The cellular localization of Na+/H+ exchanger 1, cystic fibrosis transmembrane conductance regulator,potassium channel,epithelial sodium channel γ and vacuolar-type H+-ATPase in human eccrine sweat glands

The secretory portions of human eccrine sweat glands secrete isotonic fluid into the lumen and then the primary fluid is rendered hypotonic during its passage to the skin surface. During the processes of sweat secretion and absorption, many enzymes and proteins play important roles. In the study, the cellular localizations of Na⁺/H⁺ exchanger 1 (NHE1), cystic fibrosis transmembrane conductance regulator (CFTR), potassium channel (KC), epithelial sodium channel γ (γENaC) and vacuolar-type H+-ATPase (V-ATPase) in human eccrine sweat glands and epidermis were detected using immunofluorescence labeling. The results revealed that in the secretory coils, the basolateral membranes showed evidence of CFTR, NHE1 and KC activities, the apical membranes showed the activities of KC and NHE1, and the nucleus showed γEaNC and V-ATPase activities; in the duct, the peripheral and luminal ductal cells showed evidence of CFTR, NHE1 and KC, the apical membranes showed the activities of CFTR and NHE1, and the nucleus showed γEaNC, V-ATPase and KC activities. The cellular localization of these proteins in eccrine sweat glands is helpful to better understand the mechanisms of sweat secretion and absorption.     

Immunolocalisation of 11β-HSD-1 and -2, glucocorticoid receptor, mineralocorticoid receptor and Na+ K+-ATPase during the postnatal development of the rat epididymis

Glucocorticoids have been implicated in male reproductive function and 11β-HSD-1 and -2, the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), all of which are known to modulate glucocorticoid action, have been localised in the adult rat epididymis, but their developmental expression has not been investigated. Na(+)K(+)-ATPase activity, responsible for sodium transport, is induced by both mineralocorticoids and glucocorticoids in the kidney and colon, and has been localised in epididymal epithelium. This study examined the immunolocalisation of 11β-HSD-1 and -2, GR, MR and Na(+)K(+)-ATPase in rat epididymal epithelium (n = 5) at postnatal days (pnd) 1, 7, 15, 28, 40, 60, 75 and 104, and relative mRNA expression of 11β-HSD-1 and -2, and GR at pre-puberty (pnd 28) and post-puberty (pnd 75). 11β-HSD-1, GR and MR were localised in the epididymal epithelium from pnd 1, and 11β-HSD-2 and Na(+)K(+)-ATPase reactivity from pnd 15. At pnd 28 there was maximal immunoreactivity for both the GR and MR and 11β-HSD-1 and -2. 11β-HSD-1 mRNA expression in the caput increased from pre- to post-puberty, whereas 11β-HSD-2 mRNA expression fell over the same period (P < 0.01). GR mRNA expression was similar at pre- and post-puberty in both caput and cauda. Developmental changes in expression of 11β-HSD-1 and -2 suggest that overall exposure of the epididymis to glucocorticoids increases post-puberty, but cell-specific expression of the 11β-HSD enzymes still provides a capacity for intricate local control of glucocorticoid exposure.     

Integrin α1/β1 and α2/β1 as a receptor for IgA1 in human glomerular mesangial cells in IgA nephropathy

IgA nephropathy (IgAN) is characterized by mesangial deposition of IgA1 and galactose-deficient IgA1 is expected to play a pathogenic role. However, the identity of the receptor for IgA1 is still controversial. Hence, the aim of this study was to explore the receptor for galactose-deficient IgA1. Human monoclonal IgA1 was treated with exoglycosidase and FITC-conjugated control, asialo- and agalactosyl-IgA1 was used as a probe to detect the receptor in cultured human mesangial cells. Tumor necrosis factor-α or transforming growth factor-β1 treatment accelerated IgA1-binding on mesangial cells, and these effects were diminished by the addition of dexamethasone, whereas these changes were not dependent on galactose-deficiency of IgA1. According to comprehensive gene expression analysis, we focused on integrin β1. Pre-treatment by Mn(2+), which activates integrin by changing its structure, enhanced the binding of IgA1 in cultured mesangial cells. Furthermore, pre-incubation with collagens specifically enhanced binding of IgA1 in the cultured human mesangial cells without activation by Mn(2+). Collagen type IV distributed in the mesangial region of the glomeruli as well as Bowman's capsule and tubular basal membrane in IgAN patients, and the IgA1 with collagen type IV induced proliferative signals on mesangial cells by phosphorylating extracellular signal-regulated kinase more effectively than the IgA1 alone. Immunoprecipitation assay revealed the binding of IgA1 and integrin α1/β1 and α2/β1 heterodimer and down-regulation of integrin α1, α2 and β1 expression in human mesangial cells induced by each specific small interfering RNA diminished the ability to bind IgA1 probe. Integrin α1/β1 and α2/β1 would be a candidate receptor for IgA1.     

