolated from the jellyfish Aequorea emits blue light in the presence of Ca2+ by an intramolecular process that involves chemical transformation of the coelenterazine moiety into coelenteramide and CO2. Because of its high sensitivity to Ca2+, aequorin has widely been used as a Ca2+ indicator in various biological systems. We have replaced the coelenterazine moiety in the protein with several synthetic coelenterazine analogues, providing semi-synthetic Ca2+-sensitive photoproteins. One of the semi-synthetic photoproteins, derived from coelenterazine analogue (II) (with an extra ethano group), showed highly promising properties for the measurement of Ca2+, namely (1) the rise time of luminescence in response to Ca2+ was shortened by approx. 4-fold compared with native aequorin and (2) the luminescence spectrum showed two peaks at 405 nm and 465 nm and the ratio of their peak heights was dependent on Ca2+ concentration in the range of pCa 5-7, thus allowing the determination of [Ca2+] directly from the ratio of two peak intensities. Coelenterazine analogue (I) (with a hydroxy group replaced by an amino group) was also incorporated into apo-aequorin, yielding a Ca2+-sensitive photoprotein, which indicates that an electrostatic interaction between the phenolate group in the coelenterazine moiety and some cationic centre in apo-aequorin is not important in native aequorin, contrary to a previous suggestion.

Effects of aging on prolactin release after stress in female and male rats.

Young adult and elderly male and female intact rats, as well as chronically ovariectomized (OVX) young and elderly female rats, were subjected to an acute stress by cutting the tip of the tail and prolactin (Prl) concentrations were measured in their blood collected by decapitation at various times thereafter. Maximum concentrations of the hormone were markedly lower in all the three groups of elderly rats than those found in the corresponding young animals, and appeared to occur with a delay in the females, but not in the males. In addition, the Prl-response to stress was attenuated in OVX animals regardless of their age. The result of these experiments, performed at two points on the age scale, suggests that in sexually mature rats of both sexes the stress-induced secretion of Prl is inversely related to the age of the animal and that the reverse relationship is retained in OVX females.

Recombinant aequorin and recombinant semi-synthetic aequorins. Cellular Ca2+ ion indicators.

Properties of a recombinant aequorin were investigated in comparison with those of natural aequorin. In chromatographic behaviour the recombinant aequorin did not match any of ten isoaequorins tested, although it was very similar to aequorin J. Its sensitivity to Ca2+ was found to be higher than that of any isoaequorin except aequorin D. The recombinant aequorin exhibited no toxicity when tested in various kinds of cells, even where samples of natural aequorin had been found to be toxic. Properties of four recombinant semi-synthetic aequorins (fch-, hcp-, e- and n-types), prepared from the recombinant apo-aequorin and synthetic analogues of coelenterazine, were approximately parallel with those of corresponding semi-synthetic aequorins prepared from natural apo-aequorin. Both recombinant e-aequorin and natural e-aequorin J luminesced with high values of the luminescence intensity ratio I400/I465, although the ratios were not pCa-dependent. The recombinant aequorin and recombinant semi-synthetic aequorins are highly suited for monitoring cellular Ca2+.

Luteinization of ovaries and gonadotrophin and prolactin secretion in rats with posterior hypothalamic lesions.

Posterior hypothalamic lesions restricted to the mammillary body in newborn rats evoked significantly elevated serum prolactin concentrations (P < 0.05) in adult females in the afternoon of proestrous (16.00 h), while at the same time serum LH values appeared significantly depressed (P < 0.05) as compared to controls. FSH concentrations were not affected. Parallel to changes in hormonal pattern, the ovaries of the lesioned animals grew to excessive dimensions due to the accumulation and persistence of numerous corpora lutea (CL) (syndrome of hyperluteinized ovaries). The results suggest that the posterior hypothalamus can regulate prolactin and LH secretion and that the fate of CL is associated with a quantitative ratio in the circulation of at least two hormones, prolactin and LH.

Absence of detectable phosphocreatine in rat luteal cells.

Previous studies from this laboratory showed that adenosine amplifies the action of luteinizing hormone (LH) severalfold in rat and human luteal cells by an intracellular, adenosine 5'-triphosphate (ATP)-linked process. The objective of this study was to evaluate the contribution of phosphocreatine (PCr) and creatine kinase (CK) to the dynamics of luteal ATP metabolism. Levels of PCr in luteinized rat ovaries were similar to those seen in liver but were approximately 1 and 7% of levels found in skeletal and heart muscle, respectively. In isolated rat luteal cells, little detectable PCr was seen after incubation in the presence or absence of adenosine, although cell ATP levels were increased twofold by adenosine treatment. The presence or absence of LH had no effect on either PCr or ATP levels in incubations of isolated luteal cells. Analysis of CK activity in tissue and cell homogenates showed that the specific activity of CK in luteal cells was in the same range as that seen in liver but less than 1/30 of that seen in skeletal muscle. From these studies we conclude that rat luteal cells contain little, if any, PCr and low levels of CK. Thus the rapid changes in ATP levels that are seen in rat luteal tissue and cells may occur because these cells have little capacity to buffer ATP levels with a reservoir of high-energy phosphate groups in the form of PCr.

Endocrine regulation of ascorbic acid transport and secretion in luteal cells.

Luteal ascorbic acid depletion by LH and prostaglandin (PG) F2 alpha is well known, but how such depletion occurs is not. We therefore investigated the nature and regulation of ascorbic acid uptake and depletion in the rat CL and luteal cells. In vivo studies showed that blockade of steroidogenesis by aminoglutethimide prevented ascorbate depletion by LH, but not PGF2 alpha. Also, the time course for half-maximal depletion of ascorbic acid in vivo in response to PGF2 alpha was extremely rapid (2-3 min) compared to that known for LH (60 min). Thus, ascorbate depletion by LH and PGF2 alpha appears to occur by different mechanisms. In luteal cells, ascorbate uptake was energy-, sodium-, and microfilament-dependent with a Michaelis constant (Km) of 33 microM, similar to that reported for other cells. In contrast to findings for other cells, PGF2 alpha was found to be a potent and rapid inhibitor of ascorbate uptake with a half-maximal inhibition (IC50) of about 5 nM in luteal cells. Ascorbate uptake was unaffected by LH, PGE2, glucose, bromo-cAMP, progesterone, phorbol ester, ionomycin, hydrogen peroxide (H2O2), or aminoglutethimide. Also novel was the finding that luteal cell secretion of ascorbic acid was rapidly and potently stimulated by PGF2 alpha (IC50 about 5 nM), an effect mimicked by LH, H2O2, generators of reactive oxygen, calcium ionophore, and cytochalasin B. Basal release of ascorbic acid was energy-dependent, as secretion was blocked by a mitochondrial uncoupler and lowered temperature. Phorbol ester, bromo-cAMP, progesterone, aminoglutethimide, and ouabain had no effect on ascorbic acid secretion in luteal cells. These findings indicate that the secretion of ascorbic acid induced by PGF2 alpha, and possibly LH, may be mediated by calcium, reactive oxygen, and cytoskeletal changes. The ability of PGF2 alpha to inhibit ascorbate transport and to stimulate secretion implicates these processes as the basis for the rapid depletion of ascorbic acid in the CL. Ascorbate depletion by LH is associated with stimulation of steroidogenesis and an increase in ascorbic acid secretion.