Androgens are essential for the development and maintenance of spermatogenesis, but the underlying mechanisms of androgen action in the testis remain unclear. To help clarify these mechanisms, gene expression was measured in testes of pubertal (20 d old), androgen-insensitive, testicular feminized (Tfm) mice and in normal controls. Using microarrays (Affymetrix chips 430A and 430B), initial data identified a large number of genes down-regulated in the Tfm testis (>4700). These genes were largely of germ cell origin, reflecting the arrest of spermatogenesis that is apparent in the 20-d-old Tfm testis. Subsequent screening in vitro and in silico of this gene set identified 20 genes of a somatic tubular origin that were significantly down-regulated in the Tfm testis and six genes that were significantly up-regulated. Altered expression of these genes was confirmed by real-time PCR, and genes down-regulated in the Tfm testis were shown to be up-regulated in testes of hypogonadal (hpg) mice treated with androgen. In a developmental study using real-time PCR most of the regulated genes showed normal expression during fetal and neonatal development and deviated from control only between 10 and 20 d. In all cases, expression was also reduced in the adult, although interpretation is more complex because of the inherent cryptorchidism in the adult Tfm mouse. Of the total number of somatic genes showing differential expression in the Tfm testis, 50% were associated with three separate groups of genes involved in regulation of vitamin A metabolism, solute transportation, and cytoskeletal function. Thus, effects of androgens on tubular function and spermatogenesis may be mediated in part through regulation of the tubular environment and control of retinoic acid concentrations.

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Landeen LK, Dederko DA, Kondo CS, Hu BS, Aroonsakool N, Haga JH, Giles WR. Mechanisms of the negative inotropic effects of sphingosine-1-phosphate on adult mouse ventricular myocytes. Am J Physiol Heart Circ Physiol 294: H736-H749, 2008. First published November 16, 2007 doi:10.1152/ajpheart.00316.2007.-Sphingosine-1-phosphate (S1P) induces a transient bradycardia in mammalian hearts through activation of an inwardly rectifying K ϩ current (IK ACh ) in the atrium that shortens action potential duration (APD) in the atrium. We have investigated probable mechanisms and receptor-subtype specificity for S1P-induced negative inotropy in isolated adult mouse ventricular myocytes. Activation of S1P receptors by S1P (100 nM) reduced cell shortening by ϳ25% (vs. untreated controls) in fieldstimulated myocytes. S1P 1 was shown to be involved by using the S1P 1-selective agonist SEW2871 on myocytes isolated from S1P3-null mice. However, in these myocytes, S1P 3 can modulate a somewhat similar negative inotropy, as judged by the effects of the S1P 1 antagonist VPC23019. Since S1P1 activates Gi exclusively, whereas S1P 3 activates both Gi and Gq, these results strongly implicate the involvement of mainly G i. Additional experiments using the IK ACh blocker tertiapin demonstrated that IK ACh can contribute to the negative inotropy following S1P activation of S1P 1 (perhaps through Gi␤␥ subunits). Mathematical modeling of the effects of S1P on APD in the mouse ventricle suggests that shortening of APD (e.g., as induced by I K ACh ) can reduce L-type calcium current and thus can decrease the intracellular Ca 2ϩ concentration ([Ca 2ϩ ]i) transient. Both effects can contribute to the observed negative inotropic effects of S1P. In summary, these findings suggest that the negative inotropy observed in S1P-treated adult mouse ventricular myocytes may consist of two distinctive components: 1) one pathway that acts via G i to reduce L-type calcium channel current, blunt calcium-induced calcium release, and decrease [Ca 2ϩ ]i; and 2) a second pathway that acts via Gi to activate IK ACh and reduce APD. This decrease in APD is expected to decrease Ca 2ϩ influx and reduce [Ca 2ϩ ]i and myocyte contractility.calcium; contraction; cell shortening; inhibitory G protein; acetylcholine-sensitive potassium; myocyte SPHINGOSINE-1-PHOSPHATE (S1P) is a biologically active, cell membrane-associated sphingolipid that binds with high affinity to five distinct G-coupled protein receptor isoforms (S1P 1-5 ). S1P 1 has been detected in abundance in neonatal rat cardiomyocytes (39). In these cells, exposure to S1P (500 nM) results in an initial negative inotropic effect (reduction of systolic calcium). However, this may be followed by calcium overload (increased diastolic calcium) and then a cessation of contractility. In isolated atrial myocytes, S1P has been shown to activate a weakly inwardly rectifying potassium (K ϩ ) current. This K ϩ conductance is very similar to the K ϩ current activated by ACh (I K ACh ). Activation of this current can shorte...

