Anita Aperia

Anatomical and physiological evidence for D 1 and D 2 dopamine receptor colocalization in neostriatal neurons

Despite the importance of dopamine signaling, it remains unknown if the two major subclasses of dopamine receptors exist on the same or distinct populations of neurons. Here we used confocal microscopy to demonstrate that virtually all striatal neurons, both in vitro and in vivo, contained dopamine receptors of both classes. We also provide functional evidence for such colocalization: in essentially all neurons examined, fenoldopam, an agonist of the D1 subclass of receptors, inhibited both the Na+/K+ pump and tetrodotoxin (TTX)-sensitive sodium channels, and quinpirole, an agonist of the D2 subclass of receptors, activated TTX-sensitive sodium channels. Thus D1 and D2 classes of ligands may functionally interact in virtually all dopamine-responsive neurons within the basal ganglia.

Ouabain, a steroid hormone that signals with slow calcium oscillations.

The plant-derived steroid, digoxin, a specific inhibitor of Na,K-ATPase, has been used for centuries in the treatment of heart disease. Recent studies demonstrate the presence of a digoxin analog, ouabain, in mammalian tissue, but its biological role has not been elucidated. Here, we show in renal epithelial cells that ouabain, in doses causing only partial Na,K-ATPase inhibition, acts as a biological inducer of regular, low-frequency intracellular calcium ([Ca2+]i) oscillations that elicit activation of the transcription factor, NF-κB. Partial inhibition of Na,K-ATPase using low extracellular K+ and depolarization of cells did not have these effects. Incubation of cells in Ca2+-free media, inhibition of voltage-gated calcium channels, inositol triphosphate receptor antagonism, and redistribution of actin to a thick layer adjacent to the plasma membrane abolished [Ca2+]i oscillations, indicating that they were caused by a concerted action of inositol triphosphate receptors and capacitative calcium entry via plasma membrane channels. Blockade of ouabain-induced [Ca2+]i oscillations prevented activation of NF-κB. The results demonstrate a new mechanism for steroid signaling via plasma membrane receptors and underline a novel role for the steroid hormone, ouabain, as a physiological inducer of [Ca2+]i oscillations involved in transcriptional regulation in mammalian cells.

Why rare diseases are an important medical and social issue.

Rare diseases aff ect a limited number of individuals (defi ned as no more than one in 2000 individuals in the European Union and no more than about one in 1250 in the USA), but the number of disorders that fi t this defi nition is very large (>5000 according to WHO). Therefore, the number of patients aff ected by a rare disease could be about 30 million in Europe and 25 million in North America. The true burden of rare diseases in Europe and elsewhere is diffi cult to estimate, since epidemiological data for most of these diseases are not available. Rare diseases are an important public-health issue and a challenge for the medical community. They are called health orphans, because rare diseases were neglected for many years. Similarly, before the USA passed the Orphan Drug Act in 1983, the pharmaceutical industry had neglected the development of treatments for rare diseases, hence the name orphan drugs. Public awareness about the diffi culties of patients with rare diseases was fi rst raised by the report of the National Commission on Orphan Disease of the US Government in 1989. The Commission’s hearings with hundreds of stakeholders highlighted issues that aff ected patients’ care, such as little information on rare diseases, diffi culties of fi nancing research, drawbacks of providing adequate health insurance and coverage of medical expenses, and the limited availability of eff ective treatments. The International Classifi cation of Diseases (ICD) that is used in most countries is not convenient for rare diseases. The absence of a universally recognised coding system is an obstacle for reliable registration of patients in national or international databases, preventing assessment of the economic and social eff ects of rare diseases. For some disorders, national or international registries are available, which have been set up and maintained by researchers, patients’ associations, public institutions, or drug companies. The European Rare Disease Task Force of the Health and Consumers Protection Directorate General of the European Commission has set up a working group to collaborate with WHO on ICD-10, and is considering all other existing classifi cations to provide the rare-diseases community with a uniform system. Assessment of the prevalence of rare diseases was attempted by the European Organization for Rare Diseases (Eurordis), and Orphanet, with the support of the European Commission. This study not only provided an estimate of the prevalence of several rare diseases (table), but also showed the absence of reliable data, low consistency between sources of information, and poor methodological quality of epidemiological studies. Additionally, facilities for biochemical or genetic testing are scarce. We use the collective term of rare diseases to include a very heterogeneous group of disorders that can aff ect any system. Most rare diseases are genetic disorders, which are often severely disabling, substantially aff ect life expectancy, and impair physical and mental abilities. These disabilities result in reduced quality of life, and aff ect an individual’s potential for education and earning capabilities. One example is inborn errors of metabolism, most of which are rare (prevalence between 1 in 1400 and 1 in 5000 livebirths). An Italian prospective study (1985–97) on patients aged 0–17 years revealed that, of the 1935 newborn babies with inborn errors of metabolism identifi ed by the study, only 11% reached adulthood. Rare diseases also pose a considerable burden on the aff ected families. This burden was assessed by sending a questionnaire to 2500 patients with chronic diseases (8·2% of which were rare diseases). Patients with rare disorders had the worst exper ience in terms of loss of social and economic oppor tunities, and of medical care. Patients with rare diseases face diagnostic delays. This issue was shown in a survey of eight rare diseases (Crohn’s disease, cystic fi brosis, Duchenne muscular dystrophy, Ehlers-Danlos syndrome, Marfan’s syndrome, Prader-Willi syndrome, tuberous sclerosis, and Lancet 2008; 371: 2039–41

