N-Cadherin-mediated cell adhesion is regulated by extracellular Zn(2+).

Synapses in the central nervous system (CNS) are highly dynamic structures that undergo reorganisation in response to synaptic activity. Dysfunctional structural synaptic plasticity is associated with impaired brain function and several neurological disorders. As response to synaptic activity, dendritic spines of excitatory synapses were reported to undergo alterations in their molecular structure and morphology leading to increased postsynaptic density size and spine volume. For these structural changes a transient activity-dependent weakening of synaptic adhesion will be necessary. Here, we report that zinc can modulate N-cadherin-mediated adhesion. Quantification of binding activity was performed using laser tweezer technique. Our results show that increased levels of zinc abolished N-cadherin binding without altering the number of N-cadherin molecules expressed at the cell surface. Furthermore, zinc directly interacted with N-cadherin and the regulatory role was found to take place under physiological zinc concentrations within minutes. Given that zinc is released at zincergic synapses in the CNS, our findings may contribute to mechanistic insights in the interplay between zinc signalling, activation of glutamate receptors and downstream pathways, and the coordination of pre- and postsynaptic changes via trans-synaptic cell adhesion complexes, all finally contributing to synaptic plasticity.

[The endoscopic management of the cubital tunnel syndrome--an anatomical study and first clinical results]. / Die endoskopische Dekompression des Kubitaltunnelsyndroms--Anatomische Grundlagen und erste klinische Ergebnisse.

Besides the carpal tunnel syndrome, the cubital tunnel syndrome (CuTS) represents the second most frequent nerve entrapment syndrome. The current gold standard for surgical therapy consists of simple open decompression. Recently, an endoscopic procedure involving long-distance decompression of the ulnar nerve has been developed and this is the topic of the present study. The first part of this paper describes preliminary anatomic investigations on 22 cadaver arms. In every sample we observed a thickening of the submuscular membrane between the heads of the flexor carpi ulnaris (FCU) which surrounds the ulnar nerve. This was especially the case for the first 10 cm from the medial epicondyle In the second part we report our experiences with this endoscopic decompression procedure in 36 patients. With this endoscopic decompression we achieved good to very good results according to the Bishop classification in 28 patients (78%). On the basis of anatomic considerations and our current experience, the endoscopic procedure seems to represent a promising alternative to simple decompression.

Different Ca2+ affinities and functional implications of the two synaptic adhesion molecules cadherin-11 and N-cadherin.

Cadherins of synaptic complexes are considered to be critically involved in long-term potentiation (LTP). Here we compared biophysical properties of cadherin-11 and N-cadherin, which appear to exert opposing effects on LTP, i.e., inhibition and promotion, respectively. Characterization of cadherin-11 binding by atomic force microscopy and laser tweezers revealed a significantly higher Ca(2+) affinity, with half-maximal binding (K(D)) at 0.11-0.26 mM Ca(2+), as compared to N-cadherin (K(D) approximately 0.7 mM Ca(2+)). Adhesive properties of both cadherins were modulated to a similar degree by manipulation of the actin cytoskeleton indicating to unlikely account for opposing roles in LTP induction. However, differences in Ca(2+) affinity could well explain opposing binding properties during activity-dependent transient reduction of extracellular Ca(2+) ([Ca(2+)](e)) in the synaptic cleft: whereas high frequency stimulation with drop of [Ca(2+)](e) to 0.3-0.8 mM Ca(2+) will result in significant weakening of N-cadherin adhesion, cadherin-11-based adhesion will stay mostly stable. Reduction of N-cadherin adhesion may facilitate synaptic remodeling and LTP induction, while cadherin-11 adhesion with its higher stability at low [Ca(2+)](e) may counteract this process explaining why in cadherin-11-deficient mice LTP is increased rather than decreased.

The role of VASP in regulation of cAMP- and Rac 1-mediated endothelial barrier stabilization.