Landeen LK, Aroonsakool N, Haga JH, Hu BS, Giles WR. Sphingosine-1-phosphate receptor expression in cardiac fibroblasts is modulated by in vitro culture conditions. Am J Physiol Heart Circ Physiol 292: H2698 -H2711, 2007. First published March 2, 2007; doi:10.1152/ajpheart.01065.2006.-The bioactive molecule sphingosine-1-phosphate (S1P) binds with high affinity to five recognized receptors (S1P1-5) to affect various tissues, including cellular responses of cardiac fibroblasts (CFbs) and myocytes. CFbs are essential components of myocardium, and detailed study of their cell signaling and physiology is required for a number of emerging disciplines. Meaningful studies on CFbs, however, necessitate methods for selective, reproducible cell isolations. Macrophages reside within normal cardiac tissues and often are isolated with CFbs. A protocol was therefore developed that significantly reduces macrophage levels and utilizes more CFb-specific markers (discoidin domain receptor-2) instead of, or in addition to, more commonly used cytoskeletal markers. Our results demonstrate that primary isolated, purified CFbs express predominantly S1P 1-3; however, the relative levels of these receptor subtypes are modulated with time and by culture conditions. In coculture experiments, macrophages altered CFb S1P receptor levels relative to controls. Further investigations using known macrophage-secreted factors showed that S1P and H 2O2 had minimal effects on CFb S1P1-3 expression, whereas transforming growth factor-␤1, TNF-␣, and PDGF-BB significantly altered all S1P receptor subtypes. Lowering FBS concentrations from 10% to 0.1% increased S1P2, whereas supplementation with either PDGF-BB or Rho-associated protein kinase inhibitor Y-27632 significantly elevated S1P3 levels. S1P2 and S1P3 receptor levels are known to regulate cell migration. Using cells isolated from either normal or S1P3-null mice, we demonstrate that S1P3 is important and necessary for CFb migration. These results highlight the importance of demonstrating CFb culture purity in functional studies of S1P and also identify conditions that modulate S1P receptor expression in CFbs. migration; macrophage; phenotype SPHINGOSINE-1-PHOSPHATE (S1P) is a biologically active, cell membrane-associated sphingolipid that is secreted by various cells upon activation. S1P binds with high affinity to five distinct G protein-coupled receptors, also referred to as endothelial differentiation gene (EDG) receptors. S1P receptors are ubiquitously expressed on mammalian cells and can affect such cellular responses as proliferation, differentiation, and migration (reviewed in Refs. 1,12,13,39,40,and 45). To characterize S1P regulation, it is therefore necessary to have pure populations of the target cells.Consistently obtaining highly purified cell cultures, which do not include any significant contaminating cell types, remains a considerable challenge when working with primary isolates from organs and tissues. However, this initial step is a requirement for many current molecular and c...

Mechanical force can induce a number of fundamental short- and long-term responses in myocardium. These include alterations in ECM, activation of cell-signaling pathways, altered gene regulation, changes in cell proliferation and growth, and secretion of a number of peptides and growth factors. It is now known that a number of these autocrine/paracrine factors are secreted from both cardiomyocytes and ventricular cardiac fibroblasts (CFb) in response to stretch. One such substance is IGF-I. IGF-I is an important autocrine/paracrine factor that can regulate physiological or pathophysiological responses, such as hypertrophy. In this study, we addressed the possible effects of mechanical perturbation, biaxial strain, on IGF-I secretion from adult rat CFb. CFb were subjected to either static stretch (3–10%) or cyclic stretch (10%; 0.1–1 Hz) over a 24-h period. IGF-1 secretion from CFb in response to selected stretch paradigms was examined using ELISA to measure IGF-I concentrations in conditioned media. Static stretch did not result in any measurable modulation of IGF-I secretion from CFb. However, cyclic stretch significantly increased IGF-I secretion from CFb in a frequency- and time-dependent manner compared with nonstretched controls. This stretch-induced increase in secretion was relatively insensitive to changes in extracellular [Ca2+] or to block of L-type Ca2+ channels. In contrast, thapsigargin, an inhibitor of sarco(endo)plasmic reticulum Ca2+ ATPase, remarkably decreased stretch-induced IGF-I secretion from CFb. We further show that IGF-I can upregulate mRNA expression of atrial natriuretic peptide in myocytes. In summary, cyclic stretch can significantly increase IGF-I secretion from CFb, and this effect is dependent on a thapsigargin-sensitive pool of intracellular [Ca2+].