Activation/deactivation of renal Na+,K(+)-ATPase: a final common pathway for regulation of natriuresis.

Renal sodium metabolism, a major determinant of blood pressure, is regulated with great precision by a variety of endocrine, autocrine, and neuronal factors. Although these factors arc known to regulate sodium metabolism by affecting the rate of tubular sodium reabsorption, the molecular mechanisms by which they act are poorly understood, Na+,K+‐ATPase plays a pivotal role for sodium reabsorption in all tubular segments. The activity of this enzyme can be dynamically regulated by phosphorylation and dephosphorylation, Here we summarize both old and new evidence that several major substances believed to be involved in the regulation of sodium metabolism and blood pressure, i.e., the antidiuretic agents angiotensin II and norepinephrine, and the diuretic agents dopamine and atrial natriuretic peptide (ANP), may achieve their effects through a common pathway that involves reversible activation/deactivation of renal tubular Na+,K+‐ATPase. Regulation of Na+,K+‐ATPase activity was studied using a preparation of single proximal tubule (PT) segments, dissected from rat kidneys. Na+,K+‐ATPasc activity was stimulated by angiotensin II and the α‐adrenergic agonist, oxymetazoline, at physiological, nonsaturating Na+ concentrations. These stimulatory effects were blocked by dopamine and ANP as well as by their respective second messengers, cAMP and cGMP. They were also blocked by the specific protein phosphatase 2B inhibitor FK306, These results indicate that regulation of sodium excretion by norepinephrine, angiotensin II, dopamine, and ANP can be accounted for by a bidirectionally regulated intracellular protein phosphorylation cascade that modulates the activity of renal tubular Na+,K+‐ATPase.—Aperia, A., Holtbäck, U., Syrén, M.‐L., Svensson, L.‐B., Fryckstedt, J., Greengard, P. Activation deactivation of renal Na+,K+‐ATPase: a final common pathway for regulation of natriuresis. FASEB J. 8: 436‐439; 1994.

Alpha-haemolysin of uropathogenic E. coli induces Ca2+ oscillations in renal epithelial cells.

