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This study was initiated to determine whether the noradrenergic (NE) neurons of the locus coeruleus (LC) could mediate the stimulatory action of androgens on serotonin-related gene expression in male macaques. These experiments follow our observations that serotonin neurons lack androgen receptors (ARs), and yet respond to androgens. Male Japanese macaques (Macaca fuscata) were castrated for 5-7months and then treated for 3months with [1] placebo, [2] T (testosterone), [3] DHT (dihydrotestosterone; non-aromatizable androgen) plus ATD (steroidal aromatase inhibitor), or [4] FLUT (Flutamide; androgen antagonist) plus ATD (n=5/group). The noradrenergic (NE) innervation of the raphe was determined with immunolabeling of axons with an antibody to dopamine-β-hydroxylase (DBH). Immunolabeling of tyrosine hydroxylase (TH) dendrites and corticotropin releasing hormone (CRH) axons innervating the LC was also determined. Due to the longer treatment period employed, the expression of the cognate nuclear receptors was sought. Androgen receptor (AR), estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) immunostaining was accomplished. Quantitative image analysis was applied and immunopositive neurons or axons with boutons were measured. Double-label of NE neurons for each receptor plus TH determined whether the receptors were localized in NE neurons. Androgens with or without aromatase activity significantly stimulated DBH axon density in the raphe (ANOVA, p=0.006), and LC dendritic TH (ANOVA, p<0.0001), similar to serotonin-related mRNA expression in the raphe. There were significantly more AR-positive neurons in T- and DHT+ATD-treated groups compared to placebo or FLUT+ATD-treated groups (ANOVA, p=0.0014). There was no difference in the number of positive-neurons stained for ERα or ERβ. The CRH axon density in the LC was significantly reduced with aromatase inhibition, suggesting that CRH depends on estrogen, not androgens (ANOVA, p=0.0023). Double-immunohistochemistry revealed that NE neurons did not contain AR. Rather, AR-positive nuclei were found in neighboring cells that are likely neurons. However, >80% of LC NE neurons contained ERα or ERβ. In conclusion, the LC NE neurons may transduce the stimulatory effect of androgens on serotonin-related gene expression. Since LC NE neurons lack AR, the androgenic stimulation of dendritic TH and axonal DBH may be indirectly mediated by other neurons. Estrogen, either from metabolism of T or from de novo synthesis, appears necessary for robust CRH innervation of the LC, which differs from female macaques., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We previously showed that tryptophan hydroxylase 2 (TPH2) and serotonin reuptake transporter (SERT) mRNAs are increased by the androgens, testosterone (T) and dihydrotestosterone (DHT) in serotonin neurons of male macaques. In addition, we observed that serotonin in axons of a terminal region were markedly decreased by aromatase inhibition and lack of estradiol (E) from metabolism of T. These observations implicated androgen receptors (AR) and estrogen receptors (ER) in the transduction of steroid hormone actions in serotonin neurons. Due to the longer treatment period employed, the expression of the cognate nuclear receptors was sought. We used single and double immunohistochemistry to quantitate and phenotypically localize AR, ERα and ERβ in the dorsal raphe of male macaques. Male Japanese macaques (Macaca fuscata) were castrated for 5-7 months and then treated for 3 months with [1] placebo, [2] T, [3] DHT (non-aromatizable androgen) plus ATD (steroidal aromatase inhibitor), or [4] Flutamide (FLUT; androgen antagonist) plus ATD (n = 5/group). After single labeling of each receptor, quantitative image analysis was applied and receptor positive neurons were counted. Double-label of raphe neurons for each receptor plus TPH2 determined whether the receptors were localized in serotonin neurons. There were significantly more AR-positive neurons in T- and DHT+ATD-treated groups (p = 0.0014) compared to placebo or FLUT+ATD-treated groups. There was no difference in the number of positive-neurons stained for ERα or ERβ⋅ Double-immunohistochemistry revealed that serotonin neurons did not contain AR. Rather, AR-positive nuclei were found in neighboring cells that are likely neurons. However, approximately 40% of dorsal raphe serotonin neurons contained ERα or ERβ⋅ In conclusion, the stimulatory effect of androgens on TPH2 and SERT mRNA expression is mediated indirectly by neighboring neurons contain AR. The stimulatory effect of E, derived from T metabolism, on serotonin transport is partially mediated directly via nuclear ERs., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Annexin A5 is a Ca2+-dependent phospholipid binding protein well known for its high phosphatidylserine affinity. In vitro, translocation to sarcolemma and externalization of endogenous annexin A5 in the cardiomyocyte has recently been demonstrated to exert a proapoptotic effect. To determine whether these in vitro findings occurred in vivo, we performed myocardial infarction (MI) and studied the time course of apoptosis and annexin A5 localization (0.5 to 8 h) in the border zone around the infarcted area. This zone that was defined as Evans blue unstained and triphenyltetrazolium chloride (TTC) stained, represented 42.3 +/- 5.5% of the area at risk and showed apoptotic characteristics (significant increases in caspase 3 activity 2.3-fold at 0.5 h; P < 0.05), transferase-mediated dUTP nick-end labeling-positive cardiomyocytes (15.8 +/- 0.8% at 8 h), and DNA ladder. When compared with sham-operated rats, we found that in this area, annexin A5 was translocated to the sarcolemma as early as 0.5 h after MI and that translocation increased with time. Moreover, the amount of annexin A5 was unchanged in the border zone and decreased in the infarcted area after 1 h (77.1 +/- 4.8%; P < 0.01 vs. perfused area), suggesting a release in the latter but not in the former. In conclusion, we demonstrated that annexin A5 translocation is an early and rapid event of the whole border zone, likely due to Ca2+ increase. Part of this translocation occurred in areas where apoptosis was later detected and suggests that in vivo as in vitro annexin A5 might be involved in the regulation of early apoptotic events during cardiac pathological situations.

