Non-genomic Glucocorticoid Actions Research Articles

Genomic and Non-Genomic Actions of Glucocorticoids on Adipose Tissue Lipid Metabolism.

Glucocorticoids (GCs) are hormones that aid the body under stress by regulating glucose and free fatty acids. GCs maintain energy homeostasis in multiple tissues, including those in the liver and skeletal muscle, white adipose tissue (WAT), and brown adipose tissue (BAT). WAT stores energy as triglycerides, while BAT uses fatty acids for heat generation. The multiple genomic and non-genomic pathways in GC signaling vary with exposure duration, location (adipose tissue depot), and species. Genomic effects occur directly through the cytosolic GC receptor (GR), regulating the expression of proteins related to lipid metabolism, such as ATGL and HSL. Non-genomic effects act through mechanisms often independent of the cytosolic GR and happen shortly after GC exposure. Studying the effects of GCs on adipose tissue breakdown and generation (lipolysis and adipogenesis) leads to insights for treatment of adipose-related diseases, such as obesity, coronary disease, and cancer, but has led to controversy among researchers, largely due to the complexity of the process. This paper reviews the recent literature on the genomic and non-genomic effects of GCs on WAT and BAT lipolysis and proposes research to address the many gaps in knowledge related to GC activity and its effects on disease.

Nongenomic glucocorticoid effects and their mechanisms of action in vertebrates.

Glucocorticoids (GC) act on multiple organ systems to regulate a variety of physiological processes in vertebrates. Due to their immunosuppressive and anti-inflammatory actions, glucocorticoids are an attractive target for pharmaceutical development. Accordingly, they are one of the most widely prescribed classes of therapeutics. Through the classical mechanism of steroid action, glucocorticoids are thought to mainly affect gene transcription, both in a stimulatory and suppressive fashion, regulating de novo protein synthesis that subsequently leads to the physiological response. However, over the past three decades multiple lines of evidence demonstrate that glucocorticoids may work through rapid, nonclassical mechanisms that do not require alterations in gene transcription or translation. This review assimilates evidence across the vertebrate taxa on the diversity of nongenomic actions of glucocorticoids and the membrane-associated cellular mechanisms that may underlie rapid glucocorticoid responses to include potential binding sites characterized to date.

Further evidence for a membrane receptor that binds glucocorticoids in the rodent hypothalamus

In parallel with their well-characterized delayed genomic effects, steroid hormones exhibit rapid, non-genomic effects at molecular, cellular and behavioral levels. We have proposed a model of rapid, non-genomic glucocorticoid inhibition of hypothalamic neuroendocrine cells through a putative membrane-associated glucocorticoid receptor (GR). Here we tested for plasma membrane GR immunoreactivity and binding in the hypothalamic supraoptic and paraventricular nuclei. Selective cross-linking of membrane proteins with membrane-impermeant BS3 and subsequent Western blot analysis with a monoclonal GR antibody revealed a reduction in the intensities of a ∼98kDa immunoreactive band and a ∼64kDa band in the rat paraventricular and supraoptic nuclei, and of a 64kDa band in hippocampal tissue, which suggested that these proteins are associated with the membrane. Saturation binding of [3H]-corticosterone and [3H]-dexamethasone in rat and mouse hypothalamic tissue revealed a Kd 4–24-fold lower and a Bmax 4–7-fold lower for the membrane-associated GR compared to the intracellular GR, suggesting a lower affinity and abundance of the glucocorticoid binding sites in the membrane than in the cytosol. Together, these findings suggest the presence of a low-affinity, low-abundance membrane-associated GR in the hypothalamus that shares homology with the intracellular GR, and are consistent with physiological evidence of rapid, non-genomic glucocorticoid actions in hypothalamic neuroendocrine cells that are GR dependent.

