cAMP signaling affects age-associated deterioration of pacemaker beating interval dynamics.

Sinoatrial node (SAN) beating interval variability (BIV) and the average beating interval (BI) are regulated by a coupled-clock system, driven by Ca2+-calmodulin activated adenylyl cyclase, cAMP, and downstream PKA signaling. Reduced responsiveness of the BI and BIV to submaximal, [X]50, ß-adrenergic receptor (ß-AR) stimulation, and phosphodiesterase inhibition (PDEI) have been documented in aged SAN tissue, whereas the maximal responses, [X]max, do not differ by age. To determine whether age-associated dysfunction in cAMP signaling leads to altered responsiveness of BI and BIV, we measured cAMP levels and BI in adult (2-4 months n = 27) and aged (22-26 months n = 25) C57/BL6 mouse SAN tissue in control and in response to ß-AR or PDEI at X50 and [X]max. Both cAMP and average BI in adult SAN were reduced at X50, whereas cAMP and BI at Xmax did not differ by age. cAMP levels and average BI were correlated both within and between adult and aged SAN. BIV parameters in long- and short-range terms were correlated with cAMP levels for adult SAN. However, due to reduced cAMP within aged tissues at [X]50, these correlations were diminished in advanced age. Thus, cAMP level generated by the coupled clock mechanisms is tightly linked to average BI. Reduced cAMP level at X50 in aged SAN explains the reduced responsiveness of the BI and BIV to ß-AR stimulation and PDEI.

Changes in cAMP signaling are associated with age-related downregulation of spontaneously beating atrial tissue energetic indices.

The prevalence of atria-related diseases increases exponentially with age and is associated with ATP supply-to-demand imbalances. Because evidence suggests that cAMP regulates ATP supply-to-demand, we explored aged-associated alterations in atrial ATP supply-to-demand balance and its correlation with cAMP levels. Right atrial tissues driven by spontaneous sinoatrial node impulses were isolated from aged (22-26 months) and adult (3-4 months) C57/BL6 mice. ATP demand increased by addition of isoproterenol or 3-Isobutyl-1-methylxanthine (IBMX) and decreased by application of carbachol. Each drug was administrated at the dose that led to a maximal change in beating rate (Xmax) and to 50% of that maximal change in adult tissue (X50). cAMP, NADH, NAD + NADH, and ATP levels were measured in the same tissue. The tight correlation between cAMP levels and the beating rate (i.e., the ATP demand) demonstrated in adult atria was altered in aged atria. cAMP levels were lower in aged compared to adult atrial tissue exposed to X50 of ISO or IBMX, but this difference narrowed at Xmax. Neither ATP nor NADH levels correlated with ATP demand in either adult or aged atria. Baseline NADH levels were lower in aged as compared to adult atria, but were restored by drug perturbations that increased cAMP levels. Reduction in Ca2+-activated adenylyl cyclase-induced decreased cAMP and prolongation of the spontaneous beat interval of adult atrial tissue to their baseline levels in aged tissue, brought energetics indices to baseline levels in aged tissue. Thus, cAMP regulates right atrial ATP supply-to-demand matching and can restore age-associated ATP supply-to-demand imbalance.

A remarkable adaptive paradigm of heart performance and protection emerges in response to marked cardiac-specific overexpression of ADCY8.