Regulation of actin dynamics is critical for endothelial barrier functions. We provide evidence that the actin-binding protein vasodilator-stimulated phosphoprotein (VASP) is required for endothelial barrier maintenance. Baseline permeability was significantly increased in VASP-deficient (VASP(-/-)) microvascular myocardial endothelial cells (MyEnd) in the absence of discernible alterations of immunostaining for adherens and tight junctions. We tested whether VASP is involved in the endothelium-stabilizing effects of cAMP or Rac 1. Forskolin and rolipram (F/R) to increase cAMP and cytotoxic necrotizing factor 1 (CNF-1) to activate Rac 1 were equally efficient to stabilize barrier functions in VASP(-/-) and wild-type (wt) cells. In wt cells, VASP was phosphorylated in response to F/R but did not localize to intercellular junctions. In contrast, CNF-1 and expression of constitutively active Rac 1 induced translocation of VASP to cell borders in wt cells, where it colocalized with active Rac 1. In VASP(-/-) cells, Rac 1 activity was reduced to 0.4 of wt levels in controls and increased approximately 20-fold in response to CNF-1 compared with 7-fold activation in wt cells. Moreover, inactivation of Rac 1 by lethal toxin led to a greater increase of permeability compared with wt cells. All these data suggest that VASP is involved in the regulation of Rac 1 activity. Taking these findings together, our study indicates that VASP at least in part stabilizes endothelial barrier functions by control of Rho-family GTPases.

Differential role of Rho GTPases in endothelial barrier regulation dependent on endothelial cell origin.

From studies using macrovascular endothelium, it was concluded that Rho A activation generally leads to endothelial barrier breakdown. Here, we characterized the role of Rho GTPases in endothelial barrier regulation in four different cell lines, both microvascular and macrovascular. Rho A activation by cytotoxic necrotizing factor y (CNFy) induced stress fiber formation in all cell lines. This was paralleled by gap formation and barrier breakdown in microvascular mesenteric endothelial cells (MesEnd), human dermal microvascular endothelial cells (HDMEC) as well as in macrovascular pulmonary artery endothelial cells (PAEC) but not in microvascular myocardial endothelial cells (MyEnd). In MyEnd cells, activation of Rac 1 and Cdc42 by CNF-1 strengthened barrier properties whereas in MesEnd, HDMEC and PAEC all three GTPases were activated which increased permeability in PAEC but not in MesEnd and HDMEC. In PAEC, CNF-1-induced decrease of barrier properties was blocked by the Rho kinase inhibitor Y27632 indicating that co-activation of Rho A dominated the barrier response. Inactivation of Rac 1 by toxin B or by lethal toxin (LT) compromised barrier properties in all cell lines. Taken together, Rac 1 requirement for endothelial barrier maintenance but not the destabilizing role of Rho A seems to be ubiquitous.

Ischemia-induced increase of stiffness of alphaB-crystallin/HSPB2-deficient myocardium.

The two small heat shock proteins (sHSPs), alphaB-crystallin and HSPB2, have been shown to translocate within a few minutes of cardiac ischemia from the cytosol to myofibrils; and it has been suggested that their chaperone-like properties might protect myofibrillar proteins from unfolding or aggregation during stress conditions. Further evidence of an important role for HSPs in muscle function is provided by the fact that mutations of the alphaB-crystallin gene cause myopathy and cardiomyopathy. In the present study, we subjected isolated papillary muscles of alphaB-crystallin/HSPB2-deficient mice to simulated ischemia and reperfusion. During ischemia in alphaB-crystallin/HSPB2-deficient muscles, the development of contracture started earlier and reached a higher value compared to the wildtype mice. The recovery of contracture of alphaB-crystallin/HSPB2-deficient muscles was also attenuated during the simulated reperfusion period. However, twitch force was not significantly altered at any time of the experiment. This suggests that during ischemic insults, alphaB-crystallin/HSPB2 may not be important for the contraction process itself, but rather serve to maintain muscular elasticity.

Rho and rho kinase modulation of barrier properties: cultured endothelial cells and intact microvessels of rats and mice.