Dexamethasone rapidly inhibits glucose uptake via non-genomic mechanisms in contracting myotubes

Glucocorticoids (GCs) are a class of steroid hormones that regulate multiple aspects of glucose homeostasis. In skeletal muscle, it is well established that prolonged GC excess inhibits glucose uptake and utilization through glucocorticoid receptor (GR)-mediated transcriptional changes. However, it remains obscure that whether the rapid non-genomic effects of GC on glucose uptake are involved in acute exercise stress. Therefore, we used electric pulse stimulation (EPS)-evoked contracting myotubes to determine whether the non-genomic actions of GC were involved and its underlying mechanism(s). Pretreatment with dexamethasone (Dex, 10 μM) significantly prevented contraction-stimulated glucose uptake and glucose transporter 4 (Glut4) translocation within 20 min in C2C12 myotubes. Neither GC nuclear receptor antagonist (RU486) nor protein synthesis inhibitor (cycloheximide, Chx) affected the rapid inhibition effects of Dex. AMPK and CaMKII-dependent signaling pathways were associated with the non-genomic effects of Dex. These results provide evidence that GC rapidly suppresses glucose uptake in contracting myotubes via GR-independent non-genomic mechanisms. AMPK and CaMKII-mediated Glut4 translocation may play a critical role in GC-induced rapid inhibition of glucose uptake.

Hypothalamic Nuclear Glucocorticoid Receptors: Acute Stress and Rapid Actions.

Stress, be it physical or psychological, causes the activation of a neuroendocrine cascade that involves both central and peripheral mechanisms. In the simplest terms, central release of CRH from the paraventricular nucleus of thehypothalamus (PVN)andsubsequent releaseofACTH from the pituitary result in the peripheral release of glucocorticoids (cortisol in primates and corticosterone in many rodent species) from the adrenal gland (1). Glucocorticoids then act via negative feedback both centrally and peripherally through the glucocorticoid receptor (GR) to inhibit the further release of CRH from the PVN and ACTH from the pituitary (2). This process is a delicate balance that helps restore homeostasis, but perturbation of these responses can lead to neurobehavioral and physiological dysfunctions, including mood disorders and obesity (3–5). Hence, understanding the basic mechanisms that mediate this neuroendocrine loop has broad implications for both health and disease. Two companion papers in this issue by Solomon et al (6) and Nahar et al (7) have helped to further reveal the mechanisms through which glucocorticoids influence hypothalamic-pituitary-adrenal (HPA) function. Both reports use a powerful mouse model in which Sim1-cre recombinase transgenic mice are bred with homozygous floxed GR mice. Sim1 is a transcription factor specifically expressed in the PVN, supraoptic nucleus, and periventricular nucleus (8) and is responsible for the proper maturation of neurosecretory cells in the PVN (9). As Solomon et al report, theoffspring fromthis cross showsignificantly reduced GR levels in the PVN while leaving GR levels intact in the pituitary and other brain regions important in glucocorticoid-dependent negative feedback (6), thus resulting in a relatively anatomically restricted GR reduction. Using these PVN GR knockdown (KD) mice, Solomon et al (6) find sex-specific and stressor-dependent effects on HPA reactivity. In particular, they show GR KD mice respond to a brief 30-minute session of restraint stress with significantly increased and prolonged ACTH and corticosterone responses compared with their littermate controls, suggesting reduced negative feedback on the HPA axis (6). Notably, however, these effects of GR KD were observed only in males because female GR KD mice show reduced restraint-induced ACTH and no differences in stress-induced corticosterone responsiveness. In response to chronic variable stress, neither male nor female GR KD mice show any differences in HPA reactivity. Hence, the authors conclude that there are significant sex differences in the role that PVN GR plays in response to acute stress and that changes in HPA reactivity after chronic stress in male and female mice are independent of GR in the PVN (6). Complimenting this line of research, the report by Nahar et al (7) investigated rapid nongenomic actions of glucocorticoids in modulating excitatory and inhibitory transmission in the mouse hypothalamus. They show that hypothalamic magnocellular neurons respond to dexamethasone (DEX), a synthetic glucocorticoid, with a rapid suppression of excitatory postsynaptic inputs (ie, glutamatergic synapses). The authors further demonstrate that this effect of DEX is mediated by postsynaptic membrane associated receptors and G protein signaling (7), similar to their seminal work in rats (10–12). The authors also show that DEX leads to a rapid nitric oxide-dependent increase in inhibitory postsynaptic inputs [ie, -aminobutyric acid

Rapid Nongenomic Glucocorticoid Actions in Male Mouse Hypothalamic Neuroendocrine Cells Are Dependent on the Nuclear Glucocorticoid Receptor.