Adult (3 month) mice with cardiac-specific overexpression of adenylyl cyclase (AC) type VIII (TGAC8) adapt to an increased cAMP-induced cardiac workload (~30% increases in heart rate, ejection fraction and cardiac output) for up to a year without signs of heart failure or excessive mortality. Here, we show classical cardiac hypertrophy markers were absent in TGAC8, and that total left ventricular (LV) mass was not increased: a reduced LV cavity volume in TGAC8 was encased by thicker LV walls harboring an increased number of small cardiac myocytes, and a network of small interstitial proliferative non-cardiac myocytes compared to wild type (WT) littermates; Protein synthesis, proteosome activity, and autophagy were enhanced in TGAC8 vs WT, and Nrf-2, Hsp90α, and ACC2 protein levels were increased. Despite increased energy demands in vivo LV ATP and phosphocreatine levels in TGAC8 did not differ from WT. Unbiased omics analyses identified more than 2,000 transcripts and proteins, comprising a broad array of biological processes across multiple cellular compartments, which differed by genotype; compared to WT, in TGAC8 there was a shift from fatty acid oxidation to aerobic glycolysis in the context of increased utilization of the pentose phosphate shunt and nucleotide synthesis. Thus, marked overexpression of AC8 engages complex, coordinate adaptation "circuity" that has evolved in mammalian cells to defend against stress that threatens health or life (elements of which have already been shown to be central to cardiac ischemic pre-conditioning and exercise endurance cardiac conditioning) that may be of biological significance to allow for proper healing in disease states such as infarction or failure of the heart.

Proteomes of primary skin fibroblasts from healthy individuals reveal altered cell responses across the life span.

Changes in the proteome of different human tissues with advancing age are poorly characterized. Here, we studied the proteins present in primary skin fibroblasts collected from 82 healthy individuals across a wide age spectrum (22-89 years old) who participated in the GESTALT (Genetic and Epigenetic Signatures of Translational Aging Laboratory Testing) study of the National Institute on Aging, NIH. Proteins were extracted from lysed fibroblasts and subjected to liquid chromatography-mass spectrometry analysis, and the expression levels of 9341 proteins were analyzed using linear regression models. We identified key pathways associated with skin fibroblast aging, including autophagy, scavenging of reactive oxygen species (ROS), ribosome biogenesis, DNA replication, and DNA repair. Changes in these prominent pathways were corroborated using molecular and cell culture approaches. Our study establishes a framework of the global proteome governing skin fibroblast aging and points to possible biomarkers and therapeutic targets.

Phosphoprotein Phosphatase 1 but Not 2A Activity Modulates Coupled-Clock Mechanisms to Impact on Intrinsic Automaticity of Sinoatrial Nodal Pacemaker Cells.

Spontaneous AP (action potential) firing of sinoatrial nodal cells (SANC) is critically dependent on protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent protein phosphorylation, which are required for the generation of spontaneous, diastolic local Ca2+ releases (LCRs). Although phosphoprotein phosphatases (PP) regulate protein phosphorylation, the expression level of PPs and phosphatase inhibitors in SANC and the impact of phosphatase inhibition on the spontaneous LCRs and other players of the oscillatory coupled-clock system is unknown. Here, we show that rabbit SANC express both PP1, PP2A, and endogenous PP inhibitors I-1 (PPI-1), dopamine and cyclic adenosine 3',5'-monophosphate (cAMP)-regulated phosphoprotein (DARPP-32), kinase C-enhanced PP1 inhibitor (KEPI). Application of Calyculin A, (CyA), a PPs inhibitor, to intact, freshly isolated single SANC: (1) significantly increased phospholamban (PLB) phosphorylation (by 2-3-fold) at both CaMKII-dependent Thr17 and PKA-dependent Ser16 sites, in a time and concentration dependent manner; (2) increased ryanodine receptor (RyR) phosphorylation at the Ser2809 site; (3) substantially increased sarcoplasmic reticulum (SR) Ca2+ load; (4) augmented L-type Ca2+ current amplitude; (5) augmented LCR's characteristics and decreased LCR period in intact and permeabilized SANC, and (6) increased the spontaneous basal AP firing rate. In contrast, the selective PP2A inhibitor okadaic acid (100 nmol/L) had no significant effect on spontaneous AP firing, LCR parameters, or PLB phosphorylation. Application of purified PP1 to permeabilized SANC suppressed LCR, whereas purified PP2A had no effect on LCR characteristics. Our numerical model simulations demonstrated that PP inhibition increases AP firing rate via a coupled-clock mechanism, including respective increases in the SR Ca2+ pumping rate, L-type Ca2+ current, and Na+/Ca2+-exchanger current. Thus, PP1 and its endogenous inhibitors modulate the basal spontaneous firing rate of cardiac pacemaker cells by suppressing SR Ca2+ cycling protein phosphorylation, the SR Ca2+ load and LCRs, and L-type Ca2+ current.