Bicarbonate secretion by rat bile duct brush cells indicated by immunohistochemical localization of CFTR, anion exchanger AE2, Na+/HCO3 − cotransporter, carbonic anhydrase II, Na+/H+ exchangers NHE1 and NHE3, H+/K+-ATPase, and Na+/K+-ATPase

The function of brush cells (BCs) is unknown. In a previous study, the rat common bile duct was examined by ultrastructural cytochemical methods for localizing HCO₃⁻, Cl⁻, and Na⁺ ions. All ion precipitates increased in or on BCs after secretin or meal stimulation, and it was proposed that BCs may secrete NaHCO₃. In this study, immunohistochemical localization of proteins known to be important in HCO₃⁻ secretion was investigated in the rat common bile duct. Immunoreactivity of proteins involved in Cl⁻/HCO₃⁻ exchange reaction, cystic fibrosis transmembrane conductance regulator (CFTR) and Cl⁻/HCO₃⁻ exchanger (AE2), was found on the microvilli (MV) and along the basolateral membrane (BLM) of BCs. The proteins involved in HCO₃⁻ production, Na⁺/HCO₃⁻ cotransporter (NBC), was found along the BLM but was absent on the MV, whereas carbonic anhydrase II (CA II) was observed on the MV and along the BLM. Of proteins responsible for the extrusion of H⁺, Na⁺/H⁺ exchanger 1 (NHE1) was localized along the BLM whereas Na⁺/H⁺ exchanger 3 (NHE3) was found on the MV and BLM. Activity of H⁺/K⁺-ATPase was found along the BLM and on the MV, and Na⁺/K⁺-ATPase was localized along the BLM. The immunoreactivity of most of these proteins was absent or weak in principal cells. These results strongly suggest that BCs are a significant source of HCO₃⁻ secretion.     

Comparing the expression of integrins αvβ3, αvβ5, αvβ6, αvβ8, fibronectin and fibrinogen in human brain metastases and their corresponding primary tumors

Aims: To evaluate the expression of αv-series integrins in brain metastases. Inhibitors targeting these integrins are being tested for their therapeutic potential. Material and Method: The extracellular regions of the αvβ3, αvβ5, αvβ6, αvβ8, the cytoplasmic domain of β3, the αv-chain, and the ECM molecules fibronectin and fibrinogen were studied immunohistochemically in a series of 122 carcinoma and 60 melanomas metastatic to the central nervous system. In addition, 38 matched primary and metastatic tumors to the brain were compared directly. Results: The αv-subunit was generally moderately to highly expressed in most tumors. αvβ3 and cytoplasmic β3 were weakly to moderately detectable in metastatic renal cell carcinomas and melanomas, αvβ5 was prominently expressed in metastatic renal and colorectal carcinomas, αvβ6 was most abundantly detectable in metastatic lung adenocarcinomas, but absent in melanomas. The tumor associated vessels in CNS metastases consistently expressed αvβ3, αvβ5, αv-, fibronectin and fibrinogen, however, mostly at low levels, while αvβ6, αvβ8 were lacking in vasculature. The comparative analysis of 38 matched primary tumors and brain metastases showed comparable levels of expression only for αvβ3 and αvβ8, while αvβ6 and αvβ5 were higher in primaries. Conclusion: We confirmed that integrin expression exhibits considerable heterogeneity according to tumor origin. αvβ5 is the most promising target for integrin targeted treatment in brain metastases.     