Annexins II, V, and VI belong to a family of Ca(2+)-dependent phospholipid-binding proteins that have been involved mainly in signal transduction, differentiation, membrane trafficking events, or binding to the extracellular matrix, or that might be effective as Ca(2+)-channels. They are abundant in the mammalian myocardium and might play a role in ventricular remodeling and altered calcium handling during heart failure. To test this hypothesis, we compared the expression and distribution of these annexins in nonfailing (n = 9) and failing human hearts with idiopathic dilated cardiomyopathy (n = 11). Northern blot and slot blot analysis were used to determine the annexin mRNA levels and Western blots were used to quantify the amounts of annexin proteins. Distribution of annexins was studied by immunohistofluorescence labeling and compared with that of a sarcolemmal marker (Na+/K(+)-ATPase) and of a myofibrillar protein (alpha-actinin). We showed that nonfailing hearts contained a higher amount of annexin VI than of annexin V or II (13.5 +/- 1.8, 3.7 +/- 0.2, and 2.5 +/- 0.5 microg/mg protein, respectively). In failing hearts, there was a parallel increase in both mRNA and protein levels of annexin II (146% and 132%, p < 0.05, respectively) and annexin V (152%, p < 0.01, 147%, p < 0.005, respectively); the protein level of annexin VI was also increased (117%, p < 0.05), whereas the increase of its mRNA level was statistically insignificant. We observed a predominant localization of annexin II in interstitium, and of annexins V and VI in cardiomyocytes at the level of the sarcolemma, T-tubules, and intercalated disks in nonfailing hearts, whereas in failing hearts enlarged interstitium contained all three annexins. Furthermore, annexin V staining at the level of cardiomyocytes almost disappeared. In conclusion, we showed that heart failure is accompanied by marked overexpression of annexins II and V, as well as translocation of annexin V from cardiomyocytes to interstitial tissue. The data suggest that annexins may contribute to ventricular remodeling and annexin V to impaired Ca2+ handling in failing heart.