Overexpression of a Neuronal Type Adenylyl Cyclase (Type 8) in Sinoatrial Node Markedly Impacts Heart Rate and Rhythm.

Heart rate (HR) and HR variability (HRV), predictors of over-all organism health, are widely believed to be driven by autonomic input to the sinoatrial node (SAN), with sympathetic input increasing HR and reducing HRV. However, variability in spontaneous beating intervals in isolated SAN tissue and single SAN cells, devoid of autonomic neural input, suggests that clocks intrinsic to SAN cells may also contribute to HR and HRV in vivo. We assessed contributions of both intrinsic and autonomic neuronal input mechanisms of SAN cell function on HR and HRV via in vivo, telemetric EKG recordings. This was done in both wild type (WT) mice, and those in which adenylyl cyclase type 8 (ADCY8), a main driver of intrinsic cAMP-PKA-Ca2+ mediated pacemaker function, was overexpressed exclusively in the heart (TGAC8). We hypothesized that TGAC8 mice would: (1) manifest a more coherent pattern of HRV in vivo, i.e., a reduced HRV driven by mechanisms intrinsic to SAN cells, and less so to modulation by autonomic input and (2) utilize unique adaptations to limit sympathetic input to a heart with high levels of intrinsic cAMP-Ca2+ signaling. Increased adenylyl cyclase (AC) activity in TGAC8 SAN tissue was accompanied by a marked increase in HR and a concurrent marked reduction in HRV, both in the absence or presence of dual autonomic blockade. The marked increase in intrinsic HR and coherence of HRV in TGAC8 mice occurred in the context of: (1) reduced HR and HRV responses to ß-adrenergic receptor (ß-AR) stimulation; (2) increased transcription of genes and expression of proteins [ß-Arrestin, G Protein-Coupled Receptor Kinase 5 (GRK5) and Clathrin Adaptor Protein (Dab2)] that desensitize ß-AR signaling within SAN tissue, (3) reduced transcripts or protein levels of enzymes [dopamine beta-hydorxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT)] required for catecholamine production in intrinsic cardiac adrenergic cells, and (4) substantially reduced plasma catecholamine levels. Thus, mechanisms driven by cAMP-PKA-Ca2+ signaling intrinsic to SAN cells underlie the marked coherence of TGAC8 mice HRV. Adaptations to limit additional activation of AC signaling, via decreased neuronal sympathetic input, are utilized to ensure the hearts survival and prevent Ca2+ overload.

Basal Spontaneous Firing of Rabbit Sinoatrial Node Cells Is Regulated by Dual Activation of PDEs (Phosphodiesterases) 3 and 4.