Regulation of the Na+2Cl–K+ cotransporter in isolated rat colon crypts

The Na(+2)Cl(-)K+ cotransporter accepts NH4+ at its K+-binding site. Therefore, the rate of cytosolic acidification after NH4+ addition to the bath (20 mmol/l) measured by BCECF fluorescence can be used to quantify the rate of this cotransporter. In isolated colon crypts of rat distal colon (RCC) addition of NH4+ led to an initial alkalinization, corresponding to NH3 uptake. This was followed by an acidification, corresponding to NH4+ uptake. The rate of this uptake was quantified by exponential curve fitting and is given in arbitrary units (delta fluorescence ratio units/1000 s). In pilot experiments it was shown that the pH signal caused by the Na(+)2Cl(-)K+ co-transporter could be amplified if the experiments were carried out in the presence of bath Ba2+ to inhibit NH4+ uptake via K+ channels. Therefore all subsequent experiments were performed in the presence of 1 mmol/l Ba2+. In the absence of any secretagogue, preincubation of RCC in a low-Cl- solution (4 mmol/l) for 10 min enhanced the uptake rate significantly from 1.70+/-0.11 to 2.54+/-0.27 U/1000 s (n=20). The addition of 100 mmol/l mannitol (hypertonic solution) enhanced the rate significantly from 1.93+/-0.17 to 2.84+/-0.43 U/1000 s (n=5). Stimulation of NaCl secretion by a solution containing 100 micromol/l carbachol (CCH) led to a small but significant increase in NH4+ uptake rate from 2.06+/-0.34 to 2.40+/-0.30 U/1000 s (n= 11). The increase in uptake rate observed with stimulation of the cAMP pathway by isobutylmethylxanthine (IBMX) and forskolin (100 micromol/l and 5 micromol/l, respectively) was from 2.39+/-0.24 to 3.06+/-0.36 U/1000 s (n=24). Whatever the mechanism used to increase the NH4+ uptake rate, azosemide (500 micromol/l) always reduced this rate to control values. Hence three manoeuvres enhanced loop-diuretic-inhibitable uptake rates of the Na(+)2Cl(-)K+ cotransporter: (1) lowering of cytosolic Cl- concentration; (2) cell shrinkage; (3) activation of NaCl secretion by carbachol and (4) activation of NaCl secretion by cAMP. The common denominator of all four activation pathways may be a transient fall in cell volume.     

Differences in the protein-kinase-A-dependent regulation of CFTR Cl– channels and Na+–K+ pumps in guinea-pig ventricular myocytes

Protein-kinase-A- (PKA-) dependent regulation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- current (I(CFTR)) and Na+-K+ pump current (Ip) was studied in single guinea-pig ventricular myocytes. Both currents were measured simultaneously by means of whole-cell recording at 30 degrees C. The adenylyl cyclase activator forskolin was used to stimulate PKA activity. At -20 mV, forskolin (4 microM) induced a fast activation of I(CFTR) and a delayed stimulation of Ip. Despite the strikingly different time courses, however, the potency of the drug to regulate both currents was identical. Half-maximal activation of I(CFTR) and stimulation of Ip, respectively, were observed at 9.6 x 10(-8) M and 9.9 x 10(-8) M forskolin. Inclusion of a specific peptide inhibitor of PKA in the pipette solution (PKI, 20 microM) blocked forskolin's effect on Ip. However, regardless of the time allowed for cell dialysis, there still was a marked, transient activation of I(CFTR), which could be prevented by: (1) a short pre-activation of I(CFTR) with forskolin or (2) the additional inclusion in the pipette solution of a synthetic peptide (Ht31 peptide, 60 microM) that interferes with PKA binding to its anchoring proteins. Thus, there is a tight functional coupling between PKA and CFTR Cl- channels in guinea-pig ventricular myocytes. The coupling is probably due to the close physical proximity of channels and kinases mediated by PKA anchoring proteins. Na+-K+ pumps, on the other hand, though also regulated by PKA, appear to be loosely coupled to the kinases.     