Corticosteroids act classically via cognate nuclear receptors to regulate gene transcription; however, increasing evidence supports rapid, nontranscriptional corticosteroid actions via activation of membrane receptors. Using whole-cell patch clamp recordings in hypothalamic slices from male mouse genetic models, we tested for nongenomic glucocorticoid actions at glutamate and gamma aminobutyric acid (GABA) synapses in hypothalamic neuroendocrine cells, and for their dependence on the nuclear glucocorticoid receptor (GR). In enhanced green fluorescent protein-expressing CRH neurons of the paraventricular nucleus (PVN) and in magnocellular neurons of the PVN and supraoptic nucleus (SON), dexamethasone activated postsynaptic membrane-associated receptors and G protein signaling to elicit a rapid suppression of excitatory postsynaptic inputs, which was blocked by genetic deletion of type I cannabinoid receptors and a type I cannabinoid receptor antagonist. In magnocellular neurons, dexamethasone also elicited a rapid nitric oxide-dependent increase in inhibitory postsynaptic inputs. These data indicate a rapid, synapse-specific glucocorticoid-induced retrograde endocannabinoid signaling at glutamate synapses and nitric oxide signaling at GABA synapses. Unexpectedly, the rapid glucocorticoid effects on both excitatory and inhibitory synaptic transmission were lost with conditional deletion of GR in the PVN and SON in slices from a single minded-1-cre-directed conditional GR knockout mouse. Thus, the nongenomic glucocorticoid actions at glutamate and GABA synapses on PVN and SON neuroendocrine cells are dependent on the nuclear GR. The nuclear GR, therefore, is responsible for transducing the rapid steroid response at the membrane, or is either a critical component in the signaling cascade or regulates a critical component of the signaling cascade of a distinct membrane GR.

Novel aspects of glucocorticoid actions.

Normal hypothalamic-pituitary-adrenal (HPA) axis activity leading to the rhythmic and episodic release of adrenal glucocorticoids (GCs) is essential for body homeostasis and survival during stress. Acting through specific intracellular receptors in the brain and periphery, GCs regulate behaviour, as well as metabolic, cardiovascular, immune and neuroendocrine activities. By contrast to chronic elevated levels, circadian and acute stress-induced increases in GCs are necessary for hippocampal neuronal survival and memory acquisition and consolidation, as a result of the inhibition of apoptosis, the facilitation of glutamatergic neurotransmission and the formation of excitatory synapses, and the induction of immediate early genes and dendritic spine formation. In addition to metabolic actions leading to increased energy availability, GCs have profound effects on feeding behaviour, mainly via the modulation of orexigenic and anorixegenic neuropeptides. Evidence is also emerging that, in addition to the recognised immune suppressive actions of GCs by counteracting adrenergic pro-inflammatory actions, circadian elevations have priming effects in the immune system, potentiating acute defensive responses. In addition, negative-feedback by GCs involves multiple mechanisms leading to limited HPA axis activation and prevention of the deleterious effects of excessive GC production. Adequate GC secretion to meet body demands is tightly regulated by a complex neural circuitry controlling hypothalamic corticotrophin-releasing hormone (CRH) and vasopressin secretion, which are the main regulators of pituitary adrenocorticotrophic hormone (ACTH). Rapid feedback mechanisms, likely involving nongenomic actions of GCs, mediate the immediate inhibition of hypothalamic CRH and ACTH secretion, whereas intermediate and delayed mechanisms mediated by genomic actions involve the modulation of limbic circuitry and peripheral metabolic messengers. Consistent with their key adaptive roles, HPA axis components are evolutionarily conserved, being present in the earliest vertebrates. An understanding of these basic mechanisms may lead to novel approaches for the development of diagnostic and therapeutic tools for disorders related to stress and alterations of GC secretion.

Differentiation between genomic and non-genomic feedback controls yields an HPA axis model featuring Hypercortisolism as an irreversible bistable switch