BACKGROUND: Spontaneous firing of sinoatrial node cells (SANCs) is regulated by cAMP-mediated, PKA (protein kinase A)-dependent (cAMP/PKA) local subsarcolemmal Ca2+ releases (LCRs) from RyRs (ryanodine receptors). LCRs occur during diastolic depolarization and activate an inward Na+/Ca2+ exchange current that accelerates diastolic depolarization rate prompting the next action potential. PDEs (phosphodiesterases) regulate cAMP-mediated signaling; PDE3/PDE4 represent major PDE activities in SANC, but how they modulate LCRs and basal spontaneous SANC firing remains unknown. METHODS: Real-time polymerase chain reaction, Western blot, immunostaining, cellular perforated patch clamping, and confocal microscopy were used to elucidate mechanisms of PDE-dependent regulation of cardiac pacemaking. RESULTS: PDE3A, PDE4B, and PDE4D were the major PDE subtypes expressed in rabbit SANC, and PDE3A was colocalized with α-actinin, PDE4D, SERCA (sarcoplasmic reticulum Ca2+ ATP-ase), and PLB (phospholamban) in Z-lines. Inhibition of PDE3 (cilostamide) or PDE4 (rolipram) alone increased spontaneous SANC firing by ≈20% (P<0.05) and ≈5% (P>0.05), respectively, but concurrent PDE3+PDE4 inhibition increased spontaneous firing by ≈45% (P<0.01), indicating synergistic effect. Inhibition of PDE3 or PDE4 alone increased L-type Ca2+ current (ICa,L) by ≈60% (P<0.01) or ≈5% (P>0.05), respectively, and PLB phosphorylation by ≈20% (P>0.05) each, but dual PDE3+PDE4 inhibition increased ICa,L by ≈100% (P<0.01) and PLB phosphorylation by ≈110% (P<0.05). Dual PDE3+PDE4 inhibition increased the LCR number and size (P<0.01) and reduced the SR (sarcoplasmic reticulum) Ca2+ refilling time (P<0.01) and the LCR period (time from action potential-induced Ca2+ transient to subsequent LCR; P<0.01), leading to decrease in spontaneous SANC cycle length (P<0.01). When RyRs were disabled by ryanodine and LCRs ceased, dual PDE3+PDE4 inhibition failed to increase spontaneous SANC firing. CONCLUSIONS: Basal cardiac pacemaker function is regulated by concurrent PDE3+PDE4 activation which operates in a synergistic manner via decrease in cAMP/PKA phosphorylation, suppression of LCR parameters, and prolongation of the LCR period and spontaneous SANC cycle length.

Ca(2+)/calmodulin-activated phosphodiesterase 1A is highly expressed in rabbit cardiac sinoatrial nodal cells and regulates pacemaker function.

Constitutive Ca(2+)/calmodulin (CaM)-activation of adenylyl cyclases (ACs) types 1 and 8 in sinoatrial nodal cells (SANC) generates cAMP within lipid-raft-rich microdomains to initiate cAMP-protein kinase A (PKA) signaling, that regulates basal state rhythmic action potential firing of these cells. Mounting evidence in other cell types points to a balance between Ca(2+)-activated counteracting enzymes, ACs and phosphodiesterases (PDEs) within these cells. We hypothesized that the expression and activity of Ca(2+)/CaM-activated PDE Type 1A is higher in SANC than in other cardiac cell types. We found that PDE1A protein expression was 5-fold higher in sinoatrial nodal tissue than in left ventricle, and its mRNA expression was 12-fold greater in the corresponding isolated cells. PDE1 activity (nimodipine-sensitive) accounted for 39% of the total PDE activity in SANC lysates, compared to only 4% in left ventricular cardiomyocytes (LVC). Additionally, total PDE activity in SANC lysates was lowest (10%) in lipid-raft-rich and highest (76%) in lipid-raft-poor fractions (equilibrium sedimentation on a sucrose density gradient). In intact cells PDE1A immunolabeling was not localized to the cell surface membrane (structured illumination microscopy imaging), but located approximately within about 150nm inside of immunolabeling of hyperpolarization-activated cyclic nucleotide-gated potassium channels (HCN4), which reside within lipid-raft-rich microenvironments. In permeabilized SANC, in which surface membrane ion channels are not functional, nimodipine increased spontaneous SR Ca(2+) cycling. PDE1A mRNA silencing in HL-1 cells increased the spontaneous beating rate, reduced the cAMP, and increased cGMP levels in response to IBMX, a broad spectrum PDE inhibitor (detected via fluorescence resonance energy transfer microscopy). We conclude that signaling via cAMP generated by Ca(2+)/CaM-activated AC in SANC lipid raft domains is limited by cAMP degradation by Ca(2+)/CaM-activated PDE1A in non-lipid raft domains. This suggests that local gradients of [Ca(2+)]-CaM or different AC and PDE1A affinity regulate both cAMP production and its degradation, and this balance determines the intensity of Ca(2+)-AC-cAMP-PKA signaling that drives SANC pacemaker function.

Sarcoplasmic reticulum Ca2+ cycling protein phosphorylation in a physiologic Ca2+ milieu unleashes a high-power, rhythmic Ca2+ clock in ventricular myocytes: relevance to arrhythmias and bio-pacemaker design.