αvβ3, αvβ5 and αvβ6 integrins in brain metastases of lung cancer

Integrins are transmembranous adhesion molecules postulated to be involved in the brain metastatic cascade. We investigated the correlation of alpha v beta 3 (αvβ3), alpha v beta 5 (αvβ5) and alpha v beta 6 (αvβ6) integrin isoform expression with clinical characteristics including survival times in lung cancer patients with brain metastases (BM). All BM from lung cancer operated at our institution between 1990 and 2011, were identified; where available, primary tumors were retrieved as well. Immunohistochemical analysis for αvβ3, αvβ5 and αvβ6 integrin subunits was performed and correlated with Ki67 and hypoxia-inducible factor (HIF)-1α indexes. Clinical data including survival data were obtained by chart review. 191 BM specimens of 191 patients with histologically confirmed lung cancer (172 non-small cell lung cancer and 19 small cell lung cancer) were included. In 18 patients matched primary tumor samples were available. αvβ6 expression was commonly found on BM tumor cells (103/191; 53.9 %) and showed a significant association with low Ki67 proliferation indices (46 vs. 36 %, p = 0.001, Mann–Whitney U test) and favorable survival times (p = 0.020; log rank test) in patients with non-squamous NSCLC BM. αvβ5 expression was highly expressed on vascular structures (167/191; 87.4 %) and tumor stroma in BM (151/191; 79.1 %) and associated with high HIF-1α indices (60 vs. 90, p = 0.007, Mann–Whitney U test). αvβ3 expression was more frequently found on vascular structures in BM than in primary tumors (68.1 vs. 5.6 %; p = 0.645; Chi square test) and its expression in BM tumor cells correlated with low Ki67 indices (41 vs. 28 %; p = 0.046, Mann–Whitney U test). Expression of αv integrin subunits seem to be of pathobiological and clinical relevance in patients with NSCLC BM. Further investigations of their involvement in the brain metastatic cascade and their role as biomarkers are warranted.     

Long-term modulation of Na+ and K+ channels by TGF-��1 in neonatal rat cardiac myocytes

Previous work shows that transforming growth factor-β1 (TGF-β1) promotes several heart alterations, including atrial fibrillation (AF). In this work, we hypothesized that these effects might be associated with a potential modulation of Na(+) and K(+) channels. Atrial myocytes were cultured 1-2?days under either control conditions, or the presence of TGF-β1. Subsequently, Na(+) (I(Na)) and K(+) (I(K)) currents were investigated under whole-cell patch-clamp conditions. Three K(+) currents were isolated: inward rectifier (I(Kin)), outward transitory (I(to)), and outward sustained (I(Ksus)). Interestingly, TGF-β1 decreased (50%) the densities of I(Kin) and I(Ksus) but not of I(to). In addition, the growth factor reduced by 80% the amount of I(Na) available at -80?mV. This effect was due to a significant reduction (30%) in the maximum I(Na) recruited at very negative potentials or I(max), as well as to an increased fraction of inactivated Na(+) channels. The latter effect was, in turn, associated to a -7?mV shift in V(1/2) of inactivation. TGF-β1 also reduced by 60% the maximum amount of intramembrane charge movement of Na(+) channels or Q(max), but did not affect the corresponding voltage dependence of activation. This suggests that TGF-β1 promotes loss of Na(+) channels from the plasma membrane. Moreover, TGF-β1 also reduced (50%) the expression of the principal subunit of Na(+) channels, as indicated by western blot analysis. Thus, TGF-β1 inhibits the expression of Na(+) channels, as well as the activity of K(+) channels that give rise to I(Ksus) and I(Kin). These results may contribute to explaining the previously observed proarrhythmic effects of TGF-β1.     

Macrophage motility requires distinct α5β1/FAK and α4β1/paxillin signaling events

Macrophages function as key inflammatory mediators at sites of infection and tissue damage. Integrin and growth factor receptors facilitate recruitment of monocytes/macrophages to sites of inflammation in response to numerous extracellular stimuli. We have shown recently that FAK plays a role in regulating macrophage chemotaxis and invasion. As FAK is an established downstream mediator of integrin signaling, we sought to define the molecular circuitry involving FAK and the predominant β1 integrin heterodimers expressed in these cells-α4β1 and α5β1. We show that α4β1 and α5β1 integrins are required for efficient haptotactic and chemotactic invasion and that stimulation of these integrin receptors leads to the adoption of distinct morphologies associated with motility. FAK is required downstream of α5β1 for haptotaxis toward FN and chemotaxis toward M-CSF-1 and downstream of α4β1 for the adoption of a polarized phenotype. The scaffolding molecule paxillin functions independently of FAK to promote chemotaxis downstream of α4β1. These studies expand our understanding of β1 integrin signaling networks that regulate motility and invasion in macrophages and thus, provide important new insights into mechanisms by which macrophages perform their diverse functions.     