BackgroundThe hypothalamic-pituitary-adrenal axis (HPA axis) is a major part of the neuroendocrine system responsible for the regulation of the response to physical or mental stress and for the control of the synthesis of the stress hormone cortisol. Dysfunctions of the HPA axis characterized by either low (hypocortisolism) or increased (hypercortisolism) cortisol levels are implicated in various pathological conditions. Their understanding and therapeutic correction may be supported by mathematical modeling and simulation of the HPA axis.MethodsMass action and Michaelis Menten enzyme kinetics were used to provide a mechanistic description of the feedback mechanisms within the pituitary gland cells by which cortisol inhibits its own production. A separation of the nucleus from the cytoplasm by compartments enabled a differentiation between slow genomic and fast non-genomic processes. The model in parts was trained against time resolved ACTH stress response data from an in vitro cell culture of murine AtT-20 pituitary tumor cells and analyzed by bifurcation discovery tools.ResultsA recently found pituitary gland cell membrane receptor that mediates rapid non-genomic actions of glucocorticoids has been incorporated into our model of the HPA axis. As a consequence of the distinction between genomic and non-genomic feedback processes our model possesses an extended dynamic repertoire in comparison to existing HPA models. In particular, our model exhibits limit cycle oscillations and bistable behavior associated to hypocortisolism but also features a (second) bistable switch which captures irreversible transitions in hypercortisolism to elevated cortisol levels.ConclusionsModel predictive control and inverse bifurcation analysis have been previously applied in the simulation-based design of therapeutic strategies for the correction of hypocortisolism. Given the HPA model extension presented in this paper, these techniques may also be used in the study of hypercortisolism. As an example, we show how sparsity enforcing penalization may suggest network interventions that allow the return from elevated cortisol levels back to nominal ones.

Why do we need nongenomic glucocorticoid mechanisms?

Glucocorticoids (GCs) are a class of steroid hormones that have been known to be involved in various physiological processes and to play a pivotal role in preserving basal and stress-related homeostasis. GCs are also widely used clinically as anti-inflammatory, immunosuppressive, anti-shock drugs. It is believed traditionally that GCs exert most of their effects genomically. In addition to the well-known classical genomic mechanisms, GCs also affect various functions via rapid, nongenomic mechanisms. The therapeutic benefits of nongenomic GC actions have been exploited in clinical medicine, especially with high-dose pulsed glucocorticoid administration. However, it is certainly not the case that the inherent nongenomic glucocorticoid mechanisms evolved only for their clinical utility. Here, we review the recent literature on nongenomic actions of GCs related to stress and the physiological significance of these actions, and we propose reasons why nongenomic mechanisms of GC actions are needed.

Non-genomic effect of glucocorticoids on cardiovascular system

Glucocorticoids (GCs) are essential steroid hormones for homeostasis, development, metabolism, and cognition and possess anti-inflammatory and immunosuppressive actions. Since glucocorticoid receptor II (GR) is nearly ubiquitous, chronic activation or depletion of GCs leads to dysfunction of diverse organs, including the heart and blood vessels, resulting predominantly from changes in gene expression. Most studies, therefore, have focused on the genomic effects of GC to understand its related pathophysiological manifestations. The nongenomic effects of GCs clearly differ from well-known genomic effects, with the former responding within several minutes without the need for protein synthesis. There is increasing evidence that the nongenomic actions of GCs influence various physiological functions. To develop a GC-mediated therapeutic target for the treatment of cardiovascular disease, understanding the genomic and nongenomic effects of GC on the cardiovascular system is needed. This article reviews our current understanding of the underlying mechanisms of GCs on cardiovascular diseases and stress, as well as how nongenomic GC signaling contributes to these conditions. We suggest that manipulation of GC action based on both GC and GR metabolism, mitochondrial impact, and the action of serum- and glucocorticoid-dependent kinase 1 may provide new information with which to treat cardiovascular diseases.

ZAP-70 tyrosines 315 and 492 transmit non-genomic glucocorticoid (GC) effects in T cells