Previous experiments using cultured endothelial monolayers indicate that Rho-family small GTPases are involved in modulation of endothelial monolayer permeability by regulating assembly of the cellular actin filament scaffold, activity of myosin-based contractility and junctional distribution of the Ca2+-dependent endothelial cell adhesion molecule, VE-cadherin. We investigated these mechanisms using both cultured endothelial cells (from porcine pulmonary artery and mouse heart) and vascular endothelium in situ (mouse aorta, and individually perfused venular microvessels of mouse and rat mesentery). Exposure to Clostridium difficile toxin B (100 ng x ml(-1)) inactivated 50-90% of all endothelial Rho proteins within 60-90 min. This was accompanied by considerable reduction of actin filament stress fibres and junctional F-actin in cultured endothelial monolayers and in mouse aortic endothelium in situ. Also, VE-cadherin became discontinuous along endothelial junctions. Inhibition of Rho kinase with Y-27632 (30 microM) for 90-120 min induced F-actin reduction both in vitro and in situ but did not cause redistribution or reduction of VE-cadherin staining. Perfusion of microvessels with toxin B increased basal hydraulic permeability (L(p)) but did not attenuate the transient increase in L(p) of microvessels exposed to bradykinin. Perfusion of microvessels with Y-27632 (30 microM) for up to 100 min reduced basal L(p) but did not attenuate the permeability increase induced by platelet activating factor (PAF) or bradykinin. These results show that toxin B-mediated reduction of endothelial barrier properties is due to inactivation of small GTPases other than RhoA. Rho proteins as well as RhoA-mediated contractile mechanisms are not involved in bradykinin- or PAF-induced hyperpermeability of intact microvessels.

The catenin, p120ctn, is a common membrane-associated protein in various epithelial and non-epithelial cells and tissues.

The cadherin-binding catenin p120ctn was originally identified as an Src-tyrosine kinase substrate. More recently, p120ctn has been shown in some cell types to be associated with catenin/cadherin complexes of adherens junctions. To address the question whether p120ctn is restricted to certain cell types or whether it is a general cellular component we investigated tissue distribution of p120ctn by immunohistochemistry and immunoblotting in the rat. We found p120ctn to be widely distributed in several tissues where it is mainly restricted to the plasma membrane. In various epithelia p120ctn was found in association with different adherens junctions such as the zonula adherens and puncta adherentia. In addition, p120ctn was localized along infoldings of the basal cell membrane, most prominently in renal proximal and distal tubules. pl20ctn was not restricted to epithelia. It was also found at intercalated discs of cardiomyocytes. In the nervous system, immunostaining was particularly prominent in areas rich in synapses suggesting that pl20ctn is a component of synaptic adherens junctions as well. By immunoblotting, four different isoforms of pl20ctn could be detected displaying similar electrophoretic mobilities as the isoforms 1A, 1B, 2A, and 2B reported from mice. Whereas all epithelia assayed contained at least two isoforms, testis, heart, brain, and retina contained a single 110-kDa band that corresponds to isoform 1B in mice.

Binding of the stress protein alpha B-crystallin to cardiac myofibrils correlates with the degree of myocardial damage during ischemia/reperfusion in vivo.

Stress proteins are assumed to protect cells against various kinds of stresses including ischemia. In this study, we focused on the behaviour of the most abundant myocardial stress protein, alpha B-crystallin, during ischemia and reperfusion of the pig heart in vivo, alpha B-crystallin constitutes 1-2% of the soluble protein pool and underwent, during severe but reversibly damaging ischemia (25 min), complete translocation to the Z-line area of myofibrils. Irreversibly damaging ischemia (60 min) was accompanied by extreme stretching of the majority of myofibrils, and by concomitant extension of alpha B-crystallin localization from the Z-line area to I-bands. This I-band shift correlated with displacement of the T12 epitope of titin from the vicinity of Z-lines into I-bands, indicating that the primary binding sites for alpha B-crystallin might also be located in juxtaposition to Z-lines and move into the I-bands during extreme sarcomeric stretching. During reperfusion after 25 min of ischemia, alpha B-crystallin disappeared rapidly from myofibrils: whereas reperfusion after irreversibly damaging ischemia (60 min) resulted in dissociation of alpha B-crystallin only from those myofibrils and myocardiocytes that were still able to contract, and alpha B-crystallin remained bound to the overstretched, damaged myofibrils no longer capable of contraction. The time course of translocation of alpha B-crystallin to myofibrils during ischemia correlated with phosphorylation of approximately 20% of the entire alpha B-crystallin pool. However, disappearance of alpha B-crystallin from myofibrils during reperfusion was not accompanied by dephosphorylation, indicating that phosphorylation alone does not explain myofibrillar binding of alpha B-crystallin. Ischemia-induced myofibrillar targeting of alpha B-crystallin probably requires additional structural and posttranslational modifications of myofibrillar components in juxtaposition to I-bands.

