Functional and molecular interactions between aquaporins and Na,K-ATPase.

The water channel aquaporin 4 (AQP4) is abundantly expressed in astrocytes and provides a mechanism by which water permeability of the plasma membrane can be regulated. Astrocytes play a key role in the clearance of both potassium (K(+)) and glutamate released during neuronal activity. Emerging evidence suggests that AQP4 facilitates K(+) clearance by astrocytes and contributes to recovery of neuronal excitability. Here we report that AQP4 can assemble with its regulator metabotropic glutamate receptor 5 (mGluR5) and with Na,K-ATPase; the enzyme responsible for active K(+) transport and for establishing the electrochemical gradient across the cell plasma membrane. We have, by use of pull down assays in rat brain tissue, identified the segment in the AQP4 NH(2)-terminus containing the amino acid residues 23-32 as the site for interaction with Na,K-ATPase catalytic subunit and with mGluR5. Mutagenesis studies revealed that the AQP4 amino acids K27 and W30 are of key importance for interaction with both Na,K-ATPase and mGluR5. To confirm that interaction also occurs within intact cells, we have performed fluorescence resonance energy transfer (FRET) studies in primary astrocytes derived from rat striatum. The results indicate close proximity of wild type AQP4 and Na,K-ATPase in the plasma membrane of rat astrocytes. FRET efficiencies observed with the mutants AQP4 K27A and AQP4 W30A were significantly lower, highlighting the importance of these residues for the interaction between AQP4 and Na,K-ATPase. We conclude that AQP4/Na,K-ATPase/mGluR5 can form a macromolecular complex/transporting microdomain in astrocytes. This complex may be of functional importance for the regulation of water and K(+) homeostasis in the brain, as well as for neuron-astrocyte metabolic crosstalk.

Phage display selection of Affibody molecules with specific binding to the extracellular domain of the epidermal growth factor receptor.

Affibody molecules specific for the epidermal growth factor receptor (EGFR) have been selected by phage display technology from a combinatorial protein library based on the 58-residue, protein A-derived Z domain. EGFR is overexpressed in various malignancies and is frequently associated with poor patient prognosis, and the information provided by targeting this receptor could facilitate both patient diagnostics and treatment. Three selected Affibody variants were shown to selectively bind to the extracellular domain of EGFR (EGFR-ECD). Kinetic biosensor analysis revealed that the three monomeric Affibody molecules bound with similar affinity, ranging from 130 to 185 nM. Head-to-tail dimers of the Affibody molecules were compared for their binding to recombinant EGFR-ECD in biosensor analysis and in human epithelial cancer A431 cells. Although the dimeric Affibody variants were found to bind in a range of 25-50 nM affinities in biosensor analysis, they were found to be low nanomolar binders in the cellular assays. Competition assays using radiolabeled Affibody dimers confirmed specific EGFR-binding and demonstrated that the three Affibody molecules competed for the same epitope. Immunofluorescence microscopy demonstrated that the selected Affibody dimers were initially binding to EGFR at the cell surface of A431, and confocal microscopy analysis showed that the Affibody dimers could thereafter be internalized. The potential use of the described Affibody molecules as targeting agents for radionuclide based imaging applications in various carcinomas is discussed.

Modulation of Na+,K+-ATPase activity is of importance for RVD.

AIM: This study was performed to examine the role of Na+,K+-ATPase activity for the adaptive response to cell swelling induced by hypoosmoticity, i.e. the regulatory volume decrease (RVD). METHODS: The studies were performed on COS-7 cells transfected with rat Na+,K+-ATPase. To study changes in cell volume, cells were loaded with the fluorescent dye calcein and the intensity of the dye, following exposure to a hypoosmotic medium, was recorded with confocal microscopy. RESULTS: Ouabain-mediated inhibition of Na+,K+-ATPase resulted in a dose dependent decrease in the rate of RVD. Total 86Rb+ uptake as well as ouabain dependent 86Rb+ uptake, used as an index of Na+,K+-ATPase dependent K+ uptake, was significantly increased during the first 2 min following exposure to hypoosmoticity. Since protein kinase C (PKC) plays an important role in the modulation of RVD, a study was carried out on COS-7 cells expressing rat Na+,K+-ATPase, where Ser23 in the catalytic alpha1 subunit of rat Na+,K+-ATPase had been mutated to Ala (S23A), abolishing a known PKC phosphorylation site. Cells expressing S23A rat Na+,K+-ATPase exhibited a significantly lower rate of RVD and showed no increase in 86Rb+ uptake during RVD. CONCLUSION: Taken together, these results suggest that a PKC-mediated transient increase in Na+,K+-ATPase activity plays an important role in RVD.

The newborn infant is protected by an innate antimicrobial barrier: peptide antibiotics are present in the skin and vernix caseosa.