ZAP-70 kinase is a key regulator of early T-cell signaling; moreover, it also participates in non-genomic glucocorticoid (GC) signaling. Short-term high-dose GC-analogue treatment induces the phosphorylation of the kinase, and its association with the GC receptor (GR).In the present work, first, we identified those tyrosine (Y) residues of the ZAP-70 kinase which were involved in non-genomic GC signaling using an array of P116 cells (ZAP-70-deficient Jurkat subclone) lentivirally-transfected with wild type or point-mutated ZAP-70 constructs where Y-residues were replaced with phenylalanine (F) at positions 069, 126, 178, 238, 292, 315, 492 or 493. Then, we characterized the GC-analogue-induced Y-phosphorylation of 3 key substrates of the ZAP-70 kinase: SLP-76, LAT and Cbl. Finally, we studied the cross talk between the non-genomic GC- and TcR/CD3 signaling pathways.Y-F mutations at positions 315 or 492 abolished the short high-dose Dexamethasone (DX) treatment-induced ZAP-70 phosphorylation suggesting that these Y-residues were involved in ZAP-70-mediated non-genomic GC actions. DX treatment alone induced Y-phosphorylation of LAT, SLP-76 and Cbl; moreover, in F315- and F492-ZAP-70 mutated cells decreased DX-induced Y-phosphorylation of SLP-76 and Cbl was observed indicating that these molecules might transmit downstream non-genomic GC signals in a ZAP-70 dependent manner. Short, high dose DX treatment influenced significantly the anti-CD3-induced signaling events: we observed alterations in LAT, SLP-76 and Cbl Y-phosphorylation and a decreased Ca2+-signal.These results confirm that ZAP-70 represents an important link between the non-genomic GC and TcR/CD3 signaling pathways. Importantly, the DX-induced effects on resting and activated T-cells are differentially mediated. These fine molecular details help to better understand the complex mechanism of non-genomic GC effects in T-cells.

Molecular mechanisms of glucocorticoids action: implications for treatment of rhinosinusitis and nasal polyposis

Intra-nasal glucocorticoids are the most effective drugs available for rhinosinusitis and nasal polyposis treatment. Their effectiveness depends on many factors and not all of them have been well recognized so far. The authors present the basic information on molecular mechanisms of glucocorticoid action, direct and indirect effects of glucocorticoids on transcription of genes encoding inflammatory mediators. They focus on recently proved nongenomic mechanisms which appear quickly, from several seconds to minutes after glucocorticoid administration and discuss clinical implications resulting from this knowledge. Discovery of nongenomic glucocorticoid actions allows for better use of these drugs in clinical practice.

Genomic and non-genomic actions of glucocorticoids in asthma

Glucocorticoids are the mainstay of asthma therapy. They are primarily used to suppress airway inflammation, which is the central pathological change in asthmatic patients’ airways. This is achieved by many different mechanisms. The classical mechanism is by suppression of the genetic transcription of many inflammatory cytokines that are key in asthma pathophysiology (transrepression). On the other hand, the transcription of certain inhibitory cytokines is activated by glucocorticoids (transactivation), a mechanism that also mediates many of the adverse effects of glucocorticoids. The onset of action through these mechanisms is often delayed (4-24 hours). Other mechanisms mediated through non-genomic pathways are increasingly appreciated. These are delivered in part by binding of glucocorticoids to nonclassical membrane-bound glucocorticoid receptors or by potentiating the α1-adrenergic action on the bronchial arterial smooth muscles, in addition to other mechanisms. These effects are characterized by their rapid onset and short duration of action. Understanding these different mechanisms will help in the development of new and better drugs to treat this common disease and to develop new improved strategies in our approach to its management. Here, the genomic and non-genomic mechanisms of actions of glucocorticoids in asthma are briefly reviewed, with special emphasis on the current updates of the non-genomic mechanisms.

Non-genomic glucocorticoid effects to provide the basis for new drug developments

Glucocorticoids act via genomic and non-genomic actions. The genomic glucocorticoid actions are well known and new details on processes of transactivation and transrepression have been reported recently. Here we describe the current knowledge on non-genomic glucocorticoid actions and discuss why these actions are considered to be of therapeutic relevance. It is assumed that rapid non-genomic glucocorticoid effects are mediated by three different mechanisms: (1) physicochemical interactions with cellular membranes (non-specific non-genomic effects); (2) membrane-bound glucocorticoid receptor (mGCR)-mediated non-genomic effects; and (3) cytosolic glucocorticoid receptor (cGCR)-mediated non-genomic effects. With regard to the first mechanism, we discuss here lazaroids and the novel development of drug targeting with liposomes as the carrier system for glucocorticoids. The clinical use of the latter two mechanisms is still speculative, but intriguing ideas are being discussed in this regard.