Pyelonephritis is one of the most common febrile diseases in children. If not treated appropriately, it causes irreversible renal damage and accounts for a large proportion of end stage renal failures. Renal scarring can occur in the absence of inflammatory cells, indicating that bacteria may have a direct signalling effect on renal cells. Intracellular calcium ([Ca2+]i) oscillations can protect cells from the cytotoxic effects of prolonged increases in intracellular calcium. However, no pathophysiologically relevant protein that induces such oscillations has been identified. Here we show that infection by uropathogenic Escherichia coli induces a constant, low-frequency oscillatory [Ca 2+]i response in target primary rat renal epithelial cells induced by the secreted RTX (repeats-in-toxin) toxin α-haemolysin. The response depends on calcium influx through L-type calcium channels as well as from internal stores gated by inositol triphosphate. Internal calcium oscillations induced by α-haemolysin in a renal epithelial cell line stimulated production of cytokines interleukin (IL)-6 and IL-8. Our findings indicate a novel role for α-haemolysin in pyelonephritis: as an inducer of an oscillating second messenger response in target cells, which fine-tunes gene expression during the inflammatory response.

POSTNATAL DEVELOPMENT OF RENAL FUNCTION IN PRE-TERM AND FULL-TERM INFANTS

ABSTRACT. Aperia, A., Broberger, B., Klinder, G., Herin, P. and Zetterström, R. (Department of Paediatrics, Karolinska Institute, St. Görans Childrens Hospital, Stockholm and Huddinge Hospital, Huddinge, Sweden). Postnatal deveopment of renal function in preterm and full‐term infants. Acta Paediatr Scand, 70:183, 1981. –This study has been designed to examine the effect of gestational age (GA) on the postnatal development of renal function and has been performed in pre‐term (PT) infants (GA=30–34 weeks) and in full‐term (FT) infants (GA=39–41 weeks). Postnatal age has ranged from 1–35 days. From 8 hour urine samples collected after spontaneous voiding and a capillary blood sample, determinations have been made of the clearance of creatinine (CCr), the fractional excretion of β2‐microglobulin (FEβ2) and the fractional excretion of sodium (FENa). In some infants receiving fluid parenterally, simultaneous determinations were made of the clearance of creatinine and inulin. As judged from this study, CCr is a reliable indicator of the glomerular filtration rate (GFR). GFR was almost the same in newborn PT and FT, but from 0.3–1 week of age GFR increased significantly more rapidly in FT than in PT. From 1–5 weeks of age GFR increased at approximately the same rate in PT and FT infants. The absolute value for GFR in 3–5 weeks old infants was lower in PT than in FT. FEβ2 was higher in PT than in FT infants during the entire first month of life and FENa was higher in PT than in FT infants during the first week of life, suggesting a glomerular tubular imbalance at least at the level of the proximal tubule in PT infants. It is concluded that different stages of maturation will alter the preconditions for the renal adaptation to extrauterine life during at least the first month of life. Therefore special attention must be paid to the limited renal function in PT during their entire first month of life.

Cell signaling microdomain with Na, K-ATPase and inositol 1,4,5-trisphosphate receptor generates calcium oscillations

Recent studies indicate novel roles for the ubiquitous ion pump, Na,K-ATPase, in addition to its function as a key regulator of intracellular sodium and potassium concentration. We have previously demonstrated that ouabain, the endogenous ligand of Na,K-ATPase, can trigger intracellular Ca2+ oscillations, a versatile intracellular signal controlling a diverse range of cellular processes. Here we report that Na,K-ATPase and inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) form a cell signaling microdomain that, in the presence of ouabain, generates slow Ca2+ oscillations in renal cells. Using fluorescent resonance energy transfer (FRET) measurements, we detected a close spatial proximity between Na,K-ATPase and InsP3R. Ouabain significantly enhanced FRET between Na,K-ATPase and InsP3R. The FRET effect and ouabain-induced Ca2+ oscillations were not observed following disruption of the actin cytoskeleton. Partial truncation of the NH2 terminus of Na,K-ATPase catalytic α1-subunit abolished Ca2+ oscillations and downstream activation of NF-κB. Ouabain-induced Ca2+ oscillations occurred in cells expressing an InsP3 sponge and were hence independent of InsP3 generation. Thus, we present a novel principle for a cell signaling microdomain where an ion pump serves as a receptor.