BACKGROUND: Peptide antibiotics are part of the surface defences against microbial intruders. However, the presence and significance of these innate immune effectors in the skin barrier of the newborn infant have not yet been appreciated. Erythema toxicum neonatorum is an inflammatory skin reaction of unknown aetiology and significance, commonly present in the healthy newborn infant. OBJECTIVES: As peptide antibiotics are upregulated in inflammatory skin disorders, we hypothesized that this also could be the case in erythema toxicum. We also investigated if the vernix caseosa, a cream-like white substance present on the skin of the infant at birth, might contribute to host defences. METHODS: The presence of the human antibacterial peptide LL-37 was investigated by immunohistochemistry and confocal imaging of skin biopsies from four 1-day-old infants with an erythema toxicum rash and four matched newborns without the rash. In addition, we analysed the expression of LL-37 and human beta defensin-1, an antibacterial peptide of epithelial origin, by reverse transcriptase-polymerase chain reaction. Finally, we screened for antibacterial components in vernix material obtained from six healthy newborns by inhibition zone assays. RESULTS: All biopsies from the lesions of erythema toxicum showed a dense, nodular infiltrate with numerous LL-37-expressing cells located in the dermal layer and a clear localization of the peptide within CD15-expressing neutrophils, EG2-expressing eosinophils and CD1a-expressing dendritic cells. LL-37 was also found to be located in CD1a-expressing Langerhans cells and a positive staining for the peptide was seen throughout the whole epidermal layer, both in infants with and without the rash. Skin samples from infants with the rash of erythema toxicum showed a constitutive expression of human beta defensin-1, while the expression of LL-37 seemed to be induced. Furthermore, LL-37 and lysozyme were detected in the protein fractions derived from the vernix caseosa, and these fractions exhibited a clear antibacterial activity. CONCLUSIONS: Peptide antibiotics are present in the vernix caseosa and in the skin of the healthy newborn infant, indicating effective innate immune protection already during fetal and neonatal life.

beta-Adrenoceptor agonist sensitizes the dopamine-1 receptor in renal tubular cells.

The renal effects of dopamine are mainly mediated via the dopamine-1 receptor (D1 receptor). This receptor is recruited from intracellular compartments to the plasma membrane by dopamine and atrial natriuretic peptide (ANP), via adenylyl cyclase activation. We have studied whether isoproterenol, a beta-adrenoceptor (beta-AR) agonist that may interact with dopamine in the regulation of rat renal Na+, K+-adenosine triphosphatase (ATPase) activity, can recruit D1 receptors to the plasma membrane. The spatial regulation of D1 receptors was examined using confocal microscopy techniques in LLCPK cells and the functional interaction between dopamine and isoproterenol was examined by studying their effects on Na+, K+-ATPase activity in microdissected single proximal tubular segments from rat. Isoproterenol was found to translocate the D1 receptors from the interior of the cell towards the plasma membrane. The recruitment of dopamine 1 receptors was found to be cyclic adenosine phosphate (cAMP) dependent, while protein kinase C (PKC) activation was not involved. The functional studies on Na+, K+-ATPase activity showed that the effect of isoproterenol was abolished by a D1-like receptor antagonist (SCH 23390), and mediated via protein kinase A (PKA) and PKC dependent pathways. The results provide an explanation for the interaction between G protein-coupled receptors. The effects of isoproterenol on Na+, K+-ATPase activity can be explained by a heterologous recruitment of D1 receptors to the plasma membrane.

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 ([Ca(2+)](i)) oscillations that elicit activation of the transcription factor, NF-kappa B. Partial inhibition of Na,K-ATPase using low extracellular K(+) and depolarization of cells did not have these effects. Incubation of cells in Ca(2+)-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 [Ca(2+)](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 [Ca(2+)](i) oscillations prevented activation of NF-kappa B. 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 [Ca(2+)](i) oscillations involved in transcriptional regulation in mammalian cells.

Lens growth and protein density in the rat lens after in vivo exposure to ultraviolet radiation.