Basal phosphorylation of sarcoplasmic reticulum (SR) Ca(2+) proteins is high in sinoatrial nodal cells (SANC), which generate partially synchronized, spontaneous, rhythmic, diastolic local Ca(2+) releases (LCRs), but low in ventricular myocytes (VM), which exhibit rare diastolic, stochastic SR-generated Ca(2+) sparks. We tested the hypothesis that in a physiologic Ca(2+) milieu, and independent of increased Ca(2+) influx, an increase in basal phosphorylation of SR Ca(2+) cycling proteins will convert stochastic Ca(2+) sparks into periodic, high-power Ca(2+) signals of the type that drives SANC normal automaticity. We measured phosphorylation of SR-associated proteins, phospholamban (PLB) and ryanodine receptors (RyR), and spontaneous local Ca(2+) release characteristics (LCR) in permeabilized single, rabbit VM in physiologic [Ca(2+)], prior to and during inhibition of protein phosphatase (PP) and phosphodiesterase (PDE), or addition of exogenous cAMP, or in the presence of an antibody (2D12), that specifically inhibits binding of the PLB to SERCA-2. In the absence of the aforementioned perturbations, VM could only generate stochastic local Ca(2+) releases of low power and low amplitude, as assessed by confocal Ca(2+) imaging and spectral analysis. When the kinetics of Ca(2+) pumping into the SR were increased by an increase in PLB phosphorylation (via PDE and PP inhibition or addition of cAMP) or by 2D12, self-organized, "clock-like" local Ca(2+) releases, partially synchronized in space and time (Ca(2+) wavelets), emerged, and the ensemble of these rhythmic local Ca(2+) wavelets generated a periodic high-amplitude Ca(2+) signal. Thus, a Ca(2+) clock is not specific to pacemaker cells, but can also be unleashed in VM when SR Ca(2+) cycling increases and spontaneous local Ca(2+) release becomes partially synchronized. This unleashed Ca(2+) clock that emerges in a physiological Ca(2+) milieu in VM has two faces, however: it can provoke ventricular arrhythmias; or if harnessed, can be an important feature of novel bio-pacemaker designs.

Ca²âº-dependent phosphorylation of Ca²âº cycling proteins generates robust rhythmic local Ca²âº releases in cardiac pacemaker cells.

The spontaneous beating of the heart is governed by spontaneous firing of sinoatrial node cells, which generate action potentials due to spontaneous depolarization of the membrane potential, or diastolic depolarization. The spontaneous diastolic depolarization rate is determined by spontaneous local submembrane Ca²âº releases through ryanodine receptors (RyRs). We sought to identify specific mechanisms of intrinsic Ca²âº cycling by which sinoatrial node cells, but not ventricular myocytes, generate robust, rhythmic local Ca²âº releases. At similar physiological intracellular Ca²âº concentrations, local Ca²âº releases were large and rhythmic in permeabilized sinoatrial node cells but small and random in permeabilized ventricular myocytes. Furthermore, sinoatrial node cells spontaneously released more Ca²âº from the sarcoplasmic reticulum than did ventricular myocytes, despite comparable sarcoplasmic reticulum Ca²âº content in both cell types. This ability of sinoatrial node cells to generate larger and rhythmic local Ca²âº releases was associated with increased abundance of sarcoplasmic reticulum Ca²âº-ATPase (SERCA), reduced abundance of the SERCA inhibitor phospholamban, and increased Ca²âº-dependent phosphorylation of phospholamban and RyR. The increased phosphorylation of RyR in sinoatrial node cells may facilitate Ca²âº release from the sarcoplasmic reticulum, whereas Ca²âº-dependent increase in phosphorylation of phospholamban relieves its inhibition of SERCA, augmenting the pumping rate of Ca²âº required to support robust, rhythmic local Ca²âº releases. The differences in Ca²âº cycling between sinoatrial node cells and ventricular myocytes provide insights into the regulation of intracellular Ca²âº cycling that drives the automaticity of sinoatrial node cells.