C-peptide stimulates rat renal tubular Na+, K+-ATPase activity in synergism with neuropeptide Y

SummaryThis study was performed in order to test the hypothesis that the connecting peptide of proinsulin, C-peptide, might in itself possess biological activity. Renal tubular Na+, K+-ATPase, which is a well-established target for many peptide hormones, was chosen as a model. Rat C-peptide (I) was found to stimulate Na+, K+-ATPase activity in single, proximal convoluted tubules dissected from rat kidneys. C-peptide increased the Na+ affinity of the enzyme and all subsequent studies were performed at non-saturating Na+ concentrations. C-peptide stimulation of Na+, K+-ATPase activity occurred in a concentration-dependent manner in the dose range 10−8–10−6 mol/l. The presence of neuropeptide Y, 5×10−9 mol/l, enhanced this effect and stimulation of Na+, K+-ATPase activity then occurred in the C-peptide dose range 10−11–10−8 mol/l. C-peptide stimulation of Na+, K+-ATPase activity was abolished in tubules pretreated with pertussis toxin. It was also abolished in the presence of FK 506, a specific inhibitor of the Ca2+-calmodulin-dependent protein phosphatase 2B. These results indicate that C-peptide stimulates Na+, K+-ATPase activity, probably by activating a receptor coupled to a pertussis toxin-sensitive G-protein with subsequent activation of Ca2+-dependent intracellular signalling pathways.

Selective up-regulation of dopamine D1 receptors in dendritic spines by NMDA receptor activation

Glutamate, by activating N-methyl-d-aspartate (NMDA) receptors, alters the balance between dopamine D1 and D2 receptor signaling, but the mechanism responsible for this effect has not been known. We report here, using immunocytochemistry of primary cultures of rat neostriatal neurons, that activation of NMDA receptors recruits D1 receptors from the interior of the cell to the plasma membrane while having no effect on the distribution of D2 receptors. The D1 receptors were concentrated in spines as shown by colocalization with phalloidin-labeled actin filaments. The effect of NMDA on D1 receptors was abolished by incubation of cells in calcium-free medium and was mimicked by the calcium ionophore ionomycin. Recruitment of D1 receptors from the interior of the cell to the membrane was confirmed by subcellular fractionation. The recruited D1 receptors were functional as demonstrated by an increase in dopamine-sensitive adenylyl cyclase activity in membranes derived from cells that had been pretreated with NMDA. These results provide evidence for regulated recruitment of a G protein-coupled receptor in neurons, provide a cell biological basis for the effect of NMDA on dopamine signaling, and reconcile the conflicting hyperdopaminergic and hypoglutamatergic hypotheses of schizophrenia.

20-Hydroxyeicosa-tetraenoic acid (20 HETE) activates protein kinase C. Role in regulation of rat renal Na+,K+-ATPase.

It is well documented that the activity of Na+,K+-ATPase can be inhibited by the arachidonic acid metabolite, 20-hydroxyeicosa-tetraenoic acid (20 HETE). Evidence is presented here that this effect is mediated by protein kinase C (PKC). PKC inhibitors abolished 20 HETE inhibition of rat Na+,K+-ATPase in renal tubular cells. 20 HETE caused translocation of PKC alpha from cytoplasm to membrane in COS cells. It also inhibited Na+,K+-ATPase activity in COS cells transfected with rat wild-type renal Na+,K+-ATPase alpha1 subunit, but not in cells transfected with Na+,K+-ATPase alpha1, where the PKC phosphorylation site, serine 23, had been mutated to alanine. PKC-induced phosphorylation of rat renal Na+,K+-ATPase, as well as of histone was strongly enhanced by 20 HETE at the physiologic calcium concentration of 1.3 microM, but not at the calcium concentration of 200 microM. The results indicate that phospholipase A2-arachidonic acid-20 HETE pathway can exert important biological effects via activation of PKC and that this effect may occur in the absence of a rise in intracellular calcium.