The respiration parameters of mitochondria from rat heart muscle and from fast-twitch and slow-twitch skeletal muscle skinned fibers were comparatively analyzed. Electrophoretic patterns of fiber protein composition were also compared. It was found that fibers with low affinity of mitochondria for ADP (i.e., heart and slow-twitch skeletal muscle soleus) contain a 27.5-kD protein that is absent from the fibers that exert high affinity for ADP (i.e., fast-twitch skeletal muscle gastrocnemius). Partial proteolysis, which increases the affinity of mitochondria of the heart and slow-twitch skeletal muscles for ADP, results in the disappearance of this protein. The results suggest that this protein may be an intracellular factor that controls the permeability of the outer mitochondrial membrane for ADP.

The rate of mitochondrial oxidative phosphorylation of saponin-skinned human muscle fibers from m. vastus lateralis in the presence of glutamate, malate and ATP is reported to be sensitive to caffeine and to changes of free calcium ion concentration. An approximately twofold increase in respiration was observed by the addition of 15 mM caffeine, because of the efflux of calcium from sarcoplasmic reticulum. Direct addition of a Ca2+/CaEGTA buffer, containing 1.5 microM free calcium ions had a similar effect. The ATP-splitting activity of skinned fibers was also stimulated by caffeine or calcium. These observations can be explained exclusively by the calcium-induced activation of actomyosin ATPase. (i) Thapsigargin, an inhibitor of the sarcoplasmic reticulum Ca(2+)-ATPase, had no influence. (ii) In myosin-extracted 'ghost' fibers containing intact mitochondria and an intact sarcoplasmic reticulum caffeine had a negligible effect on oxidative phosphorylation. (iii) The caffeine-induced increase in rate of fiber respiration was concomitant with a decrease in mitochondrial membrane potential and a decrease in the redox state of the mitochondrial NAD system. (iv) The calcium ionophore A 23187 caused a stimulation of respiration and ATP-splitting activity, similar to caffeine. (v) The calcium dependencies of respiration and ATP splitting activity of saponin-skinned human muscle fibers were in experimental error identical. Therefore it is concluded that calcium efflux from sarcoplasmic reticulum affects oxidative phosphorylation in skeletal muscle mostly via the stimulation of actomyosin ATPase.

Relative diffusivities of ADP and creatine in cardiomyocytes were studied. The isolated rat cardiomyocytes were lysed with saponin (40 micrograms/ml) to perforate or completely disrupt sarcolemma that was evidenced by leakage of 80-100% lactate dehydrogenase. In these cardiomyocytes mitochondria were used as 'enzymatic probes' to determine the average local concentration of substrates exerting acceptor control of respiration--ADP or creatine (the latter activates respiration via mitochondrial creatine kinase reaction)--when their concentrations in the surrounding medium were changed. The kinetic parameters for ADP and creatine in control of respiration of saponin-treated cardiomyocytes were compared with those determined in isolated mitochondria and skinned cardiac fibers. The apparent Km for creatine (at 0.2 mM ATP) was very close and in a range of 6.0-6.9 mM in all systems studied, showing the absence of diffusion difficulties for this substrate. On the contrary, the apparent Km for ADP increased from 18 +/- 1 microM for isolated mitochondria to 250 +/- 59 microM for cardiomyocytes with the lysed sarcolemma and to 264 +/- 57 microM for skinned fibers. This elevation of Km was not eliminated by inhibition of myokinase with diadenosine pentaphosphate. When 25 mM creatine was present, the apparent Km for ADP decreased to 36 +/- 6 microM. These data are taken to indicate specific restrictions of diffusion of ADP most probably due to its interaction with intermediate binding sites in cardiomyocytes. The important role of phosphocreatine-creatine kinase system of energy transport is to overcome the restrictions in regulation of energy fluxes due to decreased diffusivity of ADP.