Large Na+ influx and high Na+, K+–ATPase activity in mitochondria-rich epithelial cells of the inner ear endolymphatic sac

Fluid in the mammalian endolymphatic sac (ES) is connected to the endolymph in the cochlea and the vestibule. Since the dominant ion in the ES is Na(+), it has been postulated that Na(+) transport is essential for regulating the endolymph pressure. This study focused on the cellular mechanism of Na(+) transport in ES epithelial cells. To evaluate the Na(+) transport capability of the ES epithelial cells, changes in intracellular Na(+) concentration ([Na(+)](i)) of individual ES cells were measured with sodium-binding benzofurzan isophthalate in a freshly dissected ES sheet and in dissociated ES cells in response to either the K(+)-free or ouabain-containing solution. Analysis of the [Na(+)](i) changes by the Na(+) load and mitochondrial staining with rhodamine 123 showed that the ES cells were classified into two groups; one exhibited an intensive [Na(+)](i) increase, higher Na(+), K(+)-ATPase activity, and intensive mitochondrial staining (mitochondria-rich cells), and the other exhibited a moderate [Na(+)](i) increase, lower Na(+), K(+)-ATPase activity, and moderate mitochondrial staining (filament-rich cells). These results suggest that mitochondria-rich ES epithelial cells (ca. 30% of ES cells) endowed with high Na(+) permeability and Na(+), K(+)-ATPase activity potentially contribute to the transport of Na(+) outside of the endolymphatic sac.     

Na+–K+ pump location and translocation during muscle contraction in rat skeletal muscle

Muscle contraction may up-regulate the number of Na(+)-K(+) pumps in the plasma membrane by translocation of subunits. Since there is still controversy about where this translocation takes place from and if it takes place at all, the present study used different techniques to characterize the translocation. Electrical stimulation and biotin labeling of rat muscle revealed a 40% and 18% increase in the amounts of the Na(+)-K(+) pump alpha(2) subunit and caveolin-3 (Cav-3), respectively, in the sarcolemma. Exercise induced a 36% and 19% increase in the relative amounts of the alpha(2) subunit and Cav-3, respectively, in an outer-membrane-enriched fraction and a 41% and 17% increase, respectively, in sarcolemma giant vesicles. The Na(+)-K(+) pump activity measured with the 3-O-MFPase assay was increased by 37% in giant vesicles from exercised rats. Immunoprecipitation with Cav-3 antibody showed that 17%, 11% and 14% of the alpha(1) subunits were associated with Cav-3 in soleus, extensor digitorum longus, and mixed muscles, respectively. For the alpha(2), the corresponding values were 17%, 5% and 16%. In conclusion; muscle contraction induces translocation of the alpha subunits, which is suggested to be caused partly by structural changes in caveolae and partly by translocation from an intracellular pool.     

Synoviocytes in chronic synovitis in situ and cytokine stimulated synovial cells in vitro neo-express α1, α3 and α5 chains of β1 integrins

The expression of the 1 integrins was examined immunohistochemically in synoviocytes from normal synovial membrane and from chronic synovitis of different aetiology and intensity. Normal synoviocytes were 61-positive but lacked 1 through 5. In mild inflammation type A synoviocytes neo-expressed 1, 3, and 5 chains. In severe inflammation both type A and B synoviocytes expressed 3, 4, 5, and 6 chains. The effects of inflammatory cytokines, as single agents or in combination, on the 1 integrin expression in cultured normal synoviocytes was determined by immunocytochemistry and flow cytometry. The 1 chain, while absent in unstimulated synoviocytes, was induced by interleukin-1 (IL-1), tumour necrosis factor- (TNF-), and interferon- (INF-). This effect was enhanced by combining IL-1 and TNF-. Expression of the 3 chain was up-regulated by IL-1 and, more intensely, by IFN-. Transforming growth factor (TGF-) inhibited the up-regulating effect of IL-1 and antagonized the effect of IFN- on 3 chain expression. Expression of the 5 chain was up-regulated significantly by co-stimulation through IL-1 together with TGF- or TNF-. Thus, the 1 integrin profile of cytokine activated synoviocytes in vitro resembled that of synoviocytes in synovitis in situ. These data suggest that IL-1, TNF-, IFN-, and TGF- are likely to be among the effectors regulating 1 integrin expression in synoviocytes in vivo.     