Ischemia-induced phosphorylation and translocation of stress protein alpha B-crystallin to Z lines of myocardium.

It is becoming clear that stress proteins play a role in various aspects of postischemic myocardial recovery and that the cytoskeleton of cardiac myocytes is an important determinant for cellular survival during ischemia and energy depletion. In the present study, we addressed the question of whether the cytoskeleton-binding stress protein alpha B-crystallin may be involved in early cellular responses of rat and porcine myocardium to ischemia. Immunostaining and subcellular fractionation revealed a rapid ischemia-induced redistribution of alpha B-crystallin from a cytosolic pool to intercalated disks and Z lines of the myofibrils. This striking translocation of alpha B-crystallin from the cytosol to sites of the myofibrillar system that are known to be sensitive to ischemia-reperfusion injury was accompanied by a rapid shift of a fraction of alpha B-crystallin to a more acidic isoelectric point. This shift is caused by alpha B-crystallin phosphorylation, as identified by its augmentation in the presence of phosphatase inhibitors (vanadate, fluoride) and comigration of the acidic alpha B-crystallin form with the phosphorylated B1 form of lenticular alpha B-crystallin. In view of the chaperone-like function of alpha B-crystallin in conjunction with its high level of constitutive expression in the myocardium (1-2% of soluble protein content), we consider alpha B-crystallin an excellent candidate to play a role in early aspects of the protection of the myocardial contractile apparatus against ischemia-reperfusion injury.

Migrating transformed MDCK cells are able to structurally polarize a voltage-activated K+ channel.

Cell migration of transformed renal epithelial cells (MDCK-F) depends-in addition to cytoskeletal mechanisms-on the polarized activity of a Ca2+-sensitive K+ channel in the rear part of the cells. However, because of the lack of specific markers for this channel we are not able to determine whether a polarized distribution of the channel protein underlies its functional polarization. To determine whether the migrating MDCK-F cells have retained the ability to target K+ channels to distinct membrane areas we stably transfected the cells with the voltage-dependent K+ channel Kv1.4. Stable expression and insertion into the plasma membrane could be shown by reverse transcription-PCR, genomic PCR, Western blot, and patch-clamp techniques, respectively. The distribution of Kv1.4 was assessed with indirect immunofluorescence by using conventional and confocal microscopy. These experiments revealed that Kv1.4 is expressed only in transfected cells where it elicits the typical voltage-dependent, rapidly inactivating K+ current. The Kv1.4 protein is clustered at the leading edge of protruding lamellipodia of migrating MDCK-F cells. This characteristic distribution of Kv1.4 provides strong evidence that migrating MDCK-F cells are able to insert ion channels into the plasma membrane in an asymmetric way, which reflects the polarization of migrating cells in the plane of movement. These findings suggest that not only epithelial cells and nerve cells, but also migrating cells, can create functionally distinct plasma membrane areas.

Rapid activation of Na+/H+ exchange by aldosterone in renal epithelial cells requires Ca2+ and stimulation of a plasma membrane proton conductance.