A bioluminescence method for direct measurement of phosphodiesterase activity.

We have adapted bioluminescence methods to be able to measure phosphodiesterase (PDE) activity in a one-step technique. The method employs a four-enzyme system (PDE, adenylate kinase (AK) using excess CTP instead of ATP as substrate, pyruvate kinase (PK), and firefly luciferase) to generate ATP, with measurement of the concomitant luciferase-light emission. Since AK, PK, and luciferase reactions are coupled to recur in a cyclic manner, AMP recycling maintains a constant rate of ATP formation, proportional to the steady-state AMP concentration. The cycle can be initiated by the PDE reaction that yields AMP. As long as the PDE reaction is rate limiting, the system is effectively at steady state and the bioluminescence kinetics progresses at a constant rate proportional to the PDE activity. In the absence of cAMP and PDE, low concentrations of AMP trigger the AMP cycling, which allows standardizing the system. The sensitivity of the method enables detection of <1 µU (pmol/min) of PDE activity in cell extracts containing 0.25-10 µg protein. Assays utilizing pure enzyme showed that 0.2 mM IBMX completely inhibited PDE activity. This single-step enzyme- and substrate-coupled cyclic-reaction system yields a simplified, sensitive, reproducible, and accurate method for quantifying PDE activities in small biological samples.

Oxysterols in heart failure.

Oxysterols are biologically active molecules that result from the oxidation of cholesterol. Several oxysterols are found in macrophages and macrophage-derived 'foam cells' in atherosclerotic tissue. Lipophilic oxysterols penetrate cell membranes and, therefore, their concentrations can reach harmful levels in endothelial and smooth muscle cells located in close proximity to the atherosclerotic plaques or inflammatory zones. New findings suggest that the effects of oxysterols on cardiomyocytes can lead to cell hypertrophy and death. This may make oxysterols one of the major factors precipitating morbidity in atherosclerosis-induced cardiac diseases and inflammation-induced heart complications. The pathological actions of oxysterols on muscle cells were shown to depend on dysfunctional Ca(2+) signaling; however, the mechanisms of the effects remain to be elucidated. Understanding the effects of oxysterols could lead to therapies that modulate malfunction of cardiomyocytes. This review discusses the experimental findings and the relevance of oxysterols to heart failure, and suggests strategies for important future investigations.

Ca(2+) -stimulated basal adenylyl cyclase activity localization in membrane lipid microdomains of cardiac sinoatrial nodal pacemaker cells.

Spontaneous, rhythmic subsarcolemmal local Ca(2+) releases driven by cAMP-mediated, protein kinase A (PKA)-dependent phosphorylation are crucial for normal pacemaker function of sinoatrial nodal cells (SANC). Because local Ca(2+) releases occur beneath the cell surface membrane, near to where adenylyl cyclases (ACs) reside, we hypothesized that the dual Ca(2+) and cAMP/PKA regulatory components of automaticity are coupled via Ca(2+) activation of AC activity within membrane microdomains. Here we show by quantitative reverse transcriptase PCR that SANC express Ca(2+)-activated AC isoforms 1 and 8, in addition to AC type 2, 5, and 6 transcripts. Immunolabeling of cell fractions, isolated by sucrose gradient ultracentrifugation, confirmed that ACs localize to membrane lipid microdomains. AC activity within these lipid microdomains is activated by Ca(2+) over the entire physiological Ca(2+) range. In intact SANC, the high basal AC activity produces a high level of cAMP that is further elevated by phosphodiesterase inhibition. cAMP and cAMP-mediated PKA-dependent activation of ion channels and Ca(2+) cycling proteins drive sarcoplasmic reticulum Ca(2+) releases, which, in turn, activate ACs. This feed forward "fail safe" system, kept in check by a high basal phosphodiesterase activity, is central to the generation of normal rhythmic, spontaneous action potentials by pacemaker cells.

