Elisabeth Falkenstein

Rapid, nongenomic steroid actions: A new age?

In the traditional theory of steroid action, steroids bind to intracellular receptors and modulate nuclear transcription after translocation of steroid-receptor complexes into the nucleus. Due to similarities of molecular structure, specific receptors for steroids, vitamin D(3) derivatives, and thyroid hormone are considered to represent a superfamily of steroid receptors. While genomic steroid effects characterized by their delayed onset of action and their sensitivity to blockers of transcription and protein synthesis have been known for several decades, rapid actions of steroids have been more widely recognized and characterized in detail only recently. Rapid effects of steroids, thyroid hormones, and the steroid hormone metabolite of vitamin D(3), 1alpha, 25-dihydroxyvitamin D(3), on cellular signaling and function may be transmitted by specific membrane receptors. Binding sites in membranes have been characterized, exposing binding features compatible with an involvement in rapid steroid signaling. Characteristics of putative membrane receptors are completely distinct from intracellular steroid receptors, a fact which is further supported by the inability of classic steroid receptor antagonists to block nongenomic steroid actions. A putative progesterone membrane receptor has been cloned and functionally expressed with regard to progesterone binding. Development of drugs that specifically affect nongenomic action alone or even both modes of action may find applications in various, areas such as in the cardiovascular and central nervous systems and treatment of preterm labor, infertility, and electrolyte abnormalities.

Aldosterone, Not Estradiol, Is the Physiological Agonist for Rapid Increases in cAMP in Vascular Smooth Muscle Cells

BACKGROUND Steroid-induced gene regulation in the endocrine tissues and vascular wall is achieved through the interaction of specific receptor proteins and promoters of target genes. In addition to these delayed steroid actions, rapid effects of steroids have been reported in various tissues that were clearly incompatible with the classic theory of genomic steroid action. METHODS AND RESULTS Because high doses of 17beta-estradiol have been shown to modulate intracellular cAMP levels in vascular smooth muscle cells, steroid-induced stimulation of adenylate cyclase stimulation and phosphorylation of cAMP response element binding protein was investigated in porcine coronary artery vascular smooth muscle cells. Aldosterone induces a approximately 1.5- to 2.5-fold increase in intracellular cAMP levels (EC50 approximately 0.01 to 0.1 nmol/L) within 1 minute, whereas 17beta-estradiol and hydrocortisone act only at supraphysiological concentrations (10 micromol/L). Aldosterone-induced changes in intracellular cAMP are calcium dependent; they are not blocked by inhibitors of mineralocorticoid receptors, transcription, or protein synthesis. In addition, aldosterone induces a time-dependent phosphorylation of cAMP response element binding protein with potential transcriptional importance. CONCLUSIONS A nongenomic modulation of vascular smooth muscle cells by aldosterone is consistent with the data that aldosterone, not estrogen, is the physiological stimulus for cAMP.

Nongenomic effects of aldosterone: cellular aspects and clinical implications.

Nongenomic action of aldosterone has been observed in many cell types which often are different from the classic target tissues for mineralocorticoid action, such as vascular cells. As judged from their time scale and insensitivity toward inhibitors of protein synthesis, effects are not mediated by the classic mineralocorticoid receptor pathway. Here we summarize studies on rapid in vitro aldosterone effects, e.g. ion fluxes, and second messengers involved therein. Furthermore, several clinical studies on in vivo aldosterone action have shown rapid effects on cardiovascular parameters, among them baroreflex and vascular resistance. Taken together with the beneficial effect of aldosterone antagonism in heart failure patients that was demonstrated in the Randomized Aldactone Evaluation Study (RALES), aldosterone may be an equally important factor of the renin-angiotensin-aldosterone system in cardiovascular pathogenesis.

Nongenomic steroid actions : Fact or fantasy ?