PURPOSE: To investigate lens growth after different doses of ultraviolet radiation (UVR) and to investigate the long-term effect of a near-threshold UVR dose on the refractive index distribution in the lens. METHODS: Sprague-Dawley rats received UVR (lambda(MAX) = 300 nm) unilaterally during a 15-minute period. The exposure dose ranged from 0.1 to 20 kJ/m(2), and the rats were kept for up to 32 weeks after exposure. Intact lenses were photographed and lens wet and dry masses were measured. The protein density was estimated by quantitative microradiography. Freeze-dried lens sections were used for contact x-ray photographs. From the transmission of the microradiographs, protein density and refractive index profiles were calculated along the lens radius with a resolution of 2.5 microm. RESULTS: Lens dry mass in exposed eyes was lower than in nonexposed eyes at one week after exposure. Lens water content was decreased after low UVR doses but increased after high doses. The difference between exposed and nonexposed lenses in dry mass and water content increased with time after exposure. No significant difference was found for the mean protein density in exposed and nonexposed lenses. The protein density increased linearly in the lens cortex, from a minimum in the superficial cortex of 0.26 g/cm(3) to a maximum in the deep cortex of 0.81 g/cm(3). This corresponded to a refractive index of 1.38 and 1.48, respectively. CONCLUSIONS: Lenses exposed to UVR grew more slowly than their nonexposed contralaterals. This growth inhibition was dose dependent. Near-threshold doses led to decreased water content in the lens whereas high doses led to swelling. Six months after near-threshold UVR exposure, no global change of the refractive index was found. However, local variations of the refractive index caused a subtle cortical light scattering.

Dopamine-induced translocation of protein kinase C isoforms visualized in renal epithelial cells.

Short-term regulation of sodium metabolism is dependent on the modulation of the activity of sodium transporters by first and second messengers. In understanding diseases associated with sodium retention, it is necessary to identify the coupling between these messengers. We have examined whether dopamine, an important first messenger in tubular cells, activates and translocates various protein kinase C (PKC) isoforms. We used a proximal tubular-like cell line, LLCPK-1 cells, in which dopamine was found to inhibit Na(+)-K(+)-ATPase in a PKC-dependent manner. Translocation of PKC isoforms was studied with both subcellular fractionation and confocal microscopy. Both techniques revealed a dopamine-induced translocation from cytosol to plasma membrane of PKC-alpha and -epsilon, but not of PKC-delta, -gamma, and -zeta. The process of subcellular fractionation resulted in partial translocation of PKC-epsilon. This artifact was eliminated in confocal studies. Confocal imaging permitted detection of translocation within 20 s. Translocation was abolished by a phospholipase C inhibitor and by an antagonist against the dopamine 1 subtype (D(1)) but not the 2 subtype of receptor (D(2)). In conclusion, this study visualizes in renal epithelial cells a very rapid activation of the PKC-alpha and -epsilon isoforms by the D(1) receptor subtype.

Confocal pH imaging of microscopic specimens using fluorescence lifetimes and phase fluorometry: influence of parameter choice on system performance.

We investigate the performance of confocal pH imaging when using phase fluorometry and fluorophores with pH-dependent lifetimes. In these experiments, the specimen is illuminated by a laser beam, whose intensity is sinusoidally modulated. The lifetime-dependent phase shift in the fluorescent signal is detected by a lock-in amplifier, and converted into a pH value through a calibration procedure. A theoretical investigation is made of how the different system parameters will influence the results concerning sensitivity and noise. Experiments carried out with the fluorophore SNAFL-2 support these theoretical predictions. It is found that, under realistic experimental conditions, we can expect a pH change of 0.1 units to be easily detected in an 8-bit digital image. However, the pixel-to-pixel root mean square noise is often of the order of one pH unit. This comparatively high level of noise has its origin in photon quantum noise. pH measurements on living cells show a systematic deviation from expected values. This discrepancy appears to be the result of fluorophore interaction with various cell constituents, and is the subject of further investigation.

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 [Ca2+]i response in target primary rat renal epithelial cells induced by the secreted RTX (repeats-in-toxin) toxin alpha-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 alpha-haemolysin in a renal epithelial cell line stimulated production of cytokines interleukin (IL)-6 and IL-8. Our findings indicate a novel role for alpha-haemolysin in pyelonephritis: as an inducer of an oscillating second messenger response in target cells, which fine-tunes gene expression during the inflammatory response.

Mechanisms by which intrarenal dopamine and ANP interact to regulate sodium metabolism.

Maintenance of a normal blood pressure requires a precise and fine-tuned regulation of salt metabolism. This is accomplished by a bidirectional regulation of renal tubular sodium transporters by natriuretic and antinatriuretic hormones. Dopamine, produced in the renal proximal tubular cells, plays an important role in this interactive system. Dopamine inhibits the activity of Na+,K+ ATPase as well as of many important sodium influx pathways in the nephron. These effects of dopamine are particularly pronounced in situation of sodium loading. There is an abundance of evidence suggesting that the natriuretic effects of ANP are to a large extent mediated via renal dopamine 1 like receptors. The renal tubular dopamine 1 like receptors are, under basal conditions, mainly located intracellularly. ANP and its second messenger, cGMP, cause a rapid translocation of the dopamine 1 like receptors to the plasma membrane. This phenomenon may explain how ANP and dopamine act in concert to regulate sodium metabolism. Regulation of sodium metabolism and blood pressure is critically dependent on a normal function of the renal dopamine system. Hence, abnormalities in the interaction between dopamine and ANP may predispose to hypertension.