There is increasing evidence for an additional acute, nongenomic action of the mineralocorticoid hormone aldosterone on renal epithelial cells, leading to a two-step model of mineralocorticoid action on electrolyte excretion. We investigated the acute effect of aldosterone on intracellular free Ca2+ and on intracellular pH in an aldosterone-sensitive Madin-Darby canine kidney cell clone. Within seconds of application of aldosterone, but not of the glucocorticoid hydrocortisone, there was a 3-fold sustained increase of intracellular Ca2+ at a half-maximal concentration of 10(-10) mol/liter. Omission of extracellular Ca2+ prevented this hormone response. In the presence of extracellular Ca2+ aldosterone led to intracellular alkalinization. The Na+/H+ exchange inhibitor ethyl-isopropanol-amiloride (EIPA) prevented the aldosterone-induced alkalinization but not the aldosterone-induced increase of intracellular Ca2+. Omission of extracellular Ca2+ also prevented aldosterone-induced alkalinization. Instead, aldosterone led to a Zn(2+)-dependent intracellular acidification in the presence of EIPA, indicative of an increase of plasma membrane proton conductance. Under control conditions, Zn2+ prevented the aldosterone-induced alkalinization completely. We conclude that aldosterone stimulated net-entry of Ca2+ from the extracellular compartment and a plasma membrane H+ conductance as prerequisites for the stimulation of plasma membrane Na+/H+ exchange which in turn modulates K+ channel acitivity. It is probable that the aldosterone-sensitive H+ conductance maintains Na+/H+ exchange activity by providing an acidic environment in the vicinity of the exchanger. Thus, genomic action of aldosterone determines cellular transport equipment, whereas the nongenomic action regulates transporter activity that requires responses within seconds or minutes, which explains the rapid effects on electrolyte excretion.

Actin and villin compartmentation during ATP depletion and recovery in renal cultured cells.

ATP-depletion in renal cultured cells has been used as a model for studying various cytoskeletal and functional alterations induced by renal ischemia. This communication explores the reversibility of these effects utilizing a novel method [1] that depleted ATP (ATP-D) to 2% of control within 30 minutes and caused complete recovery (REC) of ATP in one hour. Under confocal microscopy, ATP-D (30 min) caused thinning of F-actin from the microvilli, cortical region, and basal stress fibers, with the concurrent appearance of intracellular F-actin patches. These changes were more pronounced after 60 minutes of ATP-D. One hour of REC following 30 minutes of ATP-D produced complete recovery of F-actin in each region of the cell. However, after 60 minutes of ATP-D, a heterogeneous F-actin recovery pattern was observed: almost complete recovery of the apical ring and microvilli, thinned cortical actin with occasional breaks along the basolateral membrane, and a dramatic reduction in basal stress fiber density. The time course of cortical actin and actin ring disruption and recovery coincided with a drop recovery in the transepithelial resistance and the cytoskeletal dissociation and reassociation of the Na,K-ATPase. Additionally, the microvilli retracted into the cells during ATP-D, a process that was reversed during REC. Triton extraction and confocal microscopy demonstrated that villin remained closely associated with microvillar actin during both ATP-D and REC. These distinctive regional differences in the responses of F-actin to ATP depletion and repletion in cultured renal epithelial cells may help to clarify some of the differential tubular responses to ischemia and reperfusion in the kidney.

Mercapturic acid formation in cultured opossum kidney cells.

We investigated the last step of mercapturic acid formation, the N-acetylation of cysteine S-conjugates, in the established opossum kidney (OK) cell line which exhibits characteristics of the proximal tubule. S-Benzyl-L-cysteine was used as a model substance for such a cysteine S-conjugate. We succeeded in showing that OK cells absorb S-benzyl-L-cysteine via an active transport system which is inhibitable by phenylalanine. This transport follows Michaelis-Menten kinetics and the two characterizing parameters were determined: the Michaelis-Menten constant Km = 1.8 mmol/l, and the maximum of the difference between the intracellular and the extracellular concentration of S-benzyl-L-cysteine delta Cmax = 19.4 mmol/l. S-Benzyl-L-cysteine is converted to N-acetyl-S-benzyl-L-cysteine at a constant rate, which is independent of the extracellular S-benzyl-L-cysteine concentration. Under the tested experimental conditions this is probably due to saturation of the microsomal N-acetyltransferase catalyzing this reaction. In conclusion, we have shown that OK cells are a suitable model for studying mercapturate formation. They take up S-benzyl-L-cysteine mainly via the same carrier as phenylalanine, which is known to be transported in the rat by the high-capacity, low-affinity neutral amino acid carrier, and convert it to N-acetyl-L-benzyl-S-cysteine.