JNK activation decreases PP2A regulatory subunit B56alpha expression and mRNA stability and increases AUF1 expression in cardiomyocytes.

A central feature of heart disease is a molecular remodeling of signaling pathways in cardiac myocytes. This study focused on novel molecular elements of MAPK-mediated alterations in the pattern of gene expression of the protein phosphatase 2A (PP2A). In an established model of sustained JNK activation, a 70% decrease in expression of the targeting subunit of PP2A, B56alpha, was observed in either neonatal or adult cardiomyocytes. This loss in protein abundance was accompanied by a decrease of 69% in B56alpha mRNA steady-state levels. Given that the 3'-untranslated region of this transcript contains adenylate-uridylate-rich elements known to regulate mRNA degradation, experiments explored the notion that instability of B56alpha mRNA accounts for the response. mRNA time-course analyses with real-time PCR methods showed that B56alpha transcript was transformed from a stable (no significant decay over 1 h) to a labile form that rapidly degraded within minutes. These results were supported by complementary experiments that revealed that the RNA-binding protein AUF1, known to destabilize target mRNA, was increased fourfold in JNK-activated cells. A variety of other stress-related stimuli, such as p38 MAPK activation and phorbol ester, upregulated AUF1 expression in cultured cardiac cells as well. In addition, gel mobility shift assays demonstrated that p37AUF1 binds with nanomolar affinity to segments of the B56alpha 3'-untranslated region. Thus these studies provide new evidence that signaling-induced mRNA instability is an important mechanism that underlies the changes in the pattern of gene expression evoked by stress-activated pathways in cardiac cells.

Testosterone regulates 25-hydroxycholesterol production in testicular macrophages.

Recently, we found that testicular macrophages produce 25-hydroxycholesterol (25-HC) and express 25-hydroxylase, the enzyme that converts cholesterol to 25-HC. In addition, 25-HC may be an important paracrine factor mediating the known interactions between macrophages and neighboring Leydig cells, because it is efficiently converted to testosterone by Leydig cells. The purpose of the present study was to determine if testosterone can regulate the production of 25-HC in rat testicular macrophages, representing a potential negative-feedback loop from Leydig cells. We found that expression of 25-hydroxylase mRNA and production of 25-HC by cultured testicular macrophages were significantly inhibited by testosterone at 10 micro g/ml. This dose of testosterone did not have an effect on cell viability and did not change the rate of mRNA degradation in the presence of actinomycin D. These studies indicate that production of 25-HC is negatively regulated by testosterone, which may be representative of a paracrine negative-feedback loop.

25-hydroxycholesterol is produced by testicular macrophages during the early postnatal period and influences differentiation of Leydig cells in vitro.

Leydig cells develop inappropriately in animals lacking testicular macrophages. We have recently found that macrophages from adult animals produce 25-hydroxycholesterol, an oxysterol involved in the differentiation of hepatocytes and keratinocytes. Therefore, we hypothesized that testicular macrophages also produce 25-hydroxycholesterol during the early postnatal period and that this oxysterol plays a role in the differentiation of Leydig cells. We assessed the production of 25-hydroxycholesterol and 25-hydroxylase mRNA by cultured testicular macrophages from rats at 10, 20, and 40 days of age. We also tested the long-term effects of 25-hydroxycholesterol on basal and LH-stimulated testosterone production, and 3beta-hydroxysteroid dehydrogenase activity as end points of Leydig cell differentiation in vitro. We found that testicular macrophages from animals at all ages produced both 25-hydroxycholesterol and 25-hydroxylase mRNA, with macrophages from 10-day-old animals having the highest steady-state levels of message. We also found that chronic exposure of Leydig cells to 25-hydroxycholesterol increased basal production of testosterone but decreased LH-stimulated steroidogenesis at all ages. Finally, 25-hydroxycholesterol increased 3beta-hydroxysteroid dehydrogenase activity in both progenitor and immature Leydig cells. These findings support the hypothesis that testicular macrophages play an important role in the differentiation of Leydig cells through the secretion of 25-hydroxycholesterol.