G-protein-coupled glucocorticoid receptors on the pituitary cell membrane

Rapid, nongenomic actions of glucocorticoids (GCs) have been well documented, but information about putative membrane receptors that mediate them is scarce. We used fluorescence correlation spectroscopy to search for membrane GC-binding on the mouse pituitary cell line AtT-20. A slowly diffusing fraction (tau3; diffusion constant 3x10(-10) cm2 s-1) of fluorescein-labeled dexamethasone on the cell membrane corresponds to fluorescein-dexamethasone binding. Preincubation experiments were performed to test binding specificity: a 500-fold excess of unlabeled dexamethasone abolished subsequent fluorescein-dexamethasone membrane binding from 58+/-2 (control) to 8+/-1 (% of tau3, mean+/-s.e.m.), the natural ligand corticosterone prevented it partially (29+/-2), while the sex steroids estradiol (56+/-4) and progesterone (50+/-4) and the GC-receptor antagonist RU486 (56+/-2) had no effect. Preincubation with pertussis toxin resulted in disappearance of the slowest diffusion component (11+/-4) suggesting association of the receptor with a G-protein. Varying the concentration of fluorescein-dexamethasone showed that membrane binding is highly cooperative with an apparent Kd of 180 nM and Bmax of 230 nM. Taken together, these results demonstrate high-affinity GC-binding on the cell membrane of AtT-20 cells with characteristics distinct from intracellular binding.

Aktueller Stand zur Entwicklung neuer Glucocorticoidrezeptorliganden

Recent insights into the mechanisms of genomic and non-genomic glucocorticoid actions have stimulated the search for novel glucocorticoid receptor ligands. These efforts are driven by the need to improve the benefit-risk ratio of these important drugs. Glucocorticoids are very frequently and successfully used drugs which mediate important immunosuppressive and anti-inflammatory effects, but unfortunately they have pleiotropic effects causing a number of adverse reactions which limit their clinical use, especially at higher dosages and for longer periods. For this reason, novel glucocorticoid receptor ligands are being developed, among them selective glucocorticoid receptor agonists (SEGRAs). SEGRAs are drugs that predominantly induce transrepression effects (inhibition of protein synthesis), whereas the transactivation activity (induction of protein synthesis) is significantly reduced as compared with conventional glucocorticoid drugs. This makes sense since it became evident over the last few years that many adverse effects are predominantly caused by the transactivation mechanism, whereas anti-inflammatory effects are mostly mediated by transrepression mechanisms. Other interesting examples for exciting new developments are NO-glucocorticoids and long-circulating liposomal glucocorticoids. It is, however, true of all these developments that further in vivo and in vitro investigations and clinical trials will have to define in more detail their safety-efficacy profile in order to answer the questions whether these drugs as "improved glucocorticoids" will enter clinical medicine in the near future.

The membrane glucocorticoid receptor on the pituitary is G protein coupled and interacts with the cytoskeleton

Background: While nongenomic glucocorticoid actions like fast feedback inhibition of ACTH release are well documented, only very little is known about possible receptors mediating them. We recently identified specific, positive-cooperative glucocorticoid binding sites on the cell membrane of the ACTH-producing pituitary cell line AtT-20. The bound dexamethasone molecules showed a high degree of mobility restriction suggesting tighter association e.g. with the cytoskeleton. Additionally, two fractions of fast (freely diffusing ligand) and intermediate mobility (diffusion on/inside the cell) were found. The aim of the present study was to further characterize these binding sites on living cells in relation to the intracellular glucocorticoid receptor (iGR), the actin cytoskeleton (which is involved in ACTH release) and the possible involvement of a G protein.

Mechanisms and clinical relevance of nongenomic glucocorticoid actions

Glucocorticoids have profound anti-inflammatory and immunosuppressive actions when used therapeutically. The therapeutic dose is quite variable and depends on the disease, but ranges from very low to extremely high. The rationale for the use of various dosage regimens for specific clinical indications is the existence of three distinct, therapeutically relevant effects: genomic, specific nongenomic and unspecific nongenomic. Genomic effects are mediated by cytosolic receptors that alter expression of specific genes. Specific nongenomic effects occur within a few minutes and are mediated by steroid-selective membrane receptors. Unspecific nongenomic effects occur within seconds, but only at high glucocorticoid dosages, and seem to result from direct interactions with biological membranes. For unspecific nongenomic effects, methylprednisolone and other glucocorticoids have been shown to inhibit cation cycling across the plasma membrane, but to have little effect on protein synthesis. Thus, glucocorticoids could diminish or prevent the acute immune response by interfering with processes such as the rise in intracellular Ca2+ concentration. It is proposed that the additional therapeutic benefit of higher doses is obtained via these nongenomic effects.