In the common theory of steroid action, steroids bind to intracellular receptors and modulate nuclear transcription after translocation of steroid--receptor complexes into the nucleus. Due to homologies of molecular structure, specific receptors for steroids, vitamin D3, and thyroid hormone are considered to represent a superfamily of steroid receptors. While genomic steroid effects being characterized by their delayed onset of action and their sensitivity to blockers of transcription and protein synthesis have been known for several decades, very rapid actions of steroids have been more widely recognized and characterized in detail only recently. Rapid effects of steroids, vitamin D3, and thyroid hormones on cellular signaling and function may be transmitted by specific membrane receptors. Although no receptor of this kind has been cloned up to now, binding sites in membranes have been characterized exposing binding features compatible with an involvement in rapid steroid signaling. Characteristics of putative membrane receptors were completely different from those of intracellular steroid receptors, which was further supported by the inability of classic steroid receptor antagonists to inhibit nongenomic steroid actions. Development of drugs that specifically affect nongenomic action alone or even both modes of actions may find applications in various areas such as the cardiovascular and central nervous systems and treatment of preterm labor, infertility, and electrolyte homeostasis. To acquaint the reader with major aspects of nongenomic steroid actions, these effects on cellular function will be summarized, potentially related binding sites in membranes discussed, and the physiological or pathophysiological relevance of nonclassic actions exemplified.

Recognition of a human sperm surface protein involved in the progesterone-initiated acrosome reaction by antisera against an endomembrane progesterone binding protein from porcine liver.

Antisera against a porcine liver endomembrane progesterone (P4)-binding protein inhibited the P4-initiated acrosome reaction (AR) but not the ionomycin-initiated AR of human sperm. Indirect immunofluorescence studies detected antigen in the sperm head that moved during capacitation from a posterior head region to a midhead region. Moreover, the antisera detected a 44.6 kDa protein in western blots of sperm digitonin extracts. These results suggest that a sperm protein with at least partial homology to the liver endomembrane P4-binding protein, is a putative P4-receptor on the sperm plasma membrane involved in the P4-initiated AR.

Are high-affinity progesterone binding site(s) from porcine liver microsomes members of the σ receptor family?

Membrane progesterone binding sites have been purified recently from pig liver. Since progesterone is considered as an endogenous sigma (sigma) receptor ligand, these sites were characterized pharmacologically by ligands selective for sigma receptor and dopamine receptor binding sites, and by other drugs from distinct pharmacological classes. Binding studies using the radioligand [3H]progesterone were done in crude membrane preparations and solubilized fractions to determine half-maximal inhibitory concentration (IC50) values, from which inhibitory constants (Ki values) were calculated. Radioligand binding was inhibited by the sigma receptor ligands haloperidol, carbetapentane citrate, 1,3-Di(2-tolyl)guanidine (DTG), R(-)-N-(3-phenyl-1-propyl)-1-phenyl-2 aminopropane HCl (R(-)-PPAAP HCl), or sigma receptor antagonists like (+)-3-(3-hydroxyphenyl)-N-propylpiperidine HCl (R(+)-PPP HCl) and cis-9-[3-(3,5-dimethyl-1-piperazinyl)propyl]-9H-carbazole dihydrochloride (rimcazole 2HCl). The hierarchy of inhibitory action was not fully compatible with either sigma receptor class I (moderate affinity of pentazocine, diphenylhydantoin (phenytoin) insensitivity) or II sites (high affinity of carbetapentane). The data thus suggest that progesterone binding sites in porcine liver membranes are related to the sigma receptor binding site superfamily, but may represent a particular species with progesterone specificity.

Chemical modification and structural analysis of the progesterone membrane binding protein from porcine liver membranes

In addition to the classical genomic steroid actions on modulation of transcription and protein synthesis, rapid, nongenomic effects have been described for various steroids. These effects on cellular signaling and function are supposed to be transmitted by membrane binding sites unrelated to the classical intracellular receptors. Recently, a high affinity progesterone membrane binding protein (mPR) has been characterized in porcine liver membranes. In the present study, amino acid residues that are essential for progesterone binding to porcine liver microsomal mPR have been identified by the use of protein modifying reagents. Among all reagents tested, agents with specificity for carboxyl groups, methionine and tryptophan such as N,N′-dicyclohexylcarbodiimide, chloramine T and N-bromosuccinimide induced a reduction in [3H]progesterone binding. To evaluate the presence of essential disulfide bridges, porcine liver microsomes were incubated with the disulfide reducing agent dithiothreitol (DTT) and [3H]progesterone binding was measured. This treatment also resulted in a reduction of binding activity with an IC50 of 20 mM for DTT. Western-blotting analysis in the presence or absence of the reducing agent suggested that mPR – in its binding state – consists of at least two identical subunits with an apparent molecular mass of 28 kDa which are linked by a disulfide bridge. In conclusion, in the present study evidence for an involvement of carboxyl-, tryptophan- and methionine residues in [3H]progesterone binding to porcine liver microsomes is given. In addition, it is shown that mPR can form disulfide-linked homodimers.