The purified succinate-ubiquinone reductase catalyzes the L- (or D-) malate: acceptor oxidoreductase reaction with Km for malate of about 2.10(-3) M and initial Vmax of 50 and 100 nmol per min per mg of protein for L- and D-stereoisomers, respectively (25 degrees C, pH 7.0). The reaction rate rapidly decreases both in the absence and presence of L-glutamate and L-glutamate-oxaloacetate transaminase added for trapping of oxaloacetate. Both keto and enol forms of oxaloacetate were found to be strong, slowly dissociating inhibitors of succinate dehydrogenase; the first-order rate constant for the enzyme inhibition by the enol form is about 3 times as high as that by the keto form. Oxidation of malate by succinate dehydrogenase in the presence of the oxaloacetate trapping system occurs at an indefinitely constant rate when enoloxaloacetate, which is an immediate product of the reaction, is rapidly converted into the keto isomer--a substrate for transaminase. A quantitative kinetic scheme for malate oxidation by succinate dehydrogenase which includes two kinetically distinct enzyme-oxaloacetate complexes is proposed, and the specific role of the mitochondrial oxaloacetate keto-enol-tautomerase (EC 5.3.2.2) in the regulation of succinate dehydrogenase is suggested.

Two highly purified proteins with quite different properties capable of oxaloacetate keto-enol-tautomerase activity (oxaloacetate keto-enol-isomerase, EC 5.3.2.2) were isolated from the bovine heart mitochondrial matrix. The first protein has an apparent molecular mass of 37 kDa as determined by SDS-gel electrophoresis and Sephacryl SF-200 gel filtration. It is quite stable upon storage at 40 degrees C and reaches the maximal catalytic activity at pH 8.5 with a half-maximal activity at pH 7.0. The enzyme is specifically inhibited by oxalate and diethyloxaloacetate. When assayed in the enol----ketone direction at 25 degrees C (pH 9.0), the enzyme obeys a simple substrate saturation kinetics with Km and Vmax values of 45 microM and 74 units per mg of protein, respectively; the latter value corresponds to the turnover number of 2700 min-1. The second protein has an apparent molecular mass of 80 kDa as determined by SDS-gel electrophoresis and Sephacryl SF-300 gel filtration. The enzyme is rapidly inactivated at 40 degrees C and shows a sharp pH optimum of activity at pH 9.0. The enzyme can be completely protected from thermal inactivation by oxaloacetate and dithiothreitol. The kinetic parameters of the enzyme as assayed in the enol----ketone direction at 25 degrees C (pH 9.0) are: Km = 220 microM and Vmax = 20 units per mg of protein; the latter corresponds to the turnover number of 1600 min-1. The enzyme activity is specifically inhibited by maleate and pyrophosphate. About 30% of the total oxaloacetate tautomerase activity in crude mitochondrial matrix is represented by the 37 kDa enzyme and about 70% by the 80 kDa protein.

Highly purified succinate-ubiquinone reductase catalyzes the oxidation of L- or D-malate with a Km and initial Vmax equal to approximately 10(-3) M and approximately 100 nmol/min/mg of protein, respectively. The malate dehydrogenase activity of succinate dehydrogenase rapidly decreases regardless of the presence of glutamate plus glutamate-oxaloacetate transaminase. The inhibitor trapping system, however, prevents the inactivation of succinate dehydrogenase under the conditions when the rate of tautomeric oxaloacetate enol in equilibrium oxaloacetate ketone interconversion is high. These results suggest that enol oxaloacetate is an immediate product of malate oxidation at the succinate dehydrogenase active site. Two proteins (Mr 37 and 80 kD) which catalyze the oxaloacetate tautomerase reaction were isolated from the mitochondrial matrix. Some physico-chemical and kinetic properties of these enzymes were characterized. The larger protein was identified as inactive aconitase. The system containing succinate dehydrogenase, L-malate, glutamate plus transaminase and oxaloacetate tautomerase was reconstituted. Such a system is capable of oxidizing malate to aspartate without rapid inactivation of succinate dehydrogenase. Taken together, the data obtained emphasize a significant role of enzymatic oxaloacetate tautomerization in the control of the succinate dehydrogenase activity in the mitochondrial matrix.