Fibroblast expression of α-smooth muscle actin, α2β1 integrin and αvβ3 integrin: influence of surface rigidity

Open wound contraction necessitates cell and connective tissue interactions, that produce tension. Investigating fibroblast responses to tension utilizes collagen coated polyacrylamide gels with differences in stiffness. Human foreskin fibroblasts were plated on native type I collagen-coated polyacrylamide gel cover slips with different rigidities, which were controlled by bis-acrylamide concentrations. Changes in alpha smooth muscle actin (αSMA), α2β1 integrin (CD49B) and αvβ3 integrin (CD-51) were documented by immuno-histology and Western blot analysis. Cells plated on rigid gels were longer, and expressed αvβ3 integrin and αSMA within cytoplasmic stress fibers. In contrast, cells on flexible gels were shorter, expressed α2β1 integrin and had fine cytoskeletal microfilaments without αSMA. Increased tension changed the actin makeup of the cytoskeleton and the integrin expressed on the cell's surface. These in vitro findings are in agreement with the tension buildup as an open wound closes by wound contraction. It supports the notion that cells under minimal tension in early granulation tissue express α2β1 integrin, required for organizing fine collagen fibrils into thick collagen fibers. Thicker fibers create a rigid matrix, generating more tension. With increased tension cytoskeletal stress fibers develop that contain αSMA and αvβ3 integrin that replaces α2β1 integrin, consistent with cell switching from collagen to non-collagen proteins interactions.     

Localization of the gene encoding the α2 subunit of the human VLA-2 receptor to chromosome 5q23-31

The ₂ subunit of the VLA-2 receptor (CD49B) was mapped to human chromosome 5 by several independent approaches. First, the expression of the ₂ subunit at the protein level was investigated in a panel of human-mouse hybrid cell lines. Cell surface expression was detected by indirect immunofluorescence with monoclonal anti-₂ antibody 12F1. Intracellular ₂ antigen was detected by immunostaining of whole cell extracts or of immunoprecipitated 12F1 antigen with the monoclonal antibodies 3H8 and 5C5. Second, the presence of human genomic ₂ sequences in the panel of human-mouse hybrids was detected by PCR, using primers derived from the published ₂ cDNA sequence. The specificity of the amplification product was shown by direct sequencing. The results of the PCR study were confirmed by amplifying aCD14 gene fragment, known to map to chromosome 5. Finally, in situ hybridization with a³H-labeled 1040-bp cDNA probe, also obtained by PCR, confirmed and refined the localization ofCD49B on chromosome 5 at q23-31.     

A d-amino acid containing peptide as a potent,noncovalent inhibitor of α5β1 integrin in human prostate cancer invasion and lung colonization

Primary tumors often give rise to disseminated tumor cells (DTC’s), which acquire full malignancy after invading distant site(s). Thus, DTC’s may be a productive target for preventing prostate cancer metastasis progression. Our prior research showed that PHSCN peptide (Ac-PHSCN-NH₂) targets activated α5β1 integrin to prevent invasion and metastasis in preclinical adenocarcinoma models, and disease progression in Phase I clinical trial. Here, we report that d-stereoisomer replacement of histidine and cysteine in PHSCN produces a highly potent derivative, Ac-PhScN-NH₂ (PhScN). PhScN was 27,000- to 150,000-fold more potent as an inhibitor of basement membrane invasion by DU 145 and PC-3 prostate cancer cells. A large increase in invasion–inhibitory potency occurred after covalent modification of the sulfhydryl group in PHSCN to prevent disulfide bond formation; while the potency of covalently modified PhScN was not significantly increased. Thus PhScN and PHSCN invasion inhibition occurs by a noncovalent mechanism. These peptides also displayed similar cell surface binding dissociation constants (K_d), and competed for the same site. Consistent with its increased invasion–inhibitory potency, PhScN was also a highly potent inhibitor of lung extravasation and colonization in athymic nude mice: it was several hundred- or several thousand-fold more potent than PHSCN at blocking extravasation by PC-3 or DU 145 cells, and 111,000- or 379,000-fold more potent at inhibiting lung colonization, respectively. Furthermore, systemic 5 mg/kg PhScN monotherapy was sufficient to cause complete regression of established, intramuscular DU 145 tumors. PhScN thus represents a potent new family of therapeutic agents targeting metastasis by DTC’s to prevent parallel progression in prostate cancer.     