The Human Membrane Progesterone Receptor Gene: Genomic Structure and Promoter Analysis

Rapid, nongenomic effects of steroids are likely to be mediated by membrane receptors not by intracellular steroid receptors. We recently identified a progesterone membrane binding protein (mPR) from human liver. The corresponding hmpr gene is comprised of 3 exons and 2 introns. The promoter sequence of hmpr lacks a typical TATA box but contains instead a high homology to a transcription Initiatior consensus sequence, which overlaps the experimentally determined transcriptional start site. The major proximal promoter is GC-rich and sequence analysis revealed a CpG island spanning the transcriptional start site. Several putative cis-regulatory DNA-motifs, which represent possible binding sites for transcription factors like AP2, NF-AT, Ahr/Arnt and C/EBP were identified in the genomic upstream region by sequence homology. Functional analysis of differently deleted fragments of the hmpr upstream region in a GFP-reportergene assay in transiently transfected cultured cells indicates the general testability of the hmpr promoter in vivo.

Beeinflussung der Arzneimittelwirkung durch Erbfaktoren und Erkrankungen

Zum ThemaDer genetische Polymorphismus zahlreicher Enzyme bewirkt deutliche interindividuelle und interethnische Unterschiede bei der Metabolisierung von Pharmaka, die durchaus von klinischer Relevanz sein können. Neben unerwünschten Arzneimittelwirkungen kann ein rascherer oder langsamerer Metabolismus zu ernsthaften Erkrankungen bis hin zur Entwicklung von Karzinomen führen. Die Auswirkungen dieser genetischen Variabilität, die sich mittels Genotypisierung darstellen lässt, werden in diesem Beitrag am Beispiel eines Isoenzyms des Cytochrom P450-Systems und der N-Acetyltransferase eingehend dargestellt. Auch verschiedene Erkrankungen können sich auf die Pharmakokinetik und Pharmakodynamik von Medikamenten auswirken. Besonders bei älteren, multimorbiden Patienten werden diese Interaktionen häufig nicht ausreichend berücksichtigt. Die Auswirkungen von Leberfunktionsstörungen und Niereninsuffizienz werden daher nachfolgend ausführlicher erläutert.

Novel, Membrane-Intrinsic Receptors for Progesterone and Aldosterone

In 1972, binding of [3H] aldosterone to plasma membranes from rat kidney with Kd ~ 100 nM was reported [1]. In a similar system, solubilization of binding activity was performed, but no further identification had been achieved [2]. Apart from these studies in the classical mineralocorticoid target organ, Armanini et al. [3] reported specific high-affinity binding of [3H] aldosterone to mononuclear leukocytes. The dissociation constant Kd was 2.7 nM with a binding site density of 209 sites per cell. The order of binding affinities was aldosterone ≈ deoxycorticosterone ≈ corticosterone > hydrocortisone > dexamethasone, which differs from the selectivity profile of the classic nuclear mineralocorticoid receptor (MR). Microsomal membranes from mononuclear leukocytes were subjected to photochemical crosslinking with the radioligand aldosterone-3-(0-carboxymethyl)-oximino-(2-[125I]iodohistamine) in the presence and absence of an excess of unlabeled aldosterone. After SDS electrophoresis, a prominent maximum was visible at ~50 kDa which could be displaced by aldosterone, but not Cortisol [4]. Later, plasma membrane preparations from porcine kidney were studied with regard to non-classical receptors.