Anatomical and physiological evidence for D1 and D2 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.

Receptor recruitment: a mechanism for interactions between G protein-coupled receptors.

There is a great deal of evidence for synergistic interactions between G protein-coupled signal transduction pathways in various tissues. As two specific examples, the potent effects of the biogenic amines norepinephrine and dopamine on sodium transporters and natriuresis can be modulated by neuropeptide Y and atrial natriuretic peptide, respectively. Here, we report, using a renal epithelial cell line, that both types of modulation involve recruitment of receptors from the interior of the cell to the plasma membrane. The results indicate that recruitment of G protein-coupled receptors may be a ubiquitous mechanism for receptor sensitization and may play a role in the modulation of signal transduction comparable to that of the well established phenomenon of receptor endocytosis and desensitization.

In situ microscopic analysis of asbestos and synthetic vitreous fibers retained in hamster lungs following inhalation.

Hamsters breathed, nose-only, for 13 weeks, 5 days/week, 6 hr/day, either man-made vitreous fiber (MMVF)10a, MMVF33, or long amosite asbestos at approximately 300 World Health Organization (WHO) fibers/cc or long amosite at 25 WHO fibers/cc. [World Health Organization fibers are longer than 5 microm and thicker than 3 microm, with aspect ratio >3.] After sacrifice, fiber burden was estimated (left lungs) by ashing and scanning electron microscopy (ashing/SEM) or (right middle lobes) by confocal laser scanning microscopy (CLSM) in situ. In situ CLSM also provided three-dimensional views of fibers retained, undisturbed, in lung tissue. Fibers of each type were lodged in alveoli and small airways, especially at airway bifurcations, and were seen fully or partly engulfed by alveolar macrophages. Amosite fibers penetrated into and through alveolar septa. Length densities of fibers in parenchyma (total length of fiber per unit volume of lung) were estimated stereologically from fiber transsections counted on two-dimensional optical sections and were 30.5, 25.3, 20.0, and 81.6 mm/mm3 for MMVF10a, MMVF33, and low- and high-dose amosite, respectively. Lengths of individual fibers were measured in three dimensions by tracking individual fibers through series of optical sections. Length distributions of amosite fibers aerosolized, but before inhalation versus after retention in the lung were similar, whether determined by ashing/SEM or in situ CLSM. In contrast, the fraction of short MMVF10a and MMVF33 fibers increased and the geometric mean fiber lengths of both MMVFs decreased by approximately 60% during retention. Most likely due to fiber deposition pattern and differences in sampling, fiber burdens [MMVF10a, MMVF33, and amosite (high dose; 269 WHO fibers/cc)] determined by ashing/SEM were 1.4, 1. 5, and 3.5 times greater, respectively, than those calculated from in situ CLSM data. In situ CLSM is able to provide detailed information about the anatomic sites of fiber retention and also fiber lengths and burdens in good agreement with ashing/SEM results.

Dopamine-induced recruitment of dopamine D1 receptors to the plasma membrane.

The recruitment of G protein-coupled receptors from the cytoplasm to the plasma membrane generally is believed to be a constitutive process. We show here by the use of both confocal microscopy and subcellular fractionation that, for at least one such receptor, this recruitment is regulated and not constitutive. Cells from a proximal tubular-like cell line, LLCPK1 cells, were incubated with either a D1 agonist, a dopamine precursor, or an inhibitor of dopamine metabolism to increase dopamine availability in the cell. Each of the three procedures led to a rapid translocation of dopamine D1 receptors from the cytosol to the plasma membrane.

Pain patterns in lumbar disc hernia Drawings compared to surgical findings in 159 patients.

We compared preoperative pain-drawing patterns of 159 patients with findings at lumbar disc surgery. Disc pathology was classified into 2 groups: intact anulus (negative exploration and protruding disc) and ruptured anulus (subligamentary perforation and sequestrated hernia). Patients with intact anulus drew fewer modalities, but more often marked pain in the trunk, neck and upper extremities, than those with ruptured anulus. Certain pain-drawing patterns--e.g., extra text, arrows and nonanatomical pain distribution--have previously been shown to correlate to unfavorable pathological traits in psychometric tests in populations of patients with chronic low back pain. In our study, most of these so-called nonorganic pain-pattern items occurred equally often in the 2 groups. In fact, one third of the patients with ruptured anulus produced pathological drawings according to the scoring system designed by Ransford et al. (1976). Although pain drawings help the investigator to obtain a rapid overview of the patient's pain pattern, the use of penalty points as a preoperative psychological screening instrument in patients with pain that is not chronic may be questioned.