The Na+2Cl–K+ cotransporter in the rectal gland of Squalus acanthias is activated by cell shrinkage

Effects of cAMP on Cl^– secretion, intracellular Cl^– activity and cell volume were studied in isolated perfused rectal gland tubules (RGT) of Squalus acanthias with electrophysiological and fluorescence methods. Recording of equivalent short-circuit current (I _sc) showed that cAMP stimulates Na⁺Cl^– secretion in a biphasic manner. The first and rapid phase corresponds to Cl^– exit via the respective protein-kinase-A- (PKA-) phosphorylated Cl^– conductance. The inhibitory effect of the loop diuretic furosemide (0.5 mmol/l, n=12) indicates that second phase reflects the delayed (1–2 min) activation of the Na⁺2Cl^–K⁺ cotransporter. During the first phase cytosolic Cl^– activity, as monitored by 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) fluorescence, fell to 78% (n=23) of the control value. Concomitantly, a transient fall in cell volume was recorded by calcein fluorescence to 92% (n=5) of the control value. Preincubation of the RGT with phalloidin (0.1 mmol/l, n=6) or cytochalasin D (0.1 mmol/l, n=4) almost completely prevented the development of the second phase of I _sc activation. When cytosolic Cl^– activity was increased by exposing the RGT to a high K⁺ concentration (25 mmol/l), in the presence of mannitol to prevent volume increases, stimulation was unaffected and biphasic. In contrast, when cell volume was clamped to an increased value (115%, n=8) by removing extracellular NaCl, the second phase was abolished completely (n=11). These data suggest that the primary and key process for triggering the Na⁺2Cl^–K⁺ cotransport is transient cell shrinkage. Received: 11 February 1999 / Accepted: 24 March 1999     

Role of PKC in the effects of α1-adrenergic stimulation on Ca2+ transients, contraction and Ca2+ current in guinea-pig ventricular myocytes

 The effects of α₁-adrenoceptor stimulation on intracellular Ca²⁺ transients, contractility and L-type Ca²⁺ current (I _Ca,L) were studied in single cells isolated from ventricles of guinea-pig hearts. The aim of our study was to elucidate the mechanisms of the positive inotropic effect of α₁-adrenergic stimulation by focussing on the role of protein kinase C (PKC). Phenylephrine, an α₁-adrenergic agonist, at concentrations of 50–100 μM elicited a biphasic inotropic response: a transient negative inotropic response (22.9±6.0% of control) followed by a sustained positive inotropic response (61.0±8.4%, mean±SE, n=12). The Ca²⁺ transient decreased by 10.2±3.9% during the negative inotropic phase, while it increased by 67.7±10% (n=12) during the positive inotropic phase. These effects were inhibited by prazosin (1 μM), a α₁-adrenergic antagonist. Phenylephrine increased the I _Ca,L by 60.8±21% (n=5) during the positive inotropic phase. To determine whether activation of PKC is responsible for the increases in Ca²⁺ transients, contractile amplitude and I _Ca,L during α₁-adrenoceptor stimulation, we tested the effects of 4β-phorbol 12-myristate 13-acetate (PMA), a PKC activator, and of bisindolylmaleimide I (GF109203X) and staurosporine, both of which are PKC inhibitors. PMA mimicked phenylephrine’s effects on Ca²⁺ transients, contractile amplitude and I _Ca,L. PMA (100 nM) increased the Ca²⁺ transient, contractile amplitude and I _Ca,L by 131±17%, 137±25% (n=8), and 81.1±26% (n=5), respectively. Prior exposure to GF109203X (1 μM) or staurosporine (10 nM) prevented the phenylephrine-induced increases in Ca²⁺ transients, contractile amplitude and I _Ca,L. Our study suggests that during α₁-adrenoceptor stimulation increase in I _Ca,L via PKC causes an increase in Ca²⁺ transients and thereby in the contractile force of the ventricular myocytes. Received: 16 July 1998 / Received after revision and accepted